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The Pennsylvania State University
The Graduate School
College of Agricultural Sciences
THE MAIZE RED ALEURONE1 ENCODES A FLAVONOID 3′-HYDROXYLASE
WHICH IS REQUIRED FOR THE BIOSYNTHESIS OF PURPLE
ANTHOCYANINS AND RED PHLOBAPHENES
A Dissertation in
Agronomy
by
Mandeep Sharma
© 2010 Mandeep Sharma
Submitted in Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy
May 2010
The dissertation of Mandeep Sharma was reviewed and approved* by the following:
Surinder Chopra Associate Professor of Maize Genetics Dissertation Advisor Chair of Committee
David Huff Associate Professor of Turfgrass Breeding and Genetics
David Braun Associate Professor of Biology
Kathleen Brown Professor of Postharvest Physiology
Seogchan Kang Professor of Plant Pathology
David Sylvia Professor of Soil Microbiology Head of the Department of Crop and Soil Sciences
*Signatures are on file in the Graduate School
iii
ABSTRACT
Maize is an economically important crop with most of the cultivated varieties
susceptible to corn ear worm and leaf blight fungus. One type of resistance to these
insects and pathogens has been attributed to the presence of C-glycosyl flavones and 3-
deoxyanthocyanidins. The 3'-hydroxylated products of these compounds are more toxic
than non-hydroxylated products. The phenylpropanoid pathway in plants leads to the
synthesis of several flavonoid end products that include anthocyanin and phlobaphene
pigments as well as C-glycosyl flavone and 3-deoxyanthocyanidin defense compounds in
maize. Flavonoid 3'-hydroxylase (F3'H) is the key enzyme required for generating
flavonoid compound diversity by 3'-hydroxylation of B-ring.
We have shown that red aleurone1/purple aleurone1 (pr1) gene is required for
F3'H activity in maize. Mutations in the pr1 locus lead to the accumulation of
pelargonidin (red) as oppose to cyanidin (purple) pigments in the wild-type aleurone
cells. Using heterologous probes and primers from sorghum and rice f3'hs, and sequence
information from maize genome, we isolated a putative maize f3'h1 sequence. PCR based
polymorphism analysis showed that the red kernel phenotype co-segregates with the
putative f3'h1 sequence. This polymorphism was due to the insertion of 24 dinucleotide
repeats in the upstream promoter region of the two pr1 alleles. A third mutant allele had a
17 bp deletion near the TATA box. Maize populations segregating for Pr1 and pr1 were
developed and a genetic ratio of 3:1 was observed for Pr1:pr1. Genetic mapping using
SNP markers based on the isolated f3'h1 gene sequence confirms the previously known
RFLP based map position of pr1 on chromosome 5L. Further, genetic complementation
iv
experiments using CaMV 35S::F3'H1 promoter-gene construct established that the
encoded protein product was capable of performing 3'-hydroxylation reaction both in
vitro and in vivo. The f3'h1 transcript was detected in floral as well as vegetative tissues
of plants segregating for Pr1. On the other hand, pr1 plants did not show any detectable
levels of f3'h1 mRNA indicating that the dinucleotide repeat insertion or the deletion in 5'
upstream promoter region in pr1 alleles have affected the steady state transcription of the
f3'h1 gene.
Biosynthesis of anthocyanin, phlobaphene, 3-deoxyanthocynanidin, and C-
glycosyl flavone is regulated by different regulatory genes. Here we showed that the pr1
gene plays important role in the biosynthesis of 3' hydroxylated products of these
flavonoid compounds and was regulated by two sets of transcription factors in separate
branches of the flavonoid pathway. In one pathway, pr1 was regulated by pericarp color
(p) gene to produce 3-deoxyanthocyanins and C-glycosyl flavones in maize floral tissues.
In an alternate pathway, pr1 played critical role in biosynthesis of 3-hydroxyanthocyanins
and was under the regulatory control of pair of transcription factors, red1 (r1) and
colorless1 (c1).
The expression of f3′h1 was required for the biosynthesis of luteoforol, one of the
flavan-4-ols that is precursor of the phlobaphenes. Flavan-4-ols are also the anticipated
precursors of antifungal 3-deoxyanthocyanidins. Our results indicated that Pr1 plants
accumulate significantly higher levels of luteolinidin in their silks when compared to its
levels in pr1 plants. Further analysis of silk extracts revealed accumulation of higher
amounts of maysin, a highly toxic insecticidal C-glycosyl flavone, in Pr1 plants
compared to pr1. These plants also showed enhanced resistance to corn earworm larvae.
v
Our study shows that the pr1 gene is playing an important role in generating diversity in
flavonoid compounds and has novel role in the biosynthesis of agronomically important
flavonoid defense compounds.
vi
TABLE OF CONTENTS
LIST OF FIGURES .....................................................................................................vii
LIST OF TABLES....................................................................................................... ix
ACKNOWLEDGEMENTS.........................................................................................x
Chapter 1 General Introduction ..................................................................................1
References…………………………………………………………………….19
Chapter 2 The maize red aleurone1 encodes a flavonoid 3′-hydroxylase which is required for the biosynthesis of purple anthocyanins ...........................................25
References…………………………………………………………………….61
Chapter 3 Tissue specific regulation of Zea mays pr1 gene is responsible for differential accumulation of insecticidal and antifungal flavonoid compounds...67
References……………………………………………………………………101
Chapter 4 Genetic control of purple anthocyanin formation in the aleurone tissue of maize ................................................................................................................107
References…………………………………………………………………….134
Chapter 5 Conclusions and perspectives.....................................................................138
References…………………………………………………………………….144
Appendix Corn ear worm silk feeding bioassay. ........................................................146
vii
LIST OF FIGURES
Figure 1.1: Flavonoid Biosynthetic pathway in maize……………………………8
Figure 2.1: Phenylpropanoid biosynthetic pathway leading to the production of anthocyanins. ........................................................................................................27
Figure 2.2: Phenotypes of pr1 and Pr1 maize ears......................................................29
Figure 2.3: Multiple sequence alignment of deduced amino acid sequences of F3'H from maize and other plant species..............................................................40
Figure 2.4: Un-rooted phylogenetic tree showing the evolutionary relationships of maize F3'H and other plant F3'Hs.........................................................................41
Figure 2.5: Characterization of λZT2-1 clone and pr1 lesion ....................................42
Figure 2.6: Alignment of the nucleotide sequences of Pr1 and pr1 surrounding the dinucleotide repeat region in the upstream promoter area of pr1.........................43
Figure 2.7: f3'h1 maps to the pr1 locus on chromosome 5L .......................................45
Figure 2.8: Mutant pr1 plants do not accumulate f3'h1 transcript...............................46
Figure 2.9: Mutant plants with red aleurone produce pelargonidin while wild-type plants with purple aleurone produce cyanidin ......................................................49
Figure 2.10: Complementation of mutant f3'h phenotypes of maize and Arabidopsis with f3'h1 ..........................................................................................51
Figure 2.11: Anthocyanin accumulation in wild-type and transgenic Arabidopsis plants.....................................................................................................................54
Figure 3.1: Flavonoid biosynthetic pathway in maize .................................................69
Figure 3.2: Cob pigmentation phenotype of pr1 alleles in different p1 backgrounds..........................................................................................................81
Figure 3.3: Absorption spectra of methanolic extracts from cob glumes of pr1 alleles in the presence of functional and non-functional p1 alleles ......................82
Figure 3.4: Expression of pr1 gene is up-regulated in the presence of a functional p1 and/or p2 genes ................................................................................................85
viii
Figure 3.5: Characterization of the 3-deoxyanthocyanidin in Pr1 and pr1 silk tissue. HPLC chromatograms of silk methanolic extracts from Pr1 and pr1 at 480 nm ..................................................................................................................87
Figure 3.6: Quantitative analysis of total luteolinidin levels in silk tissue of pr1 alleles in the presence of functional and non-functional p1alleles .......................88
Figure 3.7: Characterization of the C-glycosyl flavones in Pr1 and pr1 silks. HPLC chromatograms of silk methanolic extracts from Pr1 and pr1 at 340 nm .........................................................................................................................90
Figure 3.8: Maysin, apimaysin, rhamnosyl isoorientin, and chlorogenic acid levels in silk tissue of pr1 alleles in the presence of functional and non-functional p1 alleles ...............................................................................................................92
Figure 3.9: Insect silk feeding bioassay to test the effect of differential accumulation of C-glycosyl flavones in pr1 alleles on corn earworm growth and development...................................................................................................93
Figure 4.1: Anthocyanin biosynthetic pathway in maize.............................................110
Figure 4.2: Genetic analysis to study the regulation of pr1 by c1 and r1 during purple anthocyanin synthesis in kernel aleurone ..................................................120
Figure 4.3: Test cross analysis to confirm the genetic analysis that c1 and r1 are required for pr1 expression during anthocyanin biosynthesis ..............................121
Figure 4.4: Expression analysis to demonstrate that functional c1 and r1 are required for accumulation of pr1 transcript. Diagrammatic representation of pr1 promoter structure showing the repsence of C1 binding site (CBS) and anthocyanin regulatory element (ARE) ................................................................122
Figure 4.5: Genetic analysis demonstrating that the pr1 gene is required for cyanidin formation through anthocyanin pathway ...............................................124
Figure 4.6: Anthocyanin analysis of the aleurone tissue collected from purple, red, and colorless kernels of F2 segregating ears. ........................................................125
Figure 4.7: Reverse phase HPLC analysis of anthocyanin pigments extracted from kernel aleurone......................................................................................................126
Figure 4.8: Quantitative analysis of anthocyanin content of the aleurone tissue of purple, red, and colorless (cl) kernels in genetic background of mutant anthocyanin genes.................................................................................................127
Figure 4.9: Analysis of anthocyanin accumulation in maize aleurone during its development..........................................................................................................129
ix
LIST OF TABLES
Supplemental Table 2.1: Sequences of primers used in reverse transcription analysis. ................................................................................................................66
Table 3.1: Genotype and phenotype of different lines developed and used in this study......................................................................................................................75
Table 4.1: Genotype and kernel phenotype of different anthocyanin mutant stocks used in this study. .................................................................................................114
Table 4.2: PCR primers used in reverse transcription gene expression analysis.........118
x
ACKNOWLEDGEMENTS
My PhD thesis is the product of a wonderful cooperation with many people and it
is my pleasure to have the opportunity to express my gratitude to them all in my humble
acknowledgement.
I am heartily thankful to my supervisor, Dr. Surinder Chopra for the opportunity
to do this research in his laboratory and for his supervision, advice, and guidance through
out these years of my PhD study. Above all and the most needed, he provided me
unflinching encouragement and support in various ways. His truly scientist intuition has
made him a constant oasis of ideas and passion in science, which exceptionally inspire
and enrich my growth as student, researcher and scientist.
I am especially indebted to my research committee members Drs: David Braun,
Kathleen Brown, David Huff, Seogchan Kang, and Dawn Luthe, for their valuable
discussions. Their constructive criticism and helpful suggestions have been of great
impact on shaping my research ideas and improving my scientific thinking.
My deep gratitude and appreciation also goes to present and past members of
Chopra lab: Dr. Rajandeep Sekhon, Dr. Farag Ibraheem, Dr. Michael Robbins, PoHao
Wang, and Dr. Iffa Gaffoor, for their invaluable support, useful discussions during our
exciting lab meetings, and suggestions through out the course of my research and writing
times. I would also like to thank numerous talented undergraduate students for their great
help in lab and field work at different stages of my PhD studies.
I owe sincere thanks to the hard working staff in the Crop and Soil Sciences’
academic unit and the Penn State international programs for their effort to help us not
xi
worried about anything but research. I offer my regards and blessings to all of those who
supported me in any respect during the completion of the project.
I am indebted to my wife Shubra for her wonderful support, love, and care. Above
all, no words can express my gratitude to my parents whose blessings, unconditional
love, sacrifices, and never-ending support allowed me to be in this position today. I
dedicate this thesis to them.
Finally, I am thankful to almighty for everything I have.
Chapter 1
General Introduction
Different plant pigments
Plant pigments play an important role in attracting pollinators in seed dispersing
organisms. Their beauty prompted horticulturalists to develop hybrids of ornamental
plants with altered colors. Through these crosses, pioneer researchers such as Gregor
Mendel discovered many of the genetic principles that are taken for granted today. In
nature three types of chemically distinct pigments are responsible for plant colors:
betalains, carotenoids, and anthocyanins. Betalains are water soluble, nitrogen containing
compounds which are synthesized from the amino acid tyrosine. There are two major
classes of betalains: betacyanins and betaxanthins (STRACK et al. 2003). Betacyanins give
red to violet colors, such as those found in red beets (Beta vulgaris) or in the flower of
portulaca (Portulaca grandiflora), while betaxanthins give yellow to orange colors
(CHRISTINET et al. 2004). Betalains are distributed only among the members of the plant
order Caryophyllales. Carotenoids are lipid soluble C40 tetraterpenoids and are
universally distributed in the plant kingdom. Carotenoids are synthesized from
isopentenyl diposphate. They are precursors of plant hormone abscisic acid and provide
photoprotection during photosynthesis (CUNNINGHAM and GANTT 1998; HIRSCHBERG
2001). Carotenoids are also precursors of vitamin-A biosynthesis and therefore, are
important component of animal diet (FRASER and BRAMLEY 2004). Carotenoids are
responsible for most of the yellow and orange flower colors in ornamentals like marigold
2
(Tagetes patula), Narcissus, Gerbera, and Rosa. Anthocyanins are water soluble
pigments that occur in almost all vascular plants (FORKMANN 1991). They are responsible
for most of the orange, red, purple, and blue colors in plants (WINKEL-SHIRLEY 2001).
Anthocyanin biosynthesis takes place through the flavonoid pathway which is the focus
of this study.
Flavonoid compounds
Flavonoids represent a large family of low molecular weight polyphenolic
secondary metabolites that are widely spread throughout the plant kingdom, ranging from
mosses to angiosperms (KOES et al. 1994). Previously, flavonoids were regarded as
dispensable phytochemicals. Although the most visible function of flavonoids is the
formation of red and purple anthocyanin pigments, recent studies have established that
flavonoids play vital roles in the developmental processes of plants (TAYLOR and
GROTEWOLD 2005). Different flavonoid compounds share the same basic skeleton, the
flavan-nucleus, consisting of two aromatic rings with six carbon atoms (ring A and B)
interconnected by a hetero-cycle including three carbon atoms (ring C). There are more
than 6000 flavonoid compounds which constitute a relatively diverse family of aromatic
molecules (WINKEL-SHIRLEY 2001). There are six major subgroups of these compounds
that are found in higher plants: the chalcones, flavones, flavonols, flavandiols,
anthocyanins, and condensed tannins (HARBORNE and WILLIAMS 2000). Some plant
species also synthesize specialized forms of flavonoids, such as isoflavonoids in legumes
and 3-deoxyanthocyanins in sorghum, maize, and gloxinia (WINKEL-SHIRLEY 2001).
3
According to the modifications of the central C-ring they can be divided in above
mentioned structural classes.
Function of flavonoids in plants
Flavonoids have many functions in the biochemistry, physiology, and ecology of
plants and are important in both human and animal nutrition (TAYLOR and GROTEWOLD
2005). Functions of these pigments have been implicated in attracting pollinators
(GIURFA et al. 1995), protection from UV damage (LI et al. 1993; STAPLETON and
WALBOT 1994), regulation of auxin transport (BROWN et al. 2001), signaling molecules
in plant bacterium symbiosis (HIRSCH 1992), germination of pollen (NAPOLI et al. 1999;
TAYLOR and MILLER 2002), and as defense compounds against biotic stresses (BYRNE et
al. 1996; HAHLBROCK and SCHEEL 1989). Plant coloration is not only attractive for
pollinators and seed distribution, but also provides aesthetically valuable characteristics
for humans (GROTEWOLD 2006).
Flavonoids modulate auxin transport in a tissue specific manner. They directly
modulate auxin transport at the site of auxin synthesis (shoot-apex) and redirection (root-
tip) (JACOBS and RUBERY 1988). Auxin transport from shoot tip to root tip is enhanced in
the absence of flavonoids and reduced in the presence of excess flavonols, kaempferol
and quercetin (MURPHY et al. 2000). The flavonols are also required for functional pollen
formation in monocots and dicots as well as in other angiosperms and gymnosperms
(ESCRIBANO-BAILON et al. 2004; KOES et al. 1994). It has been shown that flavonol-
deficient pollen failed to produce a functional pollen tube (COE et al. 1981; NAPOLI et al.
1999). Flavonoids also play an important role in nodule formation in legumes. They are
4
exuded by roots and act as chemo-attractants to nodule forming rhizobia (ECKARDIT
2006). Flavonoids also activate rhizobial nod genes which produce nod factors
(KOBAYASHI et al. 2004). Nod factors are perceived by plant receptors to initiate root hair
curling and subsequent rhizobial entry and nodule development (WASSON et al. 2006).
Flavonoid compounds like anthocyanin are induced in response to various
environmental stresses, including high light. Since plant flavonoids have absorption
spectra in the UV light range, these pigments have been hypothesized to play role in
shielding plant DNA from UV radiation damage (WOO et al. 2005). Flavonoid mutant
maize lines show significantly more DNA damage than wild-type lines which accumulate
flavonoid compounds in the plant body (STAPLETON and WALBOT 1994).
Function of flavonoids in humans and animals
It is well known that diets rich in fruits and vegetables are protective against
cardiovascular diseases, certain forms of cancers, and other diseases (LEE et al. 2007;
SHIH et al. 2007). These protective effects have been attributed to antioxidant nutrients:
vitamin C, β-carotene, and plant phenolics such as flavonoids. Flavonoids are
multifunctional and can act as reducing agents, hydrogen donating antioxidants, and
oxygen quenchers. In some forage crops, the accumulation of moderate levels of
proanthocyanidins confer bloat safety and protein protection in ruminant systems
(FORKMANN and MARTENS 2001).
The antioxidant activity of flavonoids as hydrogen donating radical scavengers is
due to the availability of the phenolic hydrogens. Flavonol quercetin and the flavan-3-ol
epicatechin gallate, which are constituents of green tea, black tea, and red wine, have a
5
five fold higher total antioxidant activity than vitamins E and C (RICE-EVANS et al.
1997). In addition to free radical scavenging activity, flavonoids also have other
biological activities such as, vasodilatory, anticarcinogenic, anti-inflammatory,
antiallergic, antiviral, and estrogenic (RICE-EVANS et al. 1996).
It has been established that the position and degree of hydroxylation is
fundamental to the antioxidant property of flavonoids, particularly in terms of the o-
dihyroxylation of the B-ring. The flavonol Kaempferol, which has a lone 4'-OH group in
the B-ring, has 27% of antioxidant activity of quercetin which has an additional OH
group at 3' position of B-ring (RICE-EVANS et al. 1996).
Flavonoids as model system
In addition to their vital function in plant growth and development, flavonoid
pigments have been used as a tool to study very basic questions in biology (CHOPRA et al.
2006). Past utilization of flavonoids include Mendel’s use of seed coat color and flower
color in peas (Pisum sativum) to develop the laws of heredity and Nobel laureate Barbara
McClintock’s discovery of transposable elements by following anthocyanin pigment
formation in maize kernels. More recently, these compounds have helped in unraveling
the phenomenon of RNA induced gene silencing by over expressing an anthocyanin
biosynthetic gene in petunia (Petunia hybrida) (NAPOLI et al. 1990). The flavonoid
biosynthetic pathway is an extensively studied pathway which contributes to making
flavonoids an excellent model system to study gene interactions (KOES et al. 2005).
Many flavonoid structural and regulatory genes have been isolated in several plant
species based on the alteration in flower and seed pigmentation (HOLTON and CORNISH
6
1995). Flavonoid pigments provide a natural reporter system that has been used to study
the regulation of gene expression and the channeling and intracellular transport of
metabolites (CHOPRA et al. 2006; IRANI et al. 2003; LEE et al. 1998).
Flavonoid biosynthesis in maize
The flavonoid biosynthetic pathway is derived from the amino acid phenylalanine
and is one of the best understood plant secondary metabolite pathways. A diagram of the
flavonoid biosynthetic pathway, the basic structure of the flavonoid molecule, and
different organs of maize are shown in Figure 1.1. Several other major classes of plant
products in addition to flavonoids are derived from phenylalanine, including lignin,
coumarines, stilbenes, and benzoic acid derivatives (DIXON and PAIVA 1995; HOLTON
and CORNISH 1995). All these compounds share common initial steps through the general
phenylpropanoid pathway. Phenylpropanoids are derived from the shikimic acid
pathway, which participates in the biosynthesis of most plant phenolics. Shikimic acid is
also the precursor for synthesis of three essential aromatic amino acids: phenylalanine,
tyrosine, and tryptophan. Phenylalanine drives the phenylpropanoid pathway through
which tannin, flavonoid, and lignin biosynthesis originate. Phenylpropanoid compounds
are so named because of the basic structure of a three-carbon side chain on an aromatic
ring, which is derived from phenylalanine. In this pathway, phenylalanine ammonia lyase
(PAL) deaminates phenylalanine to cinnamic acid, which undergoes aromatic
hydroxylation by cinnamate 4-hydroxylase (C4H) to form p-coumaric acid (HAHLBROCK
and SCHEEL 1989). Then 4-coumarate:CoA ligase (4CL) catalyzes the activation of p-
coumaric acid to form p-coumaroyl-CoA. These compounds could be shunted to either
7
simple compounds such as chlorogenic acid and flavonoids or to complex compounds
such as lignin. The first committed step of the flavonoid pathway is the condensation of
three malonyl-CoA molecules, which are derived from carbohydrate metabolism, with p-
coumaroyl-CoA to produce a chalcone; this step is catalyzed by the enzyme chalcone
synthase (CHS). In maize, CHS is encoded by duplicate genes, colorless2 (c2) and white
pollen1 (whp1) (COE et al. 1988). The second step is isomerization of the chalcone to
naringenin, which occurs through the action of chalcone-flavanone isomerase (CHI),
encoded by chalcone isomerase1 (chi1) (GROTEWOLD and PETERSON 1994). Naringenin
is the key product of this pathway as it gives rise to different branches which leads to the
production of various flavonoid compounds, e.g., anthocyanins, phlobaphenes, 3-
deoxyanthocyanidins, and C-glycosyl flavones in maize. These compounds are
synthesized in a tissue specific fashion in maize. Anthocyanins accumulate in the kernel
aleurone and plant body, while phlobaphenes accumulate in floral tissues such as,
pericarp, cob-glumes, tassel-glumes, and silks, 3-deoxyanthocyanidins, and C-glycosyl
flavones accumulate in silks.
Anthocyanin synthesis in maize
In maize kernels, the major anthocyanin constituents are pelargonidin and
cyanidin. During the synthesis of anthocyanins, naringenin, a flavanone, is converted to
the dihydroflavonols dihydroquercetin (DHQ) and dihydrokaemferol (DHK) through
hydroxylation of the carbon 3 of the C ring by flavanone-3-hydroxylase (F3H) (DEBOO et
al. 1995). Both flavanones and dihydroflavonols can be hydroxylated on the 3' position of
the B-ring by flavonoid 3'-hydroxylase (F3'H) (LARSON et al. 1986).
8
Lemma
Palea
Cob glumesKernel
PericarpAleurone
Naringenin
4-Coumaroyl-CoA 3 Malonyl-CoA+
CHI
CHS
Chalcone
c2
chi1
Leucopelargonidin
F3'H
pr1
F3'H
pr1Eriodictyol
Dihydroquercitin
F3H
Cyanidin
DFR
ANS
GT
Leucocyanidin
fht1
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
a1
a2
bz1
Anthocyanin 3-glycoside
Vacuolar Anthocyanin
GSTbz2
MRPZmMrp
DFRa1
Apiferol LuteoforolF3'H
Apigeninidin
Phlobaphenes
F3'H
Luteolinidin
Poly
mer
izat
ion
3-Deoxyanthocyanidins
pr1
pr1
bp1?
FNS Luteolin Apimaysin
Maysin
F3'Hpr1
Lemma
Palea
Cob glumesKernel
PericarpAleurone
Lemma
Palea
Cob glumes
Lemma
Palea
Cob glumesKernel
PericarpAleurone
Kernel
PericarpAleurone
Naringenin
4-Coumaroyl-CoA 3 Malonyl-CoA+
CHI
CHS
Chalcone
c2
chi1
Naringenin
4-Coumaroyl-CoA 3 Malonyl-CoA+
CHI
CHS
Chalcone
c2
chi1
Leucopelargonidin
F3'H
pr1
F3'H
pr1Eriodictyol
Dihydroquercitin
F3H
Cyanidin
DFR
ANS
GT
Leucocyanidin
fht1
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
a1
a2
bz1
Leucopelargonidin
F3'H
pr1
F3'H
pr1Eriodictyol
Dihydroquercitin
F3H
Cyanidin
DFR
ANS
GT
Leucocyanidin
fht1
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
a1
a2
bz1
Anthocyanin 3-glycoside
Vacuolar Anthocyanin
GSTbz2
MRPZmMrp
Anthocyanin 3-glycoside
Vacuolar Anthocyanin
GSTbz2
MRPZmMrp
DFRa1
Apiferol LuteoforolF3'H
Apigeninidin
Phlobaphenes
F3'H
Luteolinidin
Poly
mer
izat
ion
3-Deoxyanthocyanidins
pr1
pr1
bp1?
DFRa1
Apiferol LuteoforolF3'H
Apigeninidin
Phlobaphenes
F3'H
Luteolinidin
Poly
mer
izat
ion
3-Deoxyanthocyanidins
pr1
pr1
bp1?
Apiferol LuteoforolF3'H
Apigeninidin
Phlobaphenes
F3'H
Luteolinidin
Poly
mer
izat
ion
3-Deoxyanthocyanidins
pr1
pr1
bp1?
FNS Luteolin Apimaysin
Maysin
F3'Hpr1FNS Luteolin Apimaysin
Maysin
F3'Hpr1
Figure 1.1: Flavonoid biosynthetic pathway in maize. Chemical structure shows basic skeleton ofa flavonoid molecule. Position of pericarp and aleurone in kernel as well as palea and lemma incob glume are shown. Gene (enzyme) abbreviations: c2 (CHS), chalcone synthase; chi (CHI), chalcone isomerase; f3h (F3H), flavanone 3-hydroxylase; pr1 (F3'H), flavonoid 3'-hydroxylase; a1 (DFR), dihydroflavanone reductase; a2 (AS), anthocyanidin synthase; bz1 (UFGT), UDP-glucose flavonoid 3-O-glucosyltransferase; bz2 (GST), glutathione S-transferase; ZmMrp (MRP), multidrug resistance-like transporter; FNS1, flavone synthase; and brown pericarp1 (bp1) (Emerson et al. 1935; Meyers 1927).
9
The red aleurone1 (pr1) gene has been suggested to encodes for F3'H. This assignment
of the pr1 locus as the f3'h gene was based on the correlation between higher cyanidin :
pelargonidin ratios with increased doses of a dominant Pr1 allele (LARSON et al. 1986).
However, no gene for F3'H enzyme has been cloned in maize prior to the work that is
discussed in the later chapters of this thesis. Dihydroflavonols (DHQ and DHK) are
reduced by dihydroflavonol 4-reductase (DFR) to respective leucoanthocyanidins, which
are then converted by anthocyanidin synthase (ANS) to the colored anthocyanidins,
cyanidin and pelargonidin, respectively. In maize, DFR and ANS are encoded by
anthocyaninless1 (a1) and anthocyaninless2 (a2) genes, respectively (O'REILLY et al.
1985; REDDY and COE 1962). Anthocyanidins are glycosylated by UDP flavonoid 3-O-
glucosyl transferase (UFGT) encoded by the bronze1 (bz1) gene in maize (DOONER and
NELSON 1977). The glycosylated molecules are transported and stored in the vacuole.
Transportation of glycosylated anthocyanins to the vacuole is mediated by glutathione-S-
transferase (GST) and a multidrug resistance-like transporter (MRP) localized in the
vacuolar membrane. GST is encoded by the bronze2 (bz2) gene and MRP by ZmMrp3
and ZmMrp4 genes (GOODMAN et al. 2004; MARRS et al. 1995). The flavonoid
biosynthetic enzymes are associated with the cytoplasmic face of the endoplasmic
reticulum (ER) and anchored to the membrane through the cytochrome P450 proteins that
participate in the pathway, e.g., F3'H and C4H (HRAZDINA. 1992; WINKEL-SHIRLEY
1999).
10
Biosynthesis of 3-deoxyanthocyanins
Two different types of anthocyanins are formed in maize: 3-hydroxyanthocyanins
and 3-deoxyanthocyanins (STYLES and CESKA 1975). In contrast to 3-
hydroxyanthocyanins (generally referred as anthocyanins) which are present in almost
every tissue of maize plant, 3-deoxyanthocyanins are restricted to the pericarp, cob, and
silk tissues. The synthesis of 3-deoxyanthocyanins is controlled by the pericarp color1
(p1) gene which also regulates the biosynthesis of C-glycosyl flavones (GROTEWOLD et
al. 1994; MCMULLEN et al. 2001; STYLES and CESKA 1977). The 3-deoxyanthocyanins
include flavon-4-ols and 3-deoxyanthocyanidins. Flavans-4-ols, luteoforol and apiferol,
are produced from naringenin through the action of DFR (GROTEWOLD et al. 1998b;
STYLES and CESKA 1975). Flavans-4-ols are polymerized to brick-red phlobaphene
pigment putatively through the action of the maize brown pericarp1 (bp1) gene
(EMERSON et al. 1935; MEYERS 1927). Flavans-4-ols, apiferol and luteoforol, are also the
anticipated precursors of 3-deoxyanthocyanidins, apigeninidin and luteolinidin,
respectively (GROTEWOLD et al. 1998b; STYLES and CESKA 1989). The 3-
deoxyanthocyanidins have been identified as reddish-brown pigments in sorghum plants
challenged with either pathogenic or nonpathogenic fungi (AGUERO et al. 2002; SNYDER
and NICHOLSON 1990). The 3-deoxyanthocyanidins accumulate in intracellular inclusion
bodies in the epidermal cells under attack. These inclusion bodies move towards the site
of fungal infection and release their contents, killing both the fungus and the cell that
synthesizes them (LO et al. 1999; SNYDER and NICHOLSON 1990). In Sorghum, 3-
deoxyanthocyanidins play an important role in plant defense as phytoalexins and their
synthesis is regulated by yellow seed1 (y1), an ortholog of the p1 gene of maize (CHOPRA
11
et al. 2002; LO et al. 1999). Apigeninidin, luteolinidin, and methoxy-luteolinidin are the
major phytoalexins of this class which are induced in response to fungal inoculation.
Luteolinidin and methoxy-luteolinidin are known to be more toxic towards the infecting
pathogens as compared to other sorghum phytoalexins (NICHOLSON et al. 1987). When
challenged with fungus, these two compounds are produced at higher levels in the
resistant cultivars as compared to the susceptible cultivars (LO et al. 1999).
Biosynthesis of C-glycosyl flavones
The corn earworm Helicoverpa zea Boddie is a major insect pest of maize. Its
larvae cause significant damage by feeding on silks, and hence interfere with pollination.
Ultimately larvae gain access to the young kernels and thus, this infestation also enhances
the chances of fungal infestation of the damaged ears. C-glycosyl flavones found in
maize silks have antibiotic activity to corn earworm (WAISS et al. 1979). Upon insect
wounding, C-glycosyl flavones in the silks are oxidized to more toxic quinones. These
quinones can bind to the –SH and –NH2 groups of free amino acids and proteins, thus
reducing their availability for insect growth (FELTON et al. 1989; WISEMAN and
CARPENTER 1995). Insect toxicity of these compounds requires a hydroxyl group on the
B-ring which may be due to the availability of an extra hydroxyl group for oxidation
during conversion to quinones (LINDROTH and PETERSON 1988). Antibiotic activity of C-
glycosyl flavones can be characterized by reduced larval and pupal weight, and extended
time to pupation (WISEMAN and ISENHOUR 1990).
Maysin, apimaysin, and methoxymaysin are three predominant C-glycosyl
flavones found in maize silks. These three C-glycosyl flavones differ by their B-ring
12
substitutions: maysin has a hydroxyl group at the 3'-position of B ring, while
methoxymaysin has a methoxy group (LINDROTH and PETERSON 1988). In apimaysin this
position remains unsubstituted (ELLIGER et al. 1980a; ELLIGER et al. 1980b). Maysin has
double the level of antibiosis against lepidopteron insects than that of apimaysin or
methoxymaysin (ELLIGER et al. 1980a; SNOOK et al. 1994). The pericarp color (p) locus
regulates the synthesis of C-glycosyl flavones in the maize silks (MCMULLEN et al. 1998;
MCMULLEN et al. 2001). The p locus contains two genes p1 and p2 (ZHANG et al. 2000).
Both p1 and p2 are major QTL for maysin biosynthesis and accounts for more than sixty
percent of its phenotypic variance (BYRNE et al. 1996; ZHANG et al. 2003). Both genes
can induce maysin synthesis in maize silks, but in contrast to p1, p2 does not express in
kernel pericarp and cob-glumes (GROTEWOLD et al. 1998a; ZHANG et al. 2003). C-
glycosyl flavones are derived from flavanones by the action of flavone synthase (FNS)
which converts naringenin or eriodictyol to respective flavones. C-glycosyl flavones are
formed from flavones by the action of C-glycosyl transferase (CGT).
Furthermore, the synthesis of maysin and rhamnosyl-isoorientin, also a flavone, is
induced in response to increased levels of UV-B radiation in the leaves of maize plants
growing at high altitudes (CASATI and WALBOT 2005). Induction of these flavones in the
leaves is controlled by a yet unidentified p-homologue, which is expressed in leaves and
is regulated by UV-B radiation (CASATI and WALBOT 2005).
Regulation of flavonoid biosynthetic genes
Accumulation of purple and red anthocyanin, and brick red phlobaphene pigments
in maize is regulated by colorless1 (c1)/purple leaf1 (pl1) and pericarp color1 (p1),
13
respectively, which are members of the R2R3 MYB family of regulatory genes (CONE et
al. 1993; RABINOWICZ et al. 1999). To regulate the transcription of biosynthetic genes,
the protein product of c1/pl1 gene interacts directly with the product of the booster1
(b1)/red1 (r1) gene, which are members of the basic helix-loop-helix (bHLH) MYC
homologous DNA binding domain gene family (GOFF et al. 1992; LUDWIG and WESSLER
1990; STYLES and CESKA 1977). Anthocyanin accumulation in the kernel aleurone
requires interaction between R1 and C1 while both B1 and PL1 are needed for
anthocyanin biosynthesis in vegetative plant tissues (CHANDLER et al. 1989). P1 activates
transcription of biosynthetic genes independent of any co-activator (GROTEWOLD et al.
1994). Flavonoid biosynthetic genes c2, chi1, and a1, which are common to both
anthocyanin and phlobaphene biosynthetic pathways, are regulated separately by
transcription factors of both pathways (GOFF et al. 1992; GROTEWOLD and PETERSON
1994). Promoter of these biosynthetic genes contain specific binding elements for these
transcription factors (TUERCK and FROMM 1994). For example, the a1 gene is
independently regulated in these two branches of the flavonoid pathway and converts
dihydroflavonols as well as flavanones to their respective products. Its promoter has a
high affinity (haPBS) and a low affinity (laPBS) p1 binding sites, and an anthocyanin
regulatory element (ARE) (LESNICK and CHANDLER 1998; TUERCK and FROMM 1994).
Transient expression experiments with maize suspension cells show that C1/R1 or P1 can
direct high level of expression from promoters containing these binding sites or cis-
elements (GROTEWOLD et al. 1994; TUERCK and FROMM 1994) and mutations in haPBS,
laPBS, and ARE result in inhibition of a1 activation by C1/R1 and P1 (SAINZ et al. 1997).
The importance of the ARE element in the control of a1 by the C1/R1 and P1 regulatory
14
factors was also demonstrated in a study of insertion/excision events of transposable
elements from the a1-m2 and a1-mum2 alleles in vivo (POOMA et al. 2002).
In addition to these regulatory genes, there are additional genes that affect
anthocyanin pigmentation in aleurone and include viviparous1 (vp1), anthocyaninless
lethal1 (anl1), intensifier1 (in1), and pale aleurone color1 (pac1). Mutation in the vp1
gene produces kernels with colorless aleurone, which fail to mature properly, and its gene
product is required for c1 expression (MCCARTY et al. 1989; ROBERTSON 1955). The
anl1 mutant lacks anthocyanin pigmentation of aleurone and was discovered as recessive
lethal (COE 1988). The in1 gene also has a regulatory role and suppresses the synthesis of
anthocyanin pigmentation in the aleurone i.e. it acts as a dominant inhibitor, while its
mutant allele enhances kernel pigmentation (BURR et al. 1996). This was evident from
the observation that in the presence of pr1, homozygous in1 kernels were black as
compared to red in In1/ pr1 kernels (FRASER 1924). The in1 gene product exhibits high
homology with that of b1/r1, the bHLH myc homolog that is involved in the regulation of
anthocyanin biosynthesis (BURR et al. 1996). pac1 encodes a WD40 protein and mutation
in pac1 results in pale pigmentation of the kernel aleurone and seedling roots and reduced
expression of anthocyanin structural genes (SELINGER and CHANDLER 1999).
Molecular basis of regulation of flavonoid biosynthesis
The DNA binding domain of the P1 protein has similarity with regions of other
MYB proteins, and the P1 MYB domain shows over 70% similarity to the C1 protein
(GROTEWOLD et al. 1991). However, C1 or Pl1 requires the R1 or B1 bHLH protein as
transcriptional co-activators and P1 does not, even though P1 regulates some structural
15
genes that are also activated by C1 (CHANDLER et al. 1989; GROTEWOLD et al. 1994). It
has been shown that there is physical interaction between proteins encoded by c1 and
r1/b1, which is mediated by the MYB domain of C1 and the N-terminal region of R1/B1
(GOFF et al. 1992).
The R3 MYB repeat of C1 is necessary for the specificity of its interaction with
R1 protein (GROTEWOLD et al. 2000). The interaction of C1 with R1 can be transferred to
P1 by substituting four residues of the first helix of R3 MYB repeat of P1 with
corresponding residues from C1. This was demonstrated by the R1 dependent
transcriptional activation of bz1 promoter by this altered P1 protein (GROTEWOLD et al.
2000). Although, the exact mechanism by which R1 or B1 controls transcription is not
known, it has been suggested that R1 not only activates C1 but also is essential for
specific regulation of its targets (GROTEWOLD et al. 2000). Further, Hernandez et al.
(2004) proposed that there are two components of C1 and R1 interaction. The first
component depends on binding of C1 to PBS and may be involved in relieving C1 from
an inhibitor. The second component recruits an R-interacting factor (RIF1) to make
contact with DNA through ARE cis-regulatory elements. RIF1 has been identified as an
ENT/AGENET domain containing nuclear protein which specifically interacts with the
bHLH region of R1 (HERNANDEZ et al. 2007). Proteins having these domains are
generally associated with histone modifications. Thus, RIF1 highlights the role of
epigenetic mechanisms in the regulation of flavonoid gene expression (HERNANDEZ et al.
2007; HERNANDEZ et al. 2004).
16
Functional conservation of anthocyanin genes in maize and Arabidopsis
The flavonoid biosynthetic pathway is present in both monocots and dicots. Since
plants in these groups are separated by large evolutionary distances; it is generally
considered that the corresponding enzymes of the flavonoid pathways are divergent in
them. One reason could be the rapid divergence of enzymes of secondary metabolic
pathways due to less stringent selective pressures. But the fact that mutations in
Arabidopsis (dicot) flavonoid biosynthetic genes can be complemented by homologs
from maize (monocot) suggests that gene function has been conserved despite sequence
divergence (DONG et al. 2001). For example, Arabidopsis transparent testa (tt) mutants;
tt4, tt5, and tt3 are complemented by the maize c2, chi1, and a1 genes, respectively, and
produce similar types and amounts of anthocyanins as found in wild-type Arabidopsis
plants (DONG et al. 2001). This was despite the fact that the maize genes had only 60-
85% sequence identity to their homologous genes in Arabidopsis. Not only structural
genes, but regulatory genes from maize can also be expressed in dicot species.
Transformation of Arabidopsis and tobacco plants with maize r1 and c1 regulatory genes
produce increased anthocyanin pigmentation in both dicot species (LLOYD et al. 1992).
Along with providing the evidence for functional conservation of enzymes involved in
the formation of specific secondary metabolites, Arabidopsis flavonoid mutants also
provides a suitable system to analyze the activity of homologous genes from other plants.
17
RESEARCH OBJECTIVES
The phenylpropanoid pathway through which tannins, flavonoids, and lignins
originate is one of the best studied metabolic pathways in nature. Phenylpropanoids are
derived from shikimic acid pathway, which participates in the biosynthesis of most plant
phenolics. We are interested in understanding the biosynthesis, genetic route and utility
of the flavonoid class of compounds. The flavonoid biosynthetic pathway is responsible
for the formation of most of the red, blue, and purple pigments in plants. Flavonoid
molecules have three ring structure (A, B, C) with a C6-C3-C6 skeleton. The
hydroxylation pattern of the B-ring of flavonoids is responsible for generating the
diversity in these pigments. The flavonoid 3′ hydroxylase (F3′H) enzyme controls the
hydroxylation at 3′ position of B-ring. In maize, the 3′-hydroxylation activity has long
been attributed to the pr1 locus. This speculation was based on the chemistry of
compounds and F3′H enzyme activity present in dominant and recessive pr1 plants (COE
1955; LARSON et al. 1986; MCCLARY 1942). Although the flavonoid pathway has been
studied extensively, the understanding of the 3′ hydroxylation activity and its regulation
is still incomplete. It has never been demonstrated that the pr1 locus encodes the F3′H
enzyme and no sequence information was available for the pr1 locus. The objective of
my research was to understand the novel role of pr1 gene in biosynthesis of antifungal
and insecticidal compounds synthesized through the flavonoid pathway and its tissue
specific regulation in maize. Chapter 2 describes the characterization of pr1 mutants,
cloning of pr1 gene, and genetic complementation studies confirming the functionality of
the isolated gene.
18
Anthocyanins form a key group of flavonoids showing bright coloration ranging
from blue to orange. Depending on the genetic constitution of plants, two types of
anthocyanins are formed in maize. The c1/r1 controls the formation of 3-
hydroxyanthocyanins, while p1 affects the biosynthesis of 3-deoxyanthocyanidins and
phlobaphenes. Studies have implicated the role of pr1 in the formation of anthocyanins as
well as in synthesis of precursors of phlobaphene pigments. There are examples present
for sharing of common structural enzymes among different branches of flavonoid
pathway and their differential regulation by regulatory genes of these pathways.
Therefore, in Chapter 3, I use genetic, biochemical, and molecular analysis to investigate
whether the pr1 gene is involved in the 3′-hydroxylation of the B-ring of different
flavonoid substrates and is regulated by two sets of transcription factors. Further, I
discovered that pr1 is required for biosynthesis of insecticidal C-glycosyl flavones and
antifungal 3-deoxyanthocyanidins. In chapter 4, I used genetic and biochemical
approaches to confirm the role and position of pr1 in anthocyanin biosynthetic pathway.
Further, kinetics of cyanidin accumulation in kernel aleurone was studied. Based on the
results of these studies it can be suggested that the pr1 gene is playing key role in generating
3′ hydroxylated products in different branches of flavonoid pathway.
19
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Chapter 2
The maize red aleurone1 encodes a flavonoid 3′-hydroxylase which is required for the biosynthesis of purple anthocyanins
INTRODUCTION
In the plant kingdom, the flavonoid biosynthetic pathway is ubiquitous and leads
to the synthesis of a variety of pigmented and non-pigmented compounds. The pigmented
flavonoid metabolites have also been used as phenotypic markers and have proven to be
an excellent model system to study genetic, molecular, and biochemical processes to
understand regulation of tissue-specific gene expression patterns. Flavonoids also have
important biological functions during the growth and development of a plant (DIXON and
STEELE 1999; SHIRLEY 1996; STAFFORD 1990; TAYLOR and GROTEWOLD 2005). In
addition to their benefits to plants, flavonoids have also shown many pharmacological
and dietary benefits for humans and animals (LIU et al. 2004; MIYAGI et al. 2000).
Flavonoid biosynthesis takes place through the phenylpropanoid pathway
(Figure 2.1), and depending upon the genetic constitution of the plant system, naringenin
can have several different fates of formation of anthocyanins, flavans, flavones,
condensed tannins, and phlobaphenes (BODDU et al. 2005; WINKEL-SHIRLEY 2001). In
maize, purple and red anthocyanins are derived from 3-hydroxyflavonoids (STYLES and
CESKA 1989) and their tissue-specific accumulation has been shown to be regulated by
pairs of duplicated transcription factors red1/booster1 (r1/b1) and colorless1/purple
plant1 (c1/pl1). The R1 and B1 are bHLH proteins with MYC homologous DNA binding
26
domain (GOFF et al. 1992; LUDWIG et al. 1990), while the C1 and PL1 are MYB-
homologous DNA binding domain proteins (CONE et al. 1993). Anthocyanin
accumulation in kernel aleurone requires joint action of R1 and C1 while B1 and PL1
together are needed for anthocyanin biosynthesis in vegetative plant parts (CHANDLER et
al. 1989).
Formation of purple anthocyanidins in kernel aleurone require the activity of
flavonoid 3'-hydroxylase (F3'H), and this activity has been attributed to the functional red
aleurone1 locus, also known as purple aleurone1 (pr1) in maize (LARSON et al. 1986).
Since the early 20th century, the pr1 locus has been used as a marker in numerous genetic
studies in maize involving identification, characterization, and mapping of several genes
(EAST 1912; EYSTER 1926; LARSON et al. 1986). During the mid 20th century, genetic and
biochemical studies established that the red and purple aleurone color difference is due to
the presence of an additional hydroxyl group in purple pigment and that a single gene is
responsible for this phenotype (COE 1955; MCCLARY 1942; ZARUDNAYA 1950). Maize
F3'H is a NADPH dependent cytochrome P450 enzyme, which can act on a wide range of
substrates: flavonols, flavones, and flavanones, in flavonoid biosynthetic pathway
(LARSON and BUSSARD 1986). In general, P450 cytochromes are membrane bound
hemoproteins that contain heme groups and are involved in electron transport. They are
generally involved in detoxification of a large variety of potentially toxic compounds but
also play important roles in a wide range of biosynthetic reactions.
27
Phenylalanine
Cinnamate
PAL
4-coumarate
pal
4CL
4-coumaroyl-CoA
c4hC4H
4cl
Chalconec2
chi1CHI
CHS3-malonyl-CoA
+
Naringenin
ANTHOCYANINS
bz1bz2
UFGTGST
f3h
F3'H
F3'H
f3h
pr1DHKDHQ
Eriodictyol
pr1F3H
F3H
Cyanidin Pelargonidin
ASa2DFRa1
ASa2DFRa1
Phenylalanine
Cinnamate
PAL
4-coumarate
pal
4CL
4-coumaroyl-CoA
c4hC4H
4cl
Chalconec2
chi1CHI
CHS3-malonyl-CoA
+
Naringenin
Phenylalanine
Cinnamate
PAL
4-coumarate
pal
4CL
4-coumaroyl-CoA
c4hC4H
4cl
Chalconec2
chi1CHI
CHS3-malonyl-CoA
+
Naringenin
ANTHOCYANINS
bz1bz2
UFGTGST
ANTHOCYANINS
bz1bz2
UFGTGST
f3h
F3'H
F3'H
f3h
pr1DHKDHQ
Eriodictyol
pr1F3H
F3H
Cyanidin Pelargonidin
ASa2DFRa1
ASa2DFRa1
f3h
F3'H
F3'H
f3h
pr1DHKDHQ
Eriodictyol
pr1F3H
F3H
Cyanidin Pelargonidin
ASa2DFRa1
ASa2DFRa1
ASa2DFRa1
ASa2DFRa1
Figure 2.1: Phenylpropanoid biosynthetic pathway leading to the production of anthocyanins.Genes (enzymes) in the pathway are: pal (PAL), phenylalanine-ammonia lyase; c4h (C4H), cinnamic acid hydroxylase; 4cl (4CL), 4-coumaryl:CoA ligase; c2 (CHS), chalcone synthase; chi(CHI), chalcone isomerase; f3h (F3H), flavanone 3-hydroxylase; pr1 (F3'H), flavonoid 3'-hydroxylase (based on enzyme activity assay); a1 (DFR), dihydroflavanone reductase; a2 (AS), anthocyanidin synthase; bz1 (UFGT), UDP-glucose flavonoid 3-O-glucosyltransferase; and bz2(GST), glutathione S-transferase.
28
Although availability of mutants at different catalytic steps has led to isolation of
most of the structural genes required for maize anthocyanin biosynthesis (DOONER et al.
1991), information has been lacking for the sequence of the functional pr1 gene.
Mutations in pr1 produce red aleurone because the dihydrokaempferol (DHK) is
metabolized into a bright red pigment, pelargonidin, as opposed to the purple cyanidin
pigment produced in maize lines carrying functional pr1 (Figure 2.2). Synthesis of
cyanidin requires conversion of DHK to dihydroquercitin (DHQ) by the action of a F3'H,
which adds a hydroxyl group at the 3'-position of the B-ring of flavanones (FORKMANN
1991). Using mutant and wild-type stocks of pr1, LARSON et al. (1986) demonstrated
that a functional pr1 locus may encode or regulate an F3'H enzyme activity that catalyzes
DHK to DHQ in vitro. To define the role of pr1 in maize anthocyanin biosynthesis, I
isolated and characterized a putative maize f3'h (f3'h1) sequence. My results provide the
historic missing link between red and purple anthocyanin biosynthesis in maize. Analysis
of f3'h1 and its transcriptional regulation in distinct tissue types was performed to
understand the intermediate steps leading to the synthesis of anthocyanins in maize.
Results presented here indicate that the pr1 gene expression is regulated by transcription
factors that control the synthesis of anthocyanins in silk, husk, and aleurone tissues.
MATERIALS AND METHODS
Maize genetic stocks: Seeds of maize inbred line W23 (genotype P1-wr c1 r-g) and
genetic stocks MGS 14273 (pr1 A1 A2 C1 R1), MGS 130543 (pr1 A1 A2 C1
29
pr1/pr1 Pr1/Pr1pr1/pr1 Pr1/Pr1 Figure 2.2: Phenotypes of pr1 and Pr1 maize ears. Pr1 has red aleurone color while Pr1 has purple colored aleurone.
R1), and MGS 14284 (pr1 A1 A2 C1 R1) segregating for pr1 were kindly provided by the
Maize Genetics Co-operation Stock Center (USDA-ARS, University of Illinois, Urbana,
IL). To develop F2 population, pr1 plants were crossed with W22 (Pr1 A1 C1 R1) and
progenies were grown from the selfed F1 plants. F2 populations showed a 3:1 segregation
for purple to red aleurone. Segregating plants were used for RNA expression and co-
segregation analysis using PCR based polymorphism. Genetic complementation studies
were performed using maize stock Mp708 (pr1 c1 r1 p1). The genotype of Zea mays
Black Mexican Sweet (BMS) cells used in this study is p1-ww A1 A2 C1 C2 R-g b1 pl1.
These cells were derived from cambial tissue in the hypocotyl where they do not express
transcription factors C1, Pl1, P1, B1 or R1 (GROTEWOLD et al. 1998). BMS cell
suspension cultures were maintained on Murashige and Skoog (MS) medium
(MURASHIGE and SKOOG 1962) containing 1.5 mg/L 2,4-D in an incubator shaker at 150
rpm in dark at 26°.
30
Genomic, cDNA cloning and sequence characterization: During the onset of this
experiment putative f3′h sequences available in the GenBank were used to design PCR
primers to isolate full length maize f3′h sequence. Oligonucleotides F2 (forward), 5′-
AGTGCGAGGTGGACGGGTTC- 3′ and R2 (reverse), 5′-GCAGACGGCA-
GCAGTCTCCCCT- 3′ were based on the alignment of the maize partial EST sequence
(accession no. BG873885) with sorghum f3′h sequence (BODDU et al. 2004), and used to
PCR amplify a 387 bp fragment named as F387. Fragment FR is a partial genomic
sequence from the 5′ half of the rice gene which was PCR amplified using forward
primer OSF1, 5′-CATACGGCCATGGACGTTGTGCCT-3′ and reverse primer ZMR4,
5′-AAACGTCTCCTTGATCACCGC-3′. All DNA fragments used as probes were
labeled with α 32P-dCTP using the Prime-a-Gene Labeling System (Promega, Madison,
WI). A λ FIX II (Stratagene, La Jolla, CA) library prepared from seedling leaf DNA of
maize inbred line W23 was screened to isolate the full length maize f3′h1 gene (f3′h1). A
total of 800,000 clones were screened and all screenings were performed using duplicate
filters (Hybond-N Nylon, Amersham Biosciences, Piscataway, NJ) hybridized either to
FR or F387 fragments as probes. Filters were exposed to X-OMAT film (KODAK,
Rochester, NY) for 8 to 12 hr before developing. Three positive clones were isolated.
The full-length maize cDNA was obtained by performing RT PCR using gene specific
primers SBF12 (forward), 5′-CTTCTAGAACCGAG-3′ and SBR22 (reverse), 5′-
TTGGAT CCCCTACTCCGCTGCGTAT-3′ on total RNA isolated from maize silks of
W23. Standard PCR buffer and reaction conditions were followed (SAMBROOK and
RUSSELL 2001) with the modified annealing temperature of 60° for 2 min, followed by
polymerization at 72° for 2 min. PCR products and restriction fragments were sub-cloned
31
into the pGemT-Easy (Promega, Madison, WI) and pBluescript II (Stratagene, La Jolla,
CA) plasmid vectors, respectively, and sequenced from both ends using vector specific
primers. All DNA sequencing were performed at the Pennsylvania State University’s
Nucleic Acid Facility using method of dye primer cycle sequencing and reactions were
run on a 3100 capillary machine (Applied Biosystems, Foster city, CA). Sequence
assembly and analysis was done using the GCG® Wisconsin Package 1 (Accelrys, Inc.,
Burlington, MA) and the BLAST and other sequence analysis tools available from the
NCBI at www.ncbi.nih.gov (ALTSCHUL et al. 1990). Predicted amino acid sequences
were obtained from the NCBI Nucleotide Data Base (http://www.ncbi.nlm.nih.gov) or by
using the ExPASy Translate Tool (http://ca.expasy.org/tools/dna.html). Conserved
domain searches were done using the NCBI CD Search tool at
http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.
Phylogenetic tree analysis: Amino acid sequences were aligned using ClustalX program
(version 1.81; multiple alignment parameters: gap opening 10, gap extension 0.20; DNA
weight matrix: IUB; Protein weight matrix: Gonnet series) (THOMPSON et al. 1994) and a
phylogenetic tree constructed using the neighbour-joining method (SAITOU and NEI
1987). Drawing of the bootstrap Philip tree was done using the NJ PLOT Program
(version 1.6.6 Win32; using 1000 replicates for nodes of the bootstrap alignment trials).
The protein sequences were obtained from Genbank. The F3'H protein sequences besides
ZmF3'H are: Arabidopsis thaliana (AF271651), Callistephus chinensis (AF313488),
Glycine max (AF499731), Matthiola incana (AF313491), Oryza sativa (AC021892),
32
Pelargonium x hortorum (AF315465), Perilla frutescens (AB045593), Petunia hybrida
(AF155332), Torenia hybrida (AB057673), and Sorghum bicolor (AY675075).
PCR amplification: Positions of PCR primers used for successful identification of
polymorphism in Pr1 and pr1 alleles are shown in Figure 2.5, and their sequences are: P-
1, 5΄-TGACTTGCACCTCCTTGTTCTGTC-3΄; P-2, 5΄-TTTAGTGCACAACCTTTAG
GG-3΄; P-3, 5΄-GTACGAAATTCCAGATCGCGGGTA-3΄; and P-4, 5΄-ATAGCCACAT
GGTGTGGTGCGG-3΄. The standard reaction (50 μl) contained 0.1 μg of genomic DNA,
25 μl of GoTaq green master mix (Promega), and 200 nM each of the two
oligonucleotide primers. A typical reaction consisted of 35 cycles of denaturation (1min,
94°), annealing (1min, 57°), and extension (2 min 30 sec, 72°) in a thermal cycler (MJ
Research Inc.). PCR products from both mutant and wild-type pr1 alleles were cloned in
pCR2.1-TOPO cloning vector (Invitrogen, Carlsbad, CA) and sequenced. Nucleotide
sequences were aligned using ClustalW program (LARKIN et al. 2007).
Mapping of f3′h1 gene: A single nucleotide polymorphism (SNP) based assay was
developed to map the f3′h1 sequence. The following primers, 5′-AGGTGGACGGGTT
CCGCATC-3′, and 5′-GTATGCCTCCTCCATGTCTAGC-3′ were used to amplify and
sequence a segment of the f3′h1 gene from DNA of the inbred lines Tx501, NC7A, and
Mp708. Examination of the sequence alignments revealed a C (NC7A and Mp708)/G
(Tx501) polymorphism. The primer 5′-GATGAGCTCGAAGTCGCT-3′ was designed to
interrogate the polymorphic site by primer extension. The SNP assays were performed as
follows: the initial amplification product was cleaned with shrimp alkaline phosphatase
33
(Roche Diagnostics, Indianapolis, IN) and Exonuclease I (USB, Cleveland, OH) to
remove residual dNTP’s and primer. Four microliter of template was enzyme treated with
2 U of both shrimp alkaline phosphatase and Exonuclease I in a final volume of 10 µl.
Samples were incubated at 37° for 60 min followed by deactivation of enzyme at 72° for
15 min. Single nucleotide polymorphism reactions were performed with the ABI
PRISM® SNaPshot ddNTP primer extension kit (Applied Biosystems, Foster City, CA)
as follows: 4 µl of template, 1 µl of interrogation primer (0.15 pmoles/µl), 1 µl of
SNaPshot mix, and sterile water added to obtain a final volume of 10 µl. Cycling
conditions of the SNP program were: 25 cycles of the sequence of 10 sec-96°, 5 sec-50°,
30 sec-60°. After the SNP reaction, cleaning of template was carried out with 0.5 U of
shrimp alkaline phosphatase. Samples were incubated at 37° for 60 min followed by
deactivation of enzyme at 72° for 15 min. The SNP samples were prepared as follows: 3
µl of SNP template, 0.4 µl of GeneScan-120 LIZ size standard (Applied Biosystems) and
deionized formamide to a final volume of 10 µl. Samples were denatured at 95° for 4 min
and cooled at 4°. Analysis of the samples was carried out using a 36-cm array and POP4
polymer on the ABI PRISM® 3100 Genetic Analyzer machine (Applied Biosystems) and
the ABI PRISM® GeneScan analysis software. The genotypes for SNP were derived
from 346 (Tx501 × NC7A)F2 individuals and 246 (Tx501 × Mp708)F2 individuals
obtained from previously characterized quantitative trait locus mapping populations
(CORTES-CRUZ et al. 2003). The linkage maps were constructed with
MAPMAKER/EXP, Version 3.0 (Whitehead Institute, Cambridge MA).
34
RNA gel blot analysis: For RNA isolation, silk and husk tissues were collected at the
silk emergence and aleurone/endosperm were collected 24 to 28 days after pollination
(DAP). To isolate total RNA, tissues were ground in liquid nitrogen and then extracted
using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). The RNA was
separated on a denaturing gel containing 5% formaldehyde (v/v), 1.2% Agarose (w/v),
and 1X RNA buffer (0.4 M MOPS, 0.1 M anhydrous sodium acetate and 0.01 M Sodium
EDTA). The fractionated RNA was transferred onto a nylon membrane (Osmonics Inc.,
Minnetonka, MN). RNA gel blot hybridizations were performed for 24 hr at 65° in a
hybridization mixture containing, 0.5 M sodium phosphate at pH 7.2, 1 mM EDTA, 7%
SDS, and 1% BSA. All membranes were washed in a solution containing 0.1 X SSC (1 X
SSC is 0.15 M sodium chloride and 0.015 M sodium citrate), and 0.5% SDS once at 50°
for 15 min, and twice at 65° for 15 to 30 min. Filters were exposed to an X-OMAT film
(KODAK, Rochester, NY) for 1 to 4 days before developing. Filters were stripped by
washing in a boiling solution of 0.1% SDS before re-hybridization.
RT-PCR expression assay: First strand cDNA was synthesized using SuperScript II
cDNA synthesis system (Invitrogen, Carlsbad, CA) and oligo-dT primer for reverse
transcription (RT) PCR. One microgram of aleurone total RNA from aleurones was used
for each reverse transcription reaction. First strand cDNA was diluted to a final volume
of 100 µl with sterile ddH2O. Five microliter of first strand cDNA was amplified using
primers 5F3H-F2 (forward) 5′-GAGCACGTGGCGTACAACTA-3′ and ZMR4 (reverse)
5′-AAACGTCTCCTTGATCACCGC-3′ spanning over exon1 and exon2, respectively.
Plasmid DNA containing complete f3′h1 genomic sequence was used as an internal
35
control, producing PCR product of 996 bp as compared to 771 bp product for f3′h1
cDNA. For detection of transcript of other anthocyanin genes, primer sets used are shown
in supplemental table 1 (PIAZZA et al. 2002). Primers used for the amplification of cDNA
of anthocyanin genes were designed such that they span over the intron of the gene in
order to detect any genomic DNA contamination. PCR reactions were performed in a
total volume of 25 µl with GoTaq green master mix (Promega) using manufacturer’s
instructions. Templates were PCR amplified with 94° for 2 min, 35 cycles of 94° for 1
min, 55°-60° for 1 min, and 72° for 2 min 30 sec, and 72° for 8 min.
Extractions, TLC, and HPLC analysis of flavonoid compounds: Thin layer
chromatography (TLC) was carried out using silica 60 plates (Sigma Aldrich, St. Louis,
MO) and a mobile solvent containing ethyl acetate:formic acid:acetic acid:water
(100:11:11:27, v/v) (DONG et al. 2001; HOLTON 1995). Approximately 2 gm of chopped
up aleurones and endosperms were allowed to soak in 1 ml of methanol with 1% HCl for
48 hr before transferring the extracts to a fresh tube. A total of 30 µl extract was spotted
per lane. For High Performance Liquid Chromatography (HPLC) analysis of
dihydroflavonols, maize silk samples (300 mg) taken at the time of silk emergence were
imbibed in 1 ml mixture of methanol:HCl (99:1,v/v) and compounds were allowed to
leach for 48 hr at 4°. The anthocyanin pigments were extracted by grinding 300 mg of
aleurone tissue in 1 ml of 80% methanol. Extracts were acid hydrolyzed with an equal
volume of 4 N HCl to prepare aglycones (BURBULIS et al. 1996; NYMAN and
KUMPULAINEN 2001). The extracts were then filtered through 0.45 µm Acrodisc LC 13
mm syringe filters (Gelman Laboratory, Ann Arbor, MI). Reverse phase HPLC analysis
36
was performed on a Shimadzu high performance liquid chromatograph (Shimadzu,
Columbia, MD) using an Ascentis C18 column (25 cm X 4.6 mm, 5 μm; Supelco,
Bellefonte, PA). Pigment separation was done at 35˚ by gradient elution using 0.2%
formic acid (solution A) and 100% methanol (solution B) at a flow rate of 1 ml/min. The
injection volume was 50 μl and spectral measurements were taken over a wavelength
range of 230 nm to 550 nm, which is known to detect flavonoid compounds
(GROTEWOLD et al. 1998).
Transient complementation of pr1 mutation: BMS cells were subcultured 72 hr prior
and placed in liquid media with osmotic stressor 0.2 M Mannitol for 4 hr prior to
bombardment. A suction flask connected to vacuum was used to filter cells onto sterilized
filter discs. Each disc with cells was then placed in the center of a petri plate containing
1X MS medium solidified with 6% phytagel. All steps were performed in sterile
conditions in a flow chamber (Labconco, Kansas city, MO). Maize kernels were surface
sterilized, soaked in sterile water overnight, and placed on solidified MS media
containing 2 M Mannitol. Plates were incubated in dark at 26° for 48 hr to begin growth
of coleoptiles. Biolistic transformation was carried out using the PDS-1000 Helium
Particle Delivery System (BioRad, Hercules, CA) using a chamber vacuum of 27 inches
Hg, and the helium pressure for rupture disk of 1100 psi, with a target distance of 9 cm
and 6 cm for BMS cells and maize coleoptiles, respectively. Tungsten particles of size
1.1 micron were coated with 0.5 µg of plasmid DNA containing the CaMV 35S::C1+R1
alone or with CaMV 35S::F3′H1 (pr1). Each tungsten-DNA preparation was used for 5
shots following the conditions described previously (GOFF et al. 1990). BMS cells and
37
kernels were placed under fluorescent white light at room temperature for 24 to 48 hr
prior to observation under the dissection microscope (Nikon, SMZ1000, Nikon
Corporation, Tokyo, Japan).
Arabidopsis seed stocks, growth conditions, and transformation: For genetic
complementation experiment, seeds of Landsberg erecta and transparent testa7 (tt7)
mutant were obtained from Arabidopsis Biological Resource Center at Ohio State
University, Columbus. Mutant tt7 used in the study was in L. erecta background. Seeds
were surface sterilized with 10% bleach and germinated in pots filled with steam
sterilized Metro Mix 300. Soil was misted with water after seeding, then covered with
plastic wrap and kept in cold room at 4° for 3 to 5 days to break seed dormancy and to
get a uniform germination. Plastic wrap was removed and pots were transferred to growth
chamber maintained at 22° and 70% relative humidity (RH). Plants were kept under short
day conditions (10 hr light and 14 hr dark) for first three weeks and then transferred to
greenhouse under long day cycle (16 hr light and 8 hr dark) to induce flowering.
Overnight grown Agrobacterium tumefaciens strain GV3101 carrying CaMV 35S::F3′H1
construct cloned in pSR3000 vector was harvested by centrifugation and resuspended in
200 ml solution containing 5% sucrose and 0.002% Silwet l-77 (Lehle Seeds, Round
Rock, TX). Plants were transformed by floral dip method (CLOUGH and BENT 1998) in
which each plant inflorescence was kept immersed for at least 10 sec. Transformation
process was repeated after one week to ensure transformation of new florets that emerged
after first cycle of transformation. Screening of transformants was done on MS medium
containing 50 µg/ml kanamycin and 100 µg/ml ampicillin. Green seedlings were
38
transferred to soil mix and grown in growth chamber at 22° with 10 hr light and 14 hr
dark cycle.
Spectrophotometric analysis: Seeds of L. erecta, tt7, and T2 seeds from two
independent transformation events were germinated on minimal medium containing 3%
sucrose and 0.5% (w/v) agar. These were kept at 25° in continuous light, seedlings were
collected after 10 days, and pigments were extracted as described in DONG et al. (2001).
Briefly, seedlings were homogenized in 1.5 ml of 1% (v/v) HCl in methanol, then 1 ml of
double distilled water was added, and chlorophyll was separated from the extracts with
chloroform. Pigment analysis was done on UV spectrophotometer, UV-mini 1240
(Shimadzu Corporation) and measurements were taken over wavelength range of 400 nm
to 600 nm. HPLC analysis of extracts was performed with the same column and solvent
program used for aleurone anthocyanin analysis.
RESULTS
Isolated sequence encodes a putative flavonoid 3'-hydroxylase: From the restriction
mapping of three positive clones, the largest λ clone (Figure 2.5) was sub-cloned to
obtain the sequence of f3'h1. Sequence alignments with other plant F3'Hs showed that the
isolated sequence encodes a predicted F3'H peptide of 514 amino acids (Figure 2.3).
Overall, the maize F3'H shows about 55% identity with dicot F3'Hs and high sequence
similarity with monocot F3'Hs: 78% with rice, 90% with sugarcane, 65% with barley,
39
and 91% with sorghum. Since, F3'Hs belong to the category of cytochrome P450-
dependent monoxygenases, domains conserved among these proteins were found in the
putative maize F3'H sequence. These domains include heme-binding site (HBS), oxygen-
binding site (OBS), and the characteristic hydrophobic membrane anchor present at the
amino-terminus. A dicot F3'H specific sequence GGEK with unknown function has
previously been reported (BRUGLIERA et al. 1999; TODA et al. 2002) and this sequence
was found to be modified to GGSH in maize F3'H. Phylogenetic analysis was performed
with deduced amino acid sequences of maize F3'H and F3'Hs from other plant species
using the ClustalX program (THOMPSON et al. 1994). Result of phylogenetic analysis is
presented in Figure 2.4. The phylogenetic tree shows that maize F3'H falls in the same
clade as other monocots such as rice and the closely related species sorghum. Also,
similar to the dicots Arabidopsis and Matthiola forming a distinct clade, F3'Hs of cereal
crops maize, sorghum, and rice do not show any evolutionary divergence within this
clade.
Dinucleotide repeats insertion and a deletion near the promoter region defines the
lesion in pr1: To characterize the lesion present in pr1 gene, we followed a PCR based
approach. Using pr1 gene specific primers (Figure 2.5), we detected polymorphism
between wild-type and mutant pr1 alleles. Three different mutant alleles of pr1 and one
inbred, MP708, which also contain recessive pr1 gene, were tested for polymorphism.
Sequencing results indicated the presence of 24 TA dinucleotide repeats in upstream
promoter region of MP708, MGS 14273 and MGS 130543 and a 17 bp deletion near the
TATA box of MGS 14284 (Figure 2.6). Figure 2.5 show the positions of dinucleotide
40
repeats and deletion in relation to transcription start site. PCR screening of genic region
did not show any sequence polymorphism between Pr1 and pr1 alleles. This shows
F3’H specific Hydrophobic anchor
F3’H specific HBS
Proline rich region
Maize -MDVPLPLLLGSVAVSLVVWCLLLRRG-----GAGKGKRPLPPGPRGWPVLGNLPQVGAKPHHTMCAMARE-YGPLFRLRFGSAEVVVAASARVAAQFL 92 Sorghum -MDVPLPLLLGSLAVSVVVWCLLLRRGGD---GKGKGKRPMPPGPRGWPVLGNLPQLGSHPHHTMCALAKK-YGPLFRLRFGSAEVVVAASARVAAQFL 94 Oryza MDVVPLPLLLGSLAVSAAVWYLVYFLRGGSGGDAARKRRPLPPGPRGWPVLGNLPQLGDKPHHTMCALARQ-YGPLFRLRFGCAEVVVAASAPVAAQFL 98 Perilla MISAAVSLIICTSILGVLVYFLFLRRGGGS------NGRPLPPGPRPWPIVGNLPQLGPKPHQSMAALARVHGPLMHLKMGFVHVVVAASATVAEKFL 92 Torenia -MSPLALMILSTLLGFLLYHSLRLLLFSGQ------GRRLLPPGPRPWPLVGNLPHLGPKPHASMAELARAYGPLMHLKMGFVHVVVASSASAAEQCL 91 Callistephus --MTILPFIFYTCITALVLYVLLNLLTRN--------PNRLPPGPTPWPIVGNLPHLGMIPHHSLAALAQKYGPLMHLRLGFVDVVVAASASVAAQFL 88 Petunia --MEILSLILYTVIFSFLLQFILRSFFRKR------YPLPLPPGPKPWPIIGNLVHLGPKPHQSTAAMAQTYGPLMYLKMGFVDVVVAASASVAAQFL 90 Arabidopsis --MATLFLTILLATVLFLILRIFSHRRNRS------HNNRLPPGPNPWPIIGNLPHMGTKPHRTLSAMVTTYGPILHLRLGFVDVVVAASKSVAEQFL 90 Mathiola ---MTTLILTILLATFLSLFIFFLLRRNRN------RNHRLPPGPNPWPIVGNLPHMGPKPHQTLAAMVTTYGPILHLRLGFVNVVVAASKSVAEQFL 89 Glycine ----MSPLIVALATIAAAILIYRIIKFITR------PSLPLPPGPKPWPIVGNLPHMGPVPHHSLAALARIHGPLMHLRLGFVDVVVAASASVAEQFL 88 Pellargonium --MYNMSLYLLLGSSALAFAAYLVLFSFSK------SRRRLPPGPKAWPIVGNLPHMGSMPHQNLAAMARTYGPLVYLRLGFVDVVVALSASMASQFL 90 Maize ARFLPGGSHAGVDVKGSDFELIP-FGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGMTADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYA---- 514 Sorghum DRFLPGGSHAGVDVKGSDFELIP-FGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGMTADKLDMEEAYGLTLQRAVPLKVRPAPRLLPSAYAAE-- 517 Oryza SRFLPGRMHADVDVKGADFGLIP-FGAGRRICAGLSWGLRMVTLMTATLVHGFDWTLANGATPDKLNMEEAYGLTLQRAVPLMVQPVPRLLPSAYGV--- 526 Perilla ERFLMGGEKPNVDVRGNDFELIP-FGSGRRICAGMNLGIRMVQLLIATMVHAFDFELANGQLAKDLNMEEAYGITLQRADPLVVHPRPRLARHVYQAQV- 523 Torenia ERFLTGGEKADVDVKGNDFELIP-FGAGRRICAGVGLGIRMVQLLTASLIHAFDLDLANGLLPQNLNMEEAYGLTLQRAEPLLVHPRLRLATHVY----- 512 Callistephus SRFLPGGEKPDADIKGNDFEVIP-FGAGRRICAGMSLGMRMVQLLIATLVQTFDWELANGLDPEKLNMEEAYGLTLQRAEPLMVHPRPRLSPHVYESR-- 518 Petunia ERFLPGGEKPKVDVRGNDFEVIP-FGAGRRICAGMNLGIRMVQLMIATLIHAFNWDLVSGQLPEMLNMEEAYGLTLQRADPLVVHPRPRLEAQAYIG--- 512 Arabidopsis ERFLPGGEKSGVDVKGSDFELIP-FGAGRRICAGLSLGLRTIQFLTATLVQGFDWELAGGVTPEKLNMEESYGLTLQRAVPLVVHPKPRLAPNVYGLGSG 513 Mathiola ERFLPGGEKFGVDVKGSDFELIP-FGAGRRICAGLSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNMEETYGITVQRAVPLIVHPKPRLALNVYGVGSG 513 Glycine ERFLLGGEKADVDVRGNDFEVIP-FGAGRRICAGLSLGLQMVQLLTAALAHSFDWELEDCMNPEKLNMDEAYGLTLQRAVPLSVHPRPRLAPHVYSMSS- 513 Pellargonium ERFLPGSEKENVDVKGNDFELIP-FGAGRRICAGMSLGLRMVQLLTATLLHAFNWDLPQGQIPQELNMDEAYGLTLQRASPLHVRPRPRLPSHLY----- 511
Figure 2.3: Multiple sequence alignment of deduced amino acid sequences of F3'H from maize and other plant species using ClustalX program (see accession numbers in material and method). Lightly shaded areas are F3'H specific sequences and regions shaded dark are conserved domainsfound in the CYP450 family of proteins. Specific regions indicated above the first sequence are ahydrophobic anchor, oxygen binding site (OBS), and heme binding site (HBS). The last amino acid is not numbered in partial sequences.
that the mutant pr1 alleles may be defective due to these insertion and deletion events in
5′ region of the gene. To determine if the isolated f3'h1 sequence is linked to the pr1
locus, we developed a population segregating for Pr1 and pr1 by crossing pr1 plants with
W22 inbred (see materials and methods). A total of 40 segregating plants from each
41
0.1
Perilla
Torenia
Oryza
Sorghum
Maize
Callistephus
Petunia
Glycine
Arabidopsis Matiola
Pelargonium
940635
1000
1000
654864
416
1000
0.1
Perilla
Torenia
Oryza
Sorghum
Maize
Callistephus
Petunia
Glycine
Arabidopsis Matiola
Pelargonium
940635
1000
1000
654864
416
1000
Figure 2.4: Un-rooted phylogenetic tree showing the evolutionary relationships of maize F3'H and other plant F3'Hs. Accession numbers of the other plant F3'Hs are mentioned in material and methods. Alignment data are from ClustalX program. Bootstrap values sampled 1000 times andused to construct the consensus tree shown. The scale bar refers to the number of substitutions per site.
population were characterized for PCR based polymorphism. A 1:2:1 ratio for
Pr1/Pr1:Pr1/pr1:pr1/pr1 showed a molecular segregation of polymorphic bands. In
conclusion, these results provide the molecular mechanism behind the pr1 mutation and
the first evidence that the isolated sequence is linked to the pr1 locus.
42
Figure 2.5: Characterization of λZT2-1 clone and pr1 lesion. Partial restriction map of the λ ZG2-1 clone from which the f3'h1 was subcloned. The split line above shows the sub-clones (pZG219 and pZG214) obtained by digesting the λ DNA with NotI and subcloning into NotI-pBluescript SK(+). FR and F387 are the probes used to screen the genomic library. Partial restriction map of asegment of λZG2-1 clone shows f3'h1 and its 5' and 3' flanking sequences. Grey boxes represent two exons that are joined by a bent line which corresponds to the single intron of the f3'h1 gene. Enlarged 5' flanking region of f3'h1 shows the location of twenty four dinucleotide repeats and deletion near TATA box present in mutant pr1 alleles are shown with respect to transcription start site (shown as bent arrow and marked +1). Small arrows below the map illustrate the orientation and position of the PCR primers used to amplify the targeted region. Restriction map of f3'h1 is also shown; restriction enzymes shown are: H, HindIII; N, NotI; P, PstI; Sc, SacI; S, SalI; X, XbaI; and Xh, XhoI.
43
Figure 2.6: Alignment of the nucleotide sequences of Pr1 and pr1 surrounding the dinucleotide repeat region in the upstream promoter area of pr1. The sequence derived from pr1 is shown below the sequence of Pr1. The dinucleotide repeats in the pr1 are marked with the boxes. P-1 and P-2 represents the PCR primers used to amplify this sequence and their position with respectto transcription start site is shown.
The f3'h1 gene sequence maps to the pr1 locus on chromosome 5L: A genetic linkage
map was constructed for chromosome 5 containing an f3'h1 SNP based on the scoring of
346 and 246 individuals for the (Tx501 × NC7A)F2 and (Tx501 × Mp708)F2 populations,
respectively (Figure 2.7). The position of the f3'h1 SNP corresponds to the genetic RFLP
placed position of the pr1 locus (MaizeGDB, http://www.maizegdb.org) and
interestingly, it was also detected as a major QTL for the synthesis of apimaysin vs.
maysin in these same populations (CORTES-CRUZ et al. 2003). Therefore, these genetic
44
mapping results support the conclusion that the putative f3'h1 gene sequence isolated in
this study corresponds to the pr1 locus on chromosome 5L, which may be responsible for
the 3' hydroxylation of the B-ring in flavonoid biosynthesis (LARSON et al. 1986).
The f3'h1 transcript is absent in tissues accumulating red anthocyanins: RT-PCR
and RNA gel blot analysis of steady state transcription of f3'h1 were performed for kernel
aleurone (Figure 2.8), young maize silk, and husk tissues collected from plants
segregating for Pr1 or pr1 alleles. Pr1 plants produced ears with all purple kernels and
showed transcript of f3'h1. An f3'h1 specific signal could not be detected in RNA from
homozygous pr1 plants that produced ears with red kernels. RT-PCR using gene specific
primers for other flavonoid pathway genes as well as for c1 and r1, the anthocyanin
regulatory genes, showed steady state transcripts of these genes in both Pr1 and pr1
plants. Similarly, RNA gel blots were stripped and re-hybridized with probes of other
genes required for anthocyanin biosynthesis. RNA gel blot analysis showed that
including c1, one of the transcription factors required for the pathway, early (c2) as well
as late (bz2) anthocyanin biosynthetic genes were expressed in both functional and
mutated pr1 plants. In summary, these results show that f3'h1 specific transcript is absent
in mutant pr1 plants and the TA dinucleotide repeats insertion may be responsible for this
lack of RNA expression.
f3'h1 is required for the formation of cyanidin anthocyanins: Methanolic extracts of
pr1 and Pr1 endosperms and aleurones were separated using thin layer chromatography
45
umc1416
umc1153
bnlg118
umc2013
bnlg278
f3′h1
umc1800
umc1098
umc1056
bnlg1660
bnlg565
umc147821.8
39.0
51.4
64.3
77.3
89.9
106.2
141.9
163.8
178.9
201.9
Population 1(Tx501 × NC7A)F2
62.1
mmc081
umc1098
70.0
umc1478
umc1019
umc1416
umc1153
umc2013
bnlg118
f3′h1
mmc282
umc1056
bnlg565
umc152315.3
35.7
51.2
58.5
87.4
107.2
145.2
132.5
176.5
18.5
bnlg14325.1
umc133267.0
bnlg278111.9
Population 2(Tx501 × Mp708)F2
umc1416
umc1153
bnlg118
umc2013
bnlg278
f3′h1
umc1800
umc1098
umc1056
bnlg1660
bnlg565
umc147821.8
39.0
51.4
64.3
77.3
89.9
106.2
141.9
163.8
178.9
201.9
Population 1(Tx501 × NC7A)F2
umc1416
umc1153
bnlg118
umc2013
bnlg278
f3′h1
umc1800
umc1098
umc1056
bnlg1660
bnlg565
umc147821.8
39.0
51.4
64.3
77.3
89.9
106.2
141.9
163.8
178.9
201.9
Population 1(Tx501 × NC7A)F2
62.1
mmc081
umc1098
70.0
umc1478
62.1
mmc081
umc1098
70.0
umc1478
62.1
mmc081
umc1098
70.0
umc1478
umc1019
umc1416
umc1153
umc2013
bnlg118
f3′h1
mmc282
umc1056
bnlg565
umc152315.3
35.7
51.2
58.5
87.4
107.2
145.2
132.5
176.5
18.5
bnlg14325.1
umc133267.0
bnlg278111.9
Population 2(Tx501 × Mp708)F2
umc1019
umc1416
umc1153
umc2013
bnlg118
f3′h1
mmc282
umc1056
bnlg565
umc152315.3
35.7
51.2
58.5
87.4
107.2
145.2
132.5
176.5
18.5
bnlg14325.1
umc133267.0
bnlg278111.9
Population 2(Tx501 × Mp708)F2
Figure 2.7: f3'h1 maps to the pr1 locus on chromosome 5L. Genetic linkage mapping of f3'h1SNP marker on chromosome 5 for (Tx501XNC7A) and (Tx501XMP708) F2 populations. The position of f3'h1 is indicated on both maps and is corresponding to the pr1 locus. Cumulative distances given in centimorgan are indicated to the left of the chromosome and marker loci are indicated to the right.
46
Figure 2.8: Mutant pr1 plants do not accumulate f3'h1 transcript. RT-PCR and RNA hybridization analyses were carried out using plants segregating for Pr1 and pr1. Total RNA was extracted from developing aleurones, young maize silk, and husk. (A) Expression of f3'h1 and other anthocyanin genes in aleurone tissues were detected by RT-PCR of homozygous pr1/pr1 mutant and Pr1/Pr1 wild-type plants. Gene specific primers for f3'h1, c2, chi1, a1, bz2, and transcriptionfactors, c1 and r1 were used to detect their transcripts. Aleurone tissues from W22 were used as positive control for anthocyanin gene expression, while α-tubulin1 was used as an internal control. (B) Gel blot of RNA isolated from silk tissues was hybridized to f3'h1, c2, bz2, c1, and α-tubulin1 specific probes. (C) Husk RNA gel blot hybridized with f3'h1 probe. Ethidium bromide stained gel picture shows loading control for RNA in each lane.
(TLC) (Figure 2.9). pr1 aleurones accumulated red pelargonidin glycosides, while Pr1
showed the presence of purple cyanidin glycosides. None to very little cyanidin pigment
47
was detected in the pr1 plants. While the yellow tetrahydroxychalcone can sometimes be
detected in the extracts, numerous other compounds in the pathway are colorless and
were not detected in our TLCs (BRUGLIERA et al. 1999; HOLTON and CORNISH 1995).
Although kaempferol and quercitin are also colorless, they can act as co-pigments to shift
the color towards intense blue (BRUGLIERA et al. 1999). There are only six chromophore
forms (aglycones) of anthocyanins (FUKUCHI-MIZUTANI et al. 2003), and the one that is
not present in maize but seen in flowers is the blue colored delphinidin (FUKUCHI-
MIZUTANI et al. 2003). Characteristic peaks for flavonoids fall into two spectral areas of
measurement; these being denoted as Band II at 230-285 nm and Band I at 300-550 nm.
Flavanones and dihydroflavonols typically produce larger peaks at 230 nm and smaller
peaks at 280 nm. Chalcones give a peak at 360 nm; flavanols, such as kaempferol and
quercitin, show a large peak at 375 nm (MABRY et al. 1970); while anthocyanidins, such
as cyanidin and pelargonidin give larger peaks at 530 nm and 515 nm, respectively
(DONG et al. 2001). HPLC of silk methanolic extracts from lines containing the mutant
pr1 gave a peak at 12 min for DHK, while lines containing Pr1 showed a peak at 8 min,
which is DHQ as measured at 230 nm. HPLC analysis of acid hydrolyzed extracts from
the aleurone tissues at 530 nm revealed that the mutant pr1 plants accumulated mainly 4'-
hydroxylated anthocyanidin, pelargonidin, which gave a single peak at 32 min. Whereas,
the wild-type pr1 plants accumulated very high levels of 3', 4'-hydroxylated
anthocyanidin, cyanidin, producing a single peak at 29 min, and very little, often below
quantification limits, pelargonidin was also observed. Heterozygous Pr1/pr1 plants show
both cyanidin and pelargonidin peaks, although, the amount of cyanidin was much higher
than that of pelargonidin (data not shown). These chromatography results showed that the
48
pr1 gene is required for formation of DHQ from DHK, and subsequently, for the
formation of cyanidin compounds, which give purple color to the aleurone tissue in wild-
type pr1 plants. The accumulation of respective anthocyanidins and dihyroflavonols in
the mutant and wild-type plants confirms that the pr1 gene is responsible for formation of
3', 4'-hydroxylated flavonoids.
Isolated f3'h1 transiently complements the pr1 mutant phenotype: To directly test the
function of the isolated f3'h1 in anthocyanin synthesis, we adopted a transient
complementation assay based on previous studies on the maize r1 gene (LLOYD et al.
1992). BMS cells and germinating seeds of maize line Mp708 were biolistically
transformed either with the plasmid constructs carrying C1+R1 alone placed under the
control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter
(35S::C1+R1) or together with 35S::F3'H1 (Figure 2.10A). When transformed with the
35S::C1+R1 alone, spots of red pelargonidin pigment due to the activation of the
anthocyanin pathway by C1 and R1 were observed. Co-bombardment of 35S::C1+R1
with 35S::F3'H1 produced purple cyanidin pigment spots. These results demonstrated
that the isolated f3'h1 encodes a functional protein that is capable of performing the 3' B-
ring hydroxylation reaction leading to the synthesis of purple cyanidin.
49
A
3 4 5 6 7 8
Pr1 Pr1 Pr1 pr1 pr1 pr1
1 2
C1 C2
PC
C
0
50
100
150
200
250
0
50
100
150
200
250
0
50
100
150
200
250
29.1
Time (min)
A51
5
20 25 30 3515
0
100
200
300
400
20 25 30 3515 20 25 30 3515
0
100
200
300
400
0
100
200
300
400
Pelargonidin
A53
0
Pr1/Pr1
Cyanidin
32.6pr1/pr1
B
A23
0
Pr1/Pr1
DHK
DHQ
0 5 10 15
pr1/pr1
DHK
A23
0
Time (min)
A
3 4 5 6 7 8
Pr1 Pr1 Pr1 pr1 pr1 pr1
1 2
C1 C2
PC
A
3 4 5 6 7 8
Pr1 Pr1 Pr1 pr1 pr1 pr1
1 2
C1 C2
PC
3 4 5 6 7 8
Pr1 Pr1 Pr1 pr1 pr1 pr1
1 2
C1 C2
3 4 5 6 7 8
Pr1 Pr1 Pr1 pr1 pr1 pr1
1 2
C1 C2
PC
C
0
50
100
150
200
250
0
50
100
150
200
250
0
50
100
150
200
250
29.1
Time (min)
A51
5
20 25 30 3515
0
100
200
300
400
20 25 30 3515 20 25 30 3515
0
100
200
300
400
0
100
200
300
400
Pelargonidin
A53
0
Pr1/Pr1
Cyanidin
32.6pr1/pr1
C
0
50
100
150
200
250
0
50
100
150
200
250
0
50
100
150
200
250
29.1
Time (min)
A51
5
20 25 30 3515
0
100
200
300
400
20 25 30 3515 20 25 30 3515
0
100
200
300
400
0
100
200
300
400
Pelargonidin
A53
0
Pr1/Pr1
Cyanidin
32.6pr1/pr1
0
50
100
150
200
250
0
50
100
150
200
250
0
50
100
150
200
250
29.1
Time (min)
A51
5A
515
20 25 30 3515
0
100
200
300
400
20 25 30 3515 20 25 30 3515
0
100
200
300
400
0
100
200
300
400
Pelargonidin
A53
0A
530
Pr1/Pr1
Cyanidin
32.6pr1/pr1
B
A23
0
Pr1/Pr1
DHK
DHQ
0 5 10 15
pr1/pr1
DHK
A23
0
Time (min)
B
A23
0
Pr1/Pr1
DHK
DHQ
0 5 10 15
A23
0A
230
Pr1/Pr1
DHK
DHQDHQ
0 5 10 15
pr1/pr1
DHK
A23
0
pr1/pr1
DHK
A23
0A
230
Time (min)
Figure 2.9: Mutant plants with red aleurone produce pelargonidin while wild-type plants with purple aleurone produce cyanidin. (A) TLC of elutes extracted from Pr1 or pr1 kernels. Methanolic extracts from kernels of plants with Pr1/Pr1 or pr1/pr1 genotype, were run on a silica 60 plate in a solvent of ethyl acetate: methanol: formic acid: acetic acid: water (45:10:5:5:14 v/v).Samples shown in each lane are: 1, control C1 for cyanidin; 2, control C2 for pelargonidin; 3-5,Pr1; 6-8, pr1. Pigments visualized in Pr1/Pr1 are cyanidin and its glycosylated derivatives. Pigments in pr1/pr1 are pelargonidin and its glycosylated derivatives. (B) HPLC analysis of silk methanolic extracts from sibling plants carrying pr1 or Pr1. Extracts were analyzed at λ 230 nm
50
for dihydrokaemferol (DHK) and dihydroquercitin (DHQ). Here pr1 shows a single peak representing DHK, while Pr1 shows two peaks representing both compounds, thus DHK is being converted into DHQ through the action of F3'H (Figure 1.1). (C) Reverse phase HPLC analysis of anthocyanidin pigments, at wavelengths 530 nm and 515 nm for cyanidin (Pr1/Pr1) and pelargonidin (pr1/pr1), respectively.
f3′h1 stably complements Arabidopsis tt7 mutant phenotype: Arabidopsis tt7 has a
mutation in the f3'h gene (PEER et al. 2001; SHIRLEY et al. 1995). tt7 mutant seeds have
yellow coat color as compared to wild-type seeds with brown seed coat. tt7 seeds fail to
accumulate brown tannins in the testa (KOORNNEEF 1990; SHIRLEY et al. 1995). In
addition, tt7 seedlings do not produce anthocyanin pigments in the cotyledon or
hypocotyl when grown on a nitrogen deficient minimum medium (HSIEH et al. 1998).
Development of anthocyanin pigments in wild-type Arabidopsis seedlings in response to
stress has been used to study anthocyanin biosynthetic pathway (GROTEWOLD et al.
1998). We have used this assay to ascertain the role of isolated f3'h1 in the development
of anthocyanins (Figure 2.10B). tt7 plants were transformed with the 35S::f3'h1 transgene
(see material and methods). The T1 seeds were screened on kanamycin containing
medium, and resistant plants were grown to maturity in the growth chamber. Seeds
collected from kanamycin-resistant T2 plants showed complete restoration of brown seed
coat color. In the seedling assay, T2 seedlings produced red cotyledons when grown on a
minimal medium. Spectrophotometric analysis of the extracts from 10 days old L. erecta
and f3'h1 complemented tt7 seedlings grown on minimal medium showed the
characteristic absorbance of anthocyanidin pigments at a wavelength of 530 nm, while tt7
mutant seedlings did not show this peak (Figure 2.11). Anthocyanin compounds present
in wild-type, mutant, and f3'h1 expressing seedlings were further characterized by HPLC.
51
A
pr1 p1 c1 r1
35S::C1+R1 35S::C1+R1 + 35S::ZmF3'H1
Col
eopt
iles
BM
S
pr1 p1-ww
C
1 R-g b pl
B
Landsberg erecta
tt7 tt7 + 35S::ZmF3'H1
Seed
lings
Se
eds
A
pr1 p1 c1 r1
35S::C1+R1 35S::C1+R1 + 35S::ZmF3'H1
Col
eopt
iles
BM
S
pr1 p1-ww
C
1 R-g b pl
pr1 p1 c1 r1
35S::C1+R1 35S::C1+R1 + 35S::ZmF3'H1
Col
eopt
iles
BM
S
pr1 p1-ww
C
1 R-g b pl
B
Landsberg erecta
tt7 tt7 + 35S::ZmF3'H1
Seed
lings
Se
eds
Landsberg erecta
tt7 tt7 + 35S::ZmF3'H1
Seed
lings
Se
eds
Figure 2.10: Complementation of mutant f3'h phenotypes of maize and Arabidopsis with f3'h1. (A) Complementation analysis was performed by biolistic transformation of BMS cells (pr1 C1 R-g b1 pl1) and germinating seeds of maize stock line Mp708 (pr1p1c1r1) with the plasmid constructs of CaMV 35S::C1+R1 alone (left) or together with CaMV 35S::F3'H1 (right). Red spots are pelargonidin pigment accumulated due to activation of the anthocyanin pathway by the transcription factors C1 and R1. Purple cyanidin pigment spots are seen with the addition ofactive f3'h1. This indicates that the isolated f3'h1 gene can perform 3' B-ring hydroxylation reaction converting pelargonidin to cyanidin. (B) Complementation of tt7 mutant plants with the f3'h1, resulted in accumulation of pigments in seedling and seed coat. Arabidopsis tt7 mutantplants deficient for F3'H were transformed via Agrobacterium inoculation using plasmid containing 35S::F3'H1. Purple seedling pigment and brown testa color accumulated in Landsberg erecta (left) and 35S:: F3'H1 complemented tt7 mutant (right) while no pigment accumulation was observed in mutant tt7 seedling and seeds (center).
52
Expression of f3'h1 in tt7 mutant restored the accumulation of anthocyanins. The peaks
for different cyanidin glycosides were detected in both wild-type and f3'h1
complemented tt7 mutant, which were absent in tt7 mutant seedlings. Overall, these
results demonstrate that the isolated f3′h1 gene encodes a functional F3'H enzyme
capable of complementing the tt7 mutant phenotype in Arabidopsis.
DISCUSSION
The pr1 gene has been subjected to a large number of studies in maize; however,
the molecular difference between Pr1 and pr1 plants has never been investigated. The
results presented in this study show that steady state transcript of f3'h1 is present in
aleurone tissue of Pr1 but pr1 shows loss of transcript. Similar is true for the f3'h1
expression in silk and husk tissue of Pr1 and pr1 plants. Gene expression pattern of f3'h1
correlates with the accumulation of anthocyanidins in aleurone and of dihydroflavonols
in silk tissue. Although, LARSON et al. (1986) in their study on F3'H enzyme in maize
did find some F3'H activity in seedlings of homozygous mutant pr1 plants. They
suggested that either the mutant pr1 allele is hypomorphic i.e. it has reduced level of pr1
gene expression or there is a duplicate f3'h gene present in maize. However, in the
present study we did not find evidence either for reduced levels of pr1 gene expression or
a second f3'h gene in any of the tissues studied. The reason of this discrepancy may be
the sequence difference between the f3'h1 and the proposed second hydroxylase gene or
it may be possible that they had detected the activity of a P450 hydroxylase, other than
53
F3'H, in mutant pr1 plants. Our results from the expression analysis confirm that the
historic purple and red aleurone color difference in Pr1 and pr1 alleles is due to the lack
of f3'h1 transcript in pr1 and not due to absence or change in the expression of
anthocyanin biosynthetic or regulatory genes.
In the current study, for anthocyanin analysis we used aleurone tissue from
homozygous Pr1 and pr1 lines developed from a segregating population and maintained
by self-pollination for five generations. TLC analysis of kernel aleurone extracts showed
that purple cyanidin-glycosides and red pelargonidin-glycosides accumulated in Pr1 and
pr1 lines, respectively. This indicates a probable block in the formation of
dihydroquercitin (DHQ) in mutant lines. DHQ can be formed from dihydrokaempferol
(DHK) by F3'H or from naringenin to eriodictyol via F3'H followed by F3H activity.
Chromatographic analysis of the silk extracts from Pr1 plants show the presence of DHQ
and DHK as well while the pr1 plants have DHK only. The correlation of Pr1 and pr1
alleles to the types of flavonoid compounds produced is consistent with the role of pr1 as
F3'H encoding gene. Through chromatographic analysis, we inferred that the pr1 gene is
able to perform 3'-hydroxylation reaction in the 3-hydroxyanthocyanin branch pathway.
This was further confirmed by the presence of dominant peaks for cyanidin and
pelargonidin (differ by one hydroxyl group attached at 3' position in cyanidin) in the
methanolic aleurone extracts from purple and red aleurones, respectively. It has been
54
A
ac
b
λmax 530nm
a : L. erecta b : tt7 c : tt7 + 35S::ZmF3'H1
B
tt7
tt7+ 35S::ZmF3'H1
L. erecta
Standards
Time (Min)
A53
0A
530
A53
0A
530 C
C
C
P
P
P
A
ac
b
λmax 530nm
a : L. erecta b : tt7 c : tt7 + 35S::ZmF3'H1
A
ac
b
λmax 530nm
a : L. erecta b : tt7 c : tt7 + 35S::ZmF3'H1
ac
b
λmax 530nm
a : L. erecta b : tt7 c : tt7 + 35S::ZmF3'H1
B
tt7
tt7+ 35S::ZmF3'H1
L. erecta
Standards
Time (Min)
A53
0A
530
A53
0A
530 C
C
C
P
P
P
B
tt7
tt7+ 35S::ZmF3'H1
L. erecta
Standards
Time (Min)
A53
0A
530
A53
0A
530 C
C
C
P
P
P
tt7
tt7+ 35S::ZmF3'H1
L. erecta
Standards
Time (Min)
A53
0A
530
A53
0A
530 C
C
C
P
P
P
Figure 2.11: Anthocyanin accumulation in wild-type and transgenic Arabidopsis plants. (A)Spectrophotometeric analysis of methanolic extracts from 10 days old seedlings grown on a minimal medium (a) Landsberg erecta, (b) f3'h1 complemented, (c) mutant tt7. L.erecta and 35S::F3'H1 complemented seedlings gave the maximum absorption peak at 530 nm whichcorresponds to cyanidin while tt7 mutant seedlings showed no absorption peak at wavelength range for anthocyanin compounds. (B) HPLC analysis of anthocyanins present in hydrolizedmethanol extracts of 10 days old seedlings from L. erecta, tt7, and f3'h1 complemented tt7mutant. The standards for cyanidin and pelargonidin are also shown. C, cyanidin; P, pelargonidin; and CG, cyanidin glycosides.
55
shown that aforementioned anthocyanin compounds have antifungal and insecticidal
activities (HAMMERSCHMIDT and NICHOLSON 1977; HEDIN et al. 1983; HIPSKIND 1996;
KARAGEORGOU and MANETAS 2006). Therefore, it will be important to test whether the
pr1 gene co-segregates with these plant resistance compounds.
F3'H enzyme activity is required for hydroxylation at the 3'-position on the B-ring
of flavonoid compounds and was observed to act on a wide range of substrates including:
flavonols, flavones, and flavanones (LARSON and BUSSARD 1986). Interestingly,
hydroxylation pattern of the B-ring is also the key determinant in the antioxidant property
of anthocyanins. Cyanidin, because of an additional hydroxyl group at its 3' B-ring
position, has a higher oxygen radical absorbing capacity as compared to pelargonidin and
therefore, has higher anticarcinogenic activity (WANG et al. 1997). The additional
hydroxyl group increases the polarity of cyanidin and is the reason why it elutes earlier
than pelargonidin during chromatographic analysis.
The pr1 locus has been used as a phenotypic marker in numerous genetic studies
and its role has been attributed in 3'-hydroxylation of various flavonoid compounds.
However, it has not been demonstrated directly that the pr1 locus in maize encodes a
F3'H enzyme, a missing link between red and purple phenotypes of pr1 alleles used in
previous studies. Using conserved signature sequences, we designed degenerate primers
to isolate a partial sequence of a putative f3'h1. Full length sequence characterizations
were accomplished by further isolating and sequencing sub-clones constructed from a λ
genomic library. Our SNP based genetic linkage analysis map the f3'h1 to pr1 locus on
chromosome 5L.
56
The proposed function of the f3'h1 is verified by both in vitro and in vivo
complementation of the pr1 phenotypes. In transient essays, co-transformation by f3'h1
and c1/r1 causes the accumulation of purple pigments in BMS cells as well as in kernel
aleurones, both carry nonfunctional pr1 gene in their genetic background. However,
transformation with c1/r1 alone results in the formation of red pigments and no pigment
accumulation was observed in tissues transformed with either empty vector or f3'h1
alone. These results are in agreement with the fact that c1/r1 regulate transcriptional
activation of anthocyanin genes for the synthesis of purple cyanidin, in the presence of
functional pr1. Furthermore, complementation of the yellow seed phenotype of the
Arabidopsis tt7 mutant by 35S-f3'h1 transgene demonstrates that the isolated gene is
capable of substituting for the function of the F3'H encoding Arabidopsis tt7 gene in vivo
(SCHOENBOHM et al. 2000). Although the flavonoid biosynthetic pathway is present in
both monocots and dicots, but it is generally considered that the corresponding enzymes
in this pathway are very divergent in these two groups of plants. One theory behind this
perception is that due to less stringent selective pressure on the corresponding enzymes of
the secondary metabolic pathways these enzymes have diverged rapidly during evolution
(PICHERSKY and GANG 2000). But in contrast, it has been found that mutations in
Arabidopsis and petunia (both are dicots) flavonoid biosynthetic genes can be
complemented by corresponding flavonoid genes from Zea mays, a monocot (DONG et al.
2001). In accordance with these observations our results also show that f3'h1 transformed
seedlings produce cyanidin and similar anthocyanin compounds accumulate in the wild-
type Arabidopsis seedlings. Similar results were obtained when maize chalcone synthase
(c2), chalcone isomerase1 (chi1), and anthocyaninless1 (a1) genes were used to
57
complement Arabidopsis transparent testa (tt) mutants; tt4, tt5, and tt3, respectively
(DONG et al. 2001). The anthocyanins formed in complemented mutants were similar in
type and amount to those found in wild-type Arabidopsis plants (DONG et al. 2001). This
is despite the fact that the maize genes, including f3'h1 (which has 57% homology to
Arabidopsis F3'H), have moderate sequence identity to homologous genes in
Arabidopsis. Consistent with this, LLOYD et al. (1992) showed that regulatory genes c1
and r1 from maize expressed in transformed Arabidopsis and tobacco plants resulted in
increased anthocyanin pigmentation in both dicot species. These results, in addition to
results from the current study, demonstrate that multiple enzymes of flavonoid
biosynthetic pathway are conserved between monocots and dicots and can be transferred
between these two divergent groups of flowering plants. Also, our results from
complementation of tt7 mutation by the f3'h1 explicitly confirm that the isolated f3'h1
gene encodes for a functional F3'H enzyme.
The deduced amino acid sequence of f3'h1 shows a 55 to 57% similarity with
F3'Hs from dicots and a higher similarity to F3'Hs from monocots: 78% with rice, 90%
with sugarcane, 65% with barley, and more than 90% with sorghum. Flavonoid-3'-
hydroxylase (F3'H) is a member of super family CYPs that catalyze NADPH and O2-
dependent hydroxylation reactions and are membrane associated. We found that maize
F3'H contains highly conserved regions of amino acid sequences which are also present
in other plant P450s (NELSON and STROBEL 1988). One of the conserved regions is a
proline rich region which functions as a hinge near the membrane anchored N-terminal
region to destabilize alpha-helixes (PPxP). Important conserved regions also include the
heme binding domain centered on a cysteine residue that is needed for catalysis of carbon
58
monoxide (FxxGxRxCxG) and an oxygen binding domain [(A/G)Gx(D/E)T (T/S)] for
proton transfer in the hydroxylation reaction at the active site (CHAPPLE 1998; NELSON
and STROBEL 1988). An additional signature region in dicots used to differentiate F3'Hs
from F3'5'Hs and other P450s is a conserved GGEK sequence motif; however, in case of
monocots such as Zea maize (current study), Sorghum bicolor, and Saccharum, this is
modified to GGSH motif (BODDU et al. 2004; BRUGLIERA et al. 1999). In another
monocot, Hordeum vulgare, this sequence has further been modified to GGTH motif.
Genetic evolution is probably the cause of these amino acid substitutions but the
significance of these GGXX motifs has not yet been established.
Sequence analysis of pr1 alleles shows an insertion of 24 TA dinucleotide repeats
in the upstream promoter region which is absent in Pr1 alleles. Three out of four pr1
alleles tested (including MP708) show this 48 bp insertion. Further, the dinucleotide
repeat insertion co-segregates with the red aleurone phenotype in a F2 population
segregating for Pr1/pr1. Presence of dinucleotide repeat insertions in the promoter region
of several genes has been shown to interfere with their expression levels. A number of
reasons have been suggested to explain the mechanism by which dinucleotide repeats can
affect the expression of a gene. For example, dinucleotide repeats can reduce the
promoter activity by changing the spacing between two binding sites, or by inserting into
a binding site and making it unavailable for binding of an important trans-factor
(RAIJMAKERS et al. 2000). We have analyzed f3'h1 sequence by MetInspector
(Genomatrix, Munich, Germany) (QUANDT et al. 1995) and by visual observation but no
important binding site has been found to be disrupted by this insertion. We also did not
find any increase in distance between two important binding elements which could result
59
in reduced promoter activity. Interestingly, there are reports that dinucleotide repeat
insertions within or in close proximity to the promoter regions modulate transcription
efficiency even when they are not affecting cis regulatory binding sites. Importantly,
sequences containing alternating purines and pyrimidines, including TA repeats, are
capable of forming left handed Z-DNA under physiological conditions and also increase
the flexibility of DNA (RICH et al. 1984). Z-DNA has a zig-zag conformation of its
phosphate backbone and has deeper minor groove but lacks a major groove. RNA
polymerase cannot transcribe through Z-DNA (PECK and WANG 1985). A significant
number of studies have shown that Z-DNA forming sequences were located at the 5' end
of genes and in some studies were present up to 1.5 kb upstream of the transcribed
sequences (COMINGS 1998; SCHROTH et al. 1992; WANG et al. 1979). An association of
Z-DNA formation with inhibition of transcription has been established (NAYLOR and
CLARK 1990; WU et al. 1994). As mentioned earlier, dinucleotide repeats are also highly
flexible, which increases their bending ability. The longer the dinucleotide repeat stretch,
the longer the bending ability reaction (GEBHARDT et al. 1999). This could favor a DNA
secondary structure that supports or hinders the binding of a factor to a neighboring
enhancer element if the polymorphic segment is prolonged (GEBHARDT et al. 1999).
Importantly, it has been found that higher the number of dinucleotide repeats present in
the promoter region of these genes, more severe is the decrease in level of gene
expression (RAIJMAKERS et al. 2000). It has been speculated that the greater number of
dinucleotide repeats can inhibit transcriptional activity of genes by affecting their ability
to form alternative DNA structures such as Z-DNA or other secondary structures
(ROTHENBURG et al. 2001).
60
Recently, it has been demonstrated that f3'h gene in sorghum is induced in
response to fungal infection and its expression is correlated with biosynthesis of
flavonoid phytoalexins (BODDU et al. 2004). These phytoalexins accumulate in
intracellular inclusion bodies and move towards the site of fungal infection, releasing
their content which kills both the fungus and the host cell (NICHOLSON et al. 1987).
Moreover, pr1 was detected as a major QTL for synthesis of C-glycosyl flavones that
have insecticidal activity against corn earworm (CORTES-CRUZ et al. 2003; LEE et al.
1998). Therefore, it will be highly interesting to investigate the role of pr1 in plant
disease and insect resistance through biosynthesis of flavonoid class of defense
compounds. By elucidating the synthesis of these chemicals we may be able to
manipulate their in vivo production in maize and other related plant species.
61
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Supplemental Table 2.1: Sequences of primers used in reverse transcription analysis.
Gene Primer sequences Product size (bp)
References
c1 PL6 5′-TCGGACGACTGCAGCTCGGC-3′ AC1 5′-CACCGTGCCTAATTTCCTGTCCGA-3′ 313 Piazza et al.,
2002
r1 OR31 5′-ATGGCTTCATGGGGCTTAGATAC-3′ OR32 5′-GAATGCAACCAAACACCTTATGCC-3′ 403 Piazza et al.,
2002
c2 CHSF 5′-TCGATCGGTCTCTCTGGTACAACGTA-3′ CHSR 5′- TACATCATGAGGCGGTTCACGGA-3′ 549 This Study
chi1 CHIF 5′-GTGCGGAATTTAACATGGCGTGC-3′ CHIR 5′-CGGCGCGAAAGTCTCTGGCTT-3′
444 This Study
a1 A1 5′- TTCTCGTCCAAGAAGCTCCAGGA-3′ A2 5′- CAATTCGTTGAACATGGAAGTAAG-3′
285 Piazza et al., 2002
bz2 BZ2F 5′-ATATGCGAGTCCGCAGTCATCGT-3′ BZ2R 5′-TCGATGAGTGAGAGGCCGTGAA-3′
379 This Study
tubulin TubulinF 5′-AGGATCCACTTCATGCTTTCCTCC-3′ TubulinR 5′-CACCTTCCTCACCCTCATCAAACT-3′ 546 This Study
Chapter 3
Tissue specific regulation of Zea mays pr1 gene is responsible for differential accumulation of insecticidal and antifungal flavonoid compounds
INTRODUCTION
Flavonoid metabolites account for much of the red, blue, and purple pigmentation
in plants (WINKEL-SHIRLEY 2001). These pigments because of their dispensable nature
have been used as tools to study basic questions in plant biology (CHOPRA et al. 2006).
For example, Mendel used flower color of peas to study the laws of heredity and Nobel
laureate Barbara McClintok discovered transposable elements while observing
anthocyanin pigments in maize kernels. More recently, these pigments have been used to
discover RNA-induced gene silencing by over expressing an anthocyanin biosynthetic
gene in petunia (NAPOLI et al. 1990). Flavonoid compounds also play vital role in plant
growth and development (TAYLOR and GROTEWOLD 2005). These pigments have been
implicated in attracting pollinators (GIURFA et al. 1995), protection from UV damage (LI
et al. 1993; STAPLETON and WALBOT 1994), and regulation of auxin transport (BROWN et
al. 2001). They also serve as signaling molecules in plant bacterium symbiosis (HIRSCH
1992) and as defense compounds against biotic stresses (BYRNE et al. 1996a;
HAHLBROCK and SCHEEL 1989).
The maize flavonoid pathway provides an excellent system to study gene
interaction in plants because of its extensive characterization at genetic, biochemical, and
molecular levels (KOES et al. 2005). Generally four different types of flavonoid
68
compounds are produced in maize: anthocyanin, phlobaphene, 3-deoxyanthocyanidin,
and C-glycosyl flavone. These compounds are synthesized in different organs of maize.
Depending on the genetic constitution of plant, anthocyanin accumulate in most plant
parts whereas phlobaphene are present in kernel pericarp (outer layer of ovary wall), cob-
glumes (palea and lemma), tassel glumes, and husk. Conversely, expression of 3-
deoxyanthocyanidin and C-glycosyl flavone is limited to silk tissue (Figure 3.1) (STYLES
and CESKA 1975; WAISS et al. 1979).
Accumulation of purple and red anthocyanin pigments in maize is regulated by
two sets of duplicated regulatory genes: colorless1 (c1)/purple leaf1 (pl1) are members of
R2R3 MYB family of transcription factors and booster1 (b1)/red1 (r1) encode basic
helix-loop-helix (bHLH) MYC homologous DNA binding domain proteins (CONE et al.
1993; RABINOWICZ et al. 1999). Studies have shown that protein product of c1 or pl1
gene interact directly with the product of b1 or r1 gene, respectively, to activate
transcription of flavonoid biosynthetic genes (GOFF et al. 1992; LUDWIG and WESSLER
1990; STYLES and CESKA 1977). These transcription factors regulate tissue specific
accumulation of anthocyanins in maize (LUDWIG and WESSLER 1990): joint action of R1
and C1 is required for pigment accumulation in kernel aleurones, while B1 and PL1 are
needed together for anthocyanin accumulation in vegetative plant tissues (CHANDLER et
al. 1989). Interestingly, pericarp color1 (p1) gene, a member of the R2R3 MYB family
of transcription factors, activates flavonoid biosynthetic genes independent of any co-
activator during the biosynthesis of brick-red phlobaphene pigments (GROTEWOLD et al.
1994).
69
Pelargonidin R = H
Cyanidin R = OH
Apigeninidin R = H
Luteolinidin R = OH
Apiferol R = H
Luteoforol R = OH
Apimaysin R = H
Maysin R = OH
Eriodictyol
pr1
F3Hfht1
DFRa1
Anthocyanins
F3'H
Dihydroflavonols
a2 ANS
bz1
bz2
UFGT
GST
Phlobaphenes
Apiferol
a1 DFR
F3'H
pr1Luteoforol
Apigeninidin
pr1
Luteolinidin
F3'H
Polymerization
bp1 ?
P1
CHSc2
Chalcone
CHIchi1
Naringenin
4-Coumaroyl CoA 3 Malonyl-CoA+
C1/Pl1+
R1/B1
PhenylalaninePAL C4H
4-Coumarate
4CL
Chlorogenic acid
pr1
FNS1
Luteolin
Isoorientin
GT
Apimaysin
RT
Rhamnosylisoorientin
Maysin
F3'H
sm2
sm1
EriodictyolF3'Hpr1
Pelargonidin R = H
Cyanidin R = OH
Apigeninidin R = H
Luteolinidin R = OH
Apiferol R = H
Luteoforol R = OH
Apimaysin R = H
Maysin R = OH
Pelargonidin R = H
Cyanidin R = OH
Pelargonidin R = H
Cyanidin R = OH
Apigeninidin R = H
Luteolinidin R = OH
Apigeninidin R = H
Luteolinidin R = OH
Apiferol R = H
Luteoforol R = OH
Apiferol R = H
Luteoforol R = OH
Apimaysin R = H
Maysin R = OH
Apimaysin R = H
Maysin R = OH
Eriodictyol
pr1
F3Hfht1
DFRa1
Anthocyanins
F3'H
Dihydroflavonols
a2 ANS
bz1
bz2
UFGT
GST
Phlobaphenes
Apiferol
a1 DFR
F3'H
pr1Luteoforol
Apigeninidin
pr1
Luteolinidin
F3'H
Polymerization
bp1 ?
P1
CHSc2
Chalcone
CHIchi1
Naringenin
4-Coumaroyl CoA 3 Malonyl-CoA+
C1/Pl1+
R1/B1
PhenylalaninePAL C4H
4-Coumarate
4CL
Chlorogenic acid
pr1
FNS1
Luteolin
Isoorientin
GT
Apimaysin
RT
Rhamnosylisoorientin
Maysin
F3'H
sm2
sm1
EriodictyolF3'Hpr1
Eriodictyol
pr1
F3Hfht1
DFRa1
Anthocyanins
F3'H
Dihydroflavonols
a2 ANS
bz1
bz2
UFGT
GST
Eriodictyol
pr1
F3Hfht1
DFRa1
Anthocyanins
F3'H
Dihydroflavonols
a2 ANS
bz1
bz2
UFGT
GST
Phlobaphenes
Apiferol
a1 DFR
F3'H
pr1Luteoforol
Apigeninidin
pr1
Luteolinidin
F3'H
Polymerization
bp1 ?
Phlobaphenes
Apiferol
a1 DFR
F3'H
pr1Luteoforol
Apigeninidin
pr1
Luteolinidin
F3'H
Polymerization
bp1 ?
P1
CHSc2
Chalcone
CHIchi1
Naringenin
4-Coumaroyl CoA 3 Malonyl-CoA+
C1/Pl1+
R1/B1
PhenylalaninePAL C4H
4-Coumarate
4CL
P1
CHSc2
Chalcone
CHIchi1
Naringenin
4-Coumaroyl CoA 3 Malonyl-CoA+
C1/Pl1+
R1/B1P1P1
CHSc2
Chalcone
CHIchi1
Naringenin
4-Coumaroyl CoA 3 Malonyl-CoA+
C1/Pl1+
R1/B1
C1/Pl1+
R1/B1
C1/Pl1+
R1/B1
PhenylalaninePAL C4H
4-CoumaratePhenylalaninePAL C4H
4-Coumarate
4CL
Chlorogenic acidChlorogenic acid
pr1
FNS1
Luteolin
Isoorientin
GT
Apimaysin
RT
Rhamnosylisoorientin
Maysin
F3'H
sm2
sm1
EriodictyolF3'Hpr1
pr1
FNS1
Luteolin
Isoorientin
GT
Apimaysin
RT
Rhamnosylisoorientin
Maysin
F3'H
sm2
sm1
EriodictyolF3'Hpr1
Figure 3.1: Flavonoid biosynthetic pathway in maize. Biosynthetic genes (enzymes) in thepathway are: c2 (CHS), chalcone synthase; chi1 (CHI), chalcone isomerase; fht1 (F3H), flavanone 3-hydroxylase; pr1 (F3'H), flavonoid 3'-hydroxylase; a1 (DFR), dihydroflavanone reductase; a2 (AS), anthocyanidin synthase; bz1 (UFGT), UDP-glucose flavonoid 3-O-glucosyltransferase; and bz2 (GST), glutathione S-transferase; FNS1, flavone synthase; GT, C-glycosyl transferase; sm2 (RT), rhamnosyl-transferase; salmon silk1 (sm1) (MCMULLEN et al.2004); and brown pericarp1 (bp1) (EMERSON et al. 1935; MEYERS 1927). The regulatory genes involved in biosynthesis of various flavonoid compounds are: colorless1 (c1)/purple leaf1 (pl1);
70
booster1 (b1)/red1 (r1); and pericarp color1 (p1).
There are more than 100 alleles of p1 which have been identified based on their
expression in the floral organs (BRINK and STYLES 1966; COCCIOLONE et al. 2001).
Alleles of p1 have been named according to their pericarp and cob-glumes pigmentation:
P1-wr (white pericarp, red cob), P1-rr (red pericarp, red cob), P1-rw (red pericarp, white
cob), and p1-ww (white pericarp, white cob) (ANDERSON 1924; BRINK and STYLES 1966;
EMERSON 1917). Three flavonoid biosynthetic genes colorless2 (c2) encodes chalcone
synthase, chalcone isomerase1 (chi1) encodes chalcone isomerase, and anthocyaninless1
(a1) encodes dihydroflavonol reductase, have been shown to be common to both
anthocyanin and phlobaphene biosynthetic pathways and are regulated independently by
transcription factors of these pathways (GOFF et al. 1992; GROTEWOLD and PETERSON
1994). Their promoters contain specific cis-elements to which these transcription factors
bind (TUERCK and FROMM 1994). The independent regulation of a1 gene in the
anthocyanin and phlobaphene branches of flavonoid pathway has been well
characterized. Its promoter has a high affinity P1 Binding Site (haPBS), a low affinity P1
Binding Site (laPBS), and an Anthocyanin Regulatory Element (ARE) (LESNICK and
CHANDLER 1998; TUERCK and FROMM 1994). In both in vitro and in vivo studies, it has
been shown that C1+R1 or P1 can direct high level of expression from promoters
containing these binding sites (GROTEWOLD et al. 1994; POOMA et al. 2002; TUERCK and
FROMM 1994). Mutations in haPBS, laPBS, and ARE result in the inhibition of a1
activation by C1+R1 or P1 (SAINZ et al. 1997).
71
The p locus regulates the biosynthesis of agronomically important, C-glycosyl
flavone and 3-deoxyanthocyanidin compounds in maize silks (MCMULLEN et al. 1998).
The p locus is a compound locus, which contains two genes p1 and p2 on chromosome 1
(ZHANG et al. 2000). Recently, an additional p3 gene has been identified on chromosome
9 (GOETTEL and MESSING 2009). The p locus has been shown to be a major QTL for
maysin biosynthesis and accounts for more than fifty percent of its phenotypic variance
for maysin (BYRNE et al. 1996b; ZHANG et al. 2003). Since the role of p3 gene is not
known, we will exclude it from further discussion here. Although, p1 and p2 can induce
synthesis of C-glycosyl flavones in transformed BMS cell cultures and maize silks, these
two genes differ in their expression patterns in other tissues (GROTEWOLD et al. 1998a;
ZHANG et al. 2003). Both p1 and p2 are expressed in silks but only p1 is expressed in the
kernel pericarp and cob glumes (ZHANG et al. 2000). p1 and p2 regulated C-glycosyl
flavones have antibiotic activity against lepidopteron insects like corn earworm,
Helicoverpa zea (Boddie), a major insect pest of maize (WAISS et al. 1979). Damage is
largely caused by larvae feeding on silks, thereby interfering with pollination. Eventually
the larvae gain access to young kernels enhancing the chance of fungal contamination
which seriously diminishes the quality of sweet corn. Upon insect wounding, C-glycosyl
flavones in silks are oxidized to quinones, which bind to the –SH and –NH2 groups of
free amino acids and proteins that insect larvae require for growth (FELTON et al. 1989;
WISEMAN and CARPENTER 1995).
Three major C-glycosyl flavones found in maize silks are maysin, apimaysin, and
methoxymaysin. To attain their full biological toxicity C-glycosyl flavones require
presence of a hydroxyl group on the B-ring: maysin has a hydroxyl group at the 3'-
72
position of B ring (see Figure 3.1), while methoxymaysin has a methoxy group
(LINDROTH and PETERSON 1988). In apimaysin this position remains un-substituted
(ELLIGER et al. 1980a; ELLIGER et al. 1980b; WAISS et al. 1979), which suggests that a
flavonoid 3'-hydroxylase (F3'H) enzyme might be involved in conversion of apimaysin to
maysin by 3' hydroxylation of its B-ring. This extra hydroxyl group may be required for
oxidation and conversion of flavones to quinones. Of these three C-glycosyl flavones,
maysin has the highest level of antibiosis against lepidopteron insects which is twice
more than that of apimaysin or methoxymaysin (ELLIGER et al. 1980a; ELLIGER et al.
1980b; SNOOK et al. 1994). Antibiotic activity of C-glycosyl flavone causes reduced
larval and pupal weight, and extends time to pupation (WISEMAN and ISENHOUR 1990).
Furthermore, maysin and another flavone called rhamnosylisoorientin, are induced in
response to increased levels of UV-B radiations and protect plants against the damaging
effects of these radiations (CASATI and WALBOT 2005).
P1-controlled phlobaphene pigments are derived from flavan-4-ols: apiferol and
luteoforol, which are also the anticipated precursors of 3-deoxyanthocyanidin,
apigeninidin and luteolinidin, respectively (GROTEWOLD et al. 1998b; STYLES and CESKA
1989). The 3-deoxyanthocyanidin are a rare class of reddish-brown pigments that play an
important role in plant defense in sorghum (AGUERO et al. 2002; CHOPRA et al. 2002;
SNYDER and NICHOLSON 1990). Biosynthesis of phlobaphene and 3-deoxyanthocyanidin
in sorghum is regulated by an ortholog of maize p1 gene, named yellow seed1 (y1)
(BODDU et al. 2005; CHOPRA et al. 1999; ZANTA et al. 1994). It has been demonstrated
that y1 encodes a R2R3 MYB transcription factor highly similar to P1 (BODDU et al.
2006; CARVALHO et al. 2005; CHOPRA et al. 1999). 3-deoxyanthocyanidin function as
73
phytoalexins, because they are low molecular weight antimicrobial compounds produced
in response to infection (HAMMERSCHIMIDT and DANN 1999). 3-deoxyantocyanidins
accumulate in intracellular inclusion bodies in the epidermal cells under attack (SNYDER
and NICHOLSON 1990). Subsequently, these inclusion bodies move towards site of fungal
infection and release their contents, killing both the fungus and the cell that synthesized
them (LO and NICHOLSON 1998; LO et al. 1999; SNYDER and NICHOLSON 1990).
Apigeninidin and luteolinidin are the major phytoalexins of this class. Luteolinidin is
known to be more toxic towards the infecting pathogens as compared to apigeninidin
(NICHOLSON et al. 1987). When challenged with fungus, luteolinidin is produced at
higher levels in the resistant cultivars as compared to the susceptible cultivars (LO et al.
1999). Luteolinidin differs from apigeninidin by 3'-hydroxyl group of the B-ring; 3'
hydroxylation reaction is probably catalyzed by a F3'H enzyme. Previously, it has been
shown that a f3'h gene was induced in response to fungal penetration in sorghum and
therefore, was correlated to accumulation of luteolinidin (BODDU et al. 2004).
We recently isolated a maize f3'h gene (f3'h1) corresponding to red aleurone1
(pr1) locus present on chromosome 5L (M. Sharma and S. Chopra, unpublished). We
showed that the f3'h1 gene encodes a cytochrome P450 F3'H protein that is involved in
the 3' hydroxylation of the B-ring of both anthocyanidins and dihydroflavonols. The pr1
locus has also been implicated in conversion of apiferol to luteoforol which then
polymerizes to form phlobaphenes (STYLES and CESKA 1975). Moreover, the pr1 locus is
a major QTL for apimaysin biosynthesis (CORTES-CRUZ et al. 2003; LEE et al. 1998).
Unlike previous studies, Cortes-Cruz et al., 2003 showed that pr1 locus is also required
for maysin synthesis. Therefore, it is reasonable to hypothesize that the pr1 gene plays a
74
key role in generating 3′ hydroxylated products in different branches of flavonoid pathway.
We have recently shown that pr1 is regulated in the anthocyanin biosynthetic pathway and
requires c1 and r1 for its expression in aleurone tissue (M. Sharma and S. Chopra,
unpublished). In the current study, we demonstrate that the pr1 is also required for the
biosynthesis of 3-deoxyflavonoids which polymerize to produce dark reddish brown
phlobaphenes. We elucidated the genetic regulation of pr1 by p1 and its role in the
biosynthesis of related flavonoid compounds known to be regulated by p locus in maize.
MATERIALS AND METHODS
Maize genetic stocks: The maize inbred lines W23 (genotype P1-wr Pr1 c1 r-g), W22
(P1-wr Pr1 C1 R1), and other genetic stocks MGS 14273 (P1-wr pr1 C1 R1) and MGS
14284 (p1-ww pr1 C1 R1) were kindly provided by the Maize Genetics Co-operation
Stock Center (USDA-ARS, University of Illinois, Urbana, IL). The p1-ww (4co63)
inbred line was obtained from the National Seed Storage Laboratory (Fort Collins, CO),
while P1-rr-4B2, p1-ww-1112 and p-del2 were obtained from Dr. Thomas Peterson,
Iowa State University, Ames, IA (ATHMA and PETERSON 1991; GROTEWOLD et al.
1991b). The p-del2 deletion mutant was derived from P1-vv9D9A and has a deletion
encompassing both p1 and p2 (ZHANG and PETERSON 1999; ZHANG et al. 2003). All p1
alleles except p-del2 and p1-ww-1112 are in 4co63 genetic background. To develop F2
populations, pr1 (MGS 14273) plants were crossed with P1-wr (W22), P1-rr-4B2, and p-
del2 and progenies were grown from selfed F1 plants. These F2 populations showed a 3:1
75
segregation for purple to red aleurones. To develop homozygous Pr1 and pr1 stocks
containing different p alleles, plants from F2 ears showing desirable pericarp, cob-glumes,
and kernel aleurone pigmentation phenotypes (see Table 3.1) were subjected to six
subsequent cycles of self-pollination and selection to develop stocks. To confirm the
presence of Pr1 vs. pr1, polymorphism was detected by PCR using primers in the
promoter region (M. Sharma and S. Chopra, unpublished). Our genetic tests had shown
that all these p stocks carry a functional pr1 gene but all except for p-del2 do not produce
3-hydroxyanthocyanins because of the recessive nature of the c1 locus (data not shown).
The p-del2 allele does have a functional c1 allele yielding purple kernels.
Table 3.1: Genotype and phenotype of different lines developed and used in this study.
Phenotype
ColorlessColorlessRedpr1/pr1; p-del2/p-del2
ColorlessColorlessPurplePr1/Pr1; p-del2/p-del2ColorlessColorlessRedpr1/pr1; p1-ww/p1-ww
ColorlessColorlessPurplePr1/Pr1; p1-ww/p1-wwLight redColorlessRedpr1/pr1; P1-wr/P1-wr
Dark redColorlessPurplePr1/Pr1; P1-wr/P1-wrLight redRedRedpr1/pr1; P1-rr/P1-rr
Dark redRedPurplePr1/Pr1; P1-rr/P1-rrCob-glumesPericarpAleuroneGenotype
Phenotype
ColorlessColorlessRedpr1/pr1; p-del2/p-del2
ColorlessColorlessPurplePr1/Pr1; p-del2/p-del2ColorlessColorlessRedpr1/pr1; p1-ww/p1-ww
ColorlessColorlessPurplePr1/Pr1; p1-ww/p1-wwLight redColorlessRedpr1/pr1; P1-wr/P1-wr
Dark redColorlessPurplePr1/Pr1; P1-wr/P1-wrLight redRedRedpr1/pr1; P1-rr/P1-rr
Dark redRedPurplePr1/Pr1; P1-rr/P1-rrCob-glumesPericarpAleuroneGenotype
RNA gel blot analysis: Silks were collected two days after emergence, and pericarp and
cob tissues were dissected 20 days after pollination (DAP). To isolate total RNA, tissues
were ground in liquid nitrogen and then extracted using Tri-Reagent (Molecular Research
Center Inc., Cincinnati, OH). The RNA was separated on a denaturing gel containing 5%
76
formaldehyde (v/v), 1.2% Agarose (w/v), and 1X RNA buffer (0.4 M MOPS, 0.1 M
anhydrous sodium acetate and 0.01 M Sodium EDTA). The fractionated RNA was
transferred onto a nylon membrane (Osmonics Inc., Minnetonka, MN) and hybridized
with α32P-dCTP probe fragments. Probe fragments used for Northern analysis were
obtained from the plasmids pC2 containing a maize c2 cDNA (WIENAND et al. 1986),
pChi1 containing a maize chi1 cDNA (GROTEWOLD and PETERSON 1994), pA1 with a
maize a1 cDNA (SCHWARZ-SOMMER et al. 1987), pf3'h1 containing maize f3'h1 cDNA,
and pP1 containing full length p1 cDNA (GROTEWOLD et al. 1991a). The p1 probe used
here can recognize both p1 and p2 transcripts (BODDU et al. 2006). RNA gel blot
hybridizations were performed for 24 h at 65° in a hybridization mixture containing, 0.5
M sodium phosphate at pH 7.2, 1 mM EDTA, 7% SDS, and 1% BSA. All membranes
were washed in a solution containing 0.1 X SSC (1 X SSC is 0.15 M sodium chloride and
0.015 M sodium citrate), and 0.5% SDS once at 50° for 15 min, and twice at 65° for 15 to
30 min. Filters were exposed to an X-OMAT film (KODAK, Rochester, NY) for 1 to 4
days before developing. Filters were stripped by washing thrice in a boiling solution of
0.1% SDS before re-hybridization.
C-glycosyl flavone and 3-deoxyanthocyanidin analysis: Primary ear shoots were
covered prior to silk emergence to prevent pollination. Silks were collected on ice two
days after emergence from the ear and subsequently freeze dried. Silk samples were then
shipped on dry ice to the Richard B. Russell Research Center (USDA-ARS, Athens,
Georgia) for biochemical analysis. Flavones were extracted with 125 ml methanol at -20o
for 14 days. Concentration of flavones were determined by reversed-phase high-
77
performance liquid chromatography (HPLC) (MCMULLEN et al. 2001; SNOOK et al.
1989) and expressed as percent dry silk weight. For 3-deoxyanthocyanidin, silks were
extracted for 24 h at -20° with 10 ml of 1% HCL-Methanol (v/v). Their levels were
detected at 495 nm by HPLC, with the same column and solvent program used for
flavone analysis. Commercial standard of luteolinidin hydrochloride was used for
quantification (Roth-Atomergic Chemicals Corp., Farmingdale, N.Y.). Chrysin was used
as internal reference standard. Total 3-deoxyanthocyanin concentration was calculated as
the sum of three distinct luteolinidin glycoside peaks (MCMULLEN et al. 2001). Mean
concentration of total luteolinidin glycosides and C-glycosyl flavones and standard error
was calculated based on number of samples for each genotype. Significant difference for
concentration of these compounds between genotypes was determined by ‘student t-test’
at 95% significance level.
Flavan-4-ols analysis: To detect the presence of flavan-4-ols, 500 mg of cob-glumes
were macerated with a plastic grinder in an Eppendorf tube containing 1 mL of 30%
HCl/70% butanol (v/v) and incubated for 60 min at 37° (WATTERSON and BUTLER 1983).
Samples were spun for 10 min at 14,000 rpm, and the absorption spectra of the
supernatants were determined using a Shimadzu UV-mini 1240 spectrophotometer
(Shimadzu Corporation, Columbia, MD) (STAFFORD 1990; STICH and FORKMANN 1988).
Apiferol and luteoforol are flavan-4-ols previously described from maize and sorghum
that give flavylium ions in acidic butanol with a λ max of 535 and 552 nm, respectively
(WATTERSON and BUTLER 1983). Since flavan-4-ols are unstable and difficult to
quantify, they can be converted to their respective 3-deoxyanthocyanidins and measured.
78
To confirm the identity of the major flavan-4-ols in cob of Pr1 and pr1 alleles in the
genetic background of different p alleles, a methanol extract was hydrolyzed with
aqueous HCl. This converts flavan-4-ols such as apiferol and luteoforol to their
corresponding 3-deoxyanthocyanidin (i.e. apigeninidin and luteolinidin). The treated Pr1
cob extract had a λ max of 498 nm that shifted in alcoholic AlCl3 to a shoulder at 546 nm.
The addition of HCl restored its absorption to 498 nm. The pr1 cob extract had a λ max
of 482 nm and did not respond to AlCl3 test. The results of our samples were verified
using commercial standards for apigeninidin and luteolinidin (Extrasynthese, Genay
Cedex, France). Commercial sample of apigeninidin had a λ max of 480 nm and did not
respond to AlCl3, whereas luteolinidin had a λ max of 498 nm that shifted in AlCl3 to 546
nm and reverted to 498 nm upon re-addition of HCl.
Insect bioassay: Corn earworm (Helicoverpa zea Boddie) eggs were obtained from
Benzon Research Company, Carlisle, PA. Eggs were incubated at 28˚ and they hatched
after 48 hours to produce neonate larvae. Maize lines having functional or mutant pr1
gene in genetic background carrying four different alleles of p gene; P1-wr, P1-rr, p1-
ww, and p-del2 were grown at State College, PA during the summer of 2007. Silks were
collected 2-3 days after emergence and were pooled from 20 field grown plants per line.
The experiment was conducted as a randomized complete block design with 30
replications and two cups per replicate. Freshly collected silks were filled into 1 oz.
plastic diet cups containing 10 ml of 2.5% agar to prevent silk drying. Fresh silk tissues
were used, instead of silk extracts added to artificial insect diet, to maximize the
resemblance to natural larval feeding conditions. One neonate larvae was introduced into
79
each cup. Larvae were allowed to feed in a controlled environment maintained at 28˚,
75% RH, and a photoperiod of 14:10 (light: dark) hours. Larval weights were recorded
after 8 days of feeding. Larvae were subsequently shifted to an artificial diet (JACOB and
CHIPPENDALE 1971) until pupation. Number of dead larvae was also counted. However,
no correlation was observed between larval mortality and pr1 genotypes (data not
shown). Mean larval weight or mean of days to pupate and standard error was calculated
based on number of surviving larvae. Significant difference between genotypes was
determined using ‘student t-test’ at 95% significance level.
RESULTS
Pr1 is required for luteoforol synthesis in cob glumes: Maize plants carrying Pr1 and
pr1 alleles have purple and red kernel aleurone phenotypes, respectively, due to
formation of cyanidin (purple) and pelargonidin (red) pigments in aleurone tissues (M.
Sharma and S. Chopra, unpublished). Cyanidin and pelargonidin are produced via the
anthocyanin branch of the flavonoid pathway. Previous studies implicated the role of pr1
gene in biosynthesis of 3-deoxyflavonoids (STYLES and CESKA 1975). We tested if pr1 is
regulated by p1, a MYB transcription factor shown to be required for the biosynthesis of
3-deoxyflavonoids and phlobaphenes. Phenotypic characterization of P1-wr ears
segregating for Pr1 and pr1 showed differential cob pigmentation phenotypes: dark red in
P1-wr/P1-wr; Pr1/Pr1 as compared to light red cobs in P1-wr/P1-wr; pr1/pr1
(Figure 3.2). Biochemical characterization for differential accumulation of flavan-4-ols
80
which are precursors of phlobaphenes was performed (WATTERSON and BUTLER 1983):
P1-wr/P1-wr; Pr1/Pr1 dark red cob tissue had maximum absorption (λ max) at 552 nm
while light red cob from P1-wr/P1-wr; pr1/pr1 plants had λ max at 535 nm. These
absorption spectra correspond to luteoforol and apiferol, respectively (GROTEWOLD et al.
1998a). To further confirm if pr1 gene affects flavan-4-ols accumulation in cob tissue, we
converted total flavan-4-ols to their corresponding 3-deoxyanthocyanidin by acid
hydrolysis of cob methanolic extracts (Figure 3.3). Interestingly, cob extracts from P1-
wr/P1-wr; Pr1/Pr1 showed presence of luteolinidin (λ max 498 nm) corresponding to
luteoforol. Conversely, cob extracts of P1-wr/P1-wr; pr1/pr1 showed the presence of
apigeninidin (λ max 482 nm) which corresponds to apiferol, as the major flavan-4-ol.
Moreover, Pr1 and pr1 plants in the presence of P1-rr allele also showed pericarp and
cob color differences and had maximum absorption wavelengths corresponding to
luteolinidin and apigeninidin, respectively (not shown). Importantly, p1-ww-1112 and p-
del2 plants, carrying null alleles of p1, and p1 and p2, respectively, did not show any
visible pigmentation or detectable flavan-4-ol accumulation in cob glumes. This was in
accordance with p1 gene function as regulator of phlobaphene pigment formation
(GROTEWOLD et al. 1994). In conclusion, our results indicate that pr1 is involved in the
conversion of apiferol to luteoforol in the presence of a functional p1 gene.
81
Pr1/Pr1
pr1/pr1
P1-wr/P1-wr p-del2/p-del2P1-rr/P1-rr
Purple aleurone
Red aleurone
Pr1/Pr1
pr1/pr1
P1-wr/P1-wr p-del2/p-del2P1-rr/P1-rrP1-wr/P1-wr p-del2/p-del2P1-rr/P1-rr
Purple aleurone
Red aleurone
Figure 3.2: Functional pr1 is required for the formation of luteoforol. The pr1 cob glumes have light red pigmentation and Pr1 has dark red cob glumes in the presence of P1-wr and P1-rr alleles. Pr1 and pr1 ears carrying p-del2 allele do not show any pigmentation.
82
Wavelength (nm)
Pr1pr1
Abs
orba
nce
Pr1/Pr1; p-del2/p-del2 pr1/pr1; p-del2/p-del2
Abs
orba
nce
Pr1/Pr1; P1-wr/P1-wr
Luteolinidin
λmax498
498
Abs
orba
nce
pr1/pr1; P1-wr/P1-wr
Apigeninidin
λmax482
480
Wavelength (nm)
Pr1pr1
Abs
orba
nce
Pr1/Pr1; p-del2/p-del2 pr1/pr1; p-del2/p-del2
Pr1pr1
Pr1pr1
Abs
orba
nce
Pr1/Pr1; p-del2/p-del2 pr1/pr1; p-del2/p-del2
Abs
orba
nce
Pr1/Pr1; P1-wr/P1-wr
Luteolinidin
λmax498
498
Abs
orba
nce
Pr1/Pr1; P1-wr/P1-wr
Luteolinidin
λmax498
498
Abs
orba
nce
pr1/pr1; P1-wr/P1-wr
Apigeninidin
λmax482
480
Abs
orba
nce
pr1/pr1; P1-wr/P1-wr
Apigeninidin
λmax482
480
Figure 3.3: Absorption spectra of dark red, light red, and colorless cob glumes. Methanolicextracts from dark cob glumes collected from Pr1 ears in genetic background of P1-wr and P1-rr alleles gave maximum absorption at 498 nm. Light red cob tissues from pr1 ears in P1-wr and P1-rr genetic background gave maximum absorption at 482 nm. This corresponds to maximumabsorption peak for standard, luteolinidin and apigeninidin, respectively. No absorption peak forflavan-4-ols was observed in p-del2 cob tissue.
f3′h1 transcript accumulation correlates with p1 expression in floral tissues: We
have previously shown that pr1 is regulated by c1/r1 transcription factors in the
83
anthocyanin pathway (M. Sharma and S. Chopra, unpublished). Along with
anthocyanins, pr1 appears to control the composition of flavan-4-ols in cob tissues.
However, p1 gene is known to regulate the phlobaphene pathway through which flavan-
4-ols are formed in these floral tissues (GROTEWOLD et al. 1994). Thus, it is possible that
the p1 gene regulates the pr1 transcript during differential accumulation of flavan-4-ols in
these tissues. To test this idea, we looked at steady state transcript levels of pr1 in
pericarp, cob-glumes, and silk tissues of plants carrying functional and null p1 alleles in a
common genetic background of 4co63 (Figure 3.4). While no f3′h1 transcript was present
in the colorless pericarp of P1-wr and p1-ww, substantial levels were detected in P1-rr
that shows red pericarp pigmentation. In red cob glumes of P1-rr and P1-wr there was an
appreciable amount of f3′h1 transcript, while p1-wws carrying the null p1 alleles did not
show f3′h1 transcript in cob glumes. Transcript levels of p1, c2, chi1, a1, and f3'h1 were
compared in silk tissues. Similar to p1 expression, f3'h1 was highly expressed in silk
tissue of P1-rr and P1-wr. Although, both p1-ww alleles (p1-ww [4co63] and p1-ww-
1112) have non-functional p1 gene, they showed detectable levels of p2 expression. The
probe used in this study can detect both p1 and p2 specific transcripts (see Materials and
Methods) and p1-ww alleles used in this study have functional p2 gene. The p1 and p2
transcripts were not detected in p-del2 silks because of deletions in both the genes.
Interestingly, f3'h1 expression was also detected at appreciable levels in p1-ww silk tissue
which can thus be attributed to its regulation by a functional p2 gene expressed in the silk
tissues (ZHANG et al. 2000). Plants carrying the p-del2 allele did not show transcript of
f3'h1 indicating that a functional p1 and/or p2 gene is necessary for its expression. Other
biosynthetic genes c2, chi1, and a1 showed expression pattern similar to that of f3'h1,
84
indicating a commonality in the expression of these genes. Overall, results indicate that
the p gene regulates the transcriptional expression of f3'h1 in maize floral tissues which
include pericarp, cob glumes, and silks.
Formation of luteolinidin in maize silks requires a functional pr1: In sorghum, 3-
deoxyanthocyanidins are suggested to be formed from flavan-4-ols and shown to have
antifungal properties (NICHOLSON et al. 1987). Although p1 is known to regulate 3-
deoxyanthocyanidins, their detailed biosynthesis is poorly understood (GROTEWOLD et al.
1998a; MCMULLEN et al. 2001). Since we showed that pr1 induces the differential
accumulation of p1 regulated flavan-4-ols, it is possible that the pr1 is also playing a role
in the 3-deoxyanthocyanidin biosynthesis. To test whether pr1 is involved in 3'
hydroxylation of the B-ring of 3-deoxyanthocyanidins, silk extracts from Pr1 and pr1
plants carrying different p1 alleles (see Materials and Methods) were analyzed by reverse
phase chromatography. Silk extracts of Pr1 plants carrying P1-wr or P1-rr alleles
showed four major peaks at 480 nm for luteolinidin glycosides labeled as a, b, c, and d
with retention time of 13.6 min, 14.1 min, 14.9 min, and 17.9 min, respectively
(Figure 3.5). The peaks labeled luteolinidin glycosides a, b, c, and d had the luteolinidin
aglycone spectra but eluted considerably before the aglycone so were more polar and
hence must be glycosylated. Based on their different elution times, these are mono-
glucosyl or di-glucosyl luteolinidins. Interestingly, silks from pr1 plants in P1-rr and P1-
wr genetic background, showed much smaller peaks for all luteolinidin glycosides and
few peaks of unknown compounds. Pr1 and pr1 plants carrying p-del2 or p1-ww alleles
did not produce any detectable levels of these compounds. This later result is consistent
85
A
P1-rr P1-wr p1-ww (4co63)
p1-ww -1112
B
Gel
c2
Pericarp Cob Glumes
f3′h1
p1-w
w (4
co63
)
P1-rr
P1-w
r
p1-w
w-11
12
p1-w
w (4
co63
)
P1-r
rP1
-wr
p1-w
w-11
12
C
Silk
p1
c2
chi1
a1
Gel
f3′h1
p1-w
w (4
co63
)P1
-rr
P1-w
r
p1-w
w-11
12
p-de
l2
A
P1-rr P1-wr p1-ww (4co63)
p1-ww -1112
P1-rr P1-wr p1-ww (4co63)
p1-ww -1112
B
Gel
c2
Pericarp Cob Glumes
f3′h1
p1-w
w (4
co63
)
P1-rr
P1-w
r
p1-w
w-11
12
p1-w
w (4
co63
)
P1-r
rP1
-wr
p1-w
w-11
12
Gel
c2
Pericarp Cob Glumes
f3′h1
Gel
c2
Pericarp Cob Glumes
f3′h1
p1-w
w (4
co63
)
P1-rr
P1-w
r
p1-w
w-11
12
p1-w
w (4
co63
)
P1-rr
P1-w
r
p1-w
w-11
12
P1-rr
P1-w
r
p1-w
w-11
12
p1-w
w (4
co63
)
P1-r
rP1
-wr
p1-w
w-11
12
p1-w
w (4
co63
)
P1-r
rP1
-wr
p1-w
w-11
12
P1-r
rP1
-wr
p1-w
w-11
12
C
Silk
p1
c2
chi1
a1
Gel
f3′h1
p1-w
w (4
co63
)P1
-rr
P1-w
r
p1-w
w-11
12
p-de
l2
C
Silk
p1
c2
chi1
a1
Gel
f3′h1
p1-w
w (4
co63
)P1
-rr
P1-w
r
p1-w
w-11
12
p-de
l2
p1-w
w (4
co63
)P1
-rr
P1-w
r
p1-w
w-11
12
p-de
l2
Figure 3.4: Expression of pr1 gene is up-regulated in the presence of a functional p1 and/or p2 genes. (A) Different p alleles with functional and nonfunctional p1 gene are shown: P1-rr, red pericarp, red cob glumes; P1-wr, white pericarp, red cob glumes; p1-ww [4co63], white pericarp and white cob glumes, and p1-ww-1112, white pericarp and white cob glumes. (B) Northern blotof RNA extracted from pericarp tissue and cob glumes of different p alleles was probed with f3′h1 and c2 probes. P1-rr lane shows f3′h1 expression in pericarp. While in cob glumes, f3′h1 expression was observed in P1-rr and P1-wr. (C) RNA gel blot of silk tissues from different p alleles was hybridized with different gene probes. Gel pictures show equal loading of RNA indifferent lanes.
86
with the fact that these alleles lack a functional p1 (p1-wws) and p1 and p2 (p-del2) genes
to induce 3-deoxyanthocyanidin accumulation in silks. Further, quantitative analysis
showed similar trends for the accumulation of luteolinidin glycosides in silks of Pr1 and
pr1 plants (Figure 3.6); Pr1 silks accumulated significantly higher levels of total
luteolinidin glycosides as compared to pr1 silks in P1-rr and P1-wr genetic backgrounds
(P = 0.010 and 0.012, respectively). None or very little amount of 3-deoxyanthocyanidins
accumulated in p-del2 or p1-ww plants. In summary, results from HPLC analysis of 3-
deoxyanthocyanidins in silks of Pr1 and pr1 plants showed that the pr1 gene is required
for the synthesis of luteolinidin. In addition, these results demonstrate that pr1 gene
expression requires a functional p allele during 3-deoxyanthocyanidin biosynthesis.
Functional pr1 gene is required for maysin biosynthesis: Our results thus far suggest
that pr1 is under regulatory control of p and is required for the biosynthesis of both
phlobaphenes and 3-deoxyanthocyanidins. To test if pr1 has a role in p-regulated C-
glycosyl flavones biosynthesis, we analyzed flavone levels in silk tissue of Pr1 and pr1
plants using reverse phase chromatography (Figure 3.7). A major peak for maysin and
small peaks for apimaysin and chlorogenic acid (CGA) were detected in Pr1/Pr1; P1-
wr/P1-wr silks at 340 nm. Chlorogenic acid, maysin, and apimaysin showed
87
pr1/pr1; P1-wr/P1-wrUnknown
a
c
d
A 480
a
b c
dPr1/Pr1; P1-wr/P1-wr
A48
0
Pr1/Pr1; p-del2/p-del2
Time (min)
A48
0
5 10 15 20 25
pr1/pr1; P1-wr/P1-wrUnknown
a
c
d
A 480
A 480
a
b c
dPr1/Pr1; P1-wr/P1-wr
A48
0A
480
Pr1/Pr1; p-del2/p-del2
Time (min)
A48
0A
480
5 10 15 20 255 10 15 20 25
Figure 3.5: Characterization of the 3-deoxyanthocyanidin in Pr1 and pr1 silk tissue. Compounds could be identified because their retention matched known standards. HPLC chromatograms ofsilk methanolic extracts from Pr1 and pr1 at 480 nm. Luteolinidin glycosides a, b, c, and d were eluted at approximately 13.6 min, 14.1 min, 14.9 min, and 17.9 min, respectively.
88
Tota
l Lut
eolin
idin
Gly
cosid
es
(ug/
gdr
y w
eight
)
Genotype
Tota
l Lut
eolin
idin
Gly
cosid
es
(ug/
gdr
y w
eight
)To
tal L
uteo
linid
in G
lyco
sides
(u
g/g
dry
weig
ht)
Genotype Figure 3.6: Total luteolinidin levels in silk tissue of pr1/P1-wr, Pr1/P1-wr, pr1/P1-rr, Pr1/P1-rr, pr1/p-del2, Pr1/p-del2, pr1/p1-ww, Pr1/p1-ww were determined by HPLC analysis at 495 nm. All data are presented as mean of six replicates.
retention times of 12.0 min, 22.0 min, and 23.5 min, respectively. Silk extracts from
pr1/pr1; P1-wr/P1-wr showed single dominant peak for apimaysin, a small peak for
CGA and no peak for maysin. Peaks for maysin and apimaysin were not observed at
appreciable level in Pr1/Pr1; p-del2/p-del2 silks. Maysin and apimaysin were identified
by comparing their retention times and UV absorption spectrum with authentic standards.
Quantitative analysis showed that Pr1 plants in P1-wr or P1-rr backgrounds has
significantly higher levels of maysin (P = 0.007, 0.023) as compared to pr1 plants in
similar backgrounds (Figure 3.8). In contrast, pr1/pr1; P1-wr/P1-wr silks produced
significantly high levels of apimaysin (P = 0.001) and very little maysin. In p-del2 (null
for p1 and p2) silk extracts, no significant levels of C-glycosyl flavones were detected
irrespective of the type of pr1 allele present. While in Pr1 and pr1 lines carrying p1-ww
(4co63) allele, very low levels of maysin and apimaysin were detected. The chemical
89
structural difference between maysin and apimaysin is the presence of an additional
hydroxyl group at 3' position of the B-ring in maysin. The pr1 encoded F3'H enzyme is
required for hydroxylation at this position of flavonoids (LARSON et al. 1986) (M.
Sharma and S. Chopra, unpublished). These results suggest that the pr1 gene plays a role
in the C-glycosyl flavones pathway and is required for the conversion of apimaysin to
maysin.
Levels of rhamnosylisoorientin, a C-glycosyl flavone present upstream of maysin,
and CGA, a phenylpropanoid, were also measured in these silk samples. CGA is
synthesized from cinnamate through the phenylpropanoid pathway and has structural
similarity to maysin (see Figure 3.1). Similar to maysin, CGA has been implicated in
resistance to corn earworm in maize (DUFFEY and STOUT 1996; SNOOK et al. 1994).
Interestingly, Pr1/Pr1; P1-rr/P1-rr plants accumulated significantly higher amount of
rhamnosylisoorientin (P = 0.030) in their silks as compared to homozygous pr1 plants
carrying P1-rr allele. This may be due to the conversion of naringenin to eriodictyol by
F3'H in the presence of Pr1 (LARSON and BUSSARD 1986). Eriodictyol then could get
converted into rhamnosylisoorientin in the flavone pathway (MCMULLEN et al. 2004).
This suggests that pr1 is not only required for conversion of apimaysin to maysin but also
plays a role upstream of this pathway.
Although, p1 is known to be a major QTL for CGA formation (BUSHMAN et al.
2002; GROTEWOLD et al. 1998a), very low levels of CGA were detected in lines with
functional p1 gene. Surprisingly, Pr1 and pr1 plants in p1-ww background accumulated
higher concentrations of CGA than that of Pr1 or pr1 lines carrying P1-rr, P1-wr, and p-
90
del2 alleles. Interestingly, Pr1/Pr1; p1-ww/p1-ww silks accumulated significantly higher
amount of CGA (P = 0.008) as compared to pr1 in p1-ww background. In conclusion,
Time (min)
pr1/pr1; P1-wr/P1-wr
Chlorogenic acid
A34
0
Pr1/Pr1; P1-wr/P1-wr
Chlorogenic acid
A34
0
Apimaysin
ISTD
ISTD
Maysin (22 min)
Apimaysin (23.5 min)
Maysin
Pr1/Pr1; p-del2/p-del2
Chlorogenic acid (12 min)
A34
0
ISTD
5 10 15 20 25 30
Time (min)
pr1/pr1; P1-wr/P1-wr
Chlorogenic acid
A34
0A
340
Pr1/Pr1; P1-wr/P1-wr
Chlorogenic acid
A34
0A
340
Apimaysin
ISTD
ISTD
Maysin (22 min)
Apimaysin (23.5 min)
Maysin
Pr1/Pr1; p-del2/p-del2
Chlorogenic acid (12 min)
A34
0
ISTD
5 10 15 20 25 30
Maysin
Pr1/Pr1; p-del2/p-del2
Chlorogenic acid (12 min)
A34
0A
340
ISTD
5 10 15 20 25 305 10 15 20 25 30
Figure 3.7: Characterization of the C-glycosyl flavones in Pr1 and pr1 silks. HPLC chromatograms of silk methanolic extracts from Pr1 and pr1 at 340 nm. Maysin, apimaysin, and chlorogenic acid were eluted at approximately 22 min, 23.5 min, and 11.8 min, respectively.
91
our chromatography results suggest a role for the pr1 gene in biosynthesis of C-glycosyl
flavones in silk tissue under the regulation of p gene.
Pr1 confers resistance against corn earworm: To determine the biological relevance of
differential accumulation of maysin and apimaysin in silk tissue of Pr1 and pr1 plants,
we performed insect silk feeding bioassays. Corn earworm larvae (neonate stage) were
fed on fresh silks collected from same Pr1 and pr1 lines that were used for HPLC
analysis. Corn earworm larvae’s growth rate was slower on Pr1 silks in contrast to larvae
fed on pr1 silks. Data analysis revealed that larvae fed on Pr1 silks had lower weight and
took longer time to pupate when compared to pr1 silks (P = 0.012 and 0.011)
(Figure 3.9). These results are in agreement with the accumulation of higher amounts of
maysin in Pr1 silks. Interestingly, larvae fed on pr1/pr1; P1-wr/P1-wr silks showed
lower weight and longer time to pupate as compared to Pr1/Pr1; P1-wr/P1-wr silks (P =
0.010 and 0.005, respectively), even though the Pr1 silks had higher levels of maysin as
compared to pr1. Overall, combined data from HPLC analysis and corn earworm larvae
feeding bioassay indicated that a functional pr1 is required for formation of maysin and is
responsible for providing higher resistance against corn earworm larvae in silk tissue.
92
Rham
nosy
lIso
orien
tin
(% d
ry w
eight
)M
aysin
(%
dry
weig
ht)
Apim
aysin
(% d
ry w
eight
)Ch
loro
geni
c ac
id
(% d
ry w
eight
)
Genotype
Rham
nosy
lIso
orien
tin
(% d
ry w
eight
)M
aysin
(%
dry
weig
ht)
Apim
aysin
(% d
ry w
eight
)Ch
loro
geni
c ac
id
(% d
ry w
eight
)
Genotype Figure 3.8: Maysin, apimaysin, rhamnosyl isoorientin, and chlorogenic acid levels in silk tissue of homozygous pr1/P1-wr, Pr1/P1-wr, pr1/P1-rr, Pr1/P1-rr, pr1/p-del2, Pr1/p-del2, pr1/p1-ww, and Pr1/p1-ww were determined by HPLC analysis. All data are presented as mean of tenbiological replicates.
93
Day
s to
Pup
ate
Genotype
Day
s to
Pup
ate
Genotype
Larv
al W
eigh
t (g)
afte
r 8
Day
s of F
eedi
ng
Genotype
Larv
al W
eigh
t (g)
afte
r 8
Day
s of F
eedi
ng
Genotype
Figure 3.9: Mean corn earworm larvae weight and days to pupation on fresh silk from pr1/P1-wr,Pr1/P1-wr, pr1/P1-rr, Pr1/P1-rr, pr1/p-del2, Pr1/p-del2, pr1/p1-ww, and Pr1/p1-ww lines. Larvae fed on silks of inbred B73 and p1-ww and p-del2, served as controls.
DISCUSSION
This study investigated the genetic regulation and role of the pr1-encoded f3'h1 in
the formation of antifungal and insecticidal flavonoid compounds in maize silk. In
sorghum, attempted penetration of Cochliobolus heterostrophus leads to up regulation of
94
f3'h gene and sequential accumulation of a 3-deoxyanthocyanidin compound called
luteolinidin (BODDU et al. 2004). The 3-deoxyanthocyanidin pathway in sorghum leaves
requires a myb protein encoded by y1, which is an ortholog of maize p1 (BODDU et al.
2005; CHOPRA et al. 2002; ZANTA et al. 1994). We wanted to understand if formation of
luteoforol (a flava-4-ol) and luteolinidin in maize silks requires the action of pr1 encoded
F3'H. It has been hypothesized but never demonstrated directly that the pr1 gene plays a
role in the synthesis of flavan-4-ols (precursor of phlobaphene) and C-glycosyl flavones
in maize (LEE et al. 1998; STYLES and CESKA 1975). It was not known if pr1 is regulated
in the pathway that is controlled by p gene. To do so, we dissected the molecular and
biochemical mechanism responsible for phlobaphene accumulation in pericarp and cob
glumes in Pr1 and pr1 alleles in the presence and absence of functional p alleles. Our
biochemical analysis revealed that the dark red cob tissue of Pr1 accumulated luteoforol
as compared to apiferol accumulating in light red cob of pr1. This further confirms
results from our gene expression analysis (see below). Several lines of evidence showed
that flavan-4-ols are the precursors of 3-deoxyanthocyanidin which are known to have
antifungal activity (AGUERO et al. 2002; CHOPRA et al. 2002; GROTEWOLD et al. 1998a;
MCMULLEN et al. 2001; SNYDER and NICHOLSON 1990). Interestingly, the sorghum f3'h
gene is required for the production of a 3-deoxyanthocyanidin called luteolinidin (BODDU
et al. 2004). And similar to what we have shown for f3'h1, sorghum f3'h is regulated by
p1 ortholog called y1 (BODDU et al. 2005). We found that silk extracts from Pr1
accumulated significantly greater amounts of luteolinidin glycosides as compared to pr1
silk extracts. Luteolinidin is known to be more toxic towards fungi and it accumulates at
higher levels in resistant as compared to susceptible sorghum cultivars (LO et al. 1999;
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NICHOLSON et al. 1987). It remains to be tested if silk extracts containing luteolinidin
glycosides show resistance to fungal pathogens of maize.
Our results established that f3'h1 transcription requires a functional p allele
expressing in floral tissues that included pericarp, cob glumes, and silk. Importantly, the
presence of f3'h1 transcript in silks of p1-ww lines suggest that in addition to p1, paralog
p2 may be independently required for pr1 gene expression in silks. These results were
further strengthened by the fact that, p-del2, with deletion of p1 and p2 genes (ZHANG et
al. 2003), do not show any transcript of f3'h1. p regulated expression of flavonoid
biosynthetic gene a1 has been well characterized and promoter cis-regulatory elements
have been delineated (GROTEWOLD et al. 1994). Characterization of f3'h1 promoter
shows the presence of consensus MYB binding sites that resemble conserved P binding
sites (PBS) of the a1 promoter (M. Sharma and S. Chopra, unpublished results). Similar
to a1, f3'h1 promoter also contains anthocyanin regulatory element (ARE), that are
binding sites of anthocyanin regulatory proteins. We have recently demonstrated that
f3'h1 is also required for the formation of purple cyanidin from red pelargonidin and its
expression is regulated by C1 and R1 proteins. Flavonoid 3' hydroxylase is a late gene
but its regulation resembles that of a1, an early gene in the biosynthesis of 3-deoxy- and
3-hydroxyflavonoids. It is interesting that maize has retained both P1 and C1
transcription factors, which are very similar in their structure and function and regulate
same set of genes (SAINZ et al. 1997).
From our biochemical analysis of silk extracts it is clear that the F3'H enzyme,
encoded by pr1 is required for the hydroxylation of apimaysin to maysin. Structurally, C-
glycosyl flavone compounds, apimaysin and maysin are highly related compounds,
96
differing only by 3' B-ring hydroxylation (ELLIGER et al. 1980a; LINDROTH and
PETERSON 1988). A previous study also found high correlation between the
concentrations of maysin and apimaysin (WIDSTROM and SNOOK 1998b). It has been
shown that p1 acts on apimaysin in an additive manner in the same way as it does on
maysin (GUO et al. 2004). Unexpectedely, the accumulation of maysin and apimaysin in
Pr1/Pr1; P1-wr/P1-wr and pr1/pr1; P1-wr/P1-wr silks do not exactly follow the inverse
correlation. Apimaysin levels in pr1/pr1; P1-wr/P1-wr silks increase to substantially
higher level as compared to its maysin level. Possible explanation of this may be that the
apimaysin is acting as substrate for another enzyme and is converted into a product we
are not able to detect in our analysis. Since C-glycosyl flavones biosynthesis is poorly
understood and many genes of this pathway have not been isolated, we can not determine
the fate of different substrates conclusively. Importantly, we found that the greater level
of C-glycosyl flavones in Pr1 verses pr1 plants was correlated with the reduction of
larval weight of corn earworm. However, pr1/ pr1; P1-wr/P1-wr perplexingly shows a
significantly lower larval weight and significantly higher time to pupate as compared to
Pr1 in similar genetic background. Perhaps this can be attributed to the exceptionally
high level of apimaysin accumulation in this particular line. As mentioned earlier,
apimaysin has insecticidal activity, although less than that of maysin (SNOOK et al. 1993).
In current study, levels of rhamnosylisoorientin and chlorogenic acid were
measured to understand the flux of flavone pathway in maize silks. Both these
compounds have also been shown to have insecticidal activity (DUFFEY and STOUT 1996;
SNOOK et al. 1994). Rhamnosylisoorientin is formed from isoorientin through the action
of rhamnosyl-transferase (RT) encoded by salmon silk2 (sm2) gene and product of sm1
97
gene acts on rhamnosylisoorientin to form maysin via unknown intermediate(s)
(MCMULLEN et al. 2004). Silks of sm1 or sm2 plants show salmon silk phenotype due to
accumulation of rhamnosylisoorientin and isoorientin, respectively (WIDSTROM and
SNOOK 1998a). In addition, functional p alleles regulate expression of salmon silk
phenotype such that there is an epistatic interaction between p and sm1 and sm2
(ANDERSON 1921; MCMULLEN et al. 2004). Since the silk tissues analyzed in our study
were green with functional p, both sm1 and sm2 genes must be functional. Importantly,
Pr1 silks have higher levels of rhamnosylisoorientin as compared to pr1 in the presence
of P1-rr allele. This result is consistent with the previous observations that pr1 encoded
F3'H enzyme is also involved in the early steps of conversion of flavanone (naringenin)
to dihydroxyflavanone (eriodictyol) (MCMULLEN et al. 2004). It is possible that
formation of C-glycosyl flavones does not entirely follow a single linear pathway as
demonstrated in other related studies of flavonoid biosynthesis (CORTES-CRUZ et al.
2003; GROTEWOLD et al. 1998a; LEE et al. 1998). Modulation of substrate flow through
alternative pathways has been demonstrated (BYRNE et al. 1998; MCMULLEN et al. 2001;
SZALMA et al. 2002).
Elevation of CGA levels in p1-ww lines was unexpected but demonstrates that
factors other than p1 can affect the levels of phenylpropanoids that are not directly
flavonoid precursors. This is in contrast to what has been found in some previous studies,
where p1 expression correlated with increased levels of CGA both in vitro and in vivo
(BUSHMAN et al. 2002; GROTEWOLD et al. 1998a). This may be because of the role of the
p2, present in p1-ww allele, in the biosynthesis of CGA. It has been shown that p2 can
activate PAL and can also induce production of phenylpropanoid compounds in maize
98
cell cultures (ZHANG et al. 2003). p2 is tightly linked to p1 and is involved in CGA
regulation, which may make it difficult to detect during QTL analysis. Interestingly, in a
previous study increase in CGA level was associated with decrease in flavones
concentration, similar to what we have observed in the current study (BUSHMAN et al.
2002). It may be possible that in the absence of p1, either metabolic flux gets shifted from
flavone towards the CGA pathway or some other loci divert substrate towards the CGA
biosynthetic pathway. Alternately, it is possible that C-glycosyl flavone biosynthesis
takes place through a separate branch where pr1 encoded F3'H can perform 3'-
hydroxylation of precursor phenylpropanoid compound at nine carbon stage rather than at
15 carbon stage. This could explain the accumulation of significantly higher levels of
CGA, rhamnosylisoorientin, and maysin in lines carrying functional Pr1.
The accumulation of different levels of flavones, 3-deoxyanthocyanidins, and
CGA in different p alleles could be attributed to polymorphic structural genes at different
loci (MCMULLEN et al. 2001; SZALMA et al. 2005). It has been shown that functional c2,
whp1, and a1 genes have a positive effect on maysin accumulation and negative effect on
CGA (SZALMA et al. 2005). Further, genetic variation at p locus was found to be
significant for maysin and CGA accumulation: p1 and p2 together result in a maximum
amounts; p1 lines were high in maysin concentration and moderate in CGA; p2 lines had
elevated levels of CGA and moderate maysin (BUSHMAN et al. 2002; SZALMA et al.
2005). This could explain the differential accumulation of flavones and CGA in different
p alleles used in the current study.
The importance of flavonoid defense compounds, 3-deoxyanthocyanidin and C-
glycosyl flavones has been well established (BYRNE et al. 1996b; NICHOLSON and
99
HAMMERSCHMIDT 1992; WAISS et al. 1979). Therefore, it is important to unravel the role
of regulatory and biosynthetic genes involved in their production in order to tailor
resistant plants. Through transgenic studies it is known that functional p1 and p2 can
induce biosynthesis of these compounds. As demonstrated in the current study, pr1 gene
is required for formation of more toxic antifungal and insecticidal compounds,
luteolinidin and maysin, respectively. In order to make transgenic plants with more
effective resistance, it is important to understand not only the regulation of this pathway
but also, the exact role of downstream genes. And also, it will be beneficial to use tissue
specific promoters to enhance consumer acceptance or to prevent the harmful effect, if
any, of high levels of these compounds on human or animal health. Studies with P1-rr
and P1-wr promoters have shown the induction of maysin in p1-ww plants, but those
transgenic plants also produce varying degree and distribution of pigmentation in
pericarp and other tissues (COCCIOLONE et al. 2005). Later, a p1 cDNA fused with a silk
specific promoter from zmgrp5 gene was used to induce C-glycosyl flavones formation in
silk tissue (JOHNSON et al. 2007). Although, transgenic plants accumulated higher levels
of maysin but they also accumulated pigmentation in kernel pericarp. This suggests that
further investigation is needed to completely understand the tissue specific gene
expression in plants.
As demonstrated in the current study and also reported previously (M. Sharma
and S. Chopra, unpublished), the pr1 gene plays a significant role in generating diversity
in anthocyanin, phlobaphenes, 3-deoxyanthocyanidin, and C-glycosyl flavone
compounds. Since pr1 encoded F3'H enzyme has demonstrated activity on such wide
100
range of substrates, it will be interesting to investigate the role of pr1 in biosynthesis of
related agronomically important phenylpropanoid compounds, such as, CGA, and lignins.
101
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WAISS, A. C. J., B. G. CHAN, C. A. ELLIGER, B. R. WISEMAN, W. W. MCMILLIAN, N. W. WIDSTROM, M. S. ZUBER and A. J. KEASTER, 1979 Maysin, a flavone glycoside from corn silks with antibiotic activity toward corn earworm. J. Econ. Entomol. 72: 256-258.
WATTERSON, J. J., and L. J. BUTLER, 1983 Occurance of an unusual lucoanthocyanidins and absence of proanthocyanidins in sorghum leaves. J Agric Food Chem 31: 41-45.
WIDSTROM, N. W., and M. E. SNOOK, 1998a A gene controlling biosynthesis of isoorientin, a compound in corn silks antibiotic to the corn earworm. Entomol Exp Appl 89: 119-124.
WIDSTROM, N. W., and M. E. SNOOK, 1998b Genetic variation for maysin and its analogues in crosses among corn inbreds. Crop Science 38: 372-375.
WIENAND, U., U. WEYDEMANN, U. NIESBACH-KLOSGEN, P. A. PETERSON and H. SAEDLER, 1986 Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone syntahse. Mol. Gen. Genet. 203: 202-207.
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WISEMAN, B. R., and D. J. ISENHOUR, 1990 Effects of resistant corn silks on corn earworm (Lepidoptera: Noctuidae) biology: a laboratory study J. Econ. Entomol. 83: 614-617.
ZANTA, C. A., X. YANG, J. D. AXTELL and J. L. BENNETZEN, 1994 The candystripe locus, y-cs, determines mutable pigmentation of the sorghum leaf, flower, and pericarp. Journal of Heredity 85: 23-29.
ZHANG, J., and T. PETERSON, 1999 Genome rearrangements by nonlinear transposons in maize. Genetics 153: 1403-1410.
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Chapter 4
Genetic control of purple anthocyanin formation in the aleurone tissue of maize
INTRODUCTION
Flavonoids are 15 carbon secondary metabolites that have many functions in plant
growth and development. They are essential for pollen tube germination, protection from
UV light and regulation of auxin transport (TAYLOR and GROTEWOLD 2005; WINKEL-
SHIRLEY 2001). Additionally, they serve as signaling molecules in plant-bacterium
symbiosis, and as phytoalexins synthesized to combat stress or pathogen infection (KOES
et al. 1994; NICHOLSON and HAMMERSCHMIDT 1992). In addition to their benefits to
plants, flavonoids also have many pharmacological and dietary benefits for humans and
animals (FORKMANN and MARTENS 2001; SHIH et al. 2007). Their potent antioxidant
activity helps fight against bacterial and viral infection, mutagenesis, and inflammations
(MIYAGI et al. 2000). The antioxidant activity of flavonoids can be accompanied by anti-
neoplastic, anti-thrombotic, and vasodilator activity (LIU et al. 2004). Naringenin,
quercetin, and cyanidin-3-glycoside are examples of maize anthocyanins with these
properties. Once we fully understand how these flavonoids are synthesized, we may be
able to produce desired quantities in maize and other related plant species.
Flavonoids are a large class of secondary metabolites, of which anthocyanins
represent most conspicuous class due to synthesis of wide range of pigments. Genetic
studies of anthocyanin biosynthesis began with Mendel’s experiments on flower color in
108
peas. Since then, genetics and biochemistry of these compounds have been studied
intensively in a number of different species, including maize (KOES et al. 2005). The
maize anthocyanin pathway provides an attractive model system for gene expression
studies, as the products of this pathway are easily observable red and purple pigments.
Anthocyanins have been used as marker for performing genetic screens for anthocyanin
pathway mutants (GAVAZZI et al. 1997). The dispensable nature and easily scorable
phenotype of anthocyanins has enabled genetic analysis and isolation of many
biosynthetic and regulatory genes of this pathway (CONE 2007). The chemical structures
of anthocyanins have been determined, which makes it possible to correlate genes with
structural alterations or with the presence or absence of particular anthocyanins (HOLTON
and CORNISH 1995). We have used a genetic and biochemical approach to further
characterize a gene that controls different types of anthocyanins synthesized in the
aleurone layer of the kernel. The aleurone is an epidermis-like layer around the
endosperm of many plant species, including maize. The aleurone layer has important
function in the accumulation and mobilization of storage compounds during seed
development and germination, respectively (STEWART et al. 1988; YOUNG and GALLIE
2000). In maize, depending on the genetic constitution of plants, aleurone cells can
accumulate anthocyanin pigments. Aleurone anthocyanins have also been used as
markers to study cell fate specification in endosperm (BECRAFT 2001; BECRAFT and
ASUNCION-CRABB 2000).
Flavonoid biosynthesis takes place through the phenylpropanoid pathway
(Figure 4.1). Through the action of phenylalanine ammonia lyase (PAL) amino acid
phenylalanine is converted into cinnamic acid (ROSLER et al. 1997), which in turn is
109
converted into 4-coumaroyl CoA by cinnamate 4 hydroxylase (C4H) (FERRER et al.
2008). Condensation of one molecule of 4-coumaroyl CoA and 3 molecules of malonyl
CoA by the action of chalcone synthase (CHS) gives rise to chalcone. This is the first
enzyme in the pathway and in maize it is encoded by colorless2 (c2)/white pollen1
(whp1) (COE et al. 1988). The c2 gene expresses in the aleurone layer of the seed and was
isolated by transposon tagging using the Spm/En element (WIENAND et al. 1986).
Subsequently, whp1, which controls CHS activity in pollen, was isolated by virtue of its
sequence similarity to c2 (FRANKEN et al. 1991). Under certain conditions, whp1
expression can substitute for c2 by producing CHS in the aleurone. Through the action of
chalcone-flavanone isomerase (CHI), chalcone isomerizes into a flavanone, naringenin.
Chalcone isomerase1 (chi1) encodes for CHI and was isolated using primers
complementary to conserved regions of CHI genes from petunia and Antirrhinum
(GROTEWOLD and PETERSON 1994). However, molecular analysis revealed that there may
be more than one chi gene in maize as the isolated sequence mapped to three different
loci (GROTEWOLD and PETERSON 1994). This genetic redundancy might be the reason
why there are no mutants identified for this enzymatic step in maize.
Naringenin is the key product of flavonoid pathway, because most of the branches
of this pathway depend on it as a substrate. In the anthocyanin pathway, naringenin is
converted into dihydroflavonol by hydroxylation of the carbon 3 of C-ring through the
action of flavanone 3-hydroxylase (F3H). The flavanone 3-hydroxylase1 (fht1) gene,
which encodes for F3H was cloned by screening a cDNA library with probe from the
Antirrhinum F3H gene (DEBOO et al. 1995). Expression of F3H correlates with the
110
Naringenin
4-Coumaroyl-CoA 3 Malonyl-CoA+
CHI
CHS
Chalcone
c2
chi1
F3'H
pr1
Anthocyanin 3-glycoside
Vacuolar Anthocyanin
GSTbz2
F3'H pr1
Eriodictyol
Dihydroquercitin
F3H
Cyanidin
DFR
ANS
GT
leucocyanidin
fht1
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
leucopelargonidin
a1
a2
bz1
Naringenin
4-Coumaroyl-CoA 3 Malonyl-CoA+
CHI
CHS
Chalcone
c2
chi1
Naringenin
4-Coumaroyl-CoA 3 Malonyl-CoA+
CHI
CHS
Chalcone
c2
chi1
F3'H
pr1
Anthocyanin 3-glycoside
Vacuolar Anthocyanin
GSTbz2
F3'H pr1
Eriodictyol
Dihydroquercitin
F3H
Cyanidin
DFR
ANS
GT
leucocyanidin
fht1
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
leucopelargonidin
a1
a2
bz1
F3'H
pr1
Anthocyanin 3-glycoside
Vacuolar Anthocyanin
GSTbz2
Anthocyanin 3-glycoside
Vacuolar Anthocyanin
GSTbz2
F3'H pr1
Eriodictyol
Dihydroquercitin
F3H
Cyanidin
DFR
ANS
GT
leucocyanidin
fht1
a1
a2
bz1
F3'H pr1
Eriodictyol
Dihydroquercitin
F3H
Cyanidin
DFR
ANS
GT
leucocyanidin
fht1
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
leucopelargonidin
a1
a2
bz1
F3Hfht1
Dihydrokaemferol
Pelargonidin
DFR
ANS
GT
leucopelargonidin
a1
a2
bz1
Figure 4.1: Anthocyanin biosynthetic pathway in maize. Biosynthetic genes (enzymes) in thepathway are: c2 (CHS), chalcone synthase; chi1 (CHI), chalcone isomerase; fht1 (F3H), flavanone 3-hydroxylase; pr1 (F3'H), flavonoid 3'-hydroxylase; a1 (DFR), dihydroflavanone reductase; a2 (AS), anthocyanidin synthase; bz1 (UFGT), UDP-glucose flavonoid 3-O-glucosyltransferase; and bz2 (GST), glutathione S-transferase. Dotted arrows indicate the putative role of a gene or enzyme.
pigmentation levels in kernel aleurone and flavonols level in anthers. Though fht1
appears to be single copy gene in maize, so far, no mutant has been reported for this gene
(DEBOO et al. 1995). Both naringenin and dihydroflavonols can be hydroxylated on the 3'
B-ring position by flavonoid 3'-hydroxylase (F3'H), thereby generating wide range of
diversity in anthocyanins. Maize F3'H enzyme, encoded by red aleurone1/purple
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aleurone1 (pr1) gene, belongs to cytochrome P450 super-family (LARSON and BUSSARD
1986)(M. Sharma and S. Chopra, unpublished). Kernel aleurones with the dominant Pr1
allele are purple due to accumulation of cyanidin, whereas homozygous recessive pr1
kernels are red due to accumulation of pelargonidin. These pigments differ in 3'
hydroxylation of the B-ring and assignment of F3'H activity to pr1 locus was based on
this observation (LARSON et al. 1986). Recently, we have validated with the cloning of
f3'h1 gene using heterologous probes and primers from sorghum and rice f3'h, and
sequence information from maize genome. The deduced amino acid sequence of f3'h1 is
similar to other plant F3'Hs and is mapped to pr1 locus on chromosome 5L (M. Sharma
and S. Chopra, unpublished). Dihydroflavonols are reduced by dihydroflavonol 4-
reductase (DFR) into leucoanthocyanidins which are precursors of anthocyanidin
pigments. Maize DFR is encoded by the anthocyaninless1 (a1) gene, which was cloned
by transposon tagging using Spm/En and Mu transposable elements (O'REILLY et al.
1985). The a1 gene was shown to encode DFR following in vitro translation of an a1
cDNA clone (REDDY et al. 1987). A duplicate gene, which is now called
anthocyaninless4 (a4), was isolated using a1 probe (BERNHARDT et al. 1998). It shows
84% amino acid identity to the a1 encoded DFR. But whether it encodes for a functional
enzyme is not clear: crosses between plants carrying recessive a1 allele produce colorless
kernels in ratios expected for a single-gene trait, which shows that a4 does not
functionally complement an a1 defect in the aleurone.
Most of the compounds produced before the anthocyanidin are colorless.
Mutation of the anthocyaninless2 (a2) which encodes for anthocyanin synthase (ANS)
gene blocks the enzymatic conversion of leucoanthocyanidins to anthocyanidins and its
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function was demonstrated by inter-tissue complementation studies (REDDY and COE
1962). This gene was isolated by transposon tagging with Spm/En (MENSSEN et al. 1990).
Based on sequence homology, it was suggested that the a2 gene encodes for enzyme that
shows homology to 2-oxo-glutarate-dependent oxygenases, like F3H (MENSSEN et al.
1990). This was later confirmed by expression of a2 cDNA in bacterial cells which
results in production of enzyme capable of converting leucoanthocyanidins to
anthocyanidins by 2-oxo-glutarate-dependent oxidation (NAKAJIMA et al. 2001).
The maize bronze1 (bz1) gene, encoding UDP flavonoid 3-O- glucosyl transferase
(UFGT) was isolated by transposon tagging with Ac (DOONER et al. 1985; FEDOROFF et
al. 1984). The UFGT adds glucose molecule to anthocyanidins. Biochemical analysis of
pigment accumulation and enzyme activity in the bronze-colored kernels carrying bz1
mutation established that bz1 encodes for UFGT (DOONER and NELSON 1977; LARSON
and COE 1977). Glucosylated anthocyanidins were transported by glutathione-S-
transferase (GST) to vacuole for storage. The bronze2 (bz2) gene acts late in the pathway
and encodes for this enzyme. Recessive mutations in the bz2 gene result in bronze
pigmentation of the aleurone layer (NEUFFER et al. 1968). The bz2 gene was cloned
simultaneously by two groups. One used transposon tagging with Ds element (THERES et
al. 1987) and other followed an approach combining Mu transposon tagging with
differential hybridization (MCLAUGHLIN and WALBOT 1987). Studies using maize
protoplasts transformed with bz2 gene show that bz2 encodes for GST (MARRS et al.
1995).
Gene-protein association for most of the steps in the anthocyanin biosynthesis has
been determined by biochemical and genetic studies. The biosynthesis of anthocyanins in
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kernel aleurone requires interaction between c1 and r1 encoded transcription factors and
biosynthetic genes, c2, a1, a2, bz1, and bz2. To dissect the enzymatic steps for which the
pr1 gene acts within the anthocyanin biosynthetic network, we have performed genetic
and biochemical analysis of plants segregating for functional and null alleles. Functional
alleles at all these loci are required for the formation of purple anthocyanins in the kernel
aleurone. It has been demonstrated that both c1 and r1 regulate anthocyanin biosynthetic
genes and its assumed but never shown that pr1 is also regulated by c1 and r1. Similarly,
some biochemical studies were done to suggest the position of the pr1 gene in the
anthocyanin biosynthetic pathway, but the genetic proof to verify this notion was lacking.
MATERIALS AND METHODS
Maize genetic stocks: Seeds of maize inbred line W22 (Pr1 A1 A2 C1 R) and genetic
stocks containing individual mutant alleles for pr1, c1, r1, c2, a1, a2, bz1, and bz2 genes
(see Table 4.1) were kindly provided by the Maize Genetics Co-operation Stock Center
(USDA-ARS, University of Illinois, Urbana, IL). The mutant stocks of the regulatory
genes, c1 and r1, carried dominant alleles of the structural genes required for anthocyanin
synthesis. Homozygous pr1 plants were crossed with each mutant stocks of anthocyanin
biosynthetic and regulatory genes and F2 progenies were grown from the selfed F1 plants.
114
Table 4.1: Genotype and kernel phenotype of different anthocyanin mutant stocks used in this study.
115
Plant material: For anthocyanin kinetics study, aleurone tissues were collected from
self-pollinated ears of W22 inbred line on seven different days after pollination (DAP): 0,
10, 15, 20, 22, 24, and 28. Three biological replicates of each developmental stage were
collected. Since at 0 DAP there was no kernel development, the young ears were scraped,
while at 10 and 15 DAP whole kernels were collected. From ears collected at 20 DAP
and later stages, pericarp was peeled off and aleurone layer was collected by scraping.
Collected tissues were flash frozen with liquid nitrogen and stored at -80˚. To collect
aleurone for biochemical analysis of mutants, dry kernels were washed thoroughly with
distilled water and soaked in double distilled deionized water overnight at room
temperature. Pericarps were peeled off and kernels representing aleurone and endosperm
were used for biochemical analysis.
Pigment extraction, quantification, and HPLC analysis: For anthocyanin extraction,
tissues collected from dry mature kernels and kernels over different developmental stages
were soaked overnight at 4˚ in freshly prepared 1% HCl-methanol (GOODMAN et al.
2004). Subsequently, the acidified methanolic extracts were centrifuged at 10,000 rpm for
10 minutes and supernatants were collected in fresh tubes. The absorbance of the
methanolic extracts at 530 nm and 515 nm for purple and red aleurone extracts was
recorded using 1 cm wide quartz cuvette and a UV mini-1240 spectrophotometer
(Shimadzu Scientific Instruments, Inc. Columbia, MD). Anthocyanins’ concentration was
expressed as mM of anthocyanidin standards based on the Lambert-Beer Law (Pigment
concentration in mM = Absorption x 1000/molar extinction coefficient) using the molar
extinction coefficient of cyanidin for purple and colorless aleurone extracts (24500 M-1
116
cm-1) and of pelargonidin for red aleurone extracts (29080 M-1 cm-1) (NUNES et al. 2006;
STAFFORD 1966).
Anthocyanin extractions for HPLC analysis were done as above except that the
tissue was ground in 1% HCl-methanol for 5 min and immediately spun at 14,000 rpm in
a microcentrifuge for 5 min. The supernatant was acid hydrolyzed with equal volume of
2N HCl at 70˚ for 45 min and then equal volume of 100% methanol was added to
stabilize extracts. The extracts were then filtered through 0.45 µm Acrodisc LC 13 mm
syringe filters (Gelman Laboratory, Ann Arbor, MI). Reverse phase HPLC analysis was
performed on a Shimadzu high performance liquid chromatograph (Shimadzu, Columbia,
MD) using an Ascentis C18 column (25 cm X 4.6 mm, 5 μm; Supelco, Bellefonte, PA).
Compounds were separated at 35˚ by gradient elution using 0.2% formic acid (solvent A)
and 100% methanol (solvent B) at a flow rate of 0.80 ml/min with the following
timetable: 5 min at 95% A, 5% B; from 95% A, 5% B to 0% A, 100% B in 20 min; at
100% B for 5 min; to 95% A, 5% B in 3 min; at 95% A, 5% B for 5 min. The injection
volume was 50 μl and spectral measurements were taken over a wavelength range of 250
nm to 550 nm, which is known to detect flavonoid compounds (GROTEWOLD et al. 1998).
Gene expression analysis: Kernel aleurones were collected at 24 DAP from c1 and r1
mutants as well from wild type W22 inbred line. RNA was isolated from three biological
replicates of two different mutant alleles of c1 and r1 each. Further, three samples were
analyzed from each replicate. To isolate total RNA, tissues were ground in liquid
nitrogen and then extracted using Tri-Reagent (Molecular Research Center, Inc.,
Cincinnati, OH). First strand cDNA was synthesized using SuperScript III cDNA
117
synthesis system (Invitrogen, Carlsbad, CA) and oligo-dT primer using conditions as
described by manufacturer. One microgram of total RNA from aleurones was used for
each reverse transcription reaction. First strand cDNA was diluted to a final volume of
100 µl with sterile ddH2O. Five microliter of first strand cDNA was PCR amplified using
gene specific primers. Genomic DNA from W22 line (carry all functional anthocyanin
genes) was used as negative control for DNA contamination in RNA (not shown) while
its cDNA was used as positive control. Primer sets used for detection of anthocyanin
genes transcript are shown in Table 4.2. Primers used for the amplification of cDNA of
anthocyanin genes were designed such that they span over an intron of the gene in order
to detect any genomic DNA contamination. PCR reactions were performed in a total
volume of 25 µl with GoTaq green master mix (Promega) using manufacturer’s
instructions. Templates were denatured at 94° for 4 min, followed by 30 cycles of 94° for
0.45 min, 55°-60° for 0.45 min, and 72° for 1 min 30 sec, and a final extension step of
72° for 10 min. PCR products were analyzed on 1.0 % agarose gel.
Dissection of pr1 promoter: The analysis of the pr1 promoter for the location and
distribution of cis regulatory sequence elements was performed using the Plant Care
database (http://bioinformatics.psb.ugent.be/webtools/plantcare.html/) and MetInspector
(Genomatrix, Munich, Germany) (QUANDT et al. 1995). The cis binding sites were
identified based on their similarity with sites present in promoters of other anthocyanin
genes as well as similarity with MYB and MYC protein binding sequences (SAINZ et al.
1997).
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Table 4.2: PCR primers used in reverse transcription gene expression analysis.
This Study202ActinF 5′-CCTTCGAATGCCCAGCAATG-3′
ActinR 5′-GAGGATCTTCATTAGGTGGT-3′actin
This Study621GAP1 5′-AGGGTGGTGCCAAGAAGGTTG-3′
GAP2 5′-GTAGCCCCACTCGTTGTCGTA-3′gapdh
This Study379BZ2F 5′-ATATGCGAGTCCGCAGTCATCGT-3′
BZ2R 5′-TCGATGAGTGAGAGGCCGTGAA-3′bz2
This Study7715F3H-F2 5′-GAGCACGTGGCGTACAACTA-3′
ZMR4 5′-AAACGTCTCCTTGATCACCGC-3′pr1
This Study549CHSF 5′-TCGATCGGTCTCTCTGGTACAAC-3′
CHSR 5′-TACATCATGAGGCGGTTCACGG-3′c2
Piazza et al., 2002
403OR31 5′-ATGGCTTCATGGGGCTTAGATAC-3′OR32 5′-AATGCAACCAAACACCTTATGC-3′r1
Piazza et al., 2002
313PL6 5′-TCGGACGACTGCAGCTCGGC-3′AC1 5′-CACCGTGCCTAATTTCCTGTCCGA-3′c1
Reference
Product size (bp)
Primer sequencesGene
This Study202ActinF 5′-CCTTCGAATGCCCAGCAATG-3′
ActinR 5′-GAGGATCTTCATTAGGTGGT-3′actin
This Study621GAP1 5′-AGGGTGGTGCCAAGAAGGTTG-3′
GAP2 5′-GTAGCCCCACTCGTTGTCGTA-3′gapdh
This Study379BZ2F 5′-ATATGCGAGTCCGCAGTCATCGT-3′
BZ2R 5′-TCGATGAGTGAGAGGCCGTGAA-3′bz2
This Study7715F3H-F2 5′-GAGCACGTGGCGTACAACTA-3′
ZMR4 5′-AAACGTCTCCTTGATCACCGC-3′pr1
This Study549CHSF 5′-TCGATCGGTCTCTCTGGTACAAC-3′
CHSR 5′-TACATCATGAGGCGGTTCACGG-3′c2
Piazza et al., 2002
403OR31 5′-ATGGCTTCATGGGGCTTAGATAC-3′OR32 5′-AATGCAACCAAACACCTTATGC-3′r1
Piazza et al., 2002
313PL6 5′-TCGGACGACTGCAGCTCGGC-3′AC1 5′-CACCGTGCCTAATTTCCTGTCCGA-3′c1
Reference
Product size (bp)
Primer sequencesGene
RESULTS
Anthocyanin regulatory genes c1 and r1 are required for pr1 expression in kernel
aleurone pigmentation: To genetically implicate pr1 in C1/R1 regulated pathway,
crosses were made independently between pr1 plants with red kernels and c1 and r1
mutants with colorless kernels (Table 4.1). Two different mutant stocks of each
regulatory gene were used. The F1 ear phenotype indicated that the c1 and r1 mutant
stocks have Pr1 allele in their genetic background. In maize, because of xenia effect and
colored anthocyanin pigments, it is possible to observe segregating kernel phenotypes in
119
the F2 ear itself. Representative F2 ear from each cross is shown in Figure 4.2. Purple and
red kernels were due to Pr1/- and pr1/pr1, respectively and it also showed presence of
functional alleles of anthocyanin regulatory and biosynthetic genes. Colorless kernels
indicated the presence of mutant alleles of c1 or r1. If pr1 was in the pathway of c1 and
r1 genes, we expect to see kernel phenotype segregating as 9 Purple: 3 red: and 4
colorless. Chi-square analysis of F2 kernel phenotype showed purple, red, and colorless
kernels segregated in 9:3:4 ratios. To confirm these results, we analyzed segregation ratio
of kernel aleurone phenotype on test cross ears. Segregation ratio of 1:1 for purple and
colorless kernels was observed on ears developed by crossing F1 plants with c1 or r1
mutant plants (see Figure 4.3). Results from these genetic assays showed that functional
c1 and r1 alleles were required for pr1 expression in the formation of purple aleurone
pigment.
These genetic results were verified at molecular level by studying expression of
c1, r1, and pr1 genes in mutant as well as wild-type plants. Steady state transcript of c1,
r1, and pr1 were measured in aleurones of wild-type W22 line and two mutant alleles of
c1 (MGS 131036, MGS 14633) and r1 (MGS 167054, MGS 14638) using reverse
transcription (RT) PCR (Figure 4.4). W22 kernel aleurone showed steady state transcript
of all three genes tested. However, c1, r1, and pr1 specific transcripts could not be
detected in mutant c1 and r1 plants that produced colorless kernels. Similarly, RT-PCR
of other anthocyanin pathway genes c2 and bz2 which are known to be regulated by c1
and r1, showed their steady state transcripts in W22 kernel aleurones. But their
transcripts were absent in mutant c1 or r1 aleurones. These results demonstrated the
requirement of c1 and r1 for pr1 transcript accumulation in kernel aleurone.
120
F1 progeny
pr1 / pr1 ; C1/C1 Pr1 / Pr1 ; c1 /c1X(Red aleurone) (Colorless aleurone)
⊗
Pr1/pr1 ; C1/c1(Purple aleurone)
pr1 / pr1 ; R1/R1 Pr1 / Pr1 ; r1 /r1X(Red aleurone) (Colorless aleurone)
⊗
Pr1/pr1 ; R1/r1(Purple aleurone)
F2 segregating ear
Number of kernels on F2 progeny ears with specific aleurone color
F1 GenotypeP-value
(χ2)
Pr1/pr1 ; C1/c1
MGS 131036 (c1 b1 pl1 R1-g) MGS 14633(c1 B1 Pl1 R1-r)
465
270
185
86
216
141
0.13
0.20
Purple (9)
Red (3)
Colorless (4) F1 Genotype
Purple (9)
Red (3)
Colorless (4)
Pr1/pr1 ; R1/r1
MGS 167054 (r1-g C1 b1 pl1)MGS 14638(r1-r B1 Pl1)
265 336688
614 232 298
0.10
0.20
P-value(χ2)
F1 progeny
pr1 / pr1 ; C1/C1 Pr1 / Pr1 ; c1 /c1X(Red aleurone) (Colorless aleurone)
⊗
Pr1/pr1 ; C1/c1(Purple aleurone)
pr1 / pr1 ; R1/R1 Pr1 / Pr1 ; r1 /r1X(Red aleurone) (Colorless aleurone)
⊗
Pr1/pr1 ; R1/r1(Purple aleurone)
F2 segregating ear
F1 progeny
pr1 / pr1 ; C1/C1 Pr1 / Pr1 ; c1 /c1X(Red aleurone) (Colorless aleurone)
⊗
Pr1/pr1 ; C1/c1(Purple aleurone)
pr1 / pr1 ; R1/R1 Pr1 / Pr1 ; r1 /r1X(Red aleurone) (Colorless aleurone)
⊗
Pr1/pr1 ; R1/r1(Purple aleurone)
F2 segregating ear
Number of kernels on F2 progeny ears with specific aleurone color
F1 GenotypeP-value
(χ2)
Pr1/pr1 ; C1/c1
MGS 131036 (c1 b1 pl1 R1-g) MGS 14633(c1 B1 Pl1 R1-r)
465
270
185
86
216
141
0.13
0.20
Purple (9)
Red (3)
Colorless (4) F1 Genotype
Purple (9)
Red (3)
Colorless (4)
Pr1/pr1 ; R1/r1
MGS 167054 (r1-g C1 b1 pl1)MGS 14638(r1-r B1 Pl1)
265 336688
614 232 298
0.10
0.20
P-value(χ2)
Figure 4.2: Regulation of pr1 by c1 and r1 during purple anthocyanin synthesis in kernel aleurone. Crossing scheme used to develop F1 and F2 progenies. Purple, red, and colorless kernel aleurone phenotypes were observed in F2 segregating ears. Chi-square analysis was performed to detect significant deviation of observed phenotypic classes from expected.
Further, we performed in silico analysis of the promoter sequence of pr1 gene for the
presence of cis-regulatory element sequences required for the binding of c1 and r1
encoded proteins. We found a consensus sequence “AGGTGGTAGCTGGGA” called C-
binding site (CBS), 126 bp upstream of transcription start site (TSS), which is known to
be required for binding of C1 MYB protein (SAINZ et al. 1997). In addition, we also
found sequence similar to anthocyanin regulatory element (ARE), present 106 bp
upstream of TSS (TUERCK and FROMM 1994). ARE has been demonstrated to play
important role in activation of anthocyanin genes. In conclusion, our results from genetic,
121
molecular, and sequence analysis showed that c1 and r1 are required for pr1 expression
in anthocyanin biosynthetic pathway.
Progeny
(Pr1/pr1; C1/c1) X Pr1/Pr1; c1/c1
Pr1/Pr1; C1/c1, Pr1/pr1; c1/c1
F1
(Purple aleurone) (Colorless aleurone)⊗
Test cross ear
(Pr1/pr1 ; R1/r1) X Pr1/Pr1 ; r1/r1
Pr1/Pr1; R1/r1 , Pr1/pr1; r1/r1
(Purple aleurone) (Colorless aleurone)⊗
F1
Number of kernels with specific aleurone color
Stock Purple (1)
Colorless (1)
446 475
430 450
0.34
0.50
MGS 131036 (c1 b1 pl1 R1-g) MGS 14633 (c1 B1 Pl1 R1-r)
Stock Purple (1)
Colorless (1)
466 447
290 325
0.53
0.16
MGS 167054 (r1-g C1 b1 pl1)MGS 14638 (r1-r B1 Pl1)
P-value(χ2)
P-value(χ2)
Progeny
(Pr1/pr1; C1/c1) X Pr1/Pr1; c1/c1
Pr1/Pr1; C1/c1, Pr1/pr1; c1/c1
F1
(Purple aleurone) (Colorless aleurone)⊗
Test cross ear
Progeny
(Pr1/pr1; C1/c1) X Pr1/Pr1; c1/c1
Pr1/Pr1; C1/c1, Pr1/pr1; c1/c1
F1
(Purple aleurone) (Colorless aleurone)⊗
Test cross ear
(Pr1/pr1 ; R1/r1) X Pr1/Pr1 ; r1/r1
Pr1/Pr1; R1/r1 , Pr1/pr1; r1/r1
(Purple aleurone) (Colorless aleurone)⊗
F1 (Pr1/pr1 ; R1/r1) X Pr1/Pr1 ; r1/r1
Pr1/Pr1; R1/r1 , Pr1/pr1; r1/r1
(Purple aleurone) (Colorless aleurone)⊗
F1
Number of kernels with specific aleurone color
Stock Purple (1)
Colorless (1)
446 475
430 450
0.34
0.50
MGS 131036 (c1 b1 pl1 R1-g) MGS 14633 (c1 B1 Pl1 R1-r)
Stock Purple (1)
Colorless (1)
466 447
290 325
0.53
0.16
MGS 167054 (r1-g C1 b1 pl1)MGS 14638 (r1-r B1 Pl1)
P-value(χ2)
P-value(χ2)
Figure 4.3: Crossing scheme used to develop the F1 and test cross progenies. Purple and colorless kernel aleurone phenotypes can be seen in representative test cross ears. Chi-square analysis performed to detect significant deviation of observed phenotypic classes from 1:1 segregation ratio for c1 and r1 loci, individually.
pr1 and its relationship with other anthocyanin genes: It has been well documented
through biochemical studies that recessive pr1 plants accumulated pelargonidin instead of
cyanidin in kernel aleurone. However, no genetic tests had been done conclusively to
show that pr1 is the required structural gene in the formation of anthocyanins via known
pathway. We utilized the available mutant stocks of anthocyanin biosynthetic genes, c2,
a1, a2, bz1, and bz2. Mutants for chi1 and fht1 have not been identified in maize and
therefore, were not included in the study. Crosses were made between the mutant allele of
122
+13'
TATA
-32-106
ARECBS
-1265'
GAPDH
Actin
bz2
pr1
c2
c1
r1
313
403
549
771
379
621
202 bp
W22
(C1
R1)
c1-M
GS 13
1036
c1-M
GS 14
633
r1-M
GS 16
7054
r1-M
GS 14
638
+13'
TATA
-32-106
ARECBS
-1265'
+13'
TATA
-32-106
ARECBS
-1265'
GAPDH
Actin
bz2
pr1
c2
c1
r1
313
403
549
771
379
621
202 bp
W22
(C1
R1)
c1-M
GS 13
1036
c1-M
GS 14
633
r1-M
GS 16
7054
r1-M
GS 14
638
GAPDH
Actin
bz2
pr1
c2
c1
r1
313
403
549
771
379
621
202 bp
GAPDH
Actin
bz2
pr1
c2
c1
r1
313
403
549
771
379
621
202 bp
W22
(C1
R1)
c1-M
GS 13
1036
c1-M
GS 14
633
r1-M
GS 16
7054
r1-M
GS 14
638
Figure 4.4: Mutant c1 and r1 plants do not accumulate pr1 transcript. Expression of pr1 and other anthocyanin genes in aleurone tissues was analyzed by RT-PCR in c1 (MGS 131036, MGS 14633) and r1 (MGS 167054, MGS 14638) mutants. Gene specific primers for pr1, c2, bz2, c1, and r1 were used to detect their transcripts. Aleurone from W22 was used as positive control for anthocyanin genes expression, while actin and glyceraldehyde 3-phosphate dehydrogenase(GAPDH) were used as internal controls. Diagrammatic representation of pr1 promoter structure is also shown. Position of the C1 binding site (CBS), the anthocyanin regulatory element (ARE), and the TATA box are shown in relation to the putative transcription start site (+1).
123
pr1 and mutants in critical structural genes in the pathway, namely c2, a1, a2, bz1, and
bz2. Mutations in c2, a1, and a2 produced colorless kernels; while bz1 and bz2 mutants
had bronze kernels (see Table 4.1). F1 plants were self pollinated to yield F2 ears with
segregating kernel aleurone phenotypes. If pr1 played a role in anthocyanin pathway
similar to these other anthocyanin genes, then we would expect a segregating kernel
aleurone phenotype of 9 purple: 3 red: 4 colorless. In all cases, this ratio was obtained
with an acceptable margin of error (p-values between 0.07- 0.36; Figure 4.5). Thus our
genetic test agrees with previous biochemical analysis, which suggested that the pr1 gene
encodes a F3'H enzyme required to form cyanidin.
In addition to above mentioned genetic tests, we utilized the segregating F2
kernels to analyze how anthocyanins are differentially accumulated in purple, red, and
colorless kernel aleurones. All purple aleurone tissue produced a sizable peak at 530 nm,
whereas red aleurone tissue exhibited a peak at 515 nm (Figure 4.6). As expected, we
could not detect any peaks for anthocyanins in colorless kernels of c2, a1, and a2
mutants. However, bronze kernels of bz1 and bz2 mutants showed small peak for
cyanidin. These results were further verified through HPLC analysis of acid hydrolyzed
aleurone extracts (Figure 4.7). Since products upstream of anthocyanidins are colorless,
thereby were not detectable at 515 nm used for anthocyanin analysis. However, at lower
wavelengths (280 nm) we detected presence of peaks corresponding to dihydroflavonols,
naringenin and related flavanones in a1 and a2 mutant kernel aleurones (data not shown).
Interestingly, the levels of these compounds were higher in these mutants as compared to
wild-type. This indicated the blockage at steps controlled by a1 and a2 in mutant kernels.
Bronze kernels of bz1 and bz2 mutants had profile similar to wild-type plants at 280 nm.
124
Segregation analysis of dihybrid crosses involving Pr1 and other flavonoid structural genes; c2, a1, a2, bz1, and bz2.
0.28
0.21
0.07
0.18
F1 Genotype
Pr1/pr1 ; C2/c2
Pr1/pr1 ; A1/a1
Pr1/pr1 ; A2/a2
Pr1/pr1 ; Bz1/bz1
Pr1/pr1 ; Bz2/bz2
535
Purple (9)
383
449
746
597
155
Red (3)
113
145
232
198
238
Colorless (4)
186
162
293
292 0.36
P-value(χ2)(Expected ratio)
Segregation analysis of dihybrid crosses involving Pr1 and other flavonoid structural genes; c2, a1, a2, bz1, and bz2.
0.28
0.21
0.07
0.18
F1 Genotype
Pr1/pr1 ; C2/c2
Pr1/pr1 ; A1/a1
Pr1/pr1 ; A2/a2
Pr1/pr1 ; Bz1/bz1
Pr1/pr1 ; Bz2/bz2
F1 Genotype
Pr1/pr1 ; C2/c2
Pr1/pr1 ; A1/a1
Pr1/pr1 ; A2/a2
Pr1/pr1 ; Bz1/bz1
Pr1/pr1 ; Bz2/bz2
535
Purple (9)
383
449
746
597
535
Purple (9)
383
449
746
597
155
Red (3)
113
145
232
198
155
Red (3)
113
145
232
198
238
Colorless (4)
186
162
293
292
238
Colorless (4)
186
162
293
292 0.36
P-value(χ2)(Expected ratio)
Figure 4.5: pr1 gene is required for cyanidin formation through anthocyanin pathway. IndividualF1 plants having purple colored kernels were test-crossed. Segregating genotypes and associated kernel phenotypes are listed. Chi-square analysis performed to detect significant deviation ofobserved phenotypic classes from expected classes in segregating F2 ears developed from crosses between mutant alleles of pr1 and other anthocyanin biosynthetic genes.
We also assayed the dosage-dependent accumulation of these compounds in kernel
aleurone containing mutations in various structural genes. Interestingly, we observed that
a decreased dose of certain structural genes had a significantly greater effect on
anthocyanin synthesis than other genes in the pathway. For example, the overall cyanidin
and pelargonidin levels in purple and red aleurones from ears segregating for the c2, a1,
and a2 mutants were significantly smaller than that of bz1 and bz2 (Figure 4.8). Thus
regardless of the type of anthocyanin being synthesized, purple and red kernels from bz1
and bz2 segregating ears had increased levels.
125
PhenotypeGene/Allele combination
Purple RedBronzeBronze
Pr1/-; Bz2/-pr1/pr1; Bz2/-Pr1/-; bz2/bz2pr1/pr1; bz2/bz2
Bronze2 (bz2)
Purple RedBronzeBronze
Pr1/-; Bz1/-pr1/pr1; Bz1/-Pr1/-; bz1/bz1pr1/pr1; bz1/bz1
Bronze1 (bz1)
Purple RedColorlessColorless
Pr1/-; A2/-pr1/pr1; A2/-Pr1/-; a2/a2pr1/pr1; a2/a2
Anthocyaninless2 (a2)
Purple RedColorlessColorless
Pr1/-; A1/-pr1/pr1; A1/-Pr1/-; a1/a1pr1/pr1; a1/a1
Anthocyaninless1 (a1)
Purple RedColorlessColorless
Pr1/-; C2/-pr1/pr1; C2/-Pr1/-; c2/c2pr1/pr1; c2/c2
Colorless2 (c2)
Gene name Spectral analysisPhenotypeGene/Allele combination
Purple RedBronzeBronze
Pr1/-; Bz2/-pr1/pr1; Bz2/-Pr1/-; bz2/bz2pr1/pr1; bz2/bz2
Bronze2 (bz2) Purple RedBronzeBronze
Purple RedBronzeBronze
Pr1/-; Bz2/-pr1/pr1; Bz2/-Pr1/-; bz2/bz2pr1/pr1; bz2/bz2
Bronze2 (bz2)
Purple RedBronzeBronze
Pr1/-; Bz1/-pr1/pr1; Bz1/-Pr1/-; bz1/bz1pr1/pr1; bz1/bz1
Bronze1 (bz1) Purple RedBronzeBronze
Purple RedBronzeBronze
Pr1/-; Bz1/-pr1/pr1; Bz1/-Pr1/-; bz1/bz1pr1/pr1; bz1/bz1
Bronze1 (bz1)
Purple RedColorlessColorless
Pr1/-; A2/-pr1/pr1; A2/-Pr1/-; a2/a2pr1/pr1; a2/a2
Anthocyaninless2 (a2) Purple RedColorlessColorless
Purple RedColorlessColorless
Pr1/-; A2/-pr1/pr1; A2/-Pr1/-; a2/a2pr1/pr1; a2/a2
Anthocyaninless2 (a2)
Purple RedColorlessColorless
Pr1/-; A1/-pr1/pr1; A1/-Pr1/-; a1/a1pr1/pr1; a1/a1
Anthocyaninless1 (a1) Purple RedColorlessColorless
Purple RedColorlessColorless
Pr1/-; A1/-pr1/pr1; A1/-Pr1/-; a1/a1pr1/pr1; a1/a1
Anthocyaninless1 (a1)
Purple RedColorlessColorless
Pr1/-; C2/-pr1/pr1; C2/-Pr1/-; c2/c2pr1/pr1; c2/c2
Colorless2 (c2) Purple RedColorlessColorless
Pr1/-; C2/-pr1/pr1; C2/-Pr1/-; c2/c2pr1/pr1; c2/c2
Colorless2 (c2)
Gene name Spectral analysisSpectral analysis
Figure 4.6: Anthocyanin analysis of the aleurone tissue collected from purple, red, and colorlesskernels of F2 segregating ears.
Notably, there were small but detectable levels of anthocyanins in the bronze aleurones of
bz1 and bz2. The rationale for decreased doses of these structural genes leading to
differences in anthocyanin concentration will be presented in the discussion section.
126
Retention Time (min)
Abs
orba
nce
at 5
15 n
m (m
Au)
P
0 8 16 24
Pelargonidin P
C P
0 8 16 24
Pelargonidin P
0 8 16 24
Pelargonidin P
-10
0
10
2030
Pr1/-; Bz2/Bz2pr1/pr1; Bz2/Bz2
Pr1/-; Bz2/-pr1/pr1; Bz2/-
C
-10
010
2030
Pr1/-; bz1bz1pr1/pr1; bz1/bz1
Pr1/-; Bz1/-pr1/pr1; Bz1/-P C
P
C
-10
010
20
30
Pr1/-; a2/a2pr1/pr1a2/a2
Pr1/-; A2/-pr1/pr1; A2/-
PC
P
-10
0
10
20
30
Pr1/-; a1/a1pr1/pr1; a1/a1
pr1/pr1; A1/- Pr1/-; A1/-P C P
-10
0
10
20
30
CPPr1/-; c2/c2
pr1/pr1; c2/c2Pr1/-; C2/-pr1/pr1; C2/- P
-10
010
2030
Retention Time (min)
Abs
orba
nce
at 5
15 n
m (m
Au)
P
0 8 16 24
Pelargonidin P
0 8 16 240 8 16 24
Pelargonidin P
C P
0 8 16 24
Pelargonidin P
0 8 16 240 8 16 24
Pelargonidin P
0 8 16 24
Pelargonidin P
-10
0
10
2030
0 8 16 240 8 16 24
Pelargonidin P
-10
0
10
2030
-10
0
10
2030
Pr1/-; Bz2/Bz2pr1/pr1; Bz2/Bz2
Pr1/-; Bz2/-pr1/pr1; Bz2/-
C
-10
010
2030
-10
010
2030
Pr1/-; bz1bz1pr1/pr1; bz1/bz1
Pr1/-; Bz1/-pr1/pr1; Bz1/-P C
P
C
-10
010
20
30 Pr1/-; bz1bz1pr1/pr1; bz1/bz1
Pr1/-; Bz1/-pr1/pr1; Bz1/-P C
P
C
-10
010
20
30
-10
010
20
30
Pr1/-; a2/a2pr1/pr1a2/a2
Pr1/-; A2/-pr1/pr1; A2/-
PC
P
-10
0
10
20
30 Pr1/-; a2/a2pr1/pr1a2/a2
Pr1/-; A2/-pr1/pr1; A2/-
PC
P
-10
0
10
20
30
-10
0
10
20
30
Pr1/-; a1/a1pr1/pr1; a1/a1
pr1/pr1; A1/- Pr1/-; A1/-P C P
-10
0
10
20
30 Pr1/-; a1/a1pr1/pr1; a1/a1
pr1/pr1; A1/- Pr1/-; A1/-P C P
-10
0
10
20
30
-10
0
10
20
30
CPPr1/-; c2/c2
pr1/pr1; c2/c2Pr1/-; C2/-pr1/pr1; C2/- P
-10
010
2030
CPPr1/-; c2/c2
pr1/pr1; c2/c2Pr1/-; C2/-pr1/pr1; C2/- P
-10
010
2030
-10
010
2030
Figure 4.7: Reverse phase HPLC analysis of anthocyanin pigments extracted from kernelaleurone. Chromatograms at 515 nm have been obtained from the same injected quantity ofsample. C-cyanidin, P-pelargonidin.
127
Tota
l Ant
hocy
anin
s (m
M)
Genotype (Phenotype)
0
0.2
0.4
0.6
0.8
1
1.2
c2 (c
l)
c2 (re
d)
c2 (p
urple)
a1 (c
l)
a1 (re
d)
a1 (p
urple)
a2 (c
l)
a2 (re
d)
a2 (p
urple)
bz1 (
cl)
bz1 (
red)
bz1 (
purple)
bz2 (
cl)
bz2 (
red)
bz2 (
purple)
Tota
l Ant
hocy
anin
s (m
M)
Genotype (Phenotype)
0
0.2
0.4
0.6
0.8
1
1.2
c2 (c
l)
c2 (re
d)
c2 (p
urple)
a1 (c
l)
a1 (re
d)
a1 (p
urple)
a2 (c
l)
a2 (re
d)
a2 (p
urple)
bz1 (
cl)
bz1 (
red)
bz1 (
purple)
bz2 (
cl)
bz2 (
red)
bz2 (
purple)
Figure 4.8: Anthocyanin content of the aleurone tissue of purple, red, and colorless (cl) kernels ingenetic background of mutant anthocyanin genes. Results are the means of three separateextractions from the same tissue type. Bars represent standard error.
Cyanidin accumulates gradually during maize aleurone maturation: The
accumulation of cyanidin in the aleurones of W22 (Pr1/Pr1) plants were monitored
throughout kernel/aleurone development and the results for this are shown in Figure 4.9.
W22 inbred possesses dominant alleles of all regulatory and structural genes required for
anthocyanin biosynthesis. Tissues were collected at seven different time intervals of
aleurone development. For technical reasons, it was not possible to obtain aleurones from
kernels younger than 15 DAP. Hence, we analyzed whole ears and/or kernels. After 15
DAP we were able to scrape aleurone layers from more developed kernels. Equal amount
of tissue was used for the anthocyanin extraction and analysis at each stage. Interestingly,
we found that there was no purple cyanidin produced at or before 15 DAP. However,
extracts from 10 and 15 DAP whole kernels showed accumulation of compounds with
128
λmax around 490 nm corresponding to 3-deoxyanthocyanidins found in pericarp
(Figure 4.9, panel II), while no compound was detected at λ530 nm. At 20 DAP there
was a small but noticeable peak at λ530 nm, demonstrating the starting point for cyanidin
accumulation. The accumulation of cyanidin markedly rose at 24 DAP and was
significantly elevated at 28 DAP. This accumulation showed a perfect correlation with
the purple pigmentation during kernel/aleurone development.
DISCUSSION
Anthocyanin biosynthesis in maize is transcriptionally regulated by interaction
between two sets of transcription factors encoded by c1/pl1 and r1/b1; c1 and r1 are
required in kernel aleurone while pl1 and b1 function to activate these genes in the plant
body (CHANDLER et al. 1989). We have recently shown that functional pr1 is required for
the formation of purple cyanidin in kernel aleurone while mutant pr1 plants show red
aleurone phenotype due to accumulation of pelargonidin (M. Sharma and S. Chopra,
unpublished). Our genetic analysis revealed that the pr1 gene plays a role in c1 and r1
regulated anthocyanin biosynthetic pathway. RNA expression of pr1 and other
biosynthetic genes was not detected in the absence of functional alleles of c1 and r1. R1
and C1 interact physically, and C1 binds directly to specific cis-regulating elements
(GROTEWOLD et al. 1994; SAINZ et al. 1997). In vitro protein-DNA binding assays using
point mutations or in vivo transposon insertions in cis-binding sites disrupt gene
129
Figure 4.9: Analysis of anthocyanin accumulation in maize aleurone during its development.Panel I shows the kernel phenotype and time of its collection. Panel II represent the comparisonof absorption spectra of methanolic extracts from the corresponding kernel/aleurone.
130
expression (GROTEWOLD et al. 1994; POOMA et al. 2002). Similar elements were found in
the pr1 promoter indicating that pr1 may be regulated by C1 and R1 in fashion similar to
other anthocyanin biosynthetic genes (LESNICK and CHANDLER 1998; SAINZ et al. 1997).
Interestingly, pr1 promoter is also regulated by P1 protein as indicated by the loss of pr1
expression in the absence of functional p1 alleles (see chapter 3). Independent
transcriptional regulation of pr1 by C1 and P1 may also indicate that these transcription
factors may bind to the similar cis-binding element (POOMA et al. 2002). As our results
clearly demonstrate that flavonoid regulatory genes are required for pr1 expression in
separate branches of flavonoid pathway, further studies demonstrating direct physical
interaction between pr1 promoter and these transcription factors will be required to
determine the interaction mechanism.
Mutations affecting different steps of anthocyanin biosynthesis have been isolated
and characterized (HOLTON and CORNISH 1995). Biochemical and genetic studies have
been extensively used in determining the enzymatic steps involved in anthocyanin
biosynthesis and modification (CONE 2007). We used this available information to
confirm the role and position of pr1 gene in this pathway. Pr1 encoded F3'H enzyme has
been implicated at different steps of anthocyanin biosynthetic pathway as well as in other
branches of flavonoid pathway including 3-deoxyanthocyanidins and C-glycosyl flavones
(M. Sharma and S. Chopra, unpublished). Genetic analysis of double mutants confirmed
that pr1 is related to other genes analyzed and is involved in anthocyanin biosynthesis.
Further, biochemical analyses for anthocyanin compounds confirmed these results.
Colorless kernels collected from F2 ears of c2, a1, and a2 did not accumulate
anthocyanins. However, small amount of cyanidin was detected in bronze aleurone
131
extracts of bz1 and bz2 mutants. Since pr1 is required for the formation of cyanidin, this
indicates that pr1 is acting upstream of bz1 and bz2. These results are in accordance with
the function of enzymes encoded by bz1 and bz2: glucosyl transferase encoded by bz1
adds glucose to anthocyanidins (LARSON and COE 1977) and glutathione-S-transferase
enzyme encoded by bz2 plays role in transport of anthocyanin glycosides to vacuole
(MARRS et al. 1995). Reduction in anthocyanin contents in the bz1 and bz2 aleurones
appears to stem from the inability of cyanidin to be glycosylated and transported into the
vacuole, respectively (GOODMAN et al. 2004). Reverse phase HPLC analysis exhibited
significant differences in the peak profiles of flavanones and dihydroflavonols in the c2,
a1, and a2 mutant and wild-type kernel aleurones. Dihydroflavonols gave major peaks at
280 nm in a1 and a2 colorless mutant kernels collected from segregating ears while
purple and red kernels from same ear produced small peaks. This is probably due to
blockage at steps controlled by a1 and a2 which prevent utilization of those compounds
by respective enzymes (NAKAJIMA et al. 2001; REDDY et al. 1987). This further defines
the proposed position of pr1 in the pathway, where pr1 is acting upstream of a1, a2, bz1,
and bz2. Thus position of pr1 encoded F3'H in maize is similar to flavonoid pathways in
other dicots including Arabidopsis (SCHOENBOHM et al. 2000), Petunia (BRUGLIERA et al.
1999), and grapevine (BOGS et al. 2006).
The differential level of anthocyanins in purple, red, and colorless kernels from F2
ears allowed us to uncover the effect of gene dosage on concentration of these
compounds. Interestingly, the concentration of anthocyanins varied with the decreased
dosage of different biosynthetic genes because extracts collected from both purple and
red aleurones segregated in a 2:1 ratio of heterozygous to homozygous dominant alleles.
132
In other words, the pooled aleurone tissues carried a decreased dose of the other
structural gene (i.e. c2, a1, a2, bz1,and bz2). Small amount of anthocyanins detected in
purple and red kernels from c2, a1, and a2 segregating ears is due the action of these
genes in the early steps of the pathway where the resulting products are colorless.
Therefore, the decreased dosage of these genes show greater reduction in total
anthocyanins as compared to bz1 and bz2 which are downstream and act on colored
anthocyanidin substrates.
F3'H enzyme activity in pr1 seedlings and leaf sheath was studied previously
(LARSON et al. 1986), but the course of accumulation of F3'H dependent cyanidin in
aleurones has not been determined. Cyanidin accumulation in aleurones begins
somewhere between 15 and 20 DAP. The amount of anthocyanins was rather low until 22
DAP but increases substantially after 24 DAP. Small amounts of 3-deoxyanthocyanidins
were also detected in extracts from 10 and 15 DAP kernels. Since 3-deoxyanthocyanidins
have not been reported to be synthesized in aleurone tissue, it has to be from the young
pericarps. In contrast to other flavonoids, the synthesis of anthocyanins generally starts
relatively late during floral tissue development (BOGS et al. 2006; STICH et al. 1992).
Study of the anthocyanin accumulation in relation to kernel aleurone development has
allowed a detailed insight into the process of anthocyanin formation during kernel
development. In grapevine and bilberry, there are two separate phases of gene expression
coinciding with synthesis of different flavonoid compounds during early and late fruit
development stages (DOWNEY et al. 2003; JAAKOLA et al. 2002). First phase of gene
expression is related to proanthocyanidin synthesis and later phase coincides with
anthocyanins (BOGS et al. 2006). It will be interesting to study the expression of pr1 and
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other anthocyanin biosynthetic genes during kernel development in maize and to find
how it is correlated to accumulation of anthocyanins and other flavonoids.
134
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Chapter 5
Conclusions and perspectives
Aleurone pigmentation has important historical relevance in the field of maize
genetics. For over a century, pr1 gene, responsible for red and purple kernel aleurones, is
a favorite phenotypic marker in maize genetics and has been used extensively in linkage
and mapping studies. But the molecular nature of this mutation has still been a curiosity
to maize geneticists. In this research, we have answered this long standing fascinating
problem by cloning and characterization of the pr1 gene. Biochemical characterization
showed that pelargonidin is produced in red aleurone of mutant pr1 plants as oppose to
cyanidin in wild-type purple aleurone. Mutant pr1 plants accumulate only DHK
(dihydrokaempferol) while DHK and DHQ (dihydroquercitin) were detected in wild-type
plants. This indicates that pr1 encoded F3'H uses DHK as substrate and converts it into
DHQ through the addition of a hydroxyl group at 3' B-ring position. Our genetic and
biochemical analysis show that through the action of downstream biosynthetic genes,
DHQ and DHK are converted into cyanidin and pelargonidin, respectively. Maize f3'h1
was cloned and it encodes F3'H, a cytochrome P450 enzyme required for 3'-
hydroxylation of B-ring of flavonoids. f3'h1 gene maps to the previously known RFLP
based position of the pr1 locus on long arm of chromosome 5. In vitro as well as in vivo
complementation studies using BMS cells and Arabidopsis tt7 mutants demonstrated that
f3'h1 encoded protein is able to perform 3'-hydroxylation of anthocyanin precursors.
Expression analysis revealed that mutant pr1 is defective due to lack of f3'h1 transcript.
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Two loss of function alleles had a 48 bp TA dinucleotide repeat insertion in the 5'
upstream region. Third non-functional pr1 alleles had a 17 bp deletion near the TATA
box. These insertion and deletion events in the 5' upstream promoter region may be
responsible for disrupting f3'h1 expression in mutant pr1 plants. We speculated that the
dinucleotide repeats form secondary structures which disrupt the expression of f3'h1 in
pr1 mutants. Promoter deletion expression assays using reporter genes, like GUS or GFP,
are needed to confirm the role of TA dinucleotide repeats in causing loss of f3'h1
transcript. Transient expression analysis using BMS cell suspension culture and promoter
constructs with and without dinucleotide repeats will be informative in answering this
question. According to our hypothesis, higher level of expression is expected in promoter
constructs without dinucleotide repeats as compared to the promoter with dinucleotide
repeats.
The pr1 gene plays role in anthocyanin biosynthesis which is under the regulation
of c1 and r1 (CHANDLER et al. 1989). Our genetic analysis demonstrates that functional
c1 and r1 are required for the pr1 expression during anthocyanin biosynthesis. c1 and r1
mutants show lack of pr1 mRNA, which indicates that c1 as well as r1 are required for
pr1 expression. Apart from its role in anthocyanin biosynthesis, this study shows that pr1
plays a significant role in p regulated phlobaphene (3-deoxyflavonoid) pathway
(GROTEWOLD et al. 1994). We have demonstrated that pr1 is required for conversion of
apiferol to luteoforol and functional p gene is required for pr1 expression in pericarp,
cob-glumes, and silks that synthesize phlobaphenes. The discovery of pr1 regulation by
c1/r1 and p gene introduces question on how single gene is regulated by the two sets of
transcription factors (TF) in separate branches of flavonoid pathway. These TFs bind to
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specific cis regulatory elements present in promoters of biosynthetic genes (LESNICK and
CHANDLER 1998; SAINZ et al. 1997). As we have found sequences similar to these cis
regulatory elements in the pr1 promoter, it is tempting to speculate that these TFs bind
directly to pr1 promoter. To investigate this, we could use transient expression analysis
for studying pr1 promoter activation by C1 and P1 transcription factors. In this assay, the
co-transformation of TFs and pr1 promoter (inserted into reporter plasmid) will allow
quantification of promoter activity by measuring reporter gene activity. Functional assays
are required to identify the active cis regulatory elements among candidate sequences that
can not be distinguished by sequence information alone. To define anthocyanin cis
regulatory elements, similar studies were performed on a2 and bz2 promoters, which
encode anthocyanin-biosynthetic enzymes (BODEAU and WALBOT 1996; LESNICK and
CHANDLER 1998). Additionally, ChIP analysis will be critical for establishing the pr1
interaction with C1 and P1 through putative cis regulatory sequences. Antibodies specific
to C1 and P1 can be used to immunoprecipitate (IP) the TF binding DNA complex. ChIP
DNA from the IP will be tested using different promoter regions of pr1. Alternatively,
electrophoretic mobility shift assay using C1 or P1 proteins and probe containing putative
binding sites of the pr1 promoter could be used to study the interaction of pr1 with these
TFs. Since it is known that R1 does not bind directly with the promoter of biosynthetic
genes (GROTEWOLD et al. 2000a; HERNANDEZ et al. 2007; HERNANDEZ et al. 2004), it
can be excluded from above mentioned assays.
As pr1 plays role in the formation of flavan-4-ols (the anticipated precursors of 3-
deoxyanthocyanidins (GROTEWOLD et al. 1998; STYLES and CESKA 1989)) in the
presence of functional p1 alleles, it was enticing to speculate that the pr1 is required for
141
the formation of 3-deoxyanthocyanidins. High concentration of luteolinidin, a potent
antifungal 3-deoxyanthocyanidin, was detected in Pr1 silks. Structural differences
between luteolinidin and apigeninidin and accumulation of higher levels of luteolinidin in
wild-type plants show that pr1 is required for conversion of apigeninidin to luteolinidin.
This is the first study in maize which specifically confirmed the role of pr1 gene in the
biosynthesis of 3-deoxyanthocyanidins.
We have found that the pr1 participates in accumulation of maysin, a C-glycosyl
flavone with high insecticidal activity in maize silk. Mutant pr1 plants accumulate low
levels of maysin and high amount of apimaysin, while inverse is true for wild-type plants.
Maysin and apimaysin differ by the presence of a hydroxyl group at 3' position of B-ring
(ELLIGER et al. 1980; LINDROTH and PETERSON 1988) and previous studies have detected
the pr1 locus as a major QTL for apimaysin biosynthesis (CORTES-CRUZ et al. 2003; LEE
et al. 1998). Interestingly, current study established the role of pr1 in biosynthesis of
maysin from apimaysin. Further, differential accumulation of these compounds in mutant
and wild-type pr1 plants result in corresponding levels of insect growth inhibition. The
accumulation of different amounts of rhamnosyl-isoorientin and chlorogenic acid in pr1
and Pr1 silks further point towards the role of the pr1 gene in other steps of flavones
biosynthesis. Our study extends the understanding of flavone synthesis and corn earworm
antibiosis in maize and suggests that the pr1 is involved at multiple steps of flavone
biosynthetic pathway. This data, together with the accumulation of different anthocyanins
and 3-deoxyanthocyanidins, suggests that pr1 encoded F3'H plays key role in generating
flavonoid diversity. The role of F3'H in biosynthesis of 3-deoxyanthocyanidins or
flavones can be further confirmed by enzyme feeding assays. Apigeninidin and
142
apimaysin can be fed as substrate to crude enzyme extracts of F3'H and the resulting
compounds can be identified by HPLC. Similarly, other putative substrates, including
phenylpropanoid compounds, can also be tested for F3'H activity to further define its role
in the flavonoid pathway. The pr1 has been extensively used as a phenotypic marker in
maize genetics since the early years of rediscovery of Mendelian genetics (BURR et al.
1996; EAST 1912; EYSTER 1926; LARSON et al. 1986; SELINGER and CHANDLER 1999).
Our study has finally established the molecular nature of the historic purple and red
kernel aleurone phenotypes associated with the pr1.
As components of colored fruits and vegetables, flavonoids are integral part of
human diet. Because of their antioxidant properties, flavonoids provide medical and
health benefits and therefore, are referred as nutraceuticals (LIN and WENG 2006).
Interestingly, anthocyanin compounds with additional hydroxyl group at 3' B-ring
position have higher anticarcinogenic activity than the ones without it (WANG et al.
1997). These observations and results from current study demonstrate the importance of
F3'H in the formation of anthocyanins with better health benefits as well as
agronomically important flavonoid defense compounds. This also highlights the
significance of single gene studies during the development of transgenic plants for
metabolic engineering. Current study has indeed provided crucial clues on how different
subclasses of flavonoids are synthesized in different organs at different developmental
stages. This is also the first study to show that pr1 gene maps in the p1 regulated
branches of flavonoid pathway. Other than pr1 (this study), only the regulation of a1
gene in both c1/r1 and p regulated pathways has been investigated previously
(GROTEWOLD et al. 2000b; POOMA et al. 2002; SAINZ et al. 1997). Results presented in
143
this study and the proposed experiments to study the gene regulation will further
elucidate the underlying mechanisms of single gene regulation by multiple TFs in the
flavonoid pathway. By defining the role of pr1 in 3-deoxyflavonoid and flavone
biosynthesis we further the understanding of these poorly defined pathways, which in
turn will be beneficial for engineering plants with enhanced natural resistance against
insects and fungal pathogens.
144
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Appendix
Corn ear worm silk feeding bioassay.
pr1/P1-wr
Pr1/P1-wr
Pr1/p-del2
pr1/P1-wr
Pr1/P1-wr
Pr1/p-del2
VITA
Mandeep Sharma
Education Ph.D., Agronomy (Maize Molecular Genetics) 05/2010 Pennsylvania State University-University Park, USA M.Sc., Plant Breeding and Genetics 2005 Punjab Agricultural University- Ludhiana, India B.Sc., Agriculture (Honors) 2002 Punjab Agricultural University- Ludhiana, India AWARDS
• Competitive grant award (2009), College of Agricultural Sciences, Pennsylvania State University-University Park, USA
• TAG along fund (2006-2007) to visit ICRISAT, India, as Research Scholar, College of Agricultural Sciences, Pennsylvania State University-University Park, USA
• Graduate Research Assistantship (2005-Present) Pennsylvania State University-University Park, USA
PUBLICATIONS Mandeep Sharma, Moises Cortes-Cruz, Michael McMullen, and Surinder Chopra (2009)
“The Maize red aleurone1 Encodes a Flavonoid 3'-Hydroxylase which is Required for the Biosynthesis of Purple Anthocyanins”. (to be submitted to Genetics).
Mandeep Sharma, Manjit S. Gill, and Satbir S. Gosal (2005) “Studies on Micropropagation and Genetic Transformation in Desi Cotton (Gossypium arboreum L.)”. (MS) thesis
Pawan Malhotra, Mandeep Sharma, and Satbir S. Gosal (2004) “Genetic Engineering for Developing Insect Resistance in some Field Crops”. International Conference of Biotechnology, December 2004, Dhaka, Bangladesh.
TECHNICAL EXPERIENCE Molecular Genetics: Genomic and plasmid DNA isolation; PCR; generation and screening of sub-genomic libraries; cloning; generation and isotope labeling of DNA probes from oligonucleotides and plasmids; Southern hybridization; RNA isolation, Northern hybridization, quantitative real time PCR, reverse transcription PCR; basic bioinformatics techniques. Biochemistry: Metabolic profiling assays, High Performance Liquid Chromatography; Thin Layer Chromatography; Spectrophotometery secondary metabolite assays. Tissue Culture and Genetic Transformation: Cell suspension culture; shoot apical meristem culture for micropropagation; particle bombardment for transient and stable transformation; Agrobacterium mediated stable transformation; maintenance of transgenic plants.
