Virtual Issue | 20th Anniversary | July 2011www.theplantjournal.com

Introduction

Celebrating 20 years of The Plant journal

Intr

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Since its inception twenty years ago, The Plant Journal has developed into

one of the premier journals in the basic plant sciences. During this period,

plant molecular biology has come of age, plant genomics has emerged as a

new driving force for discovery, and work on model species such as

Arabidopsis and rice has shaped our understanding of basic plant functions.

The papers published in The Plant Journal bear witness to these

developments in the plant sciences, and to highlight this we have decided to

assemble a ―virtual‖ special issue that brings together some of the highest

impact papers published in The Plant Journal. For simplicity, we have

chosen to focus on the most highly cited primary research articles in each

year, as well as the five most highly cited Technical Advance papers

published in The Plant Journal. Of course, citation alone is but one measure

of impact. Thus, the collection of papers presented here does not

necessarily provide a representative cross section of the many papers

describing fundamental discoveries that The Plant Journal has published

during its twenty-year history. Nevertheless, it is certainly a collection of

classic works in the plant sciences, demonstrating the progress made in our

discipline in recent times. The Plant Journal is proud to have been a key

publication for the dissemination of this important research, and as new

trends continue to emerge over the next twenty years, the editors and staff

are ready to adjust, provide the appropriate venue for those at the cutting

edge, and to build upon its already very strong reputation as one of the

leading journals in the field.

Christoph Benning

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Floral dip: a simplified method

forAgrobacterium-mediated transformation

ofArabidopsis thaliana

Steven J. Clough and Andrew F. Bent

The Agrobacterium vacuum infiltration method has made it possible to

transform Arabidopsis thaliana without plant tissue culture or

regeneration. In the present study, this method was evaluated and a

substantially modified transformation method was developed. The labor-

intensive vacuum infiltration process was eliminated in favor of simple

dipping of developing floral tissues into a solution containing

Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of

surfactant Silwet L-77. Sucrose and surfactant were critical to the success

of the floral dip method. Plants inoculated when numerous immature floral

buds and few siliques were present produced transformed progeny at the

highest rate. Plant tissue culture media, the hormone benzylamino purine

and pH adjustment were unnecessary, and Agrobacterium could be

applied to plants at a range of cell densities. Repeated application

ofAgrobacterium improved transformation rates and overall yield of

transformants approximately twofold. Covering plants for 1 day to retain

humidity after inoculation also raised transformation rates twofold.

Multiple ecotypes were transformable by this method. The modified

method should facilitate high-throughput transformation of Arabidopsis for

efforts such as T-DNA gene tagging, positional cloning, or attempts at

targeted gene replacement.

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Efficient transformation of rice (Oryza sativa L.)

mediated by Agrobacterium and sequence

analysis of the boundaries of the T-DNA

Yukoh Hiei, Shozo Ohta, Toshihiko Komari andTakashi

Kumashiro

A large number of morphologically normal, fertile, transgenic rice plants

were obtained by co-cultivation of rice tissues with Agrobacterium

tumefaciens. The efficiency of transformation was similar to that obtained

by the methods used routinely for transformation of dicotyledons with the

bacterium. Stable integration, expression and inheritance of transgenes

were demonstrated by molecular and genetic analysis of transformants in

the R0, R1 and R2 generations. Sequence analysis revealed that the

boundaries of the T-DNA in transgenic rice plants were essentially

identical to those in transgenic dicotyledons. Calli induced from scutella

were very good starting materials. A strain of A. tumefaciens that carried a

so-called ‗super-binary‘ vector gave especially high frequencies of

transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.

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A procedure for mapping Arabidopsis

mutations using co-dominant ecotype-

specific PCR-based markers

Andrzej Konieczny and Frederick M. Ausubel

A set of mapping markers have been designed for Arabidopsis thaliana

that correspond to DNA fragments amplifed by the polymerase chain

reaction (PCR). The ecotype of origin of these amplified fragments can be

determined by cleavage with a restriction endo-nuclease. Specifically, 18

sets of PCR primers were synthesized, each of which amplifies a single

mapped DNA sequence from the Columbia and Landsberg erecta

ecotypes. Also identifed was at least one restriction endonuclease for

each of these PCR products that generates ecotype-specific digestion

patterns. Using these co-dominant cleaved amplified polymorphic

sequences (CAPS), an Arabidopsis gene can be unambiguously mapped

to one of the 10 Arabidopsis chromosome arms in a single cross using a

limited number of F2 progeny.

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Efficient isolation and mapping of

Arabidopsis thaliana T-DNA insert junctions

by thermal asymmetric interlaced PCR

Yao-Guang Liu, Norihiro Mitsukawa,Teruko Oosumi and

Robert F. Whittier

Thermal asymmetric interlaced (TAIL-) PCR is an efficient technique for

amplifying insert ends from yeast artificial chromosome (YAC) and P1

clones. Highly specific amplification is achieved without resort to complex

manipulations before or after PCR. The adaptation of this method for

recovery and mapping of genomic sequences flanking T-DNA insertions in

Arabidopsis thaliana is described. Insertion-specific products were

amplified from 183 of 190 tested T-DNA insertion lines. Reconstruction

experiments indicate that the technique can recover single-copy

sequences from genomes as complex as common wheat (1.5 × 1010 bp).

RFLPs were screened using 122 unique flanking sequence probes, and

the insertion sites of 26 T-DNA transgenic lines were determined on an

RFLP map. These lines, whose mapped T-DNA insertions confer

hygromycin resistance, can be used for fine-scale mapping of linked

phenotypic loci.

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Construction of integrated genetic linkage

maps by means of a new computer package:

Join Map

Piet Stam

A computerized procedure to construct integrated genetic maps is

presented. The computer program (Join Map) can handle raw data from

F2s, backcrosses and recombinant inbred lines, as well as listed pair-wise

recombination frequencies. The procedure is useful for combining linkage

data that have been collected in different experiments; the result is a

mathematical alignment of the distinct genetic maps. Data from single

experiments can be dealt with as well. In view of the fast growing amount

of linkage information for molecular markers, which is often being

generated by different research groups, integrated maps provide useful

information on the map position of genes and DNA markers.

The procedure performs a sequential build-up of the map and, at each

step, a numerical search for the best fitting order of markers. Weighted

least squares is used for the estimation of map distances.

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Developmentally Regulated Epitopes Of Cell-

surface Arabinogalactan Proteins And Their

Relation To Root-tissue Pattern-formation

Knox, Jp; Linstead, Pj; Peart, J; Cooper, C; Roberts, K

Two polymorphic forms of an extracellular arabinogalactan protein (AGP1

and AGP2), obtained from the conditioned media of two carrot

suspension-cultured cell lines, have been identified in terms of binding of

anti-plasma membrane bodies JIM4 and MAC207. AGP1 and AGP2 have

been used as immunogens to generate further anti-AGP monoclonal

antibodies. JIM14 identified an epitope carried by AGP2 and also by

glycoproteins of low molecular weight localized to the plant cell wall. In

addition, further antibodies (JIM13 and JIM15) identified carbohydrate

epitopes of the AGPs that also occur on plasma membrane glycoproteins

and are expressed patterns of cells that reflect cell position at the carrot

root apex. Indirect immunofluorescence microscopy indicated that JIM13

recognized the surface of cells forming the epidermis and cells marking

the region and axis of the future xylem. JIM15 recgnized a pattern of cells

directly complementary to the JIM13 pattern. The panel of anti-AGP

monoclonal antibodies now available indicates groups of cells within the

root meristem that may reflect an early pre-pattern of the tissues of the

mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex.

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Volume 1 | Issue 3 | 1991

Lotus japonicus, an autogamous, diploid

legume species for classical and molecular

genetics

Kurt Handberg and Jens Stougaard

In the Leguminosae plant family, few of the individual plant species have

been used for plant molecular biology research. Among the species

investigated no obvious representative ‗model‘ legume has emerged.

Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is

proposed as a candidate. L. japonicus is a diploid, autogamous species,

with a good seed set, and a generation time of approximately 3 months.

The haploid genome consists of six chromosomes and the genome size

was estimated to be relatively small (0.5 pg per haploid complement). L.

japonicus is susceptible to Agrobacterium tumefaciens and transgenic

plants can be regenerated after hygromycin or kanamycin selection.

Tissue culture conditions and procedures for transformation and

regeneration are described. Stable transformation is demonstrated by

segregation of the hygromycin selectable marker after selfing of

transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed.

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Volume 2 | Issue 4 | 1992

Regulation of the expression of rbcS and other

photosynthetic genes by carbohydrates: a

mechanism for the ‘sink regulation’ of

photosynthesis?

Anne Krapp, Bettina Hofmann, Christian Schäfer and

Mark Stitt

These experiments were carried out to investigate whether accumulation

of carbohydrate leads to decreased expression of genes involved in

photosynthesis. Addition of glucose to autotrophic cell suspension

cultures of Chenopodium led to a large and reversible decrease of the

steady state transcript levels of rbcS, cab and atp-& within 5 h, but did not

decrease 18S rRNA or transcript for two glycolytic enzymes. Run-on

transcription in isolated nuclei showed that transcription rate had been

decreased. [35S]Methionine feeding showed that de novo synthesis of

Rubisco was inhibited. Decreased rbcS transcript was also found after

feeding glucose to detached leaves, and in transgenic plants expressing

invertase in the apoplast to inhibit phloem transport, and in leaves on

intact tobacco and potato plants which were cold-girdled to decrease

export. The decrease of rbcS transcript level occurred within 12 h of

coldgirdling. Comparison of carbohydrate content and rbcS transcript

level indicated that carbohydrate content per se is not the direct signal for

regulation of gene expression. Feeding of transported analogues

indicates that metabolism rather than transport of the sugars is required.

Over-expression of rbcS was found in low CO2, again indicating metabolic

control of expression. It is proposed that photosynthetic gene expression

is inhibited by metabolic factors related to high carbohydrate content, and

that this represents a basic mechanism for the ‗sink regulation‘ of photo-synthesis.

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Volume 3 | Issue 6 | 1993

Manipulation of lignin quality by

downregulation of cinnamyl alcohol

dehydrogenase

Claire Halpin et al.

The composition of lignin in tobacco stems has been altered by genetic

engineering. Antisense expression of sequences encoding cinnamyl

alcohol dehydrogenase (CAD), the enzyme catalysing the final step in

lignin precursor synthesis, leads to the production of a modified lignin in

otherwise normal plants. Although Klason and acetyl bromide lignin

determinations show little quantitative change in lignin deposition in CAD

antisense plants, a number of qualitative changes have been identified.

The lignin is altered in both composition and structure and is more

susceptible to chemical extraction. Consistent with a block in CAD activity,

antisense plants incorporate less cinnamyl alcohol monomers and more

cinnamyl aidehyde monomers into lignin than corresponding control

plants. Antisense plants with very low levels of CAD activity also show a

novel phenotype with the appearance of a red-brown colour in xylem

tissues. A similar phenotype is correlated with altered lignification and

improved digestibility in brownmidrib mutants of maize and sorghum. The

improved chemical extractability of lignin in CAD antisense plants

supports a role for this technology in improving the pulp and paper-

making value of forest trees while the similarity with brown-midrib mutants

suggests a route to more digestible forage crops.

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Volume 6 | Issue 3 | 1994

Enhanced quantitative resistance against

fungal disease by combinatorial expression of

different barley antifungal proteins in

transgenic tobacco

Guido Jach et al.

cDNAs encoding three proteins from barley (Hordeum vulgare), a class-II

chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-

inactivating protein (RIP) were expressed in tobacco plants under the

control of the CaMV 35S-promoter. High-level expression of the

transferred genes was detected in the transgenic plants by Northern and

Western blot analysis. The leader peptides in CHI and GLU led to

accumulation of these proteins in the intercellular space of tobacco

leaves. RIP, which is naturally deposited in the cytosol of barley

endosperm cells, was expressed either in its original cytosolic form or

fused to a plant secretion peptide (spRIP). Fungal infection assays

revealed that expression of the individual genes in each case resulted in

an increased protection against the soilborne fungal pathogen

Rhizoctonia solani, which infects a range of plant species including

tobacco. To create a situation similar to ‗multi-gene‘ tolerance, which

traditional breeding experience has shown to provide crops with a longer-

lasting protection, several of these antifungal genes were combined and

protection against fungal attack resulting from their co-expression in

planta was evaluated. Transgenic tobacco lines were generated with

tandemly arranged genes coding for RIP and CHI as well as GLU and

CHI. The performance of tobacco plants co-expressing the barley

transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay

revealed significantly enhanced protection against fungal attack when

compared with the protection levels obtained with corresponding isogenic

lines expressing a single barley transgene to a similar level. The data

indicate synergistic protective interaction of the co-expressed anti-fungal

proteins in vivo.

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Volume 8 | Issue 1 | 1995

Benzothiadiazole induces disease resistance

in Arabidopsis by activation of the systemic

acquired resistance signal transduction

pathway

Kay A. Lawton et al.

Benzothiadiazole (BTH) is a novel chemical activator of disease

resistance in tobacco, wheat and other important agricultural plants. In

this report, it is shown that BTH works by activating SAR in Arabidopsis

thaliana. BTH-treated plants were resistant to infection by turnip crinkle

virus, Pseudomonas syringae pv ‗tomato‘ DC3000 and Peronospora

parasitica. Chemical treatment induced accumulation of mRNAs from the

SAR-associated genes, PR-1, PR-2 and PR-5. BTH treatment induced

both PR-1 mRNA accumulation and resistance against P. parasitica in the

ethylene response mutants, etr1 and ein2, and in the methyl jasmonate-

insensitive mutant, jar1, suggesting that BTH action is independent of

these plant hormones. BTH treatment also induced both PR-1 mRNA

accumulation and P. parasitica resistance in transgenic Arabidopsis plants

expressing the nahG gene, suggesting that BTH action does not require

salicylic acid accumulation. However, because BTH-treatment failed to

induce either PR-1 mRNA accumulation or P. parasitica resistance in the

non-inducible immunity mutant, nim1, it appears that BTH activates the

SAR signal transduction pathway.

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Volume 10 | Issue 1 | 1996

A benzothiadiazole derivative induces

systemic acquired resistance in tobacco

Leslie Friedrich et al.

Systemic acquired resistance (SAR) is a pathogen-induced disease

resistance response in plants that is characterized by broad spectrum

disease control and an associated coordinate expression of a set of SAR

genes. Benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH)

is a novel synthetic chemical capable of inducing disease resistance in a

number of dicotyledenous and monocotyledenous plant species. In this

report, the response of tobacco plants to BTH treatment is characterized

and the fact that it controls disease by activating SAR is demonstrated.

BTH does not cause an accumulation of salicylic acid (SA), an

intermediate in the SAR signal transduction pathway. As BTH also

induces disease resistance and gene expression in transgenic plants

expressing the nahG gene, it appears to activate the SAR signal

transduction pathway at the site of or downstream of SA accumulation.

BTH, SA and TMV induce the PR-1a promoter using similar cis-acting

elements and gene expression is blocked by cycloheximide treatment.

Thus, BTH induces SAR based on all of the physiological and

biochemical criteria that define SAR in tobacco.

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Volume 10 | Issue 1 | 1996

Subcellular localization of H2O2 in plants. H2O2

accumulation in papillae and hypersensitive

response during the barley—powdery mildew

interaction

Hans Thordal-Christensen, Ziguo Zhang, Yangdou Wei

and David B. Collinge

Active oxygen species (AOS) are believed to have important roles in

plants in general and in plant—pathogen interactions in particular. They

are believed to be involved in signal transduction, cell wall reinforcement,

hypersensitive response (HR) and phytoalexin production, and to have

direct antimicrobial effects. Since current methods are inadequate for

localizing AOS in intact plant tissue, most studies have been conducted

using cell suspension culture/elicitors systems. 3,3-diaminobenzidine

(DAB) polymerizes instantly and locally as soon as it comes into contact

with H2O2 in the presence of peroxidase, and it was found that, by

allowing the leaf to take up this substrate, in-vivo and in-situ detection of

H2O2 can be made at subcellular levels. This method was successfully

used to detect H2O2 in developing papillae and surrounding haloes (cell

wall appositions) and whole cells of barley leaves interacting with the

powdery mildew fungus. Thus, H2O2 can be detected in the epidermal cell

wall subjacent to the primary germ tube from 6 h after inoculation, and

subjacent to the appressorium from 15 h. The earliest time point for

observation of H2O2 in relation to epidermal cells undergoing HR is 15 h

after inoculation, first appearing in the zones of attachment to the

mesophyll cells underneath, and eventually in the entire epidermal cell.

Furthermore, it was observed that proteins in papillae and HR cells are

cross-linked, a process believed to be fuelled by H2O2. This cross-linking

reinforces the apposition, presumably assisting the arrest of the pathogen.

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Volume 11 | Issue 1 | 1997

Stacks on tracks: the plant Golgi apparatus

traffics on an actin/ER network

Petra Boevink, Karl Oparka, Simon Santa Cruz, Barry

Martin, Alan Betteridge and Chris Hawes

We have visualized the relationship between the endoplasmic reticulum

(ER) and Golgi in leaf cells of Nicotiana clevelandii by expression of two

Golgi proteins fused to green fluorescent protein (GFP). A fusion of the

trans-membrane domain (signal anchor sequence) of a rat sialyl

transferase to GFP was targeted to the Golgi stacks. A second construct

that expressed the Arabidopsis H/KDEL receptor homologue aERD2,

fused to GFP, was targeted to both the Golgi apparatus and ER, allowing

the relationship between these two organelles to be studied in living cells

for the first time. The Golgi stacks were shown to move rapidly and

extensively along the polygonal cortical ER network of leaf epidermal

cells, without departing from the ER tubules. Co-localization of F-actin in

the GFP-expressing cells revealed an underlying actin cytoskeleton that

matched precisely the architecture of the ER network, while treatment of

cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the

dependency of Golgi movement on actin cables. These observations

suggest that the leaf Golgi complex functions as a motile system of actin-

directed stacks whose function is to pick up products from a relatively

stationary ER system. Also, we demonstrate for the first time in vivo

brefeldin A-induced retrograde transport of Golgi membrane protein to

the ER.

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Volume 15 | Issue 3 | 1998

Plants have a sensitive perception system

for the most conserved domain of bacterial

flagellin

Georg Felix, Juliana D. Duran, Sigrid Volko and

Thomas Boller

The flagellum is an important virulence factor for bacteria pathogenic to

animals and plants. Here we demonstrate that plants have a highly

sensitive chemoperception system for eubacterial flagellins, specifically

targeted to the most highly conserved domain within its N terminus.

Synthetic peptides comprising 15–22 amino acids of this domain acted as

elicitors of defence responses at sub-nanomolar concentrations in cells of

tomato and several other plant species. Peptides comprising only the

central 8 to 11 amino acids of the active domain had no elicitor activity but

acted as specific, competitive inhibitors in tomato cells. These antagonists

suppressed the plant‘s response to flagellin, crude bacterial extracts and

living bacterial cells. Thus, plants have a highly sensitive and selective

perception system for the flagellin of motile eubacteria.

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Volume 18 | Issue 3 | 1999

Over-expression of a single Ca2+-dependent

protein kinase confers both cold and

salt/drought tolerance on rice plants

Yusuke Saijo, Shingo Hata, Junko Kyozuka, Ko

Shimamoto and Katsura Izui

A rice gene encoding a calcium-dependent protein kinase (CDPK),

OsCDPK7, was induced by cold and salt stresses. To elucidate the

physiological function of OsCDPK7, we generated transgenic rice plants

with altered levels of the protein. The extent of tolerance to cold and

salt/drought stresses of these plants correlated well with the level of

OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive

regulator commonly involved in the tolerance to both stresses in rice.

Over-expression of OsCDPK7 enhanced induction of some stress-

responsive genes in response to salinity/drought, but not to cold. Thus, it

was suggested that the downstream pathways leading to the cold and

salt/drought tolerance are different from each other. It seems likely that at

least two distinct pathways commonly use a single CDPK, maintaining the

signalling specificity through unknown post-translational regulation

mechanisms. These results demonstrate that simple manipulation of

CDPK activity has great potential with regard to plant improvement.

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Volume 23 | Issue 3 | 2000

Sites and homeostatic control of auxin

biosynthesis in Arabidopsis during

vegetative growth

Karin Ljung, Rishikesh P. Bhalerao and Göran Sandberg

The distribution and biosynthesis of indole-3-acetic acid (IAA) was

investigated during early plant development in Arabidopsis. The youngest

leaves analysed, less than 0.5 mm in length, contained 250 pg mg−1 of IAA

and also exhibited the highest relative capacity to synthesize this hormone.

A decrease of nearly one hundred-fold in IAA content occurred as the young

leaves expanded to their full size, and this was accompanied by a clear shift

in both pool size and IAA synthesis capacity. The correlation between high

IAA content and intense cell division was further verified in tobacco leaves,

where a detailed analysis revealed that dividing mesophyll tissue contained

ten-fold higher IAA levels than tissue growing solely by elongation. We

demonstrated that all parts of the young Arabidopsis plant can potentially

contribute to the auxin needed for growth and development, as not only

young leaves, but also all other parts of the plant such as cotyledons,

expanding leaves and root tissues have the capacity to synthesize IAA de

novo. We also observed that naphthylphthalamic acid (NPA) treatment

induced tissue-dependent feedback inhibition of IAA biosynthesis in

expanding leaves and cotyledons, but intriguingly not in young leaves or in

the root system. This observation supports the hypothesis that there is a

sophisticated tissue-specific regulatory mechanism for auxin biosynthesis.

Finally, a strict requirement for maintaining the pool sizes of IAA was

revealed as reductions in leaf expansion followed both decreases and

increases in the IAA levels in developing leaves. This indicates that leaves

are not only important sources for IAA synthesis, but that normal leaf

expansion depends on rigorous control of IAA homeostasis.

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Volume 28 | Issue 4 | 2001

Monitoring the expression profiles of 7000

Arabidopsis genes under drought, cold and

high-salinity stresses using a full-length

cDNA microarray

Motoaki Seki et al.

Full-length cDNAs are essential for functional analysis of plant genes in

the post-sequencing era of the Arabidopsis genome. Recently, cDNA

microarray analysis has been developed for quantitative analysis of global

and simultaneous analysis of expression profiles. We have prepared a

full-length cDNA microarray containing ≈7000 independent, full-length

cDNA groups to analyse the expression profiles of genes under drought,

cold (low temperature) and high-salinity stress conditions over time. The

transcripts of 53, 277 and 194 genes increased after cold, drought and

high-salinity treatments, respectively, more than fivefold compared with

the control genes. We also identified many highly drought-, cold- or high-

salinity- stress-inducible genes. However, we observed strong

relationships in the expression of these stress-responsive genes based

on Venn diagram analysis, and found 22 stress-inducible genes that

responded to all three stresses. Several gene groups showing different

expression profiles were identified by analysis of their expression patterns

during stress-responsive gene induction. The cold-inducible genes were

classified into at least two gene groups from their expression profiles.

DREB1A was included in a group whose expression peaked at 2 h after

cold treatment. Among the drought, cold or high-salinity stress-inducible

genes identified, we found 40 transcription factor genes (corresponding to

≈11% of all stress-inducible genes identified), suggesting that various

transcriptional regulatory mechanisms function in the drought, cold or

high-salinity stress signal transduction pathways.

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Volume 31 | Issue 3 | 2002

OsDREB genes in rice, Oryza sativa L.,

encode transcription activators that function

in drought-, high-salt- and cold-responsive

gene expression

Joseph G. Dubouzet et al.

The transcription factors DREBs/CBFs specifically interact with the

dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element

(core motif: G/ACCGAC) and control the expression of many stress-

inducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB

homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and

OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by

cold, whereas expression of OsDREB2A was induced by dehydration and

high-salt stresses. The OsDREB1A and OsDREB2A proteins specifically

bound to DRE and activated the transcription of the GUS reporter gene

driven by DRE in rice protoplasts. Over-expression of OsDREB1A in

transgenic Arabidopsis induced over-expression of target stress-inducible

genes of Arabidopsis DREB1A resulting in plants with higher tolerance to

drought, high-salt, and freezing stresses. This indicated that OsDREB1A

has functional similarity to DREB1A. However, in microarray and RNA blot

analyses, some stress-inducible target genes of the DREB1A proteins

that have only ACCGAC as DRE were not over-expressed in the

OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to

GCCGAC more preferentially than to ACCGAC whereas the DREB1A

proteins bound to both GCCGAC and ACCGAC efficiently. The structures

of DREB1-type ERF/AP2 domains in monocots are closely related to

each other as compared with that in the dicots. OsDREB1A is potentially

useful for producing transgenic monocots that are tolerant to drought,

high-salt, and/or cold stresses.

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Volume 33 | Issue 4 | 2003

A wheat gene encoding an aluminum-

activated malate transporter

Takayuki Sasaki et al.

The major constraint to plant growth in acid soils is the presence of toxic

aluminum (Al) cations, which inhibit root elongation. The enhanced Al

tolerance exhibited by some cultivars of wheat is associated with the Al-

dependent efflux of malate from root apices. Malate forms a stable

complex with Al that is harmless to plants and, therefore, this efflux of

malate forms the basis of a hypothesis to explain Al tolerance in wheat.

Here, we report on the cloning of a wheat gene, ALMT1 (aluminum-

activated malate transporter), that co-segregates with Al tolerance in F2

and F3 populations derived from crosses between near-isogenic wheat

lines that differ in Al tolerance. The ALMT1 gene encodes a membrane

protein, which is constitutively expressed in the root apices of the Al-

tolerant line at greater levels than in the near-isogenic but Al-sensitive

line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and

cultured tobacco cells conferred an Al-activated malate efflux. Additionally,

ALMT1 increased the tolerance of tobacco cells to Al treatment. These

findings demonstrate that ALMT1 encodes an Al-activated malate

transporter that is capable of conferring Al tolerance to plant cells.

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Volume 37 | Issue 5 | 2004

Functional genomics by integrated analysis of

metabolome and transcriptome of Arabidopsis

plants over-expressing an MYB transcription

factor

Takayuki Tohge et al.

The integration of metabolomics and transcriptomics can provide precise

information on gene-to-metabolite networks for identifying the function of

unknown genes unless there has been a post-transcriptional modification.

Here, we report a comprehensive analysis of the metabolome and

transcriptome of Arabidopsis thaliana over-expressing the PAP1 gene

encoding an MYB transcription factor, for the identification of novel gene

functions involved in flavonoid biosynthesis. For metabolome analysis, we

performed flavonoid-targeted analysis by high-performance liquid

chromatography-mass spectrometry and non-targeted analysis by Fourier-

transform ion-cyclotron mass spectrometry with an ultrahigh-resolution

capacity. This combined analysis revealed the specific accumulation of

cyanidin and quercetin derivatives, and identified eight novel anthocyanins

from an array of putative 1800 metabolites in PAP1 over-expressing plants.

The transcriptome analysis of 22 810 genes on a DNA microarray revealed

the induction of 38 genes by ectopic PAP1 over-expression. In addition to

well-known genes involved in anthocyanin production, several genes with

unidentified functions or annotated with putative functions, encoding putative

glycosyltransferase, acyltransferase, glutathione S-transferase, sugar

transporters and transcription factors, were induced by PAP1. Two putative

glycosyltransferase genes (At5g17050 and At4g14090) induced by PAP1

expression were confirmed to encode flavonoid 3-O-glucosyltransferase and

anthocyanin 5-O-glucosyltransferase, respectively, from the enzymatic

activity of their recombinant proteins in vitro and results of the analysis of

anthocyanins in the respective T-DNA-inserted mutants. The functional

genomics approach through the integration of metabolomics and

transcriptomics presented here provides an innovative means of identifying

novel gene functions involved in plant metabolism.

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Volume 42 | Issue 2 | 2005

ABA-induced NO generation and stomatal

closure in Arabidopsis are dependent on

H2O2 synthesis

Jo Bright, Radhika Desikan, John T. Hancock, Iain S.

Weir and Steven J. Neil

Nitric oxide (NO) and hydrogen peroxide (H2O2) are key signalling

molecules produced in response to various stimuli and involved in a diverse

range of plant signal transduction processes. Nitric oxide and H2O2 have

been identified as essential components of the complex signalling network

inducing stomatal closure in response to the phytohormone abscisic acid

(ABA). A close inter-relationship exists between ABA and the spatial and

temporal production and action of both NO and H2O2 in guard cells. This

study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO

generation is in fact dependent on ABA-induced H2O2 production. Stomatal

closure induced by H2O2 is inhibited by the removal of NO with NO

scavenger, and both ABA and H2O2 stimulate guard cell NO synthesis.

Conversely, NO-induced stomatal closure does not require H2O2 synthesis

nor does NO treatment induce H2O2 production in guard cells. Tungstate

inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates

NO production in response to nitrite in vitro and in response to H2O2 and

ABA in vivo. Genetic data demonstrate that NR is the major source of NO in

guard cells in response to ABA-mediated H2O2 synthesis. In the NR double

mutant nia1, nia2 both ABA and H2O2 fail to induce NO production or

stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant,

responses to H2O2 are not impaired. Importantly, we show that in the

NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and

stomatal closure to ABA are severely reduced, indicating that endogenous

H2O2 production induced by ABA is required for NO synthesis. In summary,

our physiological and genetic data demonstrate a strong inter-relationship

between ABA, endogenous H2O2 and NO-induced stomatal closure.

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Volume 45 | Issue 1 | 2006

The AtGenExpress global stress expression

data set: protocols, evaluation and model

data analysis of UV-B light, drought and cold

stress responses

Joachim Kilian et al.

The tolerance responses of plants to many abiotic stresses are

conjectured to be controlled by complex gene networks. In the frame of

the AtGenExpress project a comprehensive Arabidopsis thaliana genome

transcript expression study was performed using the Affymetrix ATH1

microarray in order to understand these regulatory networks in detail. In

contrast to earlier studies, we subjected, side-by-side and in a high-

resolution kinetic series, Arabidopsis plants, of identical genotype grown

under identical conditions, to different environmental stresses comprising

heat, cold, drought, salt, high osmolarity, UV-B light and wounding.

Furthermore, the harvesting of tissue and RNA isolation were performed

in parallel at the same location using identical experimental protocols.

Here we describe the technical performance of the experiments. We also

present a general overview of environmental abiotic stress-induced gene

expression patterns and the results of a model bioinformatics analysis of

gene expression in response to UV-B light, drought and cold stress. Our

results suggest that the initial transcriptional stress reaction of Arabidopsis

might comprise a set of core environmental stress response genes which,

by adjustment of the energy balance, could have a crucial function in

various stress responses. In addition, there are indications that systemic

signals generated by the tissue exposed to stress play a major role in the

coordination and execution of stress responses. In summary, the

information reported provides a prime reference point and source for the

subsequent exploitation of this important resource for research into plant

abiotic stress.

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Volume 50 | Issue 2 | 2007

MicroRNA399 is a long-distance signal

for the regulation of plant phosphate

homeostasis

Bikram Datt Pant, Anja Buhtz, Julia Kehr and Wolf-

Rüdiger Scheible

The presence of microRNA species in plant phloem sap suggests

potential signaling roles by long-distance regulation of gene expression.

Proof for such a role for a phloem-mobile microRNA is lacking. Here we

show that phosphate (Pi) starvation-induced microRNA399 (miR399) is

present in the phloem sap of two diverse plant species, rapeseed and

pumpkin, and levels are strongly and specifically increased in phloem sap

during Pi deprivation. By performing micro-grafting experiments using

Arabidopsis, we further show that chimeric plants constitutively over-

expressing miR399 in the shoot accumulate mature miR399 species to

very high levels in their wild-type roots, while corresponding primary

transcripts are virtually absent in roots, demonstrating shoot-to-root

transport. The chimeric plants exhibit (i) down-regulation of the miR399

target transcript (PHO2), which encodes a critical component for

maintenance of Pi homeostasis, in the wild-type root, and (ii) Pi

accumulation in the shoot, which is the phenotype of pho2 mutants,

miR399 over-expressers or chimeric plants with a genetic knock-out of

PHO2 in the root. Hence the transported miR399 molecules retain

biological activity. This is a demonstration of systemic control of a

biological process, i.e. maintenance of plant Pi homeostasis, by a

phloem-mobile microRNA.

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Volume 53 | Issue 5 | 2008

Ferritins control interaction between iron

homeostasis and oxidative stress in

Arabidopsis

Karl Ravet, Brigitte Touraine, Jossia Boucherez, Jean-

François Briat, Frédéric Gaymard and Françoise Cellier

Ferritin protein nanocages are the main iron store in mammals. They have

been predicted to fulfil the same function in plants but direct evidence was

lacking. To address this, a loss-of-function approach was developed in

Arabidopsis. We present evidence that ferritins do not constitute the major

iron pool either in seeds for seedling development or in leaves for proper

functioning of the photosynthetic apparatus. Loss of ferritins in vegetative

and reproductive organs resulted in sensitivity to excess iron, as shown

by reduced growth and strong defects in flower development.

Furthermore, the absence of ferritin led to a strong deregulation of

expression of several metal transporters genes in the stalk, over-

accumulation of iron in reproductive organs, and a decrease in fertility.

Finally, we show that, in the absence of ferritin, plants have higher levels

of reactive oxygen species, and increased activity of enzymes involved in

their detoxification. Seed germination also showed higher sensitivity to

pro-oxidant treatments. Arabidopsis ferritins are therefore essential to

protect cells against oxidative damage.

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Volume 57 | Issue 3 | 2009

PYR/PYL/RCAR family members are

major in-vivo ABI1 protein phosphatase

2C-interacting proteins in Arabidopsis

Noriyuki Nishimura et al.

Abscisic acid (ABA) mediates resistance to abiotic stress and controls

developmental processes in plants. The group-A PP2Cs, of which ABI1 is the

prototypical member, are protein phosphatases that play critical roles as

negative regulators very early in ABA signal transduction. Because redundancy

is thought to limit the genetic dissection of early ABA signalling, to identify

redundant and early ABA signalling proteins, we pursued a proteomics

approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and

identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of

ABI1 complexes. Known ABA signalling components were isolated including

SnRK2 protein kinases. We confirm previous studies in yeast and now show

that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and

SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting

proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR

proteins, which were recently reported as ABA-binding signal transduction

proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1

in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA

treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-

immunoprecipitated equally well in the presence and absence of ABA. To

investigate the biological relevance of the PYR/PYLs, we analysed

pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in

ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These

findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR

family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly

stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-

PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-

mediated ABA signalling.

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Volume 61 | Issue 2 | 2010

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