Copyright 2000 by the Genetics Society of America

The Developmental Expression of the Maize Regulatory Gene Hopi DeterminesGermination-Dependent Anthocyanin Accumulation

Katia Petroni,* Eleonora Cominelli,* Gabriella Consonni,† Giuliana Gusmaroli,*Giuseppe Gavazzi† and Chiara Tonelli*

*Dipartimento di Genetica e di Biologia dei Microrganismi and †Dipartimento di Fisiologia delle Piante Coltivate e Chimica Agraria,Universita degli Studi di Milano, 20133 Milano, Italy

Manuscript received July 22, 1999Accepted for publication January 4, 2000

ABSTRACTThe Hopi gene is a member of the maize r1 gene family. By genetic and molecular analyses we report

that Hopi consists of a single gene residing on chromosome 10 z4.5 cM distal to r1. Hopi conditionsanthocyanin deposition in aleurone, scutellum, pericarp, root, mesocotyl, leaves, and anthers, thus repre-senting one of the broadest specifications of pigmentation pattern reported to date of all the r1 genes.A unique feature of the Hopi gene is that seeds are completely devoid of pigment at maturity but show aphotoinducible germination-dependent anthocyanin accumulation in aleurone and scutellum. Our analysishas shown that the Hopi transcript is not present in scutellum of developing seeds but is induced onlyupon germination and that the simultaneous presence of both C1 and Hopi mRNAs is necessary to achieveA1 activation in scutella. We conclude that the expression pattern of the Hopi gene accounts for thegermination-dependent anthocyanin synthesis in scutella, whereas the developmental competence ofgerminating seeds to induce anthocyanin production in scutella results from the combination of the light-inducible expression of C1 and the developmentally regulated expression of the Hopi gene.

ANTHOCYANINS represent the most widespread a maternally derived seed integument. The Pl-blotchedred and purple pigments in the plant kingdom. allele, however, leads to anthocyanin expression in the

These pigments are produced in a variety of plant tis- aleurone also (Cocciolone and Cone 1993). Dominantsues, where they serve many diverse functions, such as light-independent and recessive light-dependent “sunattraction of pollinators and dispersal agents and protec- red” alleles of pl1 are known (Cone et al. 1993b). Thetion against insects, phytopathogens, and UV irradia- c1 and pl loci probably originate from a duplicationtion. Anthocyanin accumulation in maize tissues re- event, since they share nearly identical coding regionsquires the expression of many genes (at least 20), some and are likely to be functionally equivalent (Cone et al.that are involved in biosynthesis and others in tissue- 1993a).specific regulation of biosynthetic loci (reviewed in Mol The second group of regulatory genes, the r1/b1 geneet al. 1998). The structural genes c2, chi1, f3h, a1, a2, family, consists of highly homologous genes encodingbz1, and bz2 encoding the biosynthetic enzymes are coor- exchangeable proteins with an amino acid sequencedinately controlled at the transcriptional level by the containing the basic helix-loop-helix (bHLH) DNA-products of at least two groups of regulatory genes, binding/dimerization domain found in MyoD proteinwhich are responsible for the developmental and tissue- (Davis et al. 1987; Chandler et al. 1989; Ludwig et al.specific pigmentation of plant and seed tissues. The 1989; Tonelli et al. 1991; Consonni et al. 1992, 1993).first group is represented by the c1/pl1 family, whose Although the r1 and b1 genes encode similar proteins,members encode proteins with sequence homology to their structural organization differs. While b1 allelesthe tryptophan cluster, DNA-binding domain of MYB consist of a single gene, many r1 alleles are geneticallyoncoproteins (Klempnauer et al. 1982; Cone et al. 1986, complex, consisting of many genes, each of which en-1993a; Paz-Ares et al. 1986, 1987). Despite the c1 tran- codes a distinct pigmentation pattern. All members ofscript being found at low levels in husks (Cooper and the r1/b1 gene family are thought to have been derivedCone 1997), the c1 gene acts to induce pigmentation

from an as-yet-unidentified single ancestral gene that,only in seed tissues, such as the aleurone and the embryo

following intrachromosomal duplication, gave rise to(McCarty et al. 1989), whereas pl1 controls pigmenta-the r1 complex on chromosome 10 (Eggleston et al.tion in the seedlings, the plant body, and the pericarp,1995; Walker et al. 1995), the Sn and Lc genes, lyingabout two units distal to r1 (Ludwig et al. 1989; Tonelliet al. 1991), and, after a postulated genome duplication

Corresponding author: Chiara Tonelli, Dipartimento di Genetica e di event, to the b1 locus on chromosome 2 (Chandler etBiologia dei Microrganismi, Universita degli Studi di Milano, ViaCeloria 26, 20133 Milano, Italy. E-mail: chiara.tonelli@unimi.it al. 1989). Molecular divergence during evolution may

Genetics 155: 323–336 (May 2000)

324 K. Petroni et al.

then have contributed to the establishment of distinct et al. 1993). Growth factors are also important in regulat-ing pigment production. Anthocyanin accumulates ingenes, each one performing a similar function in spe-

cific plant tissues. The duplicated origin of r1-like genes maize seed tissues starting at z22 days after pollination(DAP) (Neill et al. 1987). These events are controlledis inferred from the structural organization of the R-r

complex. Genetic and molecular studies have revealed by factors induced by abscisic acid (ABA) and by theproduct of the vp1 (viviparous1) gene that are requiredthat R-r is composed of multiple units with sequence

similarity, arranged in a tandem array: a plant (P) com- for the transcriptional activation of the c1 gene (Hat-tori et al. 1992) and also of the maturation-specificponent that consists of a single gene specifying plant

pigmentation; two seed genes (S1 and S2) arranged in wheat Em gene (McCarty 1995). vp1 mutants fail torepress precocious germination of developing seeds andopposite orientation, responsible for color in the seed;

and a third cryptic component, referred to as q, con- are devoid of pigment in aleurone and embryo tissues.However, if rescued before desiccation, vp1 mutant seedsisting of a truncated gene sequence (Robbins et al.

1991; Walker et al. 1995). The plant and seed pigmenta- is able to develop pigment in seed tissues during germi-nation in presence of light (McCarty and Carsontion components can be lost independently by mutation

or unequal crossing over, leading to different derivatives 1991), indicating that C1 expression also is regulatedby light during kernel development. Supporting this(Stadler 1948). While the expression pattern of com-

plex r1 genes, such as R-r, is determined by the presence idea, seeds homozygous for the c1-p allele are colorlessbut accumulate pigment if exposed to light during ger-of multiple promoters, each one associated with a cod-

ing region, other r1 alleles and the b1, Sn, and Lc genes mination (Chen and Coe 1977), suggesting that in thisallele of c1 the ABA/VP1 regulation has been lost butconsist of a single genetic unit with a characteristic tis-

sue-specific expression pattern probably depending on that the light regulation of the gene has been retained.Furthermore, C1 maize ears developed in total darknessdifferent controlling sequences located in the corre-

sponding promoter regions, as demonstrated for two produce colorless seeds that develop pigmentation aftersubsequent exposure to light (Dooner and Ralstonb1 alleles (Radicella et al. 1992; Selinger et al. 1998).

Biochemical (Dooner 1983) and molecular analyses 1994). It is likely that the transcription factors control-ling tissue-specific accumulation of anthocyanin also arehave revealed that the activity of a functional allele from

both these two gene families is necessary for the tran- part of the light signal transduction pathway inducingits accumulation. Accumulation of A1 and C2 transcriptsscriptional activation of the structural genes c2, chi1,

f3h, a1, a2, bz1, and bz2 implicated in the anthocyanin in seedling tissues, following light exposure, requiresthe activity of Sn (Tonelli et al. 1991). In additionbiosynthetic pathway (Taylor and Briggs 1990; Bodeau

and Walbot 1992; Grotewold and Peterson 1994; “sun red” alleles of pl1 show a light-regulated expressionpattern (Cone et al. 1993b; Procissi et al. 1997).Grotewold et al. 1998; Lesnick and Chandler 1998).

Transient expression experiments have suggested that In this article, we report the genetic and molecularanalysis of a new member of the r1/b1 gene family,the B1 and C1 proteins interact to form a complex able

to transactivate the bz1 promoter in maize embryogenic called Hopi. This gene conditions the pigmentation ofa wide variety of plant and seed tissues (Table 1). Wecalli (Goff et al. 1991, 1992). Since the B1 protein does

not contain either a transactivating domain or a DNA- show that the tissue-specific pigmentation conditionedby Hopi depends on a single gene residing on chromo-binding activity but is required for transactivation of the

bz1 promoter, Tuerck and Fromm (1994) proposed some 10 z4.5 cM distal to r1. A clear feature of the Hopigene is its germination-dependent ability to determinethat the B1 protein might be required for an efficient

binding of C1 to the MYB-like sites in the bz1 promoter, anthocyanin accumulation in seed tissues, similar to theactivity of the c1-p allele. In contrast to other r1 genes,but not for the transactivating activity of the B1/C1

complex itself. However, it has been recently demon- such as R-sc, which promote pigment production inaleurone and scutellum during maturation on the earstrated that C1 does not require B or R to bind anthocya-

nin promoter sequences (Sainz et al. 1997; Lesnick and (Table 1), homozygous Hopi seeds are completely de-void of pigment at maturity, but accumulate anthocya-Chandler 1998). Furthermore, the observation that

the a1 and a2 promoters contain sequences crucial for nins in scutellum and aleurone if germinated in thelight. Light responsiveness is most effective in the firstactivation, which are not bound by C1, suggests that

other factor(s) may be involved in activation through hours following the onset of germination. After a pro-longed period of dark growth, light irradiation does notthese promoter sequences (Sainz et al. 1997; Lesnick

and Chandler 1998). elicit pigmentation. It has been previously shown thatin pericarp, both sn1 and pl1 expression is light-modu-Hence, the pattern of pigmentation of a maize plant

reflects its allelic constitution at r1/b1 and c1/pl1 regula- lated, whereas in aleurone R-sc is constitutively ex-pressed and C1 shows light inducibility; in both tissues,tory loci. The activation of anthocyanin synthesis re-

quires either c1 (in the seed) or pl1 (in the plant), while the MYB-like genes were found to be the limiting factorsregulating the extent of pigment deposition (Procissithe r1/b1 genes, whose expression is tissue-specific, de-

termine the tissue distribution of pigments (Consonni et al. 1997). Here, we demonstrate that Hopi gene expres-

325Developmental Expression of Hopi

Hopi: A factor lying on chromosome 10, isolated from aTABLE 1maize stock given to G. Gavazzi by Dr. A. Brink and incorpo-

Pigment distribution in Hopi genotype vs. different rated by backcrossing into the background of inbred W22; itsgenotypes of the r1/b1 gene family origin is presumed to trace back to the Indian Hopi popula-

tion. Hopi confers pigmentation to a wide variety of planttissues (see Table 1), including pericarp, root, mesocotyl, andSeed Plantleaf blade in the seedling, midrib, ligule, leaf blade, and an-

Gene al sct per root msc leaf ant clp thers in the mature plant. In addition, Hopi determines antho-cyanin deposition in the scutellar and aleurone tissues of seeds,

Hopi 1b 1b 1a 1 6 1 6 1 following germination in the presence of light. In the presenceR-ch 1 6 1a 1 2 1 6 1 of the unlinked genetic factor Pl, it confers strong red (cherry)Sn 2 2 1a 1 1 1 2 2 pigmentation in the pericarp of the seed and, for this reason,R-nj 1 1 2 1 2 2 1 1 in the past it was termed r-ch:Hopi.R-g 1 6 2 2 2 2 2 2 Germination and anthocyanin extraction: Seeds were incu-r-r 2 2 1a 1 2 2 1 1 bated in sterile distilled water for 19 hr in rotating flasks keptR-st 1 tr 2 2 2 2 2 2 in darkness at 258, then plated in Plexiglas boxes on wet filterR-sc 1 1 2 2 2 2 2 6 paper and germinated for increasing time periods (from 0 toB-Peru 1 1 2 2 2 1 2 1 7 days) in the dark. Seedlings were then exposed to continuous

white light for 24 hr at 218 and subsequently transferred tor-D902 2 2 2 2 2 2 2 2darkness for an additional 48 hr at the same temperature

Data are taken from the following references: R-ch, Ker- conditions. Anthocyanins from individual excised scutellamicle (1997); Sn, Tonelli et al. (1991, 1994); R-nj, R-g, r-r, and were extracted with a fixed volume of 1% HCl in ethanol.R-sc, Kermicle (1997) and Tonelli (1994); R-st, Kermicle The extracts were centrifuged twice and their absorption de-(1997) and Consonni et al. (1997); B-Peru, Chandler et al. termined spectrophotometrically at 530 nm. Anthocyanin con-(1989) and Tonelli et al. (1994). All genotypes are in the centration is expressed as absorbance value at 530 nm perW22 background with C1. al, aleurone; sct, scutellum; per, scutellum. Mean values represent seven independent repli-pericarp; msc, mesocotyl; ant, anther; clp, coleoptile; 1, posi- cates. Standard errors of the means were below 5%.tive expression; 6, weakly pigmented; tr, traces of pigment; DNA and RNA analysis: Genomic DNA isolation and South-2, absence of pigment. ern analysis were performed as previously described (Tonelli

a With Pl or with exposure to light. et al. 1991). To perform a time course analysis of RNA accumu-b After germination in light. lation, seeds were germinated in the dark for different time

intervals (from 0 to 7 days) and then exposed to white lightfor 24 hr. Scutella were collected before and after light expo-sure. Cool white (F36T12/CW/HO) fluorescent lamps fromsion in scutellum is not enhanced by light and is limitedGTE Sylvania (Lighting Products Group, Danvers, MA) wereto the germination phase, whereas the accumulation ofused. For the analysis of RNA during seed maturation, imma-C1 transcript is under both developmental and lightture ears were harvested at 28 DAP and, after removal of husks,

control. We also present evidence on the role played scutella were excised from immature kernels. Scutella wereby Hopi and C1 in establishing the competence to induce also excised from dry seeds previously water imbibed for 1–2anthocyanin synthesis in scutella during germination hr. Total RNA was extracted from scutella by grinding in liquid

nitrogen and isolated as previously described (van Tunen etthrough control of the mRNA levels of structural andal. 1988). For Northern blot analysis, 30 mg total RNA wasregulatory genes involved in pigment accumulation.loaded on formaldehyde gels (Maniatis et al. 1982) and, afterelectrophoresis, blotted onto Biodyne nylon membrane (Pall).Hybridization and filter washing were performed as previouslyMATERIALS AND METHODSdescribed (Tonelli et al. 1991). RNA molecular weight mark-ers were from Bethesda Research Laboratory. Probes used forGenetic stocks: All seed stocks used in this study were inSouthern and Northern hybridization were: (i) pSn1.4, 1.4-the W22 background and were homozygous dominant for thekb PstI-EcoRI fragment derived from the 39 region of Sn cDNAa1, a2, c1, c2, bz1, and bz2 genes and homozygous recessive(Tonelli et al. 1991); (ii) pSn0.4, a 0.4-kb EcoRI-PstI fragmentfor the pl1 and b1 genes but differed in their r1 constitution.derived from the 59 portion of Sn cDNA (Tonelli et al. 1991);r1 allele stocks: Detailed descriptions of the origin, pheno-(iii) A1, a 700-bp BamHI fragment of A1 gene (Schwarz-type, and structural characteristics of the r1 alleles used inSommer et al. 1987); (iv) tub, a 1-kb EcoRI fragment from anthis study can be found in Dooner and Kermicle (1974,a-tubulin cDNA from maize (Dolfini et al. 1993); (v) restric-1976). Properties of the r1 alleles that are pertinent to thistion fragment length polymorphism (RFLP) probe, a 2.1-kbpresentation are outlined below: R-st determines spotted aleu-PstI fragment from bnl 7.49a, which maps 12.2 cM distal torone and green plant. This is a germinally and somaticallyr1 (kindly provided by Maize Genetics Cooperation-Stockunstable allele of r1 composed of four r1 genes in directCenter).orientation: one proximal r1 gene (Sc) producing a solid pur-

Genomic cloning and sequence analysis: For library con-ple pigmentation of aleurone and three distal r1 genes (Nc)struction, genomic DNA from homozygous Hopi plants wasthat determine near-colorless aleurone. The presence of adigested with HindIII and cloned into l NM1149 arms, astransposable element (I-R, Inhibitor of R) inserted in the Scpreviously described (Tonelli et al. 1991). The library wascoding region produces the spotted seed phenotype of R-stscreened with different Sn probes: pSn550 (a PCR fragment(Eggleston et al. 1995).derived from positions 2470 to 159 in the Sn promoter),r-D902: This symbol indicates an interstitial deletion involv-pSn0.4, and pSn1.4 (Tonelli et al. 1991). Positive recombi-ing a region of the long arm of chromosome 10 containingnant phages containing genomic fragments from Hopi (9 kb)the r1 locus (kindly provided by J. Kermicle). Plant and seedwere further purified. The 9-kb insert was subcloned into thetissues homozygous for the deficiency are totally devoid of

pigment (Alleman and Kermicle 1993). plasmid vector pBS [Stratagene (La Jolla, CA) cloning system]

326 K. Petroni et al.

and named pHopi9. Double-stranded DNA sequences were and control reactions in which reverse transcriptase was omit-ted revealed no residual DNA contamination. To quantifydetermined by the dideoxynucleotide chain termination

method (Sanger et al. 1977) using Sequenase (U.S. Biochemi- cDNA yield in each reaction, an aliquot was labeled with a[32P]dCTP and incorporated radioactivity was determined withcals, Cleveland) and employing oligonucleotides as primers.

The Sn promoter sequence was obtained by sequencing the a counter (model LS 6000 Beckman Instruments, Palo Alto,CA). The different samples of cDNA were then diluted to3.8-kb HindIII genomic clone containing the promoter and

the transcription start of Sn (Tonelli et al. 1991; Consonni obtain a uniform concentration. First-strand cDNA was usedas template for PCR amplification. Amplification reactionset al. 1993). The alignment was performed using the ClustalV

package (Higgins et al. 1992). The sequences of Hopi and Sn containing an aliquot of cDNA synthesized from 5 mg of totalRNA, 13 Promega polymerase buffer, 2.5 mm MgCl2, 200 mmpromoters are available in GenBank (Hopi: no. AJ251720; Sn:

no. X67619). Hopi cDNA sequence was determined by se- each dATP, dCTP, dGTP, and dTTP, 0.1 mm each primer,and 1 unit of Taq DNA polymerase (Promega, Madison, WI)quencing RT-PCR products (see RT-PCR analysis for experi-

mental procedure) obtained with the following sets of primers: were performed in a final volume of 50 ml. Samples wereoverlaid with 100 ml of mineral oil. After the first denaturationset 1, L1 (upstream primer 59-CGCGGAGGAGAGCTCC

TCCGGTT-39, position 121) and CT19 (downstream primer step (5 min at 948), the reaction mix underwent 20 cycles ofdenaturation at 948 for 1 min and 30 sec, annealing at 628 for59-TGGCGGCTGCAGCAAGCTGGCTC-39, position 1478);

set 2, HO2 (upstream primer 59-CAGCAGGCGCGTGAT 1 min and extension at 728 for 1 min. A final extension at 728for 15 min was performed to complete the reaction. A set ofGGCGCTT-39, position 1369) and OB2 (downstream primer

59-GGTGCTCGGCTGACCAAGT-39, position 1999); set 3, primers specific for the orange pericarp-1 (orp-1) gene, whichencodes the b subunit of tryptophan synthase (Wright et al.CT5 (upstream primer 59-GTCAATCCTCTGCATCCCG-39,

position 1906) and HO6 (downstream primer 59-AGCTCC 1992), was used to further standardize the concentration ofthe different samples. orp-1 was chosen as reference becauseTTGAGGTAGGCT-39, position 11781); set 4, HO3 (upstream

primer 59-ATCGAGGAGTTCTACAGC-39, position 11255) the abundance of its mRNA is comparable to that of R-sc and A1(Procissi et al. 1997). orp-1 specific sequences were amplifiedand CT10 (downstream primer 59-CATCTTTGCTTC

GATCCC-39, position 12296); set 5, oR31 and oR32 (position using the following primers: upstream primer, 59-AAGGACGTGCACACCGC-39, and downstream primer, 59-CAGATACAGA12129 and 12531, respectively; see RT-PCR analysis for

primer sequence). The positions of primers are given relative ACAACAACTC-39. The length of the amplified product was207 bp. To ensure that amplification reactions were withinto the Sn cDNA (Consonni et al. 1992). The sequence of Hopilinear ranges, the reactions were carried out for 20 cycles.cDNA was confirmed by sequencing the exons of the HopiThe PCR products were fractionated on 1.2% agarose gelsgenomic clone. The sequence of Hopi cDNA is available inand transferred onto Biodyne nylon membranes (Pall) andGenBank (Hopi : no. AJ251719).hybridized with random primed 32P-labeled fragments of orp-1.Transient transformation assay: The p35SC1 plasmid con-Hybridization signals were quantified by scanning the autora-tains the 2.1-kb EcoRI C1 cDNA (Paz-Ares et al. 1987) cloneddiographic films with a Umax densitometer and cDNA samplesinto the EcoRI site of the plant expression vector pRT101were standardized accordingly. For mRNA detection of thecarrying the CaMV35S promoter (Topfer et al. 1987), andgenes under analysis, the following specific primer sets werethe genomic clone pHopi9 was purified by using a Midi-Prepused: for Hopi, oR31 (upstream primer 59-ATGGCTTCATGGKit (QIAGEN, Chatsworth, CA). Homozygous r-D902 seedsGGCTTAGATAC-39) and oR32 (downstream primer 59-GAATwere surface-sterilized for 10 min in 5% sodium hypochloriteGCAACCAAACACCTTATGCC-39); for A1, A1 (upstreamand kept in sterile water at 258 for 16 hr. After removal of theprimer 59-TTCTCGTCCAAGAAGCTCCAGGA-39) and A2pericarp, seeds were allowed to germinate on MS medium(downstream primer 59-CAATTCGTTGAACATGGAAGTcontaining 1% sucrose and 7% Bacto Agar (Difco Labora-AAG-39); for C1, PL6 (upstream primer 59-TCGGACGACTGCtories, Detroit) at 258 for 3 days prior to bombardment. SeedsAGCTCGGC-39) and Ac1 (downstream primer 59-CACCGTGCwere maintained in the dark during germination and afterCTAATTTCCTGTCCGA-39). The size of the amplifiedparticle bombardment with the Biolistic PDS-1000/He particleproducts is 403 bp for oR31/oR32, 285 bp for A1/A2, andgun (Bio-Rad, Hercules, CA). For each preparation of six313 bp for PL6/Ac1. For each set of primers, pilot experimentsshots, 3 mg of p35SC1 and 3 mg of pHopi9, or 6 mg of p35SC1were performed to determine the melting temperature andalone, or 6 mg of pHopi9 alone in a final volume of 5 ml werethe number of cycles needed to ensure that amplificationadded to 50 ml of 60 mg/ml 1-mm gold microparticles (Bio-reactions were within the linear range (Procissi et al. 1997).Rad) in 50% glycerol, 50 ml of 2.5 m CaCl2, and 20 ml of 0.1 mThe PCR products were then separated on agarose gelsof spermidine-free base. After vortexing for about 3 min, the(1.2%), transferred onto Biodyne nylon membranes (Pall),particles were centrifuged and the supernatant was removed.and hybridized with random primed 32P-labeled fragments.Microparticles were washed twice with 70% ethanol and thenThe Hopi PCR products were hybridized with a 1.4-kb PstI-resuspended in 50 ml of 100% ethanol, followed by the spottingEcoRI fragment of Sn cDNA (Tonelli et al. 1991); the C1of 6 ml of coated particles onto Macrocarrier disks (Bio-Rad).products with a 1.4-kb XhoI-EcoRI of the C1 cDNA (Paz-AresDNA-coated gold microparticles were accelerated by the shocket al. 1987), and the A1 products with a 700-bp BamHI fragmentwave generated by the bursting of a rupture disk at z900 psiof the A1 gene (Schwarz-Sommer et al. 1987).of He gas. Bombarded seeds were kept in darkness for 72 hr

at 228 and then subjected to visual inspection using a dissectionmicroscope.

RT-PCR analysis: Reverse transcriptase polymerase chain RESULTSreaction (RT-PCR) was used to detect Hopi, A1, and C1 genetranscripts. Assays for transcripts were also performed. First- Hopi consists of a single gene nonallelic to the r1 genestrand cDNA was synthesized with an oligo(dT) primer from complex: The Hopi gene controls anthocyanin accumu-total RNA extracted from scutella of germinating seeds (see lation in an extensive number of plant and seed tissuesRNA isolation). The primer used was a 35-base oligonucleotide

(see Table 1), including pericarp, aleurone and scutel-with 17dT residues and an adapter (59-GGGAATTCGTCGAlum in the kernel, root, mesocotyl, and leaf blade inCAAGC-39) sequence (Frohman 1990). All RNA samples

were treated with DNase (1 unit/mg) before cDNA synthesis, the seedling, midrib, ligule, leaf blade, and anthers in

327Developmental Expression of Hopi

the mature plant. The pigmentation of these tissues The Hopi phenotype is strictly correlated to a 9-kbgenomic fragment: To establish whether Hopi consists ofcould be due to the activity of a complex locus whose

different genetic elements could account for the tissue- a single gene at the molecular level, 104 plants resultingfrom the above cross were individually analyzed byspecific pigmentation, as in the case of the R-r locus.

However, our efforts to separate genetically the plant Southern analysis (Figure 1B). Since the Hopi geneshares DNA similarity with both Sn and r1 genes (Ton-and seed phenotypic effects associated with Hopi have

so far failed. Nonetheless, we could not discount the elli et al. 1991), we used a 0.4-kb EcoRI-PstI fragmentderived from the 59 end of Sn cDNA as a probe (Tonellipossibility that Hopi has a compound structure, since

recombination in the region distal to r1 is reduced in et al. 1991). The results showed that the 5- and 3.5-kbHindIII fragments are associated with R-st (Figure 1B,Hopi-bearing chromosomes due to the presence of a

heterochromatic knob (K10) (Racchi and Gavazzi lanes 1 and 9), whereas a 9-kb HindIII fragment coseg-regates with the Hopi phenotype (lanes 1 and 3–7).1988). To define the molecular structure of Hopi, we

produced R-st Hopi/r-D902 plants, where R-st Hopi repre- In fact, recombinant progeny carrying Hopi in cis withr-D902 (11 individuals; Figure 1A, class 4) retain thesents a crossover derivative devoid of K10, selected from

the progeny of R-st/Hopi plants, and r-D902 is a viable 9-kb HindIII fragment (Figure 1B, lanes 3–7) and, inagreement, loss of this fragment in the R-st/r-D902 re-deletion comprising the entire r1 locus (Alleman and

Kermicle 1993). R-st Hopi/r-D902 plants were then used combinant plants (lane 9) is associated with absence ofpigmentation in aleurone and scutellum after germina-as females in testcrosses to male plants homozygous for

r -D902. Since a diagnostic phenotype of Hopi is the tion in light and in all other tissues normally pigmentedby Hopi (13 individuals; Figure 1A, class 3). We includedinduction of full pigmentation in the scutellum and

irregular aleurone pigmentation following germination in the analysis DNA extracted from the original homozy-gous Hopi line, which contained a 9-kb HindIII fragmentin light, seeds derived from the above cross (stippled

and colorless in a 1 to 1 ratio) were germinated in comigrating with that of r-D902 Hopi/r-D902 recombi-nant DNA (lane 8). As expected, no r1-homologousdarkness for 48 hr, exposed to continuous white light

for 24 hr, and then scored for aleurone and scutellum genomic fragments could be linked to r-D902, while the4-kb HindIII fragment, weakly detected in all samples,pigmentation (Figure 1A). Four phenotypic classes were

identified: two parental classes, one exhibiting red scu- was attributable to the recessive b1 gene. To confirmthat the Hopi gene resides on a single HindIII genomictellum and patches of color in the aleurone superim-

posed on a stippled background (Figure 1A, class 1, R-st fragment, we used a DNA probe of 1.4 kb derived fromthe 39 end of Sn cDNA to hybridize the same SouthernHopi/r-D902) and the other one completely devoid of

pigment (class 2, r-D902/r -D902), and two recombinant blot filter. Again, a 9-kb HindIII fragment cosegregatedwith the Hopi phenotype and was detectable only inclasses showing, respectively, stippled aleurone (class 3,

R-st/r -D902) and one red pigmentation in scutellum the R-st Hopi/r-D902 parental class, in the r-D902 Hopi/r-D902 recombinant class, and in the homozygous Hopiand patches of color on a colorless background in aleu-

rone (class 4, r -D902 Hopi/r -D902). Seedlings were then DNA (data not shown). These results suggest that Hopiconsists of a single gene residing on a single 9-kb HindIIIgrown and the mature plants scored to establish whether

the tissue-specific pigmentation determined by the Hopi fragment.To determine whether the Hopi gene is distal to r1gene depends on discrete separable genetic elements

(Figure 1A). Presence of leaf, anther, and root pigmen- as are the other displaced r1 genes, Sn and Lc, an RFLPanalysis was performed on individuals of the cross re-tation was observed in class 1 (R-st Hopi/r -D902) and

class 4 (r -D902 Hopi/r -D902). The progeny from these ported in Figure 1 using the probe bnl 7.49a, whichmaps 12.2 cM distal to r1 (Burr et al. 1988). This probeplants were germinated and the pattern of pigmentation

was analyzed. Progeny of class 4 (r -D902 Hopi/r -D902) distinguishes between the distal region of the R-st Hopichromosome and the r-D902 one (Figure 2, lanes 1individuals always showed colorless aleurone in dry ker-

nel and, after germination in light, pigmentation in and 3). The R-st Hopi/r-D902 parental plants used intestcrosses to r-D902/r-D902 were heterozygous for thescutellum and aleurone and all other tissues usually

pigmented in Hopi lines in a 3 to 1 ratio. Seeds derived RFLP marker, carrying both a 4.5- and a 7-kb SacI frag-ment (lane 2). As expected, all nonrecombinant prog-from R-st/r -D902 plants (class 3) showed stippled and

colorless aleurone in a 3 to 1 ratio and no pigmentation eny tested have the characteristic RFLP bands of theparental plants (lanes 4 and 7). The r-D902 Hopi/r-D902in seedling tissues (data not shown). The results indicate

that both classes (class 1 and class 4, Figure 1A) and recombinants retain both the 4.5-kb fragment of thedistal region of the R-st Hopi chromosome and the 7-kbtheir progeny show the phenotype expected on the as-

sumption of Hopi being a single gene responsible for fragment of the r-D902 chromosome (Figure 2, lane5), while R-st/r-D902 plants only retain the RFLP bandplant and seed phenotypic effects. These data, together

with the results obtained from several identical crosses characteristic of the r-D902 chromosome (Figure 2, lane6). Southern analysis using an RFLP probe specific for(Table 2), also indicate that R-st and Hopi are separate

genes lying 4.5 cM apart. the proximal region indicates no exchange of proximal

328 K. Petroni et al.

Figure 1.—Progeny obtained by mating a R-st Hopi/r-D902 heterozygous female to an r-D902/r-D902 male. r-D902 is indicatedin the figure as rD. (A) The first selection was made on the basis of dry seed pigmentation: class 1 and 3, showing a stippledaleurone, had received the R-st allele while the presence of a colorless aleurone was attributed to the r-D902 allele (class 2 and4). A second classification was based on scutellum and aleurone pigmentation after germination in the light: class 1 and 4,showing red scutellum and aleurone, received the Hopi gene. Southern analysis (B) allowed the genotypic structure of eachindividual to be reconstructed: the presence of HindIII bands that can be assigned to Hopi and R-st is indicated. The inferredchromosome structure is shown schematically and different genotypes are indicated as P (parental) and CO (crossover). Hopitraits in the mature plant were checked in a sample of individuals belonging to each class. 1, presence; 2, absence; n, numberof individuals tested. (B) Southern blot analysis. Genomic DNA was extracted from single seedlings and digested with HindIII.Filter was probed with a 0.4-kb EcoRI-PstI fragment derived from Sn cDNA. Lanes 3–7 contain DNA from recombinant Hopiplants. (C) Restriction map of the Hopi gene. The restriction map was generated by digestion of the genomic clone with thefollowing restriction enzymes: B, BamHI; H, HindIII; K, KpnI; P, PstI; R, EcoRI; X, XbaI. The hybridizing regions of the three PstIfragments (a, b, c) of Sn cDNA are indicated above the Hopi genomic clone.

329Developmental Expression of Hopi

TABLE 2

Progeny classes recovered from the testcross of R-st Hopi/r-D902 females with r-D902 males andestimated recombination frequency between R-st and Hopi

Progeny classesParentalgenotype Population R-st Hopi r-D902 R-st Hopi Rec %

R-st Hopir-D902 7657 3611 3698 167 4.5181

Rec %, recombination frequency between R-st and Hopi.

markers (data not shown). These results demonstrate Sn cDNA clone, thus confirming that Hopi resides on asingle HindIII genomic fragment (Tonelli et al. 1991).that Hopi is distal to r1 and suggest that Hopi, Sn, and

Lc may be alleles of the same displaced r1 homologous Sequence analysis of Hopi from positions 21489 to1442 (numbering as for the Sn gene; Consonni et al.locus.

Genomic cloning and sequence analysis of Hopi: Sev- 1993) revealed 95% nucleotide identity between Hopiand Sn. Sequence diversity mainly consisted of singleeral members of the r1/b1 gene family, such as b1, Lc,

and Sn, have been cloned by cross-hybridization using base substitutions, although some small insertions anddeletions were also revealed throughout the sequencer1 sequences (Chandler et al. 1989; Ludwig et al. 1989;

Tonelli et al. 1991). Using a similar approach, a HindIII (Figure 3).In the 59 untranslated leader sequence of the Sngenomic library from homozygous Hopi DNA was

screened with different Sn probes. Eighteen recombi- cDNA five ATG triplets preceding the actual start codonhave been previously described (Consonni et al. 1993).nant phages were isolated and, on the basis of their

size and restriction map, they were divided into two The first and the fourth of these are the start sites fortwo small upstream open reading frames (uORFs) ofgroups, one containing 10 9-kb HindIII fragments repre-

senting the Hopi gene (Figure 1C) and the other con- 38 and 15 residues, respectively. Compared to the Snleader region, a triplet insertion (position 172) in thetaining 8 3.5-kb fragments representing the recessive b1

gene (not shown). As shown in Figure 1C, the 9-kb Hopi sequence that results in an additional codon and asingle deletion (position 1148) that causes a frameshift,genomic fragments showed three regions that cross-

hybridized with three PstI fragments from the full-length together result in a shorter first uORF of 34 amino acids(Figure 3). The complete cDNA sequence confirmedthat the major open reading frame (ORF) begins at thefifth ATG start codon at nucleotide position 1379. Itencodes a putative protein of 611 residues, 98% identi-cal to SN (Consonni et al. 1992). Compared to the SnORF, four deletions are present in the Hopi cDNA, threeof which result in single amino acid deletions, D 255,A 379, and N 500, and one in a two amino acid deletion,G 417 and T 418 (Figure 4). Twenty-two nucleotidesubstitutions between Hopi and Sn were also foundthroughout the coding region; however, only 9 of themled to an amino acid substitution. Seven of these aminoacid changes are conservative and among them four areexclusively found in the HOPI protein, while B-PERUor R-S share the others. Two other amino acid substitu-tions are not conservative; the first one (N 418 insteadof K in SN) is present in all R1 proteins except SN,while the second one (G 564 instead of S) is presentexclusively in HOPI.

To determine whether the Hopi gene cloned was capa-ble of activating anthocyanin pigmentation in maizetissues, the 9-kb HindIII genomic fragment was tested

Figure 2.—RFLP analysis of homozygous R-st Hopi (lane 1) in transient transformation assays by microprojectileand r-D902 plants (lane 3) and of progeny (lanes 4–7) from

bombardment of colorless r-D902 germinating seeds.cross of R-st Hopi/r-D902 (lane 2) to r-D902/r-D902 (lane 3).Cotransformation of this Hopi genomic fragment, to-DNA was digested with SacI and hybridized with the RFLP

probe bnl 7.49a. r-D902 is indicated in the figure as rD. gether with a C1-expressing plasmid, p35SC1, restored

330 K. Petroni et al.

Figure 3.—Alignment of Hopi and Sn gene sequences from 2698 to 1442 obtained from sequencing Hopi genomic cloneand pSn3.8H (Consonni et al. 1993). Arrow indicates the Sn transcription start. The ATGs in the leader sequences are underlinedwhile stop codons are marked with an asterisk. The actual start codon is double underlined. White letters indicate identicalnucleotides. Gaps are indicated by dashes.

pigmentation in cells of scutellar node, mesocotyl, cole- for 24 hr. The pigment content was measured followingan additional 48 hr of darkness to allow anthocyaninoptile, and root (Figure 5). No pigmented cells were

observed when transformations were performed with synthesis to be completed. The results in Figure 6 (bot-tom) show that a peak in pigment content is reachedthe C1-expressing plasmid alone (data not shown). This

result demonstrates that the 9-kb fragment cloned in- when seeds are exposed to light after 48 hr of germina-tion in darkness (Figure 6B, bar 3). Seeds germinatedcludes the entire Hopi gene and that HOPI protein

can substitute for the r gene products to activate the in darkness for longer periods (bars 4–7) show a declinein the response to light, leading to almost no inductionanthocyanin biosynthetic pathway in different seedling

tissues. after 5 days of dark germination (Figure 6B, bar 6).Thus, competence to respond to light is maximal duringEffect of light and germination phase on anthocyanin

accumulation in the presence of Hopi: In contrast to the first four days of dark growth but is lost if darknessis prolonged. Aleurone differs from scutellum in itsother r1 genes, e.g., R-sc, which promotes pigment pro-

duction in seed tissues during maturation on the ear, requirement for light, since faint patches of aleuronepigmentation are observed even in the absence of lighthomozygous Hopi seeds are completely devoid of pig-

ment at maturity but upon germination show a photoin- irradiation after 3 days of germination. However, anenhancing effect of light irradiation on aleurone pig-ducible anthocyanin accumulation in the scutellum and

aleurone tissues of the seed. To establish more precisely mentation has also been observed (data not shown).Expression pattern of Hopi and A1 during germina-how light and germination interact to trigger anthocya-

nin production in scutellar tissues, water-imbibed Hopi tion in presence of light: To ascertain whether the com-petent phase for anthocyanin accumulation in responseseeds were allowed to germinate in darkness from 1 to

7 days and were then exposed to continuous white light to light was correlated with the expression pattern of

331Developmental Expression of Hopi

Figure 4.—Alignment of the complete protein encoded by Hopi, Sn (Consonni et al. 1992), Lc (Ludwig et al. 1989), R-S(Perrot and Cone 1989), and B-Peru (Radicella et al. 1991). White letters indicate identical nucleotides. Gaps are indicatedby dashes.

genes responsible for anthocyanin accumulation, we steady-state levels of the Hopi mRNA is closely correlatedto the developmental stage of the germinating seed butperformed Northern blot analysis, in which the tran-

script levels of the regulatory Hopi gene and of the A1 not to the induction of anthocyanin biosynthesis bylight. In contrast to Hopi, the transcript levels of the A1structural gene were tested. Imbibed Hopi seeds were

germinated in the dark for increasing time intervals and structural gene were strictly light dependent. In fact,A1 mRNA could be detected only after light exposurethen exposed to white light for 24 hr. Total RNA was

extracted from dark-grown and light-exposed scutella. of the germinating seeds (Figure 6B). The maximumlevel of induction was reached when 2 days of darkmRNA from unpigmented scutella excised from Hopi

developing seeds close to maturity (28 DAP) and dry germination preceded light irradiation (Figure 6B, lane3), thus following the same time course of transcriptkernels were also included in the analysis. Figure 6A

shows steady-state levels of mRNA in Hopi scutella during accumulation as the Hopi gene. Anthocyanin contentwas therefore precisely correlated to A1 gene expressiondark germination, while Figure 6, B and C, shows the

transcript levels in response to light and during seed and, in turn, A1 gene expression was correlated tochanging levels of Hopi transcript in the scutella duringmaturation, respectively. Increasing times of dark germi-

nation were associated with a transient increase in the germination (Figure 6B, lanes 1–5). However, expres-sion of Hopi was not sufficient to determine the expres-amount of Hopi mRNA that reached a peak after 3 days

of dark germination (Figure 6A, lane 3), while during sion of A1 during germination in darkness (Figure 6A).The absence of the Hopi transcript in dry seeds and inseed maturation and in the dry seed the Hopi transcript

was absent (Figure 6C). Following light irradiation the immature kernels could account for the dependenceof anthocyanin synthesis in scutella upon germinationexpression pattern of Hopi was similar to that observed

during the dark growth, showing an increase of tran- (Figure 6C).Analysis of the C1 expression pattern in scutellumscript level up to 3 days of germination (Figure 6B, lanes

1–3) and a decrease in the subsequent stages. These during germination: To establish whether the activity ofa MYB counterpart might be limiting the light-depen-results clearly indicate that the transient increase in the

332 K. Petroni et al.

dent pigmentation of scutella, we analyzed the expres-sion pattern of the seed-specific C1 gene in these tissues.Since MYB-related genes, including C1, exhibit ex-tremely low transcript abundance (Procissi et al. 1997),we performed a gene-specific RT-PCR analysis usingcDNAs derived from the same total RNA samples ana-lyzed in the previous Northern experiments. The steady-state levels of A1 and Hopi mRNA were also reassayedby RT-PCR to confirm the Northern data presented inFigure 6. As previously shown, the level of A1 steady-state mRNA, completely absent in scutella excised fromdry seed and dark-germinated scutella, is strongly in-duced by light, particularly after 2 days of dark growth(Figure 7B, lane 3). The time course of Hopi mRNAaccumulation is quite similar to that observed by North-ern analysis, showing a peak after 3 days in dark-germi-nated scutella (Figure 7A, lane 3) and a similar expres-sion pattern after light irradiation (Figure 7B). The lightinducibility of the A1 structural gene appears to resultfrom the activation of C1. In fact, the C1 transcript,which is completely absent in scutella excised from dark-grown seeds and barely detectable in dry seed, wasstrongly induced by light irradiation (Figure 7). Themaximum level of C1 induction was reached at the startof germination in light (Figure 7B, lane 1), whereas thelight responsiveness of C1 was less effective when lightirradiation was preceded by dark growth (Figure 7B,

Figure 5.—Transient expression analysis of Hopi in germi- lanes 2–5). Furthermore, the induction of the C1 regula-nating r-D902 seeds. (A) Coleoptile of colorless r-D902 seedstory gene after 24 hr of white light is clearly not influ-bombarded with p35SC1 and pHopi9 displaying purple spotsenced by the developmental stage within the first 4 daysindicative of restored anthocyanin synthesis. B is an enlarge-

ment of A. of dark germination, since the C1 transcript level didnot change until the subsequent stages of growth werereached. Nonetheless, the lack of C1 gene expression

Figure 6.—Gene expression in Hopi scutella. (Top)Hopi seeds were germinated in the dark from 1 to 7days (A) and then exposed to white light for 24 hr(B). Dashed boxes represent 24 hr of dark, while openboxes represent 24 hr of white light. Scutella fromseeds at 28 DAP and dry seeds were also included inthe analysis (C). Total RNA was extracted from scutellaas described in A, B, and C; Northern analysis wasperformed using 30 mg of total RNA per lane. The blotswere first probed with a 1.4-kb PstI-EcoRI fragment ofthe Sn cDNA (Tonelli et al. 1991) to reveal the Hopitranscript, then stripped and subsequently hybridizedwith a 700-bp BamHI fragment of the A1 gene(Schwarz-Sommer et al. 1987). Equal loading of thetotal mRNA was verified by hybridization of the blotswith a maize tubulin probe, tub (Dolfini et al. 1993).The sizes in kilobases of transcript are indicated onthe right. (Bottom) Effect of light and germinationon pigment accumulation. Anthocyanin accumulationin the scutellum was measured in each treatment afteran additional 48 hr of darkness. Values are expressedas absorbance at A530 per scutellum. Mean values repre-sent seven independent replicates. Confidence inter-vals at 95% are shown.

333Developmental Expression of Hopi

quency of R-ch mutants lacking one or more tissue-spe-cific genetic determinants was due to the presence ofa heterochromatic knob (K10) in the long arm of chro-mosome 10, which suppresses both genetic recombina-tion and the chiasma frequency (Rhoades 1942). Asimilar compound structure has also been postulatedfor the Hopi gene (Racchi and Gavazzi 1988). Ourgenetic analysis has been performed using a Hopi linedevoid of K10 and the recombinational data obtained,together with the RFLP analysis, show that Hopi consistsof a single genetic element, lying 4.5 cM distal to r1 onthe long arm of chromosome 10. In addition, through

Figure 7.—RT-PCR analysis of mRNA accumulation of an-genetic and molecular analysis we have demonstratedthocyanin structural and regulatory genes in Hopi scutellathat the broad pigmentation pattern conditioned byderived from seeds germinated from 1 to 7 days. cDNA wasHopi does not depend on the presence of multiplemade from total RNA extracted from scutella of dry seeds (0)

and from seeds germinated in the dark (A) and then exposed closely associated genetic units but on a single geneto continuous white light for 24 hr (B) as in Figure 6. Specific located in a 9-kb genomic fragment.primers for the Hopi, C1, and A1 genes were used to amplify the

Nucleotide sequence analysis showed that Hopi sharecDNA (see materials and methods). Amplifications werea high degree of similarity with Sn, both in the tran-carried out for 20 cycles. The amplification of the orp-1 tran-scribed region (98% identity) and in the promoter se-script was used as an internal control. The amplified band

corresponding to the Hopi transcript is 403 bp, 313 bp for the quence up to 21489 (95% identity). MicroprojectileC1 transcript, 285 bp for the A1 transcript, and 207 bp for the delivery of the Hopi genomic clone to colorless germi-orp-1 transcript. The blots were hybridized with the different

nating embryos results in pigmented cells, thus estab-probes (see materials and methods) and exposed to X-raylishing that the Hopi gene, cloned in this study, containsfilms for 3–4 hr, except for C1, which was exposed for 5 days.a functional coding region able to complement r1 regu-latory mutation. Furthermore, the purple pigmented

when dark growth is prolonged (Figure 7B, lanes 6–7) cells observed in scutellar nodes, mesocotyls, and cole-is reflected in the absence of anthocyanin pigmentation, optiles following the delivery of the Hopi gene suggestsuggesting that the developmental competence of scu- that the 9-kb clone contains cis-acting sequences respon-tella to respond to light is also determined by the devel- sible for the expression of the gene in seedling tissues.opmental window of expression of C1. The RT-PCR We have shown that sequence diversity between Hopianalyses also indicate that the simultaneous presence and Sn mainly consists of single base substitutions dis-of both C1 and Hopi mRNAs is necessary to achieve A1 tributed throughout the promoter sequence (Figure 3)activation in scutella. and the coding region (Figure 4). Compared to the SN

protein, five amino acid deletions and nine amino acidsubstitutions were found in the HOPI protein. Most

DISCUSSIONamino acid substitutions are conservative and, if not,are shared by the other R1 proteins. Similarly, mostThe r1/b1 gene family consists of a number of genes

that independently control the tissue-specific distribu- deletions are shared by other R1 proteins, except dele-tion of amino acid N500, which is located in the basiction of anthocyanin in plant and seed tissue. In this

study we report the cloning and characterization of a domain outside the bHLH motif.Sn and Hopi share some territories of expression, suchnovel member of the r1/b1 family.

The Hopi gene maps to chromosome 10L, the same as pericarp, mesocotyl, and particularly leaf, where theydisplay exactly the same temporal and cell-specific ex-chromosome arm of the r1 locus. It conditions antho-

cyanin deposition in aleurone, scutellum, mesocotyl, pression pattern and condition strong coloring in themidrib and ligule. However, Hopi conditions anthocya-leaves, and anthers and interacts with a dominant Pl

allele to produce a “cherry” phenotype in the pericarp, nin pigmentation in scutellum and aleurone, which aretissues typically pigmented by r1 alleles. Additionally,thus representing a very broad specification of pigmen-

tation pattern among all the r1 genes so far analyzed. Hopi significantly differs from these r1 alleles in thetiming of its control of anthocyanin synthesis in seedThe wide variety of tissues pigmented and the presence

of a strong red pigmentation in the pericarp of the seed tissues, since pigmentation is induced only during ger-mination in the presence of light. It would not be sur-in the presence of the Pl gene are features common to

other r1 genes, termed “cherry” for this reason (Table prising if the promoter sequence diversity revealed byour analysis is responsible for the different expression1). Genetic studies indicated that the R-cherry genes (R-

ch) are composed of a compound structure consisting of patterns conditioned by Hopi and Sn. However, it is alsopossible that Hopi is located near a regulatory elementfour genetic elements, each conferring a tissue-specific

pigmentation (Sastry 1970). The extremely low fre- strongly influencing its expression, providing new tissue-

334 K. Petroni et al.

specific expression in the absence of relevant sequence opmental competence of germinating seeds to induceanthocyanin production in scutella results from thechanges in its promoter.

Hopi seeds are completely devoid of pigments at the combination of the light-inducible expression of C1 andthe developmental regulated expression of the Hopi reg-end of seed maturation. However, if seeds are germi-

nated in the light, anthocyanins accumulate in scutel- ulatory gene.The r1/b1 gene family consists of a number of geneslum and aleurone. We have observed that anthocyanin

accumulation in scutella occurs only if germinating whose products perform similar roles in the control ofanthocyanin synthesis in different maize tissues. Com-seeds are transferred to light between the first and the

fourth day of germination (Figure 6B). It has been pre- parison of the Lc, Sn, R-S, and B-Peru cDNAs has shownthat the deduced protein sequences share .80% aminoviously shown that the different developmental compe-

tences of the pericarp and aleurone layers of immature acid identity (Consonni et al. 1993). This functionaland sequence similarity has led to the hypothesis of aseeds to respond to light result from the expression

pattern of bHLH and MYB regulatory genes at different common evolutionary origin of the r1/b1 gene familycomponents from a single ancestral gene. A detailedstages of seed development (Procissi et al. 1997). In

the aleurone, the R-sc transcript was detectable both at molecular analysis of the r1 gene family orthologs indifferent plant species has confirmed that the r1 and14 and 30 DAP, but the steady-state level of mRNA was

slightly higher at 30 DAP, when the aleurone layer is b1 genes have arisen from a common ancestral genethrough a polyploidization event, which presumably oc-more competent for light-dependent pigment accumu-

lation. In both developmental stages, R-sc expression is curred at the time of divergence of Zea mays from Sor-ghum (Purugganan and Wessler 1994). The prolifer-not enhanced by light irradiation, whereas C1 shows

light inducibility. However, the extremely low abun- ation of R sequences at and near the r1 locus on thelong arm of chromosome 10 is probably the result ofdance of C1 transcript at 14 DAP was found to be the

limiting factor for conferring the developmental compe- intrachromosomal duplicative events of independentorigin (Robbins et al. 1988). The view that the complextence of the aleurone to light responsiveness (Procissi

et al. 1997). The R-sc and C1 expression pattern in aleu- r1 alleles and the displaced r1 genes on chromosome10 represent more recent duplication events is sup-rone cells is similar to that observed for Hopi and C1 in

scutella during germination in the presence of light. As ported by the occurrence of the variable r1 constitutionof different geographic alleles as well as by the presencefor R-sc, Hopi gene expression in scutellum is influenced

by the developmental stage, but not by light. Its tran- of Lc or Sn confined to specific accessions (Coe et al.1988). In this scenario Hopi might be considered a novelscript is detectable only during germination with a simi-

lar accumulation pattern both in darkness and in light. allele of the same displaced r1 homologous locus. Inaccordance with this view is the finding that Sn, Lc, andThough the actual mechanism of regulation of Hopi

in germinating seeds remains to be determined, the Hopi cDNA sequences are more closely related to r1than r1 to b1 (Figure 4).absence of the Hopi messenger appears to be the factor

limiting anthocyanin pigmentation in developing seeds. The functional interchangeability of the R1 proteinshas led to the hypothesis that molecular divergence,In contrast to Hopi, the expression of C1 in scutellum

is clearly light inducible but is also under developmental following the duplication events, more significantly af-fected the gene promoters than coding regions, so thatcontrol. It has been previously shown that C1 is abun-

dant in embryo tissues of 22 DAP immature kernels, the diverse pigmentation pattern controlled by each r1gene reflects differences in regulatory sequences ratherwhen anthocyanins begin to appear in the aleurone

(McCarty et al. 1989). The activation of the C1 gene than their gene products (Ludwig et al. 1990; Radi-cella et al. 1992). A series of genetic tests has revealedis dependent on the vp1 regulatory gene and ABA,

which are known to act through cis-acting elements pres- that the wild progenitor of maize, teosinte, possessesfunctional alleles at both r1 and c1 regulatory loci, butent in the C1 promoter (Hattori et al. 1992). The

presence of C1 mRNA has also been detected at the predominantly alleles not expressed during kernel de-velopment (Hanson et al. 1996). A dominant functionalend of seed maturation in 35 DAP kernels (Paz-Ares

et al. 1986). Our analysis has shown that the C1 transcript allele of the r1 locus able to develop pigment duringseed maturation was not found in teosinte populations,is present in scutellum of dry seed and that the C1

transcript is not maintained after the onset of germina- although the possibility that it is present, at very lowfrequency, cannot be excluded. Similarly, at the c1 locus,tion (Figure 7A). Light inducibility in the aleurone has

been previously shown for germinating seeds of C1 mu- a dominant C1 allele was found in a single teosintepopulation, perhaps representing a more recent intro-tants, such as c1-p (Scheffler et al. 1994). Our results

clearly indicate that in the dark the absence of C1 tran- gression from maize. It has been proposed that purplekernels evolved by changes in cis-regulatory elementsscripts inhibits anthocyanin pigmentation in germinat-

ing seeds. We conclude that the expression pattern of involving both regulatory loci, followed by human selec-tion so that seed pigmentation became predominantthe Hopi gene accounts for the germination-dependent

anthocyanin synthesis in scutellum, whereas the devel- (Hanson et al. 1996). The origin of the Hopi gene can

335Developmental Expression of Hopi

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