Springer 2006
Plant Molecular Biology (2006) 60:185–199 DOI 10.1007/s11103-005-3568-1
Comparative structural and functional characterization of sorghum and maize duplications containing orthologous Myb transcription regulators of 3-deoxyflavonoid biosynthesis Jayanand Boddu1,3, Cizhong Jiang2,4, Vineet Sangar1, Terry Olson2, Thomas Peterson2 and Surinder Chopra1,* 1
Department of Crop & Soil Sciences, Pennsylvania State University, PA, 16802, USA (*author for correspondence; e-mail
[email protected]); 2Department of Genetics, Development and Cell Biology, Iowa State University, Ames IA, 50011, USA; 3Department of Agronomy and Plant Genetics, University of Minnesota, St Paul MN, 55108, USA; 4Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor NY, 11724, USA Received 22 December 2004; accepted in revised form 28 September 2005
Key words: 3-deoxyflavonoids, anthocyanins, flavonoid, gene structure, sorghum, maize, Myb, pericarp, phlobaphenes, phytoalexins
Abstract Sequence characterization of the genomic region of sorghum yellow seed1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.
Introduction Comparative structural genomics and characterization of genic regions among cereal grass species have provided knowledge of sequence divergence and its relationship to function (Moore et al., 1995; Song et al., 2001; Bennetzen and Ramakrishna, 2002). Recent comparisons of large scale sequencing efforts have also highlighted the functional com-
plexity and redundancy generated by duplicated genes and segmental duplicated genomes (Bowers et al., 2003; Paterson et al., 2004). In contrast to exhaustive large genome sequencing studies on evolutionary relationships, little is known in terms of direct comparison of functionality of orthologous genes or allelic regions from diverse races (Fu and Dooner, 2002; Ramakrishna et al., 2002; Ilic et al., 2003). Regulatory genes of the biosynthetic
186 pathway of flavonoid pigments are interesting model systems to study functional diversity derived from gene duplications and structural polymorphisms (Ludwig et al., 1990; Hanson et al., 1996; Zhang et al., 2000; Dias et al., 2003). It is well documented that tissue specific 3-hydroxyflavonoid or anthocyanin pigment biosynthesis in maize is regulated by the combined action of two groups of genes belonging to the Myb and Myc class of transcription factors, including the duplicated c1 or pl1 and r1 or b, respectively (Goff et al., 1992; Cone et al., 1993). The sequence conservation between the coding regions of the duplicated members of each family and their differential activity in different tissues have led to the identification of 5¢ and 3¢ regulatory regions as determinants of their diverse expression patterns (Radicella et al., 1992; Selinger et al., 1998). We have used the 3-deoxyflavonoid biosynthesis pathway as a model to compare the gene structures and functional diversity generated by
local sequence alterations and regulatory regions among homologs of the pericarp color1 (p1) gene of maize (Chopra et al., 1996). The maize p1 gene encodes a R2R3 type of Myb DNA binding domain protein, and has been shown to activate the transcription of at least three structural genes required for the biosynthesis of 3-deoxyflavonoid pigments (Grotewold et al., 1991, 1994, 1998). Most obvious pigmentation phenotypes of p1 can be observed in kernel pericarp and cob glumes of mature maize ears. There is considerable allelic variation at the p1 locus in maize and these alleles can be distinguished based on their pigmentation patterns in kernel pericarp and cob glumes (Styles and Ceska, 1977, 1989). Structure and functional analysis of two well-characterized alleles P1-wr (white pericarp and red cob) and P1-rr (red pericarp and red cob glumes; Figure 1) indicate that the phenotypic variation could be attributed to their differential transcriptional regulation (Chopra et al., 1996, 2003), which in turn may be
Figure 1. Comparison of phenotypes of sorghum y1 and maize p1 alleles (A) part of sorghum inflorescence of Y1-cs allele showing red variegated pericarp (B) leaf phenotype of Y1-cs (left) and Y1-rr (right) (C) part of sorghum inflorescence of Y1-rr allele showing red pericarp (D) Maize kernels of P1-vv showing red variegated pericarp (E) portions of maize ears of P1-rr (left) and P1-wr (right).
187 a function of their unique gene structures (Chopra et al., 1998). The P1-rr allele has a single coding sequence (Grotewold et al., 1991), while the P1-wr contains six gene copies arranged in a multi-copy tandem repeat (MTR) complex (Chopra et al., 1998). These alleles also differ from each other due to non-conserved 3¢ ends and several indels and transposon like sequences which may affect the expression patterns of each allele. In addition to pericarp and glume pigmentation, the two p1 alleles also condition pigmentation of varying degree in the husk, tassel glume and silk (Cocciolone et al., 2001; Chopra et al., 2003). More recently, p2, a p1 linked homologous sequence, has been characterized and, together with p1, has been shown to be contributing to the expression of the P regulated pathway in maize silks (Zhang et al., 2000, 2003). To extend our understanding of p1 functional diversity in grass species, we began genetic and biochemical characterization of the 3-deoxyflavonoid pathway in sorghum, a species closely related to maize (Gaut and Doebley, 1997). From the pioneering work of Kambal and Bate-Smith (1976), it is known that sorghum produces red phlobaphene pigments in seed pericarp. Additional genetic experiments have established that mutations in the functional y1 (yellow seed1) gene are responsible for the visibility of yellow endosperm color due to the absence of the phlobaphenes in pericarp (Hu et al., 1991; Zanta et al., 1994). Our recent genetic studies using a mutable allele of y1 have established that a functional y1 gene is required for the production of 3-deoxyflavonoid pigments (Chopra et al., 1999). The mutable Y1-candystripe (Y1-cs) allele conditions a variegated pigmentation phenotype in the seed pericarp and leaves (Figure 1). The seed phenotype of the Y1-cs is strikingly similar to that of the maize P1-vv allele, which has been extensively investigated and used as a convenient genetic and phenotypic marker (Lechelt et al., 1989; Athma et al., 1992). We have subsequently isolated the Y1-cs allele and identified a transposable element (Candystripe1) inserted within the y1 gene. Excision of the Cs1 element was genetically correlated with functional reversions of the y1 gene, thus establishing the causative role of a functional y1 gene in the biosynthesis of phlobaphene pigments (Chopra et al., 1999, 2002).
Here we present the detailed molecular structure and function of the y1 gene and its comparison with those of maize p alleles in order to draw similarities and differences for the regulation of phlobaphene biosynthesis in these two species. Our results indicate that the sorghum y1 gene is required for the transcription of at least three structural genes needed for the biosynthesis of 3deoxyflavonoid pigments. The major difference was found to be in the activity of the y1 gene leading to the accumulation of phlobaphenes in sorghum leaves as opposed to the unique regulatory properties of the p alleles in maize silks. Phylogenetic analysis was performed to infer the divergence time of y1/y2 duplicated sequences in sorghum and its comparison with maize p1/p2 duplication.
Materials and methods Sorghum and maize genetic stocks A sorghum genetic stock ‘CS8110419’ containing the Y1-cs (candystripe) allele also known as ‘Candystripe sorghum’ or the ‘mutable allele’ was kindly provided by Dr. J. Bennetzen, University of Georgia, Athens, GA. Origin and genetic characterization of Y1-cs has been described previously (Zanta et al., 1994; Chopra et al., 1999). The functional Y1-rr-30 (red pericarp and red glume) revertant allele used here was isolated from the CS8110419 line through the spontaneous excision of the Cs1 (Candystripe1) transposable element (Chopra et al., 1999, 2002). The revertant and the mutable alleles were maintained by selfing for several generations before being used in this study for molecular and expression analyses. Red pigmentation induced by functional y1 can be observed in the pericarp of seeds, inner glumes (palea and lemma), mature leaves, leaf sheaths, and ligules and auricles. A genetic linkage between the functional y1 gene and its phenotypic expression (data not shown) was confirmed in a population obtained from the test cross [(Y1-rr30 x Y1-cs) X y1-ww(BTx623)]. Inbred line Btx623 is a white seeded line and has a non-functional y1 allele (y1-ww) because of a partial deletion within y1 coding region (Boddu et al., 2005). Maize lines used in this study included inbred line W23 (genotype P1-wr c1 r-g) obtained from the Maize
188 Genetics Cooperation Stock Center, Urbana, IL. The P1-rr-4B2 and p1-ww-1112 genetic stocks carrying p1 + p2 and p2, respectively, have been previously described (Zhang et al., 2000). All maize genetic stocks have been backcrossed into a common background of p1-ww 4Co63 obtained from National Seed Storage Laboratory, Fort Collins, Colorado (Chopra et al., 2003). Plant DNA, RNA isolations and gel blot analysis Genomic DNA from seedling leaves was isolated using the CTAB method of Saghai-Maroof et al. (1984). Restriction enzyme digestions were performed using enzymes, reagents and reaction conditions from PROMEGA (Madison, WI). Sorghum tissues were collected at different growth and developmental stages (Gerik et al., 2003). Tissues included leaf 1, 3, and 5 from sorghum seedlings at the 5-leaf stage, flag leaf (named as immature leaf) and bottom-most surviving leaves (named as old or mature leaf tissue), immature seeds and glumes collected 15 days after anthesis (daa), and pericarp peeled from seeds at 20 daa. Maize tissues included leaf tissues at different stages of plant development, young husks and silks collected at the time of pollination, and pericarp and cob glumes collected from an ear 18 days after pollination (dap). Total RNA isolations were performed using the TRIZOL reagent (GIBCO-BRL, Carlsbad, CA). Fifteen lg of total RNA was denatured and separated on denaturing gels (Sambrook and Russel, 2001). DNA fragments used as probes were from: pY1, containing a sorghum y1 gene fragment corresponding to exon 3; pSbC2, containing a sorghum c2 gene isolated using gene specific primers (Lo et al., 2002); pA1, containing a maize a1 cDNA (Schwarz-Sommer et al., 1987); pChi, containing a maize chi1 cDNA (Grotewold and Peterson, 1994); pP1, containing full length p1 cDNA (Grotewold et al., 1991). Because of high sequence similarity in the coding regions of maize and sorghum flavonoid structural genes (c2, chi, a1, and f3¢h), maize gene fragments can be used as probes to identify expression of sorghum genes (Boddu et al., 2004). RNA gel blot hybridizations were performed for 24 hr at 43C in a hybridization mixture containing 50% formamide, 0.25 M sodium phosphate at pH 7.2, 0.25 M sodium chloride (NaCl), 1 mM EDTA, 7% SDS, and 0.05 mg/ml sheared salmon sperm DNA. Filters were washed in 0.1 SSC (1 SSC
is 0.15 M NaCl, 0.015 M sodium citrate), 0.5% SDS at 50 C for 15 min, and twice at 65C for 15 to 30 min. Filters were exposed to X-OMAT film (KODAK, Rochester, NY) for 1–4 days before developing. Filters were stripped by washing for 15 min in boiling solution of 0.1% SDS before re-hybridization. Isolation of genomic k clones, plasmid sub-cloning and DNA sequencing Previously we characterized the Y1-cs allele and reported the DNA sequence of the Candystripe1 transposon and a partial sequence of the y1 gene flanking the transposon insertion (Chopra et al., 1999). Using the y1-gene specific probes we further screened a sorghum genomic library prepared from the Y1-rr-30 allele. The genomic library was made from partially Sau3AI-digested leaf DNA of Y1-rr-30 genetic stock in a k-FIX II/XhoI vector following the reaction conditions from Stratagene (San Diego, CA). Three positive k clones were isolated and DNA fragments were sub-cloned into pBluescript II SK (+) plasmid vector by standard methods (Sambrook and Russel, 2001). Plasmid sub-clones were sequenced with gene- or vectorspecific primers using the Applied Biosystem’s (Foster City, CA) method of dye primer cycle sequencing and reaction products were separated on a 3100 capillary system. DNA sequence data was compiled using GCG Wisconsin Package 1 (Accerlys Inc., Burlington, MA). Database searches were performed with the BLAST suite of programs available at http://www.ncbi.nlm. nih.gov/BLAST/. Protein percent similarities were obtained from the matching segments in the BLAST output. Phylogenetic analysis Both the protein and cDNA sequences of the 5 grass y1 orthologs were retrieved from GenBank. The accession numbers of the sequences are: maize p1: AF427146, p2: AF210616, p2-t: AF210617; sorghum y1 and y2; rice p: AC137267. Sequence alignment was conducted using ClustalX 1.81 (Thompson et al., 1997). Using MEGA 2.0 (Kumar et al., 2001), we constructed the neighbor-joining phylogenetic trees based on protein and cDNA, respectively. An additional phylogenetic tree was built based on maximum likelihood
189 analysis using Phylip 3.6 (Felsenstein, 1985). Bootstrapping was performed to evaluate the statistical reliability of the inferred topologies. Comparisons indicated that all the topologies were essentially the same. Therefore, the phylogenetic tree based on maximum likelihood analysis was used as the representative tree in results reported here.
of Myb DNA binding domain (Figure 2B). Since y2 has a deletion of exon 2, it lacks the R2-helix III and helix 1 of the R3 Myb repeat. Based on these sequence comparisons, it appears that the y2 sequence may represent a duplicated pseudogene of the functional y1 sequence.
The y1/y2 arrangement resembles p2/p1 duplication in maize Results The sorghum yellow seed1 locus is composed of y1/y2 duplication To determine the molecular structure of the y1 gene, we isolated three overlapping genomic clones from a library prepared from a stock (Y1-rr-30) containing a functional y1 gene (see Materials and Methods). Sequences were obtained from each clone and assembled into a 22.4-kbp sequence contig which is presented in Figure 2A. Comparisons with the previously obtained partial y1 sequences from the Y1-cs allele and cDNA from the functional Y1-rr allele, allowed us to define the y1 gene structure (Figure 2A) which includes three exons (exon 1, 539 bp; exon 2; 131 bp; exon 3, 1197 bp) and two introns (intron 1, 263 bp; intron 2, 4214 bp). Analysis of this sequence composite showed that another homologous y2 sequence is present 3¢ to y1, in direct orientation. The y1 and putative y2 transcription units are separated by a 9084-kbp region, which is composed largely of short repeat sequence elements with high similarity to MITE like elements. At the nucleotide level, y1 and y2 are 97% identical to each other. Sequence alignments indicate that the y2 has a deletion in exon 1 that removes the putative translation initiation codon. There is a second deletion of about 3100 bp corresponding to exon 2 and the 5¢ region of intron 2. The sequences of the deletion end points do not show any unique features or suggest the involvement of transposable element activities in the deletion events. Further downstream, positions of the 3¢ boundary of intron 2 and 5¢ end of exon 3 are conserved between the two sequences. Within exon 3, the y2 sequence has short stretches of indels as compared to the y1 sequence. The deduced amino acid sequence of Y1 shows a highly conserved region for the R2R3 type
Sequence alignments and GenBank searches by BLAST established that the y1 gene may be an ortholog of the maize p1 gene (Chopra et al., 1999; Zhang et al., 2000; Jiang et al., 2004a). To characterize functional and structural similarities or differences among y1, y2 and functional alleles of p1 of maize and its ortholog in teosinte, we aligned their coding and flanking sequences using ClustalX (Figure 3A). Manual adjustments were applied in the alignment of the non-coding regions. All six sequences have a similar intron/ exon structure and all y1 exons and introns are approximately of the same size compared to those of P1-rr and P1-wr. Sequence conservation among the y and p homologs begins approximately 50 bp 5¢ of the putative transcription start site. In the transcribed region, the Y1-rr nucleotide sequence is 94% identical to P1-wr, p2 and p2-t and 92% identical to P1-rr. In contrast, the introns of y1 are greatly diverged and have only small blocks of sequences that are no more than 50% identical to p1 introns. In the 3¢ untranscribed regions, the y1 sequence has some regions that are highly similar to that of the maize p2 gene, while the 3¢ end of y2 shows more similarity with the teosinte p2-t gene as well as to the P1-wr allele of maize p1. These conserved regions include previously identified sequences termed fragments 61 and 15 which are present at the 3¢ ends of y2, P1-wr, p2 and p2-t. These regions have been used as landmarks of sequence rearrangements at the maize p locus to propose that P1-rr is derived from an ancestral p sequence which resembles the structure of the maize P1-wr and the teosinte p2-t gene (Zhang et al., 2000). Our comparative sequence analyses results indicate that the sorghum y2 gene and its arrangement of 3¢ sequences is more similar to the teosinte p2-t gene, while the sorghum y1 gene is more similar to the maize p2 gene than to other maize orthologs.
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Figure 2. Genomic organization of the y1/y2 region in sorghum. (A) Line diagram obtained by characterization of the Y1-cs allele (Chopra et al., 1999) is shown at the top. Triangle within the partial y1 sequence represents the Candystripe1 (Cs1) transposable element. Overlapping k and plasmid clones used to complete the sequence of the y1–y2 duplication are shown below the map of Y1-cs. The y1–y2 is contained within the assembled 22,400 bp sequence (accession number: AY860968) shown as a bar below the corresponding positions of the k clones. Arrangement of y1 and y2 separated by an intergenic region of 9084 bp is shown on this bar and gene structure below. A partial restriction map of the y1 gene is presented at the bottom. Clear boxes represent 5¢ UTRs, black boxes are ORFs of predicted exons. A bent arrow indicates putative transcription start sites. An asterisk indicates a deletion in the y2 sequence which removes the first AUG of the deduced translated product. Restriction enzyme sites shown are: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; S, SacI; SL, SalI; SC, ScaI. (B) Sequence comparison of the Myb domain region of p orthologs including sorghum Y1 (SbY1), sorghum Y2 (SbY2), maize P1-wr (ZmP1-wr), maize P1-rr, maize P2 (ZmP2) and teosinte P2-t (P2t). The positions of three helices forming R2 and R3 Myb repeats are shown. The evolutionary conserved proline to alanine change is indicated by an inverted arrow at amino acid position 63. Dark shaded regions indicate identical amino acids; the dashed region indicates the deletion within the Myb domain of the predicted Y2 protein of the R2R3 Myb domains of sorghum and maize deduced P and Y proteins.
To estimate the time of origin of the y1 and y2 duplication, we performed a phylogenetic tree analysis on the protein and cDNA sequences of maize, sorghum teosinte and rice p orthologs
(Figure 3B). We first compared y1 and y2 coding sequences, using the formula R = K/2T (Li and Graur, 1991), where K is the number of substitutions at synonymous sites (Nei and Gojobori
191
Figure 3. Structural comparison of sorghum y1 and y2 with maize and teosinte p homologs. (A) Diagrammatic representation of genomic sequences of sorghum y1, y2, maize P1-rr, P1-wr, p2, and teosinte p2-t aligned using ClustalX software (Thompson et al., 1997). A bent arrow indicates the putative transcription start site of y1 and other orthologous genes (Grotewold et al., 1991; Chopra et al., 1996; Zhang et al., 2000, 2003). Black boxes represent exons that are connected with bent lines as introns. Deleted region of exon 2 and part of intron 2 of the y2 gene copy are shown as a dotted rectangle. Grey boxes indicate a transcribed but un-translated leader sequence and a hatched box 5¢ of the transcription start represents a conserved 90 bp region of the promoter in all orthologs (Zhang et al., 2000). The P1-rr gene coding region is flanked by 5.2-kb direct repeats, indicated by horizontal arrows. In P1-wr, one full copy and a partial 5¢ end of the second copy are shown; the P1-wr allele contains 6 copies of the p1gene arranged in a head to tail fashion (Chopra et al., 1998). Numbered boxes/regions indicate location of p1 fragments described previously (Lechelt et al., 1989; Chopra et al., 1998; Zhang et al., 2000). To illustrate conserved or modified sequence regions, drawing is not to scale. Accession number for the y1–y2 contig for sorghum is AY860968 (this study). Accession numbers of other p homologous sequences used in this alignment are AF209212, Z11879, U57002, AF210616, and AF210617 (Zhang et al., 2000). (B) Phylogenetic tree based on the protein alignment of grass p1/p2 homologs using maximum likelihood analysis. Only bootstrap values of equal to or more than 50 are shown. The accession numbers of sequences used are: maize p1: AF427146, maize p2: AF210616, teosinte p2-t: AF210617; sorghum y1 and y2: AY860968; and rice p: AC137267.
1986), R is the rate of substitution per site per year (estimated at 6 10)9 for grass nuclear genes; Gaut, 1998), and T is time (years). However,
unlike maize p2/p1 paralogs, there is a deletion within R2R3 Myb domains in sorghum y2 and also the 24 c-terminal residues of y2 are divergent
192 b Figure 4. Steady-state transcription analysis of y1 and flavonoid biosynthetic genes and proposed route of 3-deoxyflavonoid biosynthesis in sorghum. (A) Gel blot analysis of transcripts from 20 daa pericarp of Y1-rr and Y1-cs genotypes, hybridized with y1, c2, chi, a1, and tub1 probes. Gene names are shown at the left. (B) Proposed sorghum phenylpropanoid pathway leading to the phlobaphene precursors (apiferol and luteoferol) and 3-deoxyanthocyanidin phytoalexins (apigeninidin and luteolinidin). Relevant enzyme activities and corresponding genes of maize indicated are: CHS, chalcone synthase (c2, whp1); CHI, chalcone isomerase (chi1); DFR, dihydroflavonol reductase (a1); F3¢H, flavonoid 3¢hydroxylase (pr); AS, anthocyanidin synthase; bp1, brown pericarp1 (McMullen et al., 2001). Pathway modeled after Kambal and Bate-Smith (1976); Lo and Nicholson (1998); Styles and Ceska (1989) and current study.
(dN) values in the isolated R2R3 Myb repeats of the grass p1/p2 homologs. One dataset comprised intact R2R3 Myb domain of all the genes, while the other set (P1-rr¢, p2¢, and y1¢) removes from each gene the region corresponding to the deletion in sorghum y2 gene. For example, the dN value is 0.045 for P1-rr/p2, and 0.039 for P1-rr¢/p2 (or p2¢/ P1-rr). This indicated that the removed regions are more divergent than the remaining part of R2R3 domains in the maize p1/p2 paralogs. Similarly, results were obtained for p1/p2 homologs between maize, sorghum, and rice. For example, the dN value is 0.147 for y1/p2, and 0.119 for y1¢/p2. Moreover, by comparing the above two cases, we found that the dN value within a species (P1-rr/ p2) is much less than that between species (y1/p2). This suggested that these homologous genes became more divergent after speciation. Taken together, although the deleted region reduces the total divergence, the difference is not significant. Therefore, the large deletion in y2 does not affect the estimation of the duplication time of y1/y2 because of the high conservation in the R2R3 Myb domains. Transcription of flavonoid biosynthesis structural genes require functional y1
from y1. Therefore, we estimated that the duplication time of y1/y2 is 9.08 and 11.3 mya with and without the c-terminal residues, respectively. To test the impact the deletion in y2 may have in the estimation of the duplication time of y1/y2, we calculated two sets of synonymous substitution
The maize P1 protein is a Myb transcription factor that has been shown to regulate the transcription of at least three structural genes, namely chalcone synthase, chalcone isomerase, and dihydroflavonol reductase encoded by c2, chi1, and a1, respectively. To test if the targets of Y1 include any or all of these three structural genes in sorghum, RNA gel
193 blot hybridizations were performed. Gel blots were prepared from total RNA from immature seed pericarp (20 days after anthesis) of the functional allele Y1-rr or the variegated allele Y1-cs. Gel blots were sequentially hybridized to y1, c2, chi1, and a1 gene probes (Figure 4A). An approximately 1.9 kbp transcript of y1 is present in Y1-rr and as expected, this transcript is barely visible in the Y1-cs allele. Abundant transcripts of sorghum genes hybridizing with maize c2, a1, and chi1 genes are present in Y1-rr as compared to Y1-cs RNA. These results suggest that transcription of these structural genes may require a functional y1 gene. Based on this and previous studies in sorghum (Lo and Nicholson, 1998; Chopra et al., 2002), the proposed biosynthetic pathway of 3-deoxyflavonoids is shown in Figure 4B. The placement of the f3¢h gene in this pathway is based on an experimental evidence presented elsewhere (Boddu et al., 2004). Phlobaphene accumulation in sorghum leaves is regulated by the Y1-rr allele Functional alleles of maize p1 and sorghum y1 have similar patterns of pigmentation in pericarp, while dissimilarities exist between the two for their expression in leaf tissues (Figure 5). The maize p alleles do not induce any obvious pigmentation in non-husk leaves except for the P1-rr allele which conditions an orange tinge in the leaf below the ear shoot (Cocciolone et al., 2000). However, leaves of sorghum lines with a functional y1 gene accumulate abundant amounts of phlobaphene pigments (Zanta et al., 1994; Chopra et al., 2002). To assess gene expression, we performed a survey of transcript accumulation of y1 in sorghum and p genes in maize. RNA gel blot hybridization results are presented in Figure 5. Transcripts of y1 were examined in leaves at different developmental stages (see Materials and Methods). During the vegetative phase, y1 is expressed at low level in seedling leaves 1, 3 and 5 (Figure 5A). Compared to these immature leaves, the flag leaf has an increased accumulation of y1 RNA and this message was barely detectable in the mature leaf. Immature seeds and glumes collected 15 daa show abundant y1 message correlating with the accumulation of 3-deoxyflavonoids in these tissues. Moreover, the latter result is confirmed in 20 daa pericarp RNA, in which y1 transcripts are abun-
dant in the Y1-rr genotype but were not detected in Y1-cs. To compare the expression of y1 with that of the maize p genes, transcriptional profiles of p1 and p2 in the P1-rr 4B2 stock and of p2 in the p1-ww-1112 stock were compared. RNA gel blots made from maize leaves at comparable stages did not detect p1 or p2 specific transcripts (data not shown). Similar to the previous findings (Grotewold et al., 1991; Chopra et al., 1996), we detected two alternatively spliced p1 specific transcripts of 1.9 and 1.0 kbp in pericarp (Figure 5B). Similar-sized p1 transcripts are abundant in P1-rr husks, and silks; in previous silk expression studies, this transcript could only be detected by RTPCR (Zhang et al., 2000) assays. The p1 probe also hybridizes with a transcript (about 1.7 kbp size, not resolved in this RNA gel blot) in both P1-rr and p1-ww stocks. Because the p1 gene is deleted in the p1-ww-1112 stock tested, the approximately 1.7 kbp band most likely represents a transcript of the p2 gene. The putative p2 transcript is present in both P1-rr and p1-ww silks as shown by RT-PCR assays (Zhang et al., 2000). In addition to the presence of the large sized p2 transcript, our results also show that there is another 1.0 kbp message that may represent the alternative spliced product of the p2 and this requires further investigation. In conclusion, results indicate that the y1 gene controls phlobaphene biosynthesis in several different sorghum tissues, whereas in maize tissues this function has been sub-divided between the p1 and p2 genes.
Discussion The y1 and y2 genes are members of recently duplicated Myb transcription factors The phenotypic resemblance between the mutable sorghum candystripe allele Y1-cs (Chopra et al., 1999; Carvalho et al., 2005) and maize variegated pericarp allele P1-vv (Athma et al., 1992), as well as similar biochemical nature of the flavonoid pigments in maize and sorghum suggested that p1 and y1 genes may have identical functions (Zanta et al., 1994; Chopra et al., 1999, 2002). Sequence characterization of the y1/y2 duplication and alignments of deduced amino acid sequences show that y1 is orthologous to the maize p1 gene that codes for a transcription factor of the R2R3 Myb
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Figure 5. Temporal and spatial transcript analysis of y1 and p homologs. (A) Gel blot analysis of y1 and tubulin1 RNA in leaves, immature seeds, glumes, and pericarps at different developmental times. On the right side diagram of a mature sorghum plant depicts (in red) the accumulation pattern of phlobaphenes in leaves and inflorescence. (B) Steady-state analysis of maize p1 and p2 transcripts in pericarp, cob glume, silk and husk tissue of maize stocks carrying P1-rr and p1-ww alleles. Alternatively spliced products of p1 are seen as 1.9 and 1.0 kbp bands (Grotewold et al. 1991) while the p2 transcript present in the p1-ww tissue has been reported previously by RT-PCR (Zhang et al., 2000). Hybridization with tubulin1 is shown for loading control. Diagram on the right depicts (in red) the pattern of phlobaphene accumulation in maize.
class. Multiple alignments show a strikingly high similarity among maize p1 orthologs, which are more than 92% similar to one another at the nucleotide level (Zhang et al., 2000). In addition, gene structure analysis indicates a high level of conservation in the intron locations, as well as the presence of a large intron 2 in these and several other maize and sorghum Myb orthologous genes (Jiang et al., 2004a). However, their c-termini exhibit sequence variation; possibly, these regions may play a role in the regulation of activity of the Myb domain (Dias et al., 2003). Based on the Myb R2R3 protein classification, Y1 and P1 and their
homologs belong to the Proline63 to Alanine63 clad (Dias et al., 2003; Jiang et al., 2004a). It was thought that their divergence from Proline63-type Myb genes occurred before the separation of eudicots and monocots, but after the amplification of Myb genes in plants (Jiang et al., 2004b). Besides high homology in the coding regions, it is interesting to note the similarities in the nontranscribed regions, especially fragments 61, C and 15. These regions are assumed to be functionally important and thus conserved for 20 million years since the maize/sorghum divergence. We also detected a number of rearrangements and
195 sequence polymorphisms at the 5¢ and 3¢ ends of the sorghum y1 and y2 genes; similar types of changes have been observed in other alleles of p1 in maize (Chopra et al., 1996), in the rp1 complex of maize and sorghum (Ramakrishna et al., 2002) and in bz alleles in maize (Fu and Dooner, 2002). The sequence polymorphisms detected at the ends of these genes do not indicate any definite mechanism involved in gene duplication. However, as proposed earlier in the bz (Fu and Dooner, 2002), and p1 allele (Zhang et al., 2000), the presence of retroelement-like sequences near the putative breakpoints could indicate their involvement in the deletion process. The inferred topology is reliable with high bootstrap values, indicating that the gene tree is consistent with the species tree (Figure 3B). The maize p1, p2, and teosinte p2-t genes form a monophyly indicating that they were derived from a common ancestor. In contrast, sorghum y1 and y2 did not form a monophyly very likely due to the large deletion in y2. However, our results of the sequence analysis indicate that the deletion in y2 does not affect the estimation of the duplication time of y1/y2 because of the high conservation in R2R3 Myb domains. The topology also indicates that maize p1 and p2 are the most recently duplicated genes. The duplication has been estimated to have occurred 2.75 mya (Zhang et al., 2000), which would be before the domestication of maize from teosinte which took place approximately 7500 years ago (Iltis, 1983; Doebley et al., 1984). The result that maize p2 is more closely related to teosinte p2-t rather than maize p1 is consistent with this estimation of the duplication time of maize p1 and p2. Although p1 has not been sequenced from teosinte, our previous DNA gel blot survey has shown that some accessions of teosinte may contain a p1 orthologous gene (Zhang et al., 2000). Thus the duplication events of y1/y2 and p2/p1 both occurred after the sorghum–maize separation, 20 mya (Gaut and Doebley, 1997). Interestingly, only one copy of the grass p1/p2 homologs has been found in the rice genome, and this may further suggest that duplication event of grass p1/p2 homolgs has likely occurred after speciation of rice, sorghum and maize. The latter is further supported by the observation that, common sequence blocks present at the 5¢ and 3¢ non-transcribed ends were only be found in maize p2-p1 and sorghum y1-y2 duplication.
Phylogenetic analysis and overall comparison of the maize p2-p1 and sorghum y1-y2 duplications and gene structures provided an interesting model of gene duplication and generation of diversity. We propose that the sorghum y1 gene is closely similar to the maize p2 and to the ancestral p gene. Duplication of y1–y2 took place in tandem and this may have been brought about by the reteroelements and MITES like sequences that are present in flanking regions of the y1 and y2 sequences. The tandem duplication thus created a new 3¢ end of the y1 gene while the y2 sequence harbored the old 3¢ end containing fragments 61, C, 15 and 6 (see Figure 3A). Sequences deleted within the y2 gene are presumably recent events and this hypothesis will be tested by characterizing gene structure of y2 in diverse sorghum races. Conserved targets of Y and P regulators In maize, biosynthesis of 3-deoxyflavonoids or flavan-4-ols requires a functional p gene and our results now show that the y1 gene may perform the same role in sorghum. Our expression study (see Figure 4) shows that a functional y1 gene is required for the transcription of genes encoding chalcone synthase, chalcone isomerase, and dihydroflavonol reductase. In sorghum, there are eight chalcone synthase genes, and it has been proposed that each of these genes may have differential expression properties because of variations in their promoter sequences (Lo et al., 2002). RNA gel blot hybridizing with the conserved coding region of chs shows that at least one of the chs homologs is under the regulatory control of the Y1 transcription factor. The presence of a small amount of c2, chi and a1 transcripts in the Y1-cs pericarp was expected due to the presence of few red revertant sectors caused by excision of the Cs1 transposon resulting in a functional state of y1 gene (Carvalho et al., 2005). In a recent study from our laboratory (Boddu et al., 2004), we presented evidence that a flavonoid 3¢-hydroxylase gene may also be under the regulatory control of the functional y1 gene in sorghum, indicating that there may be additional targets of the Y1 in sorghum and P1 in maize. Thus, analysis of parallel biochemical pathways in related plant species can be used to identify previously unknown targets of transcription factors. Similar conclusions have been reached in studies based on
196 genome wide expression profiling and microarray analysis transcriptional regulation by the maize p gene (Bruce et al., 2000). Expression of y1 represents a combination of p1 alleles The maize p1 gene functions in kernel pericarp, cob glume, silk and other floral organs as well as in some vegetative tissues including husk and leaf sheath, but no pigmentation is observed in the leaf blade. Whereas, no transcripts corresponding to the putative y2 gene of sorghum were detected by RNA gel blot or RT-PCR assays (data not shown). In addition to floral tissues, y1 regulated expression was observed in sorghum leaves. This floral and vegetative expression pattern of the y1regulated pathway seems to correspond in maize to the combined profile of P1-wr and P1-rr alleles. The P1-wr allele has been shown to induce pigmentation in the margins of husk and leaf sheath, while the P1-rr-induced pigments are visible throughout the husk and sheath (Chopra et al., 1996). As the sorghum plant approaches maturity, phlobaphenes accumulate in the margins of mature leaves while in the immature and the flag leaf pigmentation can be seen throughout the leaf blade. The accumulation of leaf pigments and the transcription of the y1 gene show a progressive increase from the bottom to top of the plant (see Figure 5). Our results showing that y1 expression occurs broadly in both floral and vegetative tissues may suggest that the y1 regulatory regions may
resemble the ancestral state, prior to the subcompartmentalization, which typifies the maize p1/ p2 expression pattern (Zhang et al., 2003). The mechanism of unique leaf expression property of the y1 locus may possibly be the function of y1 promoter elements that are either missing or highly modified in p1 and p2 promoters (Zhang et al., 2000). Promoter sequence alignments among p and y orthologs showed very poor conservation (Figure 6). Homologous promoter sequence blocks are present within -200 bp of the transcription start site of these orthologs. The maize p1 gene promoter has previously been shown to consist of tissue-specific domains and enhancers and several rearrangements and insertion sequences have shaped these regulatory elements in maize (Sidorenko et al., 2000). None of these functionally known cis-elements of p1 promoter were detected in y1 regulatory region. The y1 upstream promoter region carries several small insertions and MITES like repetitive sequences (not shown) which may have generated regulatory alterations in sorghum as has been proposed previously (Feuillet and Keller, 2002). Our results give an indication of the remarkable plasticity of grass genomes, and the potential for genome rearrangements in the regulatory regions of transcription factors to alter gene expression of the pathway. These results are in agreement with comparative analysis of sorghum and maize phytochromeA genes that have conserved coding regions and divergent promoter sequences (Morishige et al., 2002).
Figure 6. Comparison of 5¢ regulatory sequences of y and p genes. The 5¢ regulatory region of y1 sequence was aligned with promoters of p homologs. The conserved transcription start site as a bent arrow and TATA box sequence is shown. Other conserved sequence blocks are shown as dark shaded regions.
197 The regulation of expression of the y1 gene and the Y1-regulated phlobaphene pathway in leaf tissues may have evolved to cope with certain environmental constraints. For example, synthesis of flavonoid phytoalexins can be induced in sorghum leaves in response to Colletotrichum sublineolum (Snyder and Nicholson, 1990). These phytoalexins have been identified as 3-deoxyanthocyanindins which have structural similarities with the precursors of phlobaphenes (Figure 4B; Lo and Nicholson, 1998; Grotewold et al., 1998). Our previous results indicated that functional y1 gene may play a key role in the site specific production of phytoalexins in sorghum mesocotyls (Chopra et al., 2002). Interestingly, maize silks and transgenic cell lines carrying active p1 show constitutive but small amounts of 3-deoxyanthocyanidins but their antifungal activities have not been tested (McMullen et al., 2001; Grotewold et al., 1998). These studies in maize, together with our current results in sorghum, provide an example of evolutionary divergence of expression of regulatory genes p1 and y1 in closely related grass species.
Acknowledgements This work was supported in part by research support to SC under Hatch projects 3855, and 3905 of the College of Agricultural Sciences, Pennsylvania State University, and a USDANRI-2002-35318-12676 award. V.S. was supported by a pre-doctoral fellowship from the Department of Crop & Soil Sciences/College of Agricultural Sciences, Pennsylvania State University. We are thankful to Drs. Feng Zhang and Daniel Knievel for their suggestions during the preparation of this manuscript. Suggestions of two anonymous reviewers were very helpful in revising this manuscript.
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