Ryutaro Tao

Structural and Transcriptional Analysis of the Self-Incompatibility Locus of Almond: Identification of a Pollen-Expressed F-Box Gene with Haplotype-Specific Polymorphism

Gametophytic self-incompatibility in Rosaceae, Solanaceae, and Scrophulariaceae is controlled by the S locus, which consists of an S-RNase gene and an unidentified “pollen S” gene. An ∼70-kb segment of the S locus of the rosaceous species almond, the S haplotype–specific region containing the S-RNase gene, was sequenced completely. This region was found to contain two pollen-expressed F-box genes that are likely candidates for pollen S genes. One of them, named SFB (S haplotype–specific F-box protein), was expressed specifically in pollen and showed a high level of S haplotype–specific sequence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the S specificity of pollen because it showed little allelic sequence polymorphism and was expressed also in pistil. Three other S haplotypes were cloned, and the pollen-expressed genes were physically mapped. In all four cases, SFBs were linked physically to the S-RNase genes and were located at the S haplotype–specific region, where recombination is believed to be suppressed, suggesting that the two genes are inherited as a unit. These features are consistent with the hypothesis that SFB is the pollen S gene. This hypothesis predicts the involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based gametophytic self-incompatibility system.

Cloning and characterization of cDNAs encoding S-RNases from almond (Prunus dulcis) : primary structural features and sequence diversity of the S-RNases in Rosaceae

Abstract cDNAs encoding three S-RNases of almond (Prunus dulcis), which belongs to the family Rosaceae, were cloned and sequenced. The comparison of amino acid sequences between the S-RNases of almond and those of other rosaceous species showed that the amino acid sequences of the rosaceous S-RNases are highly divergent, and intra-subfamilial similarities are higher than inter-subfamilial similarities. Twelve amino acid sequences of the rosaceous S-RNases were aligned to characterize their primary structural features. In spite of␣their high level of diversification, the rosaceous S-RNases were found to have five conserved regions, C1, C2, C3, C5, and RC4 which is Rosaceae-specific conserved region. Many variable sites fall into one region, named RHV. RHV is located at a similar position to that of the hypervariable region a (HVa) of the solanaceous S-RNases, and is assumed to be involved in recognizing S-specificity of pollen. On the other hand, the region corresponding to another solanaceous hypervariable region (HVb) was not variable in the rosaceous S-RNases. In the phylogenetic tree of the T2/S type RNase, the rosaceous S-RNase fall into two subfamily-specific groups (Amygdaloideae and Maloideae). The results of sequence comparisons and phylogenetic analysis imply that the present S-RNases of Rosaceae have diverged again relatively recently, after the divergence of subfamilies.

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

Abstract In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (Sa, Sb, Sc, Sd). We previously reported the cDNA cloning of three of these alleles, namely Sb, Sc and Sd. In this paper we report the cloning and DNA sequence analysis of the Sa allele. The Sa-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (Sb, Sc, and Sd) and this similarity was lower than that observed among the Sb, Sc and Sd-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; Sa (602 bp), Sb (1083 bp), Sc (221 bp) and Sd (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the Sa- and Sb-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the Sa-allele and 556 bp in the Sb-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage.

Primary structural features of the S haplotype-specific F-box protein, SFB, in Prunus

The gene SFB encodes an F-box protein that has appropriate S-haplotype-specific variation to be the pollen determinant in the S-RNase-based gametophytic self-incompatibility (GSI) reaction in Prunus (Rosaceae). To further characterize Prunus SFB, we cloned and sequenced four additional alleles from sweet cherry (P. avium), SFB1, SFB2, SFB4, and SFB5. These four alleles showed haplotype-specific sequence diversity similar to the other nine SFB alleles that have been cloned. In an amino acid alignment of Prunus SFBs, including the four newly cloned alleles, 121 out of the 384 sites were conserved and an additional 65 sites had only conservative replacements. Amino acid identity among the SFBs ranged from 66.0% to 82.5%. Based on normed variability indices (NVI), 34 of the non-conserved sites were considered to be highly variable. Most of the variable sites were located at the C-terminal region. A window-averaged plot of NVI indicated that there were two variable and two hypervariable regions. These variable and hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the GSI reaction. Evidence of positive selection was detected using maximum likelihood methods with sites under positive selection concentrated in the variable and hypervariable regions.

Accumulation of Nonfunctional S-Haplotypes Results in the Breakdown of Gametophytic Self-Incompatibility in Tetraploid Prunus

The transition from self-incompatibility (SI) to self-compatibility (SC) is regarded as one of the most prevalent transitions in Angiosperm evolution, having profound impacts on the genetic structure of populations. Yet, the identity and function of mutations that result in the breakdown of SI in nature are not well understood. This work provides the first detailed genetic description of the breakdown of S-RNase-mediated gametophytic self-incompatibility (GSI) in a polyploid species that exhibits genotype-dependent loss of SI. Genetic analyses of six natural sour cherry (Rosaceae, Prunus cerasus) selections identified seven independent, nonfunctional S-haplotypes with disrupted pistil component (stylar-S) and/or pollen component (pollen-S) function. A genetic model demonstrating that the breakdown of SI in sour cherry is due to the accumulation of a minimum of two nonfunctional S-haplotypes within a single individual is developed and validated. Our finding that sour cherry is SI when only one nonfunctional S-haplotype is present has significant evolutionary implications since nonfunctional S-haplotypes would be maintained in the population without causing an abrupt shift to SC. Furthermore, we demonstrate that heteroallelic sour cherry pollen is self-incompatible, which is counter to the well-documented phenomenon in the Solanaceae where SC accompanying polyploidization is frequently due to the SC of heteroallelic pollen.

Functional and Expressional Analyses of PmDAM Genes Associated with Endodormancy in Japanese Apricot

Bud endodormancy in woody plants plays an important role in their perennial growth cycles. We previously identified a MADS box gene, DORMANCY-ASSOCIATED MADS box6 (PmDAM6), expressed in the endodormant lateral buds of Japanese apricot (Prunus mume), as a candidate for the dormancy-controlling gene. In this study, we demonstrate the growth inhibitory functions of PmDAM6 by overexpressing it in transgenic poplar (Populus tremula × Populus tremuloides). Transgenic poplar plants constitutively expressing PmDAM6 showed growth cessation and terminal bud set under environmental conditions in which control transformants continued shoot tip growth, suggesting the growth inhibitory functions of PmDAM6. In the Japanese apricot genome, we identified six tandemly arrayed PmDAM genes (PmDAM1–PmDAM6) that conserve an amphiphilic repression motif, known to act as a repression domain, at the carboxyl-terminal end, suggesting that they all may act as transcriptional repressors. Seasonal expression analysis and cold treatment in autumn indicated that all PmDAMs were repressed during prolonged cold exposure and maintained at low levels until endodormancy release. Furthermore, PmDAM4 to PmDAM6 responses to a short period of cold exposure appeared to vary between low- and high-chill genotypes. In the high-chill genotype, a short period of cold exposure slightly increased PmDAM4 to PmDAM6 expression, while in the low-chill genotype, the same treatment repressed PmDAM4 to PmDAM6 expression. Furthermore, PmDAM4 to PmDAM6 expression was negatively correlated with endodormancy release. We here discuss the genotype-dependent seasonal expression patterns of PmDAMs in relation to their involvement in endodormancy and variation in chilling requirements.

A Y-chromosome–encoded small RNA acts as a sex determinant in persimmons

Y male plants affect female development Although most plants have both male and female organs within a single flower, some produce separate male and female plants. In some cases, such as persimmons, males are determined by a Y chromosome. Akagi et al. examined the gene transcript differences between male and female persimmons. A gene on the Y chromosome regulated a non–sex chromosome–linked small RNA that suppresses female organ development. This small RNA was localized to male flowers and could affect female development in other plant species. The evolutionary history of these genes suggests that they are tied to the origin of the separation of sexes in the persimmon family. Science, this issue p. 646 The Y chromosome in date plum encodes a small RNA involved in male sex determination. In plants, multiple lineages have evolved sex chromosomes independently, providing a powerful comparative framework, but few specific determinants controlling the expression of a specific sex have been identified. We investigated sex determinants in the Caucasian persimmon, Diospyros lotus, a dioecious plant with heterogametic males (XY). Male-specific short nucleotide sequences were used to define a male-determining region. A combination of transcriptomics and evolutionary approaches detected a Y-specific sex-determinant candidate, OGI, that displays male-specific conservation among Diospyros species. OGI encodes a small RNA targeting the autosomal MeGI gene, a homeodomain transcription factor regulating anther fertility in a dosage-dependent fashion. This identification of a feminizing gene suppressed by a Y-chromosome–encoded small RNA contributes to our understanding of the evolution of sex chromosome systems in higher plants.

Expressional regulation of PpDAM5 and PpDAM6, peach (Prunus persica) dormancy-associated MADS-box genes, by low temperature and dormancy-breaking reagent treatment

The present study investigated the expressional regulation of PpDAM5 and PpDAM6, two of the six peach (Prunus persica) dormancy-associated MADS-box genes, in relation to lateral bud endodormancy. PpDAM5 and PpDAM6 were originally identified as homologues of Arabidopsis SHORT VEGETATIVE PHASE/AGAMOUS-LIKE 24 identified in the EVERGROWING locus of peach. Furthermore, PpDAM5 and PpDAM6 have recently been suggested to be involved in terminal bud dormancy. In this study, seasonal expression analyses using leaves, stems, and lateral buds of high-chill and low-chill peaches in field conditions indicated that both genes were up-regulated during the endodormancy period and down-regulated with endodormancy release. Controlled environment experiments showed that the expression of both PpDAM5 and PpDAM6 were up-regulated by ambient cool temperatures in autumn, while they were down-regulated by the prolonged period of cold temperatures in winter. A negative correlation between expression levels of PpDAM5 and PpDAM6 and bud burst percentage was found in the prolonged cold temperature treatment. Application of the dormancy-breaking reagent cyanamide to endo/ecodormant lateral buds induced early bud break and down-regulation of PpDAM5 and PpDAM6 expression at the same time. These results collectively suggest that PpDAM5 and PpDAM6 may function in the chilling requirement of peach lateral buds through growth-inhibiting functions for bud break.

Isolation of LEAFY and TERMINAL FLOWER 1 homologues from six fruit tree species in the subfamily Maloideae of the Rosaceae

Flowering is an essential stage of fruit production. To understand the molecular mechanisms controlling flowering in maloid fruit tree species, we isolated and analyzed genes homologous to Arabidopsis LEAFY (LFY; flower meristem identity gene) and TERMINAL FLOWER 1 (TFL1; inflorescence meristem identity gene) from six fruit tree species in the subfamily Maloideae of the Rosaceae; apple (Malus × domestica), Japanese pear (Pyrus pyrifolia), European pear (Pyrus communis), quince (Cydonia oblonga), Chinese quince (Chaenomeles sinensis), and loquat (Eriobotrya japonica). Two LFY homologues and two TFL1 homologues were cloned from all six maloid species by rapid amplification of 3′ and 5′ cDNA ends, reverse transcription-PCR, and PCR with genomic DNA. Phylogenetic analysis by the neighbor-joining method showed that the two LFY homologues and two TFL1 homologues were classified into two distinct clades. The presence of multiple copies of LFY and TFL1 homologues is discussed with reference to the polyploid origin of the subfamily Maloideae.

Diversity of S-RNase genes and-s-haplotypes in Japanese plum (Prunus salicina Lindl.)

Summary This report demonstrates the diversity of S-haplotypes in Japanese plum by molecular cloning of genomic DNAs and cDNAs that encode S-RNases. Nine different DNA fragments, designated as Sa–Si, were obtained from 17 Japanese plum cultivars by PCR with an S-RNase gene-specific primer set, Pru-C2 and PCE-R. Eleven different S-haplotypes were found in these cultivars. The banding patterns obtained with another S-RNase gene-specific primer set, Pru-T2 and PCE-R, corresponded to the S-haplotypes predicted from the Pru-C2 and PCE-R primer set. Several cultivars had the same S-haplotypes. Partial genomic DNAs for eight S-RNase genes and cDNAs for two S-RNases were cloned and sequenced. Deduced amino acid sequences contained conserved regions among the rosaceous S-RNases. Comparisons of the sequences from cDNAs and genomic DNAs revealed the presence of two introns in the S-RNase genes of Japanese plum as in other Prunus S-RNase genes. Pollination incompatibility groups and self-compatibility in Japanese plum were discussed with reference to the S-haplotypes.