No 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.

This report identifies S-RNases of sweet cherry (Prunus avium L.) and presents information about cDNA sequences encoding the S-RNases, which leads to the development of a molecular typing system for S-alleles in this fruit tree species. Stylar proteins of sweet cherry were surveyed by two dimensional polyaclylamide gel electrophoresis (2D-PAGE) to identify S-proteins associated with gametophytic self-incompatibility. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other rosaceous S-RNases. These glycoproteins were present at highest concentration in the upper segment of the mature style and shared immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding these glycoproteins were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences indicated that the cDNAs encode S-RNases; thus the S-proteins identified by 2D-PAGE are S-RNases. Although S1 to S6-alleles of sweet cherry cultivars could be distinguished from each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.

SummaryMany Prunus species, including sweet cherry and Japanese apricot, of the Rosaceae, display an S-RNase-based gametophytic self-incompatibility (GSI). The specificity of this outcrossing mechanism is determined by a minimum of two genes that are located in a multigene complex, termed the S locus, which controls the pistil and pollen specificities. SFB, a gene located in the S locus region, encodes an F-box protein that has appropriate S haplotype-specific variation to be the pollen determinant in the self-incompatibility reaction. This study characterizes SFBs of two self-compatible (SC) haplotypes, S 4¢ and S f , of Prunus. S 4¢ of sweet cherry is a pollen-part mutant (PPM) that was produced by X-ray irradiation, while S f of Japanese apricot is a naturally occurring SC haplotype that is considered to be a PPM. DNA sequence analysis revealed defects in both SFB 4¢and SFB f . A 4 bp deletion upstream from the HVa coding region of SFB 4¢ causes a frame-shift that produces transcripts of a defective SFB lacking the two hypervariable regions, HVa and HVb. Similarly, the presence of a 6.8 kbp insertion in the middle of the SFB f coding region leads to transcripts for a defective SFB lacking the C-terminal half that contains HVa and HVb. As all reported SFBs of functional S haplotypes encode intact SFB, the fact that the partial loss-of-function mutations in SFB are present in SC mutant haplotypes of Prunus provides additional evidence that SFB is the pollen S gene in GSI in Prunus.

;This study describes a novel F-box protein gene in the S-locus of sour cherry (Prunus cerasus) and sweet cherry (P. avium). The gene showed an S-haplotype-specific sequence polymorphism and the expression was specific to pollen. Genomic DNA blot analysis of eight sweet cherry cultivars with the probe for the F-box protein gene under low stringency conditions yielded RFLP bands specific to the S-haplotypes of each cultivar. We discuss the possibility of the gene for the F-box protein being a candidate for the male determinant of gametophytic self-incompatibility in 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 genotypedependent 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.

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), SFB 1 , SFB 2 , SFB 4 , and SFB 5 . 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 windowaveraged 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.

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.