Fluorescence from abnormally sterile pollen of the Japanese apricot.

We observed trees of the Japanese apricot, Prunus mume 'Nanko' (Rosaceae), bearing two types of flowers: 34% had blue fluorescent pollen under UV irradiation, and 66% had non-fluorescent pollen. The fluorescent pollen grains were abnormally crushed, sterile, and devoid of intine and pollenkitt. The development of microspores within anthers was investigated: in the abnormally developed anthers, tapetal cells were vacuolated at the unicellular microspore stage, and fluorescent pollen was produced. Compounds responsible for the blue fluorescence of pollen were identified as chlorogenic acid and 1-O-feruloyl-ß-D-glucose. The anthers with fluorescent pollen contained 6.7-fold higher and 3.8-fold lower amounts of chlorogenic acid and N 1,N 5,N 10-tri-p-coumaroylspermidine, respectively, compared to those with non-fluorescent pollen. The tapetal vacuolization, highly accumulated chlorogenic acid, and deficiency of N 1,N 5,N 10-tri-p-coumaroylspermidine imply that low-temperature stress during the early unicellular microspore stage caused a failure in microsporogenesis. Furthermore, potential effects of the visual difference on the bee behavior were also discussed through the colorimetry. The sterility, likely induced by low-temperature stress, and the preference of honeybees for fluorescence may reduce the pollination efficiency of P. mume.

Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear.

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB(-α-γ)) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S(4)-RNase and 378 kb around S(2)-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB(4-u1-u4, 4-d1-d2) and PpSFBB(2-u1-u5,) (2-d1-d5), respectively) were found, but PpSFBB(4-α-γ) and PpSFBB(2-γ) were absent. The PpSFBB(4) genes showed 66.2-93.1% amino acid identity with the PpSFBB(2) genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3-94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.

Deletion of a 236 kb region around S 4-RNase in a stylar-part mutant S 4sm-haplotype of Japanese pear.

Japanese pear (Pyrus pyrifolia Nakai) has a gametophytic self-incompatibility (GSI) mechanism controlled by a single S-locus with multiple S-haplotypes, each of which contains separate genes that determine the allelic identity of pistil and pollen. The pistil S gene is the S-ribonuclease (S-RNase) gene, whereas good candidates for the pollen S gene are the F-box protein genes. A self-compatible (SC) cultivar, 'Osa-Nijisseiki', which is a bud mutant of 'Nijisseiki' (S (2) S (4)), has a stylar-part mutant S(4)sm-haplotype, which lacks the S (4)-RNase gene but retains the pollen S gene. To delineate the deletion breakpoint in the S(4)sm-haplotype, we constructed a bacterial artificial chromosome (BAC) library from an S (4)-homozygote, and assembled a BAC contig of 570 kb around the S (4)-RNase. Genomic PCR of DNA from S (4)- and S(4)sm-homozygotes and the DNA sequence of the BAC contig allowed the identification of a deletion of 236 kb spanning from 48 kb upstream to 188 kb downstream of S (4)-RNase. The S(4)sm-haplotype lacks 34 predicted open reading frames (ORFs) including the S (4)-RNase and a pollen-specific F-box protein gene (termed as S (4) F-box0). Genomic PCR with a primer pair designed from the deletion junctions yielded a product specific for the S(4)sm-haplotype. The product could be useful as a maker for early selection of SC cultivars harboring the S(4)sm-haplotype.

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear (Pyrus pyrifolia).

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear (Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S(2)S(4)). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S(4)- and S(2)-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T(2) family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5-221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S(2)-RNase was 8.8-28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.

Development of a CAPS marker system for genotyping European pear cultivars harboring 17 S alleles.

The full-length cDNAs of eight S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars that could not be characterized by the cleaved amplified polymorphic sequences (CAPS) marker system for genotyping European pear cultivars harboring nine S alleles Sa, Sb, Sd, Se, Sh, Sk, Sl, Sq, and Sr. Comparison of the nucleotide sequences between these cDNAs and six putative S-RNase alleles previously amplified by genomic PCR revealed that five corresponded to the putative Sc-, Si-, Sm-, Sn-, and Sp-RNase alleles and the other three corresponded new S-RNase alleles (designated as putative Sg-, Ss-, and St-RNase alleles). Genomic PCR with a new set of primers was used to amplify 17 S-RNase alleles: 1906 bp (Sg), 1642 bp (St), 1414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb), and ca. 350 bp (Sa, Sc, Sd, Sh, Si, Sm, Sn, Sp, Sr, and Ss). Among them, S-RNase alleles of similar size were discriminated by digestion with 11 restriction endo-nucleases. The PCR amplification of 17 S-RNase alleles following digestion with the restriction endonucleases provided a new CAPS marker system for rapid S-genotyping of European pear cultivars harboring 17 S alleles. Using the CAPS analysis, Sc, Sg, Si, Sm, Sn, Sp, Ss, and St alleles were found in 32 cultivars, which were classified into 23 S-genotypes.

Molecular cloning and expression of the mannose/glucose specific lectin from Castanea crenata cotyledons.

cDNA clones encoding a mannose/glucose specific lectin, CCA, from Castanea crenata cotyledons have been isolated and sequenced. The cloned CCA cDNA had an open reading frame of 927 bp encoding 309 amino acid residues. Compared with the amino acid sequence determined for the protein chemically, it was clarified that CCA has no signal peptide and undergoes no proteolytic cleavage as do other mannose specific Jacalin-related lectins. The coding region of CCA was introduced into an expression vector, pET-22b(+), and then transferred into Escherichia coli BL21(DE3). Although recombinant CCA (rCCA) accumulated as inclusion bodies, refolded rCCA exhibited a similar CD spectrum to nCCA and regained the hemagglutination activity. In addition, a hapten inhibition assay revealed that nCCA and rCCA showed the same specificities toward sugars and glycoproteins. On measurement by GPC-MALLS in the native state, the absolute molecular mass of nCCA was found to be 332 7 kDa, which indicated that nCCA is a decamer of identical subunits having a molecular mass of 33 kDa. The same as the natural molecule, rCCA showed a molecular mass of 320 +/- 5 kDa and was judged to also be a decamer. These results indicate that the rCCA obtained in this study is equivalent to nCCA.