Huaqing Wu

S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia

Pear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca2+ current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo.

S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia in vitro.

Pear (Pyrus pyrifolia L.) has a S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. No studies, however, have examined the extent of organelle alterations during the SI response in Pyrus pyrifolia. Consequently, this study focused on the alterations to mitochondria and nuclear DNA in incompatible pollen tubes of the pear. Methylthiazolyldiphenyl-tetrazolium bromide was used to evaluate the viability of pollen tubes under S-RNase challenge. The results showed that the viability of the control and compatible pollen tubes decreased slightly, but that of the incompatible pollen and pollen tubes began to decline at 30 min. The mitochondrial membrane potential (Delta psi(mit)) was also tested with rhodamine 123 30 min after SI challenge, and was shown to have collapsed in the incompatible pollen tubes after exposure to S-RNase. Western blotting 2 h after SI challenge confirmed that the Delta psi(mit) collapse induced leakage of cytochrome c into the cytosol. Swollen mitochondria were detected by transmission electron microscopy as early as 1 h after SI challenge and the degradation of nuclear DNA was observed by both 4,6-diamidino-2-phenylindole and transferase-mediated dUTP nick-end labeling. These diagnostic features of programmed cell death (PCD) suggested that PCD may specifically occur in incompatible pollen tubes.

Competitive interaction between two functional S-haplotypes confer self-compatibility on tetraploid Chinese cherry (Prunus pseudocerasus Lindl. CV. Nanjing Chuisi)

Self-incompatibility (SI) has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility (GSI). In the present study, four PpsS-haplotypes (Prunus pseudocerasus S-haplotypes) comprising at least two genes, i.e., PpsS-RNase (P. pseudocerasus S-RNase) and PpsSFB (P. pseudocerasus S-haplotype-specific F-box) have been successfully isolated in tetraploid P. pseudocerasus Lindl. CV. Nanjing Chuisi (“NC”) which exhibited self-compatibility (SC), and its S-genotype was determined as S-1/S-3′/S-5/S-7. These PpsS-RNases, which were expressed exclusively in style, shared the typical structural features with S-RNases from other Prunus species exhibiting GSI. All PpsSFBs showed similar structure characteristics of SFBs from other Prunus species, and matched with the necessary conditions for pollen S-determinant. No mutations leading to dysfunction of S-haplotype were found in their full-length c-DNA sequences, except for PpsS-3′-haplotype which was not amplified by PCR. These four S-haplotypes complied with tetrasomic inheritance. Diploid pollen grains with S-genotypes S-7/S-1, S-7/S-5 and S-1/S-5 can grow the full length of the style after self-pollination, while pollen grains with S-3′/S-7, S-3′/S-1 and S-3′/S-5 cannot. These results suggest that PpsS-haplotypes-1, -5 and -7 are functional, and that competitive interaction between two of them confer self-compatibility on cultivar “NC”. Furthermore, in terms of recognition specificity, diploid pollen grains carrying PpsS-3′-haplotype are equal to monoploid pollen grains carrying the other functional S-haplotype.

Reciprocal regulation of Ca2+‐activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide

• The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. • In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. • Heme (1 μM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 μM intracellular free Ca²+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. • These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.

Identification of S-haplotype-specific S-RNase and SFB alleles in native Chinese apricot (Prunus armeniaca L.).

Summary Chinese apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility (GSI) controlled by a single locus containing at least two linked genes [i.e., the S-RNase gene and the pollen-expressed SFB (or SLF) gene] as do other fruit species in the family, Rosaceae. To elucidate the S-genotypes of 14 native Chinese apricot cultivars, PCR was performed using primers designed from Prunus S-RNase and SFB consensus sequences. After cloning and sequencing the PCR products, the S-genotypes of all 14 apricot cultivars were determined, and eight new S-RNase alleles and nine SFB alleles were identified. The S-RNases shared typical structural features with S-RNases from other Prunus spp. exhibiting GSI. The SFB genes showed similar structural characteristics to SFB genes in other Prunus spp. The intron sequences of the SFB genes revealed sequence and length polymorphisms. The deduced level of amino acid sequence identity for the eight new S-RNase alleles was 66.4 – 100% in P. armeniaca, while the similarity of the SFB alleles was 73.7 – 98.6%. The physical distances between the SFB and S-RNase genes was determined exactly in the S9,S11,S17, and S26-haplotypes, confirming that the S-RNase and SFB genes were linked. The range of distances between the two genes was 299 – 1,061 bp. This study increases our knowledge on the S-genotypes of apricot native to China, and enriches our genomic information on GSI in the Prunus genus.

Self-compatibility of ‘Katy’ apricot ( Prunus armeniaca L.) is associated with pollen-part mutations

Apricot (Prunus armeniaca L.) cultivars originated in China display a typical S-RNase-based gametophytic self-incompatibility (GSI). ‘Katy’, a natural self-compatible cultivar belonging to the European ecotype group, was used as a useful material for breeding new cultivars with high frequency of self-compatibility by hybridizing with Chinese native cultivars. In this work, the pollen-S genes (S-haplotype-specific F-box gene, or SFB gene) of ‘Katy’ were first identified as SFB1 and SFB8, and the S-genotype was determined as S1S8. Genetic analysis of ‘Katy’ progenies under controlled pollination revealed that the stylar S1-RNase and S8-RNase have a normal function in rejecting wild-type pollen with the same S-haplotype, while the pollen grains carrying either the SFB1 or the SFB8 gene are both able to overcome the incompatibility barrier. However, the observed segregation ratios of the S-genotype did not fit the expected ratios under the assumption that the pollen-part mutations are linked to the S-locus. Moreover, alterations in the SFB1 and SFB8 genes and pollen-S duplications were not detected. These results indicated that the breakdown of SI in ‘Katy’ occurred in pollen, and other factors not linked to the S-locus, which caused a loss of pollen S-activity. These findings support a hypothesis that modifying factors other than the S-locus are required for GSI in apricot.

Identification of S-genotypes in 17 Chinese cultivars of Japanese plum (Prunus salicina Lindl.) and molecular characterisation of 13 novel S-alleles

Summary Polymerase chain reaction (PCR) was conducted with Prunus S-RNase gene-specific primers on 17 Chinese cultivars of Japanese plum (Prunus salicina Lindl.). These primers were designed from the conserved regions of Prunus S-RNase genes. Each cultivar produced two amplicons, apart from two cultivars that had three amplicons. In all, 36 amplicons were cloned and sequenced. Analysis of these sequences revealed 13 novel S-alleles, the amino acid sequences of which showed 62% (S15 vs. S19) to 92% (S22 vs. S24) identity. The sequences also demonstrated several typical structural features of Prunus S-RNase genes: three conserved regions (C1, C2 and C3), one hypervariable region (RHV) with one intron, and another intron located at the junction between the signal peptide and the mature protein. Compared to S2 from apricot, S26 from Japanese plum had only two nucleotide substitutions in the exon region, which resulted in only one amino acid residue difference in the signal peptide. However, there were large numbers of nucleotide differences in the intron regions. Phylogenetic analysis of the 13 novel S-alleles, and those of other species in the family Rosaceae, resulted in two distinct groups which correlated with their sub-family classification (i.e., the Maloideae and the Prunoideae). These data should be useful in breeding programmes, for choosing suitable pollinators, and may also contribute to studies on S-allele function, the evolution of new allele specificities, and the taxonomy and speciation of Prunus.

Identification of S -genotypes in Chinese cherry cultivars ( Prunus pseudocerasus Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae, and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility. In the present study, we successfully isolated nine S-RNase alleles from cultivars of Chinese cherry by PCR amplification from genomic DNA and stylar cDNA combining with cleaved amplified polymorphic sequence marker. Analysis of amino acid sequences revealed five novel S-alleles, S2, S4, S6, S8, and S9, with respective accession numbers in the NCBI database of EF541168, EF541173, EF541172, FJ628598, and FJ628599. Results showed that “Dongtang” and “Yinzhu” contained six S-alleles (S1, S3, S5, S7, S8, and S9); “Taishanganying” contained four S-alleles (S1, S2, S4, and S6); “Daiba”, “Dayingzui”, and “Xiaomizi” contained four S-alleles (S1, S2, S5, and S8); “Laiyangduanzhi”, “Shuangquanchangba”, and “Daqingye” contained three S-alleles (S1, S2, and S8). It is interesting that different cultivars collected from the same place hold the same S-genotypes. Moreover, pollination tests and pollen tube growth assays showed that nine cultivars were self-compatible. Chinese cherry presented in this article are naturally polyploidy, which is a very useful material for the study of self-compatibility, and much of this information will be valuable for further work on self-(in)compatibility of fruit tree in Rosaceae.

Recognition specificity of self-incompatibility in Pyrus and Malus

Pyrus displays gametophytic self-incompatibility controlled by a single highly polymorphic gene complex termed S locus, which comprises a stylar-expressed gene (S-RNase) tighlty linked with a pollen expressed gene, that determines the specificity of the self-incompatibility locus. Deduced amino acid sequence of ‘Meigetsu’ S8-RNase in Pyrus pyrifolia and ‘Kuerlexiangli’ S28-RNase in P. sinkiangensis showed 100% identity. S3-RNase in Malusspectabilis was also found to be similar to S8-RNase in P. pyrifolia with 96.9% identity in the deduced amino acid sequence. The intron, which is generally highly polymorphic between alleles, was also remarkably well conserved within these allele pairs. The intron of PpS8-RNase showed 95.3 and 91.9% identity with PsS28-RNase and MsS3-RNase, respectively. Pollen tube growth in styles, pollen tube length in artificial media containing different S-RNases and segregation of S haplotypes in F1 plants revealed commonality of the recognition specificity between PpS8-RNase and PsS28-RNase and between PpS8-RNase and MsS3-RNase. Results suggested that PpS8-RNase, PsS28-RNase and MsS3-RNase have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found between PpS8-RNase and MsS3-RNase do not alter the recognition specificity.

Identification of S-genotypes and novel S-RNase alleles in Prunus mume

Summary Prunus mume (Japanese apricot) cultivars are self-incompatible and orchards require pollinator cultivars to guarantee optimal fruit production. Identification of the self-incompatibility (S) genotypes of P. mume cultivars is therefore useful for choosing pollinators and for breeding. To elucidate the S-genotypes of eleven Chinese and one introduced cultivar, PCR was performed with primers amplifying the variable second intron of the S-RNase gene. The S-genotyping results were as follows: S4S12 for ‘Dantaofen’; S3S4 for ‘Duoezhusha’; S12S13 for ‘Fubantiaozhi’; S15S16 for ‘Hongding’; S13S14 for ‘Longyanmei’; S5S11 for ‘Musashino’; S10S14 for ‘Xiaoyezhugan’; and S3S15 for ‘Qingfeng’,‘Qingjia’, ‘Xiyeqing’, ‘Dayeqing’ and ‘Changnong 17’. We identified seven new S-RNase alleles, S10-RNase to S16-RNase, deposited in GenBank under the accession numbers: DQ011150, DQ201191, DQ201192, DQ345781, DQ768219, DQ903312 and EF990750, respectively.