Sucrose, cell wall, and polyamine metabolisms involve in preserving postharvest quality of 'Zaosu' pear fruit by L-glutamate treatment.

'Zaosu' pear fruit is prone to yellowing of the surface and softening of the flesh after harvest. This work was performed to assess the influences of L-glutamate treatment on the quality of 'Zaosu' pears and elucidate the underlying mechanisms involved. Results demonstrated that L-glutamate immersion reduced ethylene release, respiratory intensity, weight loss, brightness (L*), redness (a*), yellowness (b*), and total coloration difference (ΔE); enhanced ascorbic acid, soluble solids, and soluble sugar contents; maintained chlorophyll content and flesh firmness of pears. L-glutamate also restrained the activities of neutral invertase and acid invertase, while enhancing sucrose phosphate synthetase and sucrose synthase activities to facilitate sucrose accumulation. The transcriptions of PbSGR1, PbSGR2, PbCHL, PbPPH, PbRCCR, and PbNYC were suppressed by L-glutamate, resulting in a deceleration of chlorophyll degradation. L-glutamate concurrently suppressed the transcription levels and enzymatic activities of polygalacturonases, pectin methylesterases, cellulase, and ß-glucosidase. It restrained polygalacturonic acid trans-eliminase and pectin methyl-trans-eliminase activities as well as inhibited the transcription levels of PbPL and Pbß-gal. Moreover, the gene transcriptions and enzymatic activities of arginine decarboxylase, ornithine decarboxylase, S-adenosine methionine decarboxylase, glutamate decarboxylase, γ-aminobutyric acid transaminase, glutamine synthetase along with the PbSPDS transcription was promoted by L-glutamate. L-glutamate also resulted in the down-regulation of PbPAO, PbDAO, PbSSADH, PbGDH, and PbGOGAT transcription levels, while enhancing γ-aminobutyric acid, glutamate, and pyruvate acid contents in pears. These findings suggest that L-glutamate immersion can effectively maintain the storage quality of 'Zaosu' pears via modulating key enzyme activities and gene transcriptions involved in sucrose, chlorophyll, cell wall, and polyamine metabolism.

Self S-RNase inhibits ABF-LRX signaling to arrest pollen tube growth to achieve self-incompatibility in pear.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

AIMS: Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. METHODS AND RESULTS: A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46-fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. CONCLUSIONS: AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. SIGNIFICANCE AND IMPACT OF STUDY: Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

A semi in vivo pollination technique to assess the level of gametophytic self-incompatibility and pollen tube growth in pear (Pyrus communis L.).

KEY MESSAGE: We describe a semi in vivo pollination technique to determine the compatibility relation between different pear cultivars. This assay provides a valuable addition to existing tools in GSI research. The gametophytic self-incompatibility (GSI) system in Pyrus inhibits fertilization by pollen that shares one of the two S-alleles of the style. Depending on their S-locus genotype, two pear cultivars therefore either show a cross-compatible, semi-compatible or incompatible interaction. Because GSI greatly influences seed and fruit set, accurate knowledge of the compatibility type of a cultivar is key for both pear fruit production and breeding. Currently, compatibility relations between different pear cultivars are generally assessed via S-genotyping. However, this approach is restricted to the currently known S-alleles in pear, and does not provide functional assessment of the level of (self-)incompatibility. We here present an optimized semi in vivo pollination assay, that enables quantitative analysis of (self-)incompatibility in pear, and that can also serve useful for more fundamental studies on pollen tube development and pollen-style interactions. This assay involves in vitro incubation of cut pollinated styles followed by microscopic counting of emerging pollen tubes at a specific time interval. The validity and selectivity of this method to determine compatibility interactions in pear is demonstrated in the cultivars "Celina" and "Packham's Triumph." Overall, this technique constitutes a valuable tool for quantitatively determining in vivo pollen tube growth and (cross-)compatibility in pear.

Characterization of the pectin methylesterase inhibitor gene family in Rosaceae and role of PbrPMEI23/39/41 in methylesterified pectin distribution in pear pollen tube.

MAIN CONCLUSION: Pectin methylesterase inhibitor gene family in the seven Rosaceae species (including three pear cultivars) is characterized and three pectin methylesterase inhibitor genes are identified to regulate pollen tube growth in pear. Pectin methylesterase inhibitor (PMEI) participates in a variety of biological processes in plants. However, the information and function of PMEI genes in Rosaceae are largely unknown. In this study, a total of 423 PMEI genes are identified in the genomes of seven Rosaceae species. The PMEI genes in pear are categorized into five subfamilies based on structural analysis and evolutionary analysis. WGD and TD are the main duplication events in the PMEI gene family of pear. Quantitative real-time PCR analysis indicates that PbrPMEI23, PbrPMEI39, and PbrPMEI41 are increasingly expressed during pear pollen tube growth. Under the treatment of recombinant proteins PbrPMEI23, PbrPMEI39 or PbrPMEI41, the content of methylesterified pectin at the region 5-20 µm from the pollen tube tip significantly increases, and the growth of pear pollen tubes is promoted. These results indicate that PMEI regulates the growth of pollen tubes by changing the distribution of methylesterified pectin in the apex.

The MIR-Domain of PbbHLH2 Is Involved in Regulation of the Anthocyanin Biosynthetic Pathway in "Red Zaosu" (PyrusBretschneideri Rehd.) Pear Fruit.

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in "Zaosu" pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri).

Pectin methyl-esterases (PMEs) play crucial roles in plant growth. In this study, we identified 81 PbrPMEs in pear. Whole-genome duplication and purifying selection drove the evolution of PbrPME gene family. The expression of 47 PbrPMEs was detected in pear pollen tube, which were assigned to 13 clusters by an expression tendency analysis. One of the 13 clusters presented opposite expression trends towards the changes of methyl-esterified pectins at the apical cell wall. PbrPMEs were localized in the cytoplasm and plasma membrane. Repression of PbrPME11, PbrPME44, and PbrPME59 resulted in the inhibition of pear pollen tube growth and abnormal deposition of methyl-esterified pectins at pollen tube tip. Pharmacological analysis confirmed that reduced PbrPME activities repressed the pollen tube growth. Overall, we have explored the evolutionary characteristics of PbrPME gene family and found the key PbrPME genes that control the growth of pollen tube, which deepened the understanding of pear fertility regulation.

Glycine betaine treatment alleviates loss of aroma-related esters in cold-stored 'Nanguo' pears by regulating the lipoxygenase pathway.

Glycine betaine (GB) is known to alleviate chilling injury in many fruit species. Therefore, we studied how GB affects the biosynthesis of esters in 'Nanguo' pears. Based on the kinds of esters, total esters, and the quantity of the main esters, it was evident that aroma losses were alleviated by GB treatment. In addition, unsaturated fatty acids contents (linoleic and linolenic acid) and the activities of lipoxygenase (LOX) and alcohol acyltransferase (AAT) enzymes were also increased. Meanwhile, comparing with the control fruit, the genes directly involved in ester synthesis were up-regulated in the GB-treated fruit. In addition, an increase in the activities and gene expression of antioxidant enzymes was observed in the treated samples. Thus, GB treatment promotes the synthesis of esters by regulating the LOX pathway and increasing antioxidant capacity, thereby effectively improving the quality of esters in cold-stored fruit.

Genome Resource for Two Stemphylium vesicarium Isolates Causing Stemphylium Leaf Blight of Onion in New York.

Stemphylium leaf blight caused by Stemphylium vesicarium was recently identified as an emerging disease and dominant in the foliar disease complex affecting onion in New York. Here, we report the genomes of two isolates of S. vesicarium, On16-63 and On16-391. The availability of the genomes will accelerate genomic studies of S. vesicarium, including population biology, sexual reproduction, and fungicide resistance. Additionally, comparative genomics with the other published genome of S. vesicarium causing brown spot of pear will help understand pathogen biology and underpin the development of management strategies for this disease.

RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of pollination.

KEY MESSAGE: At the early stage of pollination, the difference in gene expression between compatibility and incompatibility is highly significant about the pollen-specific expression of the LRR gene, resistance, and defensin genes. In Rosaceae, incompatible pollen can penetrate into the style during the gametophytic self-incompatibility response. It is therefore considered a stylar event rather than a stigmatic event. In this study, we explored the differences in gene expression between compatibility and incompatibility in the early stage of pollination. The self-compatible pear variety "Jinzhuili" is a naturally occurring bud mutant from "Yali", a leading Chinese native cultivar exhibiting typical gametophytic self-incompatibility. We collected the styles of 'Yali' and 'Jinzhuili' at 0.5 and 2 h after self-pollination and then performed high-throughput sequencing. According to the KEGG analysis of the differentially expressed genes, several metabolic pathways, such as "Plant hormone signal transduction", "Plant-pathogen interaction", are the main pathways was the most represented pathway. Quantitative PCR was used to validate these differential genes. The expression levels of genes related to pollen growth and disease inhibition, such as LRR (Leucine-rich repeat extensin), resistance, defensin, and auxin, differed significantly between compatible and incompatible pollination. Interestingly, at 0.5 h, most of these genes were upregulated in the compatible pollination system compared with the incompatible pollination system. Calcium transport, which requires ATPase, also demonstrated upregulated expression. In summary, the self-incompatibility reaction was initiated when the pollen land on the stigma.

Genetic rescue by distant trees mitigates qualitative pollen limitation imposed by fine-scale spatial genetic structure.

Restricted seed dispersal frequently leads to fine-scale spatial genetic structure (i.e., FSGS) within plant populations. Depending on its spatial extent and the mobility of pollinators, this inflated kinship at the immediate neighbourhood can critically impoverish pollen quality. Despite the common occurrence of positive FSGS within plant populations, our knowledge regarding the role of long-distance pollination preventing reproductive failure is still limited. Using microsatellite markers, we examined the existence of positive FSGS in two low-density populations of the tree Pyrus bourgaeana. We also designed controlled crosses among trees differing in their kinship to investigate the effects of increased local kinship on plant reproduction. We used six pollination treatments and fully monitored fruit production, fruit and seed weight, proportion of mature seeds per fruit, and seed germination. Our results revealed positive FSGS in both study populations and lower fruit initiation in flowers pollinated with pollen from highly-genetically related individuals within the neighbourhood, with this trend intensifying as the fruit development progressed. Besides, open-pollinated flowers exhibited lower performance compared to those pollinated by distant pollen donors, suggesting intense qualitative pollen limitation in natural populations. We found positive fine-scale spatial genetic structure is translated into impoverished pollen quality from nearby pollen donors which negatively impacts the reproductive success of trees in low-density populations. Under this scenario of intrapopulation genetic rescue by distant pollen donors, the relevance of highly-mobile pollinators for connecting spatially and genetically distant patches of trees may be crucial to safeguarding population recruitment.

Effects of melatonin treatment on the enzymatic browning and nutritional quality of fresh-cut pear fruit.

The effects of exogenous melatonin treatment on the enzymatic browning and nutritional quality of fresh-cut pear fruit were investigated. Fresh-cut fruit soaked with 0, 0.05, 0.1 and 0.5 mM melatonin were stored at 4 °C. Our results showed that 0.1 mM melatonin treatment was optimal for reducing the surface browning and maintaining the titratable acidity of the fresh-cut fruit, which significantly decreased MDA and H2O2 contents and the growth of microorganism, enhanced total phenolic content and antioxidant capacity, and delayed the reduction of ascorbic acid. Furthermore, melatonin treatment at 0.1 mM decreased the expression of genes involving in enzymatic browning pathway including POD, PPO1, PPO5 and LOX1, and reduced PPO activity. Moreover, this treatment increased the expression of PAL and CHS, and enhanced PAL and CHS activities. These results showed that melatonin treatment might be a promising strategy to alleviate browning and improve the nutritional quality of fresh-cut pear fruit.

Characterization of the Genes Involved in Malic Acid Metabolism from Pear Fruit and Their Expression Profile after Postharvest 1-MCP/Ethrel Treatment.

In this study, five genes involved in malic acid (MA) metabolism, including a cytosolic NAD-dependent malate dehydrogenase gene ( cyNAD-MDH), a cytosolic NADP-dependent malic enzyme gene ( cyNADP-ME), two vacuolar H+-ATPase genes ( vVAtp1 and vVAtp2), and one vacuolar inorganic pyrophosphatase gene ( vVPp), were characterized from pear fruit based on bioinformatic and experimental analysis. Their expression profile in "Housui" pear was tissue-specific, and their expression patterns during fruit development were diverse. During "Housui" pear storage, MA content decreased, which was associated with the downregulated transcripts of MA metabolism-related genes and cyNAD-MDH activity and higher cyNADP-ME activity. The response of MA metabolism to postharvest 1.5 µL L-1 1-MCP fumigation and 0.5 mL L-1 ethrel dipping was distinct: 1-MCP fumigation upregulated gene expression and cyNAD-MDH activity and suppressed cyNADP-ME activity, and thus maintained higher MA abundance when compared with those in the control; on the other hand, an opposite behavior was observed in ethrel-treated fruit.

iTRAQ-Based Identification of Proteins Related to Lignin Synthesis in the Pear Pollinated with Pollen from Different Varieties.

Most pears in Anhui Province are a kind of self-incompatible fruit whose quality is strongly influenced by the male pollen. The proteomic variation of Dangshan Su pollinated by different varieties was analysed using the isobaric tag for relative and absolute quantitation (iTRAQ) to investigate the effect of pollination by different varieties on the pear lignin pathway. Among the 3980 proteins identified from the two samples, 139 proteins were identified as differentially expressed proteins (DEPs). Of these proteins, laccase-4 (LAC4), was found to be related with lignin synthesis, and ß-glucosidase 15 (BGLU15) and peroxidase 47 (PER47) were involved in the phenylpropanoid pathway. Moreover, the lignin and stone cell contents were lower in DW (Dangshan Su pollinated by Wonhwang) than those in DJ (Dangshan Su pollinated by Jingbaili). The effect of pollination on the synthesis of lignin through the regulation of the expression of PER47, BGLU15 and LAC4 ultimately affects the formation of stone cells and the fruit quality. We report for the first time that different pollinations influence the protein expression profile in the Dangshan Su pear, and this result provides some new epididymal targets for regulating the synthesis of lignin, regulating the content of stone cells and improving the quality of the pears.

B-BOX genes: genome-wide identification, evolution and their contribution to pollen growth in pear (Pyrus bretschneideri Rehd.).

BACKGROUND: The B-BOX (BBX) proteins have important functions in regulating plant growth and development. In plants, the BBX gene family has been identified in several plants, such as rice, Arabidopsis and tomato. However, there still lack a genome-wide survey of BBX genes in pear. RESULTS: In the present study, a total of 25 BBX genes were identified in pear (Pyrus bretschneideri Rehd.). Subsequently, phylogenetic relationship, gene structure, gene duplication, transcriptome data and qRT-PCR were conducted on these BBX gene members. The transcript analysis revealed that twelve PbBBX genes (48%) were specifically expressed in pear pollen tubes. Furthermore, qRT-PCR analysis indicated that both PbBBX4 and PbBBX13 have potential role in pear fruit development, while PbBBX5 should be involved in the senescence of pear pollen tube. CONCLUSIONS: This study provided a genome-wide survey of BBX gene family in pear, and highlighted its roles in both pear fruits and pollen tubes. The results will be useful in improving our understanding of the complexity of BBX gene family and functional characteristics of its members in future study.

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing.

BACKGROUND: MicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures. RESULTS: Using in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots. CONCLUSIONS: This is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.

Expression and regulation of pear 1-aminocyclopropane-1-carboxylic acid synthase gene (PpACS1a) during fruit ripening, under salicylic acid and indole-3-acetic acid treatment, and in diseased fruit.

In plants, the level of ethylene is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). A gene encoding an ACC synthase protein was isolated from pear (Pyrus pyrifolia). This gene designated PpACS1a (GenBank accession no. KC632526) was 1488 bp in length with an open reading frame (ORF) encoding a protein of 495 amino acids that shared high similarity with other pear ACC synthase proteins. The PpACS1a was grouped into type-1 subfamily of plant ACS based on its conserved domain and phylogenetic status. Real-time quantitative PCR indicated that PpACS1a was differentially expressed in pear tissues and predominantly expressed in anthers. The expression signal of PpACS1a was also detected in fruit and leaves, but no signal was detected in shoots and petals. Furthermore, the PpACS1a expression was regulated during fruit ripening. In addition, the PpACS1a gene expression was regulated by salicylic acid (SA) and indole-3-acetic acid (IAA) in fruit. Moreover, the expression of the PpACS1a was up-regulated in diseased pear fruit. These results indicated that PpACS1a might be involved in fruit ripening and response to SA, IAA and disease.

A bioassessment of soil nickel genotoxic effect in orchard planted on rehabilitated coalmine overburden.

Environmental problems of non-rehabilitated overburden material are present in surrounding of open coal mines worldwide. Ecological restoration of this soil material usually deals with the improvement of its bad physico-chemical properties and its poor nutrient status, sometimes associated with heavy metal problems. Applied overburden restoration by planting orchard (1990) is assumed to be the first of its kind at opencast mines globally, so that present work was aimed at acquiring information about its efficiency of the applied measures concerning their possible use in agriculture. Various physical and chemical properties, together with the pseudo total and DTPA extractable metals (Fe, Mn, Cu, Zn, Co, Ni, Pb, Cr, Cd) as well as sequential Ni extraction analyses, was measured, in order to evaluate the impact of soil's Ni level (76.3-111.7 mg kg⁻¹) on decreasing yields of apples, pears and plums. As a general pattern, reclaimed soil was significantly enriched with organic matter (>2.5 percent) and nutrients compared to the initial (2 m depth) and non-reclaimed adjacent soil, approving this method for overburden restoration. Despite low Ni concentration in organs, Ni accumulation in a fruits' trees qualified these species as suitable for phytostabilization of present heavy metals, with a woody biomass as a large and important sink for Ni, especially in the roots. Applied cytogenetic studies evaluate the lack of genotoxic effect of nickel (Ni) on the gametic cells of investigated species, having no significant effect on meiosis and pollen germination. Most of the found anomalies were in apples, as a kind of aberrations with sticky figures and chromosome lagging, should be ascribed to the environmental and genetic interaction over the aging of trees.

Genetic features of the spontaneous self-compatible mutant, 'Jin Zhui' (Pyrus bretschneideri Rehd.).

'Jin Zhui' is a spontaneous self-compatible mutant of 'Ya Li' (Pyrus bretschneideri Rehd. S21S34 ), the latter displaying a typical S-RNase-based gametophytic self-incompatibility (GSI). The pollen-part mutation (PPM) of 'Jin Zhui' might be due to a natural mutation in the pollen-S gene (S34 haplotype). However, the molecular mechanisms behind these phenotypic changes are still unclear. In this study, we identified five SLF (S-Locus F-box) genes in 'Ya Li', while no nucleotide differences were found in the SLF genes of 'Jin Zhui'. Further genetic analysis by S-RNase PCR-typing of selfed progeny of 'Jin Zhui' and 'Ya Li' × 'Jin Zhui' progeny showed three progeny classes (S21S21 , S21S34 and S34S34 ) as opposed to the two classes reported previously (S21S34 and S34S34 ), indicating that the pollen gametes of 'Jin Zhui', bearing either the S21 - or S34 -haplotype, were able to overcome self-incompatibility (SI) barriers. Moreover, no evidence of pollen-S duplication was found. These findings support the hypothesis that loss of function of S-locus unlinked PPM expressed in pollen leads to SI breakdown in 'Jin Zhui', rather than natural mutation in the pollen-S gene (S34 haplotype). Furthermore, abnormal meiosis was observed in a number of pollen mother cells (PMCs) in 'Jin Zhui', but not in 'Ya Li'. These and other interesting findings are discussed.

Identification of a canonical SCF(SLF) complex involved in S-RNase-based self-incompatibility of Pyrus (Rosaceae).

S-RNase-based self-incompatibility (SI) is an intraspecific reproductive barrier to prevent self-fertilization found in many species of the Solanaceae, Plantaginaceae and Rosaceae. In this system, S-RNase and SLF/SFB (S-locus F-box) genes have been shown to control the pistil and pollen SI specificity, respectively. Recent studies have shown that the SLF functions as a substrate receptor of a SCF (Skp1/Cullin1/F-box)-type E3 ubiquitin ligase complex to target S-RNases in Solanaceae and Plantaginaceae, but its role in Rosaceae remains largely undefined. Here we report the identification of two pollen-specific SLF-interacting Skp1-like (SSK) proteins, PbSSK1 and PbSSK2, in Pyrus bretschneideri from the tribe Pyreae of Rosaceae. Both yeast two-hybrid and pull-down assays demonstrated that they could connect PbSLFs to PbCUL1 to form a putative canonical SCF(SLF) (SSK/CUL1/SLF) complex in Pyrus. Furthermore, pull-down assays showed that the SSK proteins could bind SLF and CUL1 in a cross-species manner between Pyrus and Petunia. Additionally, phylogenetic analysis revealed that the SSK-like proteins from Solanaceae, Plantaginaceae and Rosaceae form a monoclade group, hinting their shared evolutionary origin. Taken together, with the recent identification of a canonical SCF(SFB) complex in Prunus of the tribe Amygdaleae of Rosaceae, our results show that a conserved canonical SCF(SLF/SFB) complex is present in Solanaceae, Plantaginaceae and Rosaceae, implying that S-RNase-based self-incompatibility shares a similar molecular and biochemical mechanism.