Molecular Mechanism Of Self-incompatibility Research Articles

Comparative Physiological, Transcriptomic, and Metabolomic Analyses of Acacia mangium Provide New Insights into Its Molecular Mechanism of Self-Incompatibility

Acacia mangium is well known as a valuable commercial tree species in the Acacia genus. A. mangium was recently found to be self-incompatible (SI), but its SI mechanism is not clear, which has hindered the progress of genetic improvement of A. mangium with strong resistance. To confirm the SI type of A. mangium, pollen germination was observed via fluorescence microscopy at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h after self-pollination. We found over ninety percent of the pollen grains produced no pollen tube growth on the stigma/style. To further explore the SI molecular mechanism of A. mangium, tests of the transcriptome and metabolome were carried out after self-pollination. Observations of pollen germination after self-pollination using fluorescence microscopy suggested that the SI type of A. mangium is gametophytic self-incompatibility (GSI). A combined transcriptomic and metabolomic analysis showed that DEGs (differentially expressed genes) related to SI (6 S-glycoproteins, 93 F-box proteins, 69 26S proteasomes, 38 calcium-dependent protein kinases/calmodulin and 41 thioredoxin genes) were significantly enriched in six KEGG (sulfur metabolism, tyrosine metabolism, phenylalanine metabolism, butanoate metabolism, and valine, leucine, and isoleucine degradation). Further analysis of these six pathways revealed the enrichment of SI-related DEGs corresponding to succinate, methylmalonate, and 3-hydroxypropane. These three metabolites were significantly downregulated. The analysis of transcripts and metabolites suggested that transcripts of SI-related gene families (thioredoxin and F-box protein) were significantly upregulated under the regulation of transcription factors (TFs) after self-pollination, leading to a decrease in metabolites (such as succinate, methylmalonate, and 3-hydroxypropionate). We also further speculated that TFs (MYB, HB-HD-ZIP, AP2/ERF-ERF, and bZIP) and gene families (thioredoxin and F-box protein) were important factors related to the SI of A. mangium.

Hormonal Signaling during dPCD: Cytokinin as the Determinant of RNase-Based Self-Incompatibility in Solanaceae.

Research into molecular mechanisms of self-incompatibility (SI) in plants can be observed in representatives of various families, including Solanaceae. Earlier studies of the mechanisms of S-RNase-based SI in petunia (Petunia hybrida E. Vilm.) demonstrate that programmed cell death (PCD) is an SI factor. These studies suggest that the phytohormon cytokinin (CK) is putative activator of caspase-like proteases (CLPs). In this work, data confirming this hypothesis were obtained in two model objects-petunia and tomato (six Solanaceae representatives). The exogenous zeatin treatment of tomato and petunia stigmas before a compatible pollination activates CLPs in the pollen tubes in vivo, as shown via the intravital imaging of CLP activities. CK at any concentration slows down the germination and growth of petunia and tomato male gametophytes both in vitro and in vivo; shifts the pH of the cytoplasm (PHc) to the acid region, thereby creating the optimal conditions for CLP to function and inhibiting the F-actin formation and/or destructing the cytoskeleton in pollen tubes to point foci during SI-induced PCD; and accumulates in style tissues during SI response. The activity of the ISOPENTENYLTRANSFERASE 5 (IPT5) gene at this moment exceeds its activity in a cross-compatible pollination, and the levels of expression of the CKX1 and CKX2 genes (CK OXIDASE/DEHYDROGENASE) are significantly lower in self-incompatible pollination. All this suggests that CK plays a decisive role in the mechanism underlying SI-induced PCD.

Genetic Components of Self-Incompatibility in Brassica Vegetables

Brassica vegetables are very important to human beings. Self-incompatibility (SI) is a common phenomenon in Brassica. Breeding by SI lines is an important way to utilize heterosis of Brassica vegetables. It is believed that the SI inheritance in Brassica species is controlled by three linkage genes on the S-locus, including SRK (S-locus receptor kinase), SCR (S-locus cystine-rich protein)/SP11 (S-locus protein 11), and SLG (S-locus glycoprotein). SRK is the female determinant and SCR/SP11 is the pollen S gene. The expression of SLG is necessary for SRK, and it enhances the SRK-mediated SI reaction. In addition to these three S-locus genes, some other functional molecules also have significant regulatory effects on SI, such as ARC1 (arm repeat containing 1), MLPK (M-locus protein kinase), Exo70A1 (exocyst compounds), THLl/THL2 (thioredoxin H-like), MOD (aquaporin), SLR (S-locus-related glycoprotein), BPCI (pollen calcium-binding protein I), etc. SI is also associated with the dominant/recessive relationship between S alleles. Here, the genetic elements and molecular mechanisms of SI, mainly in Brassica vegetables, are reviewed.

Stigmatic Transcriptome Analysis of Self-Incompatible and Compatible Pollination in Corylus heterophylla Fisch. × Corylus avellana L.

Self-incompatibility (SI) protects plants from inbreeding depression due to self-pollination and promotes the outcrossing process to maintain a high degree of heterozygosity during evolution. Corylus is an important woody oil and nut species that shows sporophytic SI (SSI). Yet the molecular mechanism of SI in Corylus remains largely unknown. Here we conducted self- (“Dawei” × “Dawei”) and cross-pollination (“Dawei” × “Liaozhen No. 7”) experiments and then performed an RNA-Seq analysis to investigate the mechanism of pollen–stigma interactions and identify those genes that may be responsible for SSI in Corylus. We uncovered 19,163 up- and 13,314 downregulated genes from the comparison of different pollination treatments. These differentially expressed genes (DEGs) were significantly enriched in plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathway–plant. We found many notable genes potentially involved in pollen–stigma interactions and SSI mechanisms, including genes encoding receptor-like protein kinases (RLK), calcium-related genes, disease-resistance genes, and WRKY transcription factors. Four upregulated and five downregulated DEGs were consistently identified in those comparison groups involving self-incompatible pollination, suggesting they had important roles in pollen–pistil interactions. We further identified the S-locus region of the Corylus heterophylla genome based on molecular marker location. This predicted S-locus contains 38 genes, of which 8 share the same functional annotation as the S-locus genes of Corylus avellana: two PIX7 homologous genes (EVM0002129 and EVM0025536), three MIK2 homologous genes (EVM0002422, EVM0005666, and EVM0009820), one aldose 1-epimerase (EVM0002095), one 3-dehydroquinate synthase II (EVM0021283), and one At3g28850 homologous gene (EVM0016149). By characterizing the pistil process during the early postpollination phase via transcriptomic analysis, this study provides new knowledge and lays the foundation for subsequent analyses of pollen-pistil interactions.

Identification of Single Nucleotide Polymorphism in Red Clover (Trifolium pratense L.) Using Targeted Genomic Amplicon Sequencing and RNA-seq.

Red clover (Trifolium pratense L.) is a diploid, naturally cross-pollinated, cool-season species. As a perennial forage legume, red clover is mostly cultivated in temperate regions worldwide. Being a non-model crop species, genomic resources for red clover have been underdeveloped. Thus far, genomic analysis used in red clover has mainly relied on simple sequence repeat (SSR) markers. However, SSR markers are sparse in the genome and it is often difficult to unambiguously map them using short reads generated by next generation sequencing technology. Single nucleotide polymorphisms (SNPs) have been successfully applied in genomics assisted breeding in several agriculturally important species. Due to increasing importance of legumes in forage production, there is a clear need to develop SNP based markers for red clover that can be applied in breeding applications. In this study, we first developed an analytical pipeline that can confidently identify SNPs in a set of 72 different red clover genotypes using sequences generated by targeted amplicon sequencing. Then, with the same filtering stringency used in this pipeline, we used sequences from publicly available RNA-seq data to identify confident SNPs in different red clover varieties. Using this strategy, we have identified a total of 69,975 SNPs across red clover varieties. Among these, 28% (19,116) of them are missense mutations. Using Medicago truncatula as the reference, we annotated the regions affected by these missense mutations. We identified 2,909 protein coding regions with missense mutations. Pathway analysis of these coding regions indicated several biological processes impacted by these mutations. Specifically, three domains (homeobox domain, pentatricopeptide repeat containing plant-like, and regulator of Vps4 activity) were identified with five or more missense SNPs. These domain might also be a functional contributor in the molecular mechanisms of self-incompatibility in red clover. Future in-depth sequence diversity analysis of these three genes may yield valuable insights into the molecular mechanism involved in self-incompatibility in red clover.

Comparative transcriptional survey between self-incompatibility and self-compatibility in Citrus reticulata Blanco

Seedlessness is an excellent economical trait, and self-incompatibility (SI) is one of important factors resulting in seedless fruit in Citrus. However, SI molecular mechanism in Citrus is still unclear. In this study, RNA-Seq technology was used to identify differentially expressed genes related to SI reaction of ‘Wuzishatangju’ (Citrus reticulata Blanco). A total of 35.67GB raw RNA-Seq data was generated and was de novo assembled into 50,364 unigenes with an average length of 897bp and N50 value of 1549. Twenty-three candidate unigenes related to SI were analyzed using qPCR at different tissues and stages after self- and cross-pollination. Seven pollen S genes (Unigene0050323, Unigene0001060, Unigene0004230, Unigene0004222, Unigene0012037, Unigene0048889 and Unigene0004272), three pistil S genes (Unigene0019191, Unigene0040115, Unigene0036542) and three genes (Unigene0038751, Unigene0031435 and Unigene0029897) associated with the pathway of ubiquitin-mediated proteolysis were identified. Unigene0031435, Unigene0038751 and Unigene0029897 are probably involved in SI reaction of ‘Wuzishatangju’ based on expression analyses. The present study provides a new insight into the molecular mechanism of SI in Citrus at the transcriptional level.

Comparative transcript profiling of gene expression between self-incompatible and self-compatible mandarins by suppression subtractive hybridization and cDNA microarray

Self-incompatibility (SI) is an important trait of Citrus plants that is exploited by farmers to produce seedless fruit. However, the molecular mechanism of SI in Citrus is not well understood. Wuzishatangju (Citrus reticulata Blanco) (SI) is an excellent seedless cultivar selected from a seedy Shatangju cultivar (self-compatible, SC) through spontaneous bud mutation. The two cultivars are therefore excellent materials for studying the mechanisms of SI and/or SC in Citrus. In this study, an integrative strategy combining eight suppression subtractive hybridization libraries with cDNA microarray was used to study the molecular mechanisms that differ between Wuzishatangju and Shatangju (control) mandarins. A custom microarray screen resulted in a total of 1,830 up- or down-regulated clones (false discovery rate <0.05 and a fold change $${ \geqq }$$ 2) obtained from 9,810 positive clones. The expression of genes involved in embryonic development, ubiquitination pathway, Ca2+-signaling pathway, gibberellins, and auxin was significantly up-regulated in SI Wuzishatangju compared with SC Shatangju mandarin. The microarray analysis suggested that the ubiquitin-mediated proteolysis pathway might be involved in the SI reaction of Wuzishatangju. Additionally, our research highlighted some main genes (mitogen-activated protein kinase, SI S1 family protein, ubiquitin-conjugating factor E4-like, auxin transporter protein 1, and gibberellin receptor) that participate in the SI reaction of Wuzishatangju and could be beneficial for understanding the evolution of SI systems and for breeding seedless citrus fruits in the future.

新規な自家不和合性機構解明への挑戦

新規な自家不和合性機構解明への挑戦

Genomics and breeding of Brassicaceae crops

Brassicaceae crops include oil crops, vegetables, condiments, fodder crops, and ornamental plants. Most of these crops belong to or are closely related to the genus Brassica. The Brassicaceae also includes the most intensively studied model plant, Arabidopsis thaliana. In molecular genetic studies of Brassicaceae crops, information on A. thaliana genes is highly useful, and many genes important for plant breeding have been identified. Different species of Brassica have various numbers of chromosomes. The genome relationship of three monogenomic species and three digenomic species that are well known as U’s triangle was discovered in Japan, and relationships between different genera have also been studied (Mizushima 1980). In these studies, interspecific and intergeneric hybridizations have been performed, and some interspecific and intergeneric hybrids have been used as breeding materials. Studies on interspecific and intergeneric hybridization are reviewed by Kaneko and Bang in this special issue. Genome synteny relationships between different species in Brassica and closely related genera are complicated, as exemplified by the different numbers of chromosomes between species. The monogenomic species of Brassica and closely related genera have three copies, on average, of genes homologous to each A. thaliana gene, indicating whole genome triplication in the evolution of the ancestral species of Brassica and closely related genera. The recent development of next-generation sequencers has accelerated the determination of whole genome sequences from many species. A draft sequence of the Brassica rapa genome was published in 2011 (Wang et al. 2011), and whole genome sequences of other species of Brassicaceae crops will also be published soon. The progress with genome studies of Brassicaceae crops and genome relationships between them are described by Ashutosh et al. Many Brassicaceae crops have self-incompatibility, which is not common in other seed-propagated crops. Self-incompatibility is a commonly found trait in many angiosperm species, but self-compatible plants have been selected as crops. The genetics and molecular mechanisms of self-incompatibility have been most intensively studied using Brassica species, and this trait is also used as a tool for producing F1 hybrid seeds of Brassicaceae vegetables. Recent studies on Brassica self-incompatibility are introduced by Kitashiba and Nasrallah. On the other hand, the trait most widely used for F1 hybrid seed production in many crop species is cytoplasmic male sterility. Cytoplasmic male-sterile lines can be obtained by interspecific hybridization and repeated backcrossing with a paternal species, and this form of sterility is considered to be caused by incompatibility between the cytoplasm and nucleus. Cytoplasmic male sterility in Brassicaceae crops is discussed here by Yamagishi and Bhat. Brassicaceae crops generally contain glucosinolates, which are degraded to isothiocyanates by the endogenous enzyme myrosinase after disruption of cells. Isothiocyanates are pungent compounds, probably important for defense against herbivores, and they determine the taste or quality of vegetables, condiments, and feed. These compounds are reported to have anti-carcinogenic activity; therefore, Brassica vegetables are in the spotlight for medicinal uses. Studies on glucosinolates in Brassicaceae vegetables are presented by Ishida et al. Because of water deficit in farmland, probably due to global warming, drought tolerance has become increasingly important in rapeseed breeding. Drought is frequently accompanied by salinity; therefore, recent studies on rapeseed breeding for drought and salt tolerance are reviewed by Zhang et al. Furthermore, herbicide-tolerant cultivars developed by genetic engineering are an increasing percentage of rapeseed produced around the world. For the production of these genetically modified (GM) cultivars, pollen-mediated gene flow from GM cultivars to wild plants or landraces should be limited. Recent studies on the possibility of pollen-mediated gene flow from GM cultivars of rapeseed are described by Tsuda et al. After the Great East Japan Earthquake, the Rapeseed Project for Restoring Tsunami-Salt-Damaged Farmland was started by Tohoku University because of the high tolerance of Brassica crops to salinity. This project has been supported by Kureha Corporation and other companies. The cost of publication of these reviews is partially supported by this project.

Identifying differentially expressed genes in pollen from self-incompatible "Wuzishatangju" and self-compatible "Shatangju" mandarins.

Self-incompatibility (SI) is one of the important factors that can result in seedless fruit in Citrus. However, the molecular mechanism of SI in Citrus is not yet clear. In this study, two suppression subtractive hybridization (SSH) libraries (forward, F and reverse, R) were constructed to isolate differentially expressed genes in pollen from “Wuzishatangju” (SI) and “Shatangju” (self-compatibility, SC) mandarins. Four hundred and sixty-eight differentially expressed cDNA clones from 2077 positive clones were sequenced and identified. Differentially expressed ESTs are possibly involved in the SI reaction of “Wuzishatangju” by regulating pollen development, kinase activity, ubiquitin pathway, pollen-pistil interaction, and calcium ion binding. Twenty five SI candidate genes were obtained, six of which displayed specific expression patterns in various organs and stages after self- and cross-pollination. The expression level of the F-box gene (H304) and S1 (F78) in the pollen of “Wuzishatangju” was 5-fold higher than that in “Shatangju” pollen. The F-box gene, S1, UBE2, UBE3, RNaseHII, and PCP were obviously up-regulated in pistils at 3 d after self-pollination of “Wuzishatangju”, approximately 3-, 2-, 10-, 5-, 5-, and 2-fold higher, respectively than that at the same stage after cross-pollination of “Wuzishatangju” × “Shatangju” pistils. The potential involvement of these genes in the pollen SI reaction of “Wuzishatangju” is discussed.

Comprehensive Analysis of Expressed Proteins in the Different Stages of the Style Development of Self-incompatible ‘Hyuganatsu’ (Citrus tamurana hort. ex Tanaka)

In Citrus, self-incompatibility (SI) regulation is gametophytic, and this phenomenon is an economically critical problem for some Citrus cultivars without high parthenocarpic ability. Few molecular biological studies of SI in Citrus have been performed, and the molecular mechanism of SI has not been clarified. To investigate the effect of different stages of style development on pollen tube behavior, flower buds of self-incompatible ‘Hyuganatsu’ (Citrus tamurana hort. ex Tanaka) were histologically assayed. When the flower buds were observed 168 hours after pollination, pollen tubes in the self-pollinated flower buds 1 and 3 days before anthesis (DBA) were arrested in the upper part of the styles, while those in flower buds self-pollinated 5 DBA reached the base of the styles. These results revealed that SI in ‘Hyuganatsu’ has not yet occurred in the flower buds 5 DBA, but generated in the flower buds 3 DBA. To search SI-related pistil proteins in Citrus, we profiled a number of protein expressions in ‘Hyuganatsu’ styles of 1, 3, and 5 DBA by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. One-hundred thirty-eight protein spots were significantly different in abundance among the three stages, and 17 up-regulated and 26 down-regulated protein spots could be identified. Among the 17 up-regulated proteins, nine up-regulated proteins exhibited the expression pattern of 1 DBA ≥ 3 DBA > 5 DBA, a pattern which reflected the transmission from SC to SI, evaluated by pollen tube growth. BLASTP homology search against the peptide sequences of these proteins was carried out and predicted the proteins related to the maintenance of cell shape, the signaling of pollen tube growth, the flavonol biosynthesis, the responses to various stresses, photosynthesis, and the methionine metabolic process. Among the nine proteins, some may be the SI-related pistil proteins; however, it was not possible to determine the SI-related key protein of the style in Citrus.

Molecular mechanism of self-incompatibility and CO2 induced self-fertilization in the Brassicaceae

Molecular mechanism of self-incompatibility and CO2 induced self-fertilization in the Brassicaceae

Molecular mechanism of self-incompatibility in Bras-sica.

Molecular mechanism of self-incompatibility in Bras-sica.

Molecular mechanisms of self-incompatibility

Recent work has furthered our understanding of the molecules involved, and the diversity of mechanisms displayed, in self-incompatibility. S-RNases have been identified in two more plant families and their structure/function relationships are currently being dissected. Novel S genes have been cloned and expressed in Papaver rhoeas and Phalaris coerulescens, and the complexity of the S locus in Brassica is being unraveled. Present data implicate the involvement of protein kinase activity in all systems where pollen inhibition occurs at the stigma surface and RNase activity in those systems displaying stylar inhibition.