Self-incompatibility In Brassicaceae Research Articles

Comprehensive computational analysis of the SRK–SP11 molecular interaction underlying self-incompatibility in Brassicaceae using improved structure prediction for cysteine-rich proteins

Plants employ self-incompatibility (SI) to promote cross-fertilization. In Brassicaceae, this process is regulated by the formation of a complex between the pistil determinant S receptor kinase (SRK) and the pollen determinant S-locus protein 11 (SP11, also known as S-locus cysteine-rich protein, SCR). In our previous study, we used the crystal structures of two eSRK–SP11 complexes in Brassica rapa S8 and S9 haplotypes and nine computationally predicted complex models to demonstrate that only the SRK ectodomain (eSRK) and SP11 pairs derived from the same S haplotype exhibit high binding free energy. However, predicting the eSRK–SP11 complex structures for the other 100 + S haplotypes and genera remains difficult because of SP11 polymorphism in sequence and structure. Although protein structure prediction using AlphaFold2 exhibits considerably high accuracy for most protein monomers and complexes, 46% of the predicted SP11 structures that we tested showed < 75 mean per-residue confidence score (pLDDT). Here, we demonstrate that the use of curated multiple sequence alignment (MSA) for cysteine-rich proteins significantly improved model accuracy for SP11 and eSRK–SP11 complexes. Additionally, we calculated the binding free energies of the predicted eSRK–SP11 complexes using molecular dynamics (MD) simulations and observed that some Arabidopsis haplotypes formed a binding mode that was critically different from that of B. rapa S8 and S9. Thus, our computational results provide insights into the haplotype-specific eSRK–SP11 binding modes in Brassicaceae at the residue level. The predicted models are freely available at Zenodo, https://doi.org/10.5281/zenodo.8047768.

Generation of Arabidopsis thaliana transformants showing the self-recognition activity of Brassica rapa.

Self-incompatibility in the Brassicaceae family is governed by SRK and SCR, which are two highly polymorphic genes located at the S-locus. Previously, the Arabidopsis lyrata SRK and SCR genes were introduced into Arabidopsis thaliana to generate self-incompatible lines. However, there are no reports showing that Brassica SRK and SCR genes confer self-incompatibility in A. thaliana. Doing so would further advance the mechanistic understanding of self-incompatibility in Brassicaceae. Therefore, we attempted to generate A. thaliana transformants showing the self-recognition activity of Brassica rapa by introducing BrSCR along with a chimeric BrSRK (BrSRK chimera, in which the kinase domain of BrSRK was replaced with that of AlSKR-b). We found that the BrSRK chimera and BrSCR of B. rapa S-9 and S-46 haplotypes, but not those of S-29, S-44, and S-60 haplotypes, conferred self-recognition activity in A. thaliana. Analyses of A. thaliana transformants expressing mutant variants of the BrSRK-9 chimera and BrSCR-9 revealed that mutations at the amino acid residues involved in BrSRK9-BrSCR9 interaction caused defects in the self-incompatibility response. The method developed in this study for generating self-incompatible A. thaliana transformants showing B. rapa self-recognition activity will be useful for analysis of self-recognition mechanisms in Brassicaceae.

Progress on deciphering the molecular aspects of cell-to-cell communication in Brassica self-incompatibility response.

The sporophytic system of self-incompatibility is a widespread genetic phenomenon in plant species, promoting out-breeding and maintaining genetic diversity. This phenomenon is of commercial importance in hybrid breeding of Brassicaceae crops and is controlled by single S locus with multiple S haplotypes. The molecular genetic studies of Brassica 'S' locus has revealed the presence of three tightly linked loci viz. S-receptor kinase (SRK), S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11), and S-locus glycoprotein (SLG). On self-pollination, the allele-specific ligand-receptor interaction activates signal transduction in stigma papilla cells and leads to rejection of pollen tube on stigmatic surface. In addition, arm-repeat-containing protein 1 (ARC1), M-locus protein kinase (MLPK), kinase-associated protein phosphatase (KAPP), exocyst complex subunit (Exo70A1) etc. has been identified in Brassica crops and plays a key role in self-incompatibility signaling pathway. Furthermore, the cytoplasmic calcium (Ca2+) influx in papilla cells also mediates self-incompatibility response in Brassicaceae, but how this cytoplasmic Ca2+ influx triggers signal transduction to inhibit pollen hydration is still obscure. There are many other signaling components which are not well characterized yet. Much progress has been made in elucidating the downstream multiple pathways of Brassica self-incompatibility response. Hence, in this review, we have made an effort to describe the recent advances made on understanding the molecular aspects of genetic mechanism of self-incompatibility in Brassicaceae.

Epigenetic regulation of agronomical traits in Brassicaceae.

Epigenetic regulation, covalent modification of DNA and changes in histone proteins are closely linked to plant development and stress response through flexibly altering the chromatin structure to regulate gene expression. In this review, we will illustrate the importance of epigenetic influences by discussing three agriculturally important traits of Brassicaceae. (1) Vernalization, an acceleration of flowering by prolonged cold exposure regulated through epigenetic silencing of a central floral repressor, FLOWERING LOCUS C. This is associated with cold-dependent repressive histone mark accumulation, which confers competency of consequence vegetative-to-reproductive phase transition. (2) Hybrid vigor, in which an F1 hybrid shows superior performance to the parental lines. Combination of distinct epigenomes with different DNA methylation states between parental lines is important for increase in growth rate in a hybrid progeny. This is independent of siRNA-directed DNA methylation but dependent on the chromatin remodeler DDM1. (3) Self-incompatibility, a reproductive mating system to prevent self-fertilization. This is controlled by the S-locus consisting of SP11 and SRK which are responsible for self/non-self recognition. Because self-incompatibility in Brassicaceae is sporophytically controlled, there are dominance relationships between S haplotypes in the stigma and pollen. The dominance relationships in the pollen rely on de novo DNA methylation at the promoter region of a recessive allele, which is triggered by siRNA production from a flanking region of a dominant allele.

Analysis of S-locus and expression of S-alleles of self-compatible rapid-cycling Brassica oleracea 'TO1000DH3'.

Brassica oleracea is a strictly self-incompatible (SI) plant, but rapid-cycling B. oleracea 'TO1000DH3' is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Three S-locus genes, S-locus glycoprotein (SLG), S-locus receptor kinase (SRK) and S-locus protein 11 or S-locus cysteine-rich (SP11/SCR), have been reported to date, all of which are classified into class I and II. In this study, we investigated the molecular mechanism behind alterations of SI to SC in rapid-cycling B. olerace 'TO1000DH3'. Class I SRK were identified by genomic DNA PCR and PCR-RFLP analysis using SRK specific markers and found to be homozygous. Cloning and sequencing of class I SRK revealed a normal kinase domain without any S-domain/transmembrane domain. Moreover, S-locus sequencing analysis revealed only an SLG sequence, but no SP11/SCR. Expression analysis showed no SRK expression in the stigma, although other genes involved in the SI recognition reaction (SLG, MLPK, ARC1, THL) were found to have normal expression in the stigma. Taken together, the above results suggest that structural aberrations such as deletion of the SI recognition genes may be responsible for the breakdown of SI in rapid-cycling B. oleracea 'TO1000DH3'.

Characterization of self-incompatibility genes in the intergeneric hybrid xBrassicoraphanus

Brassica rapa and Raphanus sativus are strictly self-incompatible (SI) plants; however, xBrassicoraphanus (baemoochae) is an intergeneric hybrid synthesized following hybridization of B. rapa and R. sativus that is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Two S-locus genes, SRK (S-locus receptor kinase) and SP11/SCR (S-locus protein 11 or S-locus cysteine-rich), have been reported to date, both of which are classified into class I and II. In this study, we investigate if there is an alteration in the structure or the expression in SRK or SP11 genes behind the alteration of SI to SC in baemoochae. Class I and II SRK were identified by PCR of the genomic DNA of baemoochae using SRK-specific universal primers. Cloning and sequencing of both classes of SRK was conducted and compared with SRK genes of parental species. Comparison analysis showed no genomic alterations and both of them showed expression in the stigma. Similarly, SP11 genes also showed no genomic alterations and normally are expressed in the anther. Other SI-related genes (MLPK, ARC1, THL) also showed normal expression in the stigma and anther. Taken together, these results revealed that no structural/gene expression change in these genes was responsible for the breakdown of SI in baemoochae. Rather, the transformation from SI parents to SC descendants could be responding to epigenetic mechanisms.

Characterization of S haplotype in a new self-compatible Brassica rapa cultivar Dahuangyoucai

The most important Brassica species, B. rapa, is naturally self-incompatible. Self-compatible mutants would be useful for dissecting the molecular mechanism of self-incompatibility (SI), a process that promotes outcrossing by recognizing and refusing self-pollens. The S haplotype in a new self-compatible B. rapa cultivar, Dahuangyoucai, was characterized for the first time in this study. Sequence analysis of the S-locus genes, SLG (S-locus glycoprotein), SRK (S-locus receptor kinase) and SCR (S-locus cysteine-rich protein) revealed that Dahuangyoucai contained S haplotype highly similar to S-f2, a non-functional class I S haplotype identified in another self-compatible B.&amp;nbsp;rapa cultivar, Yellow Sarson. Mutations of MLPK (M-locus protein kinase) and non-transcription of the male determinant, SCR, were observed in this cultivar, which is similar to the situation reported in Yellow Sarson. With respect to the female determinant, SRK, no transcript was detected in Yellow Sarson but two fragments were detected in Dahuangyoucai. One fragment was highly similar to SRK-f2, but the other fragment was different from the signal factors previously identified in the SI reaction. The results suggest that Dahuangyoucai and Yellow Sarson have the same origin and a similar mechanism of self-compatibility, but diverge after mutations in SRK, SCR and MLPK. Further studying the self-compatibility of Dahuangyoucai might identify novel factors involved in the SI signalling cascade and provide new insights into the mechanisms of SI in Brassicaceae.

Molecular Genetic Aspects of Self-incompatibility in Brassicaceae

Molecular genetic studies of self-incompatibility (SI) are the most accentuating part in the way of advancement of reproductive mechanisms in flowering plants. In the Brassicaceae plants, self-incompatibility has been mapped genetically to a single chromosomal location where several closely linked genes have been identified. Recently, various studies have provided a novel insight into the basis of specificity in the S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cysteine-rich (SP11/SCR) interaction, the nature of the signaling cascade that culminates in the inhibition of ‘self’ pollen, and the physiological and morphological changes that are associated with transitions between the outbreeding and inbreeding modes of mating in the Brassicaceae. In this review, we discuss the current view of the molecular genetic aspects of the self-incompatibility in Brassicaceae.

The Tarenaya hassleriana Genome Provides Insight into Reproductive Trait and Genome Evolution of Crucifers

The Brassicaceae, including Arabidopsis thaliana and Brassica crops, is unmatched among plants in its wealth of genomic and functional molecular data and has long served as a model for understanding gene, genome, and trait evolution. However, genome information from a phylogenetic outgroup that is essential for inferring directionality of evolutionary change has been lacking. We therefore sequenced the genome of the spider flower (Tarenaya hassleriana) from the Brassicaceae sister family, the Cleomaceae. By comparative analysis of the two lineages, we show that genome evolution following ancient polyploidy and gene duplication events affect reproductively important traits. We found an ancient genome triplication in Tarenaya (Th-α) that is independent of the Brassicaceae-specific duplication (At-α) and nested Brassica (Br-α) triplication. To showcase the potential of sister lineage genome analysis, we investigated the state of floral developmental genes and show Brassica retains twice as many floral MADS (for minichromosome maintenance1, AGAMOUS, DEFICIENS and serum response factor) genes as Tarenaya that likely contribute to morphological diversity in Brassica. We also performed synteny analysis of gene families that confer self-incompatibility in Brassicaceae and found that the critical serine receptor kinase receptor gene is derived from a lineage-specific tandem duplication. The T. hassleriana genome will facilitate future research toward elucidating the evolutionary history of Brassicaceae genomes.

Molecular genetics, physiology and biology of self-incompatibility in Brassicaceae

Self-incompatibility (SI) is defined as the inability to produce zygotes after self-pollination in a fertile hermaphrodite plant, which has stamens and pistils in the same flower. This structural organization of the hermaphrodite flower increases the risk of self-pollination, leading to low genetic diversity. To avoid this problem plants have established several pollination systems, among which the most elegant system is surely SI. The SI trait can be observed in Brassica crops, including cabbage, broccoli, turnip and radish. To produce hybrid seed of these crops efficiently, the SI trait has been employed in an agricultural context. From another point of view, the recognition reaction of SI during pollen-stigma interaction is an excellent model system for cell-cell communication and signal transduction in higher plants. In this review, we describe the molecular mechanisms of SI in Brassicaceae, which have been dissected by genetic, physiological, and biological approaches, and we discuss the future prospects in relation to associated scientific fields and new technologies.

Molecular analysis of Self-incompatibility in Brassicaceae

Molecular analysis of Self-incompatibility in Brassicaceae