芸苔属自交不亲和细胞信号转导的研究进展

在芸苔属植物的自交不亲和细胞信号转导过程中,信号分子-SCR配体是由花粉粒产生的,被柱头乳突细胞SRK受体识别后,进行细胞内信号转导。这对受体-配体是两个由S位点编码的且高度多态的蛋白质,它们决定着自交不亲和反应。配体是位于花粉粒表面的一个小的胞被蛋白,由SCR基因编码;受体是位于柱头乳突细胞原生质膜上的跨膜的蛋白质激酶,由SRK基因编码。在自交授粉过程中,配体SCR和受体SRK的相互作用激活了受体SRK,被激活的SRK通过其下游组分ARC1介导底物的泛肽化,然后泛肽化的底物在蛋白酶体/CSN中被降解,从而导致了自交不亲和性反应。这些降解的底物可能是促进花粉水合、萌发和花粉管生长的雌蕊亲和因子。主要针对芸苔属自交不亲和细胞信号转导作一综述。     

甘蓝自交不亲和反应的细胞信号转导

S受体激酶(S—receptor kinase,SRK)和S位点富含半胱氨酸(S-locus cysteine-rich,SCR)分别是甘蓝柱头和花粉中导致自交不亲和反应的决定性蛋白质因子。本文就SRK、SCR的结构和功能加以综述,阐明两者在细胞信号转导中的作用。     

植物自交不亲和基因研究进展

自交不亲和性的研究是植物生殖生物学和分子生物学研究的热点之一,对自交不亲和基因和蛋白质的深入研究是解析自交不亲和性机理的关键.对控制孢子体自交不亲和性和配子体自交不亲和性的S基因及其蛋白质产物的分子生物学研究进展进行了综述.孢子体自交不亲和性植物S位点上至少存在3个基因,即SLG、SRK和SCR基因.其中SLG、SRK基因控制雌蕊自交不亲和性,而SCR控制花粉自交不亲和性.配子体自交不亲和植物雌蕊S基因产物为S-RNase,具有核酸酶活性;配子体自交不亲和植物花粉S基因产物尚未找到.     

芸薹属自交不亲和基因的分子生物学及进化模式

芸薹属的自交不亲和性是受单基因座、复等位基因控制的孢子体控制型。自交不亲和基因座位（S-locus）是由多个基因组成的复杂区域,称之为S多基因家族,其大多数成员分布于芸薹属的整个染色体组。目前已鉴定出100多个S等位基因,它们的起源分化始于一千万年前。S-座位上存在的多基因有3种：SRK,SLG和SCR/SP11;SRK和SLG在柱头中表达,SCR/SP11在雄蕊中表达。SRK蛋白在识别同类花粉的过程中起主要作用,而SLG蛋白增强了这种自交不亲和反应。SLG与SRK基因中编码S-结构域的核苷酸序列相似性程度高达85％～98%。基因转换可能是SLG和SRK的高度同源性能够得以保持的原因。SRK,SLG和SCR基因紧密相连,并表现出高水平的序列多样性。SRK与SLG基因间的距离很近,在20～25 kb之间。在柱头和花粉中,自交不亲和等位基因之间的共显性关系要比显性和隐性关系更加普遍,这是芸薹属自交不亲和性的一大特点。自交不亲和基因的进化模式存在两种假说：双基因进化模式和中性变异体进化模式;可能存在几种不同的进化方式,它们共同在自然群体中新的S等位基因进化过程中起作用。     

芸薹属自交不亲和基因的分子生物学及进化模式

芸薹属的自交不亲和性是受单基因座、复等位基因控制的孢子体控制型。自交不亲和基因座位(S-locus)是由多个基因组成的复杂区域,称之为S多基因家族,其大多数成员分布于芸薹属的整个染色体组。目前已鉴定出100多个S等位基因,它们的起源分化始于一千万年前。S-座位上存在的多基因有3种：SRK,SLG和SCR／SPII;SRK和SLG在柱头中表达,SCR／SPII在雄蕊中表达。SRK蛋白在识别同类花粉的过程中起主要作用,而SLG蛋白增强了这种自交不亲和反应。SLG与SRK基因中编码S-结构域的核苷酸序列相似性程度高达85％～98％。基因转换可能是SLG和SRK的高度同源性能够得以保持的原因。SRK,SLG和SCR基因紧密相连,并表现出高水平的序列多样性。SRK与SLG基因间的距离很近,在20～25kb之间。在柱头和花粉中,自交不亲和等位基因之间的共显性关系要比显性和隐性关系更加普遍,这是芸薹属自交不亲和性的一大特点。自交不亲和基因的进化模式存在两种假说：双基因进化模式和中性变异体进化模式;可能存在几种不同的进化方式,它们共同在自然群体中新的S等位基因进化过程中起作用。     

芸薹属中自交不亲和反应的信号转导

自交不亲和现象在芸薹属(Brassica)植物中普遍存在,芸薹属中表现的是典型的孢子体型自交不亲和性.单元特异性的S位点受体激酶(SRK)基因和S位点花粉胞被蛋白(SCR/SP11)发生识别后,一系列相关蛋白-臂重复蛋白(ARC1)、M位点蛋白激酶(MLPK)等,引发了自交不亲和反应信号的传导,最终产生自交不亲和反应.文章就这方面的研究进展作介绍.     

花粉——雌蕊相互作用的分子基础

显花植物授粉过程包含了花粉与雌蕊一系列复杂的细胞间相互作用。花粉在柱头上必须发生粘附与水合作用后才能萌发,花粉胞被蛋白可能在粘附机制中起主要作用,而水孔蛋白调节了花粉的水合过程;花粉管在花柱引导组织中定向生长,受引导组织胞间质、向化性物质及细胞粘附机制等因素的影响,也与花粉受体及子房有关。在植物的自交不亲和性反应中,配子体型自交不亲和性反应主要是由自交不亲和基因蛋白产物降解花粉RNA或以Ca^2 介导的信号级联反应实现的;孢子体型自交不亲和性反应则依赖于干性柱头以及雌蕊的S受体激酶及S位点糖蛋白与花粉S基因配体之间的相互作用。     

芸苔属植物自交不亲和性及其机理研究进展

自交不亲和性广泛存在于芸苔属植物当中,有着防止自交衰退等作用,在育种工作中意义重大。本文综述了芸苔属植物自交不亲和性及其表现,自交不亲和内在机制,如S复等位基因及相互作用,S位点糖蛋白,S多基因家族,S位点受体激酶,蛋白质可逆磷酸化等 。     

羽衣甘蓝S_(13-b)位点受体激酶基因的克隆及激酶结构域的原核表达

目的:分离羽衣甘蓝S13-b位点受体激酶(SRK13-b)基因并进行序列及结构域分析,构建SRK13-b结构域的原核表达载体并进行重组蛋白质的原核表达和纯化。方法:提取羽衣甘蓝S13-bS13-b自交不亲和系花期柱头的RNA,用RT-PCR法分离SRK13-b基因;将编码SRK13-b激酶结构域的序列插入大肠杆菌表达载体pET-14b中,构建原核表达质粒pET-SRK13-bCT,转化大肠杆菌BL21(DE3)pLysS菌株,经0.1mmol/LIPTG诱导,用Ni-NTA亲和层析柱对SRK13-b激酶结构域蛋白进行纯化。结果:分离获得羽衣甘蓝SRK13-b基因的长度为2571bp,编码856个氨基酸,GenBank收录号为EU180597;对SRK13-b激酶结构域蛋白进行诱导表达及纯化,SDS-PAGE显示相对分子质量约43×103的蛋白质特异表达,对表达产物进行分离纯化,获得了SRK13-b激酶结构域的融合蛋白。结论:羽衣甘蓝SRK13-b基因的克隆及激酶结构域的原核表达,为研究SRK的功能及自交不亲和性奠定了基础。     

芸苔属植物自交不亲和性及其机理研究进展

自交不亲和性广泛存在于芸苔属植物当中,有着防止自交衰退等作用,在育种工作中意义重大。本文综述了芸苔属植物自交不亲和性及其表现,自交不亲和内在机制,如S复等位基因及相互作用,S位点糖蛋白,S多基因家族,S位点受体激酶,蛋白质可逆磷酸化等。     

ARC1 is an E3 ubiquitin ligase and promotes the ubiquitination of proteins during the rejection of self-incompatible Brassica pollen

ARC1 is a novel U-box protein required in the Brassica pistil for the rejection of self-incompatible pollen; it functions downstream of the S receptor kinase (SRK). Here, we show that ARC1 has E3 ubiquitin ligase activity and contains several motifs that influence its subcellular localization. ARC1 can shuttle between the nucleus, cytosol, and proteasome/COP9 signalosome (CSN) when expressed in tobacco BY-2 suspension-cultured cells. However, ARC1 localization to the proteasome/CSN occurs only in the presence of an active SRK. In the pistil, ubiquitinated protein levels increase specifically with incompatible pollinations, but they do not change in ARC1 antisense-suppressed pistils. In addition, inhibition of the proteasomal proteolytic activity disrupts the self-incompatibility response. We propose that ARC1 promotes the ubiquitination and proteasomal degradation of compatibility factors in the pistil, which in turn leads to pollen rejection.     

Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.     

SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.     

The ARC1 E3 Ligase Gene Is Frequently Deleted in Self-Compatible Brassicaceae Species and Has a Conserved Role in Arabidopsis lyrata Self-Pollen Rejection

Self-pollen rejection is an important reproductive regulator in flowering plants, and several different intercellular signaling systems have evolved to elicit this response. In the Brassicaceae, the self-incompatibility system is mediated by the pollen S-locus Cys-Rich/S-locus Protein11 (SCR/SP11) ligand and the pistil S Receptor Kinase (SRK). While the SCR/SP11-SRK recognition system has been identified in several species across the Brassicaceae, less is known about the conservation of the SRK-activated cellular responses in the stigma, following self-pollen contact. The ARM Repeat Containing1 (ARC1) E3 ubiquitin ligase functions downstream of SRK for the self-incompatibility response in Brassica, but it has been suggested that ARC1 is not required in Arabidopsis species. Here, we surveyed the presence of ARC1 orthologs in several recently sequenced genomes from Brassicaceae species that had diversified ∼20 to 40 million years ago. Surprisingly, the ARC1 gene was deleted in several species that had lost the self-incompatibility trait, suggesting that ARC1 may lose functionality in the transition to self-mating. To test the requirement of ARC1 in a self-incompatible Arabidopsis species, transgenic ARC1 RNA interference Arabidopsis lyrata plants were generated, and they exhibited reduced self-incompatibility responses resulting in successful fertilization. Thus, this study demonstrates a conserved role for ARC1 in the self-pollen rejection response within the Brassicaceae.     

S cysteine-rich (SCR) binding domain analysis of the Brassica self-incompatibility S-locus receptor kinase

Brassica self-incompatibility, a highly discriminating outbreeding mechanism, has become a paradigm for the study of plant cell-cell communications. When self-pollen lands on a stigma, the male ligand S cysteine-rich (SCR), which is present in the pollen coat, is transmitted to the female receptor, S-locus receptor kinase (SRK). SRK is a membrane-spanning serine/threonine receptor kinase present in the stigmatic papillar cell membrane. Haplotype-specific binding of SCR to SRK brings about pollen rejection. The extracellular receptor domain of SRK (eSRK) is responsible for binding SCR. Based on sequence homology, eSRK can be divided into three subdomains: B lectin-like, hypervariable, and PAN. Biochemical analysis of these subdomains showed that the hypervariable subdomain is responsible for most of the SCR binding capacity of eSRK, whereas the B lectin-like and PAN domains have little, if any, affinity for SCR. Fine mapping of the SCR binding region of SRK using a peptide array revealed a region of the hypervariable subdomain that plays a key role in binding the SCR molecule. We show that residues within the hypervariable subdomain define SRK binding and are likely to be involved in defining haplotype specificity.     

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.     

Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.     

Molecular evolution of the s locus controlling mating in the brassicaceae

Flowering plants possess self-incompatibility (SI) mechanisms that promote outbreeding and thereby increase their genetic diversity. In the self-incompatible Brassicaceae, recognition and rejection of self-pollen is based on a receptor-ligand interaction between male and female SI determinants. A transmembrane receptor kinase (S locus Receptor Kinase, SRK) determines the SI specificity in stigmatic cells, whereas a pollen coat-localized ligand (S locus Cysteine-Rich, SCR) determines the SI specificity in pollen. During recent years, major advances have been made in the understanding of the molecular basis of self-pollen recognition by stigmatic cells. In this review, we will focus on evolutionary aspects of the SI system in Brassicaceae. We will describe how the study of the molecular aspect of SI, not only in the historical Brassica model but also in Arabidopsis species, has contributed to highlight certain aspects of evolution of SI in the Brassicaceae.