Expression of self-incompatibility ribonucleases of Antirrhinum in Escherichia coli

Self-incompatibility is an intraspecific reproductive barrier to prevent self-fertilization in the flowering plants.In many species,self-incompatibility is controlled by a single S locus with multiple alleles.So far,the only gene known in the S locus of the Solanaceae,Scrophulariaceae and Rosaceae encodes a class of ribonucleases,called self-incompatibility ribonucleases (S RNases),which have been shown to mediate stylar expression of self-incompatible reaction.As the first step to investigate their three-dimensional structure,we successfully expressed three biologically active S RNases of Antirrihnum (S2,S4 and S5) in Escherichia coli (E.coli).Their functional expressions caused no detrimental effect on host bacteria growth and provided a basis for a large scale preparation of S RNase proteins.Possible reasons for non-lethality of S RNases on E.coli are discussed.     

S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility

Many flowering plants avoid inbreeding through a genetic mechanism termed self-incompatibility. An extremely polymorphic S-locus controls the gametophytic self-incompatibility system that causes pollen rejection (that is, active arrest of pollen tube growth inside the style) when an S-allele carried by haploid pollen matches one of the S-alleles present in the diploid style. The only known product of the S-locus is an S-RNase expressed in the mature style. The pollen component to this cell-cell recognition system is unknown and current models propose that it either acts as a gatekeeper allowing only its cognate S-RNase to enter the pollen tube, or as an inhibitor of non-cognate S-RNases. In the latter case, all S-RNases are presumed to enter pollen tubes; thus, the two models make diametrically opposed predictions concerning the entry of S-RNases into compatible pollen. Here we use immunocytochemical labelling of pollen tubes growing in styles to show accumulation of an S-RNase in the cytoplasm of all pollen-tube haplotypes, thus providing experimental support for the inhibitor model.     

Direct ligand-receptor complex interaction controls Brassica self-incompatibility

Many higher plants have evolved self-incompatibility mechanisms to prevent self-fertilization. In Brassica self-incompatibility, recognition between pollen and the stigma is controlled by the S locus, which contains three highly polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also called S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma, and SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SP11 is localized in the pollen coat. It is thought that, during self-pollination, SP11 is secreted from the pollen coat and interacts with its cognate SRK in the papilla cell of the stigma to elicit the self-incompatibility response. SLG is a secreted stigma protein that is highly homologous to the SRK extracellular domain. Although it is not required for S-haplotype specificity of the stigma, SLG enhances the self-incompatibility response; however, how this is accomplished remains controversial. Here we show that a single form of SP11 of the S8 haplotype (S8-SP11) stabilized with four intramolecular disulphide bonds specifically binds the stigma membrane of the S8 haplotype to induce autophosphorylation of SRK8, and that SRK8 and SLG8 together form a high-affinity receptor complex for S8-SP11 on the stigma membrane.     

Expression of self-incompatibility ribonucleases of Antirrhinum inEscherichia coli

Self-incompatibility is an intraspecific reproductive barrier to prevent self-fertilization in the flowering plants. In many species, self-incompatibility is controlled by a single S locus with multiple alleles. So far, the only gene known in theS locus of the Solanaceae, Scrophulariaceae and Rosaceae encodes a class of ribonucleases, called self-incompatibility ribonucleases (S RNases), which have been shown to mediate stylar expression of self-incompatible reaction. As the first step to investigate their three-dimensional structure, we successfully expressed three biologically active S RNases of Antirrihnum (S ₂,S ₄ andS ₅) inEscherichia coli (E. coli). Their functional expressions caused no detrimental effect on host bacteria growth and provided a basis for a large scale preparation of S RNase proteins. Possible reasons for non-lethality of S RNases onE. coli are discussed.     

The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins

The self-incompatibility response in Brassica allows recognition and rejection of self-pollen by the stigmatic papillae. The transmembrane S-locus receptor kinase (SRK), a member of the receptor-like kinase superfamily in plants, mediates recognition of self-pollen on the female side, whereas the S-locus cysteine-rich protein (SCR) is the male component of the self-incompatibility response. SCR is presumably located in the pollen coat, and is thought to be the SRK ligand. Although many receptor-like kinases have been isolated in plants, the mechanisms of signal transduction mediated by these molecules remain largely unknown. Here we show that SRK is phosphorylated in vivo within one hour of self-pollination. We also show that, in vitro, autophosphorylation of SRK is prevented by the stigma thioredoxin THL1 in the absence of a ligand. This inhibition is released in a haplotype-specific manner by the addition of pollen coat proteins. Our data indicate that SRK is inhibited by thioredoxins and activated by pollen coat proteins.     

The S receptor kinase determines self-incompatibility in Brassica stigma

The self-incompatibility possessed by Brassica is an intraspecific reproductive barrier by which the stigma rejects self-pollen but accepts non-self-pollen for fertilization. The molecular/biochemical bases of recognition and rejection have been intensively studied. Self-incompatibility in Brassica is sporophytically controlled by the polymorphic S locus. Two tightly linked polymorphic genes at the S locus, S receptor kinase gene (SRK) and S locus glycoprotein gene (SLG), are specifically expressed in the papillar cells of the stigma, and analyses of self-compatible lines of Brassica have suggested that together they control stigma function in self-incompatibility interactions. Here we show, by transforming self-incompatible plants of Brassica rapa with an SRK28 and an SLG28 transgene separately, that expression of SRK28 alone, but not SLG28 alone, conferred the ability to reject self (S28)-pollen on the transgenic plants. We also show that the ability of SRK28 to reject S28 pollen was enhanced by SLG28. We conclude that SRK alone determines S haplotype specificity of the stigma, and that SLG acts to promote a full manifestation of the self-incompatibility response.     

Compartmentalization of S-RNase and HT-B degradation in self-incompatible Nicotiana

Pollen-pistil interactions are crucial for controlling plant mating. For example, S-RNase-based self-incompatibility prevents inbreeding in diverse angiosperm species. S-RNases are thought to function as specific cytotoxins that inhibit pollen that has an S-haplotype that matches one of those in the pistil. Thus, pollen and pistil factors interact to prevent mating between closely related individuals. Other pistil factors, such as HT-B, 4936-factor and the 120 kDa glycoprotein, are also required for pollen rejection but do not contribute to S-haplotype-specificity per se. Here we show that S-RNase is taken up and sorted to a vacuolar compartment in the pollen tubes. Antibodies to the 120 kDa glycoprotein label the compartment membrane. When the pistil does not express HT-B or 4936-factor, S-RNase remains sequestered, unable to cause rejection. Similarly, in wild-type pistils, compatible pollen tubes degrade HT-B and sequester S-RNase. We suggest that S-RNase trafficking and the stability of HT-B are central to S-specific pollen rejection.     

Self-incompatibility triggers programmed cell death in Papaver pollen

Sexual reproduction in many angiosperm plants involves self-incompatibility (SI), which is one of the most important mechanisms to prevent inbreeding. SI is genetically controlled by the S-locus, and involves highly specific interactions during pollination between pollen and the pistil on which it lands. This results in the rejection of incompatible ('self') pollen, whereas compatible ('non-self') pollen is allowed to fertilize the plant. In Papaver rhoeas, S-proteins encoded by the stigma component of the S-locus interact with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in the inhibition of pollen-tube growth. Programmed cell death (PCD) is a mechanism used by many organisms to destroy unwanted cells in a precisely regulated manner. Here we show that PCD is triggered by SI in an S-specific manner in incompatible pollen. This provides a demonstration of a SI system using PCD, revealing a novel mechanism to prevent self-fertilization. Furthermore, our data reveal that the response is biphasic; rapid inhibition of pollen-tube growth is followed by PCD, which is involved in a later 'decision-making' phase, making inhibition irreversible.     

F-box proteins in flowering plants

In eukaryotes, the ubiquitin-mediated protein degradation pathway has been shown to control several key biological processes such as cell division, development, metabolism and immune response. F-box proteins, as a part of SCF (Skp1-Cullin (or Cdc53)-F-box) complex, functioned by interacting with substrate proteins, leading to their subsequent degradation by the 26S proteasome. To date, several F-box proteins identified in Arabidopsis and Antirrhinum have been shown to play important roles in auxin signal transduction, floral organ formation, flowering and leaf senescence. Arabidopsis genome sequence analysis revealed that it encodes over 1000 predicted F-box proteins accounting for about 5% of total predicted proteins. These results indicate that the ubiquitin-mediated protein degradation involving the F-box proteins is an important mechanism controlling plant gene expression. Here, we review the known F-box proteins and their functionsin flowering plants.     

贺兰山5种国家级保护植物的花粉形态研究

通过扫描电镜（SEM）对贺兰山5种国家级重点保护植物的花粉形态进行了详细的观察描述，结果表明，这5种植物的花粉形态均与其所属科或属的总体性状相一致，而同属豆科（Leguminosae）的沙冬青（Ammopiptanthus mongolicus）和野大豆（Glycine soja），其花粉形态主要在大小和表面纹饰的细微性状存在差别；蒙古扁桃（Prunus mongolicus）的花粉形态符合蔷薇科（Rosaceae），除地榆属（Sanguisorba L．）外的总体特征；贺兰山丁香（Syringa pinnatifolia var．alashanensis）的花粉形态与木犀科（Oleaceae）丁香属（Syringa L．）其他种的形态大致相同，但纹饰表现非常强烈；四合木（Tetraena mongollcus）在我国是蒺藜科（Zygophyllaceae）的单种属植物，其花粉形态与蒺幕科其他属植物的花粉形态差别较大．     

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.     

山西植物一新纪录属和四个新纪录种

文中报道在编写《山西植物志》过程中发现整理的一新纪录属及种——蜂斗菜属ＰｅｔａｓｉｔｅｓＭｉｌ．（菊科Ｃｏｍｐｏｓｉｔａｅ）,毛裂蜂斗菜Ｐ．ｔｒｉｃｈｏｌｏｂｕｓＦｒａｎｃｈ,产宁武、沁源;另３个新纪录种为：柳叶旋覆花（ＩｎｕｌａｓａｌｉｃｉｎａＬ．（菊科）,产夏县,千年不烂心ＳｏｌａｎｕｍｃａｔｈａｙａｎｕｍＣ．Ｙ．ＷｕｅｔＳ．Ｃ．Ｈｕａｎｇ（茄科Ｓｏｌａｎａｃｅａｅ）,产阳城、晋城,婆婆纳ＶｅｒｏｎｉｃａｄｉｄｙｍａＴｅｎｏｒｅ（玄参科Ｓｃｒｏｐｈｕｌａｒｉａｃｅａｅ）产永济。     

VIGS技术初步鉴定RgMLO6和RlMLO7基因对白粉病的负调控功能

为了探讨蔷薇科植物MLO基因在抗白粉病中的作用,研究应用病毒诱导的基因沉默技术(virus induced gene silencing,VIGS)抑制了大花香水月季RgMLO6基因和长尖叶蔷薇RlMLO7基因的表达,随后接种白粉菌对这2个基因进行抗性鉴定. 研究发现在VIGS载体转化植株叶片20 d后,RgMLO6和RlMLO7基因的相对表达量显著下降了80%～90%,沉默效果明显. 分别对2个基因沉默后的嫩叶进行白粉病抗性鉴定,大花香水月季和长尖叶蔷薇的抗性水平较对照组均提高. 显微镜观察白粉菌接种2个基因沉默后植株叶片中菌丝体的生长情况,整体表现出沉默植株叶表皮细胞上的白粉菌生长较对照组生长缓慢. 结果表明RgMLO6与RlMLO7基因对蔷薇科植物的白粉病有负向调控作用.     

水稻温敏核不育系HD9802S不育性遗传的初步观察

以水稻温敏核不育系HD9802S配组的HD9802S／湘早92和HD9802S／荆楚15的F1和F2代为材料,对这两个杂交早籼稻组合F2群体的花粉育性进行了观察分析．结果表明,F2群体中可育株数和不育株数经卡平方测验符合3：1的理论比例,初步确定温敏核不育系HD9802S的雄性不育性由一对主效隐性核基因控制;同时根据F2群体花粉育性表现出连续分布的特征,推测其雄性不育性还受其他微效基因的影响。     

通过转基因途径获得植物雄性不育

对利用转基因的方法获得植物雄性不育的研究进展进行了概述 .小孢子发育过程涉及花药发育到花粉粒成熟过程中一系列基因的表达 .这些基因表达的异常往往导致部分或全部的花粉败育 .利用绒毡层特异性启动子或花药特异性启动子驱动一种外来基因的表达可以导致雄性不育 .育性的恢复也可通过转基因技术来实现 .控制植物育性的基因工程将在优势育种中发挥越来越重要的作用 .     

Fertility analysis of the Arabidopsis transformed with antisense rice osRACD gene

The full length osRACD cDNA sequence was subcloned into the pBI121 plasmid in the antisense orientation under the control of the CaMV35S promoter to construct the expression vector pBID, and the constructs were introduced into Arabidopsis plants by using the vacuum infiltration method. The siliques of the transformants stopped growing after anthesis, and they turned yellow or died later; and the siliques from the control plants transformed by the pBI continued growing after anthesis and matured normally. In vitro pollen germination demonstrated that the growth and elongation process of the pollens of the transgenic plants was inhibited, the pollen tubes were shorter and slightly fatter than the tubes of the control plants, which grew normally with long cyclindrical tubes. The above results suggest the function of osRACD gene involved in regulation of the growth and elongation process of pollen tube, its encoding protein may be one of the important factors in regulation of fertility transition of the photoperiod-sensitive genic male-sterile rice Nongken 58S.     

Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants

Most mitochondrial proteins are encoded by nuclear genes and are synthesized as precursors containing a presequence at the N terminus. In yeast and in mammalian cells, the function of the presequence in mitochondrial targeting has been revealed by chimaeric gene studies. Fusion of a mitochondrial presequence to a foreign protein coding sequence enables the protein to be imported into mitochondria in vitro as well as in vivo. Whether plant mitochondrial presequences function in the same way has been unknown. We have previously isolated and characterized a nuclear gene (atp2-1) from Nicotiana plumbaginifolia that encodes the beta-subunit of the mitochondrial ATP synthase. We have constructed a chimaeric gene comprising a putative atp2-1 presequence fused to the bacterial chloramphenicol acetyltransferase (CAT) coding sequence and introduced it into the tobacco genome. We report here that a segment of 90 amino acids of the N terminus of the beta-subunit precursor is sufficient for the specific targeting of the CAT protein to mitochondria in transgenic plants. Our results demonstrate a high specificity for organelle targeting in plant cells.     

A candidate spermatogenesis gene on the mouse Y chromosome is homologous to ubiquitin-activating enzyme E1.

The human X-linked gene A1S9 complements a temperature-sensitive cell-cycle mutation in mouse L cells, and encodes the ubiquitin-activating enzyme E1. The gene has been reported to escape X-chromosome inactivation, but there is some conflicting evidence. We have isolated part of the mouse A1s9 gene, mapped it to the proximal portion of the X chromosome and shown that it undergoes normal X-inactivation. We also detected two copies of the gene on the short arm of the mouse Y chromosome (A1s9Y-1 and A1s9Y-2). The functional A1s9Y gene (A1s9Y-1) is expressed in testis and is lost in the deletion mutant Sxrb. Therefore A1s9Y-1 is a candidate for the spermatogenesis gene, Spy, which maps to this region. A1s9X is similar to the Zfx gene in undergoing X-inactivation, yet having homologous sequences on the short arm of the Y chromosome, which are expressed in the testis. These Y-linked genes may form part of a coregulated group of genes which function during spermatogenesis.     

Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis

Meristem function in plants requires both the maintenance of stem cells and the specification of founder cells from which lateral organs arise. Lateral organs are patterned along proximodistal, dorsoventral and mediolateral axes. Here we show that the Arabidopsis mutant asymmetric leaves1 (as1) disrupts this process. AS1 encodes a myb domain protein, closely related to PHANTASTICA in Antirrhinum and ROUGH SHEATH2 in maize, both of which negatively regulate knotted-class homeobox genes. AS1 negatively regulates the homeobox genes KNAT1 and KNAT2 and is, in turn, negatively regulated by the meristematic homeobox gene SHOOT MERISTEMLESS. This genetic pathway defines a mechanism for differentiating between stem cells and organ founder cells within the shoot apical meristem and demonstrates that genes expressed in organ primordia interact with meristematic genes to regulate shoot morphogenesis.