植物多肽信号分子的特点和功能

近几年研究表明,植物体内存在类似动物和酵母的多肽信号分子,调控植物生长发育以及对环境的响应.介绍了植物中的系统素、迅速碱化因子(RALF)、早期结瘤蛋白40(ENOD40)、植物磺化激动素(PSK)、S位点富含半胱胺酸蛋白(SCR)、CLV3以及相应受体的特点和功能研究进展,并且对多肽信号在植物中的作用及其应用前景进行了探讨.     

快速碱化因子类基因在茄科植物中的研究进展

植物在调控生长发育、响应外界胁迫时,细胞内外的pH变化是及时、重要的反应之一。快速碱化因子类基因（RALF）属于植物多肽类信号分子,是一类大的家族基因,它的活动引起胞外pH值快速升高,有关RALF类基因的研究在几种茄科植物中开展的较多,包括RALF蛋白信号分子的结构及加工、基因表达、受体研究以及功能等方面。最近我们在白菜和紫菜薹中的研究发现有一些脚类基因与植物的育性有关,本文就茄科植物中的这些研究进展进行介绍,为RALF的进一步研究提供参考。     

拟南芥RALF多肽家族的功能多样性初步分析

绿色植物中的快速碱化因子(Rapid alkalinization factor,RALF)为一类进化保守的多肽信号分子,以基因家族形式存在。模式植物拟南芥中至少存在35个RALF基因成员,前期研究显示拟南芥RALF家族的部分成员,比如RALF1/23,RALF4/19可分别作为Cr RLK1L类蛋白受体激酶家族成员FERONIA及BUPS1/2的配体,调控细胞伸长、植物免疫应答及双受精等过程,但是RALF家族其他成员是否具有生物学活性,以及不同成员之间是否具有功能性差异均尚不清楚。因此,本研究异源表达了19个代表性的RALF,并对其生物学活性和功能性差异进行了分析。实验结果表明,19个RALF均对根的生长起到不同程度的抑制作用,进一步挑选了部分代表性RALF成员进行了活性氧(Reactive oxygen species,ROS)迸发及丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)磷酸化实验分析,实验结果表明,我们确证了11个RALF蛋白参与了MAPK信号的响应,同时,证实16个RALF蛋白抑制了由flg22引起的ROS的释放。此外,不同RALF成员在下胚轴细胞伸长上的作用也存在明显差异,比如RALF10促进下胚轴的伸长。以上研究结果表明不同RALF之间既存在功能冗余性,又存在功能性差异。本研究丰富了对RALF功能复杂性和多样性的认识。     

快速碱化因子基因BcRALF在菜心中的克隆及序列分析

快速碱化因子是近年来新发现的一种植物多肽类信号分子,广泛存在于高等植物中。通过已得到的普通白菜的快速碱化因子基因BcMF14（GenBank序列登录号EF523516）的核苷酸序列,在其编码框两侧设计引物,从菜心中克隆出该类信号分子基因,命名为BcRALF（登陆号：GU086228）。序列同源比对表明该基因与花椰菜、拟南芥等的快速碱化因子基因有很高的相似性,证明BcRALF属于快速碱化因子家族。蛋白质特征预测以及蛋白序列结构分析发现BcRALF蛋白包含有多个生物活性位点,符合其作为多肽类信号分子类蛋白的特征。     

植物多肽激素

近几年研究表明,植物体内存在类似动物和酵母的多肽信号分子,调控植物生长发育以及调节植物对环境的响应。本文介绍了植物中的系统素,RALF,ENOD40,PSK,SCR,SLV3的特点和功能研究进展。     

十字花科多肽激素类基因RALF的同源基因克隆及进化分析

多肽激素类基因是对植物生长发育起重要作用的一类基因,RALF是其中的重要一员,而十字花科在中国蔬菜作物中是重要的一大类群。为了摸清十字花科多种蔬菜作物的RALF同源基因信息,该文根据前期研究从油菜中扩增到的多肽激素类基因RALFbn的序列设计引物,从提取的十字花科芸薹属、萝卜属、蔊菜属、山芥属的7份重要蔬菜作物的基因组DNA中分别克隆到了RALF的同源序列。结果表明:7种蔬菜作物的RALF同源基因编码区均在300 bp左右,且无内含子,编码的蛋白质由79个氨基酸组成,说明RALF在十字花科4个属内的保守性较强;对RALF同源基因在十字花科4个属中的表达分析表明,该类基因在根、茎、叶、花序轴等营养器官中不表达或弱表达,但主要在生殖器官中表达,其中在总花蕾和开放花中的表达量普遍高于嫩角果中的表达量,表明该类基因在十字花科中的生理活跃期是花发育时期。同时,构建了十字花科4个属中RALF同源基因的系统树,芸薹属的油菜、甘蓝和芥蓝形成一个分支,而茎瘤芥和分蘖芥形成另外一个分支,且与萝卜属、山芥属、蔊菜属的材料聚在一支,该基因的进化途径在一定程度上也反映出这几种作物的遗传背景关系。该研究结果丰富了RALF家族信息,增添了十字花科植物的分子进化数据。     

油菜花粉发育相关基因RALFbn的克隆与表达分析

根据美国NCBI数据库中快速碱化因子(RALF)类基因序列的已知信息,克隆了油菜的快速碱化因子基因RALFbn,对其核酸序列及预测蛋白进行了生物信息学分析,并在油菜多种组织内观测其表达情况.结果表明:(1)经克隆获得油菜RALFbn基因的cDNA序列全长为510 bp,无内含子,编码79个氨基酸.(2)生物信息学分析发现,油菜RALFbn蛋白具有RALF类蛋白保守的“YIXY”区和4个保守的半胱氨酸残基,并且含有N豆蔻酰化位点、酪氨酸激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、和酪氨酸激酶磷酸化位点等多个生物活性位点,说明该蛋白在油菜中潜在的生理调节能力较为活跃.(3) RT-PCR检测RALFbn基因在油菜生殖器官中的表达结果发现,RALFbn主要在油菜雄蕊中表达,而在雌蕊、花瓣和萼片中没有表达.提示RALFbn基因极可能与油菜雄蕊中花粉的发育相关.     

新型植物生长调节物质——激素性多肽的研究进展

多肽是生物体内一种非常重要的物质,它以信号的形式调控着生物的生活周期。在动物、细菌、真菌上作为激素、信息素和生长因子已进行了广泛的研究。然而,在植物上１９９１年才首次报道名叫系统素的伤害信号物质的内生多肽。最近,已从植物中分离出多种肽性植物生长调节因子。本文简要介绍系统素、早期结瘤素、植物硫素、豆胰岛素等四种激素性多肽的发现与分离,以及其结构与生理作用。     

新型植物生长调节物质——激素性多肽的研究进展

多肽是生物体内一种非常重要的物质,它以信号的形式调控着生物的生活周期。在动物、细菌、真菌上作为激素、信息素和生长因子已进行了广泛的研究。然而,在植物上1991年才首次报道名叫系统素的伤害信号物质的内生多肽。最近,已从植物中分离出多种肽性植物生长调节因子。本文简要介绍系统素、早期结瘤素、植物硫素、豆胰岛索等四种激素性多肽的发现与分离,以及其结构与生理作用。     

植物多肽信号分子CLE家族

动物中存在众多多肽信号分子,它们在信号转导方面发挥重要作用。近几年,对植物中多肽信号分子的研究取得了重大突破,它们积极参与调控植物生长发育的众多过程,同时也表明多肽信号分子在细胞之间的"交流"过程中发挥作用在进化上是保守的。CLE(CLAVATA3/EMBRYO SURROUNDING REGION)家族是目前植物领域研究较热的多肽信号分子家族,通过对拟南芥CLV3和百日草TDIF等CLE多肽信号分子的研究发现,CLE蛋白在成为有功能活性的信号分子之前,存在翻译后蛋白剪切和修饰的过程,这方面与动物中多肽信使的成熟过程相似。对CLE家族成员的分子特征、生物学功能、翻译后的加工修饰和研究中出现的问题进行综述,并对本领域未来的发展方向作出展望。     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Characterization of five RALF-like genes from<Emphasis Type="Italic"> Solanum chacoense</Emphasis> provides support for a developmental role in plants

Five RALF (rapid alkalinization factor)-like genes, named ScRALF1 to 5, were isolated from fertilized ovule and ovary cDNA libraries of Solanum chacoense. They showed high sequence similarities with the RALF protein sequence from Nicotiana tabacum, and exhibited the characteristic architecture of RALF polypeptides. All ScRALFs were moderately to highly expressed at some stage of fruit maturation. ScRALF1 and ScRALF3 were predominantly expressed in ovaries and larger fruits, while ScRALF2, ScRALF4, and ScRALF5 were also expressed in other tissues, indicating that while some RALFs may be involved in fruit maturation, others could be involved in other developmental processes. Wounding or treatment of plants with growth regulators involved in plant defense responses had no significant impact on the mRNA level of any of these genes. These results suggest and support previous data showing that RALF peptides are more likely to act as a small peptide involved in plant development than in defense responses.GenBank accession numbers: ScRALF1, AY422824; ScRALF2, AY422825; ScRALF3, AY422826; ScRALF4, AY422827; ScRALF5, AY655141     

Family-wide evaluation of RAPID ALKALINIZATION FACTOR peptides

Plant peptide hormones are important players that control various aspects of the lives of plants. RAPID ALKALINIZATION FACTOR (RALF) peptides have recently emerged as important players in multiple physiological processes. Numerous studies have increased our understanding of the evolutionary processes that shaped the RALF family of peptides. Nevertheless, to date, there is no comprehensive, family-wide functional study on RALF peptides. Here, we analyzed the phylogeny of the proposed multigenic RALF peptide family in the model plant Arabidopsis (Arabidopsis thaliana), ecotype Col-0, and tested a variety of physiological responses triggered by RALFs. Our phylogenetic analysis reveals that two of the previously proposed RALF peptides are not genuine RALF peptides, which leads us to propose a revision to the consensus AtRALF peptide family annotation. We show that the majority of AtRALF peptides, when applied exogenously as synthetic peptides, induce seedling or root growth inhibition and modulate reactive oxygen species (ROS) production in Arabidopsis. Moreover, our findings suggest that alkalinization and growth inhibition are, generally, coupled characteristics of RALF peptides. Additionally, we show that for the majority of the peptides, these responses are genetically dependent on FERONIA, suggesting a pivotal role for this receptor kinase in the perception of multiple RALF peptides.

Synthetic RAPID ALKALINIZATION FACTOR peptides induced a variety of physiological responses, many of which depend on FERONIA, a receptor kinase with functions in development and defense     

Function and solution structure of the Arabidopsis thaliana RALF8 peptide

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N‐terminal His‐tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with ¹⁵N and ¹³C for NMR analysis and obtained near complete ¹H, ¹³C, and ¹⁵N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.     

RALF22 promotes plant immunity and amplifies the Pep3 immune signal

Rapid alkalinization factors(RALFs) in plants have been reported to dampen pathogenassociated molecular pattern(PAMP)-triggered immunity via suppressing PAMP-induced complex formation between the pattern recognition receptor(PRR) and its co-receptor BAK1. However, the direct and positive role of RALFs in plant immunity remains largely unknown. Herein, we report the direct and positive roles of a typical RALF, RALF22, in plant immunity. RALF22alone directly elicited a variety of typical immune re...     

Identification of a major soluble protein in mitochondria from nonphotosynthetic tissues as NAD-dependent formate dehydrogenase.

In many plant species, one of the most abundant soluble proteins (as judged by two-dimensional polyacrylamide gel electrophoresis) in mitochondria from nongreen tissues is a 40-kD polypeptide that is relatively scarce in mitochondria from photosynthetic tissues. cDNA sequences encoding this polypeptide were isolated from a lambda gt11 cDNA expression library from potato (Solanum tuberosum L.) by screening with a specific antibody raised against the 40-kD polypeptide. The cDNA sequence contains an open reading frame of 1137 nucleotides whose predicted amino acid sequence shows strong homology to an NAD-dependent formate dehydrogenase (EC 1.2.1.2) from Pseudomonas sp. 101. Comparison of the cDNA sequence with the N-terminal amino acid sequence of the mature 40-kD polypeptide suggests that the polypeptide is made as a precursor with a 23-amino acid presequence that shows characteristics typical of mitochondrial targeting signals. The identity of the polypeptide was confirmed by assaying the formate dehydrogenase activity in plant mitochondria from various tissues and by activity staining of mitochondrial proteins run on native gels combined with antibody recognition. The abundance and distribution of this protein suggest that higher plant mitochondria from various nonphotosynthetic plant tissues (tubers, storage roots, seeds, dark-grown shoots, cauliflower heads, and tissues grown in vitro) might contain a formate-producing fermentation pathway similar to those described in bacteria and algae.     

胞外多肽信使的氨基酸偏好特征研究及应用

植物细胞外多肽信使已经被证实是植物信号转导中重要的第一信使。通过运用生物信息学的方法,分析比较了已知的植物、动物及微生物的胞外多肽信使的氨基酸序列,发现这些来自不同种的胞外多肽信使具有共同的氨基酸偏好特征。利用在线工具对胞外多肽信使的氨基酸偏好特征进行了验证,并根据细胞外多肽信使的氨基酸偏好特征推测了新的植物胞外多肽信使,这些结果对发现新的植物胞外多肽信使将会有所帮助。