高等植物中的多肽激素

高等植物的第一个多肽激素（系统素）发现已经有10多年的历史。到目前为止,被普遍认可的植物多肽激素有4种：系统素、PSK、CLV3和SCR,分别参与了植物的防御反应、细胞的分裂、茎端生长点干细胞数目维持和花粉．柱头的识别过程。这些小分子多肽化合物以配基的形式与细胞膜表面的受体激酶相互作用,从而实现细胞之间的信号交流。本文对这4种多肽激素及其相应受体的研究进展做了简要评述,并着重介绍当前研究比较热门的CLV3多肽,最后对相关领域的发展前景进行探讨。     

植物多肽激素研究概况

目前发现的植物多肽多达9种.基于配基-受体的胞间互作模式,目前公认的植物多肽激素包括4种:系统素(Systemin)、植物硫肽激素(Phytosulfokine)、SCR/SP11和CLV3,分别参与了植食性昆虫防御反应、细胞增殖、自交不亲和的识别,以及茎分生组织干细胞分裂与分化平衡的维持.本文对四种植物多肽激素基因家族的研究进展做了较为详尽的综述,并结合本试验室的研究进展做了展望.     

植物多肽激素研究概况

目前发现的植物多肽多达9 种。基于配基- 受体的胞间互作模式, 目前公认的植物多肽激素包括4种: 系统素(Systemin) 、植物硫肽激素(Phytosulfokine) 、SCR􊄯SP11 和CLV3 , 分别参与了植食性昆虫防御反应、细胞增殖、自交不亲和的识别, 以及茎分生组织干细胞分裂与分化平衡的维持。本文对四种植物多肽激素基因家族的研究进展做了较为详尽的综述, 并结合本试验室的研究进展做了展望。     

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.     

植物中的“多肽激素”

植物激素是指在植物体的某一组织内合成后、转运到作用部位并对生长发育起调节作用的一类微量有机物质。目前公认的植物激素有５大类,即生长素类、细胞分裂素类、赤霉素类、脱落酸和乙烯。此外,油菜素内酯类、多胺类也被认为是新型的植物激素。在化学结构上,上述几类植物激素都不是蛋白质或多肽。但近年来,在植物体内相继发现了一些具有调节植物生理过程和传递细胞信号功能的活性多肽,称其为“多肽激素”,本文简要介绍以下４种。１　系统素系统素（ｓｙｓｔｅｍｉｎ）是从受伤的番茄叶片中分离的一种由１８个氨基酸组成的多肽,它是植…     

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。     

乌龟胃肠胰系统内分泌细胞的免疫组织化学研究

应用过氧化物酶标记的链霉卵白素(Streptavidin peroxidase,简称S-P法)免疫组织化学技术,使用六种特异性胃肠激素抗血清对乌龟胃肠胰系统内分泌细胞的种类、定位、分布密度及形态进行了研究。在乌龟胰腺中检测出5-羟色胺、生长抑素、胰高糖素和胰多肽等4种内分泌细胞,生长抑素、胰高糖素细胞多成簇大量分布于胰岛中;5-羟色胺、胰多肽细胞多散在少量分布于胰腺腺泡之间。在乌龟消化道中共检测出5-羟色胺、生长抑素、胃泌素、胰高糖素和P物质等5种内分泌细胞:5-羟色胺细胞在消化道各段均有分布,以十二指肠处分布密度最高(30.7±4.2),空肠其次,回肠、直肠处最低(12.0±1.0/11.2±3.0);生长抑素细胞仅分布于食道和胃中各段;胃泌素细胞分布于胃幽门部和十二指肠处;胰高糖素细胞分布于胃体至空肠段,以胃幽门部分布密度较高(11.3±1.1);P物质细胞仅布于胃幽门部;消化道各段均未检出胰多肽细胞。与其他爬行动物比较,乌龟胃肠胰系统内分泌细胞的分布既存在着一定共同点,又显示了较大的种间差异。       

甾体激素作用于靶细胞的分子机制

有关甾体激素作用于靶细胞分子机制的研究,近十几年来,由于方法技术的提高和建立,有了很大的发展。Jonsen 等(1962)用高比度的~3H-雌二醇,对其在子宫、阴道等靶器官中的积聚、滞留进行了比较,证实激素能专一性地与其靶细胞结合,这在研究激素对细胞的作用方面作出了主要贡献。嗣后,有了关于雌素受体的发现。Jansen 等(1967)见到,大鼠、小牛子宫细胞质内有一种与雌素亲和性很强的蛋白,这种蛋白称为雌素受体。迄今,有关甾体激     

Dual CLAVATA3 peptides in Arabidopsis shoot stem cell signaling

Plant shoot stem cell pool is constantly maintained by a negative feedback loop through peptide-receptor mediated signaling pathway. CLAVATA3 (CLV3) encode a 96 aminoacid protein which is processed to 12-amino-acid or arabinosylated 13-amino-acid peptides, acting as a ligand signal to regulate stem cell homeostasis in the shoot apical meristem (SAM). Although arabinosylated 13-amino-acid CLV3 peptide (CLV3p) shows more significant binding affinity to its receptors and biological activities in the SAM, the physiological function of two mature forms of CLV3p remained an unresolved puzzle in the past decade due to the technical difficulties of arabinosylation modification in the peptide synthesis. Here, we analyzed the role of two mature CLV3 peptides with newly synthesized arabinosylated peptide. Beside shoot meristem phenotypes, arabinosylated CLV3p showed the conventional trait of CLV2-dependent root growth inhibition. Moreover, both 12-amino-acid and arabinosylated 13-amino-acid CLV3 peptides have analogous activities in shoot stem cell signaling. Notably, we demonstrated that non-arabinosylated 12-amino acid CLV3p can affect shoot stem cell signaling at the physiological level unlike previously suggested (Ohyama et al. 2009; Shinohara and Matsubayashi 2013; Shinohara and Matsubayashi 2015). Therefore, these results support the physiological role of the 12-amino-acid CLV3p in shoot stem cell signaling in the deficient condition of arabinosylated 13-amino-acid CLV3p in Arabidopsis thaliana.     

The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway

CLAVATA3 (CLV3), CLV3/ESR19 (CLE19), and CLE40 belong to a family of 26 genes in Arabidopsis thaliana that encode putative peptide ligands with unknown identity. It has been shown previously that ectopic expression of any of these three genes leads to a consumption of the root meristem. Here, we show that in vitro application of synthetic 14-amino acid peptides, CLV3p, CLE19p, and CLE40p, corresponding to the conserved CLE motif, mimics the overexpression phenotype. The same result was observed when CLE19 protein was applied externally. Interestingly, clv2 failed to respond to the peptide treatment, suggesting that CLV2 is involved in the CLE peptide signaling. Crossing of the CLE19 overexpression line with clv mutants confirms the involvement of CLV2. Analyses using tissue-specific marker lines revealed that the peptide treatments led to a premature differentiation of the ground tissue daughter cells and misspecification of cell identity in the pericycle and endodermis layers. We propose that these 14-amino acid peptides represent the major active domain of the corresponding CLE proteins, which interact with or saturate an unknown cell identity-maintaining CLV2 receptor complex in roots, leading to consumption of the root meristem.     

CLE peptide ligands and their roles in establishing meristems

Research in the past decade revealed that peptide ligands, also called peptide hormones, play a crucial role in intercellular communication and defense response in plants. Recent studies demonstrated that a family of plant-specific genes, CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE), which has at least 31 members in Arabidopsis genome, are able to generate extracellular peptides to regulate cell division and differentiation. A hydroxyl 12-amino acid peptide derived from the conserved CLE motif of CLV3 promotes cell differentiation, whereas another CLE-derived peptide suppresses the differentiation. These peptides probably interact with membrane-bound, leucine-rich repeat receptor-like kinases (LRR-RLKs) to execute the decision between cell proliferation and differentiation.     

Individual amino acid residues in CLV3 peptide contribute to its stability in vitro

CLV3 acts as a peptide ligand to interact with leucine-rich repeat (LRR) receptor kinases in neighboring cells to restrict the size of shoot apical meristems (SAMs) in Arabidopsis. To examine contributions of individual amino acid residues in CLV3 peptide in SAM maintenance, 12 synthetic Ala-substituted CLV3 peptides were applied to clv3-2 seedlings cultured in vitro, and the sizes of SAMs were measured after 9 d. The result showed that Pro-9 and His-11 are the most critical residues, while Val-3 and Ser-5 are the least important ones for CLV3 functions in SAMs in vitro. With MALDI-TOF mass spectrum analyses, we further showed that Ala substitution in His-11 led to a greatly reduced stability of the peptide, leading to a complete degradation of the peptide after cultured with seedlings for only one hour. The substitution of Pro-9 by Ala also led to a complete degradation of the peptides after 2 d incubation. In contrast, Ala substitutions in Val-3 or Ser-5 gave very little changes on peptide stabilities. These results suggested that stabilities of Ala-substituted CLV3 peptides are positively correlated with their activities in SAMs. We thus propose that the stability of CLV3 may partially contribute to its function in SAM maintenance.     

Analogs of the CLV3 peptide: synthesis and structure-activity relationships focused on proline residues

CLAVATA3 (CLV3) is a plant peptide hormone in which the proline residues are post-translationally hydroxylated and glycosylated. CLV3 plays a key role in controlling the stem cell mass in the shoot meristem of Arabidopsis thaliana. In a previous report, we identified a dodecapeptide (MCLV3) from CLV3-overexpressing Arabidopsis calli; MCLV3 was the smallest functional peptide derived from the CLV3 precursor. Here, we designed a series of MCLV3 analogs in which proline residues were substituted with proline derivatives or N-substituted glycines (peptoids). Peptoid substitution at Pro9 decreased bioactivity without affecting specific binding to the CLV1-related protein in cauliflower membrane. These findings suggest that peptoid-substituted peptides would be lead compounds for developing potential agonists and antagonists of CLV3.     

Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides

The dodecapeptide MCLV3 is a functional peptide, derived from the CLV3 precursor protein, which is a candidate ligand of the CLV1/CLV2 receptor complex that restricts the stem cell population in the shoot apical meristem (SAM). MCLV3 can induce shoot and root meristem consumption, the typical phenotype of transgenic plants overexpressing CLV3. We investigated the bioactivities of a series of alanine-substituted MCLV3 and related peptides on the root growth of Arabidopsis. The structure-activity relationship (SAR) of MCLV3 had high similarity with that of tracheary element differentiation inhibitory factor (TDIF). We also evaluated the binding activities of the peptides by a competitive receptor binding assay using tritiated MCLV3 and the membrane fraction of a tobacco BY-2 cell line overexpressing the MCLV3 ectodomain. This dual assay, combining a biological and receptor binding assay for evaluating the activities of MCLV3-related peptides, uncovered the SAR of MCLV3, and indicated that the terminal residues play critical roles in exerting its activity and are important for specific binding to the receptor, CLV1.     

CLE14/CLE20 peptides may interact with CLAVATA2/CORYNE receptor-like kinases to irreversibly inhibit cell division in the root meristem of Arabidopsis

Towards an understanding of the interacting nature of the CLAVATA (CLV) complex, we predicted the 3D structures of CLV3/ESR-related (CLE) peptides and the ectodomain of their potential receptor proteins/kinases, and docking models of these molecules. The results show that the ectodomain of CLV1 can form homodimers and that the 12-/13-amino-acid CLV3 peptide fits into the binding clefts of the CLV1 dimers. Our results also demonstrate that the receptor domain of CORYNE (CRN), a recently identified receptor-like kinase, binds tightly to the ectodomain of CLV2, and this likely leads to an increased possibility for docking with CLV1. Furthermore, our docking models reveal that two CRN-CLV2 ectodomain heterodimers are able to form a tetramer receptor complex. Peptides of CLV3, CLE14, CLE19, and CLE20 are also able to bind a potential CLV2-CRN heterodimer or heterotetramer complex. Using a cell-division reporter line, we found that synthetic 12-amino-acid CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the root apical meristem, resulting in a short-root phenotype. Intriguingly, we observed that exogenous application of cytokinin can partially rescue the short-root phenotype induced by over-expression of either CLE14 or CLE20 in planta. However, cytokinin treatment does not rescue the short-root phenotype caused by exogenous application of the synthetic CLE14/CLE20 peptides, suggesting a requirement for a condition provided only in living plants. These results therefore imply that the CLE14/CLE20 peptides may act through the CLV2-CRN receptor kinase, and that their availabilities and/or abundances may be affected by cytokinin activity in planta.     

The Cysteine Pairs in CLV2 are Not Necessary for Sensing the CLV3 Peptide in Shoot and Root Meristems

Receptor-like proteins (RLPs) are involved in both plant defense and developmental processes. Previous genetic and biochemical studies show that the leucine-rich repeat (LRR) receptor-like protein CLAVATA2 (CLV2) functions together with CLAVATA1 (CLV1) and CORYNE (CRN) in Arabidopsis to limit the stem cell number in shoot apical meristem, while in root it acts with CRN to trigger a premature differentiation of the stem cells after sensing the exogenously applied peptides of CLV3p, CLE19p or CLE40p. It has been proposed that disulfide bonds might be formed through two cysteine pairs in the extracellular LRR domains of CLV1 and CLV2 to stabilize the receptor complex. Here we tested the hypothesis by replacing these cysteines with alanines and showed that depletions of one or both of the cysteine pairs do not hamper the function of CLV2 in SAM maintenance. In vitro peptide assay also showed that removal of the cysteine pairs did not affect the perception of CLV3 peptides in roots. These observations allow us to conclude that the formation of disulfide bonds is not needed for the function of CLV2.