Molecular and biochemical characterization of the<Emphasis Type="Italic"> Capsicum annuum calcium-dependent protein kinase 3</Emphasis> (<Emphasis Type="Italic">CaCDPK3</Emphasis>) gene induced by abiotic and biotic stresses

The isolated full-length Capsicum annuum calcium-dependent protein kinase 3 (CaCDPK3) cDNA clone was selected from the chili pepper expressed sequence tag database (). Phylogenetic analysis based on the deduced amino acid sequence of CaCDPK3 cDNA revealed significant sequence similarity to the winter squash (Cucurbita maxima) CmCPK2 gene (81% identity). Genomic gel blot analysis disclosed that CaCDPK3 belongs to a multigene family in the pepper genome. CaCDPK3 expression was root tissue-specific, as shown by Northern blot data. The gene was rapidly induced in response to various osmotic stress factors and exogenous abscisic acid application in pepper leaves. Moreover, CaCDPK3 RNA expression was induced by an incompatible pathogen and by plant defense-related chemicals such as ethephon, salicylic acid and jasmonic acid. The biochemical properties of CaCDPK3 were investigated using a CaCDPK3 and glutathione S-transferase (GST) fusion protein. The recombinant proteins retained calcium-binding ability, and displayed autophosphorylation activity in vitro in a calcium-dependent manner. Further transient-expression studies showed that CaCDPK3 fused with soluble modified green fluorescent protein (smGFP) localized to the cytosol in chili pepper protoplasts. We propose that CaCDPK3 is implicated in biotic and abiotic stresses in pepper plants.     

The pepper extracellular peroxidase CaPO2 is required for salt, drought and oxidative stress tolerance as well as resistance to fungal pathogens

In plants, biotic and abiotic stresses regulate the expression and activity of various peroxidase isoforms. Capsicum annuum EXTRACELLULAR PEROXIDASE 2 (CaPO2) was previously shown to play a role in local and systemic reactive oxygen species bursts and disease resistance during bacterial pathogen infection. Here, we report CaPO2 expression patterns and functions during conditions of biotic and abiotic stress. In pepper plants, CaPO2 expression was strongly induced by abscisic acid, but not by defense-related plant hormones such as salicylic acid, ethylene and jasmonic acid. CaPO2 was also strongly induced by abiotic and biotic stress treatments, including drought, cold, high salinity and infection by the hemibiotrophic fungal pathogen Colletotrichum coccodes. Loss-of-function of CaPO2 in virus-induced gene silenced pepper plants led to increased susceptibility to salt- and osmotic-induced stress. In contrast, CaPO2 overexpression in transgenic Arabidopsis thaliana plants conferred enhanced tolerance to high salt, drought, and oxidative stress, while also enhancing resistance to infection by the necrotrophic fungal pathogen Alternaria brassicicola. Taken together, these results provide evidence for the involvement of pepper extracellular peroxidase CaPO2 in plant defense responses to various abiotic stresses and plant fungal pathogens.     

ZmGns,a maize class I β-1,3-glucanase,is induced by biotic stresses and possesses strong antimicrobial activity

Plant b-1,3-glucanases are members of the pathogenesis-related protein 2(PR-2) family,which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses.One of the differentially expressed proteins(spot 842) identified in a recent proteomic comparison between five pairs of closely related maize(Zea mays L.) lines differing in aflatoxin resistance was further investigated in the present study.Here,the corresponding cDNA was cloned from maize and designated as ZmGns.ZmGns encodes a protein of338 amino acids containing a potential signal peptide.The expression of Zm Gns was detectible in all tissues studied with the highest level in silks.ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus.ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid.In vivo,ZmGns showed a significant inhibitory activity against thebacterial pathogen Pseudomonas syringae pv.tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis.Its high level of expression in the silk tissue and its induced expression by phytohormone treatment,as well as by bacterial and fungal infections,suggest it plays a complex role in maize growth,development,and defense.     

番茄低温响应转录因子SlNAC41克隆及表达分析

NAC转录因子在调控植物生长发育、生物及非生物逆境应答中发挥着重要作用。前期,我们通过对番茄幼苗在低温胁迫下的基因表达谱进行分析,发现Unigene SGN-U212711受低温诱导表达强烈。本研究从番茄中克隆了该基因,命名为Sl NAC41,其开放阅读框（ORF）1 173 bp,编码390个氨基酸,蛋白N端具有典型的NAM结构域,属于NAC转录因子家族成员。预测Sl NAC41蛋白分子量为43.5 k Da,等电点为5.2。实时荧光定量PCR分析表明,Sl NAC41在番茄各组织均有表达,在花中的表达量最高,在红熟果中的表达量最低。低温、干旱、高盐、甲基紫精（MV）、脱落酸（ABA）及乙烯利（ETH）处理均能诱导该基因的表达,其中,以低温和干旱诱导表达最为强烈。利用PLACE和Plant CARE对启动子序列进行预测分析发现,Sl NAC41启动子区含有大量响应光、病原菌侵染、激素、低温、脱水及盐胁迫的顺式作用元件。这些结果表明,Sl NAC41可能在番茄生物及非生物胁迫应答中发挥重要调控作用。     

Capsicum annuum tobacco mosaic virus-induced clone 1 expression perturbation alters the plant's response to ethylene and interferes with the redox homeostasis

Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.     

葡萄病程相关蛋白1基因的克隆和表达分析

以葡萄品种‘左优红’组培苗叶片为材料,利用同源克隆法获得其病程相关蛋白1基因VvPR1的cDNA全长序列。扩增片段大小为486bp,编码161个氨基酸,分子量17.5kDa,等电点PI=8.69,含有6个保守半胱氨酸,4个allergenV5/Tpx-1related保守结构域。VvPR1与多种植物PR1高度同源。实时定量PCR检测结果表明VvPR1在葡萄叶片中相对表达量最高;霜霉病菌、低温、盐和干旱胁迫均可显著诱导其表达;水杨酸、脱落酸、茉莉酸、一氧化氮、过氧化氢和硫化氢等亦可诱导其大量表达,据此推测,VvPR1参与了多种生物胁迫和非生物胁迫过程。     

Pathogen-induced expression of cyclo-oxygenase homologue in hot pepper (Capsicum annuum cv. Pukang).

The hypersensitive reaction (HR) in plants is typified by a rapid and localized cell death at the site of pathogen infection. To understand better the molecular and cellular defence mechanism controlling HR, hot pepper leaves (Capsicum annuum cv. Pukang) were inoculated with the soybean pustule pathogen Xanthomonas campestris pv. glycine 8ra. By using the DD-PCR technique, a cDNA fragment was identified that exhibited a sequence similarity to the recently identified tobacco pathogen-induced oxygenase (PIOX) with homology to animal cyclo-oxygenase (COX). Subsequently, the full-length cDNA clone, pCa-COX1, encoding the COX homologue from the pathogen-inoculated hot pepper leaf cDNA library was isolated. The deduced amino acid sequence of Ca-COX1 shares 85.8% identity with tobacco PIOX and displays a significant degree of sequence identity (21.7-23.7%) with mammalian COXs. The expression of Ca-COX1 was markedly induced at 4-12 h after pathogen infection, while HR cell death on pepper leaves appeared at approximately 15 h post-inoculation. These results are consistent with the notion that the lipid-derived signalling pathway is involved in the initial response of hot pepper plants to pathogen infection.