Heavy metal stress-inducible early light-inducible gene CaELIP from hot pepper (Capsicum annuum) shows broad expression patterns under various abiotic stresses and circadian rhythmicity

An early light-inducible protein gene (CaELIP) was isolated from a cDNA library of hot pepper (Capsicum annuum) that showed heavy metal stress-inducible expressions. This gene contains an open reading frame (ORF) encoding a protein of 160 amino acids, and the protein has significant homology with reported early light-inducible proteins from other plant species. Topology analysis for CaELIP suggested three transmembrane domains. Genomic DNA blot analysis showed that CaELIP is a single copy gene in hot pepper. The treatment of seedling roots of hot pepper with Cu induced ROS generation in the root, and the level of ROS generation was paralleled to the concentration of Cu that again was matched to the increase in the CaELIP expression level. Results suggested that expression of CaELIP can be induced by the ROS generated by the excessive Cu in the plant. Exogenous SA treatment significantly alleviated Cu-induced expression of CaELIP, while exogenous JA treatment aggravated expression of CaELIP under Cu stress. CaELIP showed a transient expression when exposing the plant to light for 1 h. CaELIP also showed an endogenous circadian rhythmicity with high expression level in the morning and decreased expression level thereafter. The expression of CaELIP was also induced by high or low temperature, high salinity, drought, and stress hormone ABA. Taken together, the results suggest that CaELIP would function in responding to environmental signals and possibly regulating the response to the abiotic stresses that can be related to the abiotic stress tolerance in plants.     

Effects of methyl jasmonate and salicylic acid on cell growth and cryptotanshinone formation in Ti transformed Salvia miltiorrhiza cell suspension cultures

Exogenous methyl jasmonate (MJ) or salicylic acid (SA) when applied alone failed to induce cryptotanshinone (a phytoalexin) formation in Ti-transformed Salvia miltiorrhiza cell suspension cultures. However, when applied in combination with yeast elicitor, SA at 50–500 M enhanced the yeast elicitor-induced cryptotanshinone formation while MJ reduced the yeast elicitor-induced cryptotanshinone formation. Ibuprofen at 100 M did not inhibit the yeast elicitor-induced cryptotanshinone formation. DMSO was superior to ethanol as a solvent for introducing MJ to the cell cultures.     

Differential Response of Floret Opening in Male-Sterile and Male-Fertile Rices to Methy Jasmonate

Effects of methyl jasmonate (MeJA) on floret opening were investigated in male-sterile and male-fertile rice ( Oryza sativa subsp. indica ). Excised or intact panicles with several top florets flowered one day before the experiments were soaked in MeJA, salicylic acid (SA), 24-epibrassinolide (24-epiBL) or control solution for 2 min. MeJA at the concentration of 4 mmol/L significantly promoted rice floret opening; the response of floret opening of male-sterile rice to MeJA was more sensitive than that of male-fertile rice (such as maintenance line and conventional variety). The percentage of finally opened-floret per panicle of Zhenshan 97A (cytoplasmic male sterile line) or Pei'ai 64S (thermo-sensitive genic male sterile line) treated with MeJA was over 40% in the day of experiment, that of Zhenshan 97B (maintenance) and Pei'ai 64 (conventional variety) was (21±1.5)% and (25±2.1)%, respectively; floret opening of intact Pei'ai 64S panicles in the field could also be induced by 2-mmol/L MeJA. The percentage of opened floret in all control panicles was 0% or less than 5% in the day of experiment. The percentage of fertilized-grain of Zhenshan 97A and Longtepu A could be increased over 24% by spraying panicles with 2-mmol/L MeJA solution （circa 6.0 μmol per panicle）. The lag time (time from the finishing of treatment to first floret opening) of male sterile line responsing to MeJA was 10 min shorter than that of male fertile line. MeJA induction on the rice floret opening could markedly be inhibited by SA solution at the concentration of 1 mmol/L, and the inhibition of SA on MeJA induction on floret opening of male-sterile rice could be nullified by MeJA treatment again. MeJA effect on floret opening of male-sterile rice could be clearly synergized by pretreatment with 24-epibrassinolide (24-epiBL).     

Jasmonate-inducible expression of a potato cathepsin D inhibitor-GUS gene fusion in tobacco cells

A potato gene encoding cathepsin D inhibitor (CDI) is expressed constitutively in tubers and flower buds and it is inducible in leaves upon wounding of the tissue or by treatment with methyl jasmonate (MJA). A fusion gene (CDI:GUS) in which the 2.4 kb long promoter of the CDI gene was translationaly fused with the coding sequence for -glucuronidase (GUS) showed MJA-inducible expression in transformed tobacco cells in suspension. The maximum level of induction by MJA was obtained in the absence of auxin and repression of MJA-inducible expression of the fusion gene by auxin was released by aphidicolin, the results suggesting that MJA-inducible expression is repressed during active cell division. JA and MJA showed similar activities in inducing the expression of the fusion gene, while other JA-related compounds such as cucurbic acid, tuberonic acid and dihydrojasmonic acid neither induced expression of the fusion gene nor inhibited the MJA-inducible expression of the fusion gene. Methyl dihydrojasmonate specifically stimulated the MJA-inducible expression of the fusion gene. The MJA-inducible expression of the fusion gene was observed even with a 100 bp long promoter of the CDI gene albeit with significantly decreased level of expression compared to the 2.4 kb long promoter. The 100 bp long CDI promoter did not contain a G-box or hexamer motif that had been implicated in the MJA-responsive expression of several other plant genes. Further mutagenesis of the 100 bp long promoter by deletion or oligonucleotide insertion suggested that although a sequence between –100 and –82 is required for the MJA-responsive expression, the presence of this sequence alone does not confer the MJA-responsive expression.Abbreviations BA N⁶-benzyladenine - CA cucurbic acid - CaMV cauliflower mosaic virus - CDI cathepsin D inhibitor - DMSO dimethyl sulfoxide - GUS -glucuronidase - HJA dihydrojasmonic acid - JA Jasmonic acid - MCA methyl cucurbate - MJA methyl jasmonate - MHJA methyl dihydrojasmonate - MTA methyl tuberonate - PI-II proteinase inhibitor II - TA tuberonic acid - 2,4-D 2,4-dichlorophenoxyacetic acid.     

2种植物生长调节剂诱导烟草抗链格孢菌研究

以链格孢菌Alternaria alternata和烟草Nicotiana tabacum品种云烟87为试材,研究不同浓度茉莉酸甲酯(MeJA)和水杨酸(SA)处理下,链格孢菌菌丝生长情况以及成熟期烟叶防御酶活性与多酚含量变化,探讨植物生长调节剂对烟草抗链格孢菌的影响。结果表明,1 mmol·L^–1 MeJA对链格孢菌的抑制效果最好,抑菌率高达59%以上,其次是3.5 mmol·L^–1SA;MeJA和SA对链格孢菌的抑制效果随药剂浓度升高而增强;0.1 mmol·L^–1 MeJA和2.5 mmol·L^–1 SA能诱导烟草叶片SOD、POD、CAT等防御酶活性,并能降低H₂O₂活性氧含量,尤其以MeJA诱导效果较好。2种植物生长调节剂处理并接种链格孢菌能诱导提高烟叶多酚代谢相关酶PPO及PAL活性,但对烟草多酚类物质影响较小;成熟烟叶中含量较高的前3种多酚物质是绿原酸、芸香苷、隐绿原酸。     

The rolC gene increases caffeoylquinic acid production in transformed artichoke cells

Caffeoylquinic acids are found in artichokes, and they are currently considered important therapeutic or preventive agents for treating Alzheimer’s disease and diabetes. We transformed artichoke [the cultivated cardoon or Cynara cardunculus var. altilis DC (Asteraceae)] with the rolC gene, which is a known inducer of secondary metabolism. High-performance liquid chromatography with UV and high-resolution mass spectrometry (HPLC-UV-HRMS) revealed that the predominant metabolites synthesized in the transgenic calli were 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and chlorogenic acid. The rolC-transformed calli contained 1.5 % caffeoylquinic acids by dry weight. The overall production of these metabolites was three times higher than that of the corresponding control calli. The enhancing effect of rolC remained stable over long-term cultivation.     

绿盲蝽取食诱导后棉花叶片内防御酶活力与基因表达量的变化

本研究以抗性棉皖棉小黄花为材料,室内条件下测定绿盲蝽Apolygus lucorum(Mayer-Dür)取食、机械损伤、外源水杨酸和外源茉莉酸甲酯诱导处理后棉叶中苯丙氨酸解氨酶基因(pal)、过氧化物酶基因(pod)和过氧化氢酶基因(cat)的相对表达量,并以健康植株为对照。结果表明:绿盲蝽取食诱导与外源信号物质诱导相似,苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性升高,过氧化氢酶(CAT)活性降低;PAL、POD和CAT活力变化与本文中选取的pal、pod和cat基因表达量变化趋势存在差异。本研究说明,绿盲蝽取食既激活了水杨酸介导的防御信号转导途径,也激活了茉莉酸介导的信号转导途径;PAL、POD和CAT 3种酶活力不完全由本文中选取的pal、pod和cat 3个基因所调控。     

Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene.

The Nicotiana plumbaginifolia gn1 gene encoding a beta-1,3-glucanase isoform has been characterized. The gn1 product represents an isoform distinct from the previously identified tobacco beta-1,3-glucanases. By expressing gn1 in Escherichia coli, we have determined directly that the encoded protein does, indeed, correspond to a beta-1,3-glucanase. In N. plumbaginifolia, gn1 was found to be expressed in roots and older leaves. Transgenic tobacco plants containing the 5'-noncoding region of gn1 fused to the beta-glucuronidase (GUS) reporter gene also showed maximum levels of GUS activity in roots and older leaves. No detectable activity was present in the upper part of the transgenic plants with the exception of stem cells at the bases of emerging shoots. The expression conferred by the gn1 promoter was differentially induced in response to specific plant stress treatments. Studies of three plant-bacteria interactions showed high levels of GUS activity when infection resulted in a hypersensitive reaction. Increased gene expression was confined to cells surrounding the necrotic lesions. The observed expression pattern suggests that the characterized beta-1,3-glucanase plays a role both in plant development and in the defense response against pathogen infection.     

Cloning and expression of a new Tibetan hulless barley (Hordeum vulgare) beta-1,3-glucanase gene

A new full-length -1,3-glucanase cDNA was obtained by RT-PCR and RACE techniques from Tibet hulless barley and its complete gene sequence obtained by DNA Walking. Sequence alignment with the BLAST program showed that cDNA has high similarity with barley -1,3-glucanase II. The gene was functionally expressed in E. coli and the recombinant protein catalysed the hydrolysis of -1,3-glucan with an action pattern characteristic of a -1,3-glucan endohydrolase (EC 3.2.1.39). Southern blot analysis indicated that the gene is a member of a small gene family. RT-PCR and northern blot analysis indicated it is constitutively expressed in barley shoots.     

Induction of pathogen resistance and pathogenesis-related genes in tobacco by a heat-stable Trichoderma mycelial extract and plant signal messengers

Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica . The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.     

The Structure of a New Piperidine Derivative from Buckwheat Seeds (Fagopyrum esculentum Moench)

Streptomyces hygroscopicus No. B–5050-HA, which produces a mixture of six maridomycins, yielded a mutant which produced 75% of the mixture as maridomycin III (MDM III).

Growth of S. hygroscopicus No. B–5050-HA, an improved MDM producer, was almost completely inhibited by 20 µg/ml of valine. This inhibition was counteracted by the addition of isoleucine, threonine, homoserine, methionine, α-amino-n-butyrate and α-ketobutyrate.

A valine resistant mutant, strain AV was isolated and found to produce increased level of MDM III at the expense of other maridomycins. Production of MDM III by the parent strain depended on the addition of isoleucine to the medium, but that by this mutant did not.

The properties of strain AV were discussed.     

Phospholipase signaling is modified differentially by phytoregulators in Capsicum chinense J. cells

Plant defense mechanisms respond to diverse environmental factors and play key roles in signaling pathways. The phospholipidic signaling pathway forms part of the plant response to several phytoregulators, such as salicylic acid (SA) and methyl jasmonate (MJ), which have been widely used to stimulate secondary metabolite production in cell cultures.¹ Furthermore, it has been reported that the levels of such phytoregulators as SA and MJ can increase in response to stressful conditions.^2,3 The phospholipidic signal transduction system involves the generation of second messengers by the hydrolysis of phospholipids. In this study, we examined how phospholipidic signaling can be modulated depending on the growth stage of the culture, and we focused on two key lipases having relevant roles in the signaling cascades in plants. An evaluation was made of the effects of SA and MJ on the phospholipase activities in Capsicum chinense Jacq. suspension cells at different phases of the culture cycle. The treatment with SA differentially modified the phospholipase C (PLC) (EC: 3.1.4.3) and phospholipase D (PLD) (EC: 3.1.4.4) activities in a dose-dependent manner that also depended on the day of the culture cycle. In contrast, the treatment with MJ resulted in a biphasic behavior of the PLC and PLD activities. We conclude that the enzymatic activities in the phospholipidic signaling pathways are modified differentially depending on the day of the culture’s growth cycle; accordingly, the response capacity to such environmental factors as phytoregulators is variable at different stages of growth and the physiology of the cells.     

喜树愈伤组织的诱导及诱导子对愈伤组织中喜树碱含量的影响

通过组织培养的方法分别对喜树营养器官进行愈伤组织的诱导和继代,筛选红色、黄色和褐色3个不同的细胞系;运用HPLC法测定各愈伤组织中喜树碱的含量,并在培养基中外加诱导子,分析其对愈伤组织中喜树碱含量的影响。结果表明,经幼叶所诱导的愈伤组织中喜树碱含量相对高于其它,红色和黄色的细胞系在不同的外植体中其喜树碱含量均高于褐色的细胞系。但从整体看来,愈伤组织中喜树碱的含量远远低于原植物中的含量。外加诱导子对愈伤组织中喜树碱的含量有影响,浓度为0.1 mg·L^-1,1 mg·L^-1的水杨酸和茉莉酸甲酯处理的愈伤组织中喜树碱含量提高,0.1 mg·L^-1的水杨酸对喜树碱的积累有明显的促进作用,茉莉酸甲酯的影响不大;浓度为3 mg·L^-1,5 mg·L^-1的水杨酸处理的愈伤组织中,喜树碱含量降低;3 mg·L^-1的茉莉酸甲酯处理的愈伤组织中,喜树碱含量降低,5 mg·L^-1处理的愈伤组织中未检测到喜树碱。     

Functional characterization of a novel tropinone reductase-like gene in Dendrobium nobile Lindl

Dendrobium nobile, a herbal medicine plant, contains many important alkaloids and other secondary metabolites with pharmacological and clinical effects. However, the biosynthetic pathway of these secondary metabolites is largely unknown. In present study, a cDNA sequence (DnTR2) that encodes a peptide with high similarity to known tropinone reductase (TR) was cloned from D. nobile Lindl. Sequence comparison and phylogenetic analysis showed that DnTR2 was evolutionarily distant from those well-characterized subgroups of TRs. qRT-PCR revealed that DnTR2 was expressed constitutively in all three vegetative organs (leaves, stems and roots) and was regulated by methyl jasmonate (MeJA), salicylic acid (SA) and nitrogen oxide (NO). Catalytic activity analysis using recombinant protein found that DnTR2 was not able to reduce tropinone, but reduced the two structural analogs of tropinone, 3-quinuclidinone hydrochloride and 4-methylcyclohexanone. Structural modeling and comparison suggested that the substrate specificity of TRs may not be determined by their phylogenetic relationships but by the amino acids that compose the substrate binding pocket. To verify this hypothesis, a site-directed mutagenesis was performed and it successfully restored the DnTR2 with tropinone reduction activity. Our results also showed that the substrate specificity of TRs was determined by a few residues that compose the substrate binding pocket which may have an important role for directed selecting of TRs with designated substrate specificities.     

Functional divergence in the Arabidopsis beta-1,3-glucanase gene family inferred by phylogenetic reconstruction of expression states

Plant beta-1,3-glucanases (beta-1,3-Gs) (E.C. 3.2.1.39) comprise large, highly complex gene families involved in pathogen defense as well as a wide range of normal developmental processes. In spite of previous phylogenetic analyses that classify beta-1,3-Gs by sequence relatedness, the functional evolution of beta-1,3-Gs remains unclear. Here, expression and phylogenetic analyses have been integrated in order to investigate patterns of functional divergence in the Arabidopsis beta-1,3-G gene family. Fifty beta-1,3-G genes were grouped into expression classes through clustering of microarray data, and functions were inferred based on knowledge of coexpressed genes and existing literature. The resulting expression classes were mapped as discrete states onto a phylogenetic tree and parsimony reconstruction of ancestral expression states was performed, providing a model of expression divergence. Results showed a highly nonrandom distribution of developmental expression states in the phylogeny (P = 0.0002) indicating a significant degree of coupling between sequence and developmental expression divergence. A weaker, yet significant level of coupling was found using stress response data, but not using hormone-response or pathogen-response data. According to the model of developmental expression divergence, the ancestral function was most likely involved in cell division and/or cell wall remodeling. The associated expression state is widely distributed in the phylogeny, is retained by over 25% of gene family members, and is consistent with the known functions of beta-1,3-Gs in distantly related species and gene families. Consistent with previous hypotheses, pathogenesis-related (PR) beta-1,3-Gs appear to have evolved from ancestral developmentally regulated beta-1,3-Gs, acquiring PR function through a number of evolutionary events: divergence from the ancestral expression state, acquisition of pathogen/stress-responsive expression patterns, and loss of the C-terminal region including the glycosylphosphatidylinisotol (GPI)-anchoring site thus allowing for extracellular secretion.