芥菜HDA9突变体构建及其与开花整合子SOC1、AGL24启动子互作

芥菜HDA9是去乙酰化酶家族成员,能通过开花信号整合子(SOC1、AGL24)调控开花时间,但其深入的分子调控机制仍不清楚。利用重叠延伸PCR将芥菜HDA9的3个关键活性位点(Asp~(172)、His~(174)和Asp~(261))分别突变为Ala,构建氨基酸位点突变体HDA9~(D172A)、HDA9~(H174A)和HDA9~(D261A)。进一步将突变体融合到pGADT7载体,酵母单杂交表明,HDA9突变后仍能与开花整合子SOC1、AGL24的启动子结合。双荧光素酶系统深入检测发现,尽管HDA9~(D172A)、HDA9~(H174A)和HDA9~(D261A)与SOC1、AGL24启动子的结合仍存在,但作用强度均显著减弱。由此暗示,芥菜HDA9的第172、174和261这3个关键活性位点可在一定程度上调节它与开花整合子的相互作用。这为HDA9开花分子调控及功能解析等深入研究奠定了基础。     

Effect of Methyl Ester of Jasmonic Acid and Benzylaminopurine on Growth and Protein Profile of Excised Cotyledons of Cucurbita Pepo (Zucchini)

Treatment of excised marrow (Cucurbita pepo L., zucchini) cotyledons with methyl ester of jasmonic acid (MeJA) had no effect on their growth in darkness. On the other hand, MeJA induced the synthesis of three polypeptides (69, 60 and 43 kDa) and stimulated the accumulation of other polypeptides (97.4 and 53 kDa). These changes in the polypeptide profile were accompanied by a suppression of total protein and RNA synthesis as well as the activity of nuclear RNA polymerases. In contrast to MeJA, N⁶-benzylaminopurine (BAP) significantly enhanced cotyledon growth and stimulated protein and RNA synthesis. Furthermore, BAP, when applied together with MeJA, was able to counteract some effects of MeJA including the appearance of specific MeJA-induced polypeptide bands. This revised version was published online in July 2006 with corrections to the Cover Date.     

The 9-lipoxygenase GhLOX1 gene is associated with the hypersensitive reaction of cotton Gossypium hirsutum to Xanthomonas campestris pv malvacearum.

Hypersensitive reaction (HR) cell death of cotton to the incompatible race 18 from Xanthomonas campestris pathovar malvacearum (Xcm) is associated with 9S-lipoxygenase activity (LOX) responsible for lipid peroxidation. Here, we report the cloning of cotton (Gossypium hirsutum L.) LOX gene (GhLOX1) and the sequencing of its promoter. GhLOX1 was found to be highly expressed during Xcm induced HR. Sequence analysis showed that GhLOX1 is a putative 9-LOX, and GhLOX1 promoter contains SA and JA responsive elements. Investigation on LOX signalisation on cotyledons infiltrated with salicylic acid (SA), or incubated with methyl-jasmonate (MeJA) revealed that both treatments induced LOX activity and GhLOX1 gene expression. HR-like symptoms were observed when LOX substrates were then injected in treated (MeJA and SA) cotyledons or when Xcm compatible race 20 was inoculated on MeJA treated cotyledons. Together these results support the fact that GhLOX1 encodes a 9 LOX whose activity would be involved in cell death during cotton HR.     

Three positive regulators of leaf senescence in Arabidopsis, ORE1, ORE3 and ORE9, play roles in crosstalk among multiple hormone-mediated senescence pathways

Leaf senescence is a developmentally programmed event, but the initiation and progression of leaf senescence are affected by a range of plant hormones including abscisic acid (ABA), ethylene and methyl jasmonate (MeJA). To investigate plant hormone crosstalk during leaf senescence, hormone-induced senescence phenotypes were analyzed in three leaf senescence mutants [ore1 (oresara1), ore3 and ore9] showing delayed senescence phenotypes in age-dependent and dark-induced senescence. The ore mutants exhibited delayed leaf senescence phenotypes following treatment with ABA, ACC (aminocyclo-propane-1-carboxylic acid) or MeJA. After each hormone treatment, the photochemical efficiency of photosystem II and chlorophyll content were significantly higher in the ore mutant leaves than in the wild-type leaves. The expression of CAB2 and SEN4 in the wild-type was rapidly altered following each hormone treatment. However, the decrease in CAB2 expression and the induction of SEN4 expression in the mutants were less affected by ABA, ACC or MeJA treatment. It is suggested that ORE1, ORE3 and ORE9 are required for the proper progression of leaf senescence mediated by ABA, ethylene and MeJA. This implies that ORE1, ORE3 and ORE9 may be linked to the crosstalk among senescence pathways induced by ABA, ethylene and MeJA, as well as age and darkness.     

蜜蜂化学生态学化学通讯与信息素研究进展

概括了蜜蜂化学生态学的主要研究内容和领域,重点评述了蜂群内的化学通讯和蜜蜂信息素研究的进展。迄今,已经鉴定的蜂王信息素有９－氧化－（反）－２－癸烯酸（９－ＯＤＡ）,Ｒ（一）－９－羟基癸烯酸Ｒ（一）－９ＨＤＡ）,Ｓ（＋）－９－羟基癸烯酸（Ｓ（＋）－９ＨＤＡ）,对－羟基苯甲酸甲酯（ＨＯＢ）和４－羟基－３－甲本基乙醇（ＨＶＡ）等５种;工蜂那氏信息互膛牛醇,橙花醇,（反,反）－法尼醇,（反）柠檬醛,（顺）     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.     

A leaf-specific 27 kDa protein of potato Kunitz-type proteinase inhibitor is induced in response to abscisic acid, ethylene, methyl jasmonate, and water deficit

The 22 kDa Kunitz-type potato proteinase inhibitor (22 kDa KPPI) was induced in tubers. However, the 27 kDa protein, which is immunologically related to the 22 kDa KPPI, was induced in leaves by wounding, hormones, and environmental stresses. The leaf-specific 27 kDa protein was induced in leaves that were treated with exogenous abscisic acid (ABA), ethephon, methyl jasmonate (MeJA), and water deficit. These results indicate that the 27 kDa protein in leaves could function as a defense protein against mechanical damages by herbivorous animals and abiotic environmental stresses that could induce plant hormones.     

Effects of Methyl jasmonate with indole-3-acetic acid and 6-benzylaminopurine on the secondary metabolism of cultured <Emphasis Type="Italic">Onosma paniculatum</Emphasis> cells

Summary Methyl jasmonate (MeJA) interacted significantly with both indole-3-acetic acid (IAA) and 6-benzylaminopurine (BA) to influence cell growth of cultured Onosma paniculatum cells. Cell growth decreased with increasing concentrations of MeJA from 0.004–4.45 μM with or without IAA and BA. The same concentrations of MeJA (0–4.45 μM) increased the cell growth with IAA and BA, when administered to the cultured cells in M₉ medium. This was found to enhance the production of shikonin. The optimum time for MeJA addition for enhanced shikonin formation was 4 d after cell inoculation in M₉ medium. Furthermore, shikonin formation was affected significantly by both MeJA/IAA and MeJA/BA combinations. Shikonin content was enhanced by increasing MeJA concentrations with IAA concentrations in the range of 0–28 μM and with BA concentrations in the range of 0–44.38 μM in MeJA/BA experiments, respectively. The optimal combination of MeJA and IAA was 4.45 μM and 0.28 μM, while MeJA and BA concentrations of 4.45 μM and 2.22 μM were optimal for shikonin formation. The result also showed that MeJA increased phenylalanine ammonia-lyase (PAL) and p-hydroxybenzoic acid-geranyltransferase (PHB-geranyltransferase) activites during the course of shikonin formation, but decreased the activity of PHB-O-glucosyltransferase within 9 d after inoculation. These results suggest that enhanced shikonin formation in cultured Onosma paniculatum cells induced by MeJA involves regulation of the key enzyme activities.     

Class I chitinase and beta-1,3-glucanase are differentially regulated by wounding,methyl jasmonate,ethylene, and gibberellin in tomato seeds and leaves

Class I chitinase (Chi9) and beta-1,3-glucanase (GluB) genes are expressed in the micropylar endosperm cap of tomato (Lycopersicon esculentum) seeds just before radicle emergence through this tissue to complete germination. In gibberellin (GA)-deficient mutant (gib-1) seeds, expression of Chi9 and GluB mRNA and protein is dependent upon GA. However, as expression occurs relatively late in the germination process, we investigated whether the genes are induced indirectly in response to tissue wounding associated with endosperm cap weakening and radicle protrusion. Wounding and methyl jasmonate (MeJA) induced Chi9 expression, whereas ethylene, abscisic acid, sodium salicylate, fusicoccin, or beta-aminobutyric acid were without effect. Chi9 expression occurred only in the micropylar tissues when seeds were exposed to MeJA or were wounded at the chalazal end of the seed. Expression of Chi9, but not GluB, mRNA was reduced in germinating seeds of the jasmonate-deficient defenseless1 tomato mutant and could be restored by MeJA treatment. Chi9 expression during germination may be associated with "wounding" from cell wall hydrolysis and weakening in the endosperm cap leading to radicle protrusion, and jasmonate is involved in the signaling pathway for this response. Among these treatments and chemicals (other than GA), only MeJA and wounding induced a low level of GluB expression in gib-1 seeds. However, MeJA, wounding, and particularly ethylene induced both genes in leaves, whereas GA induced only Chi9 in leaves. Although normally expressed simultaneously during tomato seed germination, Chi9 and GluB genes are regulated distinctly and tissue specifically by hormones and wounding.     

Methyl jasmonate and cis-jasmone do not dispose of the herbivore-induced jasmonate burst in Nicotiana attenuata

The oxylipin pathway mediates wound- and herbivore-induced defense reactions in Nicotiana attenuata as evidenced by a transient jasmonic acid (JA)-burst that precedes these defense responses. The fate of this induced JA-burst remains unknown. Two derivatives of JA, its methylester, methyl jasmonate (MeJA) and cis -jasmone ( cis J), are thought to be a means of disposing of JA through volatilization at the plant surface. In N. attenuata, the headspace quantities of these compounds did not change over 3 days, although levels of MeJA and cis J increased 100- and 70-fold, respectively, in surface extracts of attacked leaves after feeding of Manduca sexta larvae or application of larval regurgitant to mechanical wounds. Inhibition of the wound-induced increase in JA with indole-3-acetic acid (IAA) revealed an association between the JA accumulation and subsequent increases in MeJA and cis J. Induced systemic increases of MeJA were not of local origin and therefore do not contribute to the inactivation of the JA-burst in the wounded leaf. The total amount of MeJA and cis J produced could only account for 9% of the JA-burst elicited by herbivore attack and therefore their production do not represent major disposal pathways of JA in N. attenuata .     

HY5-HDA9 orchestrates the transcription of HsfA2 to modulate salt stress response in Arabidopsis

Integration of light signaling and diverse abiotic stress responses contribute to plant survival in a changing environment. Some reports have indicated that light signals contribute a plant's ability to deal with heat, cold, and stress. However, the molecular link between light signaling and the salt-response pathways remains unclear. We demonstrate here that increasing light intensity elevates the salt stress tolerance of plants. Depletion of HY5, a key component of light signaling, causes Arabidopsis thaliana to become salinity sensitive. Interestingly, the small heat shock protein (sHsp) family genes are upregulated in hy5-215 mutant plants, and HsfA2 is commonly involved in the regulation of these sHsps. We found that HY5 directly binds to the G-box motifs in the HsfA2 promoter, with the cooperation of HISTONE DEACETYLASE 9 (HDA9), to repress its expression. Furthermore, the accumulation of HDA9 and the interaction between HY5 and HDA9 are significantly enhanced by salt stress. On the contrary, high temperature triggers HY5 and HDA9 degradation, which leads to dissociation of HY5-HDA9 from the HsfA2 promoter, thereby reducing salt tolerance. Under salt and heat stress conditions, fine tuning of protein accumulation and an interaction between HY5 and HDA9 regulate HsfA2 expression. This implies that HY5, HDA9, and HsfA2 play important roles in the integration of light signaling with salt stress and heat shock response.     

HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis

Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases, which play key roles in the regulation of eukaryotic gene expression. Previously, we have identified a yeast histone deacetylase REDUCED POTASSIUM DEPENDENCY3 (RPD3) homolog, HISTONE DEACETYLASE19 (HDA19) (AtRPD3A), in Arabidopsis thaliana. Here, we report further study of the expression and function of HDA19. Analysis of Arabidopsis plants containing the HDA19:beta-glucuronidase fusion gene revealed that HDA19 was expressed throughout the life of the plant and in most plant organs examined. In addition, the expression of HDA19 was induced by wounding, the pathogen Alternaria brassicicola, and the plant hormones jasmonic acid and ethylene. Using green fluorescent protein fusion, we demonstrated that HDA19 accumulated in the nuclei of Arabidopsis cells. Overexpression of HDA19 in 35S:HDA19 plants decreased histone acetylation levels, whereas downregulation of HDA19 in HDA19-RNA interference (RNAi) plants increased histone acetylation levels. In comparison with wild-type plants, 35S:HDA19 transgenic plants had increased expression of ETHYLENE RESPONSE FACTOR1 and were more resistant to the pathogen A. brassicicola. The expression of jasmonic acid and ethylene regulated PATHOGENESIS-RELATED genes, Basic Chitinase and beta-1,3-Glucanase, was upregulated in 35S:HDA19 plants but downregulated in HDA19-RNAi plants. Our studies provide evidence that HDA19 may regulate gene expression involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.     

不同发育时期及外源处理后赤霞珠葡萄脂氧合酶活性及脂肪酸含量变化

以酿酒葡萄品种赤霞珠为试材,研究了果实发育时期、机械创伤处理与外源茉莉酸甲酯处理对葡萄果皮中脂肪酸组分、含量及LOX活性的变化。结果表明:(1)葡萄果皮的LOX活性在花后12周达到最大值;花后15周对果实进行创伤处理,处理3h后LOX活性达到最高点;花后17周对果实进行MeJA处理,处理后24hLOX活性达到最大值。(2)葡萄果皮脂肪酸组分中,饱和脂肪酸以棕榈酸和硬脂酸为主,而不饱和脂肪酸以亚油酸和亚麻酸为主。(3)葡萄果实脂肪酸含量随葡萄发育先增加后下降,花后15周创伤处理后的葡萄脂肪酸含量波动较大,花后17周茉莉酸甲酯处理后24h葡萄脂肪酸含量显著升高。     

Acetylation of 13-sophorosyloxydocosanoic acid by an acetyltransferase purified from Candida bogoriensis.

Acetyl-coenzyme A: 13-sophorosyloxydocosanoic acid (Glc2HDA) acetyltransferase was purified 14-fold in low yield from Candida bogoriensis cells. The enzyme catalyzes acetylation of the 6' and 6" positions of the sophorosyl group, producing the 13-[2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyloxy]-docosanoic acid 6',6"-diacetate (Ac2Glc2HDA) and monoacetate (AcGlc2HDA) in a product ratio of 5:1. Neither the purification steps nor heat denaturation studies indicated separation of the first and second acetylation steps. The acetyltransferase has a molecular weight of about 500,000 as determined by gel filtration on a Sepharose 4-B column. It shows a pH optimum range from 7 to 9, is strongly inhibited by 1 mM concentrations of the sulfhydryl reagents N-ethylmaleimide, p-hydroxymercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid), but only partly inhibited by 10 mM iodoacetamide. It has an apparent Km of 30 muM for acetyl-CoA, utilizes propionyl-CoA at 45% the rate of acetyl-CoA, and utilizes longer chain acyl-CoA derivatives much less efficiently. The critical micelle concentrations of the C. bogoriensis glycolipids in pH 7.7 phosphate buffer were estimated by pinacyanol chloride binding as follows: Glc2HDA, 50 mum; AcGlc2HDA, 30 muM; Ac2Glc2HDA, 12 muM. The Stokes radius of Ac2Glc2HDA micelles was 22 A as estimated by gel filtration on Bio-Gel P-150. Glc2HDA was a much better acceptor than its methyl ester in the acetyltransferase assay. A plateau in the Glc2HDA saturation curve at 50 muM and a corresponding break in the reciprocal plot at this concentration indicate the enzyme utilizes the monomeric form of this lipid as substrate.     

Monoclonal antibody-based enzyme linked immunosorbent assay for the analysis of jasmonates in plants

Methyl jasmonate (MeJA) and its free-acid form,jasmonic acid (JA) are naturally occurring plant growth regulators widely distributed in higher plants.In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples,a monoclonal antibody (MAb) (designated as MAb 3E5D7C4B6) against MeJA was derived from a JAbovine serum albumin (BSA) conjugate as an immunogen.The antibody belongs to the IgG1 subclass with a κ type light chain and has a dissociation constant of approximately 6.07 x 10-9 M.MAb3E5D7C4B6 is very specific to MeJA.It was used to develop a direct competitive enzyme-linked immunosorbent assay (dcELISA),conventional and simplified indirect competitive ELISAs (icELISA).JA was derivatized into MeJA for the ELISA analysis.The IC50 value and detection range for MeJA were,respectively,34 and 4-257 ng/mL by the conventional icELISA,21 and 3-226 ng/mL by the simplified icELISA and 5.0 and 0.7-97.0 ng/mL by the dcELISA.The dcELISA was more sensitive than either the conventional or simplified icELISA.The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves.The increased jasmonates content indicated its role in response to the drought stress and pathogens.