Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

Growth kinetics, polyamine pattern and biosynthesis in hairy root lines of Nicotiana tabacum

The possibility of a relation between the expression of root inducing (Ri) T-DNA genes of Agrobacterium rhizogenes and changes in polyamine metabolism has been explored in fast-growing tobacco hairy roots. Transformed root cultures have been established on hormone-fee medium; they came from transgenic plants of Nicotiana tabacum L. cv. Xanthi with different altered phenotypes, designated transformed (T) and supertransformed (T'). T and especially T' roots developed more rapidly both by elongation and lateral branching, and showed a higher growth rate than the untransformed control. After 3 weeks in culture, normal roots showed a very reduced meristematic zone, and flow cytometric analysis indicated that 2C nuclei were predominant in the apical parts in contrast to T and T' roots, in which endopolyploidisation also appeared. Putrescine, spermidine and traces of spermine were present in all the samples, both in free and in conjugated forms. Putrescine was the major polyamine detected in controls and in transformed roots. At the time of excision, the polyamine levels were similar in normal, T and T' roots. Significant differences were found during the progression of growth, particularly in the TCA-insoluble fraction in which polyamines varied differently according to the type of roots, increasing considerably in T roots on day 8, then decreasing. The lower polyamine contents found in growing transformed roots were concomitant to low arginine (EC 4.1.1.19) and ornithine (EC 4.1.1.17) decarboxylase activities. It is suggested that polyamine levels and related enzyme activities are linked to growth kinetics rather than being a consequence of foreign gene expression.     

Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.     

低温胁迫下褪黑激素对烟草悬浮细胞精氨酸脱羧酶活性的影响

研究了褪黑激素对烟草(Nicotiana tabacum)悬浮细胞在低温胁迫下精氨酸脱羧酶活性及细胞生存率的影响.发现褪黑激素可以明显提高低温胁迫下烟草悬浮细胞精氨酸脱羧酶的活性,并明显提高细胞的生存率.表明褪黑激素可能在低温条件下通过调节植物细胞内多胺的合成而提高抵御冷害的能力.     

Changes in endogenous cyanide and 1-aminocyclopropane-1-carboxylic acid levels during the hypersensitive response of tobacco mosaic virus-infected tobacco leaves

Leaves of Nicotiana tabacum L. cv. Xanthi necroticum plants form local necrotic lesions at the site of infection by tobacco mosaic virus. During the first seven days post-inoculation, endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-ACC increased in the lesion area. The time course of ACC accumulation coincided with an increase in the endogenous cyanide level which began within two days after inoculation. Concomitantly, the activity of -cyanoalanine synthase, the main HCN detoxifying enzyme, decreased. Likewise, treatment of leaf discs of uninfected plants with ACC led to cyanide accumulation. Exogenously applied KCN caused necrotic spots on tobacco leaves very similar to the whitish centers of virus-induced local lesions. Possible implications of cyanide in cell death during TMV-induced lesion development are discussed.     

Identification of peptidases in Nicotiana tabacum leaf intercellular fluid

Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration-centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum gamma-globulin as substrates. Peptidases were detected by 1- and 2-D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.     

烟草株高变异体的茎尖中内源激素含量变化及其对外源激素的响应

烟草矮秆基因型“农大202’的茎生长过程中茎尖中内源赤霉素（GA3）、生长素（IAA）含量始终低于中、高秆基因型,其内源脱落酸（ABA）和玉米素（ZR）含量则相对较高。喷施外源激素GA,和IAA的结果表明,GA,可调节矮秆基因型茎尖中各内源激素的含量,与正常株高基因型‘K326’的各激素含量相接近,从而促进茎生长,而IAA的作用较小。     

烟草ovate同源基因的全长cDNA克隆与序列分析

根据番茄中控制果实形状的主效数量性状基因ovate的序列,用生物信息学方法从茄科植物烟草中获得直系同源ovate基因(NTovate)的特异片段,经鉴定,此基因在烟草中至少有2个拷贝。在此基础上用cDNA末端快速扩增(RACE)方法,获得其中1个拷贝的1059bpNTovate全长cDNA序列。序列分析表明,NTovate cDNA序列编码352个氨基酸,其蛋白序列与番茄ovate蛋白序列和拟南芥ovate蛋白家族AtOFP7蛋白分别为70%和36%的序列一致率,而与此家族中其他蛋白以及水稻ovate蛋白仅在保守的ovate结构域有较低的同源性。此基因已在GenBank中登录(EU043369)。     

Mosaicism in the organization of Con A binding sites on the membrane surface of female cells of Nicotiana tabacum

The presence of mosaicism in the organization of concanavalin agglutinin (Con A) binding sites on murine egg cells was first reported 30 year ago. This discovery has triggered extensive studies into the roles of glycoproteins in gamete interactions in animals. This report comprises the first account of the existence of the mosaicism in higher plants. The distribution of Con A binding sites on both egg cells and central cells of tobacco (Nicotiana tabacum) was found to be polar and apparently determined by the location of the nucleus of the cell. On central cells, Con A binding sites were distributed on the section of the plasma membrane surface near the nucleus. By contrast, the binding sites on egg cells were concentrated away from the nucleus. Therefore, polarity of the plasma membrane component of female cells was confirmed for the first time. It is proposed that such polarized ConA binding sites could be involved in sperm recognition.     

烤烟SSR标记多样性及与致香物质的关联分析

了解烤烟品种的遗传多样性,寻找与烤烟致香物质含量关联的分子标记,挖掘高致香物质含量的优异等位变异与种质,对香气品质分子标记辅助育种具有重要意义。本研究检测了山东和四川2个生态区60份烤烟种质中76种致香物质的含量;筛选覆盖全基因组的1914个SSR标记,利用得到的390对多态性引物扩增供试群体。用基于Nei's(1983)遗传距离的邻接法(Neighor-joining,NJ)进行聚类分析。在分析群体结构的基础上,利用混合线性模型进行关联分析,并进一步发掘极显著关联标记的优异等位变异和种质。结果检测到928个等位变异,平均每个位点2.38个等位变异,变化范围为2~5个。PIC值的变化范围分别为0.141~0.733,平均值0.332。这说明供试群体遗传多样性较丰富。聚类分析将该群体划分为2个亚群,划分结果体现了烤烟品种间的亲缘关系;群体结构分析与聚类结果基本一致。关联分析结果显示:共有56个SSR标记位点与41种致香物质同时在2个生态区显著关联(P0.05)。共5个标记与6个性状在2个生态区极显著关联(P0.01),6种致香物质为草酸、肉豆蔻酸、2,4-庚二烯醛、芳樟醇、a-松油醇和gamma-壬内酯,关联标记分别为PT50662、PT60191、PT52263、PT60597、PT60597和PT52722。关联位点表型解释率为12.54%~42.20%。进一步分析发掘了14种致香物质的优异等位变异,并筛选出含增效等位变异较多的种质:净叶黄、抗9201、单育二号、满屋香、金星6007、秦烟95等。这对香气品质分子标记辅助选择育种以及亲本材料选配等奠定了基础。     

Markers for hypersensitive response and senescence show distinct patterns of expression

Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.     

烤烟冠层光谱参数与叶片叶绿素含量的相关分析

为了明确烤烟冠层光谱参数与叶片叶绿素含量的相关性,测定了不同氮肥施用量条件下烤烟冠层光谱特征和烤烟鲜烟叶片叶绿素a(Chl-a)、叶绿素b(Chl-b)、类胡萝卜素(Cars)含量,并对光谱参数与叶绿素含量进行了相关分析和回归分析。结果表明:随着氮肥施用量增加,团棵期和旺长期鲜烟叶片的Chl-a、Chl-b和Cars含量均增加,可见光波段反射率降低、近红外波段反射率增加;而打顶期叶片的3种色素含量和光谱特征的变化规律不明显。可见光460～670nm范围内,460nm反射率与叶片叶绿素含量呈显著正相关,其他波段反射率与叶片叶绿素的含量呈显著负相关;近红外780～1260nm范围内,所有波段与叶片叶绿素含量的都呈显著正相关,1480nm反射率与叶片叶绿素含量呈显著负相关。反映Chl-a、Chl-b、Cars含量与光谱参数——比值植被指数(ratio vegetation index,RVI)定量关系的最佳回归方程分别为幂函数、幂函数和指数函数:Chl-a=0.250RVI(730,550)1.511,Chl-b=0.049RVI(730,550)1.841,Cars=0.0998e0.379RVI(730,550)。     

Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum

The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene ( cph A_Te) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske- cph A_Te, pCP24- cph A_Te, pFNR- cph A_Te and pPsbY- cph A_Te. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY- cph A_Te led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24- cph A_Te and pFNR- cph A_Te constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY- cph A_Te produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T₀) transformants. Specific T₂ plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.     

Role of ammonium accumulation in bacteria-induced hypersensitive and compatible reactions of tobacco and cotton plants

Leaves of 12-week-old tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were infiltrated with suspensions of Pseudomonas syringae pv, pisi (DSM 50291) to induce hypersensitive reaction (HR). Cotyledons of 2-week-old cotton plants (Gossypium hirsutum L. cv. Acala 442 and Coker BR) were infiltrated with Xanthomonas campestris pv. malvacearum (race 10) to induce the disease. In tobacco, HR-related increases in NH⁺₄ levels started within 2 h after infection and continued up to the time of tissue decay. Increase of NH⁺₄ and especially K⁺ efflux were detected in intercellular washing fluids (IWF). Antibiotics stopped and later reverted NH⁺₄ production and K⁺ efflux, but only if applied within 2 h after infection. When 10 mM NH⁺₄ was injected into leaves, it was rapidly consumed from the IWF, and also, although more slowly, within the leaf cells. The concomitant K⁺ efflux was strong but delayed, and most of the K⁺ was reabsorbed after 2 h. Bacterial cell multiplication in HR stopped before the appearance of HR symptoms and cell necrosis. In the compatible reaction in cotton cotyledons, both NH₊⁴accumulation and K⁺ efflux proceeded much more slowly than in the HR with tobacco, and bacteria continued to multiply until general cell necrosis occurred. The compatible reaction developed faster in constant darkness than in a light/dark rhythm. Bacterial enzymes produced NH⁺₄, mainly from proteins of host cells, in both light and darkness. Continuous light delayed the main peak of both NH⁺₄ production and K⁺ efflux. High CO₂ concentration inhibited both processes, thus indicating that photorespiration plays a role in enhancing the release of free ammonium during bacterial pathogenesis. This is supported by shifts in the pattern of amino acids. The results demonstrate the accelerating and aggravating effect of ammonium in pathogenesis and HR, though ammonium is not the primary agent.     

DREB类转录因子介导的烟草抗非生物胁迫特性研究

检测并分析了转BnDREB1-5基因烟草叶片的持水性能,上、下表皮气孔大小和密度及叶绿素含量,并在自然失水7 h后检测了细胞的离子泄漏状况。结果表明:转基因烟草叶片单位时间内每平方厘米的失水量是野生型烟草叶片的62%;上表皮的气孔大于野生型,而气孔开度小于野生型;野生型烟草叶片上的气孔密度接近转基因烟草的1.5倍;野生型烟草叶片的叶绿素含量比转基因烟草叶片高29%;野生型烟草叶片的质膜相对透性是转基因烟草叶片的1.4倍。     

Cytoenzymological analysis of adenylyl cyclase activity and 3':5'-cAMP immunolocalization in chloroplasts of Nicotiana tabacum

In this study a combination of cytoenzymological and immunocytochemical techniques was used in order to demonstrate the presence of cyclic nucleotide metabolism in chloroplasts of higher plants. Catalytic cytochemistry was used to localize adenylyl cyclase activity by means of electron microscope investigation on Nicotiana tabacum cv. Petit Havana leaf fragments. Various immunocytochemical techniques were explored to visualize the presence of the second messenger adenosine 3':5'-cyclic monophosphate. Making use of adenylyl imidodiphosphate as a substrate, the enzyme activity was predominantly located at the intermembrane space of the chloroplast envelope. In order to provide further topographical information, intact, isolated chloroplasts were submitted to the same cytoenzymological procedure and revealed stromal adenylyl cyclase activity. Using high-pressure freezing as a physical fixative to obtain an instantaneous metabolic arrest the cellular vitrified water phase was sublimed under ultra-high vacuum by means of molecular distillation drying, avoiding recrystallization and hence redistribution of small highly diffusible molecules. This sequential combination preserved 3':5'-cAMP epitope retention in chloroplasts as was demonstrated by immunogold labelling. These results further substantiate in a unique way the growing evidence of the presence of an organelle-specific cAMP metabolism in higher plants. Furthermore the data presented support the status of chloroplasts as an excellent model to further investigate cAMP metabolism and to correlate it with a variety of physiological functions.