Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter

Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5 deletions of the osmotin promoter cloned into a promoter-less GUS-NOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from –108 to –248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to –1052) of enhancer-like sequence and then a sequence upstream of –1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the –248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.     

Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

ABam HI family of tobacco highly repeated DNA:

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.     

烟草花青素苷转运相关基因NtAN9的分离与表达分析

为研究花青素苷的转运,利用电子克隆和RT-PCR方法,从普通烟草(Nicotiana tabacum)花中分离了1个编码谷胱甘肽转移酶(GST)基因,命名为NtAN9(GenBank登录号KX356542)。NtAN9包含一个690bp的开放阅读框,编码229个氨基酸残基,属于phi型GST。NtAN9基因组结构由3个外显子和2个内含子组成。多序列比对分析表明,NtAN9与矮牵牛(Petunia hybrida)花青素苷转运相关的GST基因PhAN9具有88%的一致性。系统进化分析显示,NtAN9与花青素苷转运相关的GST基因聚为一支,是PhAN9的直系同源基因。定量PCR分析表明,NtAN9基因在含有花青素苷的四个花发育时期中均有表达,其中在开花前的第Ⅲ期(2cm花芽4cm)表达丰度达到最高,而在不含花青素苷的根、茎和叶中不表达。由此推测,分离得到的NtAN9可能具有类似PhAN9的功能,与烟草花青素苷的转运与积累相关。NtAN9基因的分离与表达分析,为进一步研究烟草花青素苷的转运奠定了基础。     

Relationship between Calcium and Pyruvate Kinase

Tobacco plants (Nicotiana tabacum L. cv. Sevilla) were grown under controlled conditions. The leaf content of Ca²⁺, Mg²⁺ and K⁺, and the activity of the pyruvate kinase were analyzed. The increased application of Ca²⁺ diminished the content of K⁺ and Mg²⁺ in leaves, and decreased the activity of pyruvate kinase. Taking into account these results, we suggest the pyruvate kinase activity as an bioindicator of the contents of the Ca²⁺, Mg²⁺ and K⁺.     

Proteins encoded by an auxin-regulated gene family of tobacco share limited but significant homology with glutathione S-transferases and one member indeed shows in vitro GST activity

A number of cDNAs corresponding to auxin-regulated mRNAs have been isolated from tobacco and found to be encoded by a multigene family consisting of three subfamilies. Homologous proteins have been isolated independently from soybean and potato. Here we report that the encoded proteins show a limited but significant homology to both plant and animal glutathione S-transferases (GST, EC 2.5.1.18). For the protein NT103, encoded by a member of the Nt103 subfamily, we demonstrate an in vitro GST activity. This is the first time a function is attributed to a member of this group of auxin-induced proteins or any of its homologues. The implications of this finding and the possible relationships between auxins and GSTs are discussed.     

Quantitative determination of mosaic <Emphasis Type="Italic">GFP</Emphasis> gene expression in tobacco

A specific form of gene silencing that was observed visually as a mosaic distribution of fluorescent and non-fluorescent cells apparently dispersed at random within tissues was found in a few green fluorescent protein (GFP)-transformed tobacco lines. To characterize this event quantitatively, we studied flow cytometric measurements in GFP-expressing and -silenced cells in T₁ and T₂ progeny of four selected plants. The proportion of silenced cells varied considerably among the T₁ lines but with notable genotype differences. Mosaic expression was inherited into the T₂ generation in which the majority of progenies tested exhibited a level of silencing similar to that of their T₁ parental plants. However, in some T₂ progenies segregation, evident as a decrease or increase in the proportion of fluorescent cells, was observed. We discuss several factors, such as copy number, promoter activity or polyploidy, that may be the possible causes of the gene silencing, but none sufficiently explain the appearance of the mosaic distribution.     

Dynamics of inorganic ions in microspore and pollen grain during development of the tobacco male gametophyte

We studied the dynamics of mobile potassium, chloride, and nitrate ions during development of the microspore and differentiation of the pollen grain inNicotiana tabacum L. by measuring their concentration in aqueous extracts from cells destroyed by freezing-thawing using ion-selective electrodes. Stage-specific changes in the ion content and intracellular concentration in the male gametophyte were found. A relationship of the dynamics of ions to growth processes and changes in metabolic activity during gametophytogenesis has been discussed. The changes in the potassium and chloride ion concentrations have been interpreted as regulatory changes controlling protein synthesis in the pollen grain vegetative cell. Deceased.     

Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum

Summary Protoplasts ofNicotiana tabacum var. Xanthi were incubated with liposomes containing the plasmid plGVneo23 encoding kanamycin resistance. Transformed protoplasts and calli and plants derived from transformed protoplasts were treated with the demethylating agent 5-azacytidine. Three lines of evidence indicate that 5-azacytidine can increase NPT II activity in transformed cell lines and plants: a) Addition of azacytidine to the protoplast medium increased the proportion of kanamycin-resistant transformants recovered. b) NPT II activity could not be detected in approximately 50% of calli derived from transformed protoplasts although such calli grew slowly on medium containing kanamycin. Treatment of NPT-negative calli with 5-azacytidine restored detectable gene activity and increased the growth rate of the callus in the presence of kanamycin. c) Shoot tips regenerated from transformed calli were either NPT-positive or NPT-negative. When shoots were NPT-negative, treatment with 5-azacytidine restored detectable gene activity and improved growth in the presence of kanamycin.     

Efficient and sensitive assay for T-DNA-dependent transient gene expression

We describe here a very sensitive and reproducible method to detect the efficiency ofAgrobacterium-mediated T-DNA transfer. This method is based on a quantitative assay of β-glucuronidase activity produced in the plant cell upon transfer of T-DNA carrying a specialuidA gene construct. Analysis of the transfer efficiency of a transfer-proficient bacterium compared with that of the same bacterium diluted at different ratios with a transfer-defective bacterium shows a high sensitivity of the β-glucuronidase activity in the plant. Five orders of magnitude in T-DNA transfer efficiency can be covered when the activity is measured combining the fluorimetric MUG assay (for high activity) and the histochemical X-Gluc assay (very sensitive for low activity).     

Alleviation of plant virus infection by humic acids

K-humates, obtained from oxihumolites, alleviate infection of tobacco with tobacco mosaic virus both in mixture with virus inoculum and by spraying of leaves before inoculation. However, applications of K-humates after inoculation did not influence the virus infectivity.     

Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Differential gene expression of chloroplast and cytosolic phosphoglycerate kinase in tobacco

Northern blot analysis of RNA extracted from leaves of increasing age and different organs, indicates that genes encoding both isoenzymes of tobacco phosphoglycerate kinase (PGK, EC 2.7.2.3) are differentially expressed in a developmental and tissue-specific manner. The genes for both chloroplast PGK (chl-PGK) and cytosolic PGK (cyt-PGK) also show light-modulated gene expression in vivo. In dark-grown developing cotyledonary leaves of tobacco both PGK mRNAs are present, but only the concentration of the chl-PGK mRNA increased on illumination. In contrast, on transfer to darkness, the concentration of both mRNAs decreased in light-grown seedlings and then increased again on resumption of illumination.     

Quantitative kinetic analysis of β-glucuronidase activities using a computer-directed microtiter plate reader

We have established a procedure for automated, kinetic analysis of β-glucuronidase (GUS) activities using a colorimetric or fluorometric microtiter plate reader connected to a computer that directs the measurements and accesses the data. Compared with end-point measurements, the procedure saves time, is more accurate, and needs 20 times less material. It allows a more precise determination of GUS activities over a range of 400,000-fold, with a limit of detection of about 0.01 units of GUS per mL in the colorimetric assay and 0.1 milliunit of GUS in the fluorometric assay. A general protocol for the determination of GUS activities in transgenic plant tissue was worked out and applied to investigate the expression of a chimeric β-glucuronidase gene in stably transformed tobacco calli.     

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.