Cytokinin stress changes the developmental regulation of several defence-related genes in tobacco

Tobacco shoots exposed to elevated endogenous or exogenous cytokinin levels are unable to develop roots and lack apical dominance. We have isolated cDNA copies of five mRNA species that accumulate to elevated levels in such cytokinin-stressed shoots via differential screening of a cDNA library of transgenic shoots which contain an active T-DNA cytokinin gene (T-cyt gene) from Agrobacterium tumefaciens. Four of the cDNA clones were found to correspond to plant defence-related mRNAs, encoding extensin, chitinase, PR-1 and a PR-1-like protein, respectively. In normal tobacco plants PR-1 mRNA is relatively rare in all organs. The other four mRNAs occur at relatively low levels in shoots, especially in leaves, but are very prevalent in roots. Extensin mRNA, for example, is not detectable in leaves, while it is an abundant mRNA in roots and stems. In normal shoots cultured on cytokinin-containing medium all five mRNAs accumulate to elevated levels, similar to those found in transgenic T-cyt shoots. We conclude that the imposed cytokinin stress causes changes in the tissue-specific control of the levels of several defence-related mRNA species in tobacco.     

Cytokinin regulation of a soybean pollen allergen gene

Cytokinin treatment of suspension-cultured soybean cells stimulated the accumulation of an mRNA, called cim 1, by a factor of ca. 20 within 4 h. Induction of cim 1 mRNA accumulation occurred at benzyladenine concentrations as low as 10^-8 M. Furthermore, cim 1 mRNA accumulation was stimulated in the absence of cytokinin by staurosporine (an inhibitor of protein kinases) and inhibited in the presence of cytokinin by okadaic acid (an inhibitor of protein phosphatases 1 and 2a), suggesting that cim 1 accumulation in response to cytokinin is dependent on cytokinin-induced dephosphorylation of one or more cellular proteins. The deduced amino acid sequence of the cim 1 protein product, derived from the complete nucleotide sequence of a cim 1 cDNA, was 40% identical to that of a perennial rye grass pollen allergen cDNA (Lol Pl). This sequence also indicated that the cim 1 protein product contains a putative signal peptide followed by predominantly hydrophilic residues, consistent with the hypothesis that it is exported to the apoplast.     

Characterization of a tobacco extensin gene and regulation of its gene family in healthy plants and under various stress conditions

A genomic clone (Ext 1.4) encoding an extensin was isolated from a Nicotiana tabacum genomic library. The encoded polypeptide showed features characteristic of extensins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of one Tyr-Leu-Tyr-Lys motif suggests the possibility for one intramolecular isodityrosine cross-link whereas numerous Val-Tyr-Lys motifs may participate in intermolecular cross-links. This extensin appears to be close to an extensin already characterized in N. tabacum but very different from the Ext 1.2 extensin of N. sylvestris. The analysis of genomic DNA gel blots using probes spanning different parts of the gene showed that the Ext 1.4 gene belongs to a complex multigene family having one member originating from N. sylvestris and three members from N. tomentosiformis. The Ext 1.4 specific probe found a 1.4 kb mRNA in stems, roots, ovaries and germinating seeds of healthy plants. The Ext 1.4 gene family is strongly induced in actively dividing cell suspension cultures and after wounding of leaves or stems in conditions where root formation occurs. On the contrary, it is not induced in leaves in response to a hyperensitive reaction to a viral infection or after elicitor treatment.     

Translational regulation of gene expression during conditions of cell stress

A number of stresses, including nutrient stress, temperature shock, DNA damage, and hypoxia, can lead to changes in gene expression patterns caused by a general shutdown and reprogramming of protein synthesis. Each of these stress conditions results in selective recruitment of ribosomes to mRNAs whose protein products are required for responding to stress. This recruitment is regulated by elements within the 5' and 3' untranslated regions of mRNAs, including internal ribosome entry segments, upstream open reading frames, and microRNA target sites. These elements can act singly or in combination and are themselves regulated by trans-acting factors. Translational reprogramming can result in increased life span, and conversely, deregulation of these translation pathways is associated with disease including cancer and diabetes.     

Oxidative stress and gene regulation

Reactive oxygen species are produced by all aerobic cells and are widely believed to play a pivotal role in aging as well as a number of degenerative diseases. The consequences of the generation of oxidants in cells does not appear to be limited to promotion of deleterious effects. Alterations in oxidative metabolism have long been known to occur during differentiation and development. Experimental perturbations in cellular redox state have been shown to exert a strong impact on these processes. The discovery of specific genes and pathways affected by oxidants led to the hypothesis that reactive oxygen species serve as subcellular messengers in gene regulatory and signal transduction pathways. Additionally, antioxidants can activate numerous genes and pathways. The burgeoning growth in the number of pathways shown to be dependent on oxidation or antioxidation has accelerated during the last decade. In the discussion presented here, we provide a tabular summary of many of the redox effects on gene expression and signaling pathways that are currently known to exist.     

Short communication. Differential regulation of a glucosyl transferase gene homologue during defence responses in tobacco

The regulation of a glucosyl transferase homologue which is rapidly induced during defence response in tobacco was investigated. Expression of the gene is induced in reasons to wounding and during a hypersensitive response to TMV, but regulation differs in these two responses.Key words: Plant defence, signalling, glucosyl transferase.      

Cytokinin affinity purification and identification of a tobacco BY-2 adenosine kinase

Adenosine kinase is one of the enzymes potentially responsible for the formation of cytokinin nucleotides in plants. Using a zeatin affinity column a 40 kDa protein was isolated from tobacco Bright Yellow 2 (TBY-2) and identified by mass spectrometry as adenosine kinase. The ligand interaction reported here can be disrupted by several other adenine- but not guanine-based purine derivatives. The observed interaction with cytokinins is discussed in view of a putative role for adenosine kinase in TBY-2 cytokinin metabolism. The presented results show for the first time a plant adenosine kinase affinity-purified to homogeneity that was identified by primary structure analysis.     

Molecular cloning of a metallothionein-like gene from Nicotiana glutinosa L. and its induction by wounding and tobacco mosaic virus infection.

The cloning and characterization of genes expressed in plant disease resistance could be an initial step toward understanding the molecular mechanisms of disease resistance. A metallothionein-like gene that is inducible by tobacco mosaic virus and by wounding was cloned in the process of subtractive cloning of disease resistance-response genes in Nicotiana glutinosa. One 530-bp cDNA clone (KC9-10) containing an open reading frame of 81 amino acids was characterized. Genomic Southern blot hybridization with the cDNA probe revealed that tobacco metallothionein-like genes are present in few or in one copy per diploid genome. Northern blot hybridization detected strong induction of a 0.5-kb mRNA by wounding and tobacco mosaic virus infection, but only mild induction was detected when copper was tested as an inducer. Methyl jasmonate, salicylic acid, and ethylene were also tested as possible inducers of this gene, but they had no effect on its expression. The possible role of this gene in wounded and pathogen-stressed plants is discussed.     

Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.     

Cytokinin effects on growth of quiescent tobacco pith cells

Excised pith tissue from Nicotiana glauca or the tumor-prone hybrid N. glauca × N. langsdorffii has no growth requirement for exogenous cytokinins. Addition of kinetin to cultures of these lines results in growth inhibition at a kinetin concentration 1000-fold lower than the optimal level for kinetin-requiring lines. Cytological comparison of the kinetin-inhibited 2N hybrid and glauca tissues with pith from the kinetin-requiring N. tabacum var. Wisconsin 38 suggests that the nature of the cytokinin action is similar in both situations and that the primary function of cytokinin, when it stimulates growth, may be to curtail cell expansion, thereby facilitating a balance of cell expansion and division requisite for maximal growth.     

Cytokinin activation of thiamine biosynthesis in tobacco callus cultures

Bioassays of tissue extracts show that high (500-1000 μg/liter) kinetin concentrations which permit growth of tobacco callus cultures on media without added thiamine activate the biosynthesis of this vitamin by the tissues. Although the tissue concentration of thiamine may fall appreciably, it is maintained at a level adequate for survival and slow growth of the cultures, and there is a large net increase in total thiamine content per culture with time. In the second and subsequent passages of tissue on a thiamine free medium, growth is obtained only when high kinetin concentrations are maintained. Effective inhibition of growth by antithiamines suggests that thiamine is utilized by the high-kinetin tissue.

In the presence of low (30-100 μg/liter) kinetin concentrations, which would be optimal for growth in the presence of thiamine, growth only occurs early in the first passage of tissue from a medium with the vitamin to one without it. The thiamine concentration in the tissues falls to low levels, and no net biosynthesis is apparent. The tissues turn dark and die after 2 to 3 weeks. In contrast with this, in the absence of both added thiamine and kinetin no appreciable growth occurs, but the tissues keep their normal appearance, retain their thiamine content, and may stay alive for several weeks.

     

RNA expression patterns of a type 2 metallothionein-like gene from rice

A type 2 metallothionein-like gene from rice, OsMT-2 (Oryza sativa metallothionein-like gene-2), was isolated in its cDNA form and sequenced. By northern analyses OsMT-2 expression was shown to be induced under stress by sucrose starvation, heat shock and, to a lesser extent, abscisic acid, but not excess metals, including copper. Its response to sucrose starvation was transient and different from OsMT-1, a type 1 metallothionein-like gene of rice inducible by copper. These results suggest that while OsMT-2 is also involved in cellular response to stress, its function may be complementary to that of OsMT-1.