Analysis of regulatory elements involved in stress-induced and organ-specific expression of tobacco acidic and basic β-1,3-glucanase genes

Infection of tobacco by tobacco mosaic virus (TMV) induces coordinate expression of genes encoding acidic and basic -1,3-glucanase isoforms. These genes are differentially expressed in response to other treatments. Salicylate treatment induces acidic glucanase mRNA to a higher level than basic glucanase mRNA. Ethylene treatment and wounding strongly induce the basic glucanase genes but have little effect on genes encoding the acidic isoforms. Furthermore, the basic glucanase genes are constitutively expressed in roots and lower leaves of healthy plants, whereas the acidic glucanase genes are not. In order to investigate how these expression patterns are established, we fused promoter regions of an acidic and a basic glucanase gene to the -glucuronidase (GUS) reporter gene and examined expression of these constructs in transgenic tobacco plants.A fragment of 1750 bp and two 5-truncated fragments of 650 bp and 300 bp of the acidic glucanase promoter were tested for induction of GUS gene expression after salicylate treatment and TMV infection. Upstream sequences of 1750 bp and 650 bp were sufficient for induction of the reporter gene by salicylate treatment and TMV infection, but the activity of the 300 bp fragment was strongly reduced. The results suggest that the 1750 bp upstream sequence of the acidic glucanase gene contains multiple regulatory elements.For the basic glucanase promoter it is shown that 1476 bp of upstream sequences were able to drive expression in response to TMV infection and ethylene treatment, but no response was found to incision wounding. Furthermore, high GUS activity was found in lower leaves and roots of healthy transgenic plants, carrying the 1476 bp basic glucanase promoter/GUS construct. When the promoter was truncated up to position –446 all activity was lost, indicating that the region between –1476 and –446 of the basic glucanase promoter is necessary for organ-specific and developmentally regulated expression as well as for induced expression in response to infection and other stress treatments.     

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.     

Pre-activating wounding response in tobacco prior to high-level ozone exposure prevents necrotic injury

In tobacco, both wounding and treatment with jasmonates prior to exposure of the tissue to high concentrations of ozone (250 to 500 p.p.b.) produce a dramatic decrease in ozone injury. A systemic pattern of increased ozone tolerance developed within 3–6 h after wounding and also after local application of jasmonates. Ozone treatment of transgenie (NahG) tobacco plants showed that the inability of these plants to accumulate salicylic acid is also accompanied by increased ozone tolerance. Expression of mRNA encoding the anti-oxidant enzyme ascorbate peroxidase is upregulated by ozone challenge, wounding and by methyl jasmonate exposure within 3–4 h, while levels of carbonic anhydrase mRNA are simultaneously depressed following ozone exposure and methyl jasmonate treatment. The pattern of these results shows that the response to ozone challenge in tobacco involves signalling mechanisms similar to those induced in plants by other environmental stresses that generate reactive oxygen species.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB,genes encoding vegetative storage protein acid phosphatases,and is regulated similarly by methyl jasmonate,wounding, sugars,light and phosphate

The soybean vegetative storage proteins, VSP and VSP, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp and Vsp, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Role of Jasmonates in the Elicitor- and Wound-Inducible Expression of Defense Genes in Parsley and Transgenic Tobacco

Jasmonates have been proposed to be signaling intermediates in the wound and/or elicitor-activated expression of plant defense genes. We used parsley (Petroselinum crispum) cell cultures and transgenic tobacco (Nicotiana tabacum) plants expressing 4CL1-GUS gene fusions to investigate the potential role played by jasmonates in mediating the wound and/or elicitor activation of phenylpropanoid and other defense-related genes. Jasmonates and [alpha]-linolenic acid strongly induced the expression of 4CL in a dose-dependent manner in parsley cells; methyl jasmonate also activated the coordinate expression of other phenylpropanoid genes and the accumulation of furanocoumarin phytoalexins. However, the response of the cells to optimal methyl jasmonate concentrations was distinct quantitatively and qualitatively from the response of elicitor-treated cells. In transgenic tobacco wound-inducible tobacco 4CL genes and a 4CL1 promoter-GUS transgene were responsive to jasmonates and [alpha]-linolenic acid in a dose-dependent manner. Pre-treatment of parsley cells or tobacco leaves with a lipoxygenase inhibitor reduced their responsiveness to the elicitor and to wounding. These results show that the elicitor response in parsley cells can be partially mimicked by jasmonate treatment, which supports a role for jasmonates in mediating wound-induced expression of 4CL and other phenylpropanoid genes.     

Overexpression of the apple alcohol acyltransferase gene alters the profile of volatile blends in transgenic tobacco leaves

Alcohol acyltransferases (AATs) are key enzymes in ester biosynthesis. Previous studies have found that AAT may be a stress-related gene. To investigate further the function of the apple alcohol acyltransferase gene (MdAAT2), transgenic tobacco plants overexpressing MdAAT2 were generated. Gas chromatography-mass spectroscopy analysis showed that the volatile blends were altered in these transgenic tobacco leaves. Although no apple-fruity volatile esters were detected in transgenic tobacco leaves, methyl caprylate, methyl caprate, and methyl dodecanoate were newly generated, and the concentrations of methyl benzoate and methyl tetradecanoate were significantly increased, suggesting that MdAAT2 may use medium-chain fatty acyl CoA and benzoyl-CoA as acyl donors together with methanol acceptors as substrates. Surprisingly, the concentrations of linalool were significantly increased in transgenic tobacco leaves, which may mediate the repellent effect on Myzus persicae (Sulzer) aphids. Using methyl jasmonate (MeJA) and wounding treatments, we found that MdAAT2 may substitute for the partial ability of MeJA to induce the production of linalool in transgenic plants. These data suggest that MdAAT2 may be involved in the response to the MeJA signal and may play a role in the response to biotic and abiotic stress.     

Developmental,hormonal, and pathogenesis-related regulation of the tobacco class I β-1,3-glucanase B promoter

The class I -1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric -glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I -1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I -1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from –1452 to –1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: –568 to –402 for ethylene induction of leaves; –402 to –211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and –211 to –60 for expression in roots.     

Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression

We have previously identified two cDNAs encoding vegetative storage proteins (VSPs) in Arabidopsis thaliana. Unlike soybean in which VSPs accumulate at high levels in leaves, A. thaliana VSP mRNAs are abundant in flowers. To understand tissue-specific expression and possible roles of VSPs on reproductive organ development, genes corresponding to VSPs (Vsp1 and Vsp2) and their putative promoters were characterized in this study. Genomic sequence analysis revealed that Vsp1 and Vsp2 resemble each other except in their introns, and that these two genes were organized in a tandem array with an interval of 6 kb in a region. The expression patterns of Vsp1 and Vsp2 were examined using transgenic A. thaliana plants carrying a promoter from Vsp1 or Vsp2 fused to a bacterial -glucuronidase (GUS) reporter gene. The promoter from Vsp1 expressed its effect in gynoecia, especially in styles, the basal and distal ends of ovaries and in siliques, whereas the promoter from Vsp2 showed its activity in vegetative shoots, petioles, peduncles and receptacles of floral organs. These results suggest that expression of Vsp1 and Vsp2 may be developmentally regulated in A. thaliana. In the transgenic plants, the GUS activity was induced by wounding in an area around the mid-rib of leaves. Therefore, Vsp1 and Vsp2 promoters appear to have elements required for both tissue specificity and wounding.