Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.     

Transformation of Lotus corniculatus Plants with Escherichia coli Asparagine Synthetase A: Effect on Nitrogen Assimilation and Plant Development

Asparagine and glutamine are major forms of nitrogen in the phloem sap of many higher plants. In vascular plants, glutamine-dependent asparagine synthetase (AS) is the primary source of asparagine. In Escherichia coli, asparagine is synthesized by the action of two distinct enzymes, AS-A which utilizes ammonia as a nitrogen donor, and AS-B which utilizes both glutamine and ammonia as substrates, but with a preference for glutamine. In this study, the possibility to endow plants with ammonia-dependent AS activity was investigated by heterologous expression of E. coli asnA gene with the aim to introduce a new ammonium assimilation pathway in plants. The bacterial gene is placed under the control of light-dependent promoters, and introduced by transformation into Lotus corniculatus plants. Analysis of transgenic plants has revealed a phenomenon of transgene silencing which has prevented asnA expression in several transgenics. The asnA-expressing plants are characterized by premature flowering and reduced growth. A significant reduction of the total free amino acid accumulation in transgenic plants is observed. Surprisingly, the content of asparagine in wild-type plants is about 2.5-fold higher than that of transgenic plants. While glutamine levels in transgenic plants are about 3–4-fold higher than those in wild-type plants, aspartate levels are significantly lower. Transformation with asnA also induced a significant reduction of photosynthesis when measured under saturated light and ambient CO₂ conditions.     

Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants

The Arabidopsis thaliana (L.) Heynh. minD gene (AtMinD1) was isolated and constitutively expressed in tobacco (Nicotiana tabacum L.) plants using the CaMV 35S promoter. Confocal and electron-microscopic analysis of the AtMinD1 transgenic tobacco lines revealed that the chloroplasts were abnormally large and fewer in number compared with wild-type tobacco plants. The abnormal chloroplasts were less prevalent in guard cells than in mesophyll cells. Chloroplast and nuclear gene expression was not significantly different in AtMinD1-overexpressing plants relative to wild-type tobacco plants. Chloroplast DNA copy number was not affected, based on the relative level of the rbcL gene in transgenic plants. Transgenic tobacco plants constitutively overexpressing AtMinD1 were completely normal phenotypically with respect to growth and development, and also displayed normal photosynthetic electron transport rates. These results show that the Arabidopsis MinD1 gene also functions in a heterologous system and confirm the role of the MinD protein in regulation of chloroplast division.     

The pea plastocyanin promoter directs cell-specific but not full light-regulated expression in transgenic tobacco plants

A series of 5′ deletions of the pea plastocyanin gene (petE) promoter fused to the β-glucuronidase (GUS) reporter gene has been examined for expression in transgenic tobacco plants. Strong positive and negative cis-elements which modulate quantitative expression of the transgene in the light and the dark have been detected within the petE promoter. Disruption of a negative regulatory element at ?784 bp produced the strongest photosynthesis-gene promoter so far described. Histochemical analysis demonstrated that all petE-GUS constructs directed expression in chloroplast-containing cells, and that a region from ?176 bp to +4 bp from the translation start site was sufficient for such cell-specific expression. The petE-promoter fusions were expressed at high levels in etiolated transgenic tobacco seedlings but there was no marked induction of GUS activity in the light. The endogenous tobacco plastocyanin genes and the complete pea plastocyanin gene in transgenic tobacco plants were also expressed in the dark, but showed a three- to sevenfold increase in the light. This indicates a requirement for sequences 3′ to the promoter for the full light response of the petE gene.     

The 5[prime] Flanking Regions of Vicilin and Napin Storage Protein Genes Are Down-Regulated by Desiccation in Transgenic Tobacco

Drying of seeds, when imposed prematurely, elicits a switch in metabolism; events unique to development, such as synthesis of storage protein, are terminated, whereas syntheses associated with germination and growth are initiated. To determine the role of desiccation in down-regulating the expression of genes for storage proteins, the desiccation responsiveness of the 5[prime] and 3[prime] regulatory regions of the genes encoding the pea storage protein vicilin and the Brassica napus storage protein napin was tested in transgenic tobacco seed. Chimeric genes were introduced into tobacco; these genes consisted of the coding region of the reporter gene for [beta]-glucuronidase (GUS) and 5[prime] and/or 3[prime] regions from the vicilin or napin genes or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation insensitive. In transgenic seed expressing the gene constructs containing the vicilin or napin promoters, GUS activities declined during late seed development, and more dramatically, after imbibition of mature dry seed or prematurely dried seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral promoter replaced the storage-protein gene 5[prime] region. Transient expression assays support the hypothesis that premature drying down-regulates the expression of the storage-protein gene promoter. Following desiccation, this region may become insensitive to positive controlling factors; alternatively, changes to trans-acting factors may occur.     

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T₂ progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.     

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Transgenic Tobacco Plants Expressing Pea Chloroplast Nmdh cDNA in Sense and Antisense Orientation (Effects on NADP-Malate Dehydrogenase Level,Stability of Transformants,and Plant Growth)

A full-length cDNA encoding light-activated chloroplast NADP-malate dehydrogenase (NADP-MDH) (EC 1.1.1.82) from pea (Pisum sativum L.) was introduced in the sense and antisense orientation into tobacco (Nicotiana tabacum L.). Transgenic plants with decreased or increased expression levels were obtained. Because of substantial age-dependent differences in individual leaves of a single plant, standardization of NADP-MDH levels was required first. Then, extent and stability of over- or under-expression of Nmdh, the gene encoding NADP-MDH, was characterized in the various transformants. Frequently, cosuppression effects were observed, indicating sufficient homology between the endogenous tobacco and the heterologous pea gene. Analysis of the T1 and T2 progeny of a series of independent transgenic lines revealed that NADP-MDH capacity ranged between 10% and [greater than or equal to]10-fold compared with the wild type. Under ambient conditions whole-plant development, growth period, and fertility were unaffected by NADP-MDH reduction to 20% of the wild-type level; below this threshold plant growth was retarded. A positive growth effect was registered in young plants with stably enhanced NADP-MDH levels within a defined developmental window.     

Reactive Oxygen Species Formation and Cell Death in Catalase-Deficient Tobacco Leaf Discs Exposed to Paraquat

In the present work, the response of tobacco (Nicotiana tabaccum L.) wild-type SR1 and transgenic CAT1AS plants (with a basal reduced CAT activity) was evaluated after exposure to the herbicide paraquat (PQ). Superoxide anion (O₂^.−) formation was inhibited at 3 or 21 h of exposure, but H₂O₂ production and ion leakage increased significantly, both in SR1 or CAT1AS leaf discs. NADPH oxidase activity was constitutively 57% lower in non-treated transgenic leaves than in SR1 leaves and was greatly reduced both at 3 or 21 h of PQ treatment. Superoxide dismutase (SOD) activity was significantly reduced by PQ after 21 h, showing a decrease from 70% to 55%, whereas catalase (CAT) activity decreased an average of 50% after 3 h of treatment, and of 90% after 21 h, in SR1 and CAT1AS, respectively. Concomitantly, total CAT protein content was shown to be reduced in non-treated CAT1AS plants compared to control SR1 leaf discs at both exposure times. PQ decreased CAT expression in SR1 or CAT1AS plants at 3 and 21 h of treatment. The mechanisms underlying PQ-induced cell death were possibly not related exclusively to ROS formation and oxidative stress in tobacco wild-type or transgenic plants.     

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.     

The indoleacetic acid-lysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants

Summary The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase that conjugates lysine to indoleacetic acid. A chimaeric gene consisting of the iaaL coding region under the control of the 35S RNA promoter from cauliflower mosaic virus (35SiaaL) has been used to test if iaaL gene expression leads to morphological alterations in tobacco and potato. Transgenic tobacco plantlets bearing this construct have been shown to synthesize IAA-[¹⁴C]lysine when fed with [¹⁴C]lysine. In late stages of development, their leaves show an increased nastic curvature (epinasty) of the petiole and midvein, a finding suggestive of an abnormal auxin metabolism. The alteration is transmitted to progeny as a dominant Mendelian trait cosegregating with the kanamycin resistance marker. Transgenic potato plants harbouring the construct are also characterised by petiole epinasty. Moreover, 35SiaaL transgenic plants have an increased internode length in potato and decreased root growth in both tobacco and potato. An increased content of IAA-conjugates in leaf blade was found to correlate with the epinastic alterations caused by iaaL gene expression in tobacco leaves. These data provide evidence that IAA conjugation is able to modulate hormone action, suggesting that the widespread endogenous auxin-conjugating activities are of physiological importance.     

Overexpression of [delta]-Pyrroline-5-Carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants

Proline (Pro) accumulation has been correlated with tolerance to drought and salinity stresses in plants. Therefore, overproduction of Pro in plants may lead to increased tolerance against these abiotic stresses. To test this possibility, we overexpressed in tobacco the mothbean [delta]-pyrroline-5-carboxylate synthetase, a bifunctional enzyme able to catalyze the conversion of glutamate to [delta]-pyrroline-5-carboxylate, which is then reduced to Pro. The transgenic plants produced a high level of the enzyme and synthesized 10- to 18-fold more Pro than control plants. These results suggest that activity of the first enzyme of the pathway is the rate-limiting factor in Pro synthesis. Exogenous supply of nitrogen further enhanced Pro production. The osmotic potentials of leaf sap from transgenic plants were less decreased under water-stress conditions compared to those of control plants. Overproduction of Pro also enhanced root biomass and flower development in transgenic plants under drought-stress conditions. These data demonstrated that Pro acts as an osmoprotectant and that overproduction of Pro results in the increased tolerance to osmotic stress in plants.