Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants

Selectable markers of bacterial origin such as the neomycin phosphotransferase type II gene, which can confer kanamycin resistance to transgenic plants, represent an invaluable tool for plant engineering. However, since all currently used antibiotic-resistance genes are of bacterial origin, there have been concerns about horizontal gene transfer from transgenic plants back to bacteria, which may result in antibiotic resistance. Here we characterize a plant gene, Atwbc19, the gene that encodes an Arabidopsis thaliana ATP binding cassette (ABC) transporter and confers antibiotic resistance to transgenic plants. The mechanism of resistance is novel, and the levels of resistance achieved are comparable to those attained through expression of bacterial antibiotic-resistance genes in transgenic tobacco using the CaMV 35S promoter. Because ABC transporters are endogenous to plants, the use of Atwbc19 as a selectable marker in transgenic plants may provide a practical alternative to current bacterial marker genes in terms of the risk for horizontal transfer of resistance genes.     

Tissue-specific expression directed by an Arabidopsis thaliana pre-ferredoxin promoter in transgenic tobacco plants

We have isolated and analyzed a pre-ferredoxin gene from Arabidopsis thaliana. This gene encodes a 148 amino acid precursor protein including a chloroplast transit peptide of 52 residues. Southern analysis shows the presence of a single copy of this ferredoxin (Fd) gene in the A. thaliana genome. Its expression is tissue-specific and positively affected by light. Response times, both to dark and light conditions, are remarkably rapid.A chimeric gene consisting of a 1.2 kb Fd promoter fragment fused to the -glucuronidase reporter gene was transferred to tobacco. This fusion gene is expressed in a tissue-specific way; it shows high levels of expression in green leaves, as compared to root tissue.     

Overexpression of Arabidopsis thaliana LTL1, a salt-induced gene encoding a GDSL-motif lipase, increases salt tolerance in yeast and transgenic plants

Genes involved in the mechanisms of plant responses to salt stress may be used as biotechnological tools for the genetic improvement of salt tolerance in crop plants. This would help alleviate the increasing problem of salinization of lands cultivated under irrigation in arid and semi-arid regions. We have isolated a novel halotolerance gene from Arabidopsis thaliana, A. thaliana Li-tolerant lipase 1 (AtLTL1), on the basis of the phenotype of tolerance to LiCl conferred by its expression in yeast. AtLTL1 encodes a putative lipase of the GDSL-motif family, which includes bacterial and a very large number of plant proteins. In Arabidopsis, AtLTL1 expression is rapidly induced by LiCl or NaCl, but not by other abiotic stresses. Overexpression of AtLTL1 increases salt tolerance in transgenic Arabidopsis plants, compared to non-transformed controls, allowing germination of seeds in the presence of toxic concentrations of LiCl and NaCl, and stimulating vegetative growth, flowering and seed set in the presence of NaCl. These results clearly point to a role of AtLTL1 in the mechanisms of salt tolerance. In addition, we show that AtLTL1 expression is also activated, although only transiently, by salicylic acid (SA), suggesting that the lipase could also be involved in defence reactions against pathogens.     

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana

Summary. Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana. Correspondence and reprints: Laboratório de Biotecnologia de Células Vegetais, ITQB, Apartado 127, Avenida da República (E.A.N.), 2781-901 Oeiras, Portugal.     

Overexpression of the repressor gene PvFRI-L from Phyllostachys violascens delays flowering time in transgenic Arabidopsis thaliana

The gene FRIGIDA (FRI) is floral repressor and plays a key role in the timing of Arabidopsis flowering. To study the function of FRI-like genes in bamboo, we isolated a FRI family gene from bamboo Phyllostachys violascens and named it PvFRI-L. Sequence alignment and phylogenetic analysis show that the PvFRI-L protein belongs to the FRL3 (III) subfamily from monocots and contains a conserved FRIGIDA domain. PvFRI-L was located in the nucleus of onion epidermal cells. PvFRI-L was expressed in all tested organs of flowering and non-flowering bamboo plants with a higher expression in non-flowering than in flowering plants. Overexpression of PvFRI-L in Arabidopsis caused late flowering by downregulating flowering locus T and upregulating flowering locus C. A P-box, the binding site involved in gibberellin response, was found only in the promoter region of PvFRI-L but not in that of FRI. Furthermore, PvFRI-L expression in the leaves of Ph. violascens seedlings was downregulated with gibberellic acid treatment. Taking together, our observation suggests that PvFRI-L may be flowering repressor and its delaying floral timing may be regulated by gibberellic acid in bamboo.     

Determination of gene copy number and genotype of transgenic Arabidopsis thaliana by competitive PCR

A simple and rapid method is described for determining the integrated T-DNA copy number and the genotype in transgenic Arabidopsis thaliana by two-step competitive PCR. First, the amount of genomic DNA in the extracts, obtained from an individual A. thaliana transformant, was accurately determined by the 1st competitive PCR using a known single copy gene, 4HPPD (4-hydroxyphenylpyruvate dioxygenase), as a target. Second, the number of T-DNA copies per genome was estimated by quantifying the NPTII gene, which was involved in the T-DNA, by the 2nd competitive PCR using exactly the same amount of genomic DNA for each sample. The estimated copy number and genotype obtained by this procedure were identical to those determined by Southern blot analysis and segregation analysis.     

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post‐symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early‐warning sentinels potentially have tremendous utility as wide‐area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis‐acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time‐course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

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.     

Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana

Tocopherol cyclase (VTE1, encoded by VTE1 gene) catalyzes the penultimate step of tocopherol synthesis. Transgenic tobacco plants overexpressing VTE1 from Arabidopsis were exposed to drought conditions during which transgenic lines had decreased lipid peroxidation, electrolyte leakage and H(2)O(2) content, but had increased chlorophyll compared with the wild type. Thus VTE1 can be used to increase vitamin E content of plants and also to enhance tolerance to environmental stresses.     

Overexpression of the nuclear protein gene AtDUF4 increases organ size in Arabidopsis thaliana and Brassica napus

正Organ size is an important trait of many crops that is influenced by internal and environmental signals and controlled by a combination of factors during organogenesis(Krizek,2009).The final size of plant organs is determined by two successive but overlapping processes:cell division,which increases cell number,and cell expansion,which determines final cell size(Anastasiou and Lenhard,     

Indole acetonitrile-sensitivity of transgenic tobacco containing Arabidopsis thaliana nitrilase genes

 The Arabidopsis thaliana genome has four nitrilase (nitrile aminohydrolase, EC 3.5.5.1) genes (NIT1 to NIT4). These nitrilases catalyze hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Growth of A. thaliana is inhibited by IAN probably due to hydrolysis of IAN to IAA, while the tobacco (Nicotiana tabacum) genome has only NIT4 homologs and is resistant to IAN. In this study, we introduced A. thaliana NIT1 to NIT4 into tobacco. Introduction of NIT1, NIT2 or NIT3 into tobacco conferred growth inhibition by IAN. NIT2 transgenic plants were highly sensitive to IAN, and NIT1 and NIT3 transgenic plants were moderately sensitive. On the other hand, NIT4 transgenic plants were less sensitive to IAN, although some morphological changes in the roots were observed as the wild-type tobacco. These findings suggest that the ability of transgenic tobacco to convert IAN to IAA in vivo is markedly different among transgenes of NIT1 to NIT4. Received: 22 November 1999 / Revision received: 28 January 2000 / Accepted: 4 February 2000     

Translation initiation by non-AUG codons in Arabidopsis thaliana transgenic plants

The efficiency of translation initiation at codons differing at one or two nucleotides from AUG was tested as initiation codons for the phosphinotricin-acetyltransferase gene in T-DNA plant transformation in Arabidopsis thaliana. With the exception of UUA codon that differs from AUG at two nucleotides and does not permit any detectable activity, all the other codons (AUC, GUG, ACG, and CUG) present a phosphinotrycin acetyltransferase activity that varies between 5 and 10% of the AUG activity. This low activity is sufficient to confer glufosinate resistance to some of the plants. These results indicate that, in plants as is the case in animals, non-AUG initiating codons may be used for translation initiation, namely when a low expression rate is needed.     

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg^?1 protein with indole3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[¹³C]-indole-3-acetonitrile to 1-[¹³C]-indole-3-acetic acid in vivo as determined by HPLC/ GC-MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole-3-acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole-3-acetonitrile, a strong auxin-overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole-3-acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out-growth and callus formation at the root/shoot interface (at 100 µM indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitrile to indole-3-acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high-auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.     

Ploidy levels in transgenic tomato plants determined by chloroplast number

We determined germline ploidy of primary tomato transformants by counting meiotic chromosomes. We then determined the number of chloroplasts in stomatal cells by cytological staining. A correlation of these values indicated that diploid transformants had significantly fewer chloroplasts than tetraploid transformants. By maximum likelihood, we estimate that less than 1% of diploid transformants will have chloroplast values in the tetraploid range. Transformed plants generally had more chloroplasts than plants derived from seed. Also, there was more variability between transformed than seed derived plants. Less than 5% of transformed plants were chimeric when comparing leaf and pollen ploidy levels. Of 129 transgenic plants examined, 29 (22%) were polyploid.