Cloning of a NaCl-induced fructose-1, 6-diphosphate aldolase cDNA from Dunaliella salina and its expression in tobacco

Itisaworld-widefocusthatsoilbecomesmoreandmoresaline.Aboutone-thirdirrigablelandbecomeslostallovertheworldforthereasonthatthesalinityistoohigh.Therefore,im-provingthesalt-toleranceofcropsseemsmoreandmoreimportant.Previousstudyonthemecha-nismofsaltresistantofplantshaspaidmoreattentiontohigherplants,whileonlyafewtoalgae.Studyintheseyearsshowedthatalgaehadthesimilarbiophysiologicalandbiochemicalreactiontoallhigherplantsinadversity[1].Soitisreliabletoapplyalgatorelatedstudy.Dunaliellasalina,akind…     

Overexpression of glutathione S-transferase gene increases salt tolerance of arabidopsis

The Suaeda salsa glutathione S-transferase gene (GST) was introduced into arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern and northern blot analyses confirmed that GST was transferred into the arabidopsis genome, and the GST and GPX activities in transgenic plants (GT) were much higher than in wild-type plants (WT). There were no obvious morphological or developmental differences between transgenic and wild-type plants. One transgenic homozygous line (GT_6–8) and WT plants were evaluated for salt tolerance and gene expression. Seed germination and seedling salt tolerance were improved after overexpression of GST in arabidopsis; the photosynthesis rate and the fresh weight of the GT_6–8 line were distinctly higher than those of WT plants after NaCl treatment. Glutathione content increased substantially in salt-stressed arabidopsis plants of both genotypes, and the glutathione pool in GT_6–8 plants was more oxidized than in WT plants under both control and stressful conditions. The MDA content, an indicator of lipid peroxidation, increased in WT plants but was not affected distinctly in GT_6–8 seedlings after NaCl treatment. Results from different tests indicated that the expression of the GST gene promoted a higher level of salt tolerance in vivo in transgenic arabidopsis plants.     

Cloning and molecular characterization of fructose-1,6-bisphosphate aldolase gene regulated by high-salinity and drought in Sesuvium portulacastrum

Sesuvium portulacastrum, a mangrove plant from seashore, is a halophyte species well adapted to salinity and drought. Some efforts have been made to describe its physiological and structural characteristics on salt and drought-tolerance, but the underlying molecular mechanism and key components have not yet been identified. Here, a fructose-1,6-bisphosphate aldolase gene, designated SpFBA, was isolated and characterized from S. portulacastrum roots in response to seawater. The SpFBA cDNA has a total length of 1452 bp with an open reading frame of 1071 bp, and is predicted to encode a precursor protein of 357 amino acid residues sharing high degree of homology with class I FBAs from other plants. Semi-quantitative RT-PCR analysis indicated that the SpFBA was more strongly expressed in roots than in leaves and stems, and the abiotic stimuli such as Seawater, NaCl, ABA, and PEG, could trigger a significant induction of SpFBA in S. portulacastrum roots within 2–12 h. Overproduction of Recombinant SpFBA resulted in an increased tolerance to salinity in transgenic Escherichia coli. All these results suggest that the SpFBA plays very important roles in responding to salt stress and related abiotic stimuli, and in improving the survival ability of S. portulacastrum under high salinity and drought. The GenBank Accession number of S. portulacastrum fructose-1,6-bisphosphate aldolase (SpFBA) is ACG68894.     

Altered expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase affects the growth of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP sub-unit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO₂ assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPβ affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes. These authors contributed equally to this study.     

Effects of drought on water content and photosynthetic parameters in potato plants expressing the trehalose-6-phosphate synthase gene of Saccharomyces cerevisiae

Two transgenic potato lines, T1 and T2, expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast were isolated. In our experimental approach, we applied two novelties, namely the fusion of the drought-inducible promoter StDS2 to TPS1 and a marker-free transformation method. In contrast to the expected drought-induced expression, only a very low constitutive TPS1 expression was detected in the transgenic lines, probably due to chromosomal position effects. The observed expression pattern, however, was sufficient to alter the drought response of plants. Detached leaves of T1 and T2 showed an 8 h delay in wilting compared to the non-transformed control. Potted plants of T1 and T2 kept water 6 days longer than control plants and maintained high stomatal conductance and a satisfactory rate of net photosynthesis. During drought treatment, CO₂ assimilation rate measured at saturating CO₂ level was maintained at maximum level for 6–9 days in transgenic plants while it decreased rapidly after 3 days in the wild type plants. Under optimal growth conditions, lower CO₂ fixation was detected in the transgenic than in the control plants. Stomatal densities of T1 and T2 leaves were reduced by 30–40%. This may have contributed to the lower CO₂ fixation rate and altered drought response. Ibolya Stiller, and Sándor Dulai contributed equally to this work.     

Enhanced salt tolerance of transgenic poplar plants expressing a manganese superoxide dismutase from <Emphasis Type="Italic">Tamarix androssowii</Emphasis>

Superoxide dismutases (SODs) play important role in stress tolerance of plants. In this study, an MnSOD gene (TaMnSOD) from Tamarix androssowii, under the control of the CaMV35S promoter, was introduced into poplar (Populus davidiana × P. bolleana). The physiological parameters, including SOD activity, malondialdehyde (MDA) content, relative electrical conductivity (REC) and relative weight gain, of transgenic lines and wild type (WT) plants, were measured and compared. The results showed that SOD activity was enhanced in transgenic plants, and the MDA content and REC were significantly decreased compared to WT plants when exposed to NaCl stress. In addition, the relative weight gains of the transgenic plants were 8- to 23-fold of those observed for WT plants after NaCl stress for 30 days. The data showed that the SOD activities that increased in transgenic lines are 1.3–4-folds of that increased in the WT plant when exposed to NaCl stress. Our analysis showed that increases in SOD activities as low as 0.15-fold can also significantly enhance salt tolerance in transgenic plants, suggesting an important role of increased SOD activity in plant salt tolerance.     

Heterologous expression of <Emphasis Type="Italic">P5CS</Emphasis> gene in chickpea enhances salt tolerance without affecting yield

Vigna Δ¹-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na⁺ was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.     

Regulation of seed germination and seedling growth by an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytocystatin isoform, <Emphasis Type="Italic">AtCYS6</Emphasis>

Phytocystatins are cysteine proteinase inhibitors in plants that are implicated in the endogenous regulation of protein turnover and defense mechanisms against insects and pathogens. A cDNA encoding a phytocystatin called AtCYS6 (Arabidopsis thaliana phytocystatin6) has been isolated. We show that AtCYS6 is highly expressed in dry seeds and seedlings and that it also accumulates in flowers. The persistence of AtCYS6 protein expression in seedlings was promoted by abscisic acid (ABA), a seed germination and post-germination inhibitory phytohormone. This finding was made in transgenic plants bearing an AtCYS6 promoter–β-glucuronidase (GUS) reporter construct, where we found that expression from the AtCYS6 promoter persisted after ABA treatment but was reduced under control conditions and by gibberellin₄₊₇ (GA₄₊₇) treatment during the germination and post-germinative periods. In addition, constitutive over-expression of AtCYS6 retarded germination and seedling growth, whereas these were enhanced in an AtCYS6 knock-out mutant (cys6-2). Additionally, cysteine proteinase activities stored in seeds were inhibited by AtCYS6 in transgenic Arabidopsis. From these data, we propose that AtCYS6 expression is enhanced by the germination inhibitory phytohormone ABA and that it participates in the control of germination rate and seedling growth by inhibiting the activity of stored cysteine proteinases.     

Response of transgenic rape plants bearing the <Emphasis Type="Italic">Osmyb4</Emphasis> gene from rice encoding a trans-factor to low above-zero temperature

Accumulation of soluble sugars (sucrose, fructose, and glucose), proline, phenols (total phenols and flavonoids), and antocyanins during adaptation to low-temperature stress (4°C) of two lines of spring rape (Brassica napus L., cv. Westar) characterized by weak (Bn-1) and strong (Bn-3) expression of the Osmyb4 transgene was studied. Vegetatively propagated transgenic and wild-type plants were grown in the hydroponic culture at 24°C; at the stage of 5–6 leaves, plants were exposed to 4°C for 5 days and then returned to the optimum temperature of 24°C for recovery. Transgenic plants were established to manifest improved cold and frost tolerance, which was evident from more active biomass accumulation at 4°C as compared with wild-type plants and from sustaining their viability after 2-day-long exposure to −6°C. Determination of MDA content showed that one of the reasons of their improved cold tolerance was their capability of maintaining oxidative homeostasis under low-temperature stress. This suggestion is supported by intense accumulation of phenols and antocyanins, manifesting pronounced antioxidant effects, by transgenic plants during their cold adaptation. Thus, during 2–5 days of plant exposure to 4°C, in transgenic plants the total content of phenols increased by 2.6–3.7 times, flavonoids — by 3.7–4.7 times, and antocyanins — by 3.5–5.3 times as compared with control plants growing at 24°C. Transgenic Bn-3 plants with strong expression of the Osmyb4 gene accumulated phenols and antocyanins at 4°C more actively than Bn-1 plants characterized by weak expression of this gene. Transgenic rape plants subjected to cold stress accumulated more proline, manifesting stress-protection effects, and lesser accumulation of soluble sugars. Before the beginning of experiment, the content of soluble sugars was approximately similar in wild-type plants and transgenic lines; at 4°C their level in transgenic plants was substantially lower than in control plants. As distinct from the process of cold adaptation, during recovery, the content of all tested stress-protection compounds dropped sharply. The results obtained indicate that active expression of the Osmyb4 gene from rice in the rape plants was accompanied not only by accumulation of compatible osmolytes but also by biosynthesis of antioxidants of phenolic nature.     

Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12

Summary We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.Abbreviations G1P glucose-1-phosphate - G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1-6DP fructose-1,6-diphosphate - PEP phosphoenol pyruvate     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Enhanced drought and salinity tolerance in transgenic potato plants with a BADH gene from spinach

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = −2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4–0.9 cm and fresh weight per plant increased by 17–29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.     

Molecular cloning and characterization of the promoter for the multiple stress-inducible gene <Emphasis Type="Italic">BjCHI1</Emphasis> from <Emphasis Type="Italic">Brassica juncea</Emphasis>

We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009–1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to β-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from −695 to −620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at −353 to −348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.     

Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey)

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for orchid Oncidium Gower Ramsey. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose selective medium. Genetically transformed plants of Oncidium were regenerated after cocultivating protocorm-like bodies with Agrobacterium tumefaciens strain GV3101 containing the vectors pEPYON-42P and pEPYON-42H with 35S::PMI and 35S::HPTII genes respectively. We observed that 35S::PMI (pEPYON-42P) produced high rate (27 plants) of mannose resistant transgenic plants compared to 35S::HPTII (pEPYON-42H) in which only fourteen hygromycin resistant transgenic plants were obtained. Mannose resistant transgenic plants were confirmed by PCR and Southern blot. The pmi gene expression in 35S::PMI (pEPYON-42P) transgenic plants was confirmed by RT-PCR. Furthermore, the duration of regeneration time of transgenic plants was significantly shorter in mannose selected system (4 months) than in hygromycin selected system (8 months). The pmi/mannose selection system is shown to be highly efficient for producing transgenic O. Gower Ramsey without using antibiotics or herbicides. For the first time, the pmi/mannose-based “positive” selection system has been used to obtain genetically engineered O. Gower Ramsey.     

Liver aldolase as a possible target for the action of N-methyl-N-nitrosourea

The effect of N-methyl-N-nitrosourea (MNU) on the activity of cytoplasmic and reversibly bound to subcellular structures liver aldolase was studied. In vitro, the activity of aldolase purified from rabbit muscles is inhibited by MNU by 70-80% relative to fructose-1,6-diphosphate and by 50-60% relative to fructose-1-phosphate. These substrates and the competitive inhibitor ATP do not protect the enzyme against the inactivation by MNU. MNU inhibits the activity of cytoplasmic aldolase by 30-40% and 20% 2-24 hours after a single injection (80 mg/kg) in vivo. The enzyme affinity for fructose-1,6-diphosphate is markedly decreased (2-fold). Activation of cytoplasmic aldolase relative to both substrates, which is especially well-pronounced with fructose-1-phosphate after inhibition of the enzyme activity, was observed. The enzyme activity relative to both substrates was found to increase in the mitochondrial and nuclear fractions within 48 hours. MNU has no effect on the activity of aldolase bound to microsomes. MNU influences the aldolase binding to organelle membranes. MNU injections at early periods (2-168 hours) accounts for the differences in the kinetic properties of cytoplasmic and reversibly bound to subcellular structures liver aldolase. These changes persist within 168 hours after MNU administration and may result in disturbances in cell metabolism as well as in the regulation of metabolic pathways, such as glycolysis and gluconeogenesis.