Arabidopsis thaliana flavoprotein AtHAL3a catalyzes the decarboxylation of 4'-Phosphopantothenoylcysteine to 4'-phosphopantetheine, a key step in coenzyme A biosynthesis

The Arabidopsis thaliana flavoprotein AtHAL3a is related to plant growth and salt and osmotic tolerance. AtHAL3a shows sequence homology to the bacterial flavoproteins EpiD and Dfp. EpiD, Dfp, and AtHAL3a are members of the homo-oligomeric flavin-containing Cys decarboxylase (HFCD) protein family. We demonstrate that AtHAL3a catalyzes the decarboxylation of (R)-4'-phospho-N-pantothenoylcysteine to 4'-phosphopantetheine. This key step in coenzyme A biosynthesis is catalyzed in bacteria by the Dfp proteins. Exchange of His-90 of AtHAL3a for Asn led to complete inactivation of the enzyme. Dfp and AtHAL3a are characterized by a shortened substrate binding clamp compared with EpiD. Exchange of the cysteine residue of the conserved ACGD motif of this binding clamp resulted in loss of (R)-4'-phospho-N-pantothenoylcysteine decarboxylase activity. Based on the crystal structures of EpiD H67N with bound substrate peptide and of AtHAL3a, we present a model for the binding of (R)-4'-phospho-N-pantothenoylcysteine to AtHAL3a.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

An Arabidopsis mutant impaired in coenzyme A biosynthesis is sugar dependent for seedling establishment

Once the plant coenzyme A (CoA) biosynthetic pathway has been elucidated by comparative genomics, it is feasible to analyze the physiological relevance of CoA biosynthesis in plant life. To this end, we have identified and characterized Arabidopsis (Arabidopsis thaliana) T-DNA knockout mutants of two CoA biosynthetic genes, HAL3A and HAL3B. The HAL3A gene encodes a 4'-phosphopantothenoyl-cysteine decarboxilase that generates 4'-phosphopantetheine. A second gene, HAL3B, whose gene product is 86% identical to that of HAL3A, is present in the Arabidopsis genome. HAL3A appears to have a predominant role over HAL3B according to their respective mRNA expression levels. The hal3a-1, hal3a-2, and hal3b mutants were viable and showed a similar growth rate as that in wild-type plants; in contrast, a hal3a-1 hal3b double mutant was embryo lethal. Unexpectedly, seedlings that were null for HAL3A and heterozygous for HAL3B (aaBb genotype) displayed a sucrose (Suc)-dependent phenotype for seedling establishment, which is in common with mutants defective in beta-oxidation. This phenotype was genetically complemented in aaBB siblings of the progeny and chemically complemented by pantethine. In contrast, seedling establishment of Aabb plants was not Suc dependent, proving a predominant role of HAL3A over HAL3B at this stage. Total fatty acid and acyl-CoA measurements of 5-d-old aaBb seedlings in medium lacking Suc revealed stalled storage lipid catabolism and impaired CoA biosynthesis; in particular, acetyl-CoA levels were reduced by approximately 80%. Taken together, these results provide in vivo evidence for the function of HAL3A and HAL3B, and they point out the critical role of CoA biosynthesis during early postgerminative growth.     

4'-phosphopantetheine and coenzyme A biosynthesis in plants

Coenzyme A is required for many synthetic and degradative reactions in intermediary metabolism and is the principal acyl carrier in prokaryotic and eukaryotic cells. Coenzyme A is synthesized in five steps from pantothenate, and recently the CoaA biosynthetic genes in bacteria and human have all been identified and characterized. Coenzyme A biosynthesis in plants is not fully understood, and to date only the AtHAL3a (AtCoaC) gene of Arabidopsis thaliana has been cloned and identified as 4'-phosphopantothenoylcysteine (PPC) decarboxylase (Kupke, T., Hernández-Acosta, P., Steinbacher, S., and Culiá?ez-Macià, F. A. (2001) J. Biol. Chem. 276, 19190-19196). Here, we demonstrate the cloning of the four missing genes, purification of the enzymes, and identification of their functions. In contrast to bacterial PPC synthetases, the plant synthetase is not CTP-but ATP-dependent. The complete biosynthetic pathway from pantothenate to coenzyme A was reconstituted in vitro by adding the enzymes pantothenate kinase (AtCoaA), 4'-phosphopantothenoylcysteine synthetase (AtCoaB), 4'-phosphopantothenoylcysteine decarboxylase (AtCoaC), 4'-phosphopantetheine adenylyltransferase (AtCoaD), and dephospho-coenzyme A kinase (AtCoaE) to a mixture containing pantothenate, cysteine, ATP, dithiothreitol, and Mg2+.     

Overexpression of <Emphasis Type="Italic">CsNMAPK</Emphasis> in tobacco enhanced seed germination under salt and osmotic stresses

In this research, biological function of CsNMAPK, encoding a mitogen-activated protein kinase of cucumber, was investigated under salt and osmotic stresses. Northern blot analysis showed that the expression of CsNMAPK was induced by salt and osmotic stresses in the cucumber root. In order to determine whether CsNMAPK was involved in plant tolerance to salt and osmotic stresses, transgenic tobacco plants constitutively overexpressing CsNMAPK were generated. Northern and Western blot analysis showed that strong signals were detected in the RNA and protein samples extracted from transgenic lines, whereas no signal was detected in the wild type tobacco, indicating that CsNMAPK was successfully transferred into tobacco genome and overexpressed. The results of seed germination showed that germination rates of transgenic lines were significantly higher than wild type under high salt and osmotic stresses. In addition, seed growth of transgenic lines was much better than wild type under salt and osmotic stresses. These results indicated that overexpression of CsNMAPK positively regulated plant tolerance to salt and osmotic stresses.     

A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast.

We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.     

Overexpression of a new rice vacuolar antiporter regulating protein OsARP improves salt tolerance in tobacco

We examined the function of the rice (Oryza sativa L.) antiporter-regulating protein OsARP by overexpressing it in tobacco (Nicotiana tabacum L.). In public databases, this protein was annotated as a putative Os02g0465900 protein of rice. The OsARP gene was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. The transformants were selected for their ability to grow on medium containing kanamycin. Incorporation of the transgene in the genome of tobacco was confirmed by PCR, and its expression was confirmed by Western blot analysis. Transgenic plants had better growth and vigor than non-transgenic plants under salt stress in vitro. Overexpression of OsARP in transgenic tobacco plants resulted in salt tolerance, and the plants had a higher rate of photosynthesis and effective PSII photon yield when compared with the wild type. The OsARP protein was localized in the tonoplast of rice plants. Transgenic plants accumulated more Na+ in their leaf tissue than did wild-type plants. It is conceivable that the toxic effect of Na+ in the cytosol might be reduced by sequestration into vacuoles. The rate of water loss was higher in the wild type than in transgenic plants under salt stress. Increased vacuolar solute accumulation and water retention could confer salt tolerance in transgenic plants. Tonoplast vesicles isolated from OsARP transgenic plants showed Na+/H+ exchange rates 3-fold higher than those of wild-type plants. These results suggest that OsARP on the tonoplasts plays an important role in compartmentation of Na+ into vacuoles. We suggest that OsARP is a new type of protein participating in Na+ uptake in vacuoles.     

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate–glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.     

The cotton GhNHX1 gene encoding a novel putative tonoplast Na(+)/H(+) antiporter plays an important role in salt stress

A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.     

Allene Oxide Cyclase from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Improves Tolerance Against Low Temperature and Salt Stress in Tobacco and Bacteria

Allene oxide cyclase (AOC, E 5.3.99.6) is an essential enzyme in jasmonate (JA) biosynthetic pathway. An AOC gene (defined as CaAOC, Database Accession No. AY863428) had been isolated from Camptotheca acuminata in previous work. Real-time quantitative PCR analysis indicated that mRNA expression of CaAOC was induced by salt stress (120 mM NaCl) and low temperature (4 degrees C). In order to further investigate the role of AOC gene in the processes, CaAOC was introduced into tobacco via Agrobacterium tumefaciens, and the transgenic lines were subjected to the examination of tolerance against salt stress and low temperature. Under salt stress, the chlorophyll content in transgenic tobacco was higher than that of in the wild plants. The electrolyte leakage test revealed that transgenic tobacco plants were more resistant to low temperature over control. Furthermore, 5'-truncated CaAOC was inserted into pET30 and then expressed in Escherichia coli strain BL21DE3 (pLysS). Interestingly, the transformants could grow on 2YT agar containing 400 mM NaCl. Although these mechanisms are not clear yet, this study suggested that CaAOC could not only be a potential target gene in the engineering of plants and bacteria for improved endurance against salt stress, but also be quite useful in enhancing plant tolerance to cold.     

An insight into functional genomics of transgenic lines of tomato cv Rio Grande harbouring yeast halotolerance genes

Tomato cv Rio Grande plants were transformed with yeast halotolerance genes (HAL I or HAL II) using pPM7HAL I or pJRM16HAL II, with p35GUSINT as control. Transformation efficiency varied in the three constructs, with highest transformation found with p35GUSINT. Final selection of the transgenic plants was made on the basis of PCR. Transgene integration and copy number were assessed by Southern hybridisation. The primary transformants were allowed to self-pollinate and the expected Mendelian ratios were studied in second-generation progeny. Five independent homozygous lines each of HAL I and HAL II, as well as the control, were characterised to study inter-transformant expression variability. The transformants showed considerable variability in expression of the respective genes, as shown by salt tolerance assays, chlorophyll content and peroxidase activity. The transgene expression in transgenic lines was analysed by semi-quantitative RT-PCR. In response to different salt concentrations, transgenic plants over-expressing HAL I and HAL II had significantly (α=0.05) better performance than the control This study presents the comparative responses of the three constructs under the same transformation conditions and suggests possible mechanisms governed by yeast HAL I and HAL II genes, which seem to work in a coordinated manner by relatively decreasing osmotic and oxidative shock at different rates. Our results suggest that the yeast HAL I increases K(+) /Na(+) selectivity and has a more functional role than HAL II in improving salt tolerance of the tomato cv Rio Grande grown in Pakistan.     

Cloning and Functional Characterization of a Novel Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferase Gene from <Emphasis Type="Italic">Limonium bicolor</Emphasis>

Plant glutathione S-transferases (GSTs) are involved in protecting plants against both diverse biotic and abiotic stresses. In the present study, a novel GST gene (LbGST1) was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). To characterize its function in salt tolerance, tobacco lines transformed with LbGST1 were generated. Compared with wild-type (WT) tobacco, transgenic plants overexpressing LbGST1 exhibited both GST and glutathione peroxidase activities. Moreover, superoxide dismutase, peroxidase (POD), and catalase activities in transgenic plants were significantly higher than those in WT plants, particularly when grown under conditions of salt stress. Similarly, levels of proline in transgenic plants were also higher than those in WT plants grown under NaCl stress conditions. Whereas, Malondialdehyde contents in transgenic plants were lower than those in WT plants under NaCl conditions. Furthermore, Na⁺ content in transgenic plants was lower than that in WT plants under these stress conditions. Subcellular localization analysis revealed that the LbGST1 protein was localized in the nucleus. These results suggested that overexpression of LbGST1 gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST1 gene can mediate many physiological pathways that enhance stress resistance in plants.     

Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance

An H(+)-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H(+)-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na(+) hypersensitivity and demonstrated the function of TsVP as an H(+)-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H(+)-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H(+)-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na(+) under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na(+) in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na(+) in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H(+)-PPase causes the accumulation of Na(+) in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na(+) in plant cells.