Significance of plant sulfite oxidase

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.     

杨树HDA902基因在低温胁迫应答反应中的功能

组蛋白去乙酰化酶在植物非生物胁迫应答反应中具有重要的调控作用。利用RT-PCR的方法从毛果杨中克隆了组蛋白去乙酰化酶基因HDA902。利用农杆菌介导法将其遗传转化到烟草中,并对转基因植株进行低温耐受性分析。研究结果表明,HDA902在烟草中的表达显著提高了转基因株系对低温的耐受性。叶片NBT和DAB染色结果表明,在低温处理后转基因烟草比野生型烟草产生较少的活性氧。丙二醛和脯氨酸含量测定结果表明,在低温条件下,转基因烟草叶片的脯氨酸含量显著高于野生型烟草,而丙二醛含量显著低于野生型烟草。这些研究结果表明,HDA902参与低温胁迫应答反应,其过量表达提高了植株耐低温的能力。     

Improved salt tolerance of transgenic tobacco expressing apoplastic yeast-derived invertase

We investigated the salt tolerance of transgenic tobacco, in which yeast invertase is expressed in the apoplastic (Apo-Inv) spaces. Whereas photosynthetic activities in wild-type tobacco in light were inhibited under salt stress, transgenic Apo-Inv tobacco maintained constant photosynthetic activities. The physical appearance of plants under salt stress also indicates that yeast invertase expression in the apoplastic space is beneficial for inducing salt tolerance. Apo-Inv tobacco had a much higher osmotic pressure increase in the cell sap than did wild-type tobacco under this type of stress. The physiological importance of sucrose metabolism under salt stress is discussed.     

BcPMI2, isolated from non-heading Chinese cabbage encoding phosphomannose isomerase,improves stress tolerance in transgenic tobacco

Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the l-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S::BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T₁ seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H₂O₂ treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition.     

Salinity and drought tolerance of mannitol-accumulating transgenic tobacco

Tobacco plants (Nicotiana tabacum L.) were transformed with a mannitol-1-phosphate dehydrogenase gene resulting in mannitol accumulation. Experiments were conducted to determine whether mannitol provides salt and/or drought stress protection through osmotic adjustment. Non-stressed transgenic plants were 20–25% smaller than non-stressed, non-transformed (wild-type) plants in both salinity and drought experiments. However, salt stress reduced dry weight in wild-type plants by 44%, but did not reduce the dry weight of transgenic plants. Transgenic plants adjusted osmotically by 0.57 MPa, whereas wild-type plants did not adjust osmotically in response to salt stress. Calculations of solute contribution to osmotic adjustment showed that mannitol contributed only 0-003-0-004 MPa to the 0.2 MPa difference in full turgor osmotic potential (π_o) between salt-stressed transgenic and wild-type plants. Assuming a cytoplasmic location for mannitol and that the cytoplasm constituted 5% of the total water volume, mannitol accounted for only 30–40% of the change in π_o of the cytoplasm. Inositol, a naturally occurring polyol in tobacco, accumulated in response to salt stress in both transgenic and wild-type plants, and was 3-fold more abundant than mannitol in transgenic plants. Drought stress reduced the leaf relative water content, leaf expansion, and dry weight of transgenic and wild-type plants. However, π_o was not significantly reduced by drought stress in transgenic or wild-type plants, despite an increase in non-structural carbohydrates and mannitol in droughted plants. We conclude that (1) mannitol was a relatively minor osmolyte in transgenic tobacco, but may have indirectly enhanced osmotic adjustment and salt tolerance; (2) inositol cannot substitute for mannitol in this role; (3) slower growth of the transgenic plants, and not the presence of mannitol per se, may have been the cause of greater salt tolerance, and (4) mannitol accumulation was enhanced by drought stress but did not affect π_o or drought tolerance.     

Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step

Sulfite oxidase (EC 1.8.3.1) from the plant Arabidopsis thaliana is the smallest eukaryotic molybdenum enzyme consisting of a molybdenum cofactor-binding domain but lacking the heme domain that is known from vertebrate sulfite oxidase. While vertebrate sulfite oxidase is a mitochondrial enzyme with cytochrome c as the physiological electron acceptor, plant sulfite oxidase is localized in peroxisomes and does not react with cytochrome c. Here we describe results that identified oxygen as the terminal electron acceptor for plant sulfite oxidase and hydrogen peroxide as the product of this reaction in addition to sulfate. The latter finding might explain the peroxisomal localization of plant sulfite oxidase. 18O labeling experiments and the use of catalase provided evidence that plant sulfite oxidase combines its catalytic reaction with a subsequent non-enzymatic step where its reaction product hydrogen peroxide oxidizes another molecule of sulfite. In vitro, for each catalytic cycle plant SO will bring about the oxidation of two molecules of sulfite by one molecule of oxygen. In the plant, sulfite oxidase could be responsible for removing sulfite as a toxic metabolite, which might represent a means to protect the cell against excess of sulfite derived from SO2 gas in the atmosphere (acid rain) or during the decomposition of sulfur-containing amino acids. Finally we present a model for the metabolic interaction between sulfite and catalase in the peroxisome.     

超表达拟南芥2-烯醛还原酶基因对烟草抗旱性的作用机理分析

为研究是否可以利用2-烯醛还原酶(AER)来清除活性氧下游的醛自由基达到提高植物的抗旱性,以超表达拟南芥AER基因烟草和野生型烟草(SR)为研究材料,利用干旱胁迫处理进行抗旱性分析,测定了干旱胁迫及复水后各个烟草株系的生物量、光合速率、叶绿素荧光参数、叶绿素含量、MDA和H2O2含量等指标。结果显示:(1)干旱胁迫下,转基因烟草株系的生物量、叶绿素含量、净光合速率、PSⅡ最大光化学效率及H2O2的清除能力均显著高于对照;(2)复水之后,烟草植株的各项生理指标都得到一定程度的恢复,而转基因株系相比于野生型恢复迅速,恢复能力更强。研究认为,超表达AER基因可以通过清除活性氧及其下游醛自由基来提高烟草的抗旱能力。     

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.     

Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25–50°C) for 4 h in the dark and under growth light intensity (300 μmol m⁻² s⁻¹). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H₂O₂, O₂⁻ and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.     

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.     

Transformation of beta-lycopene cyclase genes from Salicornia europaea and Arabidopsis conferred salt tolerance in Arabidopsis and tobacco

Inhibition of lycopene cyclization decreased the salt tolerance of the euhalophyte Salicornia europaea L. We isolated a β-lycopene cyclase gene SeLCY from S. europaea and transformed it into Arabidopsis with stable expression. Transgenic Arabidopsis on post-germination exhibited enhanced tolerance to oxidative and salt stress. After 8 and 21 d recovery from 200 mM NaCl treatment, transgenic lines had a higher survival ratio than wild-type (WT) plants. Three-week-old transgenic plants treated with 200 mM NaCl showed better growth than the WT with higher photosystem activity and less H(2)O(2) accumulation. Determination of endogenous pigments of Arabidopsis treated with 200 mM NaCl for 0, 2 or 4 d demonstrated that the transgenic plants retained higher contents of carotenoids than the WT. Furthermore, to compare the difference between SeLCY and AtLCY from Arabidopsis, we used viral vector mediating ectopic expression of SeLCY and AtLCY in Nicotiana benthamiana. Although LCY genes transformation increased the salt tolerance in tobacco, there is no significant difference between SeLCY- and AtLCY-transformed plants. These findings indicate that SeLCY transgenic Arabidopsis improved salt tolerance by increasing synthesis of carotenoids, which impairs reactive oxygen species and protects the photosynthesis system under salt stress, and as a single gene, SeLCY functionally showed no advantage for salt tolerance improvement compared with AtLCY.     

Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco

Polyamines (PAs) exert a protective effect against stress challenges, but their molecular role in this remains speculative. In order to detect the signaling role of apoplastic PA-derived hydrogen peroxide (H2O2) under abiotic stress, we developed a series of tobacco (Nicotiana tabacum cv Xanthi) transgenic plants overexpressing or downregulating apoplastic polyamine oxidase (PAO; S-pao and A-pao plants, respectively) or downregulating S-adenosyl-l-methionine decarboxylase (samdc plants). Upon salt stress, plants secreted spermidine (Spd) into the apoplast, where it was oxidized by the apoplastic PAO, generating H2O2. A-pao plants accumulated less H2O2 and exhibited less programmed cell death (PCD) than did wild-type plants, in contrast with S-pao and samdc downregulating plants. Induction of either stress-responsive genes or PCD was dependent on the level of Spd-derived apoplastic H2O2. Thus, in wild-type and A-pao plants, stress-responsive genes were efficiently induced, although in the latter at a lower rate, while S-pao plants, with higher H2O2 levels, failed to accumulate stress-responsive mRNAs, inducing PCD instead. Furthermore, decreasing intracellular PAs, while keeping normal apoplastic Spd oxidation, as in samdc downregulating transgenic plants, caused enhanced salinity-induced PCD. These results reveal that salinity induces the exodus of Spd into the apoplast, where it is catabolized by PAO, producing H2O2. The accumulated H2O2 results in the induction of either tolerance responses or PCD, depending also on the levels of intracellular PAs.     

玉米亚硫酸氧化酶基因的克隆及其结构与表达分析

为明确亚硫酸氧化酶(sulfite oxidase,SO)基因的结构特征和进化关系及其在玉米不同组织器官发育过程中的表达和分布特性,采用RACE技术克隆了玉米SO基因(ZmSO)的全长cDNA。序列分析表明,获得的ZmSO全长1 492bp,其中5′-UTR 160bp,3′-UTR 138bp,开放阅读框为1 194bp,编码397个氨基酸组成的蛋白质。对该基因编码氨基酸保守结构域的分析发现,ZmSO包含1个钼辅因子结合域、1个自身二聚化域和1个过氧化物体靶信号序列。系统进化分析显示,SO在进化上较为保守,玉米与其它植物的SO相似性较高。荧光定量RT-PCR分析表明,在玉米成株期,根、茎、叶、雄花和幼穗中,ZmSO在根部表达丰度最低,在叶片和幼穗中表达量较高。酶活性测定结果显示,不同器官中SO活性与其mRNA转录水平上的表达趋势相似。     

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants     

Over-expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses

Ascorbate peroxidase plays a key role in scavenging reactive oxygen species under environmental stresses and in protecting plant cells against toxic effects. The Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that StAPX was transferred into the tobacco genome and StAPX was induced by salt and osmotic stresses in tomato leaves. Over-expression of StAPX in tobacco improved seed germination rate and elevated stress tolerance during post-germination development. Two transgenic lines showed higher APX activity and accumulated less hydrogen peroxide than wild-type plants after stress treatments. The photosynthetic rates, the root lengths, the fresh and dry weights of the transgenic lines were distinctly higher than those of wild-type plants under stress conditions. Results indicated that the over-expression of StAPX had enhanced tolerance to salt stress and osmotic stress in transgenic tobacco plants.