A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

干旱、盐、温度对植物体NADP-苹果酸酶的影响与机理

NADP-苹果酸酶是植物体代谢的重要酶之一,参与了多个代谢过程,在植物体内广泛存在,与各种环境胁迫关系密切。目前,胁迫条件下的植物体NADP-苹果酸酶基因的表达情况以及酶活性的变化是关注的重点,同时,NADP-苹果酸酶在抗胁迫方面的机理研究也在逐渐的展开。综述了干旱、盐、高温和低温胁迫条件下NADP-苹果酸酶活性及该酶基因表达变化的特点,揭示了其在对植物体抵御各种胁迫带来的危害时所发挥的作用以及作用机理。     

Effects of growth regulators on the induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 mol quanta · m^–2 · s^–1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO₂ exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CAM Crassulacean acid metabolism - Chl chlorophyll - 2,4D 2,4-dichlorophenoxyacetic acid - NADP-ME NADP-malic enzyme - PEPCase phosphoenolpyruvate carboxylase Methyl jasmonate was generously provided by Dr. Vincent Franceschi (Botany Department, Washington State University). The anti-maize leaf PEPCase was kindly supplied by Dr. Tatsuo Sugiyama (Department of Agricultural Chemistry, Nagoya University, Japan) and the anti-Flaveria trinervia leaf PEPCase was kindly supplied by Dr. Samuel Sun (Department of Plant Molecular Physiology, University of Hawaii, Honulu). This work was funded in part by U.S. Department of Agriculture Competitive Grant 90-37280-5706 and an equipment grant (DMB 8515521) from the National Science Foundation. Ziyu Dai was supported in part by Guangxi Agricultural College and Ministry of Agriculture of the People's Republic of China     

Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme

 NADP-dependent malic enzyme (NADP-ME) is a major decarboxylating enzyme in NADP-ME-type C₄ species such as maize and Flaveria. In this study, chloroplastic NADP-ME was transferred to rice (Oryza sativa L.) using a chimeric gene composed of maize NADP-ME cDNA under the control of rice light-harvesting chlorophyll-a/b-binding protein (Cab) promoter. There was a 20- to 70-fold increase in the NADP-ME activity in leaves of transgenic rice compared to that in wild-type rice plants. Immunocytochemical studies by electron microscopy showed that maize NADP-ME was mostly localized in chloroplasts in transgenic rice plants, and that the chloroplasts were agranal without thylakoid stacking. Chlorophyll content and photosystem II activity were inversely correlated with the level of NADP-ME activity. These results suggest that aberrant chloroplasts in transgenic plants may be caused by excessive NADP-ME activity. Based on these results and the known fact that only bundle sheath cells of NADP-ME species, among all three C₄ subgroups, have agranal chloroplasts, we postulate that a high level of chloroplastic NADP-ME activity could strongly affect the development of chloroplasts. Received: 27 January 1999 / Accepted: 20 January 2000     

Diurnal Variations in the Activity of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme During the Early Steps of Interaction between Glycine max and Bradyrhizobium japonicum

Two important enzyme in organic acid metabolism, phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME), show marked diurnal rhythms in their activities during the establishment of the soybean - B. japonicum symbiosis. The pH of the nutrient solution changes in parallel with NADP-ME activity, being maximal during the night periods, whereas activity of PEPC was highest during the day periods. The results from the experiments with stem girdled plants indicated that the activity of root PEPC is modulated to a great extent by the supply of photosynthates from the shoots. It was also established that succinate application in the nutrient solution during inoculation altered significantly the pattern of assayed enzyme activities. Although our experiments did not reveal the precise mechanism of the involvement of root PEPC and NADP-ME in soybean response to inoculation with B. japonicum, they indicated the pattern of their activity during the first 72 h postinoculation which are critical for establishment and functioning of the symbiosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

干旱胁迫对2种光合类型C4荒漠植物叶片光合特征酶和抗氧化酶活性的影响

以荒漠C₄草本植物蔷薇猪毛菜(NADP苹果酸酶型,NADP-ME）和粗枝猪毛菜（NAD苹果酸酶型,NAD-ME）为研究对象,采用盆栽控水试验设置正常供水和轻度、中度、重度干旱处理（土壤含水量分别为田间持水量80%、60%、45%和35%）,通过测定不同程度干旱胁迫下叶片含水量、C₄光合特征酶和抗氧化酶活性等指标,探讨不同类型C₄荒漠植物光合特征酶和抗氧化系统对干旱逆境的适应机制。结果显示：（1）2种植物叶片含水量均随干旱胁迫的加剧不同程度降低。（2）叶片磷酸烯醇式丙酮酸羧化酶（PEPC）活性在中度干旱胁迫下显著增加而在重度干旱胁迫下急剧下降;蔷薇猪毛菜NAD-ME活性和粗枝猪毛菜NADP-ME活性都很低,且它们基本不受干旱胁迫的影响;随干旱胁迫的加剧,蔷薇猪毛菜NADP-ME活性呈下降趋势,而粗枝猪毛菜NAD-ME活性先显著增加而在重度干旱胁迫下显著降低。（3）随着干旱胁迫的加剧,叶片超氧化物歧化酶（SOD）活性呈下降趋势,过氧化物酶（POD）活性在不同程度干旱胁迫下均有不同程度增加;过氧化氢酶（CAT）活性在中度干旱胁迫下均有不同程度的增加,但在重度干旱胁迫下蔷薇猪毛菜CAT活性降低,而粗枝猪毛菜CAT活性显著增加;丙二醛（MDA）含量随干旱胁迫的加剧均有不同程度的增加。研究认为,一定程度干旱胁迫下,2种荒漠植物的PEPC活性均有增加;不同光合类型C₄植物叶片脱羧酶（NADP-ME和NAD-ME）对干旱胁迫的响应有明显的差异。POD和CAT是这两种C₄植物适应干旱胁迫的主要抗氧化酶,但蔷薇猪毛菜CAT在重度干旱胁迫下没有起到积极保护作用。     

Effects of short-term NaCl stress on calmodulin transcript levels and calmodulin-dependent NAD kinase activity in two species of tomato

NAD kinase is thought to play an important role in the plant cellular responses to biotic and abiotic stress as one of the isoforms of the enzyme is activated by the Ca^{2 +} –calmodulin (CaM) complex. NAD kinase activity was measured after short‐term NaCl stress applied to isolated cells from Lycopersicon esculentum, var. Volgogradskij (NaCl‐sensitive tomato) and L. pimpinellifolium, acc. PE2 (NaCl‐tolerant species). NAD kinase activity remained constant in the sensitive species, whereas a sharp decrease was observed in the tolerant one. After salt treatment, an induction of the calmodulin gene(s) was observed in the two species, together with a 30–50% decrease in ‘active’ CaM content, i.e. CaM able to activate purified NAD kinase, in L. pimpinellifolium. The decrease in NAD kinase activity could not, however, be fully explained by this decrease in active CaM content. A similar decrease in NAD kinase activity was also recorded with other ionic stresses and exposure to high temperatures, but not in the case of drought, exposure to low temperatures, hormonal (indole‐3‐acetic acid and abscisic acid) or H₂O₂ treatments. External Ca^{2 +} certainly plays a role in the biochemical mechanism(s) leading to NAD kinase inhibition, while no role could be shown for intracellular Ca^{2 +} . In addition, after salt stress, a modification of the redox state of NAD kinase seems to be responsible for the inhibition of the enzyme.     

Maize recombinant non-C<Subscript>4</Subscript> NADP-malic enzyme: A novel dimeric malic enzyme with high specific activity

Among the different isoforms of NADP-malic enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C₄-photosynthesis is the most studied. In the present work, the expression in E. coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented. The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the enzyme previously purified from etiolated leaves and roots. Moreover, the recombinant non-photosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a non-C₄ NADP-ME. Using antibodies against this recombinant enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed invivo. The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the enzyme is found in maize roots in vivo. In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2gene and the likely precursor to the evolution of the photosynthetic C₄ NADP-ME) and the 62 kDa isoform (implicated in C₄ photosynthesis). The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C₄ isoenzyme in maize.     

Expression of a carbonic anhydrase gene is induced by environmental stresses in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO₃ and Na₂CO₃), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses. Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO₃, 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in rice.     

Regulation Effects of Na+/H+ Antiporter (NHX1) on Nicotiana tabacum Stressed with Metals of Different Valences

Na⁺/H⁺ antiporter (NHX1) was reported to be induced by NaCl and conferred salt tolerance to transgenic plants overexpressing NHX1 gene. Cadmium (Cd) resistance and Cd accumulation in tobacco were also closely correlated with the expression of NHX1 gene. However, it was still unclear the regulation effects of NHX1 gene on plants under other metal stresses with different valence states. The present study focuses on the resistance of NHX1-overexpressing tobacco plants to Li (I), Pb (II), Cr (III), or Cr (VI) stress and elucidates the potential mechanisms involved in this process. SseNHX1 gene obtained by shuffling using SsNHX1 from Suaeda salsa and SeNHX1 from Salicornia europaea as sequence templates in our previous work, conferred higher NaCl and Cd tolerance to tobacco than SsNHX1 and SeNHX1. The overexpression of SseNHX1 gene in tobacco was used to explore the relationship between metal stresses that differed in valence states and NHX1 gene expression in plants. Our results showed that the content of metal ions in vacuole and the translocation factor from root to leaf were gradually reduced in NHX1-overexpressing tobacco under different metal stresses with the metal valence states increased from monovalent to hexavalent. Comparing with non-transgenic tobacco plants, NHX1 transgenic plants showed higher photosynthesis and transpiration rates when exposed to Li (I), Pb (II), or Cr (III) stress. Decreased ROS and MDA content and increased antioxidant enzyme activities were also found in transgenic tobacco roots than in non-transgenic roots under Li (I), Pb (II), or Cr (III) exposure.

     

Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses

l-Ascorbic acid (Vitamin C, AsA) is an important component of human nutrition. Plants and several animals can synthesize their own ascorbic acid, whereas humans lack the gene essential for ascorbic acid biosynthesis and must acquire from their diet. In the present study, we developed transgenic potato (Solanum tuberosum L. cv. Taedong Valley) over-expressing l-gulono-γ-lactone oxidase (GLOase gene; NCBI Acc. No. NM022220), isolated from rat cells driven by CaMV35S constitutive promoter that showed enhanced AsA accumulation. Molecular analyses of four independent transgenic lines performed by PCR, Southern and RT-PCR revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 7.5% and the time required for the generation of transgenic plants was 6–7 weeks. Transgenic tubers showed significantly enhanced AsA content (141%) and GLOase activity as compared to untransformed tubers. These transgenics were also found to withstand various abiotic stresses caused by Methyl Viologen (MV), NaCl or mannitol, respectively. The T₁ transgenic plants exposed to salt stress (100 mM NaCl) survived better with increased shoot and root length when compared to untransformed plants. The elevated level of AsA accumulation in transgenics was directly correlated with their ability to withstand abiotic stresses. These results further demonstrated that the overexpression of GLOase gene enhanced basal levels of AsA in potato tubers and also the transgenics showed better survival under various abiotic stresses.     

Archaeal promoter-directed expression of the Halobacterium salinarum catalase-peroxidase gene

The Halobacterium salinarum catalase-peroxidase gene was subcloned into shuttle vectors pWL102 and pWL202 and expressed under the control of different archaeal promoters. When Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of its own promoter, catalase-peroxidase activity increased twofold. Catalase-peroxidase activity increased threefold when Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of a tRNA promoter. This bifunctional enzyme in Hbt. salinarum was not induced by environmental stresses such as H₂O₂, intense light, darkness, high temperature, low temperature, redox inhibitors, heavy metals, or ions. Received: May 5, 2000 / Accepted: August 28, 2000     

The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes

The C(4) photosynthetic pathway involves the assimilation of CO(2) by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C(4) acids. The enzymes of the CO(2) concentrating mechanism could be affected under water deficit and limit C(4) photosynthesis. Three different C(4) grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C(4) acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C(4) enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of L: -malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO(2) by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.