Metabolome and Photochemical Analysis of Rice Plants Overexpressing Arabidopsis NAD Kinase Gene

The chloroplastic NAD kinase (NADK2) is reported to stimulate carbon and nitrogen assimilation in Arabidopsis (Arabidopsis thaliana), which is vulnerable to high light. Since rice (Oryza sativa) is a monocotyledonous plant that can adapt to high light, we studied the effects of NADK2 expression in rice by developing transgenic rice plants that constitutively expressed the Arabidopsis chloroplastic NADK gene (NK2 lines). NK2 lines showed enhanced activity of NADK and accumulation of the NADP(H) pool, while intermediates of NAD derivatives were unchanged. Comprehensive analysis of the primary metabolites in leaves using capillary electrophoresis mass spectrometry revealed elevated levels of amino acids and several sugar phosphates including ribose-1,5-bisphosphate, but no significant change in the levels of the other metabolites. Studies of chlorophyll fluorescence and gas change analyses demonstrated greater electron transport and CO₂ assimilation rates in NK2 lines, compared to those in the control. Analysis of oxidative stress response indicated enhanced tolerance to oxidative stress in these transformants. The results suggest that NADP content plays a critical role in determining the photosynthetic electron transport rate in rice and that its enhancement leads to stimulation of photosynthesis metabolism and tolerance of oxidative damages.NADP is a ubiquitous coenzyme, required in various metabolic processes, since these metabolites carry electrons through the reversible conversion between oxidized (NAD⁺, NADP⁺) and reduced (NADH, NADPH) forms in all organisms. NAD is highly oxidized and is involved primarily in intracellular catabolic reactions, whereas NADP is predominantly found in its reduced form and participates in anabolic reactions and defense against oxidative stress (Ziegler, 2000; Noctor et al., 2006; Pollak et al., 2007a). Since NAD(H) and NADP(H) play a variety of distinct physiological roles, the regulation of the NAD(H)/NADP(H) balance is essential for cell survival (Kawai and Murata, 2008; Hashida et al., 2009).One of the key enzymes that regulates NAD(H)/NADP(H) balance is NAD kinase (NADK; EC 2.7.1.23), which catalyzes NAD phosphorylation in the presence of ATP. The genes encoding NADK were cloned recently from all organisms investigated to date, except for Chlamydia trachomatis (Kawai and Murata, 2008). Only a single gene encoding NADK has been found in some bacteria and mammals (Kawai and Murata, 2008). In contrast, NADK activity was detected in not only the cytosol but also organelles in yeast and plant (Jarrett et al., 1982; Simon et al., 1982; Dieter and Marme, 1984; Iwahashi and Nakamura, 1989; Iwahashi et al., 1989), and three genes including cytosol-type and organelle-type NADK have been cloned in yeast (Kawai et al., 2001; Outten and Culotta, 2003) and plants (Turner et al., 2004, 2005).In Arabidopsis (Arabidopsis thaliana), one of the NADK isoforms is localized in the chloroplast (NADK2; Chai et al., 2005), the others are localized in the cytosol (NADK1 and NADK3; Chai et al., 2006). Analysis of Arabidopsis mutants revealed low chlorophyll (chl) content, low photosynthetic activity, growth inhibition, and hypersensitivity to environmental stresses in the nadk2 knockout mutant (Chai et al., 2005; Takahashi et al., 2006), whereas the nadk1 knockout mutant and the nadk3 knockout mutant did not show a significant phenotype, except for sensitivity to oxidative stress (Berrin et al., 2005; Chai et al., 2006). Moreover, the major part of NADP(H) biosynthesis in photosynthetic organ appears to be attributable to NADK2, because NADK and NADP(H) were strictly decreased in leaves of the nadk2 knockout mutant (Chai et al., 2005; Takahashi et al., 2006). In the plant cell, NADP is mainly located in the chloroplast (Heber and Santarius, 1965; Wigge et al., 1993), where NADP⁺ functions as the final electron acceptor of the photosynthetic electron transport. The reducing energy obtained is not only supplied for Calvin cycle, nitrogen assimilation, lipid and chl metabolism, but also play a crucial role in maintaining redox homeostasis through the regulation of producing and consuming reactive oxygen species (ROS) in the plant cell (Noctor, 2006; Noctor et al., 2006). Accordingly, these evidences indicate that chloroplastic NADK2 plays a central role in plant metabolism and stress tolerance through homeostasis of ROS as regulator of NADP/NAD balance.Since the alteration of NAD/NADP balance affects metabolism and ROS homeostasis, manipulation of NADK can be an attractive target for the engineering of plant metabolism. It was reported that overexpression of NADK causes perturbation of NADP(H) pool and has positive effects on stress tolerance or growth in various living things. In Asperadium nidulans, overexpression of NADH kinase improves the growth efficiency of the cell (Panagiotou et al., 2009). Overexpression of NADK in human HEK293 cells causes 4-to 5-fold increase of NADPH concentration and provides moderate protection against oxidative stress (Pollak et al., 2007b). Recently, we evaluated effects of the enhanced NADP(H) content in Arabidopsis by generating NADK2-overexpressing plants (Takahashi et al., 2009). Our results indicated that enhanced NADP(H) production by NADK2 overexpression promoted nitrogen assimilation and resulted in accumulation of metabolites associated with the Calvin cycle, accompanied by increased activity of Rubisco. Together, these studies demonstrated the potential use of NADK as candidate gene in promoting primary metabolism and/or stress tolerance in transgenic plants.Rice (Oryza sativa) is not only the primary crop for more than half of the world''s population, but also a model monocot system. Rice can adapt to more strong light intensity than Arabidopsis, because rice is a sun plant, whereas Arabidopsis is a shade plant. Therefore, it is possible that effects of an increased NADP(H) content could be more significant in rice plant than in Arabidopsis, due to a higher ability to manage reductive energy involved in NADP as an electron carrier. In this article, we describe the generation and characterization of transgenic rice plants expressing an Arabidopsis chloroplastic NADK (AtNADK2), under the control of the maize (Zea mays) ubiquitin promoter. We named the rice plant as NK2. We found that pleiotropic effects on primary metabolism in NK2 rice were similar to the result obtained in NADK2-overexpressing Arabidopsis plants. However, stimulation of carbon fixation and nitrogen assimilation were observed in NK2 rice, accompanying with significant increases in electron transport and CO₂ assimilation rates, unlike results of the previous study of Arabidopsis. Interestingly, the NK2 lines also showed enhanced tolerance to oxidative stress.     

Phosphoenolpyruvate Carboxylase in Arabidopsis Leaves Plays a Crucial Role in Carbon and Nitrogen Metabolism

Phosphoenolpyruvate carboxylase (PEPC) is a crucial enzyme that catalyzes an irreversible primary metabolic reaction in plants. Previous studies have used transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition to examine the role of PEPC in carbon and nitrogen metabolism. To date, the in vivo role of PEPC in carbon and nitrogen metabolism has not been analyzed in plants. In this study, we examined the role of PEPC in plants, demonstrating that PPC1 and PPC2 were highly expressed genes encoding PEPC in Arabidopsis (Arabidopsis thaliana) leaves and that PPC1 and PPC2 accounted for approximately 93% of total PEPC activity in the leaves. A double mutant, ppc1/ppc2, was constructed that exhibited a severe growth-arrest phenotype. The ppc1/ppc2 mutant accumulated more starch and sucrose than wild-type plants when seedlings were grown under normal conditions. Physiological and metabolic analysis revealed that decreased PEPC activity in the ppc1/ppc2 mutant greatly reduced the synthesis of malate and citrate and severely suppressed ammonium assimilation. Furthermore, nitrate levels in the ppc1/ppc2 mutant were significantly lower than those in wild-type plants due to the suppression of ammonium assimilation. Interestingly, starch and sucrose accumulation could be prevented and nitrate levels could be maintained by supplying the ppc1/ppc2 mutant with exogenous malate and glutamate, suggesting that low nitrogen status resulted in the alteration of carbon metabolism and prompted the accumulation of starch and sucrose in the ppc1/ppc2 mutant. Our results demonstrate that PEPC in leaves plays a crucial role in modulating the balance of carbon and nitrogen metabolism in Arabidopsis.Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is a crucial enzyme that functions in primary metabolism by irreversibly catalyzing the conversion of phosphoenolpyruvate (PEP) and HCO₃⁻ to oxaloacetate (OAA) and inorganic phosphate. PEPC is found in all plants, green algae, and cyanobacteria, and in most archaea and nonphotosynthetic bacteria, but not in animals or fungi (Chollet et al., 1996; O’Leary et al., 2011a). Several isoforms of PEPC are present in plants, including plant-type PEPCs and one bacterium-type PEPC (Sánchez and Cejudo, 2003; Sullivan et al., 2004; Mamedov et al., 2005; Gennidakis et al., 2007; Igawa et al., 2010). Arabidopsis (Arabidopsis thaliana) possesses three plant-type PEPC genes, AtPPC1, AtPPC2, and AtPPC3, and one bacterium-type PEPC gene, AtPPC4. Unlike plant-type PEPCs, bacterium-type PEPCs lack a seryl-phosphorylation domain near the N terminus, a typical domain conserved in plant-type PEPCs (Sánchez and Cejudo, 2003). Plant-type PEPCs form class 1 PEPCs, which exist as homotetramers. Recently, bacterium-type PEPCs have been reported to interact with plant-type PEPCs to form heterooctameric class 2 PEPCs in several species, including unicellular green algae (Selenastrum minutum), lily (Lilium longiflorum), and castor bean (Ricinus communis; O’Leary et al., 2011a).Because of the irreversible nature of the enzymatic reactions catalyzed by PEPC isoforms, they are strictly regulated by a variety of mechanisms. PEPC is an allosteric enzyme and is activated by its positive effector, Glc-6-P, and inhibited by its negative effectors, malate, Asp, and Glu (O’Leary et al., 2011a). Control by reversible phosphorylation is another important mechanism that regulates the activity of PEPC. In this reaction, phosphorylation catalyzed by PEPC kinase changes the sensitivity of PEPC to its allosteric effectors (Nimmo, 2003). In addition, monoubiquitination may also regulate plant-type PEPC activity (Uhrig et al., 2008). Recent research in castor oil seeds suggests that bacterium-type PEPC is a catalytic and regulatory subunit of class 2 PEPCs, as class 1 and class 2 PEPCs show significant differences in their sensitivity to allosteric inhibitors (O’Leary et al., 2009, 2011b).A number of studies on PEPC function have been performed in a variety of organisms (O’Leary et al., 2011a). The best described function of PEPC is in fixing photosynthetic CO₂ during C4 and Crassulacean acid metabolism photosynthesis. However, in most nonphotosynthetic tissues and the photosynthetic tissues of C3 plants, the fundamental function of PEPC is to anaplerotically replenish tricarboxylic acid cycle intermediates (Chollet et al., 1996). PEPC also functions in malate production in guard cells and legume root nodules (Chollet et al., 1996). A chloroplast-located PEPC isoform in rice (Oryza sativa) was recently found to be crucial for ammonium assimilation (Masumoto et al., 2010). In addition, previous work in Arabidopsis suggested that AtPPC4 might play a role in drought tolerance (Sánchez et al., 2006).Transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition showed an increase in overall organic nitrogen content at the expense of starch and soluble sugars (Rademacher et al., 2002; Chen et al., 2004; Rolletschek et al., 2004). However, the in vivo function of PEPC in carbon and nitrogen metabolism has not been reported previously.To further investigate the function of PEPC in higher plants, we isolated and characterized mutants of Arabidopsis deficient in the expression of the PEPC-encoding genes PPC1 and PPC2. We demonstrated that PPC1 and PPC2 were the most highly expressed PEPC genes in the leaves. To further define their role, we produced a double mutant (ppc1/ppc2) deficient in the expression of the PPC1 and PPC2 genes. We then conducted a detailed molecular, biochemical, and physiological characterization of this double mutant.     

拟南芥氮代谢相关基因T-DNA插入突变体低氮响应的初步研究

氮代谢相关基因家族的拟南芥T-DNA插入突变体幼苗期对低氮环境有不同的响应.叶绿素含量,干物重,全氮含量测定结果表明:有2个氨转运蛋白基因(ammonium transport gene mutant, AMTm)和8个硝酸还原酶基因(nitrate reductases gene mutant, NRm)的T-DNA突变体与野生型对照相比对外界低氮环境的适应能力存在显著差异.     

过量表达大叶补血草LgNHX1基因对拟南芥耐盐性的影响

利用植物表达载体pCAMBIA1301和农杆菌GV3101将LgNHX1(全长1 656 bp)基因在拟南芥中过量表达.在含30 mg/L潮霉素的培养基上筛选获得LgNHX1的纯合转化子,并对其进行了分子鉴定和耐盐性分析.结果显示,经PCR和RT-PCR鉴定,野生型植株(对照)没有出现扩增条带,而转基因株系有相应的扩增条带,表明LgNHX1的确已经整合到拟南芥的基因组中,并已正常转录.在不同盐浓度处理下,转基因株系生长情况好于野生型对照;转基因植株地上部分和根的干重、鲜重相对高于野生型对照,但差异没有达到显著水平;当盐浓度达到150-200 mmol/L时,两个特基因株系的Na+含量显著高于野生型,K+含量极显著高于野生型.以上结果表明,过量表达LgNHX1基因可能增强了拟南芥将Na+区隔化至液泡的能力,提高了转基因拟南芥的耐盐能力.     

Studies on Nitrogen Metabolism in Tobacco Plants

Δ¹-Pyrroline-5-carboxylate reductase has been considerably purified from tobacco leaves. This enzyme uses NADPH or NADH for the formation of proline, although the former is better used. This enzyme was found in washed chloroplast extract as well as in cytoplasmic fluid and utilized NADPH, formed by the photosynthetic NADP reduction, for the sythesis of proline in the light.     

外来入侵植物的氮代谢及其土壤氮特征

研究了4种外来入侵植物(五爪金龙、南美蟛蜞菊、金腰箭和马缨丹)和1种本地植物鸡矢藤(对照)的氮代谢及其土壤氮特征.结果表明:外来人侵植物的组织硝酸还原酶活性、根际土壤NH4-N、NO3-N含量、蛋白酶活性和脲酶活性均较高,分别为鸡矢藤的1.65～4.34、1.56～2.15、1.72～3.11、1.43～3.23和1.41～3.33倍,而植物组织硝态氮含量则较低,仅为鸡矢藤的17.5%～50.6%.相关分析表明:植物组织硝酸还原酶活性与根际土壤总氮、NH4-N、NO3-N含量呈显著正相关(P<0.05),与蛋白酶活性和脲酶活性呈极显著正相关(P<0.01).这说明,外来植物入侵使土壤氮代谢加快,氮的生物有效性增强,氮同化能力提高,并且较好地将植物体氮素代谢与土壤氮素代谢协调起来.因此,较强的氮素同化能力与加速土壤氮素的转化可能是植物成功入侵的重要机制之一.     

OsPHGPx基因的过量表达增强水稻抗氧化能力

植物在生物或非生物胁迫下会通过表达磷脂氢谷胱甘肽过氧化物酶(PHGPx)来抵御胁迫引起的氧化损伤,但是PHGPx在植物体内抗氧化途径中所扮演的生理角色目前尚不完全清楚。利用农杆菌遗传转化技术,构建了过表达Os PHGPx基因的转基因水稻,并对转基因水稻进行了PCR、实时定量PCR以及Western blot等检测分析,结果表明Os PHGPx基因已成功转入水稻并正常表达。与野生型水稻相比,这些过量表达Os PHGPx的转基因水稻抵御百草枯氧化伤害的能力提高。     

Studies on Nitrogen Metabolism in Tobacco Plants A

The effect of age on non-protein constituents of tobacco leaves (N. tabacum L., var. Bright Yellow) has been studied. For this purpose leaves in three different stalk positions, upper, middle and lower, which represent young, mature and over-mature leaves, respectively, were harvested in the day-time and at night.

The total amino nitrogen content both in the day-time and at night decreases from the upper leaf position downwards. As for the individual groups, the content of both upper and middle leaves increases in the day-time and that of the lower ones increases at night.

In general, the content of the individual amino acids is high in the upper leaves and low in the lower ones. Proline and γ-aminobutyric acid, as a ratio of the total amino acid content, show a marked difference with position, in other words with age of the leaves.

The levels of proline decrease very sharply from the upper leaf position downwards and that of γ-aminobutyric acid exhibits an opposite trend in both samples at night and in the day-time. These trends are very prominent in the case of the midribs.

The contents of other amino acids, regardless of position, show similar trends with time to those reported in the previous paper₁₎, and the aspartic acid content increases at night.     

Effects of Irradiance-Sulphur Interactions on Enzymes of Carbon,Nitrogen, and Sulphur Metabolism in Maize Plants

The effect of sulphur deprivation and irradiance (180 and 750 µmol m^–2 s^–1) on plant growth and enzyme activities of carbon, nitrogen, and sulphur metabolism were studied in maize (Zea mays L. Pioneer cv. Latina) plants over a 15-d-period of growth. Increase in irradiance resulted in an enhancement of several enzyme activities and generally accelerated the development of S deficiency. ATP sulphurylase (ATPs; EC 2.7.7.4) and o-acetylserine sulphydrylase (OASs; EC 4.2.99.8) showed a particular and different pattern as both enzymes exhibited maximum activity after 10 d from the beginning of deprivation period. Hence in maize leaves the enzymes of C, N, and S metabolism were differently regulated during the leaf development by irradiance and sulphur starvation.     

Loss of chloroplast-localized NAD kinase causes ROS stress in Arabidopsis thaliana

Journal of Plant Research - Chloroplast-localized NAD kinase (NADK2) is responsible for the production of NADP+, which is an electron acceptor in the linear electron flow of photosynthesis. The...     

Studies on Nitrogen Metabolism in Tobacco Plants,B

Nitrate reductase from the leaves of Burley 21 tobacco (Nicotiana tabacum L.) was partially purified by ammonium sulfate fractionation, protamine sulfate treatment and calcium-phosphate gel adsorption.

The enzyme has optimum pH at 7.4 and is specific for reduced diphosphopyridine nucleotide (DPNH) as the electron donor. The nitrite formed increased in proportion to the rate at which DPNH disappeared in the reaction mixtures. Addition of flavin adenine dinucleo-tide (FAD) to the assay system enhanced the activity. FAD content in the “highly purified” enzyme was also determined. The enzyme was sensitive to heavy metals and SH-group inhibitors.

Discussions are presented on the metal and the properties of the enzyme in comparison to those published on other higher plants.     

Gene Expression in Plant Lipid Metabolism in Arabidopsis Seedlings

Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG) synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS) was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6) in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX) lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis.     

Studies on the Nitrogen Metabolism in Tobacco Plants. A.

The nitrogenous compounds of tobacco saps have been studied both qualitatively and quantitatively and the following results were obtained.

(1) Nitrate nitrogen accounts for 40 to 70% of the total nitrogen and the rest is composed mostly of amino and alkaloid nitrogen.

(2) Amides and basic amino acids compose a large part of the amino and amide nitrogen. Among the amino acids and amides of the tobacco saps glutamine is the highest in the content and asparagine, lysine, leucine and serine follow glutamine.

(3) Topping procedure increased remarkably the alkaloid contents in the sap but decreased the amino acid nitrogen as compared with those of the untopped plant sap.     

Improving the Mineral Nutrition in Grafted Watermelon Plants: Nitrogen Metabolism

Watermelon (Citrullus lanatus [Trumb.] Mansfeld cv. Early Star), was used as scion grafted onto three cultivars of pumpkin (Cucurbita pepo L. cvs. Brava, Shintoza and Kamel) used as rootstocks and ungrafted Early Star plants were used as control. The rootstocks showed a high capacity for N uptake and transport to the scion where N reduction and assimilation improved growth of the scion in grafted plants with respect to the control.