Effects of Nitrogen Nutrition on Nitrogen Partitioning between Chloroplasts and Mitochondria in Pea and Wheat

Nitrogen partitioning among proteins in chloroplasts and mitochondria was examined in pea (Pisum sativum L.) and wheat (Triticum aestivum L.) grown hydroponically with different nitrogen concentrations. In pea leaves, chloroplast nitrogen accounted for 75 to 80% of total leaf nitrogen. We routinely found that 8% of total ribulose-1,5-bisphosphate carboxylase/oxygenase adhered to thylakoids during preparation and could be removed with Triton X-100. With this precaution, the ratio of stroma nitrogen increased from 53 to 61% of total leaf nitrogen in response to the nitrogen supply, but thylakoid nitrogen remained almost constant around 20% of total. The changes in the activities of the stromal enzymes and electron transport in response to the nitrogen supply reflected the nitrogen partitioning into stroma and thylakoids. On the other hand, nitrogen partitioning into mitochondria was appreciably smaller than that in chloroplasts, and the ratio of nitrogen allocated to mitochondria decreased with increasing leaf-nitrogen content, ranging from 7 to 4% of total leaf nitrogen. The ratio of mitochondrial respiratory enzyme activities to leaf-nitrogen content also decreased with increasing leaf-nitrogen content. These differences in nitrogen partitioning between chloroplasts and mitochondria were reflected in differences in the rates of photosynthesis and dark respiration in wheat leaves measured with an open gas-exchange system. The response of photosynthesis to nitrogen supply was much greater than that of dark respiration, and the CO₂ compensation point decreased with increasing leaf-nitrogen content.     

Effects of Meloidogyne incognita on Nitrogen Fixation in Soybean

The effects of a North Carolina population of Meloidogyne incognita on N₂ fixation on root-knot-susceptible ''Lee 68'' and moderately resistant ''Forrest'' soybean were evaluated 50, 75, I00, and 135 days after inoculation with nematodes. Nematodes stimulated N₂ fixation in Lee 68 by 50 days and in Forrest by 75 days. At all other intervals, N₂ fixation was either depressed or unaffected by nematodes. Additional observations indicate that the susceptibility of Lee 68 is associated with greater rates of penetration by larvae and more favorable responses of host tissues to nematodes than occur in Forrest. With time, however, the histological reactions of both hosts became less favorable for nematode development. Resistant or hypersensitive responses became common in Forrest by 75 days but not in Lee 68 until 90 days after inoculation. This population of M. incognita may stimulate N₂ fixation at a specific time interval and depress it at others; therefore, disease of susceptible soybeans caused by this nematode is probably not primarily due to a net loss of fixed nitrogen but to pathogenicity similar to that which occurs on nonlegume hosts.     

氮处理对大豆根瘤固氮能力及GmLbs基因表达的影响

共生根瘤的固氮效率受外界氮素的严格调控。除固氮酶活性外,豆血红蛋白(Lb)浓度亦是反应固氮能力的重要指标。为明确氮水平对生物固氮作用的影响,以大豆(Glycine max)为材料,在低氮(0.53 mmol·L–1)条件下接种根瘤菌,30天后再进行高氮(5.3、10、20、30和40 mmol·L–1)处理7天,分析L...     

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis

The signaling role of hydrogen gas (H₂) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of H₂ in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (ABA) induced stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering intracellular signaling events involving H₂, reactive oxygen species (ROS), nitric oxide (NO), and the guard cell outward-rectifying K⁺ channel (GORK). ABA elicited a rapid and sustained H₂ release and production in Arabidopsis. Exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H₂ production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that HRW stimulated significant inductions of NO and ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant nitrate reductase1/2 (nia1/2) or the NADPH oxidase-deficient mutant rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the HRW-promoted NO generation is dependent on ROS production. The rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. In addition, both HRW and hydrogen peroxide failed to induce NO production or stomatal closure in the nia1/2 mutant, while HRW-promoted ROS accumulation was not impaired. In the GORK-null mutant, stomatal closure induced by ABA, HRW, NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of H₂-regulated stomatal movement involved in the ABA signaling cascade in which RbohF-dependent ROS and nitric reductase-associated NO production, and subsequent GORK activation, were causally involved.Stomata are responsible for leaves of terrestrial plants taking in carbon dioxide for photosynthesis and likewise regulate how much water plants evaporate through the stomatal pores (Chaerle et al., 2005). When experiencing water-deficient conditions, surviving plants balance photosynthesis with controlling water loss through the stomatal pores, which relies on turgor changes by pairs of highly differentiated epidermal cells surrounding the stomatal pore, called the guard cells (Haworth et al., 2011; Loutfy et al., 2012).Besides the characterization of the significant roles of abscisic acid (ABA) in regulating stomatal movement, the key factors in guard cell signal transduction have been intensively investigated by performing forward and reverse genetics approaches. For example, both reactive oxygen species (ROS) and nitric oxide (NO) have been identified as vital intermediates in guard cell ABA signaling (Bright et al., 2006; Yan et al., 2007; Suzuki et al., 2011; Hao et al., 2012). The key ROS-producing enzymes in Arabidopsis (Arabidopsis thaliana) guard cells are the respiratory burst oxidase homologs (Rboh) D and F (Kwak et al., 2003; Bright et al., 2006; Mazars et al., 2010; Marino et al., 2012). Current available data suggest that there are at least two distinct pathways responsible for NO synthesis involved in ABA signaling in guard cells: the nitrite reductase (NR)- and l-Arg-dependent pathways (Desikan et al., 2002; Besson-Bard et al., 2008). Genetic evidence further demonstrated that removal of the major known sources of either ROS or NO significantly impairs ABA-induced stomatal closure. ABA fails to induce ROS production in the atrbohD/F double mutant (Kwak et al., 2003; Wang et al., 2012) and NO synthesis in the NR-deficient mutant nitrate reductase1/2 (nia1/2; Bright et al., 2006; Neill et al., 2008), both of which lead to impaired stomatal closure in Arabidopsis. Most importantly, ROS and NO, which function both synergistically and independently, have been established as ubiquitous signal transduction components to control a diverse range of physiological pathways in higher plants (Bright et al., 2006; Tossi et al., 2012).The guard cell outward-rectifying K⁺ channel (GORK) encodes the exclusive voltage-gated outwardly rectifying K⁺ channel protein, which was located in the guard cell membrane (Ache et al., 2000; Dreyer and Blatt, 2009). Expression profiles revealed that this gene is up-regulated upon the onset of drought, salinity, and cold stress and ABA exposure (Becker et al., 2003; Tran et al., 2013). Reverse genetic evidence further showed that GORK plays an important role in the control of stomatal movements and allows the plant to reduce transpirational water loss significantly (Hosy et al., 2003) and participates in the regulation of salinity tolerance by preventing salt-induced K⁺ loss (Jayakannan et al., 2013). Due to the high complexity of guard cell signaling cascades, whether and how ABA-triggered GORK up-regulation is attributed to the generation of cellular secondary messengers, such as ROS and NO, is less clear.Hydrogen gas (H₂) was recently revealed as a signaling modulator with multiple biological functions in clinical trails (Ohsawa et al., 2007; Itoh et al., 2009; Ito et al., 2012). It was previously found that a hydrogenase system could generate H₂ in bacteria and green algae (Meyer, 2007; Esquível et al., 2011). Although some earlier studies discovered the evolution of H₂ in several higher plant species (Renwick et al., 1964; Torres et al., 1984), it was also proposed that the eukaryotic hydrogenase-like protein does not metabolize H₂ (Cavazza et al., 2008; Mondy et al., 2014). Since the explosion limit of H₂ gas is about 4% to 72.4% (v/v, in the air), the direct application of H₂ gas in experiments is flammable and dangerous. Regardless of these problems to be resolved, the methodology, such as using exogenous hydrogen-rich water (HRW) or hydrogen-rich saline, which is safe, economical, and easily available, provides a valuable approach to investigate the physiological function of H₂ in animal research and clinical trials. For example, hydrogen dissolved in Dulbecco’s modified Eagle’s medium was found to react with cytotoxic ROS and thus protect against oxidative damage in PC12 cells and rats (Ohsawa et al., 2007). The neuroprotective effect of H₂-loaded eye drops on retinal ischemia-reperfusion injury was also reported (Oharazawa et al., 2010). In plants, corresponding results by using HRW combined with gas chromatography (GC) revealed that H₂ could act as a novel beneficial gaseous molecule in plant responses against salinity (Xie et al., 2012; Xu et al., 2013), cadmium stress (Cui et al., 2013), and paraquat toxicity (Jin et al., 2013). More recently, the observation that HRW could delay the postharvest ripening and senescence of kiwifruit (Actinidia deliciosa) was reported (Hu et al., 2014).Considering the fact that the signaling cascades for salt, osmotic, and drought stresses share a common cascade in an ABA-dependent pathway, it would be noteworthy to identify whether and how H₂ regulates the bioactivity of ABA-induced downstream components and, thereafter, biological responses, including stomatal closure and drought tolerance. To resolve these scientific questions, rbohD, rbohF, nia1/2, nitric oxide associated1 (noa1; Van Ree et al., 2011), nia1/2/noa1, and gork mutants were utilized to investigate the relationship among H₂, ROS, NO, and GORK in the guard cell signal transduction network. By the combination of pharmacological and biochemical analyses with this genetics-based approach, we provide comprehensive evidence to show that H₂ might be a newly identified bioeffective modulator involved in ABA signaling responsible for drought tolerance, that HRW-promoted stomatal closure was mainly attributed to the modulation of ROS-dependent NO generation, and that GORK might be the downstream target protein of H₂ signaling.     

Caveolin-1 Regulates Endothelial Adhesion of Lung Cancer Cells via Reactive Oxygen Species-Dependent Mechanism

The knowledge regarding the role of caveolin-1 (Cav-1) protein on endothelium adhesion of cancer cells is unclear. The present study revealed that Cav-1 plays a negative regulatory role on cancer-endothelium interaction. Endogenous Cav-1 was shown to down-regulate during cell detachment and the level of such a protein was conversely associated with tumor-endothelial adhesion. Furthermore, the ectopic overexpression of Cav-1 attenuated the ability of the cancer cells to adhere to endothelium while shRNA-mediated Cav-1 knock-down exhibited the opposite effect. We found that cell detachment increased cellular hydrogen peroxide and hydroxyl radical generation and such reactive oxygen species (ROS) were responsible for the increasing interaction between cancer cells and endothelial cells through vascular endothelial cell adhesion molecule-1 (VCAM-1). Importantly, Cav-1 was shown to suppress hydrogen peroxide and hydroxyl radical formation by sustaining the level of activated Akt which was critical for the role of Cav-1 in attenuating the cell adhesion. Together, the present study revealed the novel role of Cav-1 and underlying mechanism on tumor adhesion which explain and highlight an important role of Cav-1 on lung cancer cell metastasis.     

叶绿体中活性氧的产生和清除机制

正常情况下植物细胞内活性氧（reactive oxygen species ROS）的产生和清除是平衡的,但是,一旦植物遭受环境胁迫,ROS的积累超过抗氧化剂防护系统清除能力,就会产生氧胁迫损伤细胞。由于叶绿体作为光合作用的场所与其他细胞器相比更易遭受氧化胁迫的伤害。因此,叶绿体进化了更强的防御机制调控电子传递链的氧化还原平衡及叶绿体基质中的氧化还原状态。活性氧具有双重效应．高浓度的活性氧对植物细胞有很强的毒害作用,低浓度时可充当信号分子参与植物的某些防卫反应过程,本文就叶绿体中活性氧的产生（三线态叶绿素、PSI和PSI I电子传递链）、网络清除（抗氧化剂,SOD,As—Glu循环系统,硫氧还蛋白）机制以及功能作用进行了综述。     

叶绿体内活性氧产生及清除的酶系统

活性氧是需氧生物正常代谢的产物[1] 。在光照条件下 ,由于光合作用 ,水裂解而产生大量的分子氧 ,造成叶绿体局部氧浓度剧增而引起氧的光还原 ,形成大量的超氧化物阴离子 (Ｏ·-2 )。Ｏ·-2 的歧化及金属离子的作用则可引起Ｈ2 Ｏ2 和·ＯＨ的产生 ,危及叶绿体的膜结构。因此 ,叶绿体活性氧的清除对于维持其正常的生物功能具有重要意义。1 .叶绿体内活性氧的产生氧的光还原是叶绿体活性氧产生的主要方式[2 ] 。在叶绿体内 ,氧的光还原主要有 3条途径 :类囊体途径、叶绿体基质途径和黄素脱氢酶途径。1 .1　类囊体途径　　已经证明 ,在洗去铁…     

Utilization of Ammonium as a Nitrogen Source : Effects of Ambient Acidity on Growth and Nitrogen Accumulation by Soybean

Dry matter accumulation of plants utilizing NH₄⁺ as the sole nitrogen source generally is less than that of plants receiving NO₃⁻ unless acidity of the root-zone is controlled at a pH of about 6.0. To test the hypothesis that the reduction in growth is a consequence of nitrogen stress within the plant in response to effects of increased acidity during uptake of NH₄⁺ by roots, nonnodulated soybean plants (Glycine max [L.] Merr. cv Ransom) were grown for 24 days in flowing nutrient culture containing 1.0 millimolar NH₄⁺ as the nitrogen source. Acidities of the culture solutions were controlled at pH 6.1, 5.1, and 4.1 ± 0.1 by automatic additions of 0.01 n H₂SO₄ or Ca(OH)₂. Plants were sampled at intervals of 3 to 4 days for determination of dry matter and nitrogen accumulation. Rates of NH₄⁺ uptake per gram root dry weight were calculated from these data. Net CO₂ exchange rates per unit leaf area were measured on attached leaves by infrared gas analysis. When acidity of the culture solution was increased from pH 6.1 to 5.1, dry matter and nitrogen accumulation were reduced by about 40% within 14 days. Net CO₂ exchange rates per unit leaf area, however, were not affected, and the decreased growth was associated with a reduction in rates of appearance and expansion of new leaves. The uptake rates of NH₄⁺ per gram root were about 25% lower throughout the 24 days at pH 5.1 than at 6.1. A further increase in solution acidity from pH 5.1 to 4.1 resulted in cessation of net dry matter production and appearance of new leaves within 10 days. Net CO₂ exchange rates per unit leaf area declined rapidly until all viable leaves had abscised by 18 days. Uptake rates of NH₄⁺, which were initially about 50% lower at pH 4.1 than at 6.1, continued to decline with time of exposure until net uptake ceased at 10 days. Since these responses also are characteristic of the sequence of responses that occur during onset and progression of a nitrogen stress, they corroborate our hypothesis.     

氮素形态配比对菜用大豆籽粒膨大过程中氮碳同化的影响

以菜用大豆品种‘理想95-1'为试材,通过蛭石盆栽试验研究了氮素不同形态配比对菜用大豆籽粒膨大过程中碳氮代谢的影响.结果表明:营养液中增加适当的铵态氮比例(25%～50%)有利于菜用大豆的生长发育,菜用大豆植株和荚果干鲜重、干物率显著增加,尤以硝铵比为75∶25最为显著.在高比例的硝态氮(100%)或铵态氮(75%)处理下,菜用大豆籽粒的硝酸还原酶(NR)和谷氨酰胺合成酶(GS)活性分别显著升高,氮代谢显著增强,可溶性蛋白含量迅速增加,但同期的籽粒淀粉酶活性较低,可溶性糖和淀粉含量显著下降,碳代谢受到显著抑制.在硝铵比为75∶25时,菜用大豆籽粒氮代谢强度适中,同期碳代谢显著增强,籽粒可溶性糖和淀粉含量显著升高,能维持籽粒正常的生理代谢,有利于菜用大豆籽粒发育过程中营养物质的积累.可见,硝态氮和铵态氮配比能显著影响菜用大豆籽粒发育过程中的碳氮代谢,籽粒碳氮代谢与其产量密切相关,可通过调节硝铵比来获得理想菜用大豆产量.     

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

The influence of sodium azide on open-chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H₂O₂ as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.     

Effects of Water Stress on Energy Transduction in Chloroplasts

Water stress inhibited the photosynthetic O2 evolution rate of wheat leaves. It was shown that water stress decreased the electron transport rate, the activities of photophosphorylation and, coupling factor, and, the synthesis of ATP in chloroplasts. PS Ⅱ electron transport was more senstitive to water stress than PS Ⅰ. The reduction in photophosphorylation activity might be the results of reduction in electron transport rate and coupling factor activity, as well as the uncoupling effect of water stress on chloroplasts. The uncoupling effect could be due to the inhibition of light induced proton translocation in chloroplasts.     

Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study

Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91^phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process.     

光氧化胁迫条件下叶绿体中活性氧的产生、清除及防御

活性氧(ROS)具有双重作用,高浓度引起细胞损伤,低浓度起保护作用。在光氧化胁迫条件下,光合作用高能态的反应与O2丰富供应使叶绿体成为活性氧丰富的来源。当ROS的积累超过抗氧化剂防护系统清除能力,叶绿体及细胞不可逆的光氧化损伤就会出现。而高等植物的质粒是半自主的细胞器,有它们自己的基因组学及转录、翻译机制来控制ROS生成、保护光合作用机构免受光氧化损伤。因此,本文就光氧化胁迫期间,叶绿体中ROS的乍成、功能与防护机制进行了综述。     

Effects of Water Stress on Chlorophyll-Protein Complexes in Wheat Chloroplasts

The excitation energy transfer from light harvesting chlorophyll protein complexes to PS Ⅱ was inhibited under water stress. The contents of iriternal antennae chlorophyll-protein complexes of PS Ⅱ (CPa), light harvesting chlorophyll-protein complexes of PS Ⅱ (LHC Ⅱ ), light harvesting chlorophyll-protein of PS Ⅰ (LHC Ⅰ ) and chlorophyll a protein complex of reaction center of PS Ⅰ were decreased by water stress. The decrease of chlorophyll-protein complexes of PS Ⅱ was greater than that of PS Ⅰ . It was indicated that the amount of 25 kD polypeptide of LHC Ⅱ in particular, as well as that of 43 and 47 kD polypeptides of CPa, and 21 kD polypeptide of LHC Ⅰ , were reduced by water stress.     

光逆境对叶绿体叶绿素蛋白质复合物的影响

研究了强光照射对菠菜叶绿体的叶绿素蛋白质复合物及一些光合特性的影响。实验结果表明,随着强光照射时间的延长,首先,属光系统Ⅱ核心的叶绿素蛋白质复合物的CPa带明显减少了,进而属LHCII的寡聚体和二聚体的带有了不同程度的降低,最后,包括光系统I在内的叶绿素蛋白质复合物带大部分被分解了。结果还表明,当光逆境还未使叶绿素蛋白质复合物发生明显变化时,代表光系统Ⅱ活性的Fv/Fo值及DCIP光还原活性就已显著地降低了。     

Toxic Effects of Copper on Photosystem II of Spinach Chloroplasts

The room temperature fluorescence induction of chloroplasts was utilized as a probe to locate the site of inhibition on PSII by copper. It was found that, while the initial fluorescence yield was hardly affected, the variable fluorescence yield was lowered without significant change in its kinetics. Addition of DCMU, or abolishing oxygen evolution capability by Tris treatment, did not alter this basic inhibition pattern. Copper was also found to lower the fluorescence yield of chloroplasts treated with linolenic acid which inhibited the secondary electron transport on both oxidizing and reducing sides of PSII. The data indicate that copper adversely affects the primary charge separation at the PSII reaction center. We suggest that the inhibition is due to creation of a lesion close to the reaction center, leading to increased dissipation of incoming excitation energy to heat.     

Effects of High Light Stress on Carotenoid-Deficient Chloroplasts in Pisum sativum

The effects of high light stress on chloroplast ultrastructure and protein and mRNA composition were investigated in carotenoid-deficient peas (Pisum sativum, L.). In low light, the thylakoid membrane polypeptide pattern was altered, with several prominent chlorophyll-binding proteins present in diminished amounts. This change was found to be reflected in the ultrastructural organization of internal chloroplast membranes. In contrast to the normal grana stacking found in the controls, carotenoid-deficient plastids contained long, unstacked lamellae. Exposure to photooxidative light that resulted in destruction of >70% of chlorophyll did not lead to changes in total RNA and total cellular protein patterns. This treatment did lead to gross alterations in the chloroplast structure. Within 24 hours the plastid was seen as a swollen vesicle with only a few membrane remnants still present. Accumulation of five plastid-encoded mRNAs encoding a diverse array of photosynthetic proteins was found to be affected in different ways. While psaA mRNA was rapidly reduced by more than 75%, levels of psbF/E and atpB/E were reduced by 50%. psbA and petA mRNAs, on the other hand, appeared to be more resistant to photobleaching and remained relatively unchanged during 24 hours of high fluence-rate light treatment.     

氯丁唑和咪唑对叶绿体能量转换反应的效应

比较了氯丁唑和咪唑对叶绿体能量转换各步骤反应的效应,氯丁唑抑制光合基础的和偶联的电子传递,氯化铵可部分解除偶联的电子传递的抑制;咪唑促进基础电子传递。两者均抑制光合磷酸化、9-氨基吖啶荧光猝灭和膜上腺三磷酶活性。氯丁唑抑制质子吸收,促进游离腺三磷酶活;咪唑促进质子吸收,也促进游离腺三磷酶的活性。由此提出,氯丁唑具有能量传递抑制剂的特征,咪唑似解联剂。