田间小麦叶片光合作用的光抑制不伴随D1蛋白的净降解

通过测定田间小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）叶片Ｄ１蛋白的含量、光合放氧和叶绿素ａ 荧光,探讨了叶片光合作用的光抑制与Ｄ１蛋白净降解的关系。田间的小麦叶片受到晴天中午光照约３ ｈ 以后,表观光合量子效率（Φ）、光系统Ⅱ的光化学效率（Ｆｖ／Ｆｍ ）和初始荧光（Ｆ０）明显下降;若将叶片转入弱光下,这３个指标可在１ ｈ 内基本恢复;强光照射过程中Ｄ１蛋白的含量没有显著变化;Ｄ１蛋白合成抑制剂ＳＭ 使强光下叶片的慢驰豫的非光化学荧光猝灭（ｑＥ－ｓｌｏｗ ）明显增加;在弱光下恢复时引入链霉素（ＳＭ）不影响叶片光合功能的恢复;用二硫苏糖醇（ＤＴＴ）抑制叶黄素循环使中午强光照射后的叶片中Ｄ１蛋白的含量降低３０％ 左右。这些结果都表明,田间小麦叶片光合作用的光抑制不是由于Ｄ１蛋白的净降解,而是由于非辐射能量耗散的增加引起的。     

外源水杨酸对光抑制条件下小麦叶片光合作用的影响

以浓度为50、100、200 mg·kg-1的水杨酸(SA)预先处理灌浆期的小麦叶片,可有效防护强光所致的氧化损伤,维持较高的超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性,减轻丙二醛(MDA)积累.叶片在光抑制条件下,可维持较高的通过PSⅡ电子传递速率(Fm/Fo)、PSⅡ原初光化学效率(Fv/Fm)、PSⅡ量子效率(ΦPSⅡ)、光化学猝灭系数(qP)、净光合速率(Pn)和较低的非光化学猝灭系数(qNP).其中,以较低浓度SA(50 mg·kg-1)的效果较好.     

水稻光合日变化及光抑制的叶绿素荧光

研究了光敏核不育水稻(Oryza sativa L.)农垦58S(NK58S)的光合日变化和光抑制.06:00～09:00,NK58S的光抑制不明显,此时的光合功能下调以叶黄素循环为主;10:00～12:00,耗散比能流(DIo/RC)及光反应中心关闭净速率(dV/dto)增加,受体侧电子传递受阻(ψo下降),活性反应中心密度(Do)降低,NK58S光抑制加剧,PSⅡ反应中心发生失活.荧光暗弛豫分析与抑制剂处理结果表明,状态转换、叶黄素循环和PSⅡ反应中心失活均能有效保护NK58S免遭强光损伤.叶黄素循环相对于反应中心失活,前者是NK58S对强光胁迫的快速反应,在光强相对较弱时发挥主要作用,而后者在叶黄素循环达到饱和时对保护剩余活性反应中心起主要作用.     

水稻光合日变化及光抑制的叶绿素荧光

研究了光敏核不育水稻(OryzasativaL.)农垦58S(NK58S)的光合日变化和光抑制。06:00~09:00,NK58S的光抑制不明显,此时的光合功能下调以叶黄素循环为主;10:00~12:00,耗散比能流(DIo/RC)及光反应中心关闭净速率(dV/dto)增加,受体侧电子传递受阻(yO下降),活性反应中心密度(Do)降低,NK58S光抑制加剧,PSⅡ反应中心发生失活。荧光暗弛豫分析与抑制剂处理结果表明,状态转换、叶黄素循环和PSⅡ反应中心失活均能有效保护NK58S免遭强光损伤。叶黄素循环相对于反应中心失活,前者是NK58S对强光胁迫的快速反应,在光强相对较弱时发挥主要作用,而后者在叶黄素循环达到饱和时对保护剩余活性反应中心起主要作用。     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Role of Protein Synthesis in Recovering Nitrate Reductase Activity in Wheat Leaves Exposed to Heat Stress

Heating intact leaves of 14–15-day-old seedlings of wheat (Triticum aestivumL.), cv. Albidum 29, for 10 min at 44–45°C brought about a decrease in nitrate reductase activity by 50–90% of the initial level. The complete recovery of the enzyme activity occurred one to two days after the plants were returned to normal temperature conditions. Darkening plants or adding cycloheximide to the nutrient medium did not interfere with the recovery of nitrate reductase activity. The plants grown in darkness or on a nitrate-free medium were devoid of nitrate reductase activity. The transfer of these plants to the light or the addition of nitrate resulted in the induction of enzyme activity. In the untreated plants, nitrate reductase activity attained the control level in 48 h; in the heated plants, this process was considerably retarded. After heating, the activity of the preexisting enzyme recovered at a higher rate than the ability for enzyme induction. This means that the reactivation of nitrate reductase occurred even when the induction of the enzyme was almost entirely suppressed. We conclude that after the short-term effect of high temperatures, the functional activity of nitrate reductase may recover without the de novosynthesis of the enzyme protein.     

Photoinhibition of Photosynthesis in Vitis berlandieri and Vitis rupestris Leaves under Field Conditions

Photoinhibition of photosynthesis was investigated in Vitis berlandieri and Vitis rupestris leaves under field conditions at different sampling time in a day. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) and photosynthetic electron transport measurements. When the photochemical efficiency of PS2, F_v/F_m, markedly declined, F₀ increased significantly in leaves of V. berlandieri, while F₀ did not increase in V. rupestris leaves. Isolated thylakoids of leaves of V. berlandieri showed significant inhibition of whole chain and PS2 activities at midday. A smaller inhibition was observed for V. rupestris. Later, the leaves reached maximum PS2 efficiencies similar to those observed early in the morning during sampling at evening. The artificial exogenous electron donor Mn²⁺ failed to restore PS2 activity in both species, while DPC and NH₂OH significantly restored PS2 activity in V. rupestris midday leaf samples. Quantification of the PS2 reaction centre protein D1 and 33 kDa protein of water splitting complex following midday exposure of leaves showed pronounced differences between V. berlandieri and V. rupestris leaves. The marked loss of PS2 activity noticed in midday samples was mainly due to the marked loss of D1 protein in V. berlandieri while in V. rupestris it was the 33 kDa protein.     

D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.     

Photoinhibition of Photosynthesis in Sun and Shade Grown Leaves of Grapevine (Vitis vinifera L.)

The degree of photoinhibition of sun and shade grown leaves of grapevine was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (F_v/F_m) and electron transport measurements. The potential efficiency of photosystem 2 (PS2), F_v/F_m, markedly declined under high irradiance (HI) in shade leaves with less than 10 % of F₀ level. In contrast, F_v/F_m ratio declined with about 20 % increase of F₀ level in sun leaves. In isolated thylakoids, the rate of whole chain and PS2 activity in HI shade and sun leaves was decreased by about 60 and 40 %, respectively. A smaller inhibition of photosystem 1 (PS1) activity was also observed in both leaf types. In the subsequent dark incubation, fast recovery was observed in both leaf types that reached maximum PS2 efficiencies similar to non-photoinhibited control leaves. The artificial exogenous electron donors DPC, NH₂OH, and Mn²⁺ failed to restore the HI-induced loss of PS2 activity in sun leaves, while DPC and NH₂OH were significantly restored in shade leaves. Hence HI in shade leaves inactivates on the donor side of PS2 whereas it does at the acceptor side in sun leaves, respectively. Quantification of the PS2 reaction centre protein D1 and the 33 kDa protein of water splitting complex following HI-treatment of leaves showed pronounced differences between shade and sun leaves. The marked loss of PS2 activity in HI leaves was due to the marked loss of D1 protein of the PS2 reaction centre protein and the 33 kDa protein of the water splitting complex in sun and shade leaves, respectively.     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子。利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因。该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74％的同源性。在多肽链的N-端具有在真核生物中高度保守的UFD1结构域。我们将该基因定位在小麦的第六染色体群并将其命名为了UFD1。Southern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的。无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源。除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域。TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达。     

小麦泛素融合降解蛋白基因的克隆及特征分析

酵母UFD1基因编码的泛素融合降解蛋白是泛素依赖性降解系统或泛素融合降解途径中的一个关键因子.利用RT-PCR技术在小麦(Triticum aestivum L.)中分离到一个UFD1类似基因.该基因的编码区长948 bp,编码长315个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥UFD1类似蛋白有74%的同源性.在多肽链的N-端具有在真核生物中高度保守的UFD1结构域.我们将该基因定位在小麦的第六染色体群并将其命名为TUFD1.South-ern杂交和数据库搜索表明植物的UFD1基因是单拷贝或低拷贝的.无论是在单子叶中还是在双子叶植物中,UFD1蛋白都高度同源.除了N端UFD1结构域外,该类蛋白还有3个高度保守的C端结构域.TUFD1基因在小麦幼苗的根、茎、胚芽鞘、叶片以及幼穗和腊熟期子粒中呈组成性表达.     

叶黄素循环和D1蛋白周转在毛竹叶片光保护中的作用

利用叶绿素荧光技术,对强光胁迫下以及叶黄素循环抑制剂-二硫苏糖醇(DTT)和D1蛋白合成抑制剂-硫酸链霉素(SM)处理后毛竹(Phyllostachys edulis (Carr.) Lehaie)的光抑制特征进行研究。结果显示:在夏季中午强光或人为强光胁迫下,毛竹叶片最大光化学效率Fv/Fm均显著降低;在下午光强减弱或黑暗、弱光条件下,Fv/Fm可有效恢复。DTT和SM均可抑制毛竹叶片非光化学淬灭(NPQ),且DTT效果明显优于SM。另外,在强光下,DTT和SM处理均能使毛竹叶片Fv/Fm、实际光化学效率Y(Ⅱ)和光化学淬灭qP等荧光参数下降幅度增大。研究结果表明毛竹叶片具有完善的光破坏防御机制,NPQ与叶黄素循环和D1蛋白周转紧密关联,在叶片光保护机制中具有重要作用。     

Growth and Solute Composition in Two Wheat Species Experiencing Combined Influence of Stress Conditions1

The effects of environmental stress combinations on the soluble metabolites were investigated in several cultivars of Triticum aestivum and T. durum. The seedlings grown at optimum (24/16°C), low (5/–5°C) (LT), and high (40/30°C) (HT) day/night temperature conditions were exposed to waterlogging, drought, and salinity (0.7% NaCl, w/w) stresses for six days. Root and shoot fresh weight significantly decreased under waterlogging, drought and salt stresses. Fresh weight was most reduced at severe drought + HT combinations. The lowest relative water content was found under drought stress + HT combination. Soluble carbohydrate (SC) contents increased under LT conditions, but decreased under HT conditions. Under HT + salt combinations, T. aestivum genotypes showed higher SC content thanT. durum genotypes. Proline content significantly increased in the case of water deficit and salt stress. Under drought and salt stresses, T. aestivum genotypes had lower proline contents than T. durum genotypes. These results indicate that biochemical responses to drought, waterlogging, and salt stresses were significantly changed in wheat seedlings under LT and HT conditions.     

Photosystem II Regulation and Dynamics of the Chloroplast D1 Protein in Arabidopsis Leaves during Photosynthesis and Photoinhibition

Arabidopsis thaliana leaves were examined in short-term (1 h) and long-term (10 h) irradiance experiments involving growth, saturating and excess light. Changes in photosynthetic and chlorophyll fluorescence parameters and in populations of functional photosystem II (PSII) centers were independently measured. Xanthophyll pigments, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-binding sites, the amounts of D1 protein, and the rates of D1 protein synthesis were determined. These comprehensive studies revealed that under growth or light-saturating conditions, photosynthetic parameters remained largely unaltered. Photoprotection occurred at light saturation indicated by a dark-reversible increase in non-photochemical quenching accompanied by a 5-fold increase in antheraxanthin and zeaxanthin. No consistent change in the concentrations of functional PSII centers, DCMU-binding sites, or D1 protein pool size occurred. D1 protein synthesis was rapid. In excess irradiance, quantum yield of O2 evolution and the efficiency of PSII were reduced, associated with a 15- to 20-fold increase in antheraxanthin and zeaxanthin and a sustained increase in nonphotochemical quenching. A decrease in functional PSII center concentration occurred, followed by a decline in the concentration of D1 protein; the latter, however, was not matched by a decrease in DCMU-binding sites. In the most extreme treatments, DCMU-binding site concentration remained 2 times greater than the concentration of D1 protein recognized by antibodies. D1 protein synthesis rates remained unaltered at excess irradiances.     

干旱条件下冬小麦不同叶龄叶绿素荧光及叶黄素循环组分的变化(英文)

干旱条件下冬小麦叶片光化学效率（Ｆｖ／Ｆｍ）的降低伴随着叶黄素循环组分玉米黄质含量的增加。干旱初期,Ｆｖ／Ｆｍ 的降低约在暗置过夜后完全恢复。当干旱诱导的玉米黄质含量的增加达最大值时,Ｆｖ／Ｆｍ 不可逆下降,Ｆｏ 上升,表明发生了光破坏。与老叶相比,干旱条件下功能叶具有较高的玉米黄质含量,对光破坏的抗性较强。推测干旱条件下老叶不可逆衰老与其依赖于叶黄素循环的耗散过剩光能能力的下降有关。     

柑橘叶片中叶黄素循环对PSII反应中心D1蛋白的保护效应

用叶黄素循环抑制剂二硫苏糖醇（DTT）处理7h的柑橘离体叶片,其非光化学猝灭系数NPQ大幅度下降;在中等强度光（500μmol·m^-2·s^-1）和高强度光（1500μmol·m^-2·s^-1）下,DTT处理的叶片光化学效率（Fv/Fm）分别下降3．8％和39．7％,光合电子传递速率（ETR）分别下降12％和49．5％,D1蛋白含量也分别下降87％和92.3％;黑暗对DTT处理叶片的各种荧光参数和D1蛋白的影响不大。显示叶黄素循环在保护光系统（PS）II反应中心、抵御光抑制中有一定的积极效应,可能影响了D1蛋白周转。     

Photoinhibition and Recovery of photosystem 2 in Grapevine (Vitis vinifera L.) Leaves Grown Under Field Conditions

Photoinhibition of photosynthesis was investigated in grapevine (Vitis vinifera L.) exposed to 2 or 4h of high irradiance (HI) (1 700–1 800 mol m^–2 s^–1) leaves under field conditions at different sampling time in a day. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) and photosynthetic electron transport measurements. When the photochemical efficiency of photosystem 2 (PS2), F_v/F_m, markedly declined, F₀ increased in both 2 (HI2) and 4 h (HI4) HI leaves sampled at midday. When various photosynthetic activities were followed on isolated thylakoids, HI4 leaves showed significantly higher inhibition of whole chain and PS2 activity than the HI2 leaves sampled at midday. Later, the leaves reached maximum PS2 efficiencies similar to those observed early in the morning during sampling at evening. The artificial exogenous electron donor Mn²⁺ failed to restore PS2 activity in both variants of leaves, while DPC and NH²OH significantly restored PS2 activity in HI4 midday leaf samples. Quantification of the PS2 reaction centre protein D1 and 33 kDa protein of water splitting complex following midday exposure of leaves showed pronounced differences between HI2 and HI4 leaves. The marked loss of PS2 activity noticed in midday samples was mainly due to the marked loss of D1 protein in HI2, while in HI4 it was mainly 33-kDa protein.     

Carbohydrate and Proline Contents in Leaves, Roots, and Apices of Salt-Tolerant and Salt-Sensitive Wheat Cultivars1

Intra-specific variations in nonstructural carbohydrates and free proline were determined in leaves, apices, roots, and maturing seeds of two salt-tolerant cultivars (CR and Kharchia-65) and one salt-sensitive cv. Ghods of spring wheat (Triticum aestivum L.) grown in sand culture at various levels of salinity (0, 100, 200, and 300 mM NaCl and CaCl₂ at 5 : 1 molar ratio) under controlled environmental conditions. The levels of leaf, apex, and root ethanol-soluble carbohydrates, fructans, starch, and proline increased in line with elevating level of salinity in all three cultivars under investigation. The contents of proline, soluble and insoluble carbohydrates in the apex increased to levels exceeding those in the leaves and roots. Soluble carbohydrate content of salt-sensitive cv. Ghods was higher in the leaves, apices, and roots and lower in the maturing seeds than in the other cultivars at all levels of salinity except at 300 mM. The results show considerable variation in the amount of soluble, insoluble sugars, and proline among plant tissues and wheat genotypes in response to salinity. Higher soluble carbohydrates and fructan in leaves, roots and maturing seeds of stressed plants indicate that their accumulation may help plant to tolerate salinity. Salt-sensitive cv. Ghods accumulated less soluble sugars in the maturing seeds and higher soluble sugars in the apices, which might be used as an indicator in screening wheat genotypes for salinity tolerance.     

Regulation of D1-protein degradation during photoinhibition of photosystem II in vivo: Phosphorylation of the D1 protein in various plant groups

Photoinhibition of PSII and turnover of the D1 reaction-centre protein in vivo were studied in pumpkin leaves (Cucurbita pepo L.) acclimated to different growth irradiances and in low-light-grown moss, (Ceratodon purpureus) (Hedw.) Brid. The low-light-acclimated pumpkins were most susceptible to photoinhibition. The production rate of photoinhibited PSII centres (k_PI), determined in the presence of a chloroplast-encoded protein-synthesis inhibitor, showed no marked difference between the high- and low-light-grown pumpkin leaves. On the other hand, the rate constant for the repair cycle (k_REC) of PSII was nearly three times higher in the high-light-grown pumpkin when compared to low-light-grown pumpkin. The slower degradation rate of the damaged D1 protein in the low-light-acclimated leaves, determined by pulsechase experiments with [³⁵S]methionine suggested that the degradation of the Dl protein retards the repair cycle of PSII under photoinhibitory light. Slow degradation of the D1 protein in low-light-grown pumpkin was accompanied by accumulation of a phosphorylated form of the D1 protein, which we postulate as being involved in the regulation of D1-protein degradation and therefore the whole PSII repair cycle. In spite of low growth irradiance the repair cycle of PSII in the moss Ceratodon was rapid under high irradiance. When compared to the high- or low-light-acclimated pumpkin leaves, Ceratodon had the highest rate of D1-protein degradation at 1000 mol photons m^–2 s^–1. In contrast to the higher plants, the D1 protein of Ceratodon was not phosphorylated either under high irradiance in vivo or under in-vitro conditions, which readily phosphorylate the D1 protein of higher plants. This is consistent with the rapid degradation of the D1 protein in Ceratodon. Screening experiments indicated that D1 protein can be phosphorylated in the thylakoid membranes of angiosperms and conifers but not in lower plants. The postulated regulation mechanism of D1-protein degradation involving phosphorylation and the role of thylakoid organization in the function of PSII repair cycle are discussed.Abbreviations Chl Chlorophyll - D1^* phosphorylated form of D1 protein - F_max and F_v maximal and variable fluorescence respectively - k_PJ and k_REC rate constants of photoinhibition and concurrent recovery respectively - LHCII lightharvesting chlorophyll a/bprotein of PSII - PFD photon flux density Dr. R. Barbato (Dipartimento di Biologia, Universita di Padova, Padova, Italy), Prof. P. Böger (Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universität Konstanz, Konstanz, Germany), Prof. A. Melis (Department of Plant Biology, University of California, Berkeley, USA), Prof. I. Ohad (Department of Biological Chemistry, Hebrew University, Jerusalem, Israel) and Mr. A. Soitamo (Department of Biology, University of Turku, Turku, Finland) are gratefully acknowledged for the D1-protein-specific antibodies. The authors thank Ms. Virpi Paakkarinen for excellent technical assistance. This work was supported by the Academy of Finland and the Foundation of the University of Turku.     

条锈病对小麦(Triticum aestivum L.)叶片光合功能及光合功能蛋白D1表达的影响

测定了小麦(Triticum aestivum L.)感染小麦条锈病后的光合常数,以及叶绿素含量、类囊体膜光合电子传递速率和光合反应中心D1蛋白的变化.实验显示,条锈病侵染导致感病小麦叶片净光合速率与叶绿素含量降低;抗病小麦经侵染后净光合速率却有恢复过程,叶绿素含量先降后升.此外,感病小麦叶片被侵染后全链电子传递速率受到抑制,PSII电子传递速率的变化与全链电子传递速率的变化趋势相似,但PSI电子传递速率受到的影响较小;抗病小麦小麦叶片被侵染后电子传递速率所受影响较小.同时发现,病程中,感病和抗病小麦PSII的光合反应中心D1蛋白含量变化总是与PSII电子传递速率的变化类似,推测D1蛋白的表达量变化是引起PSII电子传递活性与全链电子传递速率变化的主要因素之一.