Overaccumulation of glycine betaine makes the function of the thylakoid membrane better in wheat under salt stress

Wheat(Triticum aestivum L.) lines T1, T4, and T6 were genetically modified to increase glycine betaine(GB) synthesis by introduction of the BADH(betaine aldehyde dehydrogenase, BADH)gene from mountain spinach(Atriplex hortensis L.). These transgenic lines and WT of wheat(T. aestivum L.) were used to study the effect of increased GB synthesis on wheat tolerance to salt stress. Salt stress due to 200 mmol L-1Na Cl impaired the photosynthesis of the four wheat lines, as indicated by declines in net photosynthetic rate(Pn), stomatal conductance(Gs),maximum photochemical efficiency of PSII(Fv/Fm), and actual photochemical efficiency of PSII(ФPSII) and an increase in intercellular CO2concentration(Ci). In comparison with WT, the effect of salinity on the three transgenic lines was mild. Salt stress caused disadvantageous changes in lipids and their fatty acid compositions in the thylakoid membrane of the transgenic lines and WT. Under salt stress, the three transgenic lines showed slightly higher chlorophyll and carotenoid contents and higher Hill reaction activities and Ca2+-ATPase activity than WT. All the results suggest that overaccumulation of GB resulting from introduction of the BADH gene can enhance the salt tolerance of transgenic plants, especially in the protection of the components and function of thylakoid membranes, thereby making photosynthesis better. Changes in lipids and fatty acid compositions in the thylakoid membrane may be involved in the increased salt stress tolerance of the transgenic lines.

Thylakoid Transit Peptide Is Related to the Expression and Localization of NdhB Subunits in Soybean

The chloroplast NAD(P)H dehydrogenase(NDH)complex,as one of the most important photosynthesis protein complexes in thylakoid membrane,is involved in photosystem I(PSI)cyclic electron transport(CEF).Under abiotic environmental stress,the photosynthetic apparatus is susceptible to the damage caused by the strong light illumination.However,the enhancement of NDHdependent CEF could facilitate the alleviation of the damage to the photosynthetic apparatus.The NdhB subunit encoded by chloroplast genome is one of most important subunits of NDH complex and consists of 510 amino acids.Here,according to cloning ndhB from Melrose(cultivated soybean),ACC547(wild salt-tolerant soybean),S113-6 and S111-9(hybrid descendant),based on the comparison and analysis of the sequences of NdhB subunits,we found that there is a novel thylakoid transit peptide of NdhB subunit in S111-9.In addition,crosslink immunoprecipitation,immunogold labeling and co-expression of GFP fusion protein indicated that the novel thylakoid transit peptide is favorable to the expression and localization of NdhB subunit in chloroplast.Therefore,we suggest that this novel thylakoid transit peptide plays the same role as chaperonin and contributes to facilitating the expression and localization of NdhB subunit.

Thylakoid engineered M2 macrophage for sonodynamic effect promoted cell therapy of early atherosclerosis

Atherosclerosis is the most common cause of cardiovascular diseases that contribute to the major morbidity worldwide,but still lacking of effective treatment strategy.Here,a hybrid cell is constructed for the sonodynamic effect promoted cell therapy of early atherosclerosis by fusing M2 macrophages with thylakoid(TK)membranes.After systemic administration,the obtained TK-M2 actively accumulates in the early atherosclerotic plaques,wherein M2 macrophages relieve the cholesterol accumulation and the inflammation in the foam cells.Meanwhile,the TK membranes decorated on the M2 macrophages exhibit both type I and type II sonodynamic effects under ultrasound(US)activation,inducing the direct apoptosis of foam cells.The cooperation of M2 and TK leads to significant outcome in eliminating atherosclerotic plaques without obvious side-effects,providing a new avenue for atherosclerosis treatment.

叶绿体类囊体腔蛋白功能研究进展

【目的】类囊体是叶绿体光合作用中光反应进行的重要场所。类囊体腔是由类囊体膜包围形成的一个狭小空间。在类囊体腔中存在多种不同的蛋白家族,包括高叶绿素荧光(high chlorophyll fluorescence,HCF)蛋白、亲免蛋白、放氧复合物(oxygen-evolving complex,OEC)蛋白、PsbP类蛋白等,它们对植物的光合作用、核酸代谢以及氧化还原反应等都起着重要作用。【评论】文章分类综述了参与光合作用调控的类囊体腔蛋白在光系统组装、植物生长发育调节和高光逆境响应等生理活动中发挥的重要作用。【展望】文章可为未来研究类囊体腔蛋白的生理功能提供理论参考。

秃杉素对水绵的抑制活性及叶绿体超微结构的影响

【目的】探究从秃杉Taiwania flousiana中提取得到的除草活性物质秃杉素(4-O-α-thevetopyranosyldiphyllin,TSC-3)对水生植物水绵Spirogyra communis的抑制活性和对其叶绿体的影响。【方法】采用乙醇萃取−分光光度法测定秃杉素对水绵叶绿素和类胡萝卜素的IC50。采用显微镜以及透射电镜技术观察不同质量浓度秃杉素对水绵显微结构和超微结构的影响。【结果】经秃杉素处理7 d后,水绵的类胡萝卜素、叶绿素a、叶绿素b及总色素的IC50分别为3.41、31.50、47.34和29.30 mg·L^(−1)。秃杉素能破坏水绵细胞的细胞壁,引起细胞膜消失、叶绿体解体、类囊体完全消失、淀粉粒分散以及蛋白核降解和碎片化。【结论】秃杉素可降低水绵叶绿素a、叶绿素b和类胡萝卜素的含量,对类胡萝卜素的抑制较其他色素更强;秃杉素主要作用于叶绿体和细胞壁。本研究为解释植物源除草化合物秃杉素的除草机理提供了基础的理论依据,也为水生藻类的防控提供了理论依据。

Changes in Unsaturated Levels of Fatty Acids in Thylakoid PSII Membrane Lipids During Chilling-induced Resistance in Rice

Temperature is one of the abiotic factors limiting growth and productivity of plants. In the present work, the effect of low non-freezing temperature, as an inducer of ＂chilling resistance＂, was studied in three cultivars of rice （Oryza sativa L.）, japonica cv. 9516 （j-9516）, the two parental lines of superhigh-yield hybrid rice between subspecies, Peiai/E32 （ji-PE）, and the traditional indica hybrid rice Shanyou 63 （i-SY63）. Leaves of chill-treated rice showed chilling-induced resistance, as an increase of their low-temperature tolerance was measured using chlorophyll fluorescence measurements, revealing a change in photosystem II （PSII） efficiency. After 5 d of exposure to 11 ~C under low light （100 pmol·m^-2·s^-1）, levels of unsaturated fatty acids in PSII thylakoid membrane lipids decreased during the initial 1-2 d, then increased slowly and reached 99.2%, 95.3% and 90.1% of the initial value （0 d） in j-9516, ji-PE and i-SY63, respectively, on the fifth day. However, under medium light （600 μmol·m^-2·s^-1）, all cultivars experienced similar substantial photoinhibition, which approached steady state levels after a decline in levels of unsaturated fatty acids in PSII thylakoid membrane lipids to about 57.1%, 53.8% and 44.5% of the initial values （0 d） in j-9516, ji-PE and i-SY63 on the fifth day. Under either chilling-induced resistance （the former） or low temperature photoinhibition （the latter） conditions, the changes of other physiological parameters such as D1 protein contents, electron transport activities of PSII （ETA）, Fv/Fm, xanthophyl cycle activities expressed by DES （deepoxide state） were consistent with that of levels of unsaturated fatty acids in PSII thylakoid membrane lipids. So there were negative correlations between saturated levels of fatty acids （16：1（3t）, 16：0, 18：0）, especially the 16：1（3t） fatty acid on thylakoid membrane and other physiological parameters, such as D1 protein contents, ETA and （A＋Z）/（A＋V＋Z）. A specific role of desaturation of fatty acids and the photoprotective pigments of the xanthophyl cycle, leading to an acclimation response in thylakoid membrane lipids may be involved. We conclude that chilling-induced resistance is accelerated by the unsaturation of thylakoid membranes, and the ability of rice plants to cold-harden can be enhanced by genetic engineering.

高温胁迫下硅添加对紫花苜蓿抗氧化系统和超微结构的影响

硅添加对缓解植物的高温胁迫具有重要作用,紫花苜蓿(Medicago sativa)作为优质豆科牧草在高温胁迫下会严重减产,因此研究硅添加对高温胁迫下紫花苜蓿生长的影响及其相关机制对于提高紫花苜蓿产量具有重要指导意义。本研究采用盆栽试验在两种温度条件(常温20℃/25℃,高温35℃/40℃)以及两个硅浓度水平(0和2 mmol·L^(−1))条件下对紫花苜蓿进行栽培处理,从抗氧化系统和叶片超微结构等方面研究了硅缓解紫花苜蓿对高温胁迫的适应机制。结果表明,紫花苜蓿的生物量、株高、叶绿素a、叶绿素b以及叶绿素总含量、叶片光系统Ⅱ实际光合效率和光系统Ⅱ的最大光合效率受高温影响显著下降(P<0.05),添加硅显著提高了这些参数。相反的是,高温胁迫使紫花苜蓿叶片的相对电导率、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性等抗氧化特性显著提高,添加硅进一步显著提高了高温下的SOD和CAT活性,但显著降低了相对电导率(P<0.05)。另外,高温胁迫使叶片的细胞膜和叶绿体类囊体出现不同程度的损伤,质体小球增多,而添加硅的叶片细胞形态完整,叶绿体类囊体形态较好,只有少量的质体小球。由此可见,在高温胁迫下硅添加能够促进紫花苜蓿幼苗的生长,通过增强抗氧化酶的活性,清除叶片受到高温胁迫过度累积的活性氧,有效保护了活性氧对细胞原生质膜的损伤,保持了叶片微观形态结构的稳定性,提高光合能力,从而提高了紫花苜蓿的耐热性。

STUDY ON SHRINKAGE OF THYLAKOIDS RELATED TO H^+-UPTAKE

It has been proved that the light-induced apparent absorbance change of thylakoid suspension at 540 nm (ΔA540)reflects the shrinkage of thylakoids. Recently, this method has been used to detect the energized state of thylakoids in vivo under various circumstances.

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant- specific protein involved in organeUar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the rash I phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppres- sion lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein-protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochon- dria results in 7%-10% of plants altered in leaf morphology, heat tolerance, and mitochondrlal genome sta- bility. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH 1. MSH1 deple- tion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

Pitt, a Novel Tetratricopeptide Repeat Protein Involved in Light-Dependent Chlorophyll Biosynthesis and Thylakoid Membrane Biogenesis in Synechocystis sp. PCC 6803

Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene （sir1644） from the cyanobacterium Synechocystis sp. PCC 6803 （Synechocystis 6803） that encodes a membrane-bound tetratricopeptide repeat （TPR） protein of formerly unknown function. Targeted inactivation of Pitt affected photosynthetic performance and light-dependent chlorophyll synthesis. Yeast two-hybrid analyses and native PAGE strongly suggest a complex formation between Pitt and the light-dependent protochlorophyllide oxidoreductase （POR）. Consistently, POR levels are approximately threefold reduced in the pitt insertion mutant. The membrane sublocalization of Pitt was found to be dependent on the presence of the periplasmic photosystem Ⅱ （PSⅡ） biogenesis factor PratA, supporting the idea that Pitt is involved in the early steps of photosynthetic pigment/protein complex formation.

THE FUNCTIONAL ROLE OF 23 kD AND 24 kD POLYPEPTIDES OF THYLAKOID IN Mg^(2+)-INDUCED CHANGE OF EXCITATION ENERGY DISTRIBUTION BETWEEN TWO PHOTOSYSTEMS IN THE CHLOROPLASTS FROM Codium fragile

In contrast to the chloroplasts from higher plants, Mg2+-induced PS--Ⅱ fluorescence inten-sity increase does not relate to Mg2+-induced surface charge density decrease of thylakoidin the chloroplasts from Codium fragile. Tbe extraction of the green alga chloroplasts withCa2+ to remove the 30--31kD polypeptide （QB protein） on the thylakoid surface does notaffect the above Mg2+-induced phenomena. If the Ca2+-treated chloroplasts are further di-gested by trypsin to remove the 23kD and 24kD polypeptides on the membrane surface,the Mg2+-induced fluorescence effect will completely disappear whereas the property ofMg2+-induced surface charge density changes remains unchanged. These results not onlyshow that the 23kDa and 24kDa polypeptides on the thylakoid surface are the specific act-ing sites of the cation that induce Chla fluorescence change, but also demonstrate that thecation-induced change of excitation energy distribution between two photosystems is not con-trolled by the

Energy transfer mechanism of PBS-thylakoid complexes——By deconvolution spectra and steady-state spectra (77 K) techniques

Up to now, the energy transfer study has been focused on the composite of isolated PBSs, PCand APC, as well as isolated PS Ⅱ and PS Ⅰparticles with time-resolved fluorescence tech-niques. Although these studies give more important information about the energy transferamong them, they cannot explain why the energy transfer from PBS to reaction centers

Energy transfer mechanism of PBS-thylakoid complexes——By time-resolved fluorescence spectra technique

IN a previous paper, we have studied the energy transfer mechanism among the PBS-thy-lakoid complex in detail by using steady-state spectra and deconvolution techniques. The ex-perimental results indicated that the energy transfer from PBS to two reaction centers of PS Ⅰand PS Ⅱ were parallel, and confirmed the model which was suggested by Mullineaxu.

Trichloroacetate affects the EPR Signal Ⅱ_(slow) and Signal Ⅰ in the thylakoid of Chlamydomonas reinhardtii

One electron paramagnetic resonance (EPR) signal, named Signal Ⅱ slow, originates from the oxidized Tyrosine 160 (YD) of D2 polypeptide of photosystem Ⅱ reaction center. After adding high concentration trichloroacetate (TCA) to the Chlamydomonas reinhardtii thylakoid suspension. this signal was abolished in a minute. Treatment of TCA also removes a few of polypeptides, including three extrinsic polypeptides of oxygen-evolving complex, from the thylakoid membrane. Based upon the analysis of the microenvironment around YD with a three-dimensional model, it is indicated that relatively high hydrophobicity of this microenvironment may be the essential prerequisite for TCA to affect YD. It has been observed that TCA treatment also retards the decay of the Signal I , produced by the oxidized reaction center chlorophyll dimer (P700+) of photosys-tem I .

Effects of calmodulin and calmodulin binding protein BP-10 on phosphorylation of thylakoid membrane protein

Light induced phosphorylation of endogenous thylakoid membrane protein can be inhibited markedly by a novel inhibitor CaMBP 10 which is discovered and isolated from plant. The inhibitory effect of BP 10 can be eliminated by addition of CaM. At the same time, the phosphorylation can also be inhibited by EGTA or CaM antagonists , such as TFP (trifluoperazine) and W 7 (N (6 aminohexyl) 5 chloro 1 naphthalene sulfonamide). This result implies that (i) Ca 2+ and CaM most likely participate in and regulate plant photosynthesis; (ii) the kinase that catalyzes thylakoid membrane protein phosphorylation can be regulated by Ca 2+ and CaM. However, the further experiments indicate that BP 10 has no effect on dephosphorylation of thylakoid phosphoproteins .

THE ROLE OF H^+-ATPase COMPLEX IN LIGHTINDUCED THYLAKOID SHRINKAGE

The increase of light scattering of thylakoid suspension in light reflects thylakoid shrinkage under membrane energization. This light scattering measurement has often been used to study the relationship between the dynamic change of thylakoid membrane structure and the energized state. Now there are two main different explanations concerning the mechanism of light-induced thylakoid shrinkage: one is that the movement of uncharged weak or-

Immunoelectron Microscopy for Locating Calvin Cycle Enzymes in the Thylakoids of Synechocystis 6803

Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing （HPF） and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle （phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase （RuBisCO）, 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase） and the catalytic portion of the chloroplast H^＋-ATP synthase （CF1） are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40 000, 90 000, and 150 000 g. Among these, the 150 000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate （R-5-P） ＋ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.

Biochemical Properties and Inhibition Kinetics of Phosphatase from Wheat Thylakoid Membranes

A phosphetase that hydrolyses phosphate monoesters has been Isolated from wheat thylakold membranes. Biochemical properties and inhibition kinetics of the phosphatase were Investigated using several Ions, organlc solvents, and Inhlbltors. Wheat （Trltlcum aestivum L. cv. PH82-2-2） thylakold membrane phosphatase activity was activated by Mg^2＋, Ca^2＋, and Fe^2＋ and was inhibited by Mn^2＋ and Cu^2＋. For example, enzyme activity was acUvated 34.81% by 2 mmol/l. Mg^2＋, but was Inhibited 22.3% and 8.5% by 2 and 1 mmol/L Cu^2＋, respectively. Methanol, ethanol and glycol were all able to activate enzyme activity. Enzyme activity was activated 58.5%, 48.2%, and 8.7% by 40% ethanol, methanol and glycol, respectively. From these results, It can be seen that the degree of actlvetlon of the phosphetase was greatest for ethanol and the type of acUvatlon was uncompetltlve. Moreover, the activity of the thylakold membrane phosphetase was Inhibited by molybdate, vanadete, phosphate, and fluoride and the type of Inhibition produced by these elements was uncompetltlve, non-competitive, competltlve and mixed, respectively.

A Thylakoid Membrane Protein Functions Synergistically with GUN5 in Chlorophyll Biosynthesis

Chlorophyll(Chl)is essential for photosynthetic reactions and chloroplast development.While the enzymatic pathway for Chl biosynthesis is well established,the regulatory mechanism underlying the homeostasis of Chl levels remains largely unknown.In this study,we identified CBD1(Chlorophyll Biosynthetic Defect1),which functions in the regulation of chlorophyll biosynthesis.The CBD1 gene was expressed specifically in green tissues and its protein product was embedded in the thylakoid membrane.Furthermore,CBD1 was precisely co-expressed and functionally correlated with GUN5(Genome Uncoupled 5).Analysis of chlorophyll metabolic intermediates indicated that cbd1 and cbd1gun5 mutants over-accumulatedmagnesium protoporphyrin Ⅸ(Mg-Proto Ⅸ).In addition,the cbd1 mutant thylakoid contained less Mg than the wild type not only as a result of lower Chl content,but also implicating CBD1 in Mg transport.This was supported by the finding that CBD1 complemented a Mg^(2+)uptake-deficient Salmonella strain under low Mg conditions.Taken together,these results indicate that CBD1 functions synergistically with CHLH/GUN5 in Mg-Proto IX processing,and may serve as a Mg-transport protein to maintain Mg homeostasis in the chloroplast.

THE PIGMENT-PROTEIN COMPLEXES OF HIGHER PLANTS MIGRATION IN THYLAKOID AND THE REGULATION OF LIGHT ENERGY DISTRIBUTION BETWEEN PHOTOSYSTEMS

Appressed and non-appressed lamella membranes of Castor bean leaf chloroplasts were separated by non-ionic detergent Triton-X 100.Appressed membranes showed a high oxygen-evolving activity and low chl a/b ratio. Examining with SDS-PTGE and liquid nitrogen temperature fluorescence measurement showed that they contained only PSII and light-harvesting pigment-protein complexes （LHCP）,and there was no detectable amount of PSI. Freeze-fracture electromicroscopic observation confirmed that this part was really an appressed lamella membrane. Through divalent cation Mg++, the thylakoid membranes were induced to unstack and restack.With the addition of Mg++, the fluorescence intensity was changed instantly. We realized that there existed two processes:One was a rapid process which was accomplished within 30 s. The other was a slow process of which the time duration was about 60 min. This dual effects of Mg++ had not been reported before.We had analyzed the change of F685/F730 and discussed the