A proteomic approach for investigation of photosynthetic apparatus in plants

The proteome of the photosynthetic apparatus of barley (Hordeum vulgare), obtained by analysis of thylakoids without any previous fractionation, was mapped by native electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension two-dimensional-blue native (2-D/BN)/SDS-PAGE). This protocol provided an excellent alternative to the 2-D-isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2-D separation of the most hydrophobic thylakoid proteins. Monocots and dicots showed significant differences in the first dimension while in the second dimension patterns appeared similar. Identification of each spot was performed by internal peptide primary sequence determination using both nano-electrospray ionization tandem mass spectrometry and, to a lesser extent, peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight using MALDI-TOF. This is due in particular to the fact that a limited number of peptides was obtained after trypsin digestion of these highly hydrophobic proteins. A larger number of peptides from hydrophilic intermembrane domains of transmembrane proteins were detected. Despite this, about 70% of the expected proteins were identified, including proteins with grand average of hydropathicity scores higher than 0.5. It is therefore reasonable to assert that protein hydrophobicity is not the limiting factor. Small proteins were not well identified with trypsin digestion. Instead some of these could be identified using acid hydrolysis. The method presented here does not require prefractionation of different thylakoid complexes and consequently gives confidence in comparing the proteome of the photosynthetic apparatus before and after treatment. It thus allows us to understand the molecular mechanisms underlying physiological adaptations of higher plants and to perform screening of photosynthetic mutants.     

Phosphoproteins analysis in plants: a proteomic approach

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants.     

高山植物光合机构耐受胁迫的适应机制

植物的光合作用是易受环境影响的重要生理过程之一.高山植物作为生长在特定极端环境(低温/强辐射)下的植物群体,其光合器官在形态结构和生理功能上形成了抵御强辐射和低温胁迫的特殊适应机制.但由于较高的生境异质性,高山植物的光保护适应机制存在较大的差异.光保护适应机制与光合作用密切关联,影响植物的碳同化能力和生物量的形成能力.本文对近年来国内外有关高山植物光合器官叶绿体的形态、超微解剖结构及光合机构光保护适应机理的研究进展进行了综述,并提出了今后高山植物光合作用生理适应性研究的方向.     

Pigment ligation to proteins of the photosynthetic apparatus in higher plants

Ligation of pigments to proteins of the thylakoid membrane is a central step in the assembly of the photosynthetic apparatus in higher plants. Because of the potentially damaging photooxidative activity of chlorophylls, it is likely that between their biosynthesis and final assembly, chlorophylls will always be bound to protein complexes in which photooxidation is prevented by quenchers such as carotenoids. Such complexes may include chlorophyll carriers and/or membrane receptors involved in protein insertion into the membrane. Many if not all pigment-protein complexes of the thylakoid are stabilised towards protease attack by bound pigments. The major light-harvesting chlorophyll a/b protein (Lhebl,2) folds into its native structure in vitro only when it binds pigments. Pigment-induced folding may also be a general feature of chlorophyll-carotenoid proteins of the photosynthetic apparatus.     

Trichoderma reduces scald and protects the photosynthetic apparatus in rice plants

Scald reduces the photosynthetic area, causing yield losses in rice. Changes in gas exchange parameters caused by the pathogen begin before the onset of symptoms. Chemical methods are most commonly applied to control this disease; further research into biological control methods is required. Since Trichoderma asperellum induces plant pathogen defences, increases growth, and improves photosynthetic capability, this study investigated the efficacy of T. asperellum (Ufra T06, UfraT09, Ufra T12, and Ufra T52 (Ta)) in reducing the scald lesion size and the area under the disease progress curve and in minimising the negative effects of scald on gas exchange, chlorophyll a fluorescence, chlorophyll content, and oxidative stress enzyme activity. The experiment was a completely randomised design with five replications and two treatments. Scald was reduced by 62% in plants treated with T. asperellum compared with that in control. There was a 62% increase in the net CO₂ assimilation rate (A) and a drop of 78% in the transpiration rate (E) in plants treated with T. asperellum. The maximum fluorescence (F_m) was 128% higher, and ascorbate peroxidase activity was also higher in plants treated with T. asperellum than in the control. This shows that the use of T. asperellum may be effective in improving the sustainability of the integrated management of rice diseases.     

Composition, architecture and dynamics of the photosynthetic apparatus in higher plants

The process of oxygenic photosynthesis enabled and still sustains aerobic life on Earth. The most elaborate form of the apparatus that carries out the primary steps of this vital process is the one present in higher plants. Here, we review the overall composition and supramolecular organization of this apparatus, as well as the complex architecture of the lamellar system within which it is harbored. Along the way, we refer to the genetic, biochemical, spectroscopic and, in particular, microscopic studies that have been employed to elucidate the structure and working of this remarkable molecular energy conversion device. As an example of the highly dynamic nature of the apparatus, we discuss the molecular and structural events that enable it to maintain high photosynthetic yields under fluctuating light conditions. We conclude the review with a summary of the hypotheses made over the years about the driving forces that underlie the partition of the lamellar system of higher plants and certain green algae into appressed and non-appressed membrane domains and the segregation of the photosynthetic protein complexes within these domains.     

植物光合机构对光环境的适应机制: 状态转换

在自然条件下,植物接受的照光量经常变化,而植物在进化过程中已形成了相应的适应机制,用以维持光环境变化过程中2个光反应之间光能转换的能量平衡.植物的调控系统不但能通过调控叶片和叶绿体的运动以及光合色素的积累调节光的吸收,还可以通过光系统的状态转换灵活地调节捕光色素蛋白复合体吸收的能量分配.特别是在低光强下,植物通过可对电子传递链的氧化还原状态做出响应的激酶和磷酸酶调控光系统Ⅱ捕光色素蛋白复合体(LHCⅡ)的可逆磷酸化,从而调节激发能在PSⅠ与PSⅡ之间的分配.植物的状态转换机制是植物适应光质等光环境变化的重要机制.本文综述了植物状态转换机制的研究进展,阐述了LHCⅡ的磷酸化及其在PSⅠ与PSⅡ两个光系统间的移动及其状态转换在植物适应光环境变化中的生理意义,并展望了今后的主要研究方向.     

An apparatus for measuring photosynthetic quantum yields and quanta absorption spectra of intact plants

Abstract An apparatus is described which consists of an assimilation chamber mounted in the centre of a light-integrating Ulbricht sphere, irradiated through two fibreoptic light pipes. This arrangement provides totally diffuse radiation in the sphere. The quantum flux density in die sphere is measured by an integrating quantaspectrometer connected to the sphere by eight fibreoptic light pipes. The quantaspectrometer gives the spectral and the total quantum flux density in the sphere. By measuring the quantum flux density in the sphere before and after the insertion of a plant the number of absorbed quanta per unit time and plant area can be determined. In addition, the spectral distribution of the absorbed quantum flux density is obtained. CO₂-assimilation per unit time and plant area is determined in the same apparatus. The totally diffuse radiation within the sphere minimizes mutual shading between branches and leaves. Hence, well-defined light-responsecurves of photosynthesis can be obtained by plotting the flux density of C0₂-assimilation as a function of the absorbed quantum flux density, and the quantum yields of intact plants can be calculated. Examples of photosynthetic quantum yield determinations and quanta absorption spectra are given for some plant species.     

Chemical composition of leaves and structure of photosynthetic apparatus in aquatic higher plants

Chemical composition of leaves (the content of carbon, nitrogen, nonstructural carbohydrates, organic acids, mineral substances, and water) and the structure of photosynthetic apparatus (specific leaf weight, cell volume, and the number of cells per unit leaf area) were investigated for 18 species of aquatic plants featuring various degrees of contact with aqueous environment and sediment. The rooted hydrophytes with floating leaves were characterized by comparatively high content of carbon and nitrogen (437 and 37 mg/g dry wt, respectively) and by low concentration of nonstructural carbohydrates, mineral substances, and organic acids (161, 54, and 60 mg/g dry wt, respectively). Unlike rooted plants, the free-floating nonrooted hydrophytes had characteristically higher content of nonstructural polysaccharides and mineral substances (by a factor of 1.3 and 1.6, respectively), while the leaf nitrogen content was 1.4 times lower, and the proportion of soluble carbohydrates in the total content of nonstructural carbohydrates was rather low (9%). The chemical composition of leaves in submerged rooted hydrophytes was intermediate between those for rooted hydrophytes with floating leaves and for nonrooted free plants. We found reliable positive correlations between the volume of photosynthesizing cells and the leaf content of organic acids (r = 0.69), as well as between specific leaf weight, the number of photosynthesizing cells per unit leaf area, and carbon content (r = 0.67 and r = 0.62, respectively). The content of nitrogen and nonstructural carbohydrates in hydrophytes was unrelated to structural characteristics of photosynthetic apparatus and depended on the absence or presence of plant attachment to the sediment. It is concluded that the structural traits of photosynthetic apparatus and the leaf chemical composition in hydrophytes featuring different degrees of plant contact with water and sediment reflect the specificity of plant adaptation to complex conditions of their habitats.     

Effect of cytokinins on the photosynthetic apparatus in water-stressed and rehydrated bean plants

Effects of the cytokinins 6-benzylaminopurine (BAP) and N-2-chloro-4-pyridyl-N′-phenylurea (4-PU-30) on the photochemical activity, oxygen flash yields, and thermoluminescence in bean plants under a water stress were studied. The cytokinins increased the photochemical (Hill reaction) activity and thermoluminescence "B"-band in control as well as in stressed and rehydrated plants, while the oxygen flash yields were affected only in the stressed and rehydrated plants.     

Effect of cytokinins on the photosynthetic apparatus in water-stressed and rehydrated bean plants

Effects of the cytokinins 6-benzylaminopurine (BAP) and N-2-chloro-4-pyridyl-N′-phenylurea (4-PU-30) on the photochemical activity, oxygen flash yields, and thermoluminescence in bean plants under a water stress were studied. The cytokinins increased the photochemical (Hill reaction) activity and thermoluminescence "B"-band in control as well as in stressed and rehydrated plants, while the oxygen flash yields were affected only in the stressed and rehydrated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of water stress on the photosynthetic apparatus of plants and the protective role of cytokinins: A review

Characteristics of photosynthetic apparatus (the pool of pigments and proteins; the activity of photosystems; the intensities of in vivo photoassimilation of carbon dioxide and in vitro activity of enzymes of carbon metabolism; leaf structure; chloroplast structure), undergoing changes under the conditions of water deficiency, have been reviewed. The protective role of cytokinins is due to their regulatory effects on the renewal of disrupted cellular structures, the condition of the stomata, and de novo synthesis and activation of proteins that are required for increasing plant resistance to water stress.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 133–147.Original Russian Text Copyright © 2005 by Chernyadev.     

A proteomic approach for exploring biofilm in Streptococcus mutans

Biofilm formation by Streptococcus mutans is considered as its principal virulence factor, causing dental caries. Mutants of S. mutans defective in biofilm formation were generated and analyzed to study the collective role of proteins in its formation. Mutants were characterized on the basis of adherence to saliva-coated surface, and biofilm formation. The confocal laser microscopy and scanning electron microscopy images showed that the control biofilms had cluster of cells covered by layer of exo-polysaccharide while the biofilms of mutants were thin and spaced. Two-dimensional protein electrophoresis data analysis identified 57 proteins that are either up (44 proteins) or down (13 proteins) regulated. These data points to the importance of up and down regulated proteins in the formation of biofilm in Streptococcus mutans.     

A protector effect of cytokinin preparations on the photosynthetic apparatus of wheat plants under water deficiency conditions

The protective effects of the cytokinin 6-benzylaminopurine and the compounds thidiazuron and kartolin, displaying a cytokinin activity, on the photosynthetic apparatus of young seedlings and leaves of adult plants of two wheat (Triticum aestivum L.) cultivars, Mironovskaya 808 (more drought tolerant) and Lutescens 758 (less tolerant to water stress), were compared on the background of an increasing water deficiency. At the stages of drought and subsequent rehydration, cartolin preparations were the most efficient protectors, enhancing a less pronounced decrease in the intensity of photosynthesis, carboxylating activity of the key enzyme of carbon metabolism--ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39)-and the activity of NADP-glyceraldehyde phosphate dehydrogenase--the enzyme complex comprising phosphoglycerate kinase (EC 2.7.2.3) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13). This effect also included an increase in the leaf specific density and plant productivity. The negative influence of water stress on the photosynthetic apparatus was more pronounced in a less tolerant cultivar Lutescens 758 and in the seedlings as compared with the adult plants.     

Polyamines in the photosynthetic apparatus

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.Abbreviations Chl chlorophyll - HPLC high performance liquid chromatography - LHC light-harvesting complex - PS II Photosystem II - PS II-RC Photosystem II reaction center - Put putrescine - Spd spermidine - Spm spermine - 10%S-core D₁-D₂-Cyt b559-47 kD-43 kD complex     

A protector effect of cytokinin preparations on the photosynthetic apparatus of wheat plants under water deficiency conditions

The protective effects of the cytokinin 6-benzylaminopurine and the compounds thidiazuron and kartolin, displaying a cytokinin activity, on the photosynthetic apparatus of young seedlings and leaves of adult plants of two wheat (Triticum aestivum L.) cultivars, Mironovskaya 808 (more drought tolerant) and Lutescens 758 (less tolerant to water stress), were compared on the background of an increasing water deficiency. At the stages of drought and subsequent rehydration, kartolin preparations were the most efficient protectors, enhancing a less pronounced decrease in the intensity of photosynthesis, carboxylating activity of the key enzyme of carbon metabolism—ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39)—and the activity of NADP—glyceraldehyde phosphate dehydrogenase—the enzyme complex comprising phosphoglycerate kinase (EC 2.7.2.3.) and glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13). This effect also included an increase in the leaf specific density and plant productivity. The negative influence of water stress on the photosynthetic apparatus was more pronounced in a less tolerant cultivar Lutescens 758 and in the seedlings as compared with the adult plants.     

Structural and functional reorganization of the photosynthetic apparatus in adaptation to cold of wheat plants

In seedlings of a cold-resistant wheat variety, the dynamics was studied of the main structural-functional parameters of the photosynthetic apparatus (PSA) and of cold resistance of leaf cells in low-temperature plant adaptation. It has been established that a complex of structural-functional PSA changes takes place in seedling leaves under the influence of cold. As a result, as early as in the first hours of hardening, the formation of chloroplasts begins to occur in mesophyll cells of larger sizes and with a thylakoid system of the “sun type.” Owing to structural and functional readjustment (a change of content of pigments, stabilization of pigment-protein complexes, and enhancement of nonphotochemical quenching of excess energy) in the process of cold adaptation, the rate of photosynthesis stabilizes. It is suggested that the observed structural-functional PSA rearrangement is a necessary condition for formation of increased cold resistance of leaf cells; this, alongside with other physiological-biochemical changes occurring in parallel in cells and tissues of the plants, provides their survival under conditions of low temperatures.     

Platelet biomarkers for a descending cognitive function: A proteomic approach

Memory loss is the most common clinical sign in Alzheimer''s disease (AD); thus, searching for peripheral biomarkers to predict cognitive decline is promising for early diagnosis of AD. As platelets share similarities to neuron biology, it may serve as a peripheral matrix for biomarkers of neurological disorders. Here, we conducted a comprehensive and in‐depth platelet proteomic analysis using TMT‐LC‐MS/MS in the populations with mild cognitive impairment (MCI, MMSE = 18–23), severe cognitive impairments (AD, MMSE = 2–17), and the age‐/sex‐matched normal cognition controls (MMSE = 29–30). A total of 360 differential proteins were detected in MCI and AD patients compared with the controls. These differential proteins were involved in multiple KEGG pathways, including AD, AMP‐activated protein kinase (AMPK) pathway, telomerase RNA localization, platelet activation, and complement activation. By correlation analysis with MMSE score, three positively correlated pathways and two negatively correlated pathways were identified to be closely related to cognitive decline in MCI and AD patients. Partial least squares discriminant analysis (PLS‐DA) showed that changes of nine proteins, including PHB, UQCRH, CD63, GP1BA, FINC, RAP1A, ITPR1/2, and ADAM10 could effectively distinguish the cognitively impaired patients from the controls. Further machine learning analysis revealed that a combination of four decreased platelet proteins, that is, PHB, UQCRH, GP1BA, and FINC, was most promising for predicting cognitive decline in MCI and AD patients. Taken together, our data provide a set of platelet biomarkers for predicting cognitive decline which may be applied for the early screening of AD.