Phase Behavior of Chloroplast and Microsomal Membranes during Leaf Senescence

Wide angle x-ray diffraction of chloroplast and microsomal membranes from primary leaves of Phaseolus vulgaris has revealed that for both types of membrane, portions of the lipid become crystalline as the tissue senesces. For young leaves the transition temperature is about 23 C for microsomes and below −30 C for chloroplast membranes, indicating that at physiological temperature the lipid is entirely liquid-crystalline. Between 2 and 3 weeks after planting the transition temperature rises to 38 C for microsomes, but for chloroplasts this increase to a point above physiological temperature does not occur until between 3 and 4 weeks. Thereafter the transition temperature continues to rise for both types of membrane with advancing senescence, although the rate of increase is greater for chloroplasts than for microsomes. The appearance at physiological temperature of gel phase lipid in the microsomes coincides temporally with the initiation of a decline in total protein in the tissue, and the incidence of crystallinity in chloroplasts coincides with loss of chlorophyll. This change in phase behavior cannot be attributed to an alteration in fatty acid composition, but for both membrane systems it correlates with an increase of about 4-fold in the sterol to phospholipid ratio.     

Effects of Polyamines on Chlorophyll and Protein Content, Photochemical Activity, and Chloroplast Ultrastructure of Barley Leaf Discs during Senescence

The polyamines putrescine, spermidine, and spermine prevent the loss of chlorophyll normally associated with senescence of excised leaf tissue maintained in darkness on water (control). Retention of chlorophyll in barley leaf discs was in the range of 90% 4 days after excision and placement on effective polyamine solutions. In contrast, the loss of soluble protein was hastened with 0.5 millimolar spermidine and spermine treatments but it was retarded by 0.5 millimolar putrescine.     

L-精氨酸与精胺延缓大麦离体叶片衰老效应的比较

L-精氨酸和精胺均具有延缓大麦离体叶片叶绿素,蛋白质和核酸含量下降的作用。L-精氨酸具有良好的渗透性和移动性。但其最适浓度高于精胺100倍,作用时间也滞后于精胺。L-精氨酸可使叶片内多胺含量增加,多胺合成抑制剂ABA则削弱L-精氨酸延缓叶片衰老的作用。L-精氨酸转变成具有强烈生理活性的多胺可能是其作用机理之一。     

Protective Action of Spermine and Spermidine against Photoinhibition of Photosystem I in Isolated Thylakoid Membranes

The photo-stability of photosystem I (PSI) is of high importance for the photosynthetic processes. For this reason, we studied the protective action of two biogenic polyamines (PAs) spermine (Spm) and spermidine (Spd) on PSI activity in isolated thylakoid membranes subjected to photoinhibition. Our results show that pre-loading thylakoid membranes with Spm and Spd reduced considerably the inhibition of O₂ uptake rates, P700 photooxidation and the accumulation of superoxide anions (O₂ ⁻) induced by light stress. Spm seems to be more effective than Spd in preserving PSI photo-stability. The correlation of the extent of PSI protection, photosystem II (PSII) inhibition and O₂ ⁻ generation with increasing Spm doses revealed that PSI photo-protection is assumed by two mechanisms depending on the PAs concentration. Given their antioxidant character, PAs scavenge directly the O₂ ⁻ generated in thylakoid membranes at physiological concentration (1 mM). However, for non-physiological concentration, the ability of PAs to protect PSI is due to their inhibitory effect on PSII electron transfer.     

Stabilization of Oat Leaf Protoplasts through Polyamine-mediated Inhibition of Senescence

Protoplasts isolated from Avena sativa L. leaves undergo progressive senescence when incubated aseptically in 0.6 m mannitol with or without added nutrients. This senescence is manifested by morphological deterioration and ultimate lysis of protoplasts, by a decrease in incorporation of [(3)H]uridine and [(3)H]leucine into macromolecules, and by a sharp increase in ribonuclease activity.The presence in the incubation medium of l-arginine, l-lysine, certain polyamines related to these amino acids (cadaverine, putrescine, spermidine), Ca(2+), or streptomycin stabilizes the protoplasts. Protoplasts incubated with 10 mml-arginine or l-lysine show an initial inhibition of [(3)H]uridine incorporation, but with time, incorporation is restored to levels greater than in control protoplasts. The rise in ribonuclease activity of protoplasts is completely inhibited if the protoplasts are incubated with 10 mml-arginine. Greater incorporation of [(3)H]uridine into RNA of aging protoplasts is also maintained by appropriate concentration of cadaverine, putrescine, spermidine, Ca(2+), or streptomycin in the incubation medium; the same concentrations of these substances stabilize the protoplasts against additional lysis.     

盐酸胍对大麦类囊体膜能量分配和电子传递的影响

本文以大麦叶片为实验材料,研究了盐酸胍修饰对类囊体膜能量分配及电子传递的影响。结果表明：盐酸胍处理类囊体膜,室温下Ｆ６８５荧光强度,随着盐酸胍浓度的增加而逐渐下降。盐酸胍处理导致类囊体膜在低温（７７Ｋ）下Ｆ６８５／Ｆ７８６比值下降,并随着盐酸胍浓度的增加而加剧。盐酸胍处理抑制类囊体膜以Ｈ２Ｏ为电子供体的ＤＣＩＰ光还原速度和Ｃｈｌａ诱导荧光产率,这种抑制作用可分别为加入ＰＳＩＩ的人工电子供体ＤＰＣ和     

Composition and Function of Thylakoid Membranes from Grana-rich and Grana-deficient Chloroplast Mutants of Barley

Chlorophyll-deficient barley (Hordeum vulgare) mutants were studied that had chlorophyll a/b ratios either higher or lower than the wild type. Mutants with high ratios (>5.2) had a reduced proportion of their photosynthetic lamellae appressed into grana (“grana-deficient” mutants) compared with wild type (chlorophyll a/b = 3.2), while the majority of lamellae in the chloroplasts with low chlorophyll a/b ratios (2.0-2.4) were organized into grana (“grana-rich” mutants).     

Chloroplast Protein Synthesis During Barley Leaf Growth and Senescence: Effect of Leaf Excision

Chloroplast protein synthesis was measured during the expansion,maturity and senescence of the oldest leaf of barley, Hordeumvulgare L., var. Hassan. A maximum rate of protein synthesisoccurred near the end of the expansion stage 9 d after sowing.Protein synthesis increased again at the beginning of senescenceand reached a new maximum at day 14 after sowing. Detachmentand incubation of leaves in the dark stimulated chioroplastprotein synthesis by fully expanded or by senescent leaves butnot by expanding leaves. If the detached leaves were kept inthe light, chloroplast protein synthesis was stimulated in fullyexpanded but not in senescent leaves. Short treatments (18 h)of leaf segments with growth substances in either light or indarkness, significantly changed the rate of protein synthesisshown by chloroplasts. The relationship between chloroplastprotein synthesis and leaf senescence is discussed. Key words: Hormones, light, maturity     

Dynamic Mechanical Responses of Arabidopsis Thylakoid Membranes during PSII-Specific Illumination

Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes.     

Light Activation of Rubisco by Rubisco Activase and Thylakoid Membranes

A reconstituted system comprising ribulose bisphosphate carboxylase/oxygenase(rubisco), rubisco activase, washed thylakoid membranes, andATP was used to demonstrate a light-dependent stimulation ofrubisco activation. ATP, ribulose bisphosphate, H⁺, and Mg²⁺concentrations are normally light-dependent variables in thechloroplast but were maintained at pre-determined levels. Resultsindicated that rubisco activase and washed thylakoid membranesare sufficient to catalyze light stimulation of rubisco activationwith the reconstituted system, and that rubisco activase isrequired for this light stimulation. The washed thylakoid membranesdid not exhibit rubisco activase activity, nor was rubisco activaseprotein detected immunologically. Light-dependent activationof rubisco in the reconstituted system was similar in whole-chainand PS I electron transport reactions, and saturated at approximately100 µmol photons m^–2 s^–1. ¹ Present address: Department of Biological Sciences, LouisianaTech University, Ruston, LA 71272, U.S.A.     

A New Method for Measuring the Permeability of Plant Cell Membranes Using Epidermis-free Leaf Discs

A method has been developed for measuring the uptake and effluxof radiochemicals by higher plant cells, using epidermis-freeleaf discs. The advantages and disadvantages of this methodare discussed and some physical characteristics of the tissuedescribed. The relative permeability (urea=1) of tobacco leafcells to nonelectrolytes was thiourea 2.3, ethylene glycol 39,-amino isobutyric acid 1.9. N-methylpyridinium chloride accumulatedin the tissue according to the Nernst Distribution, by a processwhich was temperature dependent.     

Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst Visualized by Microscopic Spectral Imaging

Some filamentous cyanobacteria carry out oxygenic photosynthesis in vegetative cells and nitrogen fixation in specialized cells known as heterocysts. Thylakoid membranes in vegetative cells contain photosystem I (PSI) and PSII, while those in heterocysts contain predominantly PSI. Therefore, the thylakoid membranes change drastically when differentiating from a vegetative cell into a heterocyst. The dynamics of these changes have not been sufficiently characterized in situ. Here, we used time-lapse fluorescence microspectroscopy to analyze cells of Anabaena variabilis under nitrogen deprivation at approximately 295 K. PSII degraded simultaneously with allophycocyanin, which forms the core of the light-harvesting phycobilisome. The other phycobilisome subunits that absorbed shorter wavelengths persisted for a few tens of hours in the heterocysts. The whole-thylakoid average concentration of PSI was similar in heterocysts and nearby vegetative cells. PSI was best quantified by selective excitation at a physiological temperature (approximately 295 K) under 785-nm continuous-wave laser irradiation, and detection of higher energy shifted fluorescence around 730 nm. Polar distribution of thylakoid membranes in the heterocyst was confirmed by PSI-rich fluorescence imaging. The findings and methodology used in this work increased our understanding of how photosynthetic molecular machinery is transformed to adapt to different nutrient environments and provided details of the energetic requirements for diazotrophic growth.The most essential pigment-protein complexes for oxygenic photosynthesis are PSI and PSII, which are embedded in the thylakoid membranes of chloroplasts and cyanobacteria. Cooperation between PSI and PSII achieves light-driven noncyclic electron transport from the oxidative splitting of water to the reduction of ferredoxin and is accompanied by the generation of a proton gradient for ATP synthesis. Phycobilisomes (PBS), another pigment-protein complex, are attached to the stromal side of the thylakoid membrane in cyanobacteria and red algae; they work as light-harvesting antennae to transfer electronic excitation energy mainly to PSII and, in some cases, to PSI (Gantt 1994). The integration of these pigment-protein complexes changes in response to light conditions, nutrient status, and developmental stage (Fujita et al., 1994; Grossman et al., 1994; Wolk et al., 1994).Some cyanobacteria, including Anabaena variabilis, are able to grow diazotrophically using the nitrogen-fixing enzyme nitrogenase. Because nitrogenase is sensitive to oxygen, oxygenic photosynthesis is not readily compatible with diazotrophic growth. When this filamentous cyanobacterium is grown under fixed nitrogen-deficient conditions, approximately 1 in 10 to 20 vegetative cells differentiates into a heterocyst, in which oxygenic photosynthesis is suppressed and nitrogenase becomes operative (Haselkorn, 1978; Wolk et al., 1994). The other vegetative cells continue oxygenic photosynthesis. The differentiation of heterocysts from chains of vegetative cells has been studied extensively (Golden and Yoon, 2003; Toyoshima et al., 2010). The abundances of PSII and PBS decrease during the transition. PSI appears to persist in the heterocyst to produce ATP by cyclic electron transport, because nitrogen fixation demands a large amount of ATP (Wolk et al., 1994). However, the mechanisms by which PBS and PSII are degraded during heterocyst differentiation remain unclear, and whether the amount of PSI per cell changes is unknown.The PBS of A. variabilis contain three types of phycobiliproteins, pigment-protein complexes with distinct absorption and fluorescence spectra. The core PBS contains allophycocyanin (APC), which absorbs around 654 nm (Ying and Xie, 1998); the core is most closely connected to PSII. More peripherally in the PBS, the so-called rod contains phycoerythrocyanin (PEC) and phycocyanin (PC), which absorb maximally around 575 and 604 to 620 nm, respectively (Switalski and Sauer, 1984; Zhang et al., 1998). Photon energy is absorbed by PEC, then transferred downhill through PC and APC and finally to PSII. The structure of PBS is probably optimized not only for efficient energy transfer to PSII and/or PSI but also for transformation and/or degradation under various nutrient conditions. However, the order in which these subunits degrade during heterocyst differentiation remains unknown. One strategy to address this question is to isolate heterocysts at several stages during differentiation and quantify their proteomes via mass spectrometry. However, such isolation procedures work well only when there is a good understanding of the properties of cells at different stages. Ideally, noninvasive methods should be used to understand changes in the integrity of PSII and PBS in intact cells in filaments.In principle, time-lapse microscopic observations can clarify the process of differentiation from a vegetative cell into a mature heterocyst. Spectral microscopy is an ideal tool to analyze physiological state and/or amounts of pigment-protein complexes under various conditions. Acquiring microscopic fluorescence spectra of individual cells is a natural extension of laser scanning confocal fluorescence microscopy, which has been applied to several types of cyanobacterial cells, including heterocysts (Peterson et al., 1981; Ying et al., 2002; Wolf and Schüssler, 2005; Kumazaki et al., 2007; Vermaas et al., 2008; Sukenik et al., 2009; Bordowitz and Montgomery, 2010; Collins et al., 2012, Sugiura and Itoh, 2012). Microscopic fluorescence spectra reflect the concentration of pigment-protein complexes and the energy transfer dynamics between photosynthetic pigments. However, to date, there have been no thorough time-lapse investigations of the fluorescence spectra of heterocysts and vegetative cells during the differentiation process.In this study, we investigated the dynamic changes in thylakoid membranes of A. variabilis during heterocyst differentiation. Our unique microscopic system can acquire fluorescence spectra from an entire linearly illuminated region with about 2-nm wavelength resolution in a single exposure (Kumazaki et al., 2007). Heterocyst formation was induced by transferring vegetative cell filaments from fixed-nitrogen-sufficient incubation medium to nitrogen-deprived medium. We conducted long-term observations (60–96 h) on identical filaments. Another unique feature of our setup is that it uses a near-infrared (NIR) excitation laser source. Our previous microspectroscopic study of chloroplasts of a higher plant, maize (Zea mays), and a green alga (Parachlorella kessleri) showed that continuous wave (CW) laser light emitting at 785 to 820 nm excited PSI with high selectivity under the one-photon excitation (OPE) mode. This enabled us to observe highly PSI-rich fluorescence spectra and images with signals around 710 to 740 nm, even at approximately 295 K (Hasegawa et al., 2010, 2011). We used this technique to quantify PSI in individual heterocysts compared with its parental and contiguous vegetative cells. Pigment fluorescence under OPE qualitatively differed from that under two-photon excitation (TPE) using a pulsed NIR laser (typically achieved with picosecond or femtosecond pulses), because TPE using 800 to 830 nm resulted in spectra with contributions from PBS, PSII, and PSI, as typically observed by visible light excitation (Kumazaki et al., 2007; Hasegawa et al., 2010, 2011). The advantages of our microscopic system are the high wavelength resolution and coverage of the entire fluorescence spectrum, the availability of fluorescence spectra at several differentiation stages, and the multiple excitation modes with different selectivities for pigment-protein complexes. Together, these analyses allowed us to characterize spectral decomposition and to understand the time dependence of different pigment-protein complexes, even at a physiological temperature. Microscopic absorption spectra were also obtained from single cells. These data were tentatively used to estimate the absolute concentrations of PSI and PSII in heterocysts and vegetative cells.     

Delay of Membrane Lipid Degradation by Calcium Treatment during Cabbage Leaf Senescence

Cabbage leaf discs (Brassica oleracea L., Capitata group) were floated adaxial side up in 0, 0.05, or 0.25 m CaCl₂ solutions at 15°C for 14 d in the dark. To assess whether the delay of senescence by calcium treatment involved protection of membrane lipids, chlorophyll and protein content and the lipid composition of the membranes were determined during incubation. Chlorophyll and protein content decreased with time, in correlation with a reduction in the amount of phospholipids. The degree of unsaturation of phospholipids and free fatty acids decreased, whereas the ratio of sterol to phospholipid increased. The proportions of phospholipid classes did not change during senescence. The catabolism of phospholipids was delayed by 0.05 m calcium, but accelerated by 0.25 m, as compared to the untreated control. Based on the levels of the lipid intermediates, phospholipase D, phosphatidic acid phosphatase, lipolytic acyl hydrolase, and lipoxygenase appeared to be involved in the breakdown of phospholipids during senescence. Phospholipase D and phosphatidic acid phosphatase may be directly influenced by calcium. The calcium treatment apparently did not affect the activity of acyl hydrolase. Lipoxygenase, responsible for the peroxidation of the polyunsaturated fatty acids, was probably indirectly influenced by calcium. We conclude that the delay of senescence of cabbage leaf discs by calcium treatment involved protection of membrane lipids from degradation.     

Photosystem 2 Efficiency and Thylakoid Protein Pattern in DCMU-Treated Wheat Seedlings during Senescence

Changes in various components of photosynthetic apparatus during the 6-d dark incubation at 25 °C of detached control and DCMU-treated Triticum aestivum L. leaves were examined. The rate of photosystem 2 (PS2) activity was decreased with increase of the time of dark incubation in control leaves. In contrast to this, DCMU-treated leaves demonstrated high stability by slowing down the inactivation processes. Diphenyl carbazide and NH₂OH restored the PS2 activity more in control leaves than in DCMU-treated leaves. Mn²⁺ failed to restore the PS2 activity in both control and DCMU-treated samples. Similar results were obtained when F_v/F_m was evaluated by chlorophyll fluorescence measurements. The marked loss of PS2 activity in dark incubated control leaves was primarily due to the loss of D1, 33, and 23 kDa extrinsic polypeptides and 28-25 kDa LHCP2 polypeptides.     

Retardation of Leaf Senescence by Ascorbic Acid

Leaf discs of Solatium melongena were floated on various concentrationsof ascorbic acid (AA), gibberellic acid (GA₃), and kinetin inorder to study their effect on senescence. AA was highly effectivein retarding senescence as shown by the arrest of the fall inlevels of chlorophyll, DNA, RNA, and proteins. AA was effectiveat a lower concentration than that of GA₃ or kinetin.     

Expression of Phospholipase D during Castor Bean Leaf Senescence

Membrane deterioration in plant senescence is commonly associated with progressive decreases in membrane phospholipid content. This study investigated the expression and regulation of phospholipase D (PLD; EC 3.1.4.4) during senescence in castor bean (Ricinus communis L. cv Hale) leaf discs. The rate of leaf senescence was accelerated by 50 [mu]M abscisic acid and was attenuated by 50 [mu]M cytokinin during incubation at 23[deg]C for up to 5 d. Leaf senescence was indicated by decreases in the content of total proteins, chlorophyll, and phospholipids. PLD activity in both membrane-associated and cytosolic fractions showed a gradual increase in the absence of phytohormones. Abscisic acid stimulated an increase in membrane-associated PLD and had little effect on the soluble form. On the other hand, cytokinin retarded the increase in membrane-associated PLD. Immunoblotting analysis using PLD-specific antibodies revealed that the changes in PLD activity were correlated with those of PLD protein. Analysis of PLD by nondenaturing PAGE showed the appearance of a PLD structural variant, PLD 3, in abscisic acid-treated leaf discs. Northern blotting analysis using a PLD cDNA probe revealed an increase in PLD mRNA in senescing leaf discs. These data indicate complex mechanisms for the regulation of PLD during senescence, which include increases in membrane-associated PLD, differential expression of PLD isoforms, and changes in amounts of PLD protein and mRNA. Such controlled expression points to a role for PLD in membrane deterioration and plant senescence.