Overexpression of chlorophyllide a oxygenase (CAO) enlarges the antenna size of photosystem II in Arabidopsis thaliana

The light-harvesting efficiency of a photosystem is thought to be largely dependent on its photosynthetic antenna size. It has been suggested that antenna size is controlled by the biosynthesis of chlorophyll b. To verify this hypothesis, we overexpressed the enzyme for chlorophyll b biosynthesis, chlorophyllide a oxygenase (CAO), in Arabidopsis thaliana by transforming the plant with cDNA for CAO under the control of the 35S cauliflower mosaic virus promoter. In the early de-etiolation phase, when the intrinsic CAO expression is very low, the chlorophyll a: b ratio was drastically decreased from 28 to 7.3, indicating that enhancement of chlorophyll b biosynthesis had been successfully achieved. We made the following observations in full-green rosette leaves of transgenic plants. (1) The chlorophyll a : b ratio was reduced from 2.85 to 2.65. (2) The ratio of the peripheral light-harvesting complexes (LHCII) to the core antenna complex (CPa) resolved with the green-gel system increased by 20%. (3) The ratio of the light-harvesting complex II apoproteins (LHCP) to 47-kDa chlorophyll a protein (CP47), which was estimated by the results of immunoblotting, increased by 40%. These results indicated that the antenna size increased by at least 10-20% in transgenic plants, suggesting that chlorophyll b biosynthesis controls antenna size. To the best of our knowledge, this is the first report on enlargement of the antenna size by genetic manipulations.     

The chlorophyllases AtCLH1 and AtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana

One important reaction of chlorophyll (chl) breakdown during plant senescence is the removal of the lipophilic phytol moiety by chlorophyllase. AtCLH1 and AtCLH2 were considered to be required for this reaction in Arabidopsis thaliana. Here we present evidence against this assumption. Using green fluorescent protein fusions, neither AtCLH isoform localizes to chloroplasts, the predicted site of chlorophyll breakdown. Furthermore, clh1 and clh2 single and double knockout lines are still able to degrade chlorophyll during senescence. From our data we conclude that AtCLHs are not required for senescence-related chlorophyll breakdown in vivo and propose that genuine chlorophyllase has not yet been molecularly identified.     

Consecutive binding of chlorophylls a and b during the assembly in vitro of light-harvesting chlorophyll-a/b protein (LHCIIb)

The apoprotein of the major light-harvesting chlorophyll a/b complex (LHCIIb) is post-translationally imported into the chloroplast, where membrane insertion, protein folding, and pigment binding take place. The sequence and molecular mechanism of the latter steps is largely unknown. The complex spontaneously self-organises in vitro to form structurally authentic LHCIIb upon reconstituting the unfolded recombinant protein with the pigments chlorophyll a, b, and carotenoids in detergent micelles. Former measurements of LHCIIb assembly had revealed two apparent kinetic phases, a faster one (tau1) in the range of 10 s to 1 min, and a slower one (tau2) in the range of several min. To unravel the sequence of events we analysed the binding of chlorophylls into the complex by using time-resolved fluorescence measurements of resonance energy transfer from chlorophylls to an acceptor dye attached to the apoprotein. Chlorophyll a, offered in the absence of chlorophyll b, bound with the faster kinetics (tau1) exclusively whereas chlorophyll b, in the absence of chlorophyll a, bound predominantly with the slower kinetics (tau2). In double-jump experiments, LHCIIb assembly could be dissected into a faster chlorophyll a and a subsequent, predominantly slower chlorophyll b-binding step. The assignment of the faster and the slower kinetic phase to predominantly chlorophyll a and exclusively chlorophyll b binding, respectively, was verified by analysing the assembly kinetics with a circular dichroism signal in the visible domain presumably reflecting the establishment of pigment-pigment interactions. We propose that slow chlorophyll binding is confined to the exclusively chlorophyll b binding sites whereas faster binding occurs to the chlorophyll a binding sites. The latter sites can bind both chlorophylls a and b but in a reversible fashion as long as the complex is not stabilised by proper occupation of the chlorophyll b sites. The resulting two-step model of LHCIIb assembly is able to reconcile the highly specific binding sites containing either chlorophyll a or b, as seen in the recent crystal structures of LHCIIb, with the observation of promiscuous binding sites able to bind both chlorophyll a and b in numerous reconstitution analyses of LHCIIb assembly.     

Chlorophyll-protein-complexes of thylakoids of wild type and chlorophyll b mutants of Arabidopsis thaliana

Pigment-protein-complexes of two chlorophyll b deficient mutants of Arabidopsis and from the wild type were separated electrophoretically. Light-harvesting proteins were absent in the chlorophyll b free mutant ch¹ and their amount was reduced in the mutant ch² which has a reduced content of chlorophyll b. The ratio of CPa:CP I increased with decreasing chlorophyll b content which indicated that the stoichiometry of photosystem II to photosystem I is not constant.Abbreviations Chl chlorophyll - CPa chlorophyll a-protein - CP I P-700 chlorophyll a-protein - LHCP light-harvesting chlorophyll a/b-protein - PAGE polyacrylamide gel electrophoresis - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Chlorophyll-protein-complexes of thylakoids of wild type and chlorophyll b mutants of Arabidopsis thaliana

Pigment-protein-complexes of two chlorophyll b deficient mutants of Arabidopsis and from the wild type were separated electrophoretically. Light-harvesting proteins were absent in the chlorophyll b free mutant ch¹ and their amount was reduced in the mutant ch² which has a reduced content of chlorophyll b. The ratio of CPa:CP I increased with decreasing chlorophyll b content which indicated that the stoichiometry of photosystem II to photosystem I is not constant.Abbreviations Chl chlorophyll - CPa chlorophyll a-protein - CP I P-700 chlorophyll a-protein - LHCP light-harvesting chlorophyll a/b-protein - PAGE polyacrylamide gel electrophoresis - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Chlorophyll fluorescence and photoinhibition in a tropical rainforest understory plant

The data presented here deal with the effects of high-light exposure on the 77 K fluorescence characteristics of Elatostema repens. It is shown that the decrease of the variable fluorescence during the treatment is biphasic. The reactions responsible for the first phase of fluorescence quenching are saturated under 700 mol photon m^-2 s^-1 and insensitive to streptomycin, whereas those responsible for the second phase are not yet saturated under 700 mol photon m^-2 s^-1 and sensitive to streptomycin. It is concluded that only the second phase of fluorescence quenching is associated with photoinhibitory processes. Rate and amplitude of recovery from photoinhibition are maximum under very low light (3.5 mol photon m^-2 s^-1), and very small at a moderate light (160 mol photon m^-2 s^-1) which does not cause photoinhibition. It is concluded that recovery processes are inhibited during photoinhibition. It is suggested that they could be associated with damage occuring on the oxidizing side of PSII.Abbreviations F_o, F_v, F_m initial, variable and maximum fluorescence, respectively - PFD photon flux density - PS II photosystem II     

Nicotinamide is a specific inhibitor of dark-operative protochlorophyllide oxidoreductase,a nitrogenase-like enzyme,from Rhodobacter capsulatus

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of two components, L-protein as a reductase component and NB-protein as a catalytic component. Elucidation of the crystal structures of NB-protein (Muraki et al., Nature 2010, 465: 110–114) has enabled us to study its reaction mechanism in combination with biochemical analysis. Here we demonstrate that nicotinamide (NA) inhibits DPOR activity by blocking the electron transfer from L-protein to NB-protein. A reaction scheme of DPOR, in which the binding of protochlorophyllide (Pchlide) to the NB-protein precedes the electron transfer from the L-protein, is proposed based on the NA effects.     

Photoinhibition Characteristics of a Low Chlorophyll b Mutant of High Yield Rice

The low chlorophyll b mutant of high yield rice had a lower light-harvesting complex 2 content than the wild type. The stability of oxygen evolution side of photosystem 2 was only slightly lower. A lower photon absorption rate and a stronger xanthophyll cycle capacity of this mutant led to a higher endurance to strong irradiance and a lower photoinhibition as compared with the wild type rice.     

Biogenesis of thylakoid membranes with emphasis on the process in Chlamydomonas

Recent results obtained by electron microscopic and biochemical analyses of greening Chlamydomonas reinhardtii y1 suggest that localized expansion of the plastid envelope is involved in thylakoid biogenesis. Kinetic analyses of the assembly of light-harvesting complexes and development of photosynthetic function when degreened cells of the alga are exposed to light suggest that proteins integrate into membrane at the level of the envelope. Current information, therefore, supports the earlier conclussion that the chloroplast envelope is a major biogenic structure, from which thylakoid membranes emerge. Chloroplast development in Chlamydomonas provides unique opportunities to examine in detail the biogenesis of thylakoids.Abbreviations Rubisco ribulose bisphosphate carboxylase/oxygenase - CAB Chl a/b-binding (proteins) - Chlide chlorophyllide - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - Pchlide protochlorophyllide     

The Wound-Inducible Lls1 Gene from Maize is an Orthologue of the Arabidopsis Acd1 Gene,and the LLS1 Protein is Present in Non-Photosynthetic Tissues

Previous studies indicated that the lethal leaf spot 1 lesion mimic locus of maize ( ZmLls1 ) encodes a novel cell protective function in plants. Here we show that the accelerated cell death 1 ( acd1 ) locus of Arabidopsis thaliana corresponds to gene At3g44880 on chromosome 3. Proof that the Acd1 gene is an orthologue of ZmLls1 is provided by in vivo complementation of the acd1 mutant by the ZmLls1 gene. The Atlls1 lesion mimic phenotype was delayed in a chlorophyll a oxygenase (CAO) mutant chlorina1 background which is deficient in chlorophyll b synthesis. The interpretation that the cell protective function of LLS1 is linked with the removal of a phototoxic chlorophyll intermediate is supported by the recent report that the maize Lls1 gene encodes pheophorbide a oxygenase (PaO). Western blot analysis demonstrates that the LLS1 protein is present constitutively in all photosynthetic plant tissues. A transient increase in Lls1 gene expression by about 50-fold upon physical wounding of maize leaves indicates that the function of Lls1 is regulated in response to stress. We show that the LLS1 protein is also present at low levels in non-photosynthetic tissues including etiolated leaves suggesting that the ability to degrade chlorophyll exists in a standby mode in plant cells.     

极端干旱区疏叶骆驼刺叶绿素荧光对人工水分干扰的响应特征

选取塔里木河下游天然植被恢复示范区的疏叶骆驼刺(Alhagi sparsifolia Shap.)为研究对象,测定每年引水灌溉2次(每次0.42m3/m2)、1次和不灌溉(CK)处理下疏叶骆驼刺的实际光化学量子产量(ΦPSⅡ)、电子传输速率(ETR)和光化学猝灭系数(qP)等叶绿素荧光参数及其叶水势变化,探讨疏叶骆驼刺对人工水分干扰的叶绿素荧光响应特征。结果表明:(1)随着灌溉量的减少,疏叶骆驼刺叶水势呈显著降低的趋势,并在CK下达到最低。(2)同期疏叶骆驼刺qP、ΦPSⅡ、ETR、最大光量子产量(Fv/Fm)、叶绿素含量和光饱和点均随着灌溉量的减少呈先增加后降低的趋势,非光化学淬灭系数(NPQ)和调节性能量耗散(YNPQ)则呈先降低后增加的趋势。(3)与每年1次灌溉量处理相比,不灌溉和每年2次的灌溉量处理下疏叶骆驼刺发生了光抑制,光能捕获效率与光化学反应能量下降,热耗散能力提高。研究认为,灌溉量过高(每年2次,0.84m3/m2)或不灌溉均会限制疏叶骆驼刺光化学效率和光和活性,适时适量的(春季灌水1次,0.42m3/m2)水分补给更有利于疏叶骆驼刺适应干旱胁迫并维持正常光合生长。     

Heat Stress and Spermidine: Effect on Chlorophyll Fluorescence in Tomato Plants

Two tomato (Lycopersicon esculentum L.) cultivars: Robin (tolerant) and Roma (sensitive to heat stress) were studied. Chlorophyll fluorescence induction parameters (F_v/F_p, A_max, and Rfd) at 25 °C showed that the PS2 activity was similar for both cultivars. The parameters, measured at 38 °C, decreased in both cultivars, but more in cv. Roma. Exogenous application of 4 mM spermidine improved the plant heat-resistance in both cultivars, and especially in cv. Roma. Analysis of chlorophyll fluorescence changes during linear increase in temperature showed that cv. Robin plants have higher ability to hardening and higher resistance to thermal damage of the pigment-protein complexes structure and the activity of PS2 than cv. Roma.     

Light-induced fluorescence quenching in the light-harvesting chlorophyll a/b protein complex

Summary Irradiation of the principal photosystem II light-harvesting chlorophyll-protein antenna complex, LHC II, with high light intensities brings about a pronounced quenching of the chlorophyll fluorescence. Illumination of isolated thylakoids with high light intensities generates the formation of quenching centres within LHC II in vivo, as demonstrated by fluorescence excitation spectroscopy. In the isolated complex it is demonstrated that the light-induced fluorescence quenching: a) shows a partial, biphasic reversibility in the dark; b) is approximately proportional to the light intensity; c) is almost independent of temperature in the range 0–30°C; d) is substantially insensitive to protein modifying reagents and treatments; e) occurs in the absence of oxygen. A possible physiological importance of the phenomenon is discussed in terms of a mechanism capable of dissipating excess excitation energy within the photosystem II antenna.Abbreviations chla chlorophyll a - chlb chlorophyll b - F₀ fluorescence yield with reaction centers open - F_m fluorescence yield with reaction centres closed - F_i fluorescence at the plateau level of the fast induction phase - LHC II light-harvesting chlorophyll a/b protein complex II - PS II photosystem II - PSI photosystem I - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Synergistic Effect of AlCl3 and Kinetin on Chlorophyll and Protein Contents and Photochemical Activities in Detached Wheat Primary Leaves during Dark Incubation

Al³⁺ in combination with kinetin showed more protection against degradation of chlorophyll (Chl) and protein than Al³⁺ or kinetin alone during dark-induced senescence in wheat primary leaf segments. MV-dependent whole chain electron transport, photosystem (PS) 2 mediated oxygen evolution, and PS1 activities were also delayed to a greater extent. Absorbed excitation energy distribution was more in favour of PS1 in Al³⁺ plus kinetin-treated leaf thylakoids at 72 h.     

Chlorophyll a/b-binding protein genes are differentially expressed during soybean development

The levels of chlorophyll a/b-binding protein (Cab) gene polysomal poly(A)⁺ mRNA were quantitated throughout the development of Glycine max L. Cab mRNAs were abundant in young expanding leaves, representing 6.1% of the leaf mRNA population. Lower Cab mRNA levels were present in embryos, stems, and cotyledons of developing seedlings; the lowest levels were found in roots where they accounted for 0.04% of the polysomal poly(A)⁺ mRNA of this organ. To determine the contribution of different members of the Cab gene family to the Cab mRNA populations, a quantitative S1 nuclease reconstruction assay was developed. Cab3, Cab4, and Cab5 mRNAs were detected in all stages examined during soybean development but their levels underwent differential changes. Cab3 encodes the most abundant Cab mRNA in young leaves, developing embryos, and in Stage VII cotyledons from the developing soybean seedling. The levels of Cab mRNAs were compared to the levels of ribulose-1,5-bisphosphate carboxylase small subunit gene mRNA and differences in their patterns of accumulation were noted. Collectively these data indicate that during soybean embryogenesis developmental control mechanisms supersede light-regulatory signals.     

Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism

The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compounds in Arabidopsis thaliana. By combining homology and sequence motif searches with knowledge regarding the phylogenetic distribution of pathways of isoprenoid metabolism across species, candidate genes for these pathways in A. thaliana were obtained. A detailed analysis of the vicinity of chromosome loci for genes of isoprenoid metabolism in A. thaliana provided evidence for the clustering of genes involved in common pathways. Multiple sequence alignments were used to estimate the number of genes in gene families and sequence relationship trees were utilized to classify their individual members. The integration of all these datasets allows the generation of a knowledge-based metabolic map of isoprenoid metabolic pathways in A. thaliana and provides a substantial improvement of the currently available gene annotation.     

Analysis of an Arabidopsis thaliana protein family, structurally related to cytochromes b561 and potentially involved in catecholamine biochemistry in plants

Cytochromes b561 (cyts b561) constitute a family of eukaryotic membrane proteins, catalysing ascorbate (Asc)-mediated trans-membrane electron transport, and hence likely involved in Asc regeneration. A class of proteins (DoH-CB) has been identified in plants and animals, containing the cyt b561 electron-transport domain (CB), combined with the catecholamine-binding regulatory domain of dopamine-beta-hydroxylase (DoH). A mammalian DoH-CB protein was previously reported to function as a cell-derived growth factor receptor (SDR2). We have performed an in silico analysis on DoH-CB proteins from Arabidopsis thaliana and demonstrate that structural features of both CB and DoH domains are well conserved. The combination of both domains may have evolved from a functional interaction between a cyt b561 and a DoH-containing protein, illustrating the so-called "Rosetta Stone" evolutionary principle, and this hypothesis is supported by sequence comparisons. DoH-CB proteins form a newly identified group of proteins, likely to play a key role in catecholamine action in plants. It is suggested that these proteins may function as trans-membrane electron shuttles, possibly regulated by catecholamines. The role and action of catecholamines in plants is poorly documented, but it is clear that they are involved in many aspects of growth and development. Whether the DoH-CB proteins functionally interact with Asc, as is the case for cyts b561, remains to be determined.