Chlororespiration is involved in the adaptation of Brassica plants to heat and high light intensity

Two species of Brassica were used to study their acclimation to heat and high illumination during the first stages of development. One, Brassica fruticulosa, is a wild species from south-east Spain and is adapted to both heat and high light intensity in its natural habitat, while the other, Brassica oleracea, is an agricultural species that is widely cultivated throughout the world. Growing Brassica plants under high irradiance and moderate heat was seen to affect the growth parameters and the functioning of the photosynthetic apparatus. The photosystem II (PSII) quantum yields and the capacity of photosynthetic electron transport, which were lower in B. fruticulosa than in B. oleracea, decreased in B. oleracea plants when grown under stress conditions, indicating inhibition of PSII. However, in B. fruticulosa, the values of these parameters were similar to the values of control plants. Photosystem I (PSI) activity was higher in B. fruticulosa than in B. oleracea, and in both species this activity increased in plants exposed to heat and high illumination. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed a higher amount of both proteins in B. fruticulosa than in B. oleracea. In addition, PTOX activity in plastoquinone oxidation, and NADH DH activity in thylakoid membranes were higher in the wild species (B. fruticulosa) than in the agricultural species (B. oleracea). The results indicate that tolerance to high illumination and heat of the photosynthetic activity was higher in the wild species than in the agricultural species, suggesting that plant adaptation to these stresses in natural conditions favours subsequent acclimation, and that the chlororespiration process is involved in adaptation to heat and high illumination in Brassica.     

Contribution to the structural characterization of eucaryotic PSI reaction centre—I. Critical analysis of the polypeptidic composition of different P700 enriched fractions

In thylakoid membranes, several peptides of high MW are present which may interfere with the study of CP1's components. Modifying Cleveland's technique [7] for limited proteolysis, we have characterized the polypeptides found in the 60 kD region. Some may result from incomplete washing of the CF1 while others come from the CP1; indeed, this chlorophyll protein complex, which has a higher MW (above 100 kD), very often undergoes a dissociation into smaller components of about 60 KD MW.Analysis of the protein content of different preparations commonly used to obtain PSI reaction centre enriched fractions has been performed. The and subunits of CF1 are among the main contaminants of most of these preparations. A further purification step is described which can be applied to all these preparations, but numerous peptides are still present in the active fractions. It is most unlikely that all these polypeptides are required for the primary photochemical event, and this emphasizes the necessity to find a new simple method to purify PSI reaction centres.     

菠菜能量转化复合体的人工组装

从菠菜叶绿体中分离了H~+-ATP酶复合体,其SDS-聚丙烯酰胺凝胶电泳呈九条亚单位带,为纯度较高的酶复合体。将H~+-ATP酶复合体重组于人工膜(脂质体)上,表现出PiATP交换活力。Mg~(++)-ATP酶活力也明显提高。并表现对DCCD,寡霉素的敏感性。 重组H~+-ATP酶复合体表现有ATP诱导的H~+转移。但脂质体也出现类似现象,为此对以pH改变为标准的检测方法提出商榷。     

Involvement of chlororespiration in chilling stress in the tropical species Spathiphyllum wallisii

Spathiphyllum wallisii plants were used to study the effect of chilling stress under high illumination on photosynthesis and chlororespiration. Leaves showed different responses that depended on root temperature. When stem, but not root, was chilled, photosystem II (PSII) was strongly photoinhibited. However, when the whole plant was chilled, the maximal quantum yield of PSII decreased only slightly below the normal values and cyclic electron transport was stimulated. Changes were also observed in the chlororespiration enzymes and PGR5. In whole plants chilled under high illumination, the amounts of NADH dehydrogenase (NDH) complex and plastid terminal oxidase (PTOX) remained similar to control and increased when only stem was chilled. In contrast, the amount of PGR5 polypeptide was higher in plants when both root and stem were chilled than in plants in which only stem was chilled. The results indicated that the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when the whole plant is chilled under high light, and that another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PSII activity is strongly photoinhibited in plants in which only stem is chilled, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.     

Stimulation of chlororespiration by heat and high light intensity in oat plants

High irradiance and moderate heat inhibit the activity of the photosynthetic apparatus of oat (Avena sativa L.) leaves. The incubation of oat leaves under high light intensity in conjunction with high temperatures strongly decreased the maximal quantum yield of photosystem (PS) II, indicating the close synergistic effect of both stress factors on PS II inhibition and the subsequent irreversible damage to the photosynthetic apparatus. The PS I A/B protein levels remained similar to control values in leaves incubated under high light intensity or moderate heat, and decreased only when both stress factors were simultaneously applied. Immunoblot analysis of thylakoid membranes using specific antibodies raised against the NDH-K subunit of the thylakoidal NADH dehydrogenase complex (NADH DH) and against plastid terminal oxidase (PTOX) revealed an increase in the amount of both proteins in response to high light intensity and/or heat treatments. In addition, these stress treatments were seen to stimulate the activity of electron donation by NADPH and ferredoxin to plastoquinone, the PTOX activity in plastoquinone oxidation and the NADH DH activity in thylakoid membranes. Incubation with n-propyl gallate (an inhibitor of PTOX) inhibited the increase of NDH-K and PTOX levels under high light intensity and heat, and slightly stimulated the activity of electron donation by NADPH and ferredoxin to plastoquinone. Antimycin A (an inhibitor of cyclic electron flow) increased the NADH DH activity and preserved the levels of NDH-K and PTOX in thylakoid membranes from leaves incubated under high light intensity and heat. The up-regulation of the PTOX and the thylakoidal NADH DH complex under these stress conditions supports a role for chlororespiration in the protection against high irradiance and moderate heat.     

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.     

Extreme variation in rates of evolution in the plastid Clp protease complex

Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi‐subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid‐encoded gene ( clpP1 ) and multiple nuclear genes. The clpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid‐encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history of clpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find that clpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns and RNA ‐editing sites) within seed plants. Although clpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genus Silene , which has highly elevated and heterogeneous rates of clpP1 evolution. We confirmed that clpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear‐encoded Clp subunits, even in one of the most divergent Silene species. Additionally, there is a tight correlation between amino acid substitution rates in clpP1 and the nuclear‐encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex.     

Proton gradient-driven import of the 16 kDa oxygen-evolving complex protein as the full precursor protein by isolated thylakoids

The biogenesis of the lumenal 16 kDa protein of the photosynthetic oxygen-evolving complex was analysed using an assay for the import of proteins by isolated thylakoids. The precursor protein is imported with high efficiency in the light in both the presence and absence of stromal extract. Import is almost completely blocked in the dark or if the uncoupler nigericin is present in the light. The data indicate that transport across the thylakoid membrane is driven by a proton motive force in which the proton gradient is the dominant component, and that the full precursor protein can be transported across the thylakoid membrane without prior cleavage by the stromal processing peptidase.     

Specific substitutions of light‐harvesting complex I proteins associated with photosystem I are required for supercomplex formation with chloroplast NADH dehydrogenase‐like complex

In Arabidopsis, the chloroplast NADH‐dehydrogenase‐like (NDH) complex is sandwiched between two copies of photosystem I (PSI) supercomplex, consisting of a PSI core and four light‐harvesting complex I (LHCI) proteins (PSI‐LHCI) to form the NDH–PSI supercomplex. Two minor LHCI proteins, Lhca5 and Lhca6, contribute to the interaction of each PSI–LHCI copy with the NDH complex. Here, large‐pore blue‐native gel electrophoresis revealed that, in addition to this complex, there were at least two types of higher‐order association of more LHCI copies with the NDH complex. In single‐particle images, this higher‐order association of PSI–LHCI preferentially occurs at the left side of the NDH complex when viewed from the stromal side, placing subcomplex A at the top (Yadav et al., Biochim. Biophys. Acta ‐ Bioenerg., 1858, 2017, 12). The association was impaired in the lhca6 mutant but not in the lhca5 mutant, suggesting that the left copy of PSI–LHCI was linked to the NDH complex via Lhca6. From an analysis of subunit compositions of the NDH–PSI supercomplex in lhca5 and lhca6 mutants, we propose that Lhca6 substitutes for Lhca2 in the left copy of PSI–LHCI, whereas Lhca5 substitutes for Lhca4 in the right copy. In the lhca2 mutant, Lhca3 was specifically stabilized in the NDH–PSI supercomplex through heterodimer formation with Lhca6. In the left copy of PSI–LHCI, subcomplex B, Lhca6 and NdhD likely formed the core of the supercomplex interaction. In contrast, a larger protein complex, including at least subcomplexes B and L and NdhB, was needed to form the contact site with Lhca5 in the right copy of PSI–LHCI.     

LIGHT-HARVESTING COMPLEX AF'OPROTEINS IN CYTOPLASMIC VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Cells of Chlamydomonas reinhardtii Dangeard strain cw15arg7A contain electron-opaque material, often in the form of large granules, within cytoplasmic vacuoles. Immunoelectron microscopy with antibodies to polypeptide 11, a component of the major light-harvesting chlorophyll (Chl) a/b-protein complex (LHCII,) of thylakoid membranes, revealed the presence of LHCII Polypeptides within the chloroplast and in vacuolar material in cells grown in the light. Vacuolar material was also heavily immunodecorated in dark-grown cells that did not synthesize Chl. Accumulation of LHCII polypeptides was further studied in greening and light-grown cells of a pale green mutant, deficient in LHCII, that was derived from cu15arg7A by insertional mutagenesis. Light-grown cells of this mutant strain contained relatively few thylakoid membranes and synthesized LHCII polypeptides at a low rate. However, cytoplasmic vacuoles were immunoreactive. Appearance of mature-sized LHCII polypeptides in vacuoles suggested that these proteins were partially translocated across the envelope but not retained by the chloroplast without assembly of LHCII.     

利用整合分类学方法进行蕨类植物复合体的物种划分: 以线裂铁角蕨复合体为例

广泛的杂交和多倍化使得铁角蕨属(Asplenium)下存在着许多分类困难的物种复合体, 针对这些类群进行整合分类学的研究, 有助于我们更加全面和深入地理解物种的界限以及形成机制。线裂铁角蕨复合体(Asplenium coenobiale complex)是铁角蕨属下一个形态多样性较高的类群, 由于缺乏全面取样和系统研究, 该复合体的物种划分长期存在争议。本研究选取线裂铁角蕨复合体中形态变异和地理分布具有代表性的个体, 通过孢粉学研究确定该类群的生殖特性, 运用流式细胞分析获取倍性信息, 同时结合叶绿体和核基因组片段系统发生分析的证据, 对该类群的系统演化关系和起源方式进行了探讨。结果表明: (1)虽然部分孢子囊败育的情况在线裂铁角蕨复合体中十分普遍, 但正常孢子囊内形成的64个孢子说明该类群植物仍能进行正常的有性生殖; (2)该复合体中存在着倍性变异, 其中多角铁角蕨(A. cornutissimum)是二倍体, 而其他成员均为四倍体; (3)依据母系遗传的叶绿体序列所构建的系统发生关系将该类群划为4个分支, 与基于核基因序列构建的系统树存在冲突, 这暗示杂交可能在该复合体的形成过程中起到了重要的推动作用。综上所述, 我们建议将线裂铁角蕨复合体划分为4个物种, 即同源四倍体新种马关铁角蕨(A. maguanense sp. nov.), 二倍体多角铁角蕨, 以及两个由同一对亲本正反交产生的异源四倍体线裂铁角蕨(A. coenobiale)和叶基宽铁角蕨(A. pulcherrimum)。     

Orientation of plgments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. IV. The 100 K linear dichroism spectra of thylakoids from wild-type and chlorophyll b-less barley thylakoids

Using a polyacrylamide gel squeezing technique, linear dichroism spectra of thylakoids from wild-type and chlorophyll-b less barley have been obtained at 100 K. The calculated difference linear dichroism spectra, based on normalization at 690–695 nm, are identical to those of the light-harvesting complex (LHC) isolated by Triton solubilization. This observation is in agreement with previous conclusions (Tapie, P., Haworth, P., Hervo, G. and Breton, J. (1982) Biochim. Biophys. Acta 682, 339–344) regarding: (i) scattering artifacts are absent in linear dichroism spectra determined using polyacrylamide gels, (ii) the in vivo orientation of LHC pigments is maintained in the isolated complex and (iii) the largest dimension(s) of the isolated LHC is (are), in vivo, parallel to the plane of the photosynthetic membrane.     

Regulatory factors for the assembly of thylakoid membrane protein complexes

Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.     

植物基因工程新途径：叶绿体转化

在植物基因工程研究中,叶绿体是继核转化之后又一新的遗传转化和表达受体,叶绿体转化体系具有可同时进行多基因转化,表达原核性,超量表达,后代遗传稳定,定点整合,不会产生基因沉默及母性遗传和安全性好等特点,本文着重介绍叶绿体转化体系的特点,国内外研究动态,存在的问题及未来发展方向。     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Isolation of biochemically active chloroplasts from chlamydomonas

Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits.The isolated chloroplasts are active in photosynthetic O₂-evolution and CO₂-fixation, with the highest rates obtained in the presence of ATP.The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts.     

Retrospective of a botanist

An autobiographic report of studies on the elucidation of the structure of the chloroplast is presented here. It deals with the discovery of the lamellar structure of chloroplasts by polarization-microscopy, the representation of their layer-like structure with the ultraviolet microscope and the results obtained by the electron-microscope leading to the discovery of the structural elements of the lamellar system. These lamellar structures were in the form of vesicles, and were named thylakoids. Isolation of the chloroplasts made it possible to determine their chemical composition. Amphiphilic lipids, together with water, create bimolecular layers and, therefore, are responsible for the structure of the thylakoid-membranes. The role of membrane proteins was emphasized. The isolation of the individual polypeptides was possible after dissociation in sodium-dodecyl-sulfate. Antisera to these polypeptides were used to localize them in the membrane. These antisera are able to inhibit special steps in the electron transport. Our results of the spectroscopic examination of photosynthetic membrane components are also discussed.This article was written at the invitation of Dr Govindjee.     

衣藻叶绿体遗传转化的研究

叶绿体遗传转人是近几年发展起来的新领域。本文主要介绍了叶绿体遗传转化的特点、基本原理和衣藻叶绿体遗传转化的方法与技术;     

Regulation of proton leakage from broken chloroplasts by CF0.

Measurements of proton translocation in CF₁-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF₀ is still dependent on light intensity with a first-order rate constant equal to mR₀, where R₀ is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF₀ is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF₁.CF₀ complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities.     

Functional analysis of the two Arabidopsis homologues of Toc34, a component of the chloroplast protein import apparatus

Two Arabidopsis Toc34 homologues, atToc34 and atToc33, components of the chloroplast protein import machinery located in the outer envelope membrane, were recently isolated. Both proteins insert into the outer envelope, are supposed to bind GTP and to interact with Toc75 as demonstrated by in vitro import assays. We studied the expression of the two genes by RNA gel blot analysis, promoter-GUS plants and in situ hybridisations as well as immunoblot analysis. The atToc34 and atToc33 genes are expressed in green as well as non-green tissues and are developmentally regulated. Despite these similarities, however, the two Arabidopsis Toc34 homologues are differentially expressed in various plant organs. To gain more insight into the in vivo function of both proteins, antisense plants were created. While antisense plants of atToc33 are characterized by a pale yellowish phenotype, antisense plants of atToc34 show a weaker phenotype. Protein interaction studies using an in vitro translated precursor protein and heterologously expressed atToc34 and atToc33 proteins showed a direct GTP-dependent interaction, but demonstrated different affinities of the two atToc proteins towards the precursor protein. Thus, our results indicate a more specialized function for both atToc34 and atToc33, suggesting specificity for certain imported precursor proteins.