Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis

The interconversion of chlorophyll a and chlorophyll b, referred to as the chlorophyll cycle, plays a crucial role in the processes of greening, acclimation to light intensity, and senescence. The chlorophyll cycle consists of three reactions: the conversions of chlorophyll a to chlorophyll b by chlorophyllide a oxygenase, chlorophyll b to 7-hydroxymethyl chlorophyll a by chlorophyll b reductase, and 7-hydroxymethyl chlorophyll a to chlorophyll a by 7-hydroxymethyl chlorophyll a reductase. We identified 7-hydroxymethyl chlorophyll a reductase, which is the last remaining unidentified enzyme of the chlorophyll cycle, from Arabidopsis thaliana by genetic and biochemical methods. Recombinant 7-hydroxymethyl chlorophyll a reductase converted 7-hydroxymethyl chlorophyll a to chlorophyll a using ferredoxin. Both sequence and biochemical analyses showed that 7-hydroxymethyl chlorophyll a reductase contains flavin adenine dinucleotide and an iron-sulfur center. In addition, a phylogenetic analysis elucidated the evolution of 7-hydroxymethyl chlorophyll a reductase from divinyl chlorophyllide vinyl reductase. A mutant lacking 7-hydroxymethyl chlorophyll a reductase was found to accumulate 7-hydroxymethyl chlorophyll a and pheophorbide a. Furthermore, this accumulation of pheophorbide a in the mutant was rescued by the inactivation of the chlorophyll b reductase gene. The downregulation of pheophorbide a oxygenase activity is discussed in relation to 7-hydroxymethyl chlorophyll a accumulation.     

Chlorophyll cycle regulates the construction and destruction of the light-harvesting complexes

Chlorophyll a and chlorophyll b are the major constituents of the photosynthetic apparatus in land plants and green algae. Chlorophyll a is essential in photochemistry, while chlorophyll b is apparently dispensable for their photosynthesis. Instead, chlorophyll b is necessary for stabilizing the major light-harvesting chlorophyll-binding proteins. Chlorophyll b is synthesized from chlorophyll a and is catabolized after it is reconverted to chlorophyll a. This interconversion system between chlorophyll a and chlorophyll b refers to the chlorophyll cycle. The chlorophyll b levels are determined by the activity of the three enzymes participating in the chlorophyll cycle, namely, chlorophyllide a oxygenase, chlorophyll b reductase, and 7-hydroxymethyl-chlorophyll reductase. This article reviews the recent progress on the analysis of the chlorophyll cycle and its enzymes. In particular, we emphasize the impact of genetic modification of chlorophyll cycle enzymes on the construction and destruction of the photosynthetic machinery. These studies reveal that plants regulate the construction and destruction of a specific subset of light-harvesting complexes through the chlorophyll cycle. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.     

Energy transfer between Chlorophyll C and Chlorophyll A

Spectral properties of solutions containing mixtures of chlorophyll a and chlorophyll c are investigated. The yield of excitation energy migration from chlorophyll c to chlorophyll a is obtained ranging from 23 to 48% dependent on the used dye concentrations. The back transfer from chlorophyll a to chlorophyll c is negligible. The shape of the polarization excitation spectrum of chlorophyll c in the Soret band region is less composed than that of chlorophyll a. Depolarization of chlorophyll a fluorescence by chlorophyll c is in agreement with the conclusion drawn from fluorescence quenching that excitation energy migrates from chlorophyll c to chlorophyll a.     

海岛棉与陆地棉叶绿素含量变化的差异研究

以5份陆地棉和4份海岛棉为材料,研究了整个发育进程中陆地棉和海岛棉叶绿素的变化规律之间的差异,结果显示：陆地棉与海岛棉的叶绿素a、叶绿素b和叶绿素a＋b各有不同动态变化规律,其叶绿素a/b在整个发育进程中的变化也有差异。海岛棉与陆地棉的叶绿素a变化趋势有较大差别,陆地棉的叶绿素a含量在6月18日（苗期）最高,峰值出现在8月6日,海岛棉峰值、最大值均在7月4日（现蕾期）;叶绿素b含量表现为陆地棉、海岛棉峰值均在8月6日（花铃期）,但海岛棉平均值高于陆地棉;陆地棉的叶绿素a＋b和叶绿素a曲线相似,海岛棉的叶绿素a＋b和叶绿素b曲线相似;陆地棉叶绿素a/b值表现苗期最大,然后迅速下降,海岛棉a/b值表现普遍低于陆地棉,其变化趋势为前期在7月4日出现峰值,然后下降再升高。实验说明陆地棉和海岛棉叶绿素的合成机制、调控机理可能不同。     

Biosynthesis and accumulation of microgram quantities of chlorophyll by developing chloroplasts in vitro

Developing chloroplasts were incubated under conditions previously shown to induce protochlorophyll and chlorophyll biosynthesis, as well as chloroplast maintenance and partial differentiation in vitro. In the presence of air, δ-aminolevulinic acid, coenzyme A, glutathione, potassium phosphate, methyl alcohol, magnesium, nicotinamide adenine dinucleotide, and adenosine triphosphate, microgram quantities of chlorophyll accumulated after 1 hour of incubation. Part of the chlorophyll was not extractable in organic solvents; it is referred to as bound chlorophyll. The amount of bound chlorophyll depended on the degree of cotyledon greening at the time of plastid isolation. Etioplasts with or without a lag phase of chlorophyll biosynthesis synthesized nonphototransformable protochlorophyll and smaller amounts of extractable chlorophyll. As the greening of excised cotyledons progressed, more of the chlorophyll became bound before and after in vitro incubation. It is suggested that this increase in the fraction of bound chlorophyll reflects the biosynthesis of membrane-bound chlorophyll receptor sites. In the absence of cofactors, chlorophyll biosynthesis was blocked and porphyrins accumulated, indicating damage of the chlorophyll biosynthetic chain. It is concluded that chlorophyll accumulation constitutes a potentially convenient tool for the study of thylakoid membrane biogenesis in vitro.     

Conversion of Chlorophyll b to Chlorophyll a and the Assembly of Chlorophyll with Apoproteins by Isolated Chloroplasts

The photosynthetic apparatus is reorganized during acclimation to various light environments. During adaptation of plants grown under a low-light to high-light environment, the light-harvesting chlorophyll a/b-protein complexes decompose concomitantly with an increase in the core complex of photosystem II. To study the mechanisms for reorganization of photosystems, the assembly of chlorophyll with apoproteins was investigated using isolated chloroplasts. When [14C]chlorophyllide b was incubated with chloroplasts in the presence of phytyl pyrophosphate, it was esterified and some of the [14C]chlorophyll b was converted to [14C]chlorophyll a via 7-hydroxymethyl chlorophyll. [14C]Chlorophyll a and b were incorporated into chlorophyll-protein complexes. Light-harvesting chlorophyll a/b-protein complexes of PSII had a lower [14C]chlorophyll a to [14C]chlorophyll b ratio than P700-chlorophyll a-protein complexes, indicating the specific binding of chlorophyll to apoproteins in our systems. 7-Hydroxymethyl chlorophyll, an intermediate molecule from chlorophyll b to chlorophyll a, did not become assembled with any apoproteins. These results indicate that chlorophyll b is released from light-harvesting chlorophyll a/b-protein complexes of photosystem II and converted to chlorophyll a via 7-hydroxymethyl chlorophyll in the lipid bilayer and is then used for the formation of core complexes of photosystems. These mechanisms provide the fast, fine regulation of the photosynthetic apparatus during construction of photosystems.     

不同海拔短肋羽藓叶绿素含量与光合效率研究

以云南高黎贡山百花岭不同海拔梯度(1500～3100 m)的短肋羽藓(Thuidium kanedae)为材料,通过测定其叶绿素含量和实际光合量子产量,研究其光合特性随海拔梯度变化的规律。结果表明,短肋羽藓的实际光合量子产量Y(Ⅱ)与叶绿素总量、叶绿素a、叶绿素b含量及叶绿素a/b比值之间都呈负相关关系。随着海拔升高,其叶绿素总含量、叶绿素a及叶绿素b含量都呈先升高而后下降的“单峰”分布模式,但随着海拔进一步升高,叶绿素总含量及叶绿素a含量变化不明显,而叶绿素b含量呈现明显上升趋势。叶绿素a/b比值随着海拔升高先呈现“单峰”分布模式,然后随着海拔继续升高呈现缓慢下降的趋势。实际光合量子产量Y(Ⅱ)整体表现出随海拔升高而增加的趋势,并在高海拔处出现急剧上升的趋势。本研究为揭示苔藓植物在不同海拔梯度下适应生态环境的生理机制提供参考。     

Early steps in the assembly of light-harvesting chlorophyll a/b complex: time-resolved fluorescence measurements

The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.     

Identification of Genes Associated with Chlorophyll Accumulation in Flower Petals

Plants have an ability to prevent chlorophyll accumulation, which would mask the bright flower color, in their petals. In contrast, leaves contain substantial amounts of chlorophyll, as it is essential for photosynthesis. The mechanisms of organ-specific chlorophyll accumulation are unknown. To identify factors that determine the chlorophyll content in petals, we compared the expression of genes related to chlorophyll metabolism in different stages of non-green (red and white) petals (very low chlorophyll content), pale-green petals (low chlorophyll content), and leaves (high chlorophyll content) of carnation (Dianthus caryophyllus L.). The expression of many genes encoding chlorophyll biosynthesis enzymes, in particular Mg-chelatase, was lower in non-green petals than in leaves. Non-green petals also showed higher expression of genes involved in chlorophyll degradation, including STAY-GREEN gene and pheophytinase. These data suggest that the absence of chlorophylls in carnation petals may be caused by the low rate of chlorophyll biosynthesis and high rate of degradation. Similar results were obtained by the analysis of Arabidopsis microarray data. In carnation, most genes related to chlorophyll biosynthesis were expressed at similar levels in pale-green petals and leaves, whereas the expression of chlorophyll catabolic genes was higher in pale-green petals than in leaves. Therefore, we hypothesize that the difference in chlorophyll content between non-green and pale-green petals is due to different levels of chlorophyll biosynthesis. Our study provides a basis for future molecular and genetic studies on organ-specific chlorophyll accumulation.     

An LED-based fluorometer for chlorophyll quantification in the laboratory and in the field

The chlorophyll content is an important experimental parameter in agronomy and plant biology research. In this report, we explore the feasibility of determining total concentration of extracts containing chlorophyll a and chlorophyll b by chlorophyll fluorescence. We found that an excitation at 457?nm results in the same integrated fluorescence emission for a molecule of chlorophyll a and a molecule of chlorophyll b. The fluorescence yield induced by 457?nm is therefore proportional to total molar chlorophyll concentration. Based on this observation, we designed an instrument to determine total chlorophyll concentrations. A single light emitting diode (LED) is used to excite chlorophyll extracts. After passing through a long-pass filter, the fluorescence emission is assessed by a photodiode. We demonstrate that this instrument facilitates the determination of total chlorophyll concentrations. We further extended the functionality of the instrument by including LEDs emitting at 435 and 470?nm wavelengths, thereby preferentially exciting chlorophyll a and chlorophyll b. This instrument can be used to determine chlorophyll a and chlorophyll b concentrations in a variety of organisms containing different ratios of chlorophylls. Monte-Carlo simulations are in agreement with experimental data such that a precise determination of chlorophyll concentrations in carotenoid-containing biological samples containing a concentration of less than 5?nmol/mL total chlorophyll can be achieved.     

Theoretical study on the thermodynamic properties of chlorophyll d-peptides coordinating ligand

The chlorophyll d containing cyanobacterium, Acaryochloris marina has provided a model system for the study of chlorophyll replacement in the function of oxygenic photosynthesis. Chlorophyll d replaces most functions of chlorophyll a in Acaryochloris marina. It not only functions as the major light-harvesting pigment, but also acts as an electron transfer cofactor in the primary charge separation reaction in the two photosystems. The Mg-chlorophyll d-peptide coordinating interaction between the amino acid residues and chlorophylls using the latest semi-empirical PM5 method were examined. It is suggested that chlorophyll d possesses similar coordination ligand properties to chlorophyll a, but chlorophyll b possesses different ligand properties. Compared with other studies involving theoretical correlation and our prior experiments, this study suggests that the chlorophyll a-bound proteins will bind chlorophyll d without difficulty when chlorophyll d is available.     

锰胁迫对垂序商陆光合特性及叶绿素荧光参数的影响

垂序商陆(Phytolacca americana L.)是我国首次发现的锰超富集植物。通过温室营养液培养实验,研究垂序商陆锰耐性与光合特性及叶绿素荧光参数的关系。结果表明,随着生长介质中锰浓度的升高,垂序商陆叶片的叶绿素a、叶绿素b和总叶绿素含量下降,净光合速率(Pn)和气孔导度(Gs)呈下降趋势,而胞间CO2浓度(Ci)却逐渐升高;叶绿素a/b值和蒸腾速率(Tr)在1 000μmol.L-1锰供应水平时达到最大值;叶片PSⅡ的最大量子产量(Fv/Fm)、PSⅡ有效光化学量子产量(EQY)和光合电子传递速率(ETR)呈下降趋势,而光化学淬灭系数(qP)和光合有效辐射(PAR)高于239μmol photons.m-.2s-1下的非光化学淬灭系数(NPQ)在5 000μmol.L-1锰供应水平时达到最大值。因此,垂序商陆在锰胁迫下可能具有一定的光合保护机制。     

东海赤潮高发区沉积物中叶绿素的分析

研究分析了2002年8～9月“赤潮973”航次所采集的沉积物中叶绿素a及其降解产物脱镁叶绿酸含量与分布．结果表明。叶绿素a和脱镁叶绿酸是同源的;随深度增加叶绿素a和脱镁叶绿酸的含量呈递减趋势,到一定深度后含量不再变化。个别站位叶绿素a和脱镁叶绿酸的垂直分布出现了许多小突跃,可能是由生物扰动引起;表层沉积物(0～0．5cm)中的叶绿素a和脱镁叶绿酸的变化幅度分别为0．14～1．17和0．83～5．58μg·g-1。平均值分别为0．54和2．45μg·g-1;并初步探讨水深(光强)、盐度、温度、含水量对叶绿素水平分布的影响;脱镁叶绿酸作为叶绿素的主要降解产物,到一定深度后,成为叶绿素的主要存在形式,约占80％～90％;由于水温的不同,夏季沉积物中的叶绿素a和脱镁叶绿酸含量是春季的3倍之多;比较了上层水柱中的叶绿素和沉积物中的叶绿素的相对含量,平均而言,沉积物中叶绿素占上层水柱中叶绿素的31％．     

Chlorophyll b expressed in Cyanobacteria functions as a light-harvesting antenna in photosystem I through flexibility of the proteins

Photosynthetic pigments bind to their specific proteins to form pigment-protein complexes. To investigate the pigment-binding activities of the proteins, chlorophyll b was for introduced the first time to a cyanobacterium that did not synthesize that pigment, and expression of its function in the native pigment-protein complex of cyanobacterium was confirmed by energy transfer. Arabidopsis CAO (chlorophyll a oxygenase) cDNA was introduced into the genome of Synechocystis sp. PCC6803. The transformant cells accumulated chlorophyll b, with the chlorophyll b content being in the range of 1.4 to 10.6% of the total chlorophyll depending on the growth phase. Polyacrylamide gel electrophoresis analysis of the chlorophyll-protein complexes of transformant cells showed that chlorophyll b was incorporated preferentially into the P700-chlorophyll a-protein complex (CP1). Furthermore, chlorophyll b in CP1 transferred light energy to chlorophyll a, indicating a functional transformation. We also found that CP1 of Chlamydomonas reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b with a chlorophyll b content of approximately 4.4%. On the basis of these results, the evolution of pigment systems in an early stage of cyanobacterial development is discussed in this paper.     

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     

Biosynthesis of chlorophyll b in pigment mutant C-2A' of Scenedesmus obliquus

It is demonstrated that chlorophyll b does not only derive from chlorophyll a , but is also formed separately from an in vivo-occurring chlorophyllide b . The branching point for the latter synthesis is at the level of chlorophyllide, since no protochlorophyllide b was detectable. We have indications that the enzyme oxidizing chlorophyll a to chlorophyll b accepts also non-phytylated 17,18 dihydroporphyrins and is not restricted to chlorophylls. Preparations of chlorophyllide a and chlorophyll a could both be transferred with the same enzyme fraction to chlorophyllide b and chlorophyll b , respectively. Preliminary experiments show this enzyme to be membrane bound and light independent. An updated scheme for chlorophyll b biosynthesis is presented.     

Cerium Relieves the Inhibition of Chlorophyll Biosynthesis of Maize Caused by Magnesium Deficiency

Lanthanoids (Ln) were demonstrated to improve chlorophyll formation and the growth of plants. But the mechanism of the fact that Ln promotes chlorophyll biosynthesis of plants is poorly understood. The main aim of the study was to determine Ln effects in chlorophyll formation of maize under magnesium (Mg) deficiency. Maize plants were cultivated in Hoagland’s solution. They were subjected to Mg deficiency and to cerium administered in Mg-deficient Hoagland’s media, and then the contents of various chlorophyll precursors and gen expressions of the key enzymes of chlorophyll biosynthesis were examined. The decrease of chlorophyll contents in maize leaves caused by Mg deficiency suggested an inhibition of chlorophyll synthesis that was inhibited by a reduction of the precursors as measured by analyzing the contents of δ-aminolevulinic acid, porphobilinogen, uroporphyrinogen III, Mg–protoporphyrin IX, and protochlorophyll, as well as the expression levels of magnesium chelatase, magnesium-protoporphyrin IX methyltransferase, and chlorophyll synthase; Mg deficiency significantly inhibited the transformation from coproporphyrinogen III or protoporphyrin IX to chlorophyll. However, cerium addition significantly relieved the inhibition of chlorophyll biosynthesis in maize caused by Mg deficiency and increased chlorophyll content and promoted a series of transformations from δ-aminolevulinic acid to chlorophyll and maize growth under Mg deficiency. It implied that cerium might partly substitute for the role of Mg.     

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     

Cd~(2+)胁迫对小麦幼苗生理生化特性的影响

以小麦品种'西旱2号'和'宁春4号'幼苗为材料,采用室内水培实验研究了不同浓度Cd(NO3)2处理对叶绿素含量、抗氧化酶活性及渗透性调节物含量等的影响.结果表明,两小麦品种幼苗的叶绿素a(Chl a)、叶绿素b(Chl b)及叶绿素总量在镉胁迫下均比对照降低,且高浓度镉对'西旱2号'幼苗叶片Chl b的破坏程度强于Chl a;两品种镉胁迫幼苗的过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性均比对照不同程度地升高;两品种镉胁迫幼苗的可溶性糖含量均与对照无显著差异,但'宁春4号'幼苗叶片的脯氨酸含量却比对照显著升高,且此效应具有浓度依赖性;镉胁迫下两品种小麦幼苗叶片的MDA含量变化与对照无显著差异.研究显示,两品种小麦幼苗叶片的光合色素在镉胁迫下均受到不同程度的破坏,但它们均能通过增加体内保护酶活性和渗透调节物质含量来缓解膜脂过氧化伤害,从而对镉胁迫均表现出较强的耐受能力.