Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

PsbS enhances nonphotochemical fluorescence quenching in the absence of zeaxanthin

Leaves and chloroplasts from Arabidopsis plants with increased amounts of PsbS protein showed the same percentage increase in nonphotochemical quenching in comparison to the wild type both in the presence and absence of zeaxanthin. The absorption change at 525-535 nm was also more pronounced in both cases. It is suggested that PsbS alone can cause the quenching, supporting the model in which zeaxanthin acts as an allosteric activator and is not the primary cause of the process. It is proposed that PsbS acts as a trigger of the conformational change that leads to the establishment of nonphotochemical quenching.     

Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids

The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. In this work possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. The interacting proteins, as identified by mass spectrometry analysis of the immunoaffinity eluate, include CP29, some LHCII components, but also components of Photosystem I, of the cytochrome b₆f complex as well as of ATP synthase. These proteins can be co-immunoprecipitated by using highly specific anti-PsbS antibodies and, vice-versa, PsbS is co-immunoprecipitated by antisera against components of the interacting complexes. We also find that PsbS co-migrates with bands containing PSII, ATP synthase and cytochrome b₆f as well as with LHCII-containing bands on non-denaturing Deriphat PAGE. These results suggest multiple location of PsbS in the thylakoid membrane and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae, even though the exact physiological condition(s) under which these interactions may take place remain to be clarified.     

Novel Evidence for a Reversible Dissociation of Light-harvesting Complex Ⅱ from Photosystem Ⅱ Reaction Center Complex Induced by Saturating Light Illumination in Soybean Leaves

After saturating light illumination for 3 h the potential photochemical efficiency of photosystem Ⅱ (PSⅡ) (Fv/Fm, the ratio of variable to maximal fluorescence) decreased markedly and recovered basically to the level before saturating light illumination after dark recovery for 3 h in both soybean and wheat leaves, indicating that the decline in Fv/Fm is a reversible down-regulation. Also, the saturating light illumination led to significant decreases in the low temperature (77 K) chlorophyll fluorescence parameters F685 (chlorophyll a fluorescence peaked at 685 nm ) and F685/F735 (F735, chlorophyll a fluorescence peaked at 735 nm) in soybean leaves but not in wheat leaves. Moreover,trypsin (a protease) treatment resulted in a remarkable decrease in the amounts of PsbS protein (a nuclear gene psbS-encoded 22 kDa protein) in the thylakoids from saturating light-illuminated (SI), but not in those from darkadapted (DT) and dark-recovered (DRT) soybean leaves. However, the treatment did not cause such a decrease in amounts of the PsbS protein in the thylakoids from saturating light-illuminated wheat leaves. These results support the conclusion that saturating light illumination induces a reversible dissociation of some light-harvesting complex Ⅱ (LHCⅡ) from PSⅡ reaction center complex in soybean leaf but not in wheat leaf.     

毛竹PsbS基因的克隆及其原核表达特征研究

采用RT-PCR技术从毛竹(Phyllostachys edulis)叶片中克隆到1个PsbS基因,命名为PePsbS (GenBank No. FJ600727),其编码区为810 bp,编码269个氨基酸。序列分析表明,PePsbS编码的蛋白与其它单子叶植物的PsbS蛋白有很高的相似性。蛋白结构分析表明,PePsbS基因编码蛋白包含导肽部分和成熟蛋白,其中成熟蛋白包含4个跨膜结构域。将PePsbS基因编码成熟蛋白的序列构建到原核表达载体pET23a中,并转入大肠杆菌,用IPTG进行诱导表达。结果表明, 41℃下诱导4 h的表达效果最好,目的蛋白含量约占总蛋白的21.5%,分子量约为22.0 kD。这说明温度和诱导时间明显影响PePsbS基因的表达。     

Multiple feedbacks between chloroplast and whole plant in the context of plant adaptation and acclimation to the environment

This review focuses on feedback pathways that serve to match plant energy acquisition with plant energy utilization, and thereby aid in the optimization of chloroplast and whole-plant function in a given environment. First, the role of source–sink signalling in adjusting photosynthetic capacity (light harvesting, photochemistry and carbon fixation) to meet whole-plant carbohydrate demand is briefly reviewed. Contrasting overall outcomes, i.e. increased plant growth versus plant growth arrest, are described and related to respective contrasting environments that either do or do not present opportunities for plant growth. Next, new insights into chloroplast-generated oxidative signals, and their modulation by specific components of the chloroplast''s photoprotective network, are reviewed with respect to their ability to block foliar phloem-loading complexes, and, thereby, affect both plant growth and plant biotic defences. Lastly, carbon export capacity is described as a newly identified tuning point that has been subjected to the evolution of differential responses in plant varieties (ecotypes) and species from different geographical origins with contrasting environmental challenges.     

The relationship between maximum tolerated light intensity and photoprotective energy dissipation in the photosynthetic antenna: chloroplast gains and losses

The principle of quantifying the efficiency of protection of photosystem II (PSII) reaction centres against photoinhibition by non-photochemical energy dissipation (NPQ) has been recently introduced by Ruban & Murchie (2012 Biochim. Biophys. Acta 1817, 977–982 (doi:10.1016/j.bbabio.2012.03.026)). This is based upon the assessment of two key parameters: (i) the relationship between the PSII yield and NPQ, and (ii) the fraction of intact PSII reaction centres in the dark after illumination. In this paper, we have quantified the relationship between the amplitude of NPQ and the light intensity at which all PSII reaction centres remain intact for plants with different levels of PsbS protein, known to play a key role in the process. It was found that the same, nearly linear, relationship exists between the levels of the protective NPQ component (pNPQ) and the tolerated light intensity in all types of studied plants. This approach allowed for the quantification of the maximum tolerated light intensity, the light intensity at which all plant leaves become photoinhibited, the fraction of (most likely) unnecessary or ‘wasteful’ NPQ, and the fraction of photoinhibited PSII reaction centres under conditions of prolonged illumination by full sunlight. It was concluded that the governing factors in the photoprotection of PSII are the level and rate of protective pNPQ formation, which are often in discord with the amplitude of the conventional measure of photoprotection, the quickly reversible NPQ component, qE. Hence, we recommend pNPQ as a more informative and less ambiguous parameter than qE, as it reflects the effectiveness and limitations of the major photoprotective process of the photosynthetic membrane.     

Two different strategies for light utilization in photosynthesis in relation to growth and cold acclimation

Seedlings of Lodgepole pine (Pinus contorta L.) and winter wheat (Triticum aestivum L. cv. Monopol) were cold acclimated under controlled conditions to induce frost hardiness. Lodgepole pine responded to cold acclimation by partial inhibition of photosynthesis with an associated partial loss of photosystem II reaction centres, and a reduction in needle chlorophyll content. This was accompanied by a low daily carbon gain, and the development of a high and sustained capacity for non‐photochemical quenching of absorbed light. This sustained dissipation of absorbed light as heat correlated with an increased de‐epoxidation of the xanthophyll cycle pigments forming the quenching forms antheraxanthin and zeaxanthin. In addition, the PsbS protein known to bind chlorophyll and the xanthophyll cycle pigments increased strongly during cold acclimation of pine. In contrast, winter wheat maintained high photosynthetic rates, showed no loss of chlorophyll content per leaf area, and exhibited a high daily carbon gain and a minimal non‐photochemical quenching after cold acclimation. In accordance, cold acclimation of wheat neither increased the de‐epoxidation of the xanthophylls nor the content of the PsbS protein. These different responses of photosynthesis to cold acclimation are correlated with pine, reducing its need for assimilates when entering dormancy associated with termination of primary growth, whereas winter wheat maintains a high need for assimilates as it continues to grow and develop throughout the cold‐acclimation period. It appears that without evolving a sustained ability for controlled dissipation of absorbed light as heat throughout the winter, winter green conifers would not have managed to adapt and establish themselves so successfully in the cold climatic zones of the northern hemisphere.     

Up-regulation of a photosystem II core protein phosphatase inhibitor and sustained D1 phosphorylation in zeaxanthin-retaining, photoinhibited needles of overwintering Douglas fir

Overwintering needles of the evergreen conifer Douglas fir exhibited an association between arrest of the xanthophyll cycle in the dissipating state (as zeaxanthin + antheraxanthin; Z + A) with a strongly elevated predawn phosphorylation state of the D1 protein of the photosystem II (PSII) core. Furthermore, the high predawn phosphorylation state of PSII core proteins was associated with strongly increased levels of TLP40, the cyclophilin-like inhibitor of PSII core protein phosphatase, in winter versus summer. In turn, decreases in predawn PSII efficiency, F_v/F_m, in winter were positively correlated with pronounced decreases in the non-phosphorylated form of D1. In contrast to PSII core proteins, the light-harvesting complex of photosystem II (LHCII) did not exhibit any nocturnally sustained phosphorylation. The total level of the D1 protein was found to be the same in summer and winter in Douglas fir when proteins were extracted in a single step from whole needles. In contrast, total D1 protein levels were lower in thylakoid preparations of overwintering needles versus needles collected in summer, indicating that D1 was lost during thylakoid preparation from overwintering Douglas fir needles. In contrast to total D1, the ratio of phosphorylated to non-phosphorylated D1 as well as the levels of the PsbS protein were similar in thylakoid versus whole needle preparations. The level of the PsbS protein, that is required for pH-dependent thermal dissipation, exhibited an increase in winter, whereas LHCII levels remained unchanged.