Mapping Intercellular CO2 Mole Fraction (Ci) in Rosa rubiginosa Leaves Fed with Abscisic Acid by Using Chlorophyll Fluorescence Imaging1 : Significance of Ci Estimated from Leaf Gas Exchange

Imaging of photochemical yield of photosystem II (PSII) computed from leaf chlorophyll fluorescence images and gas-exchange measurements were performed on Rosa rubiginosa leaflets during abscisic acid (ABA) addition. In air ABA induced a decrease of both the net CO2 assimilation (An) and the stomatal water vapor conductance (gs). After ABA treatment, imaging in transient nonphotorespiratory conditions (0.1% O2) revealed a heterogeneous decrease of PSII photochemical yield. This decline was fully reversed by a transient high CO2 concentration (7400 μmol mol−1) in the leaf atmosphere. It was concluded that ABA primarily affected An by decreasing the CO2 supply at ribulose-1,5-bisphosphate carboxylase/oxygenase. Therefore, the An versus intercellular mole fraction (Ci) relationship was assumed not to be affected by ABA, and images of Ci and gs were constructed from images of PSII photochemical yield under nonphotorespiratory conditions. The distribution of gs remained unimodal following ABA treatment. A comparison of calculations of Ci from images and gas exchange in ABA-treated leaves showed that the overestimation of Ci estimated from gas exchange was only partly due to heterogeneity. This overestimation was also attributed to the cuticular transpiration, which largely affects the calculation of the leaf conductance to CO2, when leaf conductance to water is low.

Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure.

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n fluorescence. When quenching in the light-harvesting complex of photosystem II was induced by a lowering of pH to 5.5, carotenoids with n fluorescence quenching relative to the control. Of the 10 carotenoids tested, quenching induced by the addition of the tertiary amine compound, dibucaine, to isolated light-harvesting complex of photosystem II could only be reversed by violaxanthin. These results are discussed in terms of the two theories developed to explain the role of zeaxanthin and violaxanthin in nonphotochemical quenching of chlorophyll fluorescence.

Altered xanthophyll compositions adversely affect chlorophyll accumulation and nonphotochemical quenching in Arabidopsis mutants

Collectively, the xanthophyll class of carotenoids perform a variety of critical roles in light harvesting antenna assembly and function. The xanthophyll composition of higher plant photosystems (lutein, violaxanthin, and neoxanthin) is remarkably conserved, suggesting important functional roles for each. We have taken a molecular genetic approach in Arabidopsis toward defining the respective roles of individual xanthophylls in vivo by using a series of mutant lines that selectively eliminate and substitute a range of xanthophylls. The mutations, lut1 and lut2 (lut = lutein deficient), disrupt lutein biosynthesis. In lut2, lutein is replaced mainly by a stoichiometric increase in violaxanthin and antheraxanthin. A third mutant, aba1, accumulates normal levels of lutein and substitutes zeaxanthin for violaxanthin and neoxanthin. The lut2aba1 double mutant completely lacks lutein, violaxanthin, and neoxanthin and instead accumulates zeaxanthin. All mutants were viable in soil and had chlorophyll a/b ratios ranging from 2.9 to 3.5 and near wild-type rates of photosynthesis. However, mutants accumulating zeaxanthin exhibited a delayed greening virescent phenotype, which was most severe and often lethal when zeaxanthin was the only xanthophyll present. Chlorophyll fluorescence quenching kinetics indicated that both zeaxanthin and lutein contribute to nonphotochemical quenching; specifically, lutein contributes, directly or indirectly, to the rapid rise of nonphotochemical quenching. The results suggest that the normal complement of xanthophylls, while not essential, is required for optimal assembly and function of the light harvesting antenna in higher plants.

Protection and storage of chlorophyll in overwintering evergreens

How evergreen species store and protect chlorophyll during exposure to high light in winter remains unexplained. This study reveals that the evergreen snow gum (Eucalyptus pauciflora Sieb. ex Spreng.) stores and protects its chlorophylls by forming special complexes that are unique to the winter-acclimated state. Our in vivo spectral and kinetic characterizations reveal a prominent component of the chlorophyll fluorescence spectrum around 715 nm at 77 K. This band coincides structurally with a loss of chlorophyll and an increase in energy-dissipating carotenoids. Functionally, the band coincides with an increased capacity to dissipate excess light energy, absorbed by the chlorophylls, as heat without intrathylakoid acidification. The increased heat dissipation helps protect the chlorophylls from photo-oxidative bleaching and thereby facilitates rapid recovery of photosynthesis in spring.

The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach1

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.

Differential Control of Xanthophylls and Light-Induced Stress Proteins, as Opposed to Light-Harvesting Chlorophyll a/b Proteins, during Photosynthetic Acclimation of Barley Leaves to Light Irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 μmol m−2 s−1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.

The roles of specific xanthophylls in photoprotection

Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the α- or β-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (α-carotene branch), zeaxanthin, and antheraxanthin (β-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), α-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.

Modification of Carbon Partitioning, Photosynthetic Capacity, and O2 Sensitivity in Arabidopsis Plants with Low ADP-Glucose Pyrophosphorylase Activity1

Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO2 assimilation, true rates of photosynthetic O2 evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO2 partial pressure. The extent of stimulation of CO2 assimilation by increasing CO2 or by reducing O2 partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO2, the rates of CO2 assimilation and O2 evolution and the percentage inhibition of photosynthesis by low O2 were higher for the wild type than for the mutants. The relative rates of 14CO2 incorporation into starch under high light and high CO2 followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO2 assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.

Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function in Chlamydomonas reinhardtii1

The D2 polypeptide of the photosystem II (PSII) complex in the green alga Chlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco1

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.

Acridones: a chemically new group of protonophores.

Although the interaction of proton-conducting ionophores (protonophores) with photosynthetic electron transport has been extensively studied during the past decade, the mode of action of protonophores remained uncertain. For a better understanding of the molecular mechanism of the action of protonophores, the introduction of chemically new types of molecules will be required. In this work, we demonstrate that acridones (9-azaanthracene-10-ones) completely fulfill this requirement. At low concentrations of acridones, the thermoluminescence bands at +20 degrees C and +10 degrees C were strongly inhibited, while normal electron transport activity was retained. This indicates that the concentrations of S2 and S3 states involved in the generation of these bands are reduced. At higher concentrations, an increased activity of electron transport was observed, which is attributed to the typical uncoupler effect of protonophores. Indeed, acridones accelerate the decay of the electrochromic absorbance change at 515 nm and also inhibit the generation of the transmembrane proton gradient, measured as an absorbance transient of neutral red. Variable fluorescence induction was quenched even at low concentrations of acridones but was restored by either a long-term illumination or high light intensity. Acridones, similarly to other protonophores, promoted the autooxidation of the high-potential form of cytochrome b559 and partially converted it to lower potential forms. These results suggest that acridones, acting as typical protonophores, uncouple electron transport, accelerate the deactivation of the S2 and S3 states on the donor side, and facilitate the oxidation of cytochrome b559 on the acceptor side of photosystem II.

Relationship between photosynthetic electron transport and pH gradient across the thylakoid membrane in intact leaves.

Under conditions (0.2% CO2; 1% O2) that allow high rates of photosynthesis, chlorophyll fluorescence was measured simultaneously with carbon assimilation at various light intensities in spinach (Spinacia oleracea) leaves. Using a stoichiometry of 3 ATP/CO2 and the known relationship between ATP synthesis rate and driving force (Delta pH), we calculated the light-dependent pH gradient (Delta pH) across the thylakoid membrane in intact leaves. These Delta pH values were correlated with the photochemical (qP) and nonphotochemical (qN) quenching of chlorophyll fluorescence and with the quantum yield of photosystem II (phiPSII). At Delta pH > 2.1 all three parameters (qP, qN, and phiPSII) changed very steeply with increasing DeltapH (decreasing pH in the thylakoid). The observed pH dependences followed hexacooperative titration curves with slightly different pKa values. The significance of the steep pH dependences with slightly different pKa values is discussed in relation to the regulation of photosynthetic electron transport in intact leaves.

Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate

Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase from Chenopodium album, screened the C. album cDNA library, and cloned a cDNA (CaCLH, C. album chlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop). CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) in Arabidopsis thaliana (AtCLH1). We expressed the gene products of AtCLH1 and of CaCLH in Escherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on an Arabidopsis genomic fragment and expressed it in E. coli, demonstrating the presence of the second Arabidopsis CLH gene (AtCLH2). No typical feature of signal sequence was identified in AtCLH1, whereas AtCLH2 had a typical signal sequence for chloroplast. AtCLH1 mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h. AtCLH2, however, did not respond to MeJA.

Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes

Coordination between the activities of organelles and the nucleus requires the exchange of signals. Using Chlamydomonas, we provide evidence that plastid-derived chlorophyll precursors may replace light in the induction of two nuclear heat-shock genes (HSP70A and HSP70B) and thus qualify as plastidic signal. Mutants defective in the synthesis of Mg-protoporphyrin IX were no longer inducible by light. Feeding of Mg-protoporphyrin IX or its dimethyl ester to wild-type or mutant cells in the dark resulted in induction. The analysis of HSP70A promoter mutants that do or do not respond to light revealed that these chlorophyll precursors specifically activate the light signaling pathway. Activation of gene expression was not observed when protoporphyrin IX, protochlorophyllide, or chlorophyllide were added. A specific interaction of defined chlorophyll precursors with factor(s) that regulate nuclear gene expression is suggested.

Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/b Protein of Photosystem II in Response to Elevated Light Intensities1

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.