High light induced disassembly of photosystem II supercomplexes in Arabidopsis requires STN7-dependent phosphorylation of CP29

Photosynthetic oxidation of water and production of oxygen by photosystem II (PSII) in thylakoid membranes of plant chloroplasts is highly affected by changes in light intensities. To minimize damage imposed by excessive sunlight and sustain the photosynthetic activity PSII, organized in supercomplexes with its light harvesting antenna, undergoes conformational changes, disassembly and repair via not clearly understood mechanisms. We characterized the phosphoproteome of the thylakoid membranes from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutant plants exposed to high light. The high light treatment of the wild type and stn8 caused specific increase in phosphorylation of Lhcb4.1 and Lhcb4.2 isoforms of the PSII linker protein CP29 at five different threonine residues. Phosphorylation of CP29 at four of these residues was not found in stn7 and stn7stn8 plants lacking the STN7 protein kinase. Blue native gel electrophoresis followed by immunological and mass spectrometric analyses of the membrane protein complexes revealed that the high light treatment of the wild type caused redistribution of CP29 from PSII supercomplexes to PSII dimers and monomers. A similar high-light-induced disassembly of the PSII supercomplexes occurred in stn8, but not in stn7 and stn7stn8. Transfer of the high-light-treated wild type plants to normal light relocated CP29 back to PSII supercomplexes. We postulate that disassembly of PSII supercomplexes in plants exposed to high light involves STN7-kinase-dependent phosphorylation of the linker protein CP29. Disruption of this adaptive mechanism can explain dramatically retarded growth of the stn7 and stn7stn8 mutants under fluctuating normal/high light conditions, as previously reported.     

Low pH-induced regulation of excitation energy between the two photosystems

Earlier studies have proposed that low pH causes state transitions in spinach thylakoid membranes. Several Arabidopsis mutants (stn7 incapable in phosphorylation of LHC II, stn8 incapable in phosphorylation of PSII core proteins, stn7 stn8 double mutant and npq4 lacking PsbS and hence qE) were used to investigate the mechanisms involved in low pH induced changes in the thylakoid membrane. We propose that protonation of PsbS at low pH is involved in enhancing energy spillover to PS I.     

Phosphoproteomics of Arabidopsis chloroplasts reveals involvement of the STN7 kinase in phosphorylation of nucleoid protein pTAC16

Light-regulated protein kinases STN7 and STN8 phosphorylate thylakoid membrane proteins and also affect expression of several chloroplast proteins via yet unknown mechanisms. Comparative phosphoproteomics of acetic acid protein extracts of chloroplasts from Arabidopsis thaliana wild type, stn7, stn8 and stn7stn8 mutants yielded two previously unknown findings: (i) neither STN7 nor STN8 kinase was required for phosphorylation of Ser-48 in Lhcb1.1-1.3 proteins; and (ii) phosphorylation of Thr-451 in pTAC16 protein was STN7-dependent. pTAC16 was found distributed between thylakoids and nucleoid. Its knockout did not affect the nucleoid protein composition and the Thr-451 phosphorylated protein was excluded from the nucleoid. Thr-451 of pTAC16 is conserved in all studied plants and its phosphorylation may regulate membrane-anchoring functions of the nucleoid.     

Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress

Acclimation to changing environments, such as increases in light intensity, is necessary, especially for the survival of sedentary organisms like plants. To learn more about the importance of ascorbate in the acclimation of plants to high light (HL), vtc2, an ascorbate-deficient mutant of Arabidopsis, and the double mutants vtc2npq4 and vtc2npq1 were tested for growth in low light and HL and compared with the wild type. The vtc2 mutant has only 10% to 30% of wild-type levels of ascorbate, vtc2npq4 has lower ascorbate levels and lacks non-photochemical quenching of chlorophyll fluorescence (NPQ) because of the absence of the photosystem II protein PsbS, and vtc2npq1 is NPQ deficient and also lacks zeaxanthin in HL but has PsbS. All three genotypes were able to grow in HL and had wild-type levels of Lhcb1, cytochrome f, PsaF, and 2-cysteine peroxiredoxin. However, the mutants had lower electron transport and oxygen evolution rates and lower quantum efficiency of PSII compared with the wild type, implying that they experienced chronic photooxidative stress. The mutants lacking NPQ in addition to ascorbate were only slightly more affected than vtc2. All three mutants had higher glutathione levels than the wild type in HL, suggesting a possible compensation for the lower ascorbate content. These results demonstrate the importance of ascorbate for the long-term acclimation of plants to HL.     

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg⁺⁺) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg⁺⁺. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg⁺⁺-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.     

Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes in Arabidopsis

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to high light. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.The use of captured sunlight energy to split water and drive oxygenic photosynthesis by photosystem II (PSII) (Barber, 2006) inevitably generates reactive oxygen species and causes oxidative damage to the PSII protein pigment complex. The light-induced damage to PSII, in particular to the D1 reaction center protein, requires PSII repair to sustain its photosynthetic function (Takahashi and Murata, 2008). Impairment and degradation of D1 increase with rising light intensities, and this protein has the fastest turnover rate among the photosynthetic proteins of plants, algae, and cyanobacteria (Yokthongwattana and Melis, 2006). However, in plants, the PSII is segregated in highly stacked membrane layers of very large thylakoid membranes (Andersson and Anderson, 1980; Kirchhoff et al., 2008), which are densely folded to fit inside chloroplasts (Mullineaux, 2005; Shimoni et al., 2005). As a consequence, the PSII repair cycle in plants is slower than in cyanobacteria (Yokthongwattana and Melis, 2006), and it includes migration of the PSII complex from the stacked membrane domains (grana) to the unstacked membranes (stroma lamellae), where proteolysis and insertion of a newly synthesized D1 protein occurs (Baena-Gonzalez and Aro, 2002; Yokthongwattana and Melis, 2006). High light also causes quantitative phosphorylation of the membrane surface–exposed regions of D1, D2, CP43, and PsbH proteins of PSII in plants (Rintamäki et al., 1997; Vener et al., 2001), but the function of this phosphorylation is largely unknown and reports on its importance for the D1 protein turnover are conflicting (Bonardi et al., 2005; Tikkanen et al., 2008).Phosphorylation of the PSII proteins in Arabidopsis thaliana depends mostly on the light-activated protein kinase STN8 (Vainonen et al., 2005), while the STN7 kinase is essential for phosphorylation of the light-harvesting proteins of PSII (Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006). An earlier study on Arabidopsis mutants lacking both STN7 and STN8 (stn7xstn8), as well as only STN8, concluded that protein phosphorylation was not essential for PSII repair (Bonardi et al., 2005), while more recent work revealed a dramatic retardation in D1 degradation under high light in the stn8 and stn7xstn8 mutants (Tikkanen et al., 2008). Moreover, it was shown that the lack of PSII phosphorylation resulted in accumulation of photodamaged PSII complexes and in general oxidative damage of photosynthetic proteins in the thylakoid membranes under high light (Tikkanen et al., 2008). The other study revealed that the stn7xstn8 double mutant grown under natural field conditions produced 41% less seeds than wild-type plants (Frenkel et al., 2007), which also indicated physiological importance of thylakoid protein phosphorylation in maintenance of plant fitness.To uncover the function of light-dependent protein phosphorylation in plant photosynthetic membranes, we performed a detailed analysis of the Arabidopsis mutants deficient in the protein kinases STN7 and STN8. The earlier published results on protein phosphorylation analyses in the stn7xstn8 mutant of Arabidopsis were restricted to antiphosphothreonine antibody-based immunodetection and did not reveal any phosphorylation of PSII core proteins (Bonardi et al., 2005; Tikkanen et al., 2008). Using a mass spectrometry (MS) approach and immunoblot analyses with two complementary antiphosphothreonine antibodies, we find remaining light-independent phosphorylation of PsbH and D2 proteins of PSII in stn7xstn8. We demonstrate that degradation and aggregation patterns of the D1 protein in stn7xstn8 differ from those in wild-type, stn7, and stn8 plants. We also observe a reproducible delay in the degradation of D1 in high light–treated leaves of stn7xstn8 and stn8 compared with the wild-type and stn7 plants. Finally, we show that phosphorylation of PSII proteins modulates macroscopic rearrangements of the entire membrane network of plant thylakoids, which facilitates lateral mobility of membrane proteins, required for repair and sustained activity of PSII.     

Identification of STN7/STN8 kinase targets reveals connections between electron transport,metabolism and gene expression

The thylakoid‐associated kinases STN7 and STN8 are involved in short‐ and long‐term acclimation of photosynthetic electron transport to changing light conditions. Here we report the identification of STN7/STN8 in vivo targets that connect photosynthetic electron transport with metabolism and gene expression. Comparative phosphoproteomics with the stn7 and stn8 single and double mutants identified two proteases, one RNA‐binding protein, a ribosomal protein, the large subunit of Rubisco and a ferredoxin‐NADP reductase as targets for the thylakoid‐associated kinases. Phosphorylation of three of the above proteins can be partially complemented by STN8 in the stn7 single mutant, albeit at lower efficiency, while phosphorylation of the remaining three proteins strictly depends on STN7. The properties of the STN7‐dependent phosphorylation site are similar to those of phosphorylated light‐harvesting complex proteins entailing glycine or another small hydrophobic amino acid in the ?1 position. Our analysis uncovers the STN7/STN8 kinases as mediators between photosynthetic electron transport, its immediate downstream sinks and long‐term adaptation processes affecting metabolite accumulation and gene expression.     

STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins

Combination of reversed genetics with analyses of in vivo protein phosphorylation in Arabidopsis thaliana revealed that STN8 protein kinase is specific in phosphorylation of N-terminal threonine residues in D1, D2, and CP43 proteins, and Thr-4 in the PsbH protein of photosystem II. Phosphorylation of D1, D2, and CP43 in the light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50-60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.     

Loss of psbS expression reduces vegetative growth, reproductive output, and light-limited, but not light-saturated, photosynthesis in Arabidopsis thaliana (Brassicaceae) grown in temperate light environments

Plants protect themselves against the deleterious effects of high light intensities by inducing a mechanism ubiquitous among plants known as energy dissipation, which safely converts excess light to heat before it can lead to the formation of free radicals. Mutants possessing a deletion of the psbS gene, such as the npq4 mutant, cannot perform energy dissipation and thus offer an opportunity to assess the importance of this process to plant function. In a temperate light environment, greenhouse-grown npq4 mutants of Arabidopsis thaliana had smaller rosette diameters and leaf numbers. The reduction in size observed in npq4 plants was associated with fewer floral stalks, fewer fruits, lower whole-plant and individual seed masses, and lower germination rates. In the field, npq4 mutants developed fewer fruits. After a controlled exposure to high light stress, both PSII efficiency and CO(2) assimilation were more significantly compromised in npq4 mutants at low light intensities, but not at high light intensities. Thus, the protective nature of energy dissipation manifests in light environments that include periods of high light, which predispose plants to PSII photoinactivation, and periods of low light, when PSII photoinactivation decreases the rate of photosynthesis.     

The transiently generated nonphotochemical quenching of excitation energy in Arabidopsis leaves is modulated by zeaxanthin

Upon the transition of dark-adapted plants to low light, the energy-dependent quenching (qE) of excitation energy is only transiently induced due to the only transient generation of the transthylakoid pH gradient. We investigated the transient qE (qE(TR)) in different Arabidopsis (Arabidopsis thaliana) mutants. In dark-adapted plants, qE(TR) was absent in the npq4 mutant (deficient in the PsbS protein) and the pgr1 mutant (restricted in lumen acidification). In comparison with wild-type plants, qE(TR) was reduced in the zeaxanthin (Zx)-deficient npq1 mutant and increased in the Zx-accumulating npq2 mutant. After preillumination of plants (to allow the synthesis of large amounts of Zx), the formation and relaxation of qE(TR) was accelerated in all plants (except for npq4) in comparison with the respective dark-adapted plants. The extent of qE(TR), however, was unchanged in npq1 and npq4, decreased in npq2, but increased in wild-type and pgr1 plants. Even in presence of high levels of Zx, qE(TR) in pgr1 mutants was still lower than that in wild-type plants. In the presence of the uncoupler nigericin, qE(TR) was completely abolished in all genotypes. Thus, the transient qE(TR) shows essentially the same characteristics as the steady-state qE; it is strictly dependent on the PsbS protein and a low lumen pH, but the extent of qE(TR) is largely modulated by Zx. These results indicate that qE(TR) does not represent a different quenching mechanism in comparison with the steady-state qE, but simply reflects the response of qE to the dynamics of the lumen pH during light activation of photosynthesis.     

Molecular and global time-resolved analysis of a psbS gene dosage effect on pH- and xanthophyll cycle-dependent nonphotochemical quenching in photosystem II

Photosynthetic light harvesting in plants is regulated by a pH- and xanthophyll-dependent nonphotochemical quenching process (qE) that dissipates excess absorbed light energy and requires the psbS gene product. An Arabidopsis thaliana mutant, npq4-1, lacks qE because of a deletion of the psbS gene, yet it exhibits a semidominant phenotype. Here it is shown that the semidominance is due to a psbS gene dosage effect. Diploid Arabidopsis plants containing two psbS gene copies (wild-type), one psbS gene (npq4-1/NPQ4 heterozygote), and no psbS gene (npq4-1/npq4-1 homozygote) were compared. Heterozygous plants had 56% of the wild-type psbS mRNA level, 58% of the wild-type PsbS protein level, and 60% of the wild-type level of qE. Global analysis of the chlorophyll a fluorescence lifetime distributions revealed three components in wild-type and heterozygous plants, but only a single long lifetime component in npq4-1. The short lifetime distribution associated with qE was inhibited by more than 40% in heterozygous plants compared with the wild type. Thus, the extent of qE measured as either the fractional intensities of the PSII chlorophyll a fluorescence lifetime distributions or steady state intensities was stoichiometrically related to the amount of PsbS protein.     

Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

The CST (Cdc13/CTC1-STN1-TEN1) complex was proposed to have evolved kingdom specific roles in telomere capping and replication. To shed light on its evolutionary conserved function, we examined the effect of STN1 dysfunction on telomere structure in plants. STN1 inactivation in Arabidopsis leads to a progressive loss of telomeric DNA and the onset of telomeric defects depends on the initial telomere size. While EXO1 aggravates defects associated with STN1 dysfunction, it does not contribute to the formation of long G-overhangs. Instead, these G-overhangs arise, at least partially, from telomerase-mediated telomere extension indicating a deficiency in C-strand fill-in synthesis. Analysis of hypomorphic DNA polymerase α mutants revealed that the impaired function of a general replication factor mimics the telomeric defects associated with CST dysfunction. Furthermore, we show that STN1-deficiency hinders re-replication of heterochromatic regions to a similar extent as polymerase α mutations. This comparative analysis of stn1 and pol α mutants suggests that STN1 plays a genome-wide role in DNA replication and that chromosome-end deprotection in stn1 mutants may represent a manifestation of aberrant replication through telomeres.     

Phosphorylation of photosystem II core proteins prevents undesirable cleavage of D1 and contributes to the fine‐tuned repair of photosystem II

Photosystem II (PSII) is a primary target for light‐induced damage in photosynthetic protein complexes. To avoid photoinhibition, chloroplasts have evolved a repair cycle with efficient degradation of the PSII reaction center protein, D1, by the proteases FtsH and Deg. Earlier reports have described that phosphorylated D1 is a poor substrate for proteolysis, suggesting a mechanistic role for protein phosphorylation in PSII quality control, but its precise role remains elusive. STN8, a protein kinase, plays a central role in this phosphorylation process. To elucidate the relationship between phosphorylation of D1 and the protease function we assessed in this study the involvement of STN8, using Arabidopsis thaliana mutants lacking FtsH2 [yellow variegated2 (var2)] and Deg5/Deg8 (deg5 deg8). In support of our presumption we found that phosphorylation of D1 increased more in var2. Furthermore, the coexistence of var2 and stn8 was shown to recover the delay in degradation of D1, resulting in mitigation of the high vulnerability to photoinhibition of var2. Partial D1 cleavage fragments that depended on Deg proteases tended to increase, with concomitant accumulation of reactive oxygen species in the mutants lacking STN8. We inferred that the accelerated degradation of D1 in var2 stn8 presents a tradeoff in that it improved the repair of PSII but simultaneously enhanced oxidative stress. Together, these results suggest that PSII core phosphorylation prevents undesirable cleavage of D1 by Deg proteases, which causes cytotoxicity, thereby balancing efficient linear electron flow and photo‐oxidative damage. We propose that PSII core phosphorylation contributes to fine‐tuned degradation of D1.     

Moderate heat stress induces state transitions in Arabidopsis thaliana

The effect of temperature on the photosynthetic machinery is crucial for the fundamental understanding of plant physiology and the bioengineering of heat-tolerant varieties. In our study, Arabidopsis thaliana was exposed to mild (40°C), short-term heat stress in the dark to evaluate the heat-triggered phosphorylation and migration of light harvesting complex (LHC) II in both wild-type (wt) and mutant lacking STN7 kinase. The 77K emission spectra revealed an increase in PSI relative to PSII emission similar to increases observed in light-induced state I to state II transitions in wt but not in stn7 mutant. Immunoblotting results indicated that the major LHCII was phosphorylated at threonine sites under heat stress in wt plants but not in the mutant. These results support the proposition that mild heat stress triggers state transitions in the dark similar to light-induced state transitions, which involve phosphorylation of LHCII by STN7 kinase. Pre-treatment of Arabidopsis leaves with inhibitor DBMIB, altered the extent of LHCII phosphorylation and PSI fluorescence emission suggests that activation of STN7 kinase may be dependent on Cyt b(6)/f under elevated temperatures in dark. Furthermore, fast Chl a transient of temperature-exposed leaves of wt showed a decrease in the F(v)/F(m) ratio due to both an increase in F(o) and a decrease in F(m). In summary, our findings indicate that a mild heat treatment (40°C) induces state transitions in the dark resulting in the migration of phosphorylated LHCII from the grana to the stroma region.     

The role of the PsbS protein in the protection of photosystems I and II against high light in Arabidopsis thaliana

The PsbS protein is recognised in higher plants as an important component in dissipating excess light energy via its regulation of non-photochemical quenching. We investigated photosynthetic responses in the arabidopsis npq4 mutant, which lacks PsbS, and in a mutant over-expressing PsbS (oePsbS). Growth under low light led to npq4 and wild-type plants being visibly indistinguishable, but induced a phenotype in oePsbS plants, which were smaller and had shorter flowering spikes. Here we report that chloroplasts from npq4 generated more singlet oxygen (¹O₂) than those from oePsbS. This accompanied a higher extent of photosystem II photoinhibition of leaves from npq4 plants. In contrast, oePsbS was more damaged by high light than npq4 and the wild-type at the level of photosystem I. The plastoquinone pool, as measured by thermoluminescence, was more oxidised in the oePsbS than in npq4, whilst the amount of photo-oxidisable P₇₀₀, as probed with actinic light or saturating flashes, was higher in oePsbS compared to wild-type and npq4. Taken together, this indicates that the level of PsbS has a regulatory role in cyclic electron flow. Overall, we show that under high light oePsbS plants were more protected from ¹O₂ at the level of photosystem II, whereas lack of cyclic electron flow rendered them susceptible to damage at photosystem I. Cyclic electron flow is concluded to be essential for protecting photosystem I from high light stress.     

Light-harvesting mutants show differential gene expression upon shift to high light as a consequence of photosynthetic redox and reactive oxygen species metabolism

The amount of light energy that is harvested and directed to the photosynthetic machinery is regulated in order to control the production of reactive oxygen species (ROS) in leaf tissues. ROS have important roles as signalling factors that instigate and mediate a range of cellular responses, suggesting that the mechanisms regulating light-harvesting and photosynthetic energy transduction also affect cell signalling. In this study, we exposed wild-type (WT) Arabidopsis and mutants impaired in the regulation of photosynthetic light-harvesting (stn7, tap38 and npq4) to transient high light (HL) stress in order to study the role of these mechanisms for up- and downregulation of gene expression under HL stress. The mutants, all of which have disturbed regulation of excitation energy transfer and distribution, responded to transient HL treatment with surprising similarity to the WT in terms of general ‘abiotic stress-regulated’ genes associated with hydrogen peroxide and 12-oxo-phytodienoic acid signalling. However, we identified distinct expression profiles in each genotype with respect to induction of singlet oxygen and jasmonic acid-dependent responses. The results of this study suggest that the control of excitation energy transfer interacts with hormonal regulation. Furthermore, the photosynthetic pigment–protein complexes appear to operate as receptors that sense the energetic balance between the photosynthetic light reactions and downstream metabolism.     

The long-term response to fluctuating light quality is an important and distinct light acclimation mechanism that supports survival of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under low light conditions

The long-term response (LTR) of higher plants to varying light qualities increases the photosynthetic yield; however, the benefit of this improvement for physiology and survival of plants is largely unknown, and its functional relation to other light acclimation responses has never been investigated. To unravel positive effects of the LTR we acclimated Arabidopsis thaliana for several days to light sources, which preferentially excite photosystem I (PSI) or photosystem II (PSII). After acclimation, plants revealed characteristic differences in chlorophyll fluorescence, thylakoid membrane stacking, phosphorylation state of PSII subunits and photosynthetic yield of PSII and PSI. These LTR-induced changes in the structure, function and efficiency of the photosynthetic machinery are true effects by light quality acclimation, which could not be induced by light intensity variations in the low light range. In addition, high light stress experiments indicated that the LTR is not involved in photoinhibition; however, it lowers non-photochemical quenching (NPQ) by directing more absorbed light energy into photochemical work. NPQ in turn is not essential for the LTR, since npq mutants performed a normal acclimation. We quantified the beneficial potential of the LTR by comparing wild-type plants with the LTR-deficient mutant stn7. The mutant exhibited a decreased effective quantum yield and produced only half of seeds when grown under fluctuating light quality conditions. Thus, the LTR represents a distinct acclimation response in addition to other already known responses that clearly improves plant physiology under low light conditions resulting in a pronounced positive effect on plant fitness.     

Isolation and characterization of mutants of common ice plant deficient in crassulacean acid metabolism

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.     

The Arabidopsis aba4-1 mutant reveals a specific function for neoxanthin in protection against photooxidative stress

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.