Loss of peroxisomal NAD kinase 3 (NADK3) affects photorespiration metabolism in Arabidopsis.

Nicotinamide adenine dinucleotides (NAD+ and NADP+) are electron mediators involved in various metabolic pathways. NADP(H) are produced by NAD kinase (NADK) through the phosphorylation of NAD(H). The Arabidopsis NADK3 (AtNADK3) is reported to preferentially phosphorylate NADH to NADPH and is localized in the peroxisome. To elucidate the biological function of AtNADK3 in Arabidopsis, we compared metabolites of nadk1, nadk2 and nadk3 Arabidopsis T-DNA inserted mutants. Metabolome analysis revealed that glycine and serine, which are intermediate metabolites of photorespiration, both increased in the nadk3 mutants. Plants grown for 6 weeks under short-day conditions showed increased NAD(H), indicating a decrease in the phosphorylation ratio in the NAD(P)(H) equilibrium. Furthermore, high CO2 (0.15%) treatment induced a decrease in glycine and serine in nadk3 mutants. The nadk3 showed a significant decrease in post-illumination CO2 burst, suggesting that the photorespiratory flux was disrupted in the nadk3 mutant. In addition, an increase in CO2 compensation points and a decrease in CO2 assimilation rate were observed in the nadk3 mutants. These results indicate that the lack of AtNADK3 causes a disruption in the intracellular metabolism, such as in amino acid synthesis and photorespiration.

Mixed population hypothesis of the active and inactive PSII complexes opens a new door for photoinhibition and fluorescence studies: an ecophysiological perspective.

The current hypotheses for the mechanisms of photosystem II (PSII) photodamage in vivo remain split on the primary damage site. However, most researchers have considered that PSII is inhibited by a sole mechanism and that the photoinhibited PSII consists of one population. In this perspective, we propose 'the mixed population hypothesis', in which there are four PSII populations: PSII with active/inactive Mn4 CaO5 oxygen-evolving complex respectively with functional/damaged primary quinone (QA ) reduction activity. This hypothesis provides a new insight into not only the PSII photoinhibition/photoprotection studies but also the repair process. We discuss our new data implying that the repair rate differs in the respective PSII populations.

Green Tea Catechins, (-)-Catechin Gallate, and (-)-Gallocatechin Gallate are Potent Inhibitors of ABA-Induced Stomatal Closure.

Stomatal movement is indispensable for plant growth and survival in response to environmental stimuli. Cytosolic Ca2+ elevation plays a crucial role in ABA-induced stomatal closure during drought stress; however, to what extent the Ca2+ movement across the plasma membrane from the apoplast to the cytosol contributes to this process still needs clarification. Here the authors identify (-)-catechin gallate (CG) and (-)-gallocatechin gallate (GCG), components of green tea, as inhibitors of voltage-dependent K+ channels which regulate K+ fluxes in Arabidopsis thaliana guard cells. In Arabidopsis guard cells CG/GCG prevent ABA-induced: i) membrane depolarization; ii) activation of Ca2+ permeable cation (ICa ) channels; and iii) cytosolic Ca2+ transients. In whole Arabidopsis plants co-treatment with CG/GCG and ABA suppressed ABA-induced stomatal closure and surface temperature increase. Similar to ABA, CG/GCG inhibited stomatal closure is elicited by the elicitor peptide, flg22 but has no impact on dark-induced stomatal closure or light- and fusicoccin-induced stomatal opening, suggesting that the inhibitory effect of CG/GCG is associated with Ca2+ -related signaling pathways. This study further supports the crucial role of ICa channels of the plasma membrane in ABA-induced stomatal closure. Moreover, CG and GCG represent a new tool for the study of abiotic or biotic stress-induced signal transduction pathways.

A new method for separate evaluation of PSII with inactive oxygen evolving complex and active D1 by the pulse-amplitude modulated chlorophyll fluorometry.

A method that separately quantifies the PSII with inactive oxygen-evolving complex (OEC) and active D1 retaining the primary quinone acceptor (QA )-reducing activity from the PSII with damaged D1 in the leaf was developed using PAM fluorometry. It is necessary to fully reduce QA to obtain F m , the maximum fluorescence. However, QA in PSII with inactive OEC and active D1 would not be fully reduced by a saturating flash. We used the acceptor-side inhibitor DCMU to fully reduce QA . Leaves of cucumber (Cucumis sativus L.) were chilled at 4°C in dark or illuminated with UV-A to selectively inactivate OEC. After these treatments, F v /F m , the maximum quantum yield, in the leaves vacuum-infiltrated with DCMU were greater than those in water-infiltrated leaves. In contrast, when the leaves were illuminated by red light to photodamage D1, F v /F m did not differ between DCMU- and water-infiltrated leaves. These results indicate relevance of the present evaluation of the fraction of PSII with inactive OEC and active D1. Several examinations in the laboratory and glasshouse showed that PSII with inactive OEC and active D1 was only rarely observed. The present simple method would serve as a useful tool to clarify the details of the PSII photoinhibition.

Effects of High Irradiance and Low Water Temperature on Photoinhibition and Repair of Photosystems in Marimo (Aegagropila linnaei) in Lake Akan, Japan.

The green alga Aegagropila linnaei often forms spherical aggregates called "marimo" in Lake Akan in Japan. In winter, marimo are exposed to low water temperatures at 1-4 °C but protected from strong sunlight by ice coverage, which may disappear due to global warming. In this study, photoinhibition in marimo was examined at 2 °C using chlorophyll fluorescence and 830 nm absorption. Filamentous cells of A. linnaei dissected from marimo were exposed to strong light at 2 °C. Photosystem II (PSII) was markedly photoinhibited, while photosystem I was unaffected. When the cells with PSII damaged by the 4 h treatment were subsequently illuminated with moderate repair light at 2 °C, the maximal efficiency of PSII was recovered to the level before photoinhibition. However, after the longer photoinhibitory treatments, PSII efficiency did not recover by the repair light. When the cells were exposed to simulated diurnal light for 12 h per day, which was more ecological, the cells died within a few days. Our results showed new findings of the PSII repair at 2 °C and serious damage at the cellular level from prolonged high-light treatments. Further, we provided a clue to what may happen to marimo in Lake Akan in the near future.

Lack of plastid-encoded Ycf10, a homolog of the nuclear-encoded DLDG1 and the cyanobacterial PxcA, enhances the induction of non-photochemical quenching in tobacco.

pH homeostasis in the chloroplast is crucial for the control of photosynthesis and other metabolic processes in plants. Recently, nuclear-encoded Day-Length-dependent Delayed Greening1 (DLDG1) and Fluctuating-Light Acclimation Protein1 (FLAP1) that are required for the light-inducible optimization of plastidial pH in Arabidopsis thaliana were identified. DLDG1 and FLAP1 homologs are specifically conserved in oxygenic phototrophs, and a DLDG1 homolog, Ycf10, is encoded in the chloroplast genome in plant cells. However, the function of Ycf10 and its physiological significance are unknown. To address this, we constructed ycf10 tobacco Nicotiana tabacum mutants and characterized their phenotypes. The ycf10 tobacco mutants grown under continuous-light conditions showed a pale-green phenotype only in developing leaves, and it was suppressed in short-day conditions. The ycf10 mutants also induced excessive non-photochemical quenching (NPQ) compared with those in the wild-type at the induction stage of photosynthesis. These phenotypes resemble those of Arabidopsis dldg1 mutants, suggesting that they have similar functions. However, there are distinct differences between the two mutant phenotypes: The highly induced NPQ in tobacco ycf10 and the Arabidopsis dldg1 mutants are diminished and enhanced, respectively, with increasing duration of the fluctuating actinic-light illumination. Ycf10 and DLDG1 were previously shown to localize in chloroplast envelope-membranes, suggesting that Ycf10 and DLDG1 differentially control H+ exchange across these membranes in a light-dependent manner to control photosynthesis.

Genetic Dissection of Growth and Eco-Physiological Traits Associated with Altitudinal Adaptation in Sakhalin Fir (Abies sachalinensis) Based on QTL Mapping.

(1) Background: The genetic basis of local adaptation in conifers remains poorly understood because of limited research evidence and the lack of suitable genetic materials. Sakhalin fir (Abies sachalinensis) is an ideal organism for elucidating the genetic basis of local adaptation because its altitudinal adaptation has been demonstrated, and suitable materials for its linkage mapping are available. (2) Method: We constructed P336 and P236 linkage maps based on 486 and 516 single nucleotide polymorphisms, respectively, that were derived from double digest restriction site-associated DNA sequences. We measured the growth and eco-physiological traits associated with morphology, phenology, and photosynthesis, which are considered important drivers of altitudinal adaptation. (3) Results: The quantitative trait loci (QTLs) for growth traits, phenology, needle morphology, and photosynthetic traits were subsequently detected. Similar to previous studies on conifers, most traits were controlled by multiple QTLs with small or moderate effects. Notably, we detected that one QTL for the crown area might be a type-A response regulator, a nuclear protein responsible for the cytokinin-induced shoot elongation. (4) Conclusion: The QTLs detected in this study include potentially important genomic regions linked to altitudinal adaptation in Sakhalin fir.

Photosystem I in low light-grown leaves of Alocasia odora, a shade-tolerant plant, is resistant to fluctuating light-induced photoinhibition.

When intact green leaves are exposed to the fluctuating light, in which high light (HL) and low light (LL) alternate, photosystem I (PSI) is readily damaged. This PSI inhibition is mostly alleviated by the addition of far-red (FR) light. Here, we grew Alocasia odora, a shade-tolerant species, at several light levels and examined their photosynthetic traits in relation to the fluctuating light-induced PSI inhibition. We found that, even in the absence of FR, PSI in LL-grown leaves was resistant to the fluctuating light. LL leaves showed higher chlorophyll (Chl) contents on leaf area basis, lower Chl a/b ratios, lower cytochrome f/P700 ratios, and lower PSII/PSI excitation ratios assessed by the 77 K fluorescence. Also, P700 in the HL phase of the fluctuating light was more oxidized. The results of the regression analyses of the PSI photoinhibition to these traits indicate that the lower electron flow rate to P700 and more excitation energy transfer to PSI protect PSI in LL-grown leaves. Both of these contribute oxidization of P700 to the efficient quencher form P700+. These features may be common in LL-grown shade-tolerant species, which are often exposed to strong sunflecks in their natural habitats.

The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I.

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

Far-Red Light Accelerates Photosynthesis in the Low-Light Phases of Fluctuating Light.

It is well known that far-red light (FR; >700 nm) drives PSI photochemistry, but its effect on photosynthetic performance has received little attention. In this study, the effects of the addition of FR to red fluctuating light (FL) have on photosynthesis were examined in the leaves of Arabidopsis thaliana. Light-activated leaves were illuminated with FL [alternating high light/low light (HL/LL) at 800/30 µmol m-2 s-1] for 10-15 min without or with FR at intensities that reflected natural conditions. The CO2 assimilation rates upon the transition from HL to LL were significantly greater with FR than without FR. The enhancement of photosynthesis by FR was small under the steady-state conditions and in the HL phases of FL. Proton conductivity through the thylakoid membrane (gH+) in the LL phases of FL, estimated from the dark relaxation kinetics of the electrochromic absorbance shift, was greater with FR than without FR. The relaxation of non-photochemical quenching (NPQ) in the PSII antenna system and the increase in PSII photochemistry in the LL phases accelerated in the presence of FR. Similar FR-effects in FL were confirmed in typical sun and shade plants. On the basis of these results, we concluded that FR exerted beneficial effects on photosynthesis in FL by exciting PSI and accelerating NPQ relaxation and PSII-yield increase. This was probably because of the increased gH+, which would reflect faster ΔpH dissipation and ATP synthesis.

DAY-LENGTH-DEPENDENT DELAYED-GREENING1, the Arabidopsis Homolog of the Cyanobacterial H+-Extrusion Protein, Is Essential for Chloroplast pH Regulation and Optimization of Non-Photochemical Quenching.

Plants convert solar energy into chemical energy through photosynthesis, which supports almost all life activities on earth. Because the intensity and quality of sunlight can change dramatically throughout the day, various regulatory mechanisms help plants adjust their photosynthetic output accordingly, including the regulation of light energy accumulation to prevent the generation of damaging reactive oxygen species. Non-photochemical quenching (NPQ) is a regulatory mechanism that dissipates excess light energy, but how it is regulated is not fully elucidated. In this study, we report a new NPQ-regulatory protein named Day-Length-dependent Delayed-Greening1 (DLDG1). The Arabidopsis DLDG1 associates with the chloroplast envelope membrane, and the dldg1 mutant had a large NPQ value compared with wild type. The mutant also had a pale-green phenotype in developing leaves but only under continuous light; this phenotype was not observed when dldg1 was cultured in the dark for ≥8 h/d. DLDG1 is a homolog of the plasma membrane-localizing cyanobacterial proton-extrusion-protein A that is required for light-induced H+ extrusion and also shows similarity in its amino-acid sequence to that of Ycf10 encoded in the plastid genome. Arabidopsis DLDG1 enhances the growth-retardation phenotype of the Escherichia coli K+/H+ antiporter mutant, and the everted membrane vesicles of the E. coli expressing DLDG1 show the K+/H+ antiport activity. Our findings suggest that DLDG1 functionally interacts with Ycf10 to control H+ homeostasis in chloroplasts, which is important for the light-acclimation response, by optimizing the extent of NPQ.

FLUCTUATING-LIGHT-ACCLIMATION PROTEIN1, Conserved in Oxygenic Phototrophs, Regulates H+ Homeostasis and Non-Photochemical Quenching in Chloroplasts.

Plants have mechanisms allowing them to acclimate to intense light conditions, which involves the dissipation of excess light energy. These mechanisms allow plants to perform photosynthesis efficiently and, therefore, must be accurately and precisely controlled. However, how plants dissipate excess light energy has yet to be fully elucidated. Herein we report the identification of a gene, which we named Fluctuating-Light-Acclimation Protein1 (FLAP1), that is conserved in oxygenic phototrophs. We show that Arabidopsis FLAP1 is associated with chloroplast thylakoid and envelope membranes and that the flap1 mutant shows delayed non-photochemical quenching (NPQ) relaxation during induction of photosynthesis at moderate light intensity. Under fluctuating light conditions, NPQ levels in the flap1 mutant were higher than those in the wild type during the high light period, and the mutant exhibited a pale-green phenotype. These findings suggest that FLAP1 is involved in NPQ control, which is important for an acclimation response to fluctuating light.

Photoprotection of PSI by Far-Red Light Against the Fluctuating Light-Induced Photoinhibition in Arabidopsis thaliana and Field-Grown Plants.

It has been reported that PSI photoinhibition is induced even in wild-type plants of Arabidopsis thaliana, rice and other species by exposure of leaves to fluctuating light (FL) for a few hours. Because plants are exposed to FL in nature, they must possess protective mechanisms against the FL-induced photodamage. Here, using A. thaliana grown at various irradiances, we examined PSI photoprotection by far-red (FR) light at intensities comparable with those observed in nature. Dark-treated leaves were illuminated by red FL alternating high/low light at 1,200/30 µmol m-2 s-1 for 800 ms/10 s. By this FL treatment without FR light for 120 min, the level of photo-oxidizable P700 was decreased by 30% even in the plants grown at high irradiances. The addition of continuous FR light during the FL suppressed this damage almost completely. With FR light, P700 was kept in a more oxidized state in both low- and high-light phases. The protective effect of FR light was diminished more in mutants of the NADH dehydrogenase-like complex (NDH)-mediated cyclic electron flow around PSI (CEF-PSI) than in the PGR5 (proton gradient regulation 5)-mediated CEF-PSI, indicating that the NDH-mediated CEF-PSI would be a major contributor to PSI photoprotection in the presence of FR light. We also confirmed that PSI photoinhibition decreased with the increase in growth irradiance in A. thaliana and field-grown plants, and that this PSI photodamage was largely suppressed by addition of FR light. These results clearly indicate that the most effective PSI protection is realized in the presence of FR light.

Elucidation of Photoprotective Mechanisms of PSI Against Fluctuating Light photoinhibition.

It has been claimed that the cyclic electron flow around PSI (CEF-PSI) plays an important role in protection of PSI against fluctuating light photoinhibition. However, the photoprotective mechanism of PSI is not fully elucidated. Here, we examined the mechanism, using two CEF-PSI mutants of Arabidopsis thaliana, and antimycin A, an inhibitor of the PGR5 (proton gradient regulation 5)-mediated CEF-PSI. Dark-adapted leaves in these plants were illuminated in fluctuating light alternating between high light at 1,200 µmol m-2 s-1 and low light at 30 µmol m-2 s-1 every 2 min, and PSI and PSII parameters were simultaneously measured for 160 min with 830 nm absorption and Chl fluorescence, respectively. When CEF-PSI, especially PGR5-mediated CEF-PSI, did not operate, the acceptor-side limitation of PSI, Y(NA), increased stepwise, leading to marked PSI photoinhibition. The deficiency of CFE-PSI decreased not only the electron transport rate through PSI but also the donor-side limitation of PSI, Y(ND), in high light phases. These results showed that the large Y(ND), observed only when CEF-PSI operated, contributed to suppression of PSI photoinhibition. Taken together with our previous report that high Y(NA) was alleviated by the enhancement of CEF-PSI, a model for the protective mechanisms of PSI is proposed. In this model, both alleviation of Y(NA) and acceleration of Y(ND) are indispensable, and for realization of such a situation, regulation of the electron flows, especially the PGR5-mediated CEF-PSI, plays a key role. It is important for effective protection to regulate the balance of Y(ND) and Y(NA) through CEF-PSI.

Light-harvesting complex Lhcb9 confers a green alga-type photosystem I supercomplex to the moss Physcomitrella patens.

Light-harvesting complex (LHC) proteins in chloroplast thylakoid membranes not only transfer absorbed light energy to the two photosystems but also regulate the rate of energy transfer to avoid photodamage. Here we demonstrate that Lhcb9, a recently discovered LHC protein in the moss Physcomitrella patens, functions to connect LHC proteins with photosystem I (PSI), resulting in the formation of two different types of PSI supercomplexes in thylakoid membranes. We observed that the Lhcb9-containing PSI supercomplex is disassembled in response to excess light conditions. On the basis of our phylogenetic analysis, it appears that P. patens acquired Lhcb9 by horizontal gene transfer from the earlier green algal lineage, leading to the presence of both green alga-type and vascular plant-type PSI supercomplexes, which would have been crucial for conquering the dynamic environmental interface between aquatic and terrestrial conditions it faced during evolution.

Long-term and short-term responses of the photosynthetic electron transport to fluctuating light.

Light energy absorbed by chloroplasts drives photosynthesis. When absorbed light is in excess, the thermal dissipation systems of excess energy are induced and the photosynthetic electron flow is regulated, both contributing to suppression of reactive oxygen species production and photodamages. Various regulation mechanisms of the photosynthetic electron flow and energy dissipation systems have been revealed. However, most of such knowledge has been obtained by the experiments conducted under controlled conditions with constant light, whereas natural light condition is drastically fluctuated. To understand photosynthesis in nature, we need to clarify not only the mechanisms that raise photosynthetic efficiency but those for photoprotection in fluctuating light. Although these mechanisms appear to be well balanced, regulatory mechanisms achieving the balance is little understood. Recently, some pioneering studies have provided new insight into the regulatory mechanisms in fluctuating light. In this review, firstly, the possible mechanisms involved in regulation of the photosynthetic electron flow in fluctuating light are presented. Next, we introduce some recent studies focusing on the photosynthetic electron flow in fluctuating light. Finally, we discuss how plants effectively cope with fluctuating light showing our recent results.

Roles of the cyclic electron flow around PSI (CEF-PSI) and O2-dependent alternative pathways in regulation of the photosynthetic electron flow in short-term fluctuating light in Arabidopsis thaliana.

To assess the roles of the cyclic electron flow around PSI (CEF-PSI) and O2-dependent alternative pathways including the water-water cycle in fluctuating light, we grew the wild type and pgr5 mutant of Arabidopsis thaliana in constant light, and measured Chl fluorescence and P700 parameters in their leaves in the fluctuating light alternating between 240 (HL) and 30 µmol photons m⁻² s⁻² (LL) every 2 min. At 20% O2, the photochemical quantum yield of PSII decreased, in particular in the pgr5 plants, soon after the start of the fluctuating light treatment. PSI of the pgr5 plants was markedly photoinhibited by this treatment for 42 min. Slight PSI photoinhibition was also observed in the wild type. We measured energy sharing between PSII and PSI and estimated the PSI and PSII electron transport rates (ETRs). pgr5 showed larger energy allocation to PSI. In contrast to the wild type, the ratio of the PSI to the PSII ETR in pgr5 was higher in LL but lower in HL at 20% O2 due to PSI acceptor-side limitation. At 2.7% or 0% O2, the CEF-PSI of the pgr5 plants was enhanced, the acceptor-side limitation of PSI electron flow was released and PSI photoinhibition was not observed. The results suggest that the light fluctuation is a potent stress to PSI and that the CEF-PSI is essential to protect PSI from this stress.