CO2 -induced chloroplast movement in one cell-layer moss leaves.

CO2 -induced chloroplast movement was reported in the monograph by Gustav Senn in 1908: unilateral CO2 supply to the one cell-layered moss leaves induced the positively CO2 -tactic periclinal arrangement of chloroplasts. Here, using the model moss plant Physcomitrium patens, we examined basic features of chloroplast CO2 -tactic relocation with a modernized experimental system. The CO2 relocation was light-dependent and, especially, CO2 relocation in red light was substantially dependent on photosynthetic activity. In blue light, CO2 relocation was mainly dependent on microfilaments while microtubule-based movement was insensitive to CO2 , whereas in red light, both cytoskeletons contributed redundantly to CO2 relocation. The CO2 relocation was observed not only when the two leaf surfaces were exposed to CO2 -free air versus CO2 -containing air, but also by exposing them physiologically relevant differences in CO2 concentrations. In the leaves placed on the surface of a gel sheet, chloroplasts avoided the gel side and positioned in the air-facing surface, and this phenomenon was also shown to be photosynthesis dependent. Based on these observations, we propose a hypothesis that the threshold light intensity between the light-accumulation and -avoidance responses of the photorelocation would be increased by CO2 , resulting in the CO2 -tactic relocation of chloroplasts.

A rapid and simple spectroscopic method for the determination of yeast cell viability using methylene blue.

Determination of cell viability is important in various microbiological studies. The microscopic method, counting dead cells stained by methylene blue (MB), has often been used for the determination of viability, although it is not efficient for the measurement of a large number of samples. Alternatively, some spectroscopic methods have been proposed to avoid tedious cell counting. One of these proposed methods detects the decrease in MB absorbance in the supernatant of cell suspension, because dead cells incorporate MB more efficiently than viable cells. However, at present, this spectroscopic method is rarely used due to its low throughput. Therefore, we devised a small-scale, rapid and simple method by improving several points as follows. (1) The peak wavelength of MB absorbance, 665 nm, was used to detect MB efficiently at the microtube scale. (2) The composition of the MB solution was improved by adding trisodium citrate. (3) The reaction time was shortened. And (4) the concentration ranges of both MB and cells, with which absorbance is linearly related to cell viability, were determined. The improved method enabled us to evaluate the dose-dependent toxicities of alcohols, antifungal/antimalarial quinacrine, and UV-C irradiation. The results were compatible with those of conventional microscopic counting and colony formation. The method would be applicable to automated determination and to various organisms such as bacteria and filamentous fungi which are difficult to be counted microscopically.

Elevated CO2 induces rapid dephosphorylation of plasma membrane H+ -ATPase in guard cells.

Light induces stomatal opening, which is driven by plasma membrane (PM) H+ -ATPase in guard cells. The activation of guard-cell PM H+ -ATPase is mediated by phosphorylation of the penultimate C-terminal residue, threonine. The phosphorylation is induced by photosynthesis as well as blue light photoreceptor phototropin. Here, we investigated the effects of cessation of photosynthesis on the phosphorylation level of guard-cell PM H+ -ATPase in Arabidopsis thaliana. Immunodetection of guard-cell PM H+ -ATPase, time-resolved leaf gas-exchange analyses and stomatal aperture measurements were carried out. We found that light-dark transition of leaves induced dephosphorylation of the penultimate residue at 1 min post-transition. Gas-exchange analyses confirmed that the dephosphorylation is accompanied by an increase in the intercellular CO2 concentration, caused by the cessation of photosynthetic CO2 fixation. We discovered that CO2 induces guard-cell PM H+ -ATPase dephosphorylation as well as stomatal closure. Interestingly, reverse-genetic analyses using guard-cell CO2 signal transduction mutants suggested that the dephosphorylation is mediated by a mechanism distinct from the established CO2 signalling pathway. Moreover, type 2C protein phosphatases D6 and D9 were required for the dephosphorylation and promoted stomatal closure upon the light-dark transition. Our results indicate that CO2 -mediated dephosphorylation of guard-cell PM H+ -ATPase underlies stomatal closure.

Cost-benefit analysis of mesophyll conductance: diversities of anatomical, biochemical and environmental determinants.

BACKGROUND: Plants invest photosynthates in construction and maintenance of their structures and functions. Such investments are considered costs. These costs are recovered by the CO2 assimilation rate (A) in the leaves, and thus A is regarded as the immediate, short-term benefit. In photosynthesizing leaves, CO2 diffusion from the air to the carboxylation site is hindered by several structural and biochemical barriers. CO2 diffusion from the intercellular air space to the chloroplast stroma is obstructed by the mesophyll resistance. The inverses is the mesophyll conductance (gm). Whether various plants realize an optimal gm, and how much investment is needed for a relevant gm, remain unsolved. SCOPE: This review examines relationships among leaf construction costs (CC), leaf maintenance costs (MC) and gm in various plants under diverse growth conditions. Through a literature survey, we demonstrate a strong linear relationship between leaf mass per area (LMA) and leaf CC. The overall correlation of CC vs. gm across plant phylogenetic groups is weak, but significant trends are evident within specific groups and/or environments. Investment in CC is necessary for an increase in LMA and mesophyll cell surface area (Smes). This allows the leaf to accommodate more chloroplasts, thus increasing A. However, increases in LMA and/or Smes often accompany other changes, such as cell wall thickening, which diminishes gm. Such factors that make the correlations of CC and gm elusive are identified. CONCLUSIONS: For evaluation of the contribution of gm to recover CC, leaf life span is the key factor. The estimation of MC in relation to gm, especially in terms of costs required to regulate aquaporins, could be essential for efficient control of gm over the short term. Over the long term, costs are mainly reflected in CC, while benefits also include ultimate fitness attributes in terms of integrated carbon gain over the life of a leaf, plant survival and reproductive output.

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.

Escaping from the Cutoff Paradox by Accumulating Long-Chain Alcohols in the Cell Membrane.

The mechanism for the cutoff, an activity cliff at which long-chain alcohols lose their biological effects, has not been elucidated. Highly hydrophobic oleyl alcohol (C18:1) exists as a mixture of monomers and aggregated droplets in water. C18:1 did not inhibit the yeast growth but inhibited the growth of the slime mold without a cell wall. C18:1 exhibited toxicity to the yeast protoplast, which was enhanced by polyethylene glycol, a fusogen. Therefore, direct interactions of C18:1 with the membrane are crucial for the toxicity. The cutoff alcohols, C14 and C16, also exhibited strong toxicity obeying the Meyer-Overton correlation, in intact yeast cells whose membrane growth was suppressed in water. Taken together, the cutoff is avoidable by securing sufficient accumulation of the wall-permeable monomers in the membrane, which supports the lipid theory. It would be important to distinguish the effective drug structure localizing in the membrane and deal with the amount in the membrane.

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.

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.

Overexpression of both Rubisco and Rubisco activase rescues rice photosynthesis and biomass under heat stress.

Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially Rubisco activation. We examined whether co-overexpression of Rubisco and Rubisco activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a Rubisco activase-overexpressing line (oxRCA) and a Rubisco- and Rubisco activase-co-overexpressing line (oxRCA-RBCS)-and analysed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the Rubisco activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the Rubisco content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to Rubisco deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the Rubisco activase content without the reduction of Rubisco content could improve yield and sustainability in rice at high temperature.

Antimalarial Quinacrine and Chloroquine Lose Their Activity by Decreasing Cationic Amphiphilic Structure with a Slight Decrease in pH.

Quinacrine (QC) and chloroquine (CQ) have antimicrobial and antiviral activities as well as antimalarial activity, although the mechanisms remain unknown. QC increased the antimicrobial activity against yeast exponentially with a pH-dependent increase in the cationic amphiphilic drug (CAD) structure. CAD-QC localized in the yeast membranes and induced glucose starvation by noncompetitively inhibiting glucose uptake as antipsychotic chlorpromazine (CPZ) did. An exponential increase in antimicrobial activity with pH-dependent CAD formation was also observed for CQ, indicating that the CAD structure is crucial for its pharmacological activity. A decrease in CAD structure with a slight decrease in pH from 7.4 greatly reduced their effects; namely, these drugs would inefficiently act on falciparum malaria and COVID-19 pneumonia patients with acidosis, resulting in resistance. The decrease in CAD structure at physiological pH was not observed for quinine, primaquine, or mefloquine. Therefore, restoring the normal blood pH or using pH-insensitive quinoline drugs might be effective for these infectious diseases with acidosis.

The effect of different spectral light quality on the photoinhibition of Photosystem I in intact leaves.

Light energy causes damage to Photosystem I (PSI) and Photosystem II (PSII). The majority of the previous photoinhibition studies have been conducted with PSII, which shows much larger photoinhibition than PSI; therefore, relatively little is known about the mechanism of PSI photoinhibition so far. A previous report showed that the photoinhibition action spectrum measured with PSI activity of isolated thylakoid is similar to the absorption spectrum of chlorophyll. However, it is known that the extent of PSI photoinhibition is much smaller in vivo compared to in vitro. It is also possible that the different extent of PSII photoinhibition, caused by different spectral light qualities, can affect the photoinhibition of PSI in vivo because PSI receives electrons from PSII. In the present research, to study the effect of light quality and the effect of the extent of PSII photoinhibition on the PSI photoinhibition in vivo, intact leaves were photoinhibited under four different light qualities. The rate coefficient of PSI photoinhibition was significantly higher in blue and red light compared to white light. The rate of PSI photoinhibition at the same photon-exposure was the largest in blue and red light and followed by white and green light. These results support the notion that light absorption by chlorophyll is responsible for the PSI photoinhibition, even in intact leaves. The variation among light colors in the relationships between the extent of photoinhibition of PSII and that of PSI indicate that PSI and PSII are independently photoinhibited with different mechanisms in the early stage of in vivo photoinhibition.

Effects of root morphology, respiration and carboxylate exudation on carbon economy in two non-mycorrhizal lupines under phosphorus deficiency.

Under phosphorus (P) deficiency, Lupinus albus develops cluster roots that allow efficient P acquisition, while L. angustifolius without cluster roots also grows well. Both species are non-mycorrhizal. We quantitatively examined the carbon budgets to investigate the different strategies of these species. Biomass allocation, respiratory rates, protein amounts and carboxylate exudation rates were examined in hydroponically-grown plants treated with low (1 µM; P1) or high (100 µM; P100) P. At P1, L. albus formed cluster roots, and L. angustifolius increased biomass allocation to the roots. The respiratory rates of the roots were faster in L. albus than in L. angustifolius. The protein amounts of the non-phosphorylating alternative oxidase and uncoupling protein were greater in the cluster roots of L. albus at P1 than in the roots at P100, but similar between the P treatments in L. angustifolius roots. At P1, L. albus exuded carboxylates at a faster rate than L. angustifolius. The carbon budgets at P1 were surprisingly similar between the two species, which is attributed to the contrasting root growth and development strategies. L. albus developed cluster roots with rapid respiratory and carboxylate exudation rates, while L. angustifolius developed a larger root system with slow respiratory and exudation rates.

Increased Cuticle Permeability Caused by a New Allele of ACETYL-COA CARBOXYLASE1 Enhances CO2 Uptake.

Carbon dioxide (CO2) is an essential substrate for photosynthesis in plants. CO2 is absorbed mainly through the stomata in land plants because all other aerial surfaces are covered by a waxy layer called the cuticle. The cuticle is an important barrier that protects against extreme water loss; however, this anaerobic layer limits CO2 uptake. Simply, in the process of adapting to a terrestrial environment, plants have acquired drought tolerance in exchange for reduced CO2 uptake efficiency. To evaluate the extent to which increased cuticle permeability enhances CO2 uptake efficiency, we investigated the CO2 assimilation rate, carbon content, and dry weight of the Arabidopsis (Arabidopsis thaliana) mutant excessive transpiration1 (extra1), whose cuticle is remarkably permeable to water vapor. We isolated the mutant as a new allele of ACETYL-COA CARBOXYLASE1, encoding a critical enzyme for fatty acid synthesis, thereby affecting cuticle wax synthesis. Under saturated water vapor conditions, the extra1 mutant demonstrated a higher CO2 assimilation rate, carbon content, and greater dry weight than did the wild-type plant. On the other hand, the stomatal mutant slow-type anion channel-associated1, whose stomata are continuously open, also exhibited a higher CO2 assimilation rate than the wild-type plant; however, the increase was only half of the amount exhibited by extra1 These results indicate that the efficiency of CO2 uptake via a permeable cuticle is greater than the efficiency via stomata and confirm that land plants suffer a greater loss of CO2 uptake efficiency by developing a cuticle barrier.

A Decrease in Mesophyll Conductance by Cell-Wall Thickening Contributes to Photosynthetic Downregulation.

It has been argued that accumulation of nonstructural carbohydrates triggers a decrease in Rubisco content, which downregulates photosynthesis. However, a decrease in the sink-source ratio in several plant species leads to a decrease in photosynthesis and increases in both structural and nonstructural carbohydrate content. Here, we tested whether increases in cell-wall materials, rather than starch content, impact directly on photosynthesis by decreasing mesophyll conductance. We measured various morphological, anatomical, and physiological traits in primary leaves of soybean (Glycine max) and French bean (Phaseolus vulgaris) grown under high- or low-nitrogen conditions. We removed other leaves 2 weeks after sowing to decrease the sink-source ratio and conducted measurements 0, 1, and 2 weeks after defoliation.

Improved stomatal opening enhances photosynthetic rate and biomass production in fluctuating light.

It has been reported that stomatal conductance often limits the steady-state photosynthetic rate. On the other hand, the stomatal limitation of photosynthesis in fluctuating light remains largely unknown, although in nature light fluctuates due to changes in sun position, cloud cover, and the overshadowing canopy. In this study, we analysed three mutant lines of Arabidopsis with increased stomatal conductance to examine to what extent stomatal opening limits photosynthesis in fluctuating light. The slac1 (slow anion channel-associated 1) and ost1 (open stomata 1) mutants with stay-open stomata, and the PATROL1 (proton ATPase translocation control 1) overexpression line with faster stomatal opening responses exhibited higher photosynthetic rates and plant growth in fluctuating light than the wild-type, whereas these four lines showed similar photosynthetic rates and plant growth in constant light. The slac1 and ost1 mutants tended to keep their stomata open in fluctuating light, resulting in lower water-use efficiency (WUE) than the wild-type. However, the PATROL1 overexpression line closed stomata when needed and opened stomata immediately upon irradiation, resulting in similar WUE to the wild-type. The present study clearly shows that there is room to optimize stomatal responses, leading to greater photosynthesis and biomass accumulation in fluctuating light in nature.

Increased stomatal conductance induces rapid changes to photosynthetic rate in response to naturally fluctuating light conditions in rice.

A close correlation between stomatal conductance and the steady-state photosynthetic rate has been observed for diverse plant species under various environmental conditions. However, it remains unclear whether stomatal conductance is a major limiting factor for the photosynthetic rate under naturally fluctuating light conditions. We analysed a SLAC1 knockout rice line to examine the role of stomatal conductance in photosynthetic responses to fluctuating light. SLAC1 encodes a stomatal anion channel that regulates stomatal closure. Long exposures to weak light before treatments with strong light increased the photosynthetic induction time required for plants to reach a steady-state photosynthetic rate and also induced stomatal limitation of photosynthesis by restricting the diffusion of CO2 into leaves. The slac1 mutant exhibited a significantly higher rate of stomatal opening after an increase in irradiance than wild-type plants, leading to a higher rate of photosynthetic induction. Under natural conditions, in which irradiance levels are highly variable, the stomata of the slac1 mutant remained open to ensure efficient photosynthetic reaction. These observations reveal that stomatal conductance is important for regulating photosynthesis in rice plants in the natural environment with fluctuating light.

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.

Whole Irradiated Plant Leaves Showed Faster Photosynthetic Induction Than Individually Irradiated Leaves via Improved Stomatal Opening.

Rapid photosynthetic induction is crucial for plants under fluctuating light conditions in a crop canopy as well as in an understory. Most previous studies have focused on photosynthetic induction responses in a single leaf, whereas the systemic responses of the whole plant have not been considered. In a natural environment, however, both single leaves and whole plants are exposed to sunlight, since the light environment is not uniform even within a given plant. In the present study, we examined whether there is any difference between the photosynthetic induction response of a leaf of a whole irradiated plant and an individually irradiated leaf in Arabidopsis thaliana to consider photosynthetic induction as the response of a whole plant. We used two methods, the visualization of photosynthesis and direct measurements of gas-exchange and Chl fluorescence, to demonstrate that whole irradiated plant promoted its photosynthetic induction via improved stomatal opening compared with individually irradiated leaf. Furthermore, using two Arabidopsis knockout mutants of abscisic acid transporter, abcg25 and abcg40, the present study suggests that abscisic acid could be involved in this systemic response for stomatal opening, allowing plants to optimize the use of light energy at minimal cost in plants in a dynamic light environment.

Mitochondrial AOX Supports Redox Balance of Photosynthetic Electron Transport, Primary Metabolite Balance, and Growth in Arabidopsis thaliana under High Light.

When leaves receive excess light energy, excess reductants accumulate in chloroplasts. It is suggested that some of the reductants are oxidized by the mitochondrial respiratory chain. Alternative oxidase (AOX), a non-energy conserving terminal oxidase, was upregulated in the photosynthetic mutant of Arabidopsis thaliana, pgr5, which accumulated reductants in chloroplast stroma. AOX is suggested to have an important role in dissipating reductants under high light (HL) conditions, but its physiological importance and underlying mechanisms are not yet known. Here, we compared wild-type (WT), pgr5, and a double mutant of AOX1a-knockout plant (aox1a) and pgr5 (aox1a/pgr5) grown under high- and low-light conditions, and conducted physiological analyses. The net assimilation rate (NAR) was lower in aox1a/pgr5 than that in the other genotypes at the early growth stage, while the leaf area ratio was higher in aox1a/pgr5. We assessed detailed mechanisms in relation to NAR. In aox1a/pgr5, photosystem II parameters decreased under HL, whereas respiratory O2 uptake rates increased. Some intermediates in the tricarboxylic acid (TCA) cycle and Calvin cycle decreased in aox1a/pgr5, whereas γ-aminobutyric acid (GABA) and N-rich amino acids increased in aox1a/pgr5. Under HL, AOX may have an important role in dissipating excess reductants to prevent the reduction of photosynthetic electron transport and imbalance in primary metabolite levels.

Elevated CO2-induced changes in mesophyll conductance and anatomical traits in wild type and carbohydrate-metabolism mutants of Arabidopsis.

Decreases in photosynthetic rate, stomatal conductance (gs), and mesophyll conductance (gm) are often observed under elevated CO2 conditions. However, which anatomical and/or physiological factors contribute to the decrease in gm is not fully understood. Arabidopsis thaliana wild-type and carbon-metabolism mutants (gwd1, pgm1, and cfbp1) with different accumulation patterns of non-structural carbohydrates were grown at ambient (400 ppm) and elevated (800 ppm) CO2. Anatomical and physiological traits of leaves were measured to investigate factors causing the changes in gm and in the mesophyll resistance (expressed as the reciprocal of mesophyll conductance per unit chloroplast surface area facing to intercellular space, Sc/gm). When grown at elevated CO2, all the lines showed increases in cell wall mass, cell wall thickness, and starch content, but not in leaf thickness. gm measured at 800 ppm CO2 was significantly lower than at 400 ppm CO2 in all the lines. Changes in Sc/gm were associated with thicker cell walls rather than with excess starch content. The results indicate that the changes in gm and Sc/gm that occur in response to elevated CO2 are independent of non-structural carbohydrates, and the cell wall represents a greater limitation factor for gm than starch.