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.     

Ozone‐triggered rapid stomatal response involves the production of reactive oxygen species,and is controlled by SLAC1 and OST1

The air pollutant ozone can be used as a tool to unravel in planta processes induced by reactive oxygen species (ROS). Here, we have utilized ozone to study ROS‐dependent stomatal signaling. We show that the ozone‐triggered rapid transient decrease (RTD) in stomatal conductance coincided with a burst of ROS in guard cells. RTD was present in 11 different Arabidopsis ecotypes, suggesting that it is a genetically robust response. To study which signaling components or ion channels were involved in RTD, we tested 44 mutants deficient in various aspects of stomatal function. This revealed that the SLAC1 protein, essential for guard cell plasma membrane S‐type anion channel function, and the protein kinase OST1 were required for the ROS‐induced fast stomatal closure. We showed a physical interaction between OST1 and SLAC1, and provide evidence that SLAC1 is phosphorylated by OST1. Phosphoproteomic experiments indicated that OST1 phosphorylated multiple amino acids in the N terminus of SLAC1. Using TILLING we identified three new slac1 alleles where predicted phosphosites were mutated. The lack of RTD in two of them, slac1‐7 (S120F) and slac1‐8 (S146F), suggested that these serine residues were important for the activation of SLAC1. Mass‐spectrometry analysis combined with site‐directed mutagenesis and phosphorylation assays, however, showed that only S120 was a specific phosphorylation site for OST1. The absence of the RTD in the dominant‐negative mutants abi1‐1 and abi2‐1 also suggested a regulatory role for the protein phosphatases ABI1 and ABI2 in the ROS‐induced activation of the S‐type anion channel.     

Mutation of the rice TCM12 gene encoding 2,3‐bisphosphoglycerate‐independent phosphoglycerate mutase affects chlorophyll synthesis,photosynthesis and chloroplast development at seedling stage at low temperatures

• Glycolysis is a central metabolic pathway that provides energy and products of primary metabolites. 2,3‐Biphosphoglycerate‐independent phosphoglycerate mutase (iPGAM) is a key enzyme that catalyses the reversible interconversion of 3‐phosphoglycerate (3‐PGA) to 2‐phosphoglycerate (2‐PGA) in glycolysis. Low temperature is a common abiotic stress in rice production. However, the mechanism for rice iPGAM genes is not fully understood at low temperature.
• In this study, the rice mutant tcm12, with chlorosis, malformed chloroplasts and impaired photosynthesis, was grown at a low temperature (<20 °C) to the three‐leaf stage, while the normal phenotype at 32 °C was used. Chlorophyll fluorescence analysis and transmission electron microscopy were used to examine features of the tcm12 mutant. The inheritance behaviour and function of TCM12 were then analysed thorough map‐based cloning, transgenic complementation and subcellular localisation.
• The thermo‐sensitive chlorosis phenotype was caused by a single nucleotide mutation (T→C) on the fifth exon of TCM12 (LOC_Os12g35040) encoding iPGAM, localised to both nucleus and membranes. In addition, TCM12 was constitutively expressed, and its disruption resulted in down‐regulation of some genes associated with chlorophyll biosynthesis and photosynthesis at low temperatures (20 °C).
• This is the first report of the involvement of rice iPGAM gene in chlorophyll synthesis, photosynthesis and chloroplast development, providing new insights into the mechanisms underlying early growth of rice at low temperatures.

     

Diffusional conductance to CO2 is the key limitation to photosynthesis in salt‐stressed leaves of rice (Oryza sativa)

Salinity significantly limits leaf photosynthesis but the factors causing the limitation in salt‐stressed leaves remain unclear. In the present work, photosynthetic and biochemical traits were investigated in four rice genotypes under two NaCl concentration (0 and 150 mM) to assess the stomatal, mesophyll and biochemical contributions to reduced photosynthetic rate (A) in salt‐stressed leaves. Our results indicated that salinity led to a decrease in A, leaf osmotic potential, electron transport rate and CO₂ concentrations in the chloroplasts (C_c) of rice leaves. Decreased A in salt‐stressed leaves was mainly attributable to low C_c, which was determined by stomatal and mesophyll conductance. The increased stomatal limitation was mainly related to the low leaf osmotic potential caused by soil salinity. However, the increased mesophyll limitation in salt‐stressed leaves was related to both osmotic stress and ion stress. These findings highlight the importance of considering mesophyll conductance when developing salinity‐tolerant rice cultivars.     

Non‐invasive,whole‐plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants

Leaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high‐sensitivity, non‐invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image‐based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less‐mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1‐5 phot2‐1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual‐imaging platform also allowed us to develop a straightforward approach to correct non‐photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy‐dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the overall photosynthetic performance of higher plants.     

Three‐dimensional microscale modelling of CO2 transport and light propagation in tomato leaves enlightens photosynthesis

We present a combined three‐dimensional (3‐D) model of light propagation, CO₂ diffusion and photosynthesis in tomato (Solanum lycopersicum L.) leaves. The model incorporates a geometrical representation of the actual leaf microstructure that we obtained with synchrotron radiation X‐ray laminography, and was evaluated using measurements of gas exchange and leaf optical properties. The combination of the 3‐D microstructure of leaf tissue and chloroplast movement induced by changes in light intensity affects the simulated CO₂ transport within the leaf. The model predicts extensive reassimilation of CO₂ produced by respiration and photorespiration. Simulations also suggest that carbonic anhydrase could enhance photosynthesis at low CO₂ levels but had little impact on photosynthesis at high CO₂ levels. The model confirms that scaling of photosynthetic capacity with absorbed light would improve efficiency of CO₂ fixation in the leaf, especially at low light intensity.     

Proteomic analysis of rice seedlings infected by Sinorhizobium meliloti 1021

Rhizobial endophytes infect and colonize not only leguminous plants, but several non‐leguminous species as well. Using green fluorescent protein tagging technique, it has been shown that Rhizobia infect different varieties of rice species and migrate from plant roots to aerial tissues such as leaf sheaths and leaves. The interaction between them was found to promote the growth of rice. The growth promotion is the cumulative result of enhanced photosynthesis and stress resistance. In addition, indole‐3‐acetic acid also contributes to the promotion. Gel‐based comparative proteomic approaches were applied to analyze the protein profiles of three different tissues (root, leaf sheath and leaf) of Sinorhizobium meliloti 1021 inoculated rice in order to get an understanding about the molecular mechanism. Upon the inoculation of rhizobia, proteins involved in nine different functional categories were either up‐regulated or down‐regulated. Photosynthesis related proteins were up‐regulated only in leaf sheath and leaf, while the up‐regulated proteins in root were exclusively defense related. The results implied that there might have been an increase in the import and transport of proteins involved in light and dark reactions to the chloroplast as well as more efficient distribution of nutrients, hence enhanced photosynthesis. Although the initiation of defensive reactions mainly occurred in roots, some different defense mechanisms were also evoked in the aerial tissues.     

Mutation of the chloroplast‐localized phosphate transporter OsPHT2;1 reduces flavonoid accumulation and UV tolerance in rice

Phosphorus (P) is an essential macronutrient required for plant development and production. The mechanisms regulating phosphate (Pi) uptake are well established, but the function of chloroplast Pi homeostasis is poorly understood in Oryza sativa (rice). PHT2;1 is one of the transporters/translocators mediating Pi import into chloroplasts. In this study, to gain insight into the role of OsPHT2;1‐mediated stroma Pi, we analyzed OsPHT2;1 function in Pi utilization and photoprotection. Our results showed that OsPHT2;1 was induced by Pi starvation and light exposure. Cell‐based assays showed that OsPHT2;1 localized to the chloroplast envelope and functioned as a low‐affinity Pi transporter. The ospht2;1 had reduced Pi accumulation, plant growth and photosynthetic rates. Metabolite profiling revealed that 52.6% of the decreased metabolites in ospht2;1 plants were flavonoids, which was further confirmed by 40% lower content of total flavonoids compared with the wild type. As a consequence, ospht2;1 plants were more sensitive to UV‐B irradiation. Moreover, the content of phenylalanine, the precursor of flavonoids, was also reduced, and was largely associated with the repressed expression of ADT1/MTR1. Furthermore, the ospht2;1 plants showed decreased grain yields at relatively high levels of UV‐B irradiance. In summary, OsPHT2;1 functions as a chloroplast‐localized low‐affinity Pi transporter that mediates UV tolerance and rice yields at different latitudes.     

C4 photosynthesis in C3 rice: a theoretical analysis of biochemical and anatomical factors

Engineering C₄ photosynthesis into rice has been considered a promising strategy to increase photosynthesis and yield. A question that remains to be answered is whether expressing a C₄ metabolic cycle into a C₃ leaf structure and without removing the C₃ background metabolism improves photosynthetic efficiency. To explore this question, we developed a 3D reaction diffusion model of bundle‐sheath and connected mesophyll cells in a C₃ rice leaf. Our results show that integrating a C₄ metabolic pathway into rice leaves with a C₃ metabolism and mesophyll structure may lead to an improved photosynthesis under current ambient CO₂ concentration. We analysed a number of physiological factors that influence the CO₂ uptake rate, which include the chloroplast surface area exposed to intercellular air space, bundle‐sheath cell wall thickness, bundle‐sheath chloroplast envelope permeability, Rubisco concentration and the energy partitioning between C₃ and C₄ cycles. Among these, partitioning of energy between C₃ and C₄ photosynthesis and the partitioning of Rubisco between mesophyll and bundle‐sheath cells are decisive factors controlling photosynthetic efficiency in an engineered C₃–C₄ leaf. The implications of the results for the sequence of C₄ evolution are also discussed.     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

The making of a chloroplast

Since its endosymbiotic beginning, the chloroplast has become fully integrated into the biology of the host eukaryotic cell. The exchange of genetic information from the chloroplast to the nucleus has resulted in considerable co‐ordination in the activities of these two organelles during all stages of plant development. Here, we give an overview of the mechanisms of light perception and the subsequent regulation of nuclear gene expression in the model plant Arabidopsis thaliana, and we cover the main events that take place when proplastids differentiate into chloroplasts. We also consider recent findings regarding signalling networks between the chloroplast and the nucleus during seedling development, and how these signals are modulated by light. In addition, we discuss the mechanisms through which chloroplasts develop in different cell types, namely cotyledons and the dimorphic chloroplasts of the C₄ plant maize. Finally, we discuss recent data that suggest the specific regulation of the light‐dependent phases of photosynthesis, providing a means to optimize photosynthesis to varying light regimes.     

The acclimation of leaf photosynthesis of wheat and rice to seasonal temperature changes in T‐FACE environments

Crops show considerable capacity to adjust their photosynthetic characteristics to seasonal changes in temperature. However, how photosynthesis acclimates to changes in seasonal temperature under future climate conditions has not been revealed. We measured leaf photosynthesis (A_n) of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) grown under four combinations of two levels of CO₂ (ambient and enriched up to 500 µmol/mol) and two levels of canopy temperature (ambient and increased by 1.5–2.0°C) in temperature by free‐air CO₂ enrichment (T‐FACE) systems. Parameters of a biochemical C₃‐photosynthesis model and of a stomatal conductance (g_s) model were estimated for the four conditions and for several crop stages. Some biochemical parameters related to electron transport and most g_s parameters showed acclimation to seasonal growth temperature in both crops. The acclimation response did not differ much between wheat and rice, nor among the four treatments of the T‐FACE systems, when the difference in the seasonal growth temperature was accounted for. The relationships between biochemical parameters and leaf nitrogen content were consistent across leaf ranks, developmental stages, and treatment conditions. The acclimation had a strong impact on g_s model parameters: when parameter values of a particular stage were used, the model failed to correctly estimate g_s values of other stages. Further analysis using the coupled g_s–biochemical photosynthesis model showed that ignoring the acclimation effect did not result in critical errors in estimating leaf photosynthesis under future climate, as long as parameter values were measured or derived from data obtained before flowering.     

LALF32‐51‐E7, a HPV‐16 therapeutic vaccine candidate,forms protein body‐like structures when expressed in Nicotiana benthamiana leaves

High‐risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV‐16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF_32‐51), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF_32‐51‐E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF_32‐51‐E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF_32‐51‐E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast‐targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB‐like structures were also seen for the cytoplasmic LG. PB‐like structure formation was confirmed for both LG and LALF_32‐51‐E7 by TEM. LALF_32‐51‐E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB‐like structures. This study could open a new avenue for the use of LALF_32‐51 as a PB‐inducing peptide.     

Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation‐induced genotoxicity was investigated via gain‐of‐functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast‐targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild‐type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV‐treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV‐C radiation, the dark‐grown E. coli RecA‐overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation‐induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV‐C radiation‐induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA‐overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.     

Retracted: Each of the chloroplast potassium efflux antiporters affects photosynthesis and growth of fully developed Arabidopsis rosettes under short‐day photoperiod

In Arabidopsis thaliana, the chloroplast harbors three potassium efflux antiporters (KEAs), namely KEA1 and KEA2 in the inner envelope and KEA3 in the thylakoid membrane. They may play redundant physiological roles as in our previous analyses of young developing Arabidopsis rosettes under long‐day photoperiod (16 h light per day), chloroplast kea single mutants resembled the wild‐type plants, whereas kea1kea2 and kea1kea2kea3 mutants were impaired in chloroplast development and photosynthesis resulting in stunted growth. Here, we aimed to study whether chloroplast KEAs play redundant roles in chloroplast function of older Arabidopsis plants with fully developed rosettes grown under short‐day photoperiod (8 h light per day). Under these conditions, we found defects in photosynthesis and growth in the chloroplast kea single mutants, and most dramatic defects in the kea1kea2 double mutant. The mechanism behind these defects in the single mutants involves reduction in the electron transport rate (kea1 and kea3), and stomata conductance (kea1, kea2 and kea3), which in turn affect CO₂ fixation rates. The kea1kea2 mutant, in addition to these alterations, displayed reduced levels of photosynthetic machinery. Taken together, our data suggest that, in addition to the previously reported roles in chloroplast development in young rosettes, each chloroplast KEA affects photosynthesis and growth of Arabidopsis fully developed rosettes.