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Volatile compounds (VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin (CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Herbivore selection of plant hosts and plant responses to insect colonization have been subjects of intense investigations. A growing body of evidence suggests that, for successful colonization to occur, (effector/virulence) proteins in insect saliva must modulate plant defense responses to the benefit of the insect. A range of insect saliva proteins that modulate plant defense responses have been identified, but there is no direct evidence that these proteins are delivered into specific plant tissues and enter plant cells. Aphids and other sap-sucking insects of the order Hemiptera use their specialized mouthparts (stylets) to probe plant mesophyll cells until they reach the phloem cells for long-term feeding. Here, we show, by immunogold-labeling of ultrathin sections of aphid feeding sites, that an immuno-suppressive aphid effector localizes in the cytoplasm of mesophyll cells near aphid stylets but not in cells further away from aphid feeding sites. In contrast, another aphid effector protein localizes in the sheaths composed of gelling saliva that surround the aphid stylets. Thus, insects deliver effectors directly into plant tissue. Moreover, different aphid effectors locate extracellularly in the sheath saliva or are introduced into the cytoplasm of plant cells. [Formula: see text] Copyright © 2016 The Author(s). This is an open-access article distributed under the CC BY-NC-ND 4.0 International license .

CO 2 -responsive CCT protein (CRCT) is the suggested positive regulator of starch synthesis in vegetative organs, particularly the leaf sheath of rice. In this study, we analyzed the effects of the starch level in the leaf sheath on the photosynthetic rate in the leaf blade using CRCT overexpression and RNA interference (RNAi) knockdown transgenic rice grown under ambient (38 Pa) or elevated (100 Pa) CO 2 conditions. In leaf sheath, the starch content was markedly changed in relation to CRCT expression levels under both CO 2 conditions. In contrast, the soluble sugar and starch contents of the leaf blade were markedly increased in the knockdown line grown under elevated CO 2 conditions. The overexpression or RNAi knockdown of CRCT did not cause large effects on the photosynthetic rate of the transgenic lines grown under ambient CO 2 condition. However, the photosynthetic rate of the overexpression line was enhanced, while that of the knockdown line was substantially decreased under elevated CO 2 conditions. These photosynthetic rates were weakly correlated with the nitrogen contents and negatively correlated with the total non-structural carbohydrate contents. Thus, the capacity for starch synthesis in leaf sheath, which is controlled by CRCT, can indirectly affect the carbohydrate content, and then the photosynthetic rate in the leaf blade of rice grown under elevated CO 2 conditions., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Leaves exposed to potassium (K) deficiency usually present decreased mesophyll conductance (g m ) and photosynthesis (A). The relative contributions of leaf anatomical traits in determining g m have been quantified; however, anatomical variabilities related to low g m under K starvation remain imperfectly known. A one-dimensional model was used to quantify anatomical controls of the entire CO 2 diffusion pathway resistance within a leaf on two Brassica napus L. cultivars in response to K deficiency. Leaf photosynthesis of both cultivars was significantly decreased under K deficiency in parallel with down-regulated g m . The mesophyll conductance limitation contributed to more than one-half of A decline. The decreased internal air space in K-starved leaves was associated with the increase of gas-phase resistance. Potassium deficiency reduced liquid-phase conductance by decreasing the exposed surface area of chloroplasts per unit leaf area (S c /S), and enlarging the resistance of the cytoplasm that can be interpreted by the increasing distance of chloroplast from cell wall, and between adjacent chloroplasts. Additionally, the discrepancies of A between two cultivars were in part because of g m variations, ascribing to an altered S c /S. These results emphasize the important role of K on the regulation of g m by enhancing S c /S and reducing cytoplasm resistance., (© 2016 John Wiley & Sons Ltd.)

Increased mesophyll conductance (gm) has been suggested as a target for selection for high productivity and high water-use efficiency in crop plants, and genotypic variability in gm has been reported in several important crop species. However, effective selection requires an understanding of how gm varies with growth conditions, to ensure that the ranking of genotypes is consistent across environments. We assessed the genotypic variability in gm and other leaf gas exchange traits, as well as growth and biomass allocation for six wheat genotypes under different water and nitrogen availabilities. The wheat genotypes differed in their response of gm to growth conditions, resulting in genotypic differences in the mesophyll limitation to photosynthesis and a significant increase in the mesophyll limitation to photosynthesis under drought. In this experiment, leaf intrinsic water-use efficiency was more closely related to stomatal conductance than to mesophyll conductance, and stomatal limitation to photosynthesis increased more in some genotypes than in others in response to drought. Screening for gm should be carried out under a range of growth conditions., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Plants are simultaneously exposed to a combination of biotic and abiotic stresses in field conditions. Crops respond to the combined stress in a unique way which cannot be understood by extrapolating the results of individual stress. In the present study, effects of individual and combined stress of herbicide (2,4-dichlorophenoxyacetic acid) and salinity (NaCl) on two Oryza sativa cultivars (ZJ 88 and XS 134) were investigated. Both herbicide and saline stress affected the plant growth differentially and produced oxidative stress in rice cultivars. Interestingly, the combination of herbicide and salinity showed a significant protection to both rice cultivars by reducing ROS (H2O2, O2(-)) and lipid peroxidation through modulation of enzymatic (SOD, POD, CAT and APX) and non-enzymatic (TSP, sugars, phenolic and proline) antioxidants. In addition, active regulation of transcript levels of genes encoding Na(+) and K(+) (OsHKT1;5, OsLti6a,b, OsHKT2;1, OsSOS1, OsCNGC1, OsNHX1 and OsAKT1) transporter proteins reduced sodium and enhanced potassium accumulation under combined stress, resulted a better growth and ionic homeostasis in both rice cultivars. The production of ABA and IAA was significantly higher in cultivar XS 134 compared to cultivar ZJ 88 under control conditions. However, combined herbicide and saline stress enhanced the accumulation of phytohormones (IAA and ABA) and transcription of ethylene in cultivar ZJ 88, which might be one of the factors responsible for poor salt tolerance in sensitive cultivar. These findings indicated that herbicide application under saline stress confers tolerance to salinity in rice cultivars, likely by reducing oxidative damage, modulating mineral absorption, upgradation of antioxidant defense and by dynamic regulation of key genes involved in Na(+) and K(+) homeostasis in plants., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

We studied the effects of cold stress (5°C) and re-warming (25°C) on gas exchange, photosystem II, key photosynthetic enzyme activities, gene expression, and carbohydrate metabolite concentrations in two Elymus nutans genotypes differing in cold resistance (DX, cold-tolerant and ZD, cold-sensitive). Cold stress led to irreversible reductions in photosynthetic rate. This reduction was accompanied by declining stomatal and mesophyll conductance (gs and gm), transpiration rate (Tr) and photochemical efficiency in both genotypes, however there were smaller decreases in DX than in ZD. Cold-tolerant DX maintained higher photosynthetic enzyme activities and transcript levels, as well as higher reducing sugar concentrations and sucrose accumulation. The relationship between Pn and internal leaf CO2 concentration (Pn/Ci curve) during cold and re-warming was analyzed to estimate the relative influence of stomatal and non-stomatal components on photosynthesis. Stomatal limitation, non-stomatal limitation, and CO2 compensation point (CP) increased in both genotypes under cold stress, but to a lesser extent in DX. Maximum CO2 assimilation rate (Pmax), and carboxylation efficiency (CE) declined, but DX had significantly higher levels of Pmax and CE than ZD. Following cold-stress recovery, the maximum quantum yield of PSII (Fv/Fm), apparent electron transport rate (ETR), Rubisco activity, Rubisco activation state and CE in DX resumed to the control levels. In contrast, Pn, Pmax, gs, gm, and Tr recovered only partially for DX, suggesting that incomplete recovery of photosynthesis in DX may be mainly related to diffusion limitations. Higher Rubisco large subunit (RbcL) and Rubisco activase (RCA) transcript levels, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, and carbohydrate accumulation contributed to higher photosynthetic recovery in DX. These results indicate that the maintenance of higher Pn and Pmax under cold stress and recovery in cold-tolerant DX could be attributed to reduced diffusion limitations and rapid recuperation of metabolic factors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

There is an urgent need for simultaneously increasing photosynthesis/yields and water use efficiency (WUE) in C3 crops. Potentially, this can be achieved by genetic manipulation of the key traits involved. However, despite significant efforts in the past two decades very limited success has been achieved. Here I argue that this is mostly due to the fact that single gene/single trait approaches have been used thus far. Photosynthesis models demonstrate that only limited improving of photosynthesis can be expected by large improvements of any of its single limiting factors, i.e. stomatal conductance, mesophyll conductance, and the biochemical capacity for photosynthesis, the latter co-limited by Rubisco and the orchestrated activity of thylakoid electron transport and the Calvin cycle enzymes. Accordingly, only limited improvements of photosynthesis have been obtained by genetic manipulation of any of these single factors. In addition, improving photosynthesis by genetic manipulation in general reduced WUE, and vice-versa, and in many cases pleiotropic effects appear that cancel out some of the expected benefits. I propose that success in genetic manipulation for simultaneous improvement of photosynthesis and WUE efficiency may take longer than suggested in previous reports, and that it can be achieved only by joint projects addressing multi-gene manipulation for simultaneous alterations of all the limiting factors of photosynthesis, including the often neglected phloem capacity for loading and transport the expected surplus of carbohydrates in plants with improved photosynthesis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

During the transition from seed to seedling, emerging embryos strategically balance available resources between building up defenses against environmental threats and initiating the developmental program that promotes the switch to autotrophy. We present evidence of a critical role for the phenylalanine (Phe) biosynthetic activity of AROGENATE DEHYDRATASE3 (ADT3) in coordinating reactive oxygen species (ROS) homeostasis and cotyledon development in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. We show that ADT3 is expressed in the cotyledon and shoot apical meristem, mainly in the cytosol, and that the epidermis of adt3 cotyledons contains higher levels of ROS Genome-wide proteomics of the adt3 mutant revealed a general down-regulation of plastidic proteins and ROS-scavenging enzymes, corroborating the hypothesis that the ADT3 supply of Phe is required to control ROS concentration and distribution to protect cellular components. In addition, loss of ADT3 disrupts cotyledon epidermal patterning by affecting the number and expansion of pavement cells and stomata cell fate specification; we also observed severe alterations in mesophyll cells, which lack oil bodies and normal plastids. Interestingly, up-regulation of the pathway leading to cuticle production is accompanied by an abnormal cuticle structure and/or deposition in the adt3 mutant. Such impairment results in an increase in cell permeability and provides a link to understand the cell defects in the adt3 cotyledon epidermis. We suggest an additional role of Phe in supplying nutrients to the young seedling., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

A field experiment was performed to study the effects of waterlogging on the leaf mesophyll cell ultrastructure, chlorophyll content, gas exchange parameters, chlorophyll fluorescence, and malondialdehyde (MDA) content of summer maize (Zea mays L.) hybrids Denghai605 (DH605) and Zhengdan958 (ZD958). The waterlogging treatments were implemented for different durations (3 and 6 days) at the third leaf stage (V3), the sixth leaf stage (V6), and the 10th day after the tasseling stage (10VT). Leaf area index (LAI), chlorophyll content, photosynthetic rate (Pn), and actual photochemical efficiency (ΦPSII) were reduced after waterlogging, indicating that waterlogging significantly decreased photosynthetic capacity. The chloroplast shapes changed from long and oval to elliptical or circular after waterlogging. In addition, the internal structures of chloroplasts were degenerated after waterlogging. After waterlogging for 6 d at V3, the number of grana and grana lamellae of the third expanded leaf in DH605 were decreased by 26.83% and 55.95%, respectively, compared to the control (CK). Those in ZD958 were reduced by 30.08% and 31.94%, respectively. Waterlogging increased MDA content in both hybrids, suggesting an impact of waterlogging on membrane integrity and thus membrane deterioration. Waterlogging also damaged the biological membrane structure and mitochondria. Our results indicated that the physiological reactions to waterlogging were closely related to lower LAI, chlorophyll content, and Pn and to the destruction of chloroplast ultrastructure. These negative effects resulted in the decrease of grain yield in response to waterlogging. Summer maize was the most susceptible to damage when waterlogging occurred at V3, followed by V6 and 10VT, with damage increasing in the wake of waterlogging duration increasing., Competing Interests: The authors have declared that no competing interests exist.

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The control of stomatal aperture involves reversible changes in the concentration of osmolytes in guard cells. Sucrose has long been proposed to have an osmolytic role in guard cells. However, direct evidence for such a role is lacking. Furthermore, recent evidence suggests that sucrose may perform additional roles in guard cells. Here, we provide an update covering the multiple roles of sucrose in guard cell regulation, highlighting the knowledge accumulated regarding spatiotemporal differences in the synthesis, accumulation, and degradation of sucrose as well as reviewing the role of sucrose as a metabolic connector between mesophyll and guard cells. Analysis of transcriptomic data from previous studies reveals that several genes encoding sucrose and hexose transporters and genes involved in gluconeogenesis, sucrose and trehalose metabolism are highly expressed in guard cells compared with mesophyll cells. Interestingly, this analysis also showed that guard cells have considerably higher expression of C4 -marker genes than mesophyll cells. We discuss the possible roles of these genes in guard cell function and the role of sucrose in stomatal opening and closure. Finally, we provide a perspective for future experiments which are required to fill gaps in our understanding of both guard cell metabolism and stomatal regulation., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Phytochromes recognize light signals and control diverse developmental processes. In rice, all three phytochrome genes-OsphyA, OsphyB, and OsphyC-are involved in regulating flowering time. We investigated the role of OsPhyA by comparing the osphyA osphyB double mutant to an osphyB single mutant. Plants of the double mutant flowered later than the single under short days (SD) but bolted earlier under long days (LD). Under SD, this delayed-flowering phenotype was primarily due to the decreased expression of Oryza sativa GIGANTEA (OsGI), which controls three flowering activators: Heading date 1 (Hd1), OsMADS51, and Oryza sativa Indeterminate 1 (OsId1). Under LD, although the expression of several repressors, e.g., Hd1, Oryza sativa CONSTANS-like 4 (OsCOL4), and AP2 genes, was affected in the double mutant, that of Grain number, plant height and heading date 7 (Ghd7) was the most significantly reduced. These results indicated that OsPhyA influences flowering time mainly by affecting the expression of OsGI under SD and Ghd7 under LD when phytochrome B is absent. We also demonstrated that far-red light delays flowering time via both OsPhyA and OsPhyB.

The photoreceptor phytochrome A acts as a light-dependent molecular switch and regulates responses initiated by very low fluences of light (VLFR) and high fluences (HIR) of far-red light. PhyA is expressed ubiquitously, but how phyA signaling is orchestrated to regulate photomorphogenesis is poorly understood. To address this issue, we generated transgenic Arabidopsis thaliana phyA-201 mutant lines expressing the biologically active phyA-YFP photoreceptor in different tissues, and analyzed the expression of several reporter genes, including ProHY5:HY5-GFP and Pro35S:CFP-PIF1, and various FR-HIR-dependent physiological responses. We show that phyA action in one tissue is critical and sufficient to regulate flowering time and root growth; control of cotyledon and hypocotyl growth requires simultaneous phyA activity in different tissues; and changes detected in the expression of reporters are not restricted to phyA-containing cells. We conclude that FR-HIR-controlled morphogenesis in Arabidopsis is mediated partly by tissue-specific and partly by intercellular signaling initiated by phyA. Intercellular signaling is critical for many FR-HIR induced responses, yet it appears that phyA modulates the abundance and activity of key regulatory transcription factors in a tissue-autonomous fashion., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (<0.5 μm), some previously studied by electron microscopy and speculated to be for cuticle formation. In vanilla leaves, transcripts of oleosins of the U lineage were present in both epidermis and mesophyll, but oleosin occurred only in epidermis. Immuno-confocal laser scanning microscopy revealed that the LDs were coated with oleosins. LDs in isolated fractions did not coalesce, and the fractions contained heterogeneous proteins including oleosins and diverse lipids. These findings reflect the in situ structure and possible functions of the LDs. Fruit mesocarp of avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 μm) and at their periphery, numerous small LDs (<0.5 μm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Photosynthesis occurs in mesophyll cells of specialized organs such as leaves. The rigid cell wall encapsulating photosynthetic cells controls the expansion and distribution of cells within photosynthetic tissues. The relationship between photosynthesis and plant growth is affected by leaf area. However, the underlying genetic mechanisms affecting carbon partitioning to different aspects of leaf growth are not known. To fill this gap, we analyzed Arabidopsis plants with altered levels of pectin methylesterification, which is known to modulate cell wall plasticity and plant growth. Pectin methylesterification levels were varied through manipulation of cotton Golgi-related (CGR) 2 or 3 genes encoding two functionally redundant pectin methyltransferases. Increased levels of methylesterification in a line over-expressing CGR2 (CGR2OX) resulted in highly expanded leaves with enhanced intercellular air spaces; reduced methylesterification in a mutant lacking both CGR-genes 2 and 3 (cgr2/3) resulted in thin but dense leaf mesophyll that limited CO2 diffusion to chloroplasts. Leaf, root, and plant dry weight were enhanced in CGR2OX but decreased in cgr2/3. Differences in growth between wild type and the CGR-mutants can be explained by carbon partitioning but not by variations in area-based photosynthesis. Therefore, photosynthesis drives growth through alterations in carbon partitioning to new leaf area growth and leaf mass per unit leaf area; however, CGR-mediated pectin methylesterification acts as a primary factor in this relationship through modulation of the expansion and positioning of the cells in leaves, which in turn drive carbon partitioning by generating dynamic carbon demands in leaf area growth and leaf mass per unit leaf area., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

C4 photosynthesis is characterized by a CO2-concentrating mechanism between mesophyll (M) and bundle sheath (BS) cells of leaves. This generates high metabolic fluxes between these cells, through interconnecting plasmodesmata (PD). Quantification of these symplastic fluxes for modeling studies requires accurate quantification of PD, which has proven difficult using transmission electron microscopy. Our new quantitative technique combines scanning electron microscopy and 3D immunolocalization in intact leaf tissues to compare PD density on cell interfaces in leaves of C3 (rice [Oryza sativa] and wheat [Triticum aestivum]) and C4 (maize [Zea mays] and Setaria viridis) monocot species. Scanning electron microscopy quantification of PD density revealed that C4 species had approximately twice the number of PD per pitfield area compared with their C3 counterparts. 3D immunolocalization of callose at pitfields using confocal microscopy showed that pitfield area per M-BS interface area was 5 times greater in C4 species. Thus, the two C4 species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD µm(-2); maize, 7.5 PD µm(-2); rice 1.0 PD µm(-2); wheat, 2.6 PD µm(-2)). Using these anatomical data and measured photosynthetic rates in these C4 species, we have now calculated symplastic C4 acid flux per PD across the M-BS interface. These quantitative data are essential for modeling studies and gene discovery strategies needed to introduce aspects of C4 photosynthesis to C3 crops., (© 2016 American Society of Plant Biologists. All rights reserved.)

Plant metabolism drives plant development and plant-environment responses, and data readouts from this cellular level could provide insights in the underlying molecular processes. Existing studies have already related key in vivo leaf gas-exchange parameters with structural traits and nutrient components across multiple species. However, insights in the relationships of leaf gas-exchange with leaf primary metabolism are still limited. We investigated these relationships through a multispecies meta-analysis approach based on data sets from 17 published studies describing net photosynthesis (A) and stomatal (gs) and mesophyll (gm) conductances, alongside the 53 data profiles from primary metabolism of 14 species grown in different experiments. Modeling results highlighted the conserved patterns between the different species. Consideration of species-specific effects increased the explanatory power of the models for some metabolites, including Glc-6-P, Fru-6-P, malate, fumarate, Xyl, and ribose. Significant relationships of A with sugars and phosphorylated intermediates were observed. While gs was related to sugars, organic acids, myo-inositol, and shikimate, gm showed a more complex pattern in comparison to the two other traits. Some metabolites, such as malate and Man, appeared in the models for both conductances, suggesting a metabolic coregulation between gs and gm The resulting statistical models provide the first hints for coregulation patterns involving primary metabolism plus leaf water and carbon balances that are conserved across plant species, as well as species-specific trends that can be used to determine new biotechnological targets for crop improvement., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Drought slows net photosynthetic rate (AN) but increases water use efficiency (WUE). Farmers give an artificial drought pretreatment to some crops in the early growth stage and find that yield increases accompanying with the improvement of WUE. We conducted well-watered, non-drought, mild drought and moderate drought pretreatments of potted cotton cultivars. The aims of the present study were to analyse the importance of mesophyll conductance (gm) as a factor that may simultaneously improve AN and WUE under drought pretreatment conditions, and to analyse the role of anatomical structure and biochemical mechanism in the variability of gm. Our results showed that significant variability of gm estimated by gas exchange and chlorophyll fluorescence was observed between non-drought pretreatment and drought pretreatment associated with change in AN and WUE. There was great difference in anatomical structure and expression of aquaporins (GhAQP1) among all the treatments. In addition, expression of carbonic anhydrase (CA) may not be important in the regulation of gm under drought pretreatment conditions. We concluded that the variability of gm offers a potential target for improving leaf AN and WUE simultaneously by the regulation of anatomical structure and GhAQP1., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Mesophyll conductance significantly, and variably, limits photosynthesis but we currently have no reliable method of measurement for C4 plants. An online oxygen isotope technique was developed to allow quantification of mesophyll conductance in C4 plants and to provide an alternative estimate in C3 plants. The technique is compared to an established carbon isotope method in three C3 species. Mesophyll conductance of C4 species was similar to that in the C3 species measured, and declined in both C4 and C3 species as leaves aged from fully expanded to senescing. In cotton leaves, simultaneous measurement of carbon and oxygen isotope discrimination allowed the partitioning of total conductance to the chloroplasts into cell wall and plasma membrane versus chloroplast membrane components, if CO2 was assumed to be isotopically equilibrated with cytosolic water, and the partitioning remained stable with leaf age. The oxygen isotope technique allowed estimation of mesophyll conductance in C4 plants and, when combined with well-established carbon isotope techniques, may provide additional information on mesophyll conductance in C3 plants., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Engineering C3 plants to increase their efficiency of carbon fixation as well as of nitrogen and water use simultaneously may be facilitated by understanding the mechanisms that underpin the C4 syndrome. Existing experimental studies have indicated that the emergence of the C4 syndrome requires co-ordination between several levels of cellular organization, from gene regulation to metabolism, across two co-operating cell systems-mesophyll and bundle sheath cells. Yet, determining the extent to which the structure of the C4 plant metabolic network may constrain gene expression remains unclear, although it will provide an important consideration in engineering C4 photosynthesis in C3 plants. Here, we utilize flux coupling analysis with the second-generation maize metabolic models to investigate the correspondence between metabolic network structure and transcriptomic phenotypes along the maize leaf gradient. The examined scenarios with publically available data from independent experiments indicate that the transcriptomic programs of the two cell types are co-ordinated, quantitatively and qualitatively, due to the presence of coupled metabolic reactions in specific metabolic pathways. Taken together, our study demonstrates that precise quantitative coupling will have to be achieved in order to ensure a successfully engineered transition from C3 to C4 crops., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Water limitation is a major global constraint for plant productivity that is likely to be exacerbated by climate change. Hence, improving plant water use efficiency (WUE) has become a major goal for the near future. At the leaf level, WUE is the ratio between photosynthesis and transpiration. Maintaining high photosynthesis under water stress, while improving WUE requires either increasing mesophyll conductance (gm ) and/or improving the biochemical capacity for CO2 assimilation-in which Rubisco properties play a key role, especially in C3 plants at current atmospheric CO2 . The goals of the present analysis are: (1) to summarize the evidence that improving gm and/or Rubisco can result in increased WUE; (2) to review the degree of success of early attempts to genetically manipulate gm or Rubisco; (3) to analyse how gm , gsw and the Rubisco's maximum velocity (Vcmax ) co-vary across different plant species in well-watered and drought-stressed conditions; (4) to examine how these variations cause differences in WUE and what is the overall extent of variation in individual determinants of WUE; and finally, (5) to use simulation analysis to provide a theoretical framework for the possible control of WUE by gm and Rubisco catalytic constants vis-à-vis gsw under water limitations., (© 2015 John Wiley & Sons Ltd.)

Crop biomass production is a function of the efficiencies with which sunlight can be intercepted by the canopy and then converted into biomass. Conversion efficiency has been identified as a target for improvement to enhance crop biomass and yield. Greater conversion efficiency in modern soybean [Glycine max (L.) Merr.] cultivars was documented in recent field trials, and this study explored the physiological basis for this observation. In replicated field trials conducted over three successive years, diurnal leaf gas exchange and photosynthetic CO2 response curves were measured in 24 soybean cultivars with year of release dates (YOR) from 1923 to 2007. Maximum photosynthetic capacity, mesophyll conductance and nighttime respiration have not changed consistently with cultivar release date. However, daily carbon gain was periodically greater in more recently released cultivars compared with older cultivars. Our analysis suggests that this difference in daily carbon gain primarily occurred when stomatal conductance and soil water content were high. There was also evidence for greater chlorophyll content and greater sink capacity late in the growing season in more recently released soybean varieties. Better understanding of the mechanisms that have improved conversion efficiency in the past may help identify new, promising targets for the future., (© 2016 John Wiley & Sons Ltd.)

CO2 exchange in leaves of maize (Zea mays L.) was examined using a microscale model of combined gas diffusion and C4 photosynthesis kinetics at the leaf tissue level. Based on a generalized scheme of photosynthesis in NADP-malic enzyme type C4 plants, the model accounted for CO2 diffusion in a leaf tissue, CO2 hydration and assimilation in mesophyll cells, CO2 release from decarboxylation of C4 acids, CO2 fixation in bundle sheath cells and CO2 retro-diffusion from bundle sheath cells. The transport equations were solved over a realistic 2-D geometry of the Kranz anatomy obtained from light microscopy images. The predicted responses of photosynthesis rate to changes in ambient CO2 and irradiance compared well with those obtained from gas exchange measurements. A sensitivity analysis showed that the CO2 permeability of the mesophyll-bundle sheath and airspace-mesophyll interfaces strongly affected the rate of photosynthesis and bundle sheath conductance. Carbonic anhydrase influenced the rate of photosynthesis, especially at low intercellular CO2 levels. In addition, the suberin layer at the exposed surface of the bundle sheath cells was found beneficial in reducing the retro-diffusion. The model may serve as a tool to investigate CO2 diffusion further in relation to the Kranz anatomy in C4 plants., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, ion homeostasis and resistance to biotic or abiotic stresses. We characterized AtCLCg, a member of the chloride channel (CLC) family in Arabidopsis localized in the vacuolar membrane. When grown on NaCl or KCl, atclcg knock-out mutants showed a decrease in biomass. In the presence of NaCl, these mutants overaccumulate chloride in shoots. No difference in growth was detected in response to osmotic stress by mannitol. These results suggest a physiological function of AtCLCg in the chloride homeostasis during NaCl stress. AtCLCg shares a high degree of identity (62%) with AtCLCc, another vacuolar CLC essential for NaCl tolerance. However, the atclcc atclccg double mutant is not more sensitive to NaCl than single mutants. As the effects of both mutations are not additive, gene expression analyses were performed and revealed that: (i)AtCLCg is expressed in mesophyll cells, hydathodes and phloem while AtCLCc is expressed in stomata; and (ii)AtCLCg is repressed in the atclcc mutant background, and vice versa. Altogether these results demonstrate that both AtCLCc and AtCLCg are important for tolerance to excess chloride but not redundant, and form part of a regulatory network controlling chloride sensitivity., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Growth and developmental processes are occasionally accompanied by multiple rounds of DNA replication, known as endoreduplication. Coordination between endoreduplication and cell size regulation often plays a crucial role in proper organogenesis and cell differentiation. Here, we report that the level of correlation between ploidy and cell volume is different in the outer and inner cell layers of leaves of Arabidopsis thaliana using a novel imaging technique. Although there is a well-known, strong correlation between ploidy and cell volume in pavement cells of the epidermis, this correlation was extremely weak in palisade mesophyll cells. Induction of epidermis cell identity based on the expression of the homeobox gene ATML1 in mesophyll cells enhanced the level of correlation between ploidy and cell volume to near that of wild-type epidermal cells. We therefore propose that the correlation between ploidy and cell volume is regulated by cell identity., (© 2016. Published by The Company of Biologists Ltd.)

Leaves growing in the forest understory usually present a decreased mesophyll conductance (gm) and photosynthetic capacity. The role of leaf anatomy in determining the variability in gm among species is known, but there is a lack of information on how the acclimation of gm to shade conditions is driven by changes in leaf anatomy. Within this context, we demonstrated that Abies pinsapo Boiss. experienced profound modifications in needle anatomy to drastic changes in light availability that ultimately led to differential photosynthetic performance between trees grown in the open field and in the forest understory. In contrast to A. pinsapo, its congeneric Abies alba Mill. did not show differences either in needle anatomy or in photosynthetic parameters between trees grown in the open field and in the forest understory. The increased gm values found in trees of A. pinsapo grown in the open field can be explained by occurrence of stomata at both needle sides (amphistomatous needles), increased chloroplast surface area exposed to intercellular airspace, decreased cell wall thickness and, especially, decreased chloroplast thickness. To the best of our knowledge, the role of such drastic changes in ultrastructural needle anatomy in explaining the response of gm to the light environment has not been demonstrated in field conditions., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus., Methods: Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy., Results: In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells., Conclusions: The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril alignment, spatially controlled cell wall expansion, allowing MCs to acquire their particular shape., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins., (© 2015 Scandinavian Plant Physiology Society.)

C4 photosynthesis represents an excellent example of convergent evolution that results in the optimization of both carbon and water usage by plants. In C4 plants, a carbon-concentrating mechanism divided between bundle sheath and mesophyll cells increases photosynthetic efficiency. Compared with C3 leaves, the carbon-concentrating mechanism of C4 plants allows photosynthetic operation at lower stomatal conductance, and as a consequence, transpiration is reduced. Here, we characterize transcriptomes from guard cells in C3 Tareneya hassleriana and C4 Gynandropsis gynandra belonging to the Cleomaceae. While approximately 60% of Gene Ontology terms previously associated with guard cells from the C3 model Arabidopsis (Arabidopsis thaliana) are conserved, there is much less overlap between patterns of individual gene expression. Most ion and CO2 signaling modules appear unchanged at the transcript level in guard cells from C3 and C4 species, but major variations in transcripts associated with carbon-related pathways known to influence stomatal behavior were detected. Genes associated with C4 photosynthesis were more highly expressed in guard cells of C4 compared with C3 leaves. Furthermore, we detected two major patterns of cell-specific C4 gene expression within the C4 leaf. In the first, genes previously associated with preferential expression in the bundle sheath showed continually decreasing expression from bundle sheath to mesophyll to guard cells. In the second, expression was maximal in the mesophyll compared with both guard cells and bundle sheath. These data imply that at least two gene regulatory networks act to coordinate gene expression across the bundle sheath, mesophyll, and guard cells in the C4 leaf., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Analyzing the proportions of stomatal (SL), mesophyll conductance (MCL) and biochemical limitations (BL) imposed by potassium (K) deficit, and evaluating their relationships to leaf K status will be helpful to understand the mechanism underlying the inhibition of K deficiency on photosynthesis (A). A quantitative limitation analysis of K deficiency on photosynthesis was performed on leaf margins and centers under K deficiency and sufficient K supply treatments of Brassica napus L. Potassium deficiency decreased A, stomatal (gs) and mesophyll conductance (gm) of margins, SL, MCL and BL accounted for 23.9%, 33.0% and 43.1% of the total limitations. While for leaf centers, relatively low limitations occurred. Nonlinear curve fitting analysis indicated that each limiting factor generated at same leaf K status (1.07%). Although MCL was the main component of limitations when A began to fall, BL replaced it at a leaf K concentration below 0.78%. Up-regulated MCL was related to lower surface area of chloroplasts exposed to intercellular airspaces (Sc/S) and larger cytosol diffusion resistance but not the cell wall thickness. Our results highlighted that photosynthetic limitations appear simultaneously under K deficiency and vary with increasing K deficiency intensity.

The rate of photosynthesis (A) of plants exposed to water deficit is a function of stomatal (gs) and mesophyll (gm) conductance determining the availability of CO2 at the site of carboxylation within the chloroplast. Mesophyll conductance often represents the greatest impediment to photosynthetic uptake of CO2, and a crucial determinant of the photosynthetic effects of drought. Abscisic acid (ABA) plays a fundamental role in signalling and co-ordination of plant responses to drought; however, the effect of ABA on gm is not well-defined. Rose, cherry, olive and poplar were exposed to exogenous ABA and their leaf gas exchange parameters recorded over a four hour period. Application with ABA induced reductions in values of A, gs and gm in all four species. Reduced gm occurred within one hour of ABA treatment in three of the four analysed species; indicating that the effect of ABA on gm occurs on a shorter timescale than previously considered. These declines in gm values associated with ABA were not the result of physical changes in leaf properties due to altered turgor affecting movement of CO2, or caused by a reduction in the sub-stomatal concentration of CO2 (Ci). Increased [ABA] likely induces biochemical changes in the properties of the interface between the sub-stomatal air-space and mesophyll layer through the actions of cooporins to regulate the transport of CO2. The results of this study provide further evidence that gm is highly responsive to fluctuations in the external environment, and stress signals such as ABA induce co-ordinated modifications of both gs and gm in the regulation of photosynthesis.

We characterized the function of the rice cytosolic hexokinase OsHXK7 (Oryza sativa Hexokinase7), which is highly upregulated when seeds germinate under O2 -deficient conditions. According to transient expression assays that used the promoter:luciferase fusion construct, OsHXK7 enhanced the glucose (Glc)-dependent repression of a rice α-amylase gene (RAmy3D) in the mesophyll protoplasts of maize, but its catalytically inactive mutant alleles did not. Consistently, the expression of OsHXK7, but not its catalytically inactive alleles, complemented the Arabidopsis glucose insensitive2-1 (gin2-1) mutant, thereby resulting in the wild type characteristics of Glc-dependent repression, seedling development, and plant growth. Interestingly, OsHXK7-mediated Glc-dependent repression was abolished in the O2 -deficient mesophyll protoplasts of maize. This result provides compelling evidence that OsHXK7 functions in sugar signaling via a glycolysis-dependent manner under normal conditions, but its signaling role is suppressed when O2 is deficient. The germination of two null OsHXK7 mutants, oshxk7-1 and oshxk7-2, was affected by O2 deficiency, but overexpression enhanced germination in rice. This result suggests the distinct role that OsHXK7 plays in sugar metabolism and efficient germination by enforcing glycolysis-mediated fermentation in O2 -deficient rice., (© 2015 Institute of Botany, Chinese Academy of Sciences.)

Abscisic acid (ABA) and phototropins act antagonistically to control stomatal movements. Here, we investigated the role of ABA in phototropin-directed chloroplast movements in mesophyll cells of Arabidopsis thaliana. We analyzed the expression of phototropins at mRNA and protein level under the influence of ABA. PHOT1 mRNA level was decreased by ABA in the dark while it was insensitive to ABA in light. PHOT2 mRNA level was independent of the hormone treatment. The levels of phototropin proteins were down-regulated by ABA, both in darkness and light. No impact of exogenous ABA on amplitudes and kinetics of chloroplast movements was detected. Chloroplast responses in wild type Arabidopsis and three mutants, abi4, abi2 (abscisic acid insensitive4, 2) and aba1 (abscisic acid1), were measured to account for endogenous ABA signaling. The chloroplast responses were slightly reduced in abi2 and aba1 mutants in strong light. To further investigate the effect, abi2 and aba1 mutants were supplemented with exogenous ABA. In the aba1 mutant, the reaction was rescued but in abi2 it was unaffected. Our results show that ABA is not directly involved in phototropin-controlled chloroplast responses in mature leaves of Arabidopsis. However, the disturbance of ABA biosynthesis and signaling in mutants affects some elements of the chloroplast movement mechanism. In line with its role as a stress hormone, ABA appears to enhance plant sensitivity to light and promote the chloroplast avoidance response.

A premature senescence and death 128 (psd128) mutant was isolated from an ethyl methane sulfonate-induced rice IR64 mutant bank. The premature senescence phenotype appeared at the six-leaf stage and the plant died at the early heading stage. psd128 exhibited impaired chloroplast development with significantly reduced photosynthetic ability, chlorophyll and carotenoid contents, root vigor, soluble protein content and increased malonaldehyde content. Furthermore, the expression of senescence-related genes was significantly altered in psd128. The mutant trait was controlled by a single recessive nuclear gene. Using map-based strategy, the mutation Oryza sativa cell division cycle 48 (OsCDC48) was isolated and predicted to encode a putative AAA-type ATPase with 809 amino-acid residuals. A single base substitution at position C2347T in psd128 resulted in a premature stop codon. Functional complementation could rescue the mutant phenotype. In addition, RNA interference resulted in the premature senescence and death phenotype. OsCDC48 was expressed constitutively in the root, stem, leaf and panicle. Subcellular analysis indicated that OsCDC48:YFP fusion proteins were located both in the cytoplasm and nucleus. OsCDC48 was highly conserved with more than 90% identity in the protein levels among plant species. Our results indicated that the impaired function of OsCDC48 was responsible for the premature senescence and death phenotype., (© 2015 The Authors. Journal of Integrative Plant Biology published by Wiley Publishing Asia Pty Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3-). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3- responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3-. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage.

The newly characterized elicitor AsES obtained from Acremonium strictum induces a strong defence response in strawberry plants and confers plants resistance against the fungal pathogen Colletotricum acutatum the casual agent of anthracnose disease. Previous studies showed that AsES causes the accumulation of reactive oxygen species (ROS) that peaked 4 h post treatment (hpt), but due to the experimental approach used it was not clear whether the accumulation of ROS observed was intracellular or extracellular or took place as a single peak. By using a different experimental setup, a more complex early events associated to the activation of the innate immunity were observed. In this paper we report that strawberry plant cells treated with AsES exhibits a triphasic production of H2O2 and a rapid intracellular accumulation of NO. The first phase consists in a progressive extracellular accumulation of H2O2 that starts immediately after the treatment with AsES and is preceded by a rapid and transient cell membrane depolarization. During this phase takes place also a rapid intracellular accumulation of NO. Microscopic observations of mesophyll cells treated with AsES reveals that NO accumulates at the chloroplast. After the first extracellular H2O2 production phase, two intracellular H2O2 accumulation events occur, the first 2 hpt, and the second 7 hpt. Cells treated with AsES also show a transient increase of ion leakage, and a progressive alkalinization of the extracellular medium., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.

In land plants, the NAD(P)H dehydrogenase (NDH) complex reduces plastoquinones and drives cyclic electron flow (CEF) around PSI. It also produces extra ATP for photosynthesis and improves plant fitness under conditions of abiotic environmental stress. To elucidate the role of CEF in salt tolerance of the photosynthetic apparatus, Na(+) concentration, chlorophyll fluorescence, and expression of NDH B and H subunits, as well as of genes related to cellular and vacuolar Na(+) transport, were monitored. The salt-tolerant Glycine max (soybean) variety S111-9 exhibited much higher CEF activity and ATP accumulation in light than did the salt-sensitive variety Melrose, but similar leaf Na(+) concentrations under salt stress. In S111-9 plants, ndhB and ndhH were highly up-regulated under salt stress and their corresponding proteins were maintained at high levels or increased significantly. Under salt stress, S111-9 plants accumulated Na(+) in the vacuole, but Melrose plants accumulated Na(+) in the chloroplast. Compared with Melrose, S111-9 plants also showed higher expression of some genes associated with Na(+) transport into the vacuole and/or cell, such as genes encoding components of the CBL10 (calcineurin B-like protein 10)-CIPK24 (CBL-interacting protein kinase 24)-NHX (Na(+)/H(+) antiporter) and CBL4 (calcineurin B-like protein 4)-CIPK24-SOS1 (salt overly sensitive 1) complexes. Based on the findings, it is proposed that enhanced NDH-dependent CEF supplies extra ATP used to sequester Na(+) in the vacuole. This reveals an important mechanism for salt tolerance in soybean and provides new insights into plant resistance to salt stress., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

To find a way to promote the rate of carbon flux and further improve the photosynthetic rate in rice, two CO2-transporting and fixing relevant genes, Ictb and FBP/Sbpase, which were derived from cyanobacteria with the 35SCaMV promotor in the respective constructs, were transformed into rice. Three homologous transgenic groups with Ictb, FBP/Sbpase and the two genes combined were constructed in parallel, and the functional effects of these two genes were investigated by physiological, biochemical and leaf anatomy analyses. The results indicated that the mesophyll conductance and net photosynthetic rate were higher at approximately 10.5-36.8% and 13.5-34.6%, respectively, in the three groups but without any changes in leaf anatomy structure compared with wild type. Other physiological and biochemical parameters increased with the same trend in the three groups, which showed that the effect of FBP/SBPase on improving photosynthetic capacity was better than that of ICTB and that there was an additive effect in ICTB+FBP/SBPase. ICTB localized in the cytoplasm, whereas FBP/SBPase was successfully transported to the chloroplast. The two genes might show a synergistic interaction to promote carbon flow and the assimilation rate as a whole. The multigene transformation engineering and its potential utility for improving the photosynthetic capacity and yield in rice were discussed.

Chloroplasts regulate their growth to optimize photosynthesis. Quantitative data show that the ratio of total chloroplast area to mesophyll cell area is constant across different cells within a single species and also across species. Wild-type chloroplasts exhibit little scatter around this trend; highly irregularly shaped mutant chloroplasts exhibit more scatter. Here we propose a model motivated by a bacterial quorum-sensing model consisting of a switch-like signaling network that turns off chloroplast growth. We calculated the dependence of the location of the relevant saddle-node bifurcation on the geometry of the chloroplasts. Our model exhibits a linear trend, with linearly growing scatter dependent on chloroplast shape, consistent with the data. When modeled chloroplasts are of a shape that grows with a constant area-to-volume ratio (disks, cylinders), we find a linear trend with minimal scatter. Chloroplasts with area and volume that do not grow proportionally (spheres) exhibit a linear trend with additional scatter.

According to microscopic observations, germinating hyphae of Botrytis cinerea, though easily penetrating Mesembryanthemum crystallinum mesophyll tissue, are limited in growth in mid-ribs and only occasionally reach vascular bundles. In mid-ribs of C3 and CAM leaves, we found significantly lower rbcL (large RubisCO subunit) abundance. Moreover, in CAM leaves, minute transcript contents for pepc1 (phosphoenolpyruvate carboxylase) and nadpme1 (malic enzyme) genes found in the mid-ribs suggest that they perform β-carboxylation at a low rate. The gene of the main H2O2-scavenging enzyme, catL (catalase), showed lower expression in C3 mid-rib parts in comparison to mesophyll. This allows maintenance of higher H2O2 quantities in mid-rib parts. In C3 leaves, pathogen infection does not impact photosynthesis. However, in CAM plants, the expression profiles of rbcL and nadpme1 were similar under biotic stress, with transcript down-regulation in mid-ribs and up-regulation in mesophyll (however, in case of rbcL not significant). After B. cinerea infection in C3 plants, transcripts for both antioxidative proteins strongly increased in mid-ribs, but not in mesophyll. In infected CAM plants, a significant transcript increase in the mesophyll was parallel to its decrease in the mid-rib region (however, in the case of catL this was not significant). Pathogen infection modified the expression of carbon and ROS metabolism genes in mid-ribs and mesophyll, resulting in the establishment of successful leaf defense., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The membrane-bound Brassinosteroid insensitive1-associated receptor kinase1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-interacting receptor-like kinase) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of suppressor of BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β-ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the β-ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10(6)  Da. These results are a major step forward in engineering a high biomass C4 grass for the commercial production of PHB., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

The appearance of dehydrins (DHNs) in cells is required for the development of cold resistance. DHNs are therefore considered specific markers of cold resistance by some authors. DHNs accumulate in plants concomitantly with a reduction of intracellular water content, and presumably protect membranes and proteins from damage caused by moisture loss. DHN content in pine needles increases in spring and autumn when moisture availability and temperatures are most unfavorable. The present work is focused on seasonal changes in DHN content in various mesophyll-cell compartments of pine (Pinus sylvestris L.) needles in association with changes in environmental factors. In spring, the number of thylakoid membranes per granum was lower than in summer and autumn. An increase in needle content of DHNs with approximate masses of 76, 73, 72, 35, and 17 kD in spring and autumn, associated with needle dehydration during this period, is shown here. The largest increase in DHN content was observed in spring, with the highest amount of DHNs presented in chloroplast membrane system including grana thylakoids, stromal thylakoids, and the two chloroplast envelope membranes and in cell walls. In the autumn, most DHNs were localized in chloroplasts and mitochondria.

Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins, including Rubisco, are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes, and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis (Arabidopsis thaliana). Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared with the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. (15)N pulse chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves., (© 2015 American Society of Plant Biologists. All Rights Reserved.)