Induction of stomatal opening following a night-chilling event alleviates physiological damage in mango trees.

Chilling events have become more frequent with climate change and are a significant abiotic factor causing physiological damage to plants and, consequently, reducing crop yield. Like other tropical and subtropical plants, mango (Mangifera indica L.) is particularly sensitive to chilling events, especially if they are followed by bright sunny days. It was previously shown that in mango leaves stomatal opening is restricted in the morning following a night-chilling event. This impairment results in restraint of carbon assimilation and subsequently, photoinhibition and reactive oxygen species production, which leads to chlorosis and in severe cases, cell death. Our detailed physiological analysis showed that foliar application of the guard cell H+-ATPase activator, fusicoccin, in the morning after a cold night, mitigates the physiological damage from 'cold night-bright day' abiotic stress. This application restored stomatal opening, thereby enabling gas exchange, releasing the photosynthetic machinery from harmful excess photon energy, and improving the plant's overall physiological state. The mechanisms by which plants react to this abiotic stress are examined in this work. The foliar application of compounds that cause stomatal opening as a potential method of minimizing physiological damage due to night chilling is discussed.

A role for ethylene signaling and biosynthesis in regulating and accelerating CO2 - and abscisic acid-mediated stomatal movements in Arabidopsis.

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO2 ]-shifts. According to our research, higher [CO2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO2 and ABA.

Physiological characterization of the wild almond Prunus arabica stem photosynthetic capability.

Leaves are the major plant tissue for transpiration and carbon fixation in deciduous trees. In harsh habitats, atmospheric CO2 assimilation via stem photosynthesis is common, providing extra carbon gain to cope with the detrimental conditions. We studied two almond species, the commercial Prunus dulcis cultivar "Um-el-Fahem" and the rare wild Prunus arabica. Our study revealed two distinctive strategies for carbon gain in these almond species. While, in P. dulcis, leaves possess the major photosynthetic surface area, in P. arabica, green stems perform this function, in particular during the winter after leaf drop. These two species' anatomical and physiological comparisons show that P. arabica carries unique features that support stem gas exchange and high-gross photosynthetic rates via stem photosynthetic capabilities (SPC). On the other hand, P. dulcis stems contribute low gross photosynthesis levels, as they are designed solely for reassimilation of CO2 from respiration, which is termed stem recycling photosynthesis (SRP). Results show that (a) P. arabica stems are covered with a high density of sunken stomata, in contrast to the stomata on P. dulcis stems, which disappear under a thick peridermal (bark) layer by their second year of development. (b) P. arabica stems contain significantly higher levels of chlorophyll compartmentalized to a mesophyll-like, chloroplast-rich, parenchyma layer, in contrast to rounded-shape cells of P. dulcis's stem parenchyma. (c) Pulse amplitude-modulated (PAM) fluorometry of P. arabica and P. dulcis stems revealed differences in the chlorophyll fluorescence and quenching parameters between the two species. (d) Gas exchange analysis showed that guard cells of P. arabica stems tightly regulate water loss under elevated temperatures while maintaining constant and high assimilation rates throughout the stem. Our data show that P. arabica uses a distinctive strategy for tree carbon gain via stem photosynthetic capability, which is regulated efficiently under harsh environmental conditions, such as elevated temperatures. These findings are highly important and can be used to develop new almond cultivars with agriculturally essential traits.

Revealing the Genetic Components Responsible for the Unique Photosynthetic Stem Capability of the Wild Almond Prunus arabica (Olivier) Meikle.

Almond [Prunus dulcis (Mill.) D. A. Webb] is a major deciduous fruit tree crop worldwide. During dormancy, under warmer temperatures and inadequate chilling hours, the plant metabolic activity increases and may lead to carbohydrate deficiency. Prunus arabica (Olivier) Meikle is a bushy wild almond species known for its green, unbarked stem, which stays green even during the dormancy period. Our study revealed that P. arabica green stems assimilate significantly high rates of CO2 during the winter as compared to P. dulcis cv. Um el Fahem (U.E.F.) and may improve carbohydrate status throughout dormancy. To uncover the genetic inheritance and mechanism behind the P. arabica stem photosynthetic capability (SPC), a segregated F1 population was generated by crossing P. arabica to U.E.F. Both parent's whole genome was sequenced, and SNP calling identified 4,887 informative SNPs for genotyping. A robust genetic map for U.E.F. and P. arabica was constructed (971 and 571 markers, respectively). QTL mapping and association study for the SPC phenotype revealed major QTL [log of odd (LOD) = 20.8] on chromosome 7 and another minor but significant QTL on chromosome 1 (LOD = 3.9). As expected, the P. arabica allele in the current loci significantly increased the SPC phenotype. Finally, a list of 64 candidate genes was generated. This work sets the stage for future research to investigate the mechanism regulating the SPC trait, how it affects the tree's physiology, and its importance for breeding new cultivars better adapted to high winter temperatures.

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2 , abscisic acid, darkness, vapor pressure deficit and ozone.

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO2 , light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO2 , light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO2 induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5-2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO2 and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).

A role for calcium-dependent protein kinases in differential CO2 - and ABA-controlled stomatal closing and low CO2 -induced stomatal opening in Arabidopsis.

Low concentrations of CO2 cause stomatal opening, whereas [CO2 ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO2 -induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+ -binding proteins that function in the stomatal CO2 response remain unknown. Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO2 , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO2 -induced stomatal closure and, unexpectedly, also in low CO2 -induced stomatal opening. Furthermore, K+ -uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2 -regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO2 control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.

Insights into the Molecular Mechanisms of CO2-Mediated Regulation of Stomatal Movements.

Plants must continually balance the influx of CO2 for photosynthesis against the loss of water vapor through stomatal pores in their leaves. This balance can be achieved by controlling the aperture of the stomatal pores in response to several environmental stimuli. Elevation in atmospheric [CO2] induces stomatal closure and further impacts leaf temperatures, plant growth and water-use efficiency, and global crop productivity. Here, we review recent advances in understanding CO2-perception mechanisms and CO2-mediated signal transduction in the regulation of stomatal movements, and we explore how these mechanisms are integrated with other signaling pathways in guard cells.

Eukaryotic lipid metabolic pathway is essential for functional chloroplasts and CO2 and light responses in Arabidopsis guard cells.

Stomatal guard cells develop unique chloroplasts in land plant species. However, the developmental mechanisms and function of chloroplasts in guard cells remain unclear. In seed plants, chloroplast membrane lipids are synthesized via two pathways: the prokaryotic and eukaryotic pathways. Here we report the central contribution of endoplasmic reticulum (ER)-derived chloroplast lipids, which are synthesized through the eukaryotic lipid metabolic pathway, in the development of functional guard cell chloroplasts. We gained insight into this pathway by isolating and examining an Arabidopsis mutant, gles1 (green less stomata 1), which had achlorophyllous stomatal guard cells and impaired stomatal responses to CO2 and light. The GLES1 gene encodes a small glycine-rich protein, which is a putative regulatory component of the trigalactosyldiacylglycerol (TGD) protein complex that mediates ER-to-chloroplast lipid transport via the eukaryotic pathway. Lipidomic analysis revealed that in the wild type, the prokaryotic pathway is dysfunctional, specifically in guard cells, whereas in gles1 guard cells, the eukaryotic pathway is also abrogated. CO2-induced stomatal closing and activation of guard cell S-type anion channels that drive stomatal closure were disrupted in gles1 guard cells. In conclusion, the eukaryotic lipid pathway plays an essential role in the development of a sensing/signaling machinery for CO2 and light in guard cell chloroplasts.

Starch biosynthesis by AGPase, but not starch degradation by BAM1/3 and SEX1, is rate-limiting for CO2 -regulated stomatal movements under short-day conditions.

Starch in guard cells functions in osmoregulation during stomatal movements. Starch metabolism is controlled by the circadian clock. We investigated the role of starch metabolism in stomatal responses to CO2 under different photoperiodic conditions. Guard cell starch levels correlate with low/high [CO2 ] exposure. Starch biosynthesis-deficient AGPase (ADG1) mutants but, unexpectedly, not the starch degradation-deficient BAM1, BAM3, and SEX1 mutants alone, are rate-limiting for stomatal conductance responses to [CO2 ]-shifts. Interestingly, AGPase is rate-limiting solely under short- but not long-day conditions. These findings suggest a model of enhanced AGPase activity in guard cells under short days such that starch biosynthesis becomes rate-limiting for CO2 -induced stomatal closing.

Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing.

Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing.

CO2 Sensing and CO2 Regulation of Stomatal Conductance: Advances and Open Questions.

Guard cells form epidermal stomatal gas-exchange valves in plants and regulate the aperture of stomatal pores in response to changes in the carbon dioxide (CO2) concentration ([CO2]) in leaves. Moreover, the development of stomata is repressed by elevated CO2 in diverse plant species. Evidence suggests that plants can sense [CO2] changes via guard cells and via mesophyll tissues in mediating stomatal movements. We review new discoveries and open questions on mechanisms mediating CO2-regulated stomatal movements and CO2 modulation of stomatal development, which together function in the CO2 regulation of stomatal conductance and gas exchange in plants. Research in this area is timely in light of the necessity of selecting and developing crop cultivars that perform better in a shifting climate.

Guard cell photosynthesis is critical for stomatal turgor production, yet does not directly mediate CO2 - and ABA-induced stomatal closing.

Stomata mediate gas exchange between the inter-cellular spaces of leaves and the atmosphere. CO2 levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO2 ]. [CO2 ] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll-deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll-deficient. Interestingly, approximately 45% of stomata had an unusual, previously not-described, morphology of thin-shaped chlorophyll-less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole-leaf photosynthetic parameters (PSII, qP, qN, FV '/FM' ) were comparable with wild-type plants. Time-resolved intact leaf gas-exchange analyses showed a reduction in stomatal conductance and CO2 -assimilation rates of the transgenic plants. Normalization of CO2 responses showed that stomata of transgenic plants respond to [CO2 ] shifts. Detailed stomatal aperture measurements of normal kidney-shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO2 ] elevation and abscisic acid (ABA), while thin-shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO2 ] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO2 and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll-less stomata cause a 'deflated' thin-shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.

Dual N- and C-terminal processing of citrus chlorophyllase precursor within the plastid membranes leads to the mature enzyme.

Chl, the central player in harvesting light energy for photosynthesis, is enzymatically degraded during natural turnover, leaf senescence, fruit ripening or following biotic/abiotic stress induction. The photodynamic properties of Chl and its metabolites call for tight regulation of the catabolic pathway enzymes to avoid accumulation of intermediate breakdown products. Chlorophyllase, the Chl dephytilation enzyme, was previously demonstrated to be an initiator of Chl breakdown when transcriptionally induced to be expressed during ethylene-induced citrus fruit color break or when heterologously expressed in different plant systems. Citrus chlorophyllase was previously shown to be translated as a precursor protein, which is subsequently post-translationally processed to a mature form. We demonstrate that maturation of citrus chlorophyllase involves dual N- and C-terminal processing which appear to be rate-limiting post-translational events when chlorophyllase expression levels are high. The chlorophyllase precursor and intermediate forms were shown to be of transient nature, while the mature form accumulates over time, suggesting that processing may be involved in post-translational regulation of enzyme in vivo function. This notion is further supported by the finding that neither N- nor C-terminal processed domains are essential for chloroplast targeting of the enzyme, and that both processing events occur within the chloroplast membranes. Studies on the processing of chlorophyllase versions truncated at the N- or C-termini or mutated to abolish C-terminal processing suggest that each of the processing events is independent. Dual N- and C-terminal processing, not involving an organellar targeting signal, has rarely been documented in plants and is unique for a plastid protein.

Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization.

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.