Plasma membrane aquaporins regulate root hydraulic conductivity in the model plant Setaria viridis.

The high rate of productivity observed in panicoid crops is in part due to their extensive root system. Recently, green foxtail (Setaria viridis) has emerged as a genetic model system for panicoid grasses. Natural accessions of S. viridis originating from different parts of the world, with differential leaf physiological behavior, have been identified. This work focused on understanding the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas exchange signaling in S. viridis. We identified 2 accessions, SHA and ZHA, with contrasting behavior at the leaf, root, and whole-plant levels. Our results indicated a role for root aquaporin (AQP) plasma membrane (PM) intrinsic proteins in the differential behavior of SHA and ZHA. Moreover, a different root hydraulic response to low levels of abscisic acid between SHA and ZHA was observed, which was associated with root AQPs. Using cell imaging, biochemical, and reverse genetic approaches, we identified PM intrinsic protein 1;6 (PIP1;6) as a possible PIP1 candidate that regulates radial root hydraulics and root-to-shoot signaling of gas exchange in S. viridis. In heterologous systems, PIP1;6 localized in the endoplasmic reticulum, and upon interaction with PIP2s, relocalization to the PM was observed. PIP1;6 was predominantly expressed at the root endodermis. Generation of knockout PIP1;6 plants (KO-PIP1;6) in S. viridis showed altered root hydraulic conductivity, altered gas exchange, and alteration of root transcriptional patterns. Our results indicate that PIPs are essential in regulating whole-plant water homeostasis in S. viridis. We conclude that root hydraulic conductivity and gas exchange are positively associated and are regulated by AQPs.

Guard cell activity of PIF4 and HY5 control transpiration.

Whole-plant transpiration, controlled by plant hydraulics and stomatal movement, is regulated by endogenous and environmental signals, with the light playing a dominant role. Stomatal pore size continuously adjusts to changes in light intensity and quality to ensure optimal CO2 intake for photosynthesis on the one hand, together with minimal water loss on the other. The link between light and transpiration is well established, but the genetic knowledge of how guard cells perceive those signals to affect stomatal conductance is still somewhat limited. In the current study, we evaluated the role of two central light-responsive transcription factors; a bZIP-family transcription factor ELONGATED HYPOCOTYL5 (HY5) and the basic helix-loop-helix (BHLH) transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4), in the regulation of steady-state transpiration. We show that overexpression of PIF4 exclusively in guard cells (GCPIF4) decreases transpiration, and can restrain the high transpiration of the pif4 mutant. Expression of HY5 specifically in guard cells (GCHY5) had the opposite effect of enhancing transpiration rates of WT- Arabidopsis and tobacco plants and of the hy5 mutant in Arabidopsis. In addition, we show that GCHY5 can reverse the low transpiration caused by guard cell overexpression of the sugar sensor HEXOKINASE1 (HXK1, GCHXK), an established low transpiring genotype. Finally, we suggest that the GCHY5 reversion of low transpiration by GCHXK requires the auto-activation of the endogenous HY5 in other tissues. These findings support the existence of an ongoing diurnal regulation of transpiration by the light-responsive transcription factors HY5 and PIF4 in the stomata, which ultimately determine the whole-plant water use efficiency.

Guard cells control hypocotyl elongation through HXK1, HY5, and PIF4.

The hypocotyls of germinating seedlings elongate in a search for light to enable autotrophic sugar production. Upon exposure to light, photoreceptors that are activated by blue and red light halt elongation by preventing the degradation of the hypocotyl-elongation inhibitor HY5 and by inhibiting the activity of the elongation-promoting transcription factors PIFs. The question of how sugar affects hypocotyl elongation and which cell types stimulate and stop that elongation remains unresolved. We found that overexpression of a sugar sensor, Arabidopsis hexokinase 1 (HXK1), in guard cells promotes hypocotyl elongation under white and blue light through PIF4. Furthermore, expression of PIF4 in guard cells is sufficient to promote hypocotyl elongation in the light, while expression of HY5 in guard cells is sufficient to inhibit the elongation of the hy5 mutant and the elongation stimulated by HXK1. HY5 exits the guard cells and inhibits hypocotyl elongation, but is degraded in the dark. We also show that the inhibition of hypocotyl elongation by guard cells' HY5 involves auto-activation of HY5 expression in other tissues. It appears that guard cells are capable of coordinating hypocotyl elongation and that sugar and HXK1 have the opposite effect of light on hypocotyl elongation, converging at PIF4.

Expression of Hexokinase in Stomata of Citrus Fruit Reduces Fruit Transpiration and Affects Seed Development.

The temporal formation and spatial distribution of stomata on the surface of citrus floral organs and, specifically, on the ovule from which the fruit develops, were analyzed using citrus plants that express green fluorescent protein (GFP) under the guard cell-specific KST1 promoter. Stomata are found on the style, sepal, and anther of the closed flower and on ovules from the stage of anthesis. It has previously been shown that hexokinase (HXK) mediates sugar-sensing in leaf guard cells and stimulates stomatal closure. The activity and response of citrus fruit stomata to sugar-sensing by HXK was examined using plants that express HXK under the KST1 promoter. Those plants are referred to as GCHXK plants. The transpiration of young green GCHXK citrus fruits was significantly reduced, indicating that their stomata respond to sugar similar to leaf stomata. Toward fruit maturation, fruit stomata are plugged and stop functioning, which explains why WT and GCHXK mature yellow fruits exhibited similar water loss. Seeds of the GCHXK plants were smaller and germinated more slowly than the WT seeds. We suggest that the stomata of young green citrus fruits, but not mature yellow fruits, respond to sugar levels via HXK and that fruit stomata are important for proper seed development.

Guard-Cell Hexokinase Increases Water-Use Efficiency Under Normal and Drought Conditions.

Water is a limiting resource for many land plants. Most of the water taken up by plants is lost to the atmosphere through the stomata, which are adjustable pores on the leaf surface that allow for gas exchange between the plant and the atmosphere. Modulating stomatal activity might be an effective way to reduce plants' water consumption and enhance their productivity under normal, as well as water-limiting conditions. Our recent discovery of stomatal regulation by sugars that is mediated by guard-cell hexokinase (HXK), a sugar-sensing enzyme, has raised the possibility that HXK might be used to increase plant water-use efficiency (WUE; i.e., carbon gain per unit of water). We show here that transgenic tomato and Arabidopsis plants with increased expression of HXK in their guard cells (GCHXK plants) exhibit reduced transpiration and higher WUE without any negative effects on growth under normal conditions, as well as drought avoidance and improved photosynthesis and growth under limited-water conditions. Our results demonstrate that exclusive expression of HXK in guard cells is an effective tool for improving WUE, and plant performance under drought.

Mesophyll Abscisic Acid Restrains Early Growth and Flowering But Does Not Directly Suppress Photosynthesis.

Abscisic acid (ABA) levels increase significantly in plants under stress conditions, and ABA is thought to serve as a key stress-response regulator. However, the direct effect of ABA on photosynthesis and the effect of mesophyll ABA on yield under both well-watered and drought conditions are still the subject of debate. Here, we examined this issue using transgenic Arabidopsis (Arabidopsis thaliana) plants carrying a dominant ABA-signaling inhibitor under the control of a mesophyll-specific promoter (FBPase::abi1-1, abbreviated to fa). Under normal conditions, fa plants displayed slightly higher stomatal conductance and carbon assimilation than wild-type plants; however, these parameters were comparable following ABA treatment. These observations suggest that ABA does not directly inhibit photosynthesis in the short term. The fa plants also exhibited a variety of altered phenotypes under optimal conditions, including more vigorous initial growth, earlier flowering, smaller flowers, and delayed chlorophyll degradation. Furthermore, under optimal conditions, fa plant seed production was less than a third of that observed for the wild type. However, under drought conditions, wild-type and fa seed yields were similar due to a significant reduction in wild-type seed and no reduction in fa seed. These findings suggest that endogenous basal ABA inhibits a stress-escape response under nonstressed conditions, allowing plants to accumulate biomass and maximize yield. The lack of a correlation between flowering time and plant biomass combined with delayed chlorophyll degradation suggests that this stress-escape behavior is regulated independently and upstream of other ABA-induced effects such as rapid growth and flowering.

Evolution of Guard-Cell Theories: The Story of Sugars.

Stomata are dynamic pores in the impermeable cuticle that coats the aerial parts of vascular plants, allowing the entry of CO2 for photosynthesis and controlling water loss. They are composed of two guard cells that can swell or shrink due to an increase or decrease in their osmotic pressure, respectively. Swelling opens the stomata and shrinking closes the stomata. For more than a century, scientists have been working to uncover the nature of the osmolytes that modulate osmotic pressure in guard cells. Recent discoveries have undermined long-standing theories in this area, reversing the understood roles of sugars and demonstrating the evolution of scientific theories. Here, we describe the evolution of guard-cell osmoregulation theories with an emphasis on the role of sugars.

Expression of Arabidopsis Hexokinase in Tobacco Guard Cells Increases Water-Use Efficiency and Confers Tolerance to Drought and Salt Stress.

Abiotic stresses such as drought and saline water impose major limitations on plant growth. Modulation of stomatal behavior may help plants cope with such stresses by reducing both water loss and salt uptake. Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in guard cells' sugar-sensing, mediating stomatal closure and coordinating photosynthesis with transpiration. We generated transgenic tobacco lines expressing the Arabidopsis hexokinase1 (AtHXK1) under the guard cell-specific promoter KST1 and examined those plants using growth room and greenhouse experiments. The expression of AtHXK1 in tobacco guard cells reduced stomatal conductance and transpiration by about 25% with no negative effects on photosynthesis or growth, leading to increased water-use efficiency. In addition, these plants exhibited tolerance to drought and salt stress due to their lower transpiration rate, indicating that improved stomatal function has the potential to improve plant performance under stress conditions.

Sucrose-induced stomatal closure is conserved across evolution.

As plants evolved to function on land, they developed stomata for effective gas exchange, for photosynthesis and for controlling water loss. We have recently shown that sugars, as the end product of photosynthesis, close the stomata of various angiosperm species, to coordinate sugar production with water loss. In the current study, we examined the sugar responses of the stomata of phylogenetically different plant species and species that employ different photosynthetic mechanisms (i.e., C3, C4 and CAM). To examine the effect of sucrose on stomata, we treated leaves with sucrose and then measured their stomatal apertures. Sucrose reduced stomatal aperture, as compared to an osmotic control, suggesting that regulation of stomata by sugars is a trait that evolved early in evolutionary history and has been conserved across different groups of plants.

Transcriptome analysis of Pinus halepensis under drought stress and during recovery.

Forest trees use various strategies to cope with drought stress and these strategies involve complex molecular mechanisms. Pinus halepensis Miller (Aleppo pine) is found throughout the Mediterranean basin and is one of the most drought-tolerant pine species. In order to decipher the molecular mechanisms that P. halepensis uses to withstand drought, we performed large-scale physiological and transcriptome analyses. We selected a mature tree from a semi-arid area with suboptimal growth conditions for clonal propagation through cuttings. We then used a high-throughput experimental system to continuously monitor whole-plant transpiration rates, stomatal conductance and the vapor pressure deficit. The transcriptomes of plants were examined at six physiological stages: pre-stomatal response, partial stomatal closure, minimum transpiration, post-irrigation, partial recovery and full recovery. At each stage, data from plants exposed to the drought treatment were compared with data collected from well-irrigated control plants. A drought-stressed P. halepensis transcriptome was created using paired-end RNA-seq. In total, ~6000 differentially expressed, non-redundant transcripts were identified between drought-treated and control trees. Cluster analysis has revealed stress-induced down-regulation of transcripts related to photosynthesis, reactive oxygen species (ROS)-scavenging through the ascorbic acid (AsA)-glutathione cycle, fatty acid and cell wall biosynthesis, stomatal activity, and the biosynthesis of flavonoids and terpenoids. Up-regulated processes included chlorophyll degradation, ROS-scavenging through AsA-independent thiol-mediated pathways, abscisic acid response and accumulation of heat shock proteins, thaumatin and exordium. Recovery from drought induced strong transcription of retrotransposons, especially the retrovirus-related transposon Tnt1-94. The drought-related transcriptome illustrates this species' dynamic response to drought and recovery and unravels novel mechanisms.

The Solanum tuberosum KST1 partial promoter as a tool for guard cell expression in multiple plant species.

To date, guard cell promoters have been examined in only a few species, primarily annual dicots. A partial segment of the potato (Solanum tuberosum) KST1 promoter (KST1 partial promoter, KST1ppro) has previously been shown to confer guard cell expression in potato, tomato (Solanum lycopersicum), citrus [Troyer citrange (C. sinensis×Poncirus trifoliata)], and Arabidopsis (Arabidopsis thaliana). Here, we describe an extensive analysis of the expression pattern of KST1ppro in eight (previously reported, as well as new) species from five different angiosperm families, including the Solanaceae and the Cucurbitaceae, Arabidopsis, the monocot barley (Hordeum vulgare), and two perennial species: grapevine (Vitis vinifera) and citrus. Using confocal imaging and three-dimensional movies, we demonstrate that KST1ppro drives guard cell expression in all of these species, making it the first dicot-originated guard cell promoter shown to be active in a monocot and the first promoter reported to confer guard cell expression in barley and cucumber (Cucumis sativus). The results presented here indicate that KST1ppro can be used to drive constitutive guard cell expression in monocots and dicots and in both annual and perennial plants. In addition, we show that the KST1ppro is active in guard cells shortly after the symmetric division of the guard mother cell and generates stable expression in mature guard cells. This allows us to follow the spatial and temporal distribution of stomata in cotyledons and true leaves.

Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential.

Sugars affect central aspects of plant physiology, including photosynthesis, stomatal behavior and the loss of water through the stomata. Yet, the potential effects of sugars on plant aquaporins (AQPs) and water conductance have not been examined. We used database and transcriptional analyses, as well as cellular and whole-plant functional techniques to examine the link between sugar-related genes and AQPs. Database analyses revealed a high level of correlation between the expression of AQPs and that of sugar-related genes, including the Arabidopsis hexokinases 1 (AtHXK1). Increased expression of AtHXK1, as well as the addition of its primary substrate, glucose (Glc), repressed the expression of 10 AQPs from the plasma membrane-intrinsic proteins (PIP) subfamily (PIP-AQPs) and induced the expression of two stress-related PIP-AQPs. The osmotic water permeability of mesophyll protoplasts of AtHXK1-expressing plants and the leaf hydraulic conductance of those plants were significantly reduced, in line with the decreased expression of PIP-AQPs. Conversely, hxk1 mutants demonstrated a higher level of hydraulic conductance, with increased water potential in their leaves. In addition, the presence of Glc reduced leaf water potential, as compared with an osmotic control, indicating that Glc reduces the movement of water from the xylem into the mesophyll. The production of sugars entails a significant loss of water and these results suggest that sugars and AtHXK1 affect the expression of AQP genes and reduce leaf water conductance, to coordinate sugar levels with the loss of water through transpiration.

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration.

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

The role of plasma membrane aquaporins in regulating the bundle sheath-mesophyll continuum and leaf hydraulics.

Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (K(leaf)), in particular, is still fairly limited. We hypothesized that the AQPs of the vascular bundle sheath (BS) cells regulate K(leaf). To examine this hypothesis, AQP genes were silenced using artificial microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (P(f)), mesophyll conductance of CO2, photosynthesis, K(leaf), transpiration, and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS Pf and decreased K(leaf) but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA mesophyll exhibited reduced P(f), but not reduced mesophyll conductance of CO2, suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed.

Mesophyll photosynthesis and guard cell metabolism impacts on stomatal behaviour.

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere. Guard cells determine stomatal aperture and must operate to ensure an appropriate balance between CO2 uptake for photosynthesis (A) and water loss, and ultimately plant water use efficiency (WUE). A strong correlation between A and stomatal conductance (gs ) is well documented and often observed, but the underlying mechanisms, possible signals and metabolites that promote this relationship are currently unknown. In this review we evaluate the current literature on mesophyll-driven signals that may coordinate stomatal behaviour with mesophyll carbon assimilation. We explore a possible role of various metabolites including sucrose and malate (from several potential sources; including guard cell photosynthesis) and new evidence that improvements in WUE have been made by manipulating sucrose metabolism within the guard cells. Finally we discuss the new tools and techniques available for potentially manipulating cell-specific metabolism, including guard and mesophyll cells, in order to elucidate mesophyll-derived signals that coordinate mesophyll CO2 demands with stomatal behaviour, in order to provide a mechanistic understanding of these processes as this may identify potential targets for manipulations in order to improve plant WUE and crop yield.

Relationship between hexokinase and the aquaporin PIP1 in the regulation of photosynthesis and plant growth.

Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel for CO2 and water, increases the rate of both photosynthesis and transpiration. In contrast, increased expression of Arabidopsis hexokinase1 (AtHXK1), a dual-function enzyme that mediates sugar sensing, decreases the expression of photosynthetic genes and the rate of transpiration and inhibits growth. Here, we show that AtHXK1 also decreases root and stem hydraulic conductivity and leaf mesophyll CO2 conductance (g(m)). Due to their opposite effects on plant development and physiology, we examined the relationship between NtAQP1 and AtHXK1 at the whole-plant level using transgenic tomato plants expressing both genes simultaneously. NtAQP1 significantly improved growth and increased the transpiration rates of AtHXK1-expressing plants. Reciprocal grafting experiments indicated that this complementation occurs when both genes are expressed simultaneously in the shoot. Yet, NtAQP1 had only a marginal effect on the hydraulic conductivity of the double-transgenic plants, suggesting that the complementary effect of NtAQP1 is unrelated to shoot water transport. Rather, NtAQP1 significantly increased leaf mesophyll CO2 conductance and enhanced the rate of photosynthesis, suggesting that NtAQP1 facilitated the growth of the double-transgenic plants by enhancing mesophyll conductance of CO2.

Substantial roles of hexokinase and fructokinase in the effects of sugars on plant physiology and development.

The basic requirements for plant growth are light, CO2, water, and minerals. However, the absorption and utilization of each of these requires investment on the part of the plant. The primary products of plants are sugars, and the hexose sugars glucose and fructose are the raw material for most of the metabolic pathways and organic matter in plants. To be metabolized, hexose sugars must first be phosphorylated. Only two families of enzymes capable of catalysing the essential irreversible phosphorylation of glucose and fructose have been identified in plants, hexokinases (HXKs) and fructokinases (FRKs). These hexose-phosphorylating enzymes appear to coordinate sugar production with the abilities to absorb light, CO2, water, and minerals. This review describes the long- and short-term effects mediated by HXK and FRK in various tissues, as well as the role of these enzymes in the coordination of sugar production with the absorption of light, CO2, water, and minerals.

Hexokinase mediates stomatal closure.

Stomata, composed of two guard cells, are the gates whose controlled movement allows the plant to balance the demand for CO2 for photosynthesis with the loss of water through transpiration. Increased guard-cell osmolarity leads to the opening of the stomata and decreased osmolarity causes the stomata to close. The role of sugars in the regulation of stomata is not yet clear. In this study, we examined the role of hexokinase (HXK), a sugar-phosphorylating enzyme involved in sugar-sensing, in guard cells and its effect on stomatal aperture. We show here that increased expression of HXK in guard cells accelerates stomatal closure. We further show that this closure is induced by sugar and is mediated by abscisic acid. These findings support the existence of a feedback-inhibition mechanism that is mediated by a product of photosynthesis, namely sucrose. When the rate of sucrose production exceeds the rate at which sucrose is loaded into the phloem, the surplus sucrose is carried toward the stomata by the transpiration stream and stimulates stomatal closure via HXK, thereby preventing the loss of precious water.

Hexose kinases and their role in sugar-sensing and plant development.

Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms and are the origin of most of the organic matter found in nature. To be utilized, hexose sugars must first be phosphorylated. The central role of hexose-phosphorylating enzymes has attracted the attention of many researchers, leading to novel discoveries. Only two families of enzymes capable of phosphorylating glucose and fructose have been identified in plants; hexokinases (HXKs), and fructokinases (FRKs). Intensive investigations of these two families in numerous plant species have yielded a wealth of knowledge regarding the genes number, enzymatic characterization, intracellular localization, and developmental and physiological roles of several HXKs and FRKs. The emerging picture indicates that HXK and FRK enzymes found at specific intracellular locations play distinct roles in plant metabolism and development. Individual HXKs were shown for the first time to be dual-function enzymes - sensing sugar levels independent of their catalytic activity and controlling gene expression and major developmental pathways, as well as hormonal interactions. FRK, on the other hand, seems to play a central metabolic role in vascular tissues, controlling the amounts of sugars allocated for vascular development. While a clearer picture of the roles of these two types of enzymes is emerging, many questions remain unsolved, such as the specific tissues and types of cells in which these enzymes function, the roles of individual HXK and FRK genes, and how these enzymes interact with hormones in the regulation of developmental processes. It is anticipated that ongoing efforts will broaden our knowledge of these important plant enzymes and their potential uses in the modification of plant traits.