Arabidopsis transcriptome responses to low water potential using high-throughput plate assays.

Soil-free assays that induce water stress are routinely used to investigate drought responses in the plant Arabidopsis thaliana. Due to their ease of use, the research community often relies on polyethylene glycol (PEG), mannitol, and salt (NaCl) treatments to reduce the water potential of agar media, and thus induce drought conditions in the laboratory. However, while these types of stress can create phenotypes that resemble those of water deficit experienced by soil-grown plants, it remains unclear how these treatments compare at the transcriptional level. Here, we demonstrate that these different methods of lowering water potential elicit both shared and distinct transcriptional responses in Arabidopsis shoot and root tissue. When we compared these transcriptional responses to those found in Arabidopsis roots subject to vermiculite drying, we discovered many genes induced by vermiculite drying were repressed by low water potential treatments on agar plates (and vice versa). Additionally, we also tested another method for lowering water potential of agar media. By increasing the nutrient content and tensile strength of agar, we show the 'hard agar' (HA) treatment can be leveraged as a high-throughput assay to investigate natural variation in Arabidopsis growth responses to low water potential.

A tale of two pumps: Blue light and abscisic acid alter Arabidopsis leaf hydraulics via bundle sheath cell H + -ATPases.

The bundle sheath cell (BSC) layer tightly enveloping the xylem throughout the leaf is recognized as a major signal-perceiving "valve" in series with stomata, regulating leaf hydraulic conductance (Kleaf) and thereby radial water flow via the transpiring leaf. The BSC blue light (BL) signaling pathway increases Kleaf and the underlying BSC water permeability. Here, we explored the hypothesis that BSCs also harbor a Kleaf-downregulating signaling pathway related to the stress phytohormone abscisic acid (ABA). We employed fluorescence imaging of xylem sap in detached leaves and BSC protoplasts from different genotypes of Arabidopsis (Arabidopsis thaliana) plants, using pH and membrane potential probes to monitor physiological responses to ABA and BL in combination with pharmacological agents. We found that BL-enhanced Kleaf required elevated BSC cytosolic Ca2+. ABA inhibited BL-activated xylem-sap-acidifying BSC H + -ATPase AHA2 (Arabidopsis H + -ATPase 2), resulting in depolarized BSCs and alkalinized xylem sap. ABA also stimulated BSC vacuolar H + -ATPase (VHA), which alkalinized the BSC cytosol. Each pump stimulation, AHA2 by BL and VHA by ABA (under BL), also required Ca2+. ABA stimulated VHA in the dark depending on Ca2+, but only in an alkaline external medium. Taken together with earlier findings on the pH sensitivity of BSC osmotic water permeability (i.e., aquaporin activity), our results suggest a Ca2+-dependent and pH-mediated causative link between the BL- and ABA-regulated activities of two BSC H + -ATPases and Kleaf.

Null mutants of a tomato Rho of plants exhibit enhanced water use efficiency without a penalty to yield.

Improving water use efficiency in crops is a significant challenge as it involves balancing water transpiration and CO2 uptake through stomatal pores. This study investigates the role of SlROP9, a tomato Rho of Plants protein, in guard cells and its impact on plant transpiration. The results reveal that SlROP9 null mutants exhibit reduced stomatal conductance while photosynthetic CO2 assimilation remains largely unaffected. Notably, there is a notable decrease in whole-plant transpiration in the rop9 mutants compared to the wild type, especially during noon hours when the water pressure deficit is high. The elevated stomatal closure observed in rop9 mutants is linked to an increase in reactive oxygen species formation. This is very likely dependent on the respiratory burst oxidase homolog (RBOH) NADPH oxidase and is not influenced by abscisic acid (ABA). Consistently, activated ROP9 can interact with RBOHB in both yeast and plants. In diverse tomato accessions, drought stress represses ROP9 expression, and in Arabidopsis stomatal guard cells, ABA suppresses ROP signaling. Therefore, the phenotype of the rop9 mutants may arise from a disruption in ROP9-regulated RBOH activity. Remarkably, large-scale field experiments demonstrate that the rop9 mutants display improved water use efficiency without compromising fruit yield. These findings provide insights into the role of ROPs in guard cells and their potential as targets for enhancing water use efficiency in crops.

Illuminating plant water dynamics: the role of light in leaf hydraulic regulation.

Light intensity and quality influence photosynthesis directly but also have an indirect effect by increasing stomatal apertures and enhancing gas exchange. Consequently, in areas such as the upper canopy, a high water demand for transpiration and temperature regulation is created. This paper explores how light intensity and the natural high Blue-Light (BL) : Red-Light (RL) ratio in these areas, is important for controlling leaf hydraulic conductance (Kleaf ) by BL signal transduction, increasing water permeability in cells surrounding the vascular tissue, in supporting the enormous water demands. Conversely, shaded inner-canopy areas receive less radiation, have lower water and cooling demands, and exhibit reduced Kleaf due to diminished intensity and BL induction. Intriguingly, shaded leaves display higher water-use efficiency (compared with upper-canopy) due to decreased transpiration and cooling requirements while the presence of RL supports photosynthesis.

Leaf hydraulic maze: Abscisic acid effects on bundle sheath, palisade, and spongy mesophyll conductance.

Leaf hydraulic conductance (Kleaf) facilitates the supply of water, enabling continual CO2 uptake while maintaining plant water status. We hypothesized that bundle sheath and mesophyll cells play key roles in regulating the radial flow of water out of the xylem by responding to abscisic acid (ABA). Thus, we generated transgenic Arabidopsis thaliana plants that are insensitive to ABA in their bundle sheath (BSabi) and mesophyll (MCabi) cells. We also introduced tissue-specific fluorescent markers to distinguish between cells of the palisade mesophyll, spongy mesophyll, and bundle sheath. Both BSabi and MCabi plants showed greater Kleaf and transpiration under optimal conditions. MCabi plants had larger stomatal apertures, higher stomatal index, and greater vascular diameter and biomass relative to the wild-type (WT) and BSabi plants. In response to xylem-fed ABA, both transgenic and WT plants reduced their Kleaf and transpiration. The membrane osmotic water permeability (Pf) of the WT's spongy mesophyll was higher than that of the WT's palisade mesophyll. While the palisade mesophyll maintained a low Pf in response to high ABA, the spongy mesophyll Pf was reduced. Compared to the WT, BSabi bundle sheath cells had a higher Pf, but MCabi spongy mesophyll had an unexpected lower Pf. These results suggest that tissue-specific regulation of Pf by ABA may be confounded by whole-leaf hydraulics and transpiration. ABA increased the symplastic permeability, but its contribution to Kleaf was negligible. We suggest that the bundle sheath spongy mesophyll pathway dynamically responds to the fluctuations in water availability, while the palisade mesophyll serves as a hydraulic buffer.

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.

Arabidopsis leaf hydraulic conductance is regulated by xylem sap pH, controlled, in turn, by a P-type H+ -ATPase of vascular bundle sheath cells.

The leaf vascular bundle sheath cells (BSCs) that tightly envelop the leaf veins, are a selective and dynamic barrier to xylem sap water and solutes radially entering the mesophyll cells. Under normal conditions, xylem sap pH below 6 is presumably important for driving and regulating the transmembranal solute transport. Having discovered recently a differentially high expression of a BSC proton pump, AHA2, we now test the hypothesis that it regulates the xylem sap pH and leaf radial water fluxes. We monitored the xylem sap pH in the veins of detached leaves of wild-type Arabidopsis, AHA mutants and aha2 mutants complemented with AHA2 gene solely in BSCs. We tested an AHA inhibitor (vanadate) and stimulator (fusicoccin), and different pH buffers. We monitored their impact on the xylem sap pH and the leaf hydraulic conductance (Kleaf ), and the effect of pH on the water osmotic permeability (Pf ) of isolated BSCs protoplasts. We found that AHA2 is necessary for xylem sap acidification, and in turn, for elevating Kleaf . Conversely, AHA2 knockdown, which alkalinized the xylem sap, or, buffering its pH to 7.5, reduced Kleaf , and elevating external pH to 7.5 decreased the BSCs Pf . All these showed a causative link between AHA2 activity in BSCs and leaf radial hydraulic water conductance.

Wide vessels sustain marginal transpiration flux and do not optimize inefficient gas exchange activity under impaired hydraulic control and salinity.

Plants optimize water use and carbon assimilation via transient regulation of stomata resistance and by limiting hydraulic conductivity in a long-term response of xylem anatomy. We postulated that without effective hydraulic regulation plants would permanently restrain water loss and photosynthetic productivity under salt stress conditions. We compared wild-type tomatoes to a transgenic type (TT) with impaired stomatal control. Gas exchange activity, biomass, starch content, leaf area and root traits, mineral composition and main stems xylem anatomy and hydraulic conductivity were analyzed in plants exposed to salinities of 1 and 4 dS m-1 over 60 days. As the xylem cannot easily readjust to different environmental conditions, shifts in its anatomy and the permanent effect on plant hydraulic conductivity kept transpiration at lower levels under unstressed conditions and maintained it under salt-stress, while sustaining higher but inefficient assimilation rates, leading to starch accumulation and decreased plant biomass, leaf and root area and root length. Narrow conduits in unstressed TT plants were related to permanent restrain of hydraulic conductivity and plant transpiration. Under salinity, TT plants followed the atmospheric water demand, sustained similar transpiration rate from unstressed to salt-stressed conditions and possibly maintained hydraulic integrity, due to likely impaired hydraulic regulation, wider conduits and higher hydraulic conductivity. The accumulation of salts and starch in the TT plants was a strong evidence of salinity tolerance via osmotic regulation, also thought to help to maintain the assimilation rates and transpiration flux under salinity, although it was not translated into higher growth.

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.

Role of guard-cell ABA in determining steady-state stomatal aperture and prompt vapor-pressure-deficit response.

Abscisic acid (ABA) is known to be involved in stomatal closure. However, its role in stomatal response to rapid increases in the vapor pressure deficit (VPD) is unclear. To study this issue, we generated guard cell-specific ABA-insensitive Arabidopsis plants (guard-cell specific abi1-1; GCabi). Under non-stressed conditions, the stomatal conductance (gs) and apertures of GCabi plants were greater than those of control plants. This supports guard-cell ABA role as limiting steady-state stomatal aperture under non-stressful conditions. When there was a rapid increase in VPD (0.15 to 1 kPa), the gs and stomatal apertures of GCabi decreased in a manner similar that observed in the WT control, but different from that observed in WT plants treated with fusicoccin. Low VPD increased the size of the stomatal apertures of the WT, but not of GCabi. We conclude that guard-cell ABA does not play a significant role in the initial, rapid stomatal closure that occurs in response to an increase in VPD, but is important for stomatal adaptation to ambient VPD. We propose a biphasic angiosperm VPD-sensing model that includes an initial ABA-independent phase and a subsequent ABA-dependent steady-state phase in which stomatal behavior is optimized for ambient VPD conditions.

Role of Aquaporins in a Composite Model of Water Transport in the Leaf.

Water-transport pathways through the leaf are complex and include several checkpoints. Some of these checkpoints exhibit dynamic behavior that may be regulated by aquaporins (AQPs). To date, neither the relative weight of the different water pathways nor their molecular mechanisms are well understood. Here, we have collected evidence to support a putative composite model of water pathways in the leaf and the distribution of water across those pathways. We describe how water moves along a single transcellular path through the parenchyma and continues toward the mesophyll and stomata along transcellular, symplastic and apoplastic paths. We present evidence that points to a role for AQPs in regulating the relative weight of each path in the overall leaf water-transport system and the movement of water between these paths as a result of the integration of multiple signals, including transpiration demand, water potential and turgor. We also present a new theory, the hydraulic fuse theory, to explain effects of the leaf turgor-loss-point on water paths alternation and the subsequent reduction in leaf hydraulic conductivity. An improved understating of leaf water-balance management may lead to the development of crops that use water more efficiently, and responds better to environmental changes.

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.

Differential tissue-specific expression of NtAQP1 in Arabidopsis thaliana reveals a role for this protein in stomatal and mesophyll conductance of CO2 under standard and salt-stress conditions.

The regulation of plant hydraulic conductance and gas conductance involves a number of different morphological, physiological and molecular mechanisms working in harmony. At the molecular level, aquaporins play a key role in the transport of water, as well as CO2, through cell membranes. Yet, their tissue-related function, which controls whole-plant gas exchange and water relations, is less understood. In this study, we examined the tissue-specific effects of the stress-induced tobacco Aquaporin1 (NtAQP1), which functions as both a water and CO2 channel, on whole-plant behavior. In tobacco and tomato plants, constitutive overexpression of NtAQP1 increased net photosynthesis (A(N)), mesophyll CO2 conductance (g(m)) and stomatal conductance (g(s)) and, under stress, increased root hydraulic conductivity (L(pr)) as well. Our results revealed that NtAQP1 that is specifically expressed in the mesophyll tissue plays an important role in increasing both A(N) and g(m). Moreover, targeting NtAQP1 expression to the cells of the vascular envelope significantly improved the plants' stress response. Surprisingly, NtAQP1 expression in the guard cells did not have a significant effect under any of the tested conditions. The tissue-specific involvement of NtAQP1 in hydraulic and gas conductance via the interaction between the vasculature and the stomata is discussed.