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.

Stomatal responses to VPD utilize guard cell intracellular signaling components.

Stomatal pores, vital for CO2 uptake and water loss regulation in plants, are formed by two specialized guard cells. Despite their importance, there is limited understanding of how guard cells sense and respond to changes in vapor pressure difference (VPD). This study leverages a selection of CO2 hyposensitive and abscisic acid (ABA) signaling mutants in Arabidopsis, including heterotrimeric G protein mutants and RLK (receptor-like kinase) mutants, along with a variety of canola cultivars to delve into the intracellular signaling mechanisms prompting stomatal closure in response to high VPD. Stomatal conductance response to step changes in VPD was measured using the LI-6800F gas exchange system. Our findings highlight that stomatal responses to VPD utilize intracellular signaling components. VPD hyposensitivity was particularly evident in mutants of the ht1 (HIGH LEAF TEMPERATURE1) gene, which encodes a protein kinase expressed mainly in guard cells, and in gpa1-3, a null mutant of the sole canonical heterotrimeric Gα subunit, previously implicated in stomatal signaling. Consequently, this research identifies a nexus in the intricate relationships between guard cell signal perception, stomatal conductance, environmental humidity, and CO2 levels.

The α subunit of the heterotrimeric G protein regulates mesophyll CO2 conductance and drought tolerance in rice.

Mesophyll conductance gm determines CO2 diffusion rates from mesophyll intercellular air spaces to the chloroplasts and is an important factor limiting photosynthesis. Increasing gm in cultivated plants is a potential strategy to increase photosynthesis and intrinsic water use efficiency (WUEi ). The anatomy of the leaf and metabolic factors such as aquaporins and carbonic anhydrases have been identified as important determinants of gm . However, genes involved in the regulation and modulation of gm remain largely unknown. In this work, we investigated the role of heterotrimeric G proteins in gm and drought tolerance in rice d1 mutants, which harbor a null mutation in the Gα subunit gene, RGA1. d1 mutants in both cv Nipponbare and cv Taichung 65 exhibited increased gm , fostering improvement in photosynthesis, WUEi , and drought tolerance compared with wild-type. The increased surface area of mesophyll cells and chloroplasts exposed to intercellular airspaces and the reduced cell wall and chloroplast thickness in the d1 mutant are evident contributors to the increase in gm . Our results indicate that manipulation of heterotrimeric G protein signaling has the potential to improve crop WUEi and productivity under drought.

Multi-seasonal water-stress memory versus temperature-driven dynamic structural changes in grapevine.

Perennial plants perpetually adapt to environmental changes in complex and yet insufficiently understood manner. We aimed to separate the intra-seasonal temperature effects on structure and function from perennial and annual water stress effects. This study focused on grapevine (Vitis vinifera L. 'Cabernet Sauvignon') petioles, which being a continuously produced organ, represent the current status of the plant. Field-grown mature plants subjected to multi-annual irrigation treatments (severe water stress, mild water stress and non-stressed) throughout the growing season were compared with greenhouse-grown plants under three temperature regimes (22, 28 and 34 °C). Physiological and functional anatomy parameters were measured. A generalized additive model (GAM) based on meteorological and lysimeter-based field data was applied to determine the relative influence of various meteorological parameters on evapotranspiration (ETc) during the growing season in the field experiment. At the beginning of the growing season, in May, petioles in the severe stress treatment showed a stress-related structure (decreased length, safer hydraulic structure and increased lignification), though having high values of stem water potential (SWP). As the season progressed and temperatures increased, all water availability treatments petioles showed similar changes, and at the end of season, in August, were structurally very similar. Those changes were independent of SWP and were comparable to high temperature-induced changes in the greenhouse. In contrast, stems hydraulic structure was strongly influenced by water availability. Regression analyses indicated a relationship between petioles xylem structure and stomatal conductance (gs), whereas gs (but not SWP) was temperature-dependent. The GAM showed that ETc was mainly dependent on temperature. Our results indicate a perennial water-stress memory response, influencing the petiole structure at the beginning of the following season. Intra-seasonally, the petiole's structure becomes independent of water status, whereas temperature drives the structural changes. Thus, ongoing climate change might disrupt plant performance by purely temperature-induced effects.

Elucidating the limiting factors for regeneration and successful establishment of the thermophilic tree Ziziphus spina-christi under a changing climate.

Due to climate change, winter temperatures are predicted to increase worldwide. For thermophilic trees, highly sensitive to low temperatures, an increase in winter temperatures may be beneficial for survival and regeneration. Ziziphus spina-christi is a thermophilic tree that has recently become more abundant and widespread in the eastern Mediterranean, presumably due to a gradual increase in winter temperatures. We aim to define the temperature limitations for seed germination and the growth and survival of young seedlings to broaden our understanding of the future geographical distribution of this species. We studied effects of temperature on germination, growth, and photosynthesis in a controlled environment with four different day/night temperature regimes (34/28 °C, 28/22 °C, 22/16 °C and 16/10 °C). Effects of endocarp on germination and seed germination in the field were also studied. Results showed that germination has a lower thermal optimum (34-22 °C, 63.5-67.5% germination) than growth and photosynthesis (34-28 °C). Moderate cold stress (22/16 °C), did not affect germination capacity, but strongly reduced seedling growth (71%) and photosynthetic capacity (44.6%). Under severe cold stress (16/10 °C), germination still occurs (22%), but seedlings cannot perform growth and photosynthesis. We conclude that slow seedling growth, not germination, is the main barrier for successful establishment of Z. spina-christi under low temperature. Warmer winters could lead to earlier establishment of seedlings and increase their chance of survival the following summer. This may explain the recent increase in the tree's relative abundance and further highlight the potential spread of this species at higher altitudes and latitudes across the Mediterranean.

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.

Long-term acclimation to drought, salinity and temperature in the thermophilic tree Ziziphus spina-christi: revealing different tradeoffs between mesophyll and stomatal conductance.

Photosynthesis is limited by three main factors: stomatal conductance (gs), mesophyll conductance (gm) and maximum capacity for Rubisco carboxylation (Vcmax). It is unclear how limiting factors vary under stress, particularly during long-term stress acclimation. In this work, we compared for the first time photosynthesis limitation resulting from long-term acclimation to three major abiotic stresses: drought, salinity and temperature. We used saplings of Ziziphus spina-christi, a thermophilic and drought-tolerant tree, which recently became more abundant in the Mediterranean, presumably due to increased winter temperatures. Stress acclimation was investigated by measuring growth, gas exchange, chlorophyll fluorescence and leaf structure. For each stress, photosynthesis-limiting factors were compared. We developed an integrative stress index that allowed us to precisely define stress level, enabling a comparison between stress types. Photosynthesis under all stresses was limited mostly by gs and gm (80-90%); whereas biochemistry (Vcmax) made a minor contribution (10-20%). The relative contribution of gs and gm on photosynthetic limitation was influenced by stress type. During acclimation to drought or salinity, photosynthesis was limited by a decline in gs, while intolerance to low temperatures was driven by decline in gm. In all the stresses, gm decreased only under progressive reduction in leaf physiological functionality and was associated with low turgor under drought, an increase in leaf Na+ under salinity and low leaf hydraulic conductance (Kleaf) at low temperatures. Mesophyll structure (mesophyll surface area exposed to the intercellular air spaces, leaf thickness, % intercellular air spaces) did not explain gm acclimation to stress. Current work gives methodology for stress studies, and defines the main factors underlying the plant response to climate change. The ability to minimize mesophyll-imposed limitations on photosynthesis was found as a strong indicator of progressive stress tolerance. Moreover, the results demonstrate how warming climate benefits the photosynthetic function in thermophilic species, such as Ziziphus spina-christi.

Hyperspectral Technologies for Assessing Seed Germination and Trifloxysulfuron-methyl Response in Amaranthus palmeri (Palmer Amaranth).

Weed infestations in agricultural systems constitute a serious challenge to agricultural sustainability and food security worldwide. Amaranthus palmeri S. Watson (Palmer amaranth) is one of the most noxious weeds causing significant yield reductions in various crops. The ability to estimate seed viability and herbicide susceptibility is a key factor in the development of a long-term management strategy, particularly since the misuse of herbicides is driving the evolution of herbicide response in various weed species. The limitations of most herbicide response studies are that they are conducted retrospectively and that they use in vitro destructive methods. Development of a non-destructive method for the prediction of herbicide response could vastly improve the efficacy of herbicide applications and potentially delay the evolution of herbicide resistance. Here, we propose a toolbox based on hyperspectral technologies and data analyses aimed to predict A. palmeri seed germination and response to the herbicide trifloxysulfuron-methyl. Complementary measurement of leaf physiological parameters, namely, photosynthetic rate, stomatal conductence and photosystem II efficiency, was performed to support the spectral analysis. Plant response to the herbicide was compared to image analysis estimates using mean gray value and area fraction variables. Hyperspectral reflectance profiles were used to determine seed germination and to classify herbicide response through examination of plant leaves. Using hyperspectral data, we have successfully distinguished between germinating and non-germinating seeds, hyperspectral classification of seeds showed accuracy of 81.9 and 76.4%, respectively. Sensitive and resistant plants were identified with high degrees of accuracy (88.5 and 90.9%, respectively) from leaf hyperspectral reflectance profiles acquired prior to herbicide application. A correlation between leaf physiological parameters and herbicide response (sensitivity/resistance) was also demonstrated. We demonstrated that hyperspectral reflectance analyses can provide reliable information about seed germination and levels of susceptibility in A. palmeri. The use of reflectance-based analyses can help to better understand the invasiveness of A. palmeri, and thus facilitate the development of targeted control methods. It also has enormous potential for impacting environmental management in that it can be used to prevent ineffective herbicide applications. It also has potential for use in mapping tempo-spatial population dynamics in agro-ecological landscapes.

The effect of blue light on stomatal oscillations and leaf turgor pressure in banana leaves.

Stomatal oscillations are cyclic opening and closing of stomata, presumed to initiate from hydraulic mismatch between leaf water supply and transpiration rate. To test this assumption, mismatches between water supply and transpiration were induced using manipulations of vapour pressure deficit (VPD) and light spectrum in banana (Musa acuminata). Simultaneous measurements of gas exchange with changes in leaf turgor pressure were used to describe the hydraulic mismatches. An increase of VPD above a certain threshold caused stomatal oscillations with variable amplitudes. Oscillations in leaf turgor pressure were synchronized with stomatal oscillations and balanced only when transpiration equaled water supply. Surprisingly, changing the light spectrum from red and blue to red alone at constant VPD also induced stomatal oscillations - while the addition of blue (10%) to red light only ended oscillations. Blue light is known to induce stomatal opening and thus should increase the hydraulic mismatch, reduce the VPD threshold for oscillations and increase the oscillation amplitude. Unexpectedly, blue light reduced oscillation amplitude, increased VPD threshold and reduced turgor pressure loss. These results suggest that additionally, to the known effect of blue light on the hydroactive opening response of stomata, it can also effect stomatal movement by increased xylem-epidermis water supply.