γ-Aminobutyric acid plays a key role in plant acclimation to a combination of high light and heat stress.

Plants are frequently subjected to different combinations of abiotic stresses, such as high light (HL) intensity, and elevated temperatures. These environmental conditions pose a threat to agriculture production, affecting photosynthesis, and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources toward stress responses, and suppressing growth. Here we show that the primary metabolic response of Arabidopsis (Arabidopsis thaliana) plants to HL or heat stress (HS) is different from that of plants subjected to a combination of HL and HS (HL+HS). We further demonstrate that the combined stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids coupled with decreased levels of metabolites participating in the tricarboxylic acid cycle. Among the amino acids exclusively accumulated during HL+HS, we identified the nonproteinogenic amino acid γ-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis (GLUTAMATE DESCARBOXYLASE 3 [gad3]) as well as mutants impaired in autophagy (autophagy-related proteins 5 and 9 [atg5 and atg9]), revealed that GABA plays a key role in the acclimation of plants to HL+HS, potentially by promoting autophagy. Taken together, our findings identify a role for GABA in regulating plant responses to combined stress.

Plant size directly correlates with water use efficiency in Arabidopsis.

Plant transpiration is a fundamental process that determines plant water use efficiency (WUE), thermoregulation, nutrition, and growth. How transpiration impacts on such essential physiological aspects and how the environment modulates these effects are fundamental questions about which little is known. We investigated the genetic and environmental factors underlying natural variation in plant transpiration and water use efficiency in a population of natural Arabidopsis thaliana accessions grown under homogeneous conditions. As expected, we observed large variation of total transpiration capacity, transpiration per surface unit, and WUE among A. thaliana accessions. Despite the variation of stomatal density and ABA content in the population, WUE did not correlate with any of these parameters. On the contrary, a surprising direct correlation was found between WUE and projected leaf area, with bigger plants displaying a more efficient use of water. Importantly, genome-wide association studies further supported our observations through the identification of several loci involved in WUE variation, mutations in which caused a simultaneous reduction in plant size and a decrease in WUE. Altogether, our results strongly suggest that, although WUE depends on many parameters, plant size is an adaptive trait with respect to water use in A. thaliana.

Bi-directional, long-distance hormonal signalling between roots and shoots of soil water availability.

While the importance of plant water relations in determining crop response to soil water availability is difficult to over-emphasise, under many circumstances, plants maintain their leaf water status as the soil dries yet shoot gas exchange and growth is restricted. Such observations lead to development of a paradigm that root-to-shoot signals regulate shoot physiology, and a conceptual framework to test the importance of different signals such as plant hormones in these physiological processes. Nevertheless, shoot-to-root (hormonal) signalling also plays an important role in regulating root growth and function and may dominate when larger quantities of a hormone are produced in the shoots than the roots. Here, we review the evidence for acropetal and basipetal transport of three different plant hormones (abscisic acid, jasmonates, strigolactones) that have antitranspirant effects, to indicate the origin and action of these signalling systems. The physiological importance of each transport pathway likely depends on the specific environmental conditions the plant is exposed to, specifically whether the roots or shoots are the first to lose turgor when exposed to drying soil or elevated atmospheric demand, respectively. All three hormones can interact to influence each other's synthesis, degradation and intracellular signalling to augment or attenuate their physiological impacts, highlighting the complexity of unravelling these signalling systems. Nevertheless, such complexity suggests crop improvement opportunities to select for allelic variation in the genes affecting hormonal regulation, and (in selected crops) to augment root-shoot communication by judicious selection of rootstock-scion combinations to ameliorate abiotic stresses.

PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA.

The identification of those prevalent abscisic acid (ABA) receptors and molecular mechanisms that trigger drought adaptation in crops well adapted to harsh conditions such as date palm (Phoenix dactylifera, Pd) sheds light on plant-environment interactions. We reveal that PdPYL8-like receptors are predominantly expressed under abiotic stress, with Pd27 being the most expressed receptor in date palm. Therefore, subfamily I PdPYL8-like receptors have been selected for ABA signaling during abiotic stress response in this crop. Biochemical characterization of PdPYL8-like and PdPYL1-like receptors revealed receptor- and ABA-dependent inhibition of PP2Cs, which triggers activation of the pRD29B-LUC reporter in response to ABA. PdPYLs efficiently abolish PP2C-mediated repression of ABA signaling, but loss of the Trp lock in the seed-specific AHG1-like phosphatase PdPP2C79 markedly impairs its inhibition by ABA receptors. Characterization of Arabidopsis transgenic plants that express PdPYLs shows enhanced ABA signaling in seed, root, and guard cells. Specifically, Pd27-overexpressing plants showed lower ABA content and were more efficient than the wild type in lowering transpiration at negative soil water potential, leading to enhanced drought tolerance. Finally, PdPYL8-like receptors accumulate after ABA treatment, which suggests that ABA-induced stabilization of these receptors operates in date palm for efficient boosting of ABA signaling in response to abiotic stress.

Root exudates: from plant to rhizosphere and beyond.

KEY MESSAGE: This article describes the composition of root exudates, how these metabolites are released to the rhizosphere and their importance in the recruitment of beneficial microbiota that alleviate plant stress. Metabolites secreted to the rhizosphere by roots are involved in several processes. By modulating the composition of the root exudates, plants can modify soil properties to adapt and ensure their survival under adverse conditions. They use several strategies such as (1) changing soil pH to solubilize nutrients into assimilable forms, (2) chelating toxic compounds, (3) attracting beneficial microbiota, or (4) releasing toxic substances for pathogens, etc. In this work, the composition of root exudates as well as the different mechanisms of root exudation have been reviewed. Existing methodologies to collect root exudates, indicating their advantages and disadvantages, are also described. Factors affecting root exudation have been exposed, including physical, chemical, and biological agents which can produce qualitative and quantitative changes in exudate composition. Finally, since root exudates play an important role in the recruitment of mycorrhizal fungi and plant growth-promoting rhizobacteria (PGPR), the mechanisms of interaction between plants and the beneficial microbiota have been highlighted.

Attenuated accumulation of jasmonates modifies stomatal responses to water deficit.

To determine whether drought-induced root jasmonate [jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile)] accumulation affected shoot responses to drying soil, near-isogenic wild-type (WT) tomato (Solanum lycopersicum cv. Castlemart) and the def-1 mutant (which fails to accumulate jasmonates during water deficit) were self- and reciprocally grafted. Rootstock hydraulic conductance was entirely rootstock dependent and significantly lower in def-1, yet def-1 scions maintained a higher leaf water potential as the soil dried due to their lower stomatal conductance (gs). Stomatal sensitivity to drying soil (the slope of gsversus soil water content) was low in def-1 self-grafts but was normalized by grafting onto WT rootstocks. Although soil drying increased 12-oxo-phytodienoic acid (OPDA; a JA precursor and putative antitranspirant) concentrations in def-1 scions, foliar JA accumulation was negligible and foliar ABA accumulation reduced compared with WT scions. A WT rootstock increased drought-induced ABA and JA accumulation in def-1 scions, but decreased OPDA accumulation. Xylem-borne jasmonates were biologically active, since supplying exogenous JA via the transpiration stream to detached leaves decreased transpiration of WT seedlings but had the opposite effect in def-1. Thus foliar accumulation of both ABA and JA at WT levels is required for both maximum (well-watered) gs and stomatal sensitivity to drying soil.

Long-distance ABA transport can mediate distal tissue responses by affecting local ABA concentrations.

Environmental stresses that perturb plant water relations influence abscisic acid (ABA) concentrations, but it is unclear whether long-distance ABA transport contributes to changes in local ABA levels. To determine the physiological relevance of ABA transport, we made reciprocal- and self-grafts of ABA-deficient flacca mutant and wild-type (WT) tomato plants, in which low phosphorus (P) conditions decreased ABA concentrations while salinity increased ABA concentrations. Whereas foliar ABA concentrations in the WT scions were rootstock independent under conditions, salinity resulted in long-distance transport of ABA: flacca scions had approximately twice as much ABA when grafted on WT rootstocks compared to flacca rootstocks. Root ABA concentrations were scion dependent: both WT and flacca rootstocks had less ABA with the flacca mutant scion than with the WT scion under conditions. In WT scions, whereas rootstock genotype had limited effects on stomatal conductance under conditions, a flacca rootstock decreased leaf area of stressed plants, presumably due to attenuated root-to-shoot ABA transport. In flacca scions, a WT rootstock decreased stomatal conductance but increased leaf area of stressed plants, likely due to enhanced root-to-shoot ABA transport. Thus, long-distance ABA transport can affect responses in distal tissues by changing local ABA concentrations.

Physiological impacts of ABA-JA interactions under water-limitation.

Plant responses to drought stress depend on highly regulated signal transduction pathways with multiple interactions. This complex crosstalk can lead to a physiological outcome of drought avoidance or tolerance/resistance. ABA is the principal mediator of these responses due to the regulation of stomatal closure that determines plant growth and survival, but also other strategies of drought resistance such as osmotic adjustment. However, other hormones such as JA seem responsible for regulating a subset of plant responses to drought by regulating ABA biosynthesis and accumulation and ABA-dependent signalling, but also by ABA independent pathways. Here, we review recent reports of ABA-JA hormonal and molecular interactions within a physiological framework of drought tolerance. Understanding the physiological significance of this complex regulation offers opportunities to find strategies of drought tolerance that avoid unwanted side effects that limit growth and yield, and may allow biotechnological crop improvement.

Terpene down-regulation triggers defense responses in transgenic orange leading to resistance against fungal pathogens.

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.

Jasmonoyl isoleucine accumulation is needed for abscisic acid build-up in roots of Arabidopsis under water stress conditions.

Phytohormones are central players in sensing and signalling numerous environmental conditions like drought. In this work, hormone profiling together with gene expression of key enzymes involved in abscisic acid (ABA) and jasmonate biosynthesis were studied in desiccating Arabidopsis roots. Jasmonic acid (JA) content transiently increased after stress imposition whereas progressive and concomitant ABA and Jasmonoyl Isoleucine (JA-Ile) accumulations were detected. Molecular data suggest that, at least, part of the hormonal regulation takes place at the biosynthetic level. These observations also point to a possible involvement of jasmonates on ABA biosynthesis under stress. To test this hypothesis, mutants impaired in jasmonate biosynthesis (opr3, lox6 and jar1-1) and in JA-dependent signalling (coi1) were employed. Results showed that the early JA accumulation leading to JA-Ile build up was necessary for an ABA increase in roots under two different water stress conditions. Signal transduction between water stress-induced JA-Ile accumulation and COI1 is necessary for a full induction of the ABA biosynthesis pathway and subsequent hormone accumulation in roots of Arabidopsis plants. The present work adds a level of interaction between jasmonates and ABA at the biosynthetic level.

Jasmonic acid transient accumulation is needed for abscisic acid increase in citrus roots under drought stress conditions.

Phytohormones are central players in sensing and signaling numerous environmental conditions like drought stress. In this work, an experimental system based on severe drought was established and hormone profiling together with gene expression of key enzymes involved in abscisic acid (ABA) and jasmonic acid (JA) biosynthesis was studied in roots of citrumelo CPB 4475 (a commercial citrus rootstock) plants. JA concentration transiently increased after a few hours of stress, returning to control levels 30 h after the onset of the condition. A more progressive ABA accumulation was observed, with the onset of this increase at the same time or right after the JA transient accumulation. Molecular data suggested that, at least, part of the hormonal regulation takes place at the biosynthetic level. These observations also pointed to a possible involvement of JA on ABA biosynthesis under stress. To test this hypothesis, JA and ABA biosynthesis were chemically inhibited and subsequently phenotypes rescued by the addition of exogenous hormones. Results showed that the early JA accumulation was necessary for the subsequent ABA increase in roots under stress whereas the opposite could not be stated. The model includes a burst of JA in roots of citrus under severe drought stress conditions that leads to a more progressive ABA accumulation that will induce later plant responses. The present work adds a new level of interaction between JA and ABA at the biosynthetic level that together with the previously described interaction between signal transduction cascades of the two hormones would allow plants to fine-tune specific responses to different stimuli.

Carrizo citrange plants do not require the presence of roots to modulate the response to osmotic stress.

The study of the effects of a specific stress condition on the performance of plants grown under field conditions is difficult due to interactions among multiple abiotic and biotic factors affecting the system. In vitro tissue-culture-based techniques allow the study of each adverse condition independently and also make possible to investigate the performance of genotypes of interest under stress conditions avoiding the effect of the root. In this paper, the response of Carrizo citrange, a commercial citrus rootstock, to osmotic stress was evaluated by culturing in vitro intact plants and micropropagated shoots. The osmotic stress was generated by adding two different concentrations of polyethyleneglycol to the culture media. Different parameters such as plant performance, organ length, antioxidant activities, and endogenous contents of proline, malondialdehyde, and hormones were determined. Differently to that observed under high salinity, when subjected to osmotic stress conditions, Carrizo citrange showed increased endogenous levels of MDA, proline, and ABA. These results evidence that the mechanisms of response of Carrizo citrange to saline or osmotic stress are different. The presence of roots was not necessary to activate any of the plant responses which indicates that the organs involved in the stress perception and signaling depends on the type of adverse condition to which plants are subjected.

Identification of ABA-Mediated Genetic and Metabolic Responses to Soil Flooding in Tomato (Solanum lycopersicum L. Mill).

Soil flooding is a compound abiotic stress that alters soil properties and limits atmospheric gas diffusion (O2 and CO2) to the roots. The involvement of abscisic acid (ABA) in the regulation of soil flooding-specific genetic and metabolic responses has been scarcely studied despite its key importance as regulator in other abiotic stress conditions. To attain this objective, wild type and ABA-deficient tomatoes were subjected to short-term (24 h) soil waterlogging. After this period, gas exchange parameters were reduced in the wild type but not in ABA-deficient plants that always had higher E and g s . Transcript and metabolite alterations were more intense in waterlogged tissues, with genotype-specific variations. Waterlogging reduced the ABA levels in the roots while inducing PYR/PYL/RCAR ABA receptors and ABA-dependent transcription factor transcripts, of which induction was less pronounced in the ABA-deficient genotype. Ethylene/O2-dependent genetic responses (ERFVIIs, plant anoxia survival responses, and genes involved in the N-degron pathway) were induced in hypoxic tissues independently of the genotype. Interestingly, genes encoding a nitrate reductase and a phytoglobin involved in NO biosynthesis and scavenging and ERFVII stability were induced in waterlogged tissues, but to a lower extent in ABA-deficient tomato. At the metabolic level, flooding-induced accumulation of Ala was enhanced in ABA-deficient lines following a differential accumulation of Glu and Asp in both hypoxic and aerated tissues, supporting their involvement as sources of oxalacetate to feed the tricarboxylic acid cycle in waterlogged tissues and constituting a potential advantage upon long periods of soil waterlogging. The promoter analysis of upregulated genes indicated that the production of oxalacetate from Asp via Asp oxidase, energy processes such as acetyl-CoA, ATP, and starch biosynthesis, and the lignification process were likely subjected to ABA regulation. Taken together, these data indicate that ABA depletion in waterlogged tissues acts as a positive signal, inducing several specific genetic and metabolic responses to soil flooding.

Soil moisture heterogeneity regulates water use in Populus nigra L. by altering root and xylem sap phytohormone concentrations.

Soil moisture heterogeneity in the root zone is common both during the establishment of tree seedlings and in experiments aiming to impose semi-constant soil moisture deficits, but its effects on regulating plant water use compared with homogenous soil drying are not well known in trees. Pronounced vertical soil moisture heterogeneity was imposed on black poplar (Populus nigra L.) grown in soil columns by altering irrigation frequency, to test whether plant water use, hydraulic responses, root phytohormone concentrations and root xylem sap chemical composition differed between wet (well-watered, WW), and homogeneously (infrequent deficit irrigation, IDI) and heterogeneously dry soil (frequent deficit irrigation, FDI). At the same bulk soil water content, FDI plants had greater water use than IDI plants, probably because root abscisic acid (ABA) concentration was low in the upper wetter layer of FDI plants, which maintained root xylem sap ABA concentration at basal levels in contrast with IDI. Soil drying did not increase root xylem concentration of any other hormone. Nevertheless, plant-to-plant variation in xylem jasmonic acid (JA) concentration was negatively related to leaf stomatal conductance within WW and FDI plants. However, feeding detached leaves with high (1200 nM) JA concentrations via the transpiration stream decreased transpiration only marginally. Xylem pH and sulphate concentration decreased in FDI plants compared with well-watered plants. Frequent deficit irrigation increased root accumulation of the cytokinin trans-zeatin (tZ), especially in the dry lower layer, and of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), in the wet upper soil layer. Root hormone accumulation might explain the maintenance of high root hydraulic conductance and water use in FDI plants (similar to well-watered plants) compared with IDI plants. In irrigated tree crops, growers could vary irrigation scheduling to control water use by altering the hormone balance.

A customizable method to characterize Arabidopsis thaliana transpiration under drought conditions.

BACKGROUND: Characterization of the dynamic response of plant transpiration to decreasing soil water content in a reproducible way is required for the correct phenotyping of traits related to water saving strategies. Nowadays, an increasing number of automated high throughput platforms are available, but their development requires a great economic investment and it is not always desirable/feasible to outsource these analyses. We propose a medium-throughput protocol to characterize transpiration responses to decreasing soil moisture in a quantitative and highly reproducible way with a minimum investment of resources. RESULTS: The quantitative characterization of plant responses to a decreasing soil water content using our phenotyping platform has showed high reproducibility between different experiments. The proposed irrigation strategy allowed us to harvest plants ranging from a well-watered condition to the loss-of-turgor point in a predictable and controlled way. Coupling this protocol with hormone profiling allows investigation of hormonal responses (metabolite accumulation as well as plant sensitivity) to water stress. As a proof-of-concept, we have characterized the dynamic responses of leaf transpiration to decreasing soil water contents in an abscisic acid (ABA) deficient genotype (aba1-1) as well as in genotypes with altered sensitivity to ABA (abi1-1 and hab1-1abi1-1), which are insensitive and hypersensitive to ABA, respectively. CONCLUSIONS: This protocol allows for assessment of quantitative differences in rosette transpiration responses to water depletion in both ABA biosynthesis mutants and genotypes with altered sensitivity to the hormone. Data indicate a correlation between ABA levels and/or hormone perception and growth rate and/or water content. The protocol guarantees the correct application of water stress to adult plants, which is essential to understand responses of mutants and/or natural accessions.

Facing Climate Change: Biotechnology of Iconic Mediterranean Woody Crops.

The Mediterranean basin is especially sensitive to the adverse outcomes of climate change and especially to variations in rainfall patterns and the incidence of extremely high temperatures. These two concurring adverse environmental conditions will surely have a detrimental effect on crop performance and productivity that will be particularly severe on woody crops such as citrus, olive and grapevine that define the backbone of traditional Mediterranean agriculture. These woody species have been traditionally selected for traits such as improved fruit yield and quality or alteration in harvesting periods, leaving out traits related to plant field performance. This is currently a crucial aspect due to the progressive and imminent effects of global climate change. Although complete genome sequence exists for sweet orange (Citrus sinensis) and clementine (Citrus clementina), olive tree (Olea europaea) and grapevine (Vitis vinifera), the development of biotechnological tools to improve stress tolerance still relies on the study of the available genetic resources including interspecific hybrids, naturally occurring (or induced) polyploids and wild relatives under field conditions. To this respect, post-genomic era studies including transcriptomics, metabolomics and proteomics provide a wide and unbiased view of plant physiology and biochemistry under adverse environmental conditions that, along with high-throughput phenotyping, could contribute to the characterization of plant genotypes exhibiting physiological and/or genetic traits that are correlated to abiotic stress tolerance. The ultimate goal of precision agriculture is to improve crop productivity, in terms of yield and quality, making a sustainable use of land and water resources under adverse environmental conditions using all available biotechnological tools and high-throughput phenotyping. This review focuses on the current state-of-the-art of biotechnological tools such as high throughput -omics and phenotyping on grapevine, citrus and olive and their contribution to plant breeding programs.

Jasmonic acid interacts with abscisic acid to regulate plant responses to water stress conditions.

Phytohormones are key players in signaling environmental stress conditions. Hormone profiling together with proline accumulation were studied in leaves and roots of different mutant lines of Arabidopsis. Regulation of proline accumulation in this system seems complex and JA-deficient (jar1-1) and JA-insensitive (jai1) lines accumulating high levels of proline despite their very low ABA levels seems to discard an ABA-dependent response. However, the pattern of proline accumulation in jai1 seedlings parallels that of ABA. Under stress conditions, there is an opposite pattern of ABA accumulation in roots of jar1-1/coi1-16 (in which ABA only slightly increase) and jai1 (in which ABA increase is even higher than in WT plants). This also makes JA-ABA crosstalk complex and discards any lineal pathway that could explain this hormonal interaction.