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.

Inhibition of gibberellin accumulation by water deficiency promotes fast and long-term 'drought avoidance' responses in tomato.

Plants reduce transpiration to avoid dehydration during drought episodes by stomatal closure and inhibition of canopy growth. Previous studies have suggested that low gibberellin (GA) activity promotes these 'drought avoidance' responses. Using genome editing, molecular, physiological and hormone analyses, we examined if drought regulates GA metabolism in tomato (Solanum lycopersicum) guard cells and leaves, and studied how this affects water loss. Water deficiency inhibited the expression of the GA biosynthesis genes GA20 oxidase1 (GA20ox1) and GA20ox2 and induced the GA deactivating gene GA2ox7 in guard cells and leaf tissue, resulting in reduced levels of bioactive GAs. These effects were mediated by abscisic acid-dependent and abscisic acid-independent pathways, and by the transcription factor TINY1. The loss of GA2ox7 attenuated stomatal response to water deficiency and during soil dehydration, ga2ox7 plants closed their stomata later, and wilted faster than wild-type (WT) M82 cv. Mutations in GA20ox1 and GA20ox2, had no effect on stomatal closure, but reduced water loss due to the mutants' smaller canopy areas. The results suggested that drought-induced GA deactivation in guard cells, contributes to stomatal closure at the early stages of soil dehydration, whereas inhibition of GA synthesis in leaves suppresses canopy growth and restricts transpiration area.

The Tomato DELLA Protein PROCERA Promotes Abscisic Acid Responses in Guard Cells by Upregulating an Abscisic Acid Transporter.

Plants reduce transpiration through stomatal closure to avoid drought stress. While abscisic acid (ABA) has a central role in the regulation of stomatal closure under water-deficit conditions, we demonstrated in tomato (Solanum lycopersicum) that a gibberellin response inhibitor, the DELLA protein PROCERA (PRO), promotes ABA-induced stomatal closure and gene transcription in guard cells. To study how PRO affects stomatal closure, we performed RNA-sequencing analysis of isolated guard cells and identified the ABA transporters ABA-IMPORTING TRANSPORTER1 1 (AIT1 1) and AIT1 2, also called NITRATE TRANSPORTER1/PTR TRANSPORTER FAMILY4 6 in Arabidopsis (Arabidopsis thaliana), as being upregulated by PRO. Tomato has four AIT1 genes, but only AIT1 1 and AIT1 2 were upregulated by PRO, and only AIT1 1 exhibited high expression in guard cells. Functional analysis of AIT1 1 in yeast (Saccharomyces cerevisiae) confirmed its activity as an ABA transporter, possibly an importer. A clustered regularly interspaced short palindromic repeats-Cas9-derived ait1 1 mutant exhibited an increased transpiration, a larger stomatal aperture, and a reduced stomatal response to ABA. Moreover, ait1 1 suppressed the promoting effects of PRO on ABA-induced stomatal closure and gene expression in guard cells, suggesting that the effects of PRO on stomatal aperture and transpiration are AIT1.1-dependent. Previous studies suggest a negative crosstalk between gibberellin and ABA that is mediated by changes in hormone biosynthesis and signaling. The results of this study suggest this crosstalk is also mediated by changes in hormone transport.

Mutations in the tomato gibberellin receptors suppress xylem proliferation and reduce water loss under water-deficit conditions.

Low gibberellin (GA) activity in tomato (Solanum lycopersicum) inhibits leaf expansion and reduces stomatal conductance. This leads to lower transpiration and improved water status under transient drought conditions. Tomato has three GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptors with overlapping activities and high redundancy. We tested whether mutation in a single GID1 reduces transpiration without affecting growth and productivity. CRISPR-Cas9 gid1 mutants were able to maintain higher leaf water content under water-deficit conditions. Moreover, while gid1a exhibited normal growth, it showed reduced whole-plant transpiration and better recovery from dehydration. Mutation in GID1a inhibited xylem vessel proliferation, which led to lower hydraulic conductance. In stronger GA mutants, we also found reduced xylem vessel expansion. These results suggest that low GA activity affects transpiration by multiple mechanisms: it reduces leaf area, promotes stomatal closure, and reduces xylem proliferation and expansion, and as a result, xylem hydraulic conductance. We further examined if gid1a performs better than the control M82 in the field. Under these conditions, the high redundancy of GID1s was lost and gid1a plants were semi-dwarf, but their productivity was not affected. Although gid1a did not perform better under drought conditions in the field, it exhibited a higher harvest index.

Multiple Gibberellin Receptors Contribute to Phenotypic Stability under Changing Environments.

The pleiotropic and complex gibberellin (GA) response relies on targeted proteolysis of DELLA proteins mediated by a GA-activated GIBBERELLIN-INSENSITIVE DWARF1 (GID1) receptor. The tomato (Solanum lycopersicum) genome encodes for a single DELLA protein, PROCERA (PRO), and three receptors, SlGID1a (GID1a), GID1b1, and GID1b2, that may guide specific GA responses. In this work, clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR associated protein 9-derived gid1 mutants were generated and their effect on GA responses was studied. The gid1 triple mutant was extremely dwarf and fully insensitive to GA. Under optimal growth conditions, the three receptors function redundantly and the single gid1 mutants exhibited very mild phenotypic changes. Among the three receptors, GID1a had the strongest effects on germination and growth. Yeast two-hybrid assays suggested that GID1a has the highest affinity to PRO. Analysis of lines with a single active receptor demonstrated a unique role for GID1a in protracted response to GA that was saturated only at high doses. When the gid1 mutants were grown in the field under ambient changing environments, they showed phenotypic instability, the high redundancy was lost, and gid1a exhibited dwarfism that was strongly exacerbated by the loss of another GID1b receptor gene. These results suggest that multiple GA receptors contribute to phenotypic stability under environmental extremes.