Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress-related proteins.

Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such gene targeting/gene tagging (GT) can alter expression of the target gene. The enzyme encoded by Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localisation have been difficult to assay. We used GT to insert YFP in frame with the 5' or 3' ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3' end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localisation and tissue-specific expression. In contrast, insertion of YFP at the 5' end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene.

Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

Time for a drought experiment: Do you know your plants' water status?

Drought stress is an increasing concern because of climate change and increasing demands on water for agriculture. There are still many unknowns about how plants sense and respond to water limitation, including which genes and cellular mechanisms are impactful for ecology and crop improvement in drought-prone environments. A better understanding of plant drought resistance will require integration of several research disciplines. A common set of parameters to describe plant water status and quantify drought severity can enhance data interpretation and research integration across the research disciplines involved in understanding drought resistance and would be especially useful in integrating the flood of genomic data being generated in drought studies. Water potential (ψw) is a physical measure of the free energy status of water that, along with related physiological measurements, allows unambiguous description of plant water status that can apply across various soil types and environmental conditions. ψw and related physiological parameters can be measured with relatively modest investment in equipment and effort. Thus, we propose that increased use of ψw as a fundamental descriptor of plant water status can enhance the insight gained from many drought-related experiments and facilitate data integration and sharing across laboratories and research disciplines.

Natural variation identifies new effectors of water-use efficiency in Arabidopsis.

Water-use efficiency (WUE) is the ratio of biomass produced per unit of water consumed; thus, it can be altered by genetic factors that affect either side of the ratio. In the present study, we exploited natural variation for WUE to discover loci affecting either biomass accumulation or water use as factors affecting WUE. Genome-wide association studies (GWAS) using integrated WUE measured through carbon isotope discrimination (δ13C) of Arabidopsis thaliana accessions identified genomic regions associated with WUE. Reverse genetic analysis of 70 candidate genes selected based on the GWAS results and transcriptome data identified 25 genes affecting WUE as measured by gravimetric and δ13C analyses. Mutants of four genes had higher WUE than wild type, while mutants of the other 21 genes had lower WUE. The differences in WUE were caused by either altered biomass or water consumption (or both). Stomatal density (SD) was not a primary cause of altered WUE in these mutants. Leaf surface temperatures indicated that transpiration differed for mutants of 16 genes, but generally biomass accumulation had a greater effect on WUE. The genes we identified are involved in diverse cellular processes, including hormone and calcium signaling, meristematic activity, photosynthesis, flowering time, leaf/vasculature development, and cell wall composition; however, none of them had been previously linked to WUE. Thus, our study successfully identified effectors of WUE that can be used to understand the genetic basis of WUE and improve crop productivity.

Size and activity of the root meristem: A key for drought resistance and a key model of drought-related signaling.

Plants make many adjustments to their growth and development in response to even small changes in water availability. Under such conditions, root elongation can be actively restricted by stress-related signaling mechanisms. Here we look at how the Arabidopsis thaliana root meristem can be affected by moderate water limitation (low water potential, ψw ). Recent characterization of the clade E Growth-Regulating (EGR) protein phosphatases and Microtubule Associated Stress Protein 1 (MASP1) provides an example of how active restriction of root meristem size allows the plant to downregulate root elongation during low ψw stress. EGR2 protein accumulation in cortex cells of the transition zone at the distal end of the root meristem illustrates how the balance of cell division versus cell expansion signals at this critical location can determine meristem size and root elongation during low ψw . These characteristics of EGRs also raise the question of whether they may also be involved in hydrotropism, and, more broadly, whether hydrotropism is a distinct response or a specific manifestation of more general mechanisms used to adjust root growth under moderate severity low ψw whether or not a gradient of water availability is present. These questions, as well as a better understanding of how specific cell layers (cortex and endodermis) seem to have an outsized role in growth regulation and better understanding the roles of plasma membrane-based signaling and polar-localized proteins in the regulation of root meristem size and cell division activity are key to elucidating the cellular mechanisms that determine root growth behavior during soil drying.

Spatial differences in stoichiometry of EGR phosphatase and Microtubule-associated Stress Protein 1 control root meristem activity during drought stress.

During moderate severity drought and low water potential (ψw) stress, poorly understood signaling mechanisms restrict both meristem cell division and subsequent cell expansion. We found that the Arabidopsis thaliana Clade E Growth-Regulating 2 (EGR2) protein phosphatase and Microtubule-Associated Stress Protein 1 (MASP1) differed in their stoichiometry of protein accumulation across the root meristem and had opposing effects on root meristem activity at low ψw. Ectopic MASP1 or EGR expression increased or decreased, respectively, root meristem size and root elongation during low ψw stress. This, along with the ability of phosphomimic MASP1 to overcome the EGR-mediated suppression of root meristem size and the observation that ectopic EGR expression had no effect on unstressed plants, indicated that during low ψw EGR activation and attenuation of MASP1 phosphorylation in their overlapping zone of expression determines root meristem size and activity. Ectopic EGR expression also decreased root cell size at low ψw. Conversely, both the egr1-1 egr2-1 and egr1-1 egr2-1 masp1-1 mutants had similarly increased root cell size but only egr1-1egr2-1 had increased cell division. These observations demonstrated that EGRs affect meristem activity via MASP1 but affect cell expansion via other mechanisms. Interestingly, EGR2 was highly expressed in the root cortex, a cell type important for growth regulation and environmental response.

Highly ABA-Induced 1 (HAI1)-Interacting protein HIN1 and drought acclimation-enhanced splicing efficiency at intron retention sites.

The Highly ABA-Induced 1 (HAI1) protein phosphatase is a central component of drought-related signaling. A screen for HAI1-interacting proteins identified HAI1-Interactor 1 (HIN1), a nuclear protein of unknown function which could be dephosphorylated by HAI1 in vitro. HIN1 colocalization and interaction with serine-arginine rich (SR) splicing factors and appearance of nuclear speckle-localized HIN1 during low water potential (ψw) stress suggested a pre-mRNA splicing-related function. RNA sequencing of Arabidopsis Col-0 wild type identified more than 500 introns where moderate severity low ψw altered intron retention (IR) frequency. Surprisingly, nearly 90% of these had increased splicing efficiency (decreased IR) during stress. For one-third of these introns, ectopic HIN1 expression (35S:HIN1) in unstressed plants mimicked the increased splicing efficiency seen in stress-treated wild type. HIN1 bound to a GAA-repeat, Exonic Splicing Enhancer-like RNA motif enriched in flanking sequence around HIN1-regulated introns. Genes with stress and HIN1-affected splicing efficiency were enriched for abiotic stress and signaling-related functions. The 35S:HIN1 plants had enhanced growth maintenance during low ψw, while hin1 mutants had reduced growth, further indicating the role of HIN1 in drought response. HIN1 is annotated as an MYB/SANT domain protein but has limited homology to other MYB/SANT proteins and is not related to known yeast or metazoan RNA-binding proteins or splicing regulators. Together these data identify HIN1 as a plant-specific RNA-binding protein, show a specific effect of drought acclimation to promote splicing efficiency of IR-prone introns, and also discover HAI1-HIN1 interaction and dephosphorylation that connects stress signaling to splicing regulation.

The flip side of phospho-signalling: Regulation of protein dephosphorylation and the protein phosphatase 2Cs.

Protein phosphorylation is a key signalling mechanism and has myriad effects on protein function. Phosphorylation by protein kinases can be reversed by protein phosphatases, thus allowing dynamic control of protein phosphorylation. Although this may suggest a straightforward kinase-phosphatase relationship, plant genomes contain five times more kinases than phosphatases. Here, we examine phospho-signalling from a protein phosphatase centred perspective and ask how relatively few phosphatases regulate many phosphorylation sites. The most abundant class of plant phosphatases, the protein phosphatase 2Cs (PP2Cs), is surrounded by a web of regulation including inhibitor and activator proteins as well as posttranslational modifications that regulate phosphatase activity, control phosphatase stability, or determine the subcellular locations where the phosphatase is present and active. These mechanisms are best established for the Clade A PP2Cs, which are key components of stress and abscisic acid signalling. We also describe other PP2C clades and illustrate how these phosphatases are highly regulated and involved in a wide range of physiological functions. Together, these examples of multiple layers of phosphatase regulation help explain the unbalanced kinase-phosphatase ratio. Continued use of phosphoproteomics to examine phosphatase targets and phosphatase-kinase relationships will be important for deeper understanding of phosphoproteome regulation.

Low Water Potential and At14a-Like1 (AFL1) Effects on Endocytosis and Actin Filament Organization.

At14a-Like1 (AFL1) is a stress-induced protein of unknown function that promotes growth during low water potential stress and drought. Previous analysis indicated that AFL1 may have functions related to endocytosis and regulation of actin filament organization, processes for which the effects of low water potential are little known. We found that low water potential led to a decrease in endocytosis, as measured by uptake of the membrane-impermeable dye FM4-64. Ectopic expression of AFL1 reversed the decrease in FM4-64 uptake seen in wild type, while reduced AFL1 expression led to further inhibition of FM4-64 uptake. Increased AFL1 also made FM4-64 uptake less sensitive to the actin filament disruptor Latrunculin B (LatB). LatB decreased AFL1-Clathrin Light Chain colocalization, further indicating that effects of AFL1 on endocytosis may be related to actin filament organization or stability. Consistent with this hypothesis, ectopic AFL1 expression made actin filaments less sensitive to disruption by LatB or Cytochalasin D and led to increased actin filament skewness and decreased occupancy, indicative of more bundled actin filaments. This latter effect could be partially mimicked by the actin filament stabilizer Jasplakinolide (JASP). However, AFL1 did not substantially inhibit actin filament dynamics, indicating that AFL1 acts via a different mechanism than JASP-induced stabilization. AFL1 partially colocalized with actin filaments but not with microtubules, further indicating actin-filament-related function of AFL1. These data provide insight into endocytosis and actin filament responses to low water potential stress and demonstrate an involvement of AFL1 in these key cellular processes.

Natural Variation in 9-Cis-Epoxycartenoid Dioxygenase 3 and ABA Accumulation.

The stress hormone abscisic acid (ABA) is critical for drought resistance; however, mechanisms controlling ABA levels are unclear. At low water potential, ABA accumulation in the Arabidopsis (Arabidopsis thaliana) accession Shahdara (Sha) was less than that in Landsberg erecta (Ler) or Columbia. Analysis of a Ler × Sha recombinant inbred line population revealed a single major-effect quantitative trait locus for ABA accumulation, which included 9-cis-epoxycarotenoid dioxygenase3 (NCED3) as a candidate gene. NCED3 encodes a rate-limiting enzyme for stress-induced ABA synthesis. Complementation experiments indicated that Sha has a reduced-function NCED3 allele. Compared with Ler, Sha did not have reduced NCED3 gene expression or protein level but did have four amino acid substitutions within NCED3. Sha differed from Ler in the apparent molecular mass of NCED3, indicative of altered NCED3 proteolytic processing in the chloroplast. Site-directed mutagenesis demonstrated that substitution at amino acid 271 was critical for the altered NCED3 molecular mass pattern, while the other Sha NCED3 polymorphisms were also involved in the reduced ABA accumulation. Sha did not have a reduced level of thylakoid-bound NCED3 but did differ from Ler in the apparent molecular mass of stromal NCED3. As Sha was not impaired in known factors critical for NCED3 function in ABA synthesis (expression, chloroplast import, and thylakoid binding), the differences between Ler and Sha NCED3 may affect NCED3 activity or other factors influencing NCED3 function. These results identify functionally important sites on NCED3 and indicate a complex pattern of NCED3 posttranslational regulation in the chloroplast.

Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook-Like10 phosphorylation required for stress growth regulation.

The clade A protein phosphatase 2C Highly ABA-Induced 1 (HAI1) plays an important role in stress signaling, yet little information is available on HAI1-regulated phosphoproteins. Quantitative phosphoproteomics identified phosphopeptides of increased abundance in hai1-2 in unstressed plants and in plants exposed to low-water potential (drought) stress. The identity and localization of the phosphoproteins as well as enrichment of specific phosphorylation motifs indicated that these phosphorylation sites may be regulated directly by HAI1 or by HAI1-regulated kinases including mitogen-activated protein kinases, sucrose non-fermenting-related kinase 2, or casein kinases. One of the phosphosites putatively regulated by HAI1 was S313/S314 of AT-Hook-Like10 (AHL10), a DNA-binding protein of unclear function. HAI1 could directly dephosphorylate AHL10 in vitro, and the level of HAI1 expression affected the abundance of phosphorylated AHL10 in vivo. AHL10 S314 phosphorylation was critical for restriction of plant growth under low-water potential stress and for regulation of jasmonic acid and auxin-related gene expression as well as expression of developmental regulators including Shootmeristemless These genes were also misregulated in hai1-2 AHL10 S314 phosphorylation was required for AHL10 complexes to form foci within the nucleoplasm, suggesting that S314 phosphorylation may control AHL10 association with the nuclear matrix or with other transcriptional regulators. These data identify a set of HAI1-affected phosphorylation sites, show that HAI1-regulated phosphorylation of AHL10 S314 controls AHL10 function and localization, and indicate that HAI1-AHL10 signaling coordinates growth with stress and defense responses.

Natural variation identifies genes affecting drought-induced abscisic acid accumulation in Arabidopsis thaliana.

Accumulation of the stress hormone abscisic acid (ABA) in response to drought and low water-potential controls many downstream acclimation mechanisms. However, mechanisms controlling ABA accumulation itself are less known. There was a 10-fold range of variation in ABA levels among nearly 300 Arabidopsis thaliana accessions exposed to the same low water-potential severity. Genome-wide association analysis (GWAS) identified genomic regions containing clusters of ABA-associated SNPs. Candidate genes within these regions included few genes with known stress or ABA-related function. The GWAS data were used to guide reverse genetic analysis, which found effectors of ABA accumulation. These included plasma-membrane-localized signaling proteins such as receptor-like kinases, aspartic protease, a putative lipid-binding START domain protein, and other membrane proteins of unknown function as well as a RING U-box protein and possible effect of tonoplast transport on ABA accumulation. Putative loss-of-function polymorphisms within the START domain protein were associated with climate factors at accession sites of origin, indicating its potential involvement in drought adaptation. Overall, using ABA accumulation as a basis for a combined GWAS-reverse genetic strategy revealed the broad natural variation in low-water-potential-induced ABA accumulation and was successful in identifying genes that affect ABA levels and may act in upstream drought-related sensing and signaling mechanisms. ABA effector loci were identified even when each one was of incremental effect, consistent with control of ABA accumulation being distributed among the many branches of ABA metabolism or mediated by genes with partially redundant function.

Epigenetics and RNA Processing: Connections to Drought, Salt, and ABA?

There have been great research advances in epigenetics, RNA splicing, and mRNA processing over recent years. In parallel, there have been many advances in abiotic stress and Abscisic Acid (ABA) signaling. Here we overview studies that have examined stress-induced changes in the epigenome and RNA processing as well as cases where disrupting these processes changes the plant response to abiotic stress. We also highlight some examples where specific connections of stress or ABA signaling to epigenetics or RNA processing have been found. By implication, this also points out cases where such mechanistic connections are likely to exist but are yet to be characterized. In the absence of such specific connections to stress signaling, it should be kept in mind that stress sensitivity phenotypes of some epigenetic or RNA processing mutants maybe the result of indirect, pleiotropic effects and thus may perhaps not indicate a direct function in stress acclimation.

Rapid Quantification of Abscisic Acid by GC-MS/MS for Studies of Abiotic Stress Response.

Drought and low water potential induce large increases in Abscisic Acid (ABA ) content of plant tissue. This increased ABA content is essential to regulate downstream stress resistance responses; however, the mechanisms regulating ABA accumulation are incompletely known. Thus, the ability to accurately quantify ABA at high throughput and low cost is important for plant stress research. We have combined and modified several previously published protocols to establish a rapid ABA analysis protocol using gas chromatography-tandem mass spectrometry (GC-MS/MS). Derivatization of ABA is performed with (trimethylsilyl)-diazomethane rather than the harder to prepare diazomethane. Sensitivity of the analysis is sufficient that small samples of low water potential treated Arabidopsis thaliana seedlings can be routinely analyzed in reverse genetic studies of putative stress regulators as well as studies of natural variation in ABA accumulation.

Comparative Analysis of Phosphoproteome Remodeling After Short Term Water Stress and ABA Treatments versus Longer Term Water Stress Acclimation.

Several studies have used short term dehydration, osmotic stress or Abscisic Acid (ABA) treatments to identify the initial protein phosphorylation-dephosphorylation responses to drought and low water potential or ABA treatments. However, longer term drought acclimation leads to altered expression of many kinases and phosphatases suggesting that it may also produce unique changes in phosphoproteome composition. To get a better overview of the state of drought-related phosphoproteomics and investigate this question of short versus longer term phosphoproteome regulation, we compared three Arabidopsis thaliana studies analyzing short term phosphoproteome changes to recent data from our laboratory analyzing phosphoproteome changes after a longer drought acclimation treatment. There was very little overlap of phosphoproteins with putative stress-induced phosphorylation or dephosphorylation among these studies. While some of this is due to technical limitations and limited coverage of the phosphoproteome achieved by each study, biological differences and the type of stress treatment used also play a role. This comparative analysis emphasized how both short and long term analysis of physiologically relevant stress treatments, as well as validation of phosphoproteomic data, will be needed to move past just scratching the surface of the stress phosphoproteome. In drought acclimation experiments, distinguishing between changes in protein abundance versus phosphorylation stoichiometry is a key challenge. We discuss initial work in using Arabidopsis seedling transient expression combined with Phos-tag gel analysis as a way to validate drought-induced phosphorylation-dephosphorylation of candidate proteins.

Protein Phosphatase 2Cs and Microtubule-Associated Stress Protein 1 Control Microtubule Stability, Plant Growth, and Drought Response.

Plant growth is coordinated with environmental factors, including water availability during times of drought. Microtubules influence cell expansion; however, the mechanisms by which environmental signals impinge upon microtubule organization and whether microtubule-related factors limit growth during drought remains unclear. We found that three Clade E Growth-Regulating (EGR) Type 2C protein phosphatases act as negative growth regulators to restrain growth during drought. Quantitative phosphoproteomics indicated that EGRs target cytoskeleton and plasma membrane-associated proteins. Of these, Microtubule-Associated Stress Protein 1 (MASP1), an uncharacterized protein, increased in abundance during stress treatment and could bind, bundle, and stabilize microtubules in vitro. MASP1 overexpression enhanced growth, in vivo microtubule stability, and recovery of microtubule organization during drought acclimation. These MASP1 functions in vivo were dependent on phosphorylation of a single serine. For all EGR and MASP1 mutants and transgenic lines examined, enhanced microtubule recovery and stability were associated with increased growth during drought stress. The EGR-MASP1 system selectively regulates microtubule recovery and stability to adjust plant growth and cell expansion in response to changing environmental conditions. Modification of EGR-MASP1 signaling may be useful to circumvent negative growth regulation limiting plant productivity. EGRs are likely to regulate additional proteins involved in microtubule stability and stress signaling.

Time to grow: factors that control plant growth during mild to moderate drought stress.

This article comments on: Time of day determines Arabidopsis transcriptome and growth dynamics under mild drought.

Interactive effects of water limitation and elevated temperature on the physiology, development and fitness of diverse accessions of Brachypodium distachyon.

An enduring question in plant physiology and evolution is how single genotypes of plants optimize performance in diverse, often highly variable, environments. We grew 35 natural accessions of the grass Brachypodium distachyon in four environments in the glasshouse, contrasting soil water deficit, elevated temperature and their interaction. We modeled treatment, genotype and interactive effects on leaf-level and whole-plant traits, including fecundity. We also assessed the relationship between glasshouse-measured traits and parameters related to climate at the place of origin. We found abundant genetic variation in both constitutive and induced traits related to plant-water relations. Most traits showed strong interaction between temperature and water availability, and we observed genotype-by-environment interaction for several traits. Notably, leaf free proline abundance showed a strong effect of genotype × temperature × water. We found strong associations between phenology, biomass and water use efficiency (WUE) with parameters describing climate of origin. Plants respond to multiple stressors in ways not directly predictable from single stressors, underscoring the complex and trait-specific mechanisms of environmental response. Climate-trait correlations support a role for WUE and phenology in local adaptation to climate in B. distachyon.