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.

Correction: The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

[This corrects the article DOI: 10.1371/journal.pgen.1010766.].

The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

The floral transition occurs at the shoot apical meristem (SAM) in response to favourable external and internal signals. Among these signals, variations in daylength (photoperiod) act as robust seasonal cues to activate flowering. In Arabidopsis, long-day photoperiods stimulate production in the leaf vasculature of a systemic florigenic signal that is translocated to the SAM. According to the current model, FLOWERING LOCUS T (FT), the main Arabidopsis florigen, causes transcriptional reprogramming at the SAM, so that lateral primordia eventually acquire floral identity. FT functions as a transcriptional coregulator with the bZIP transcription factor FD, which binds DNA at specific promoters. FD can also interact with TERMINAL FLOWER 1 (TFL1), a protein related to FT that acts as a floral repressor. Thus, the balance between FT-TFL1 at the SAM influences the expression levels of floral genes targeted by FD. Here, we show that the FD-related bZIP transcription factor AREB3, which was previously studied in the context of phytohormone abscisic acid signalling, is expressed at the SAM in a spatio-temporal pattern that strongly overlaps with FD and contributes to FT signalling. Mutant analyses demonstrate that AREB3 relays FT signals redundantly with FD, and the presence of a conserved carboxy-terminal SAP motif is required for downstream signalling. AREB3 shows unique and common patterns of expression with FD, and AREB3 expression levels are negatively regulated by FD thus forming a compensatory feedback loop. Mutations in another bZIP, FDP, further aggravate the late flowering phenotypes of fd areb3 mutants. Therefore, multiple florigen-interacting bZIP transcription factors have redundant functions in flowering at the SAM.

Histone Deacetylase Complex 1 and histone 1 epigenetically moderate stress responsiveness of Arabidopsis thaliana seedlings.

Early responses of plants to environmental stress factors prevent damage but can delay growth and development in fluctuating conditions. Optimising these trade-offs requires tunability of plant responsiveness to environmental signals. We have previously reported that Histone Deacetylase Complex 1 (HDC1), which interacts with multiple proteins in histone deacetylation complexes, regulates the stress responsiveness of Arabidopsis seedlings, but the underlying mechanism remained elusive. Here, we show that HDC1 attenuates transcriptome re-programming in salt-treated seedlings, and we identify two genes (LEA and MAF5) that inhibit seedling establishment under salt stress downstream of HDC1. HDC1 attenuates their transcriptional induction by salt via a dual mechanism involving H3K9/14 deacetylation and H3K27 trimethylation. The latter, but not the former, was also abolished in a triple knockout mutant of the linker histone H1, which partially mimics the hypersensitivity of the hdc1-1 mutant to salt stress. Although stress-induced H3K27me3 accumulation required both H1 and HDC1, it was not fully recovered by complementing hdc1-1 with a truncated, H1-binding competent HDC1 suggesting other players or independent inputs. The combined findings reveal a dual brake function of HDC1 via regulating both active and repressive epigenetic marks on stress-inducible genes. This natural 'anti-panic' device offers a molecular leaver to tune stress responsiveness in plants.

Maize adaptation across temperate climates was obtained via expression of two florigen genes.

Expansion of the maize growing area was central for food security in temperate regions. In addition to the suppression of the short-day requirement for floral induction, it required breeding for a large range of flowering time that compensates the effect of South-North gradients of temperatures. Here we show the role of a novel florigen gene, ZCN12, in the latter adaptation in cooperation with ZCN8. Strong eQTLs of ZCN8 and ZCN12, measured in 327 maize lines, accounted for most of the genetic variance of flowering time in platform and field experiments. ZCN12 had a strong effect on flowering time of transgenic Arabidopsis thaliana plants; a path analysis showed that it directly affected maize flowering time together with ZCN8. The allelic composition at ZCN QTLs showed clear signs of selection by breeders. This suggests that florigens played a central role in ensuring a large range of flowering time, necessary for adaptation to temperate areas.

GIGANTEA Is a Negative Regulator of Abscisic Acid Transcriptional Responses and Sensitivity in Arabidopsis.

Transcriptional reprogramming plays a key role in drought stress responses, preceding the onset of morphological and physiological acclimation. The best-characterized signal regulating gene expression in response to drought is the phytohormone abscisic acid (ABA). ABA-regulated gene expression, biosynthesis and signaling are highly organized in a diurnal cycle, so that ABA-regulated physiological traits occur at the appropriate time of day. The mechanisms that underpin such diel oscillations in ABA signals are poorly characterized. Here we uncover GIGANTEA (GI) as a key gatekeeper of ABA-regulated transcriptional and physiological responses. Time-resolved gene expression profiling by RNA sequencing under different irrigation scenarios indicates that gi mutants produce an exaggerated ABA response, despite accumulating wild-type levels of ABA. Comparisons with ABA-deficient mutants confirm the role of GI in controlling ABA-regulated genes, and the analysis of leaf temperature, a read-out for transpiration, supports a role for GI in the control of ABA-regulated physiological processes. Promoter regions of GI/ABA-regulated transcripts are directly targeted by different classes of transcription factors (TFs), especially PHYTOCHROME-INTERACTING FACTOR and -BINDING FACTOR, together with GI itself. We propose a model whereby diel changes in GI control oscillations in ABA responses. Peak GI accumulation at midday contributes to establishing a phase of reduced ABA sensitivity and related physiological responses, by gating DNA binding or function of different classes of TFs that cooperate or compete with GI at target regions.

Abscisic Acid and Flowering Regulation: Many Targets, Different Places.

Plants can react to drought stress by anticipating flowering, an adaptive strategy for plant survival in dry climates known as drought escape (DE). In Arabidopsis, the study of DE brought to surface the involvement of abscisic acid (ABA) in controlling the floral transition. A central question concerns how and in what spatial context can ABA signals affect the floral network. In the leaf, ABA signaling affects flowering genes responsible for the production of the main florigen FLOWERING LOCUS T (FT). At the shoot apex, FD and FD-like transcription factors interact with FT and FT-like proteins to regulate ABA responses. This knowledge will help separate general and specific roles of ABA signaling with potential benefits to both biology and agriculture.

Hormonal control of the floral transition: Can one catch them all?

The transition to flowering marks a key adaptive developmental switch in plants which impacts on their survival and fitness. Different signaling pathways control the floral transition, conveying both endogenous and environmental cues. These cues are often relayed and/or modulated by different hormones, which might confer additional developmental flexibility to the floral process in the face of varying conditions. Among the different hormonal pathways, the phytohormone gibberellic acid (GA) plays a dominant role. GA is connected with the other floral pathways through the GA-regulated DELLA proteins, acting as versatile interacting modules for different signaling proteins. In this review, I will highlight the role of DELLAs as spatial and temporal modulators of different consolidated floral pathways. Next, building on recent data, I will provide an update on some emerging themes connecting other hormone signaling cascades to flowering time control. I will finally provide examples for some established as well as potential cross-regulatory mechanisms between hormonal pathways mediated by the DELLA proteins.

A novel role for STOMATAL CARPENTER 1 in stomata patterning.

BACKGROUND: Guard cells (GCs) are specialised cells within the plant epidermis which form stomatal pores, through which gas exchange can occur. The GCs derive through a specialised lineage of cell divisions which is specified by the transcription factor SPEECHLESS (SPCH), the expression of which can be detected in undifferentiated epidermal cells prior to asymmetric division. Other transcription factors may act before GC specification and be required for correct GC patterning. Previously, the DOF transcription factor STOMATAL CARPENTER 1 (SCAP1) was shown to be involved in GC function, by activating a set of GC-specific genes required for GC maturation and activity. It is thus far unknown whether SCAP1 can also affect stomatal development. RESULTS: Here we show that SCAP1 expression can also be observed in young leaf primordia, before any GC differentiation occurs. The study of transgenic plants carrying a proSCAP1:GUS-GFP transcriptional fusion, coupled with qPCR analyses, indicate that SCAP1 expression peaks in a temporal window which is coincident with expression of stomatal patterning genes. Independent scap1 loss-of-function mutants have a reduced number of GCs whilst SCAP1 over expression lines have an increased number of GCs, in addition to altered GC distribution and spacing patterns. The study of early markers for stomatal cell lineage in a background carrying gain-of-function alleles of SCAP1 revealed that, compared to the wild type, an increased number of protodermal cells are recruited in the GC lineage, which is reflected in an increased number of meristemoids. CONCLUSIONS: Our results suggest an early role for SCAP1 in GC differentiation. We propose that a function of SCAP1 is to integrate different aspects of GC biology including specification, spacing, maturation and function.

ABA-dependent control of GIGANTEA signalling enables drought escape via up-regulation of FLOWERING LOCUS T in Arabidopsis thaliana.

One strategy deployed by plants to endure water scarcity is to accelerate the transition to flowering adaptively via the drought escape (DE) response. In Arabidopsis thaliana, activation of the DE response requires the photoperiodic response gene GIGANTEA (GI) and the florigen genes FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF). The phytohormone abscisic acid (ABA) is also required for the DE response, by promoting the transcriptional up-regulation of the florigen genes. The mode of interaction between ABA and the photoperiodic genes remains obscure. In this work we use a genetic approach to demonstrate that ABA modulates GI signalling and consequently its ability to activate the florigen genes. We also reveal that the ABA-dependent activation of FT, but not TSF, requires CONSTANS (CO) and that impairing ABA signalling dramatically reduces the expression of florigen genes with little effect on the CO transcript profile. ABA signalling thus has an impact on the core genes of photoperiodic signalling GI and CO by modulating their downstream function and/or activities rather than their transcript accumulation. In addition, we show that as well as promoting flowering, ABA simultaneously represses flowering, independent of the florigen genes. Genetic analysis indicates that the target of the repressive function of ABA is the flowering-promoting gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a transcription factor integrating floral cues in the shoot meristem. Our study suggests that variations in ABA signalling provide different developmental information that allows plants to co-ordinate the onset of the reproductive phase according to the available water resources.

Stability of small ubiquitin-like modifier (SUMO) proteases OVERLY TOLERANT TO SALT1 and -2 modulates salicylic acid signalling and SUMO1/2 conjugation in Arabidopsis thaliana.

Small ubiquitin-like modifier proteases 1 and 2 (SUMO1/2) have been linked to the regulation of salicylic acid (SA)-mediated defence signalling in Arabidopsis thaliana. In order to define the role of the SUMO proteases OVERLY TOLERANT TO SALT1 and -2 (OTS1/2) in defence and to provide insight into SUMO1/2-mediated regulation of SA signalling, we examined the status of SA-mediated defences in ots1/2 mutants. The ots1 ots2 double mutant displayed enhanced resistance to virulent Pseudomonas syringae and higher levels of SA compared with wild-type (WT) plants. Furthermore, ots1 ots2 mutants exhibited upregulated expression of the SA biosynthesis gene ICS1 in addition to enhanced SA-responsive ICS1 expression beyond that of WT. SA stimulated OTS1/2 degradation and promoted accumulation of SUMO1/2 conjugates. These results indicate that OTS1 and -2 act in a feedback loop in SA signalling and that de novo OTS1/2 synthesis works antagonistically to SA-promoted degradation, adjusting the abundance of OTS1/2 to moderate SA signalling. Accumulation of SUMO1/2 conjugates coincides with SA-promoted OTS degradation and may play a positive role in SA-mediated signalling in addition to its repressive roles reported elsewhere.

Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture.

Models for the control of above-ground plant architectures show how meristems can be programmed to be either shoots or flowers. Molecular, genetic, transgenic, and mathematical studies have greatly refined these models, suggesting that the phase of the shoot reflects different genes contributing to its repression of flowering, its vegetativeness ('veg'), before activators promote flower development. Key elements of how the repressor of flowering and shoot meristem gene TFL1 acts have now been tested, by changing its spatiotemporal pattern. It is shown that TFL1 can act outside of its normal expression domain in leaf primordia or floral meristems to repress flower identity. These data show how the timing and spatial pattern of TFL1 expression affect overall plant architecture. This reveals that the underlying pattern of TFL1 interactors is complex and that they may be spatially more widespread than TFL1 itself, which is confined to shoots. However, the data show that while TFL1 and floral genes can both act and compete in the same meristem, it appears that the main shoot meristem is more sensitive to TFL1 rather than floral genes. This spatial analysis therefore reveals how a difference in response helps maintain the 'veg' state of the shoot meristem.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

GIGANTEA enables drought escape response via abscisic acid-dependent activation of the florigens and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS.

Modulation of the transition to flowering plays an important role in the adaptation to drought. The drought-escape (DE) response allows plants to adaptively shorten their life cycle to make seeds before severe stress leads to death. However, the molecular basis of the DE response is unknown. A screen of different Arabidopsis (Arabidopsis thaliana) flowering time mutants under DE-triggering conditions revealed the central role of the flower-promoting gene GIGANTEA (GI) and the florigen genes FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) in the DE response. Further screens showed that the phytohormone abscisic acid is required for the DE response, positively regulating flowering under long-day conditions. Drought stress promotes the transcriptional up-regulation of the florigens in an abscisic acid- and photoperiod-dependent manner, so that early flowering only occurs under long days. Along with the florigens, the floral integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 is also up-regulated in a similar fashion and contributes to the activation of TSF. The DE response was recovered under short days in the absence of the floral repressor SHORT VEGETATIVE PHASE or in GI-overexpressing plants. Our data reveal a key role for GI in connecting photoperiodic cues and environmental stress independently from the central FT/TSF activator CONSTANS. This mechanism explains how environmental cues may act upon the florigen genes in a photoperiodically controlled manner, thus enabling plastic flowering responses.

The Arabidopsis thaliana MYB60 promoter provides a tool for the spatio-temporal control of gene expression in stomatal guard cells.

Plants have evolved different strategies to resist drought, of which the best understood is the abscisic acid (ABA)-induced closure of stomatal pores to reduce water loss by transpiration. The availability of useful promoters that allow for precise spatial and temporal control of gene expression in stomata is essential both for investigating stomatal regulation in model systems and for biotechnological applications in field crops. Previous work indicated that the regulatory region of the transcription factor AtMYB60 specifically drives gene expression in guard cells of Arabidopsis, although its activity is rapidly down-regulated by ABA. Here, the activity of the full-length and minimal AtMYB60 promoters is reported in rice (Oryza sativa), tobacco (Nicotiana tabacum), and tomato (Solanum lycopersicum), using a reporter gene approach. In rice, the activity of both promoters was completely abolished, whereas it was spatially restricted to guard cells in tobacco and tomato. To overcome the negative effect of ABA on the AtMYB60 promoter, a chimeric inducible system was developed, which combined the cellular specificity of the AtMYB60 minimal promoter with the positive responsiveness to dehydration and ABA of the rd29A promoter. Remarkably, the synthetic module specifically up-regulated gene expression in guard cells of Arabidopsis, tobacco, and tomato in response to dehydration or ABA. The comparative analysis of different native and synthetic regulatory modules derived from the AtMYB60 promoter offers new insights into the functional conservation of the cis-mechanisms that mediate gene expression in guard cells in distantly related dicotyledonous species and provides novel tools for modulating stomatal activity in plants.

The AtMYB60 transcription factor regulates stomatal opening by modulating oxylipin synthesis in guard cells.

Stomata are epidermal pores formed by pairs of specialized guard cells, which regulate gas exchanges between the plant and the atmosphere. Modulation of transcription has emerged as an important level of regulation of stomatal activity. The AtMYB60 transcription factor was previously identified as a positive regulator of stomatal opening, although the details of its function remain unknown. Here, we propose a role for AtMYB60 as a negative modulator of oxylipins synthesis in stomata. The atmyb60-1 mutant shows reduced stomatal opening and accumulates increased levels of 12-oxo-phytodienoic acid (12-OPDA), jasmonic acid (JA) and jasmonoyl-L-isoleucine (JA-Ile) in guard cells. We provide evidence that 12-OPDA triggers stomatal closure independently of JA and cooperatively with abscisic acid (ABA) in atmyb60-1. Our study highlights the relevance of oxylipins metabolism in stomatal regulation and indicates AtMYB60 as transcriptional integrator of ABA and oxylipins responses in guard cells.

DOF-binding sites additively contribute to guard cell-specificity of AtMYB60 promoter.

BACKGROUND: We previously demonstrated that the Arabidopsis thaliana AtMYB60 protein is an R2R3MYB transcription factor required for stomatal opening. AtMYB60 is specifically expressed in guard cells and down-regulated at the transcriptional levels by the phytohormone ABA. RESULTS: To investigate the molecular mechanisms governing AtMYB60 expression, its promoter was dissected through deletion and mutagenesis analyses. By studying different versions of AtMYB60 promoter::GUS reporter fusions in transgenic plants we were able to demonstrate a modular organization for the AtMYB60 promoter. Particularly we defined: a minimal promoter sufficient to confer guard cell-specific activity to the reporter gene; the distinct roles of different DOF-binding sites organised in a cluster in the minimal promoter in determining guard cell-specific expression; the promoter regions responsible for the enhancement of activity in guard cells; a promoter region responsible for the negative transcriptional regulation by ABA. Moreover from the analysis of single and multiple mutants we could rule out the involvement of a group of DOF proteins, known as CDFs, already characterised for their involvement in flowering time, in the regulation of AtMYB60 expression. CONCLUSIONS: These findings shed light on the regulation of gene expression in guard cells and provide new promoter modules as useful tools for manipulating gene expression in guard cells, both for physiological studies and future biotechnological applications.

The grapevine guard cell-related VvMYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress.

BACKGROUND: Under drought, plants accumulate the signaling hormone abscisic acid (ABA), which induces the rapid closure of stomatal pores to prevent water loss. This event is trigged by a series of signals produced inside guard cells which finally reduce their turgor. Many of these events are tightly regulated at the transcriptional level, including the control exerted by MYB proteins. In a previous study, while identifying the grapevine R2R3 MYB family, two closely related genes, VvMYB30 and VvMYB60 were found with high similarity to AtMYB60, an Arabidopsis guard cell-related drought responsive gene. RESULTS: Promoter-GUS transcriptional fusion assays showed that expression of VvMYB60 was restricted to stomatal guard cells and was attenuated in response to ABA. Unlike VvMYB30, VvMYB60 was able to complement the loss-of-function atmyb60-1 mutant, indicating that VvMYB60 is the only true ortholog of AtMYB60 in the grape genome. In addition, VvMYB60 was differentially regulated during development of grape organs and in response to ABA and drought-related stress conditions. CONCLUSIONS: These results show that VvMYB60 modulates physiological responses in guard cells, leading to the possibility of engineering stomatal conductance in grapevine, reducing water loss and helping this species to tolerate drought under extreme climatic conditions.

Increased phenylpropanoids production in UV-B irradiated Salvia verticillata as a consequence of altered genes expression in young leaves.

Ultraviolet-B (UV-B) radiation as an environmental potential elicitor induces the synthesis of plant secondary metabolites. The effects of UV-B radiation on photosynthetic pigments and dry weight, biochemical and molecular features of old and young leaves of Salvia verticillata were investigated. Plants were exposed to 10.97 kJ m-2 day-1 of biologically effective UV-B radiation for up to 10 days. The sampling process was performed in four steps: 1, 5, 10, and 13 days (recovery time) after the start of irradiation. As a result of plant investment in primary and secondary metabolism, the production of phenolic compounds increased, while chlorophyll levels and leaf dry weight (%) declined. Under long-term UV-B exposure, young leaves exhibited the most significant reduction in chlorophyll a and b content and leaf dry weight. The highest level of total phenol (1.34-fold) and flavonoid concentration (2-fold) relative to the control was observed on the 5th day and recovery time, respectively. Young leaves demonstrated the highest amount of phenolic acids in recovery time. Young leaves on the 5th day of the experiment exerted the highest level of antioxidant activity when compared to the control. A positive correlation was observed between antioxidant activity and the amount of phenolic compounds. Regarding the expression of phenylpropanoid pathway genes, UV-B enhanced the expression of phenylalanine ammonia-lyase, tyrosine aminotransferase, and rosmarinic acid synthase with the highest level in young leaves on the 10th day. Overall, young leaves of S. verticillata indicated higher sensitivity to UV-B radiation and developed more tangible reactions to such radiation.