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Salinity stress constrains lateral root (LR) growth and severely impacts plant growth. Auxin signaling is indispensable for the regulation of LR formation. Nevertheless, the molecular mechanism of how salinity affects root auxin signaling and whether salt would steer alternative pathway(s) to regulate LR development is unknown. Here we show that the auxin- regulated transcription factor LATERAL ORGAN BOUNDARY DOMAIN (LBD)16, known as an essential player for LR development under control conditions, is regulated by an alternative non-canonical pathway under salinity. Salt represses auxin signaling but in parallel activates an upstream transcriptional activator of LBD16, ZINC FINGER OF ARABIDOPSIS THALIANA 6 (ZAT6). ZAT6 modulates the activity ofLBD16to contribute to downstream cell wall remodeling, and promotes LR development under salinity stress. Our study thus shows that root developmental plasticity in response to salt stress is achieved by integration of auxin- dependent repressive and salt-activated auxin-independent pathways converging on LBD16 to modulate root branching modulation under salinity.

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. 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.

Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargoes and coated vesicle accessory proteins. As in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargoes of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

During salt stress, the root system architecture of a plant is important for survival. Different accessions ofArabidopsis thalianahave adopted different strategies in remodeling their root architecture during salt stress. Salt induces a multiphase growth response in roots, consisting of a stop phase, quiescent phase, recovery phase and eventually a new level of homeostasis. We explored natural variation in the length of and growth rate during these phases in both main and lateral roots and find that some accessions lack the quiescent phase. Using mathematical models and correlation-based network, allowed us to correlate dynamic traits to overall root architecture and discover that both the main root growth rate during homeostasis and lateral root appearance are the strongest determinants of overall root architecture. In addition, this approach revealed a trade-off between investing in main or lateral root length during salt stress. By studying natural variation in high-resolution temporal root growth using mathematical modeling, we gained new insights in the interactions between dynamic root growth traits and we identified key traits that modulate overall root architecture during salt stress.Summary statementBy studying natural variation in salt-induced root growth phases inArabidopsis, we show that main root growth rate during homeostasis and lateral root appearance contribute most to root architecture and we reveal a trade-off between investing in main and lateral root growth during salt stress.

Acclimation of root growth is vital for plants to survive salt stress. Halophytes are great examples of plants that thrive under high salt concentrations but their salt tolerance mechanisms, especially those mediated by root responses, are still largely unknown. We compared root growth responses of the halophyte Schrenkiella parvula with its glycophytic relative species Arabidopsis thaliana under salt stress, and performed root transcriptomic analysis to identify differences in gene regulatory networks underlying their physiological responses. Primary root growth of S. parvula is less sensitive to salt compared with Arabidopsis. The root transcriptomic analysis of S. parvula revealed the induction of sugar transporters and genes regulating cell expansion and suberization under salt stress. 14C-labelled carbon partitioning analyses consistently showed that S. parvula had a higher incorporation rate of soluble sugars in roots under salt stress compared to Arabidopsis. Further physiological investigation revealed that S. parvula roots do not show a halotropic response and maintain root cell expansion and enhanced suberization even under severe salt stress. In summary, our study demonstrates that roots of S. parvula deploy multiple physiological and developmental adjustments under salt stress to maintain growth, providing new avenues to improve salt tolerance of plants using root-specific strategies.

Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargos and coated vesicle accessory proteins. Since in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargos of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins, and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.

Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploit here a real-time salt-induced tilting assay (SITA). This method follows root growth upon both gravitropic and salt challenges and revealed that root bending is modulated by salinity, but not by osmotic stress. Next, this salt-specific response was measured in 345 natural Arabidopsis accessions and we discovered a genetic locus, encoding for the cell-wall modifying enzyme Extensin Arabinose Deficient arabinosyltransferase (ExAD), to be associated with root bending in salt. Extensins are a class of structural cell wall glycoproteins [hydroxyproline-rich glycoproteins (HRGPs)] which are post-translationally modified by O-glycosylation mostly in the form of hydroxyproline (Hyp)-arabinosylation. Here we show that salt induces ExAD-dependent Hyp-arabinosylation, influencing root bending responses and cell wall thickness. We report that exad mutants, which lack all cell wall Hyp-Araf4 modifications, display increased root epidermal cell wall thickness, and are impaired in the salt-dependent root bending response. Our study thus demonstrates a requirement for extensin Hyp-arabinosylation as a novel cell wall modification for the salt-specific directional response of roots and provides new insight into the process of how plant roots sense and respond to salinity.

Soil salinity is an increasing worldwide problem for agriculture, affecting plant growth and yield. To understand the molecular mechanisms activated in response to salt in plants, we investigated the Catharanthus roseus Receptor like Kinase 1 Like (CrRLK1L) family, which contains well described sensors previously shown to be involved in maintaining and sensing the structural integrity of the cell walls. We found that herk1the1-4 double mutants, lacking the function of the Arabidopsis thaliana Receptor like Kinase HERKULES1 combined with a gain-of-function allele of THESEUS1, phenocopied the phenotypes previously reported in plants lacking FERONIA (FER) function. We report that both fer-4 and herk1the1-4 mutants respond strongly to salt application, resulting in a more intense activation of early and late stress responses. We also show that salt triggers de-methyl esterification of loosely bound pectins, responsible for the activation of several salt response signaling pathways. Addition of calcium chloride or chemically inhibiting pectin methyl esterase (PME) activity reduced activation of the early signaling protein Mitogen Activated Protein Kinase 6 (MPK6) as well as amplitude of salt-induced marker gene induction. MPK6 is required for the full induction of the salt-induced gene expression markers we tested. The sodium specific root halotropism response on the other hand, appears independent of MPK6 or calcium application, and is only mildly influenced by the cell wall sensors FER/HERK1/THE1-4 or alteration of PME activity. We hypothesize a model where salt-triggered modification of pectin requires the functionality of FER alone or the HERK1/THE1 combination to attenuate salt responses. Collectively, our results show the complexity of salt stress responses and salt sensing mechanisms and their connection to cell wall modifications, responsible for several salt response pathways and ultimately plant resilience to salinity.

How do environmental cues steer the branching of plant roots? Insights into how water availability shapes root growth reveal an unexpected mechanism behind the hormone-mediated regulation of this process.

Increasing soil salinization largely impacts crop yield worldwide. To deal with salinity stress, plants exhibit an array of responses, including root system architecture remodelling. Here, we review recent progress in physiological, developmental and cellular mechanisms of root growth responses to salinity. Most recent research in modulation of root branching, root tropisms, as well as in root cell wall modifications under salinity stress, is discussed in the context of the contribution of these responses to overall plant performance. We highlight the power of natural variation approaches revealing novel potential pathways responsible for differences in root salt stress responses. Together, these new findings promote our understanding of how salt shapes the root phenotype, which may provide potential avenues for engineering crops with better yield and survival in saline soils.

Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its life cycle under multiple environmental stresses, including high salinity. While foundational genomic resources have been created for S. parvula, a comprehensive physiological or structural characterization of its salt stress responses is absent. We aimed to identify the influential traits that lead to stress-resilient growth of this species. We examined salt-induced changes in the physiology and anatomy of S. parvula throughout its lifecycle across multiple tissues. We found that S. parvula maintains or even exhibits enhanced growth during various developmental stages at salt stress levels known to inhibit growth in Arabidopsis and most crops. The resilient growth of S. parvula was associated with key traits that synergistically allow continued primary root growth, expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing larger and thicker leaves while facilitating continued photosynthesis during salt stress. These traits at the vegetative phase were followed by a successful transition to the reproductive phase via early flowering, development of larger siliques, and production of viable seeds during salt stress. Additionally, the success of self-fertilization during early flowering stages was dependent on salt-induced filament elongation in flowers that aborted in the absence of salt. Our results suggest that the maintenance of leaf water status and enhancement of selfing in early flowers to ensure reproductive success, are among the most influential traits that contribute to the extremophyte lifestyle of S. parvula in its natural habitat.One sentence summarySchrenkiella parvula salt-resilient growth is facilitated by uncompromised primary root growth, expansion of xylem vessels, maintenance of leaf water status and photosynthesis, and early flowering.

Abiotic stresses increasingly threaten existing ecological and agricultural systems across the globe. Plant roots perceive these stresses in the soil and adapt their architecture accordingly. This review provides insights into recent discoveries showing the importance of root system architecture (RSA) and plasticity for the survival and development of plants under heat, cold, drought, salt, and flooding stress. In addition, we review the molecular regulation and hormonal pathways involved in controlling RSA plasticity, main root growth, branching and lateral root growth, root hair development, and formation of adventitious roots. Several stresses affect root anatomy by causing aerenchyma formation, lignin and suberin deposition, and Casparian strip modulation. Roots can also actively grow toward favorable soil conditions and avoid environments detrimental to their development. Recent advances in understanding the cellular mechanisms behind these different root tropisms are discussed. Understanding root plasticity will be instrumental for the development of crops that are resilient in the face of abiotic stress., Recent discoveries show the importance of root system architecture plasticity for the survival and growth of plants under several abiotic stresses.

Plants react to a myriad of biotic and abiotic environmental signals through specific cellular mechanisms required for survival under stress. Although pathogen perception has been widely studied and characterized, salt stress perception and signaling remain largely elusive. Recent observations, obtained in the model plant Arabidopsis thaliana, show that perception of specific features of pathogens also allows plants to mount salt stress resilience pathways, highlighting the possibility that salt sensing and pathogen perception mechanisms partially overlap. We discuss these overlapping strategies and examine the emerging role of A. thaliana cell wall and plasma membrane components in activating both salt- and pathogen-induced responses, as part of exquisite mechanisms underlying perception of damage and danger. This knowledge helps understanding the complexity of plant responses to pathogens and salinity, leading to new hypotheses that could explain why plants evolved similar strategies to respond to these, at first sight, very different types of stimuli.

When plants face an environmental stress such as water deficit, soil salinity, high temperature, or shade, good communication between above- and belowground organs is necessary to coordinate growth and development. Various signals including hormones, peptides, proteins, hydraulic signals, and metabolites are transported mostly through the vasculature to distant tissues. How shoots and roots synchronize their response to stress using mobile signals is an emerging field of research. We summarize recent advances on mobile signals regulating shoot stomatal movement and root development in response to highly localized environmental cues. In addition, we highlight how the vascular system is not only a conduit but is also flexible in its development in response to abiotic stress.

Plants are exposed to an ever-changing environment to which they have to adjust accordingly. Their response is tightly regulated by complex signaling pathways that all start with stimulus perception. Here, we give an overview of the latest developments in the perception of various abiotic stresses, including drought, salinity, flooding, and temperature stress. We discuss whether proposed perception mechanisms are true sensors, which is well established for some abiotic factors but not yet fully elucidated for others. In addition, we review the downstream cellular responses, many of which are shared by various stresses but result in stress-specific physiological and developmental output. New sensing mechanisms have been identified, including heat sensing by the photoreceptor phytochrome B, salt sensing by glycosylinositol phosphorylceramide sphingolipids, and drought sensing by the specific calcium influx channel OSCA1. The simultaneous occurrence of multiple stress conditions shows characteristic downstream signaling signatures that were previously considered general signaling responses. The integration of sensing of multiple stress conditions and subsequent signaling responses is a promising venue for future research to improve the understanding of plant abiotic stress perception.

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis (Arabidopsis thaliana) SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the posttranscriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser-645 and Ser-1156 of VCS, whereas SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser-692 and Ser-680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5, and SnRK2.9 controlling root growth under nonstress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.

Plant acclimation to environmental stresses requires fast signaling to initiate changes in developmental and metabolic responses. Regulation of gene expression by transcription factors and protein kinases acting upstream are important elements of responses to salt and drought. Gene expression can be also controlled at the post-transcriptional level. Recent analyses on mutants in mRNA metabolism factors suggest their contribution to stress signaling. Here we highlight the components of mRNA decay pathways that contribute to responses to osmotic and salt stress. We hypothesize that phosphorylation state of proteins involved in mRNA decapping affect their substrate specificity.

Salinity of the soil is highly detrimental to plant growth. Plants respond by a redistribution of root mass between main and lateral roots, yet the genetic machinery underlying this process is still largely unknown. Here, we describe the natural variation among 347 Arabidopsis thaliana accessions in root system architecture (RSA) and identify the traits with highest natural variation in their response to salt. Salt-induced changes in RSA were associated with 100 genetic loci using genome-wide association studies. Two candidate loci associated with lateral root development were validated and further investigated. Changes in CYP79B2 expression in salt stress positively correlated with lateral root development in accessions, and cyp79b2 cyp79b3 double mutants developed fewer and shorter lateral roots under salt stress, but not in control conditions. By contrast, high HKT1 expression in the root repressed lateral root development, which could be partially rescued by addition of potassium. The collected data and multivariate analysis of multiple RSA traits, available through the Salt_NV_Root App, capture root responses to salinity. Together, our results provide a better understanding of effective RSA remodeling responses, and the genetic components involved, for plant performance in stress conditions.

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.

Endocytosis and relocalization of auxin carriers represent important mechanisms for adaptive plant growth and developmental responses. Both root gravitropism and halotropism have been shown to be dependent on relocalization of auxin transporters. Following their homology to mammalian phospholipase Ds (PLDs), plant PLDζ‐type enzymes are likely candidates to regulate auxin carrier endocytosis. We investigated root tropic responses for an Arabidopsis pldζ1‐KO mutant and its effect on the dynamics of two auxin transporters during salt stress, that is, PIN2 and AUX1. We found altered root growth and halotropic and gravitropic responses in the absence of PLDζ1 and report a role for PLDζ1 in the polar localization of PIN2. Additionally, irrespective of the genetic background, salt stress induced changes in AUX1 polarity. Utilizing our previous computational model, we found that these novel salt‐induced AUX1 changes contribute to halotropic auxin asymmetry. We also report the formation of “osmotic stress‐induced membrane structures.” These large membrane structures are formed at the plasma membrane shortly after NaCl or sorbitol treatment and have a prolonged presence in a pldζ1 mutant. Taken together, these results show a crucial role for PLDζ1 in both ionic and osmotic stress‐induced auxin carrier dynamics during salt stress., This work shows that both ionic and osmotic stress affect auxin carrier dynamics during salinity stress, a response that requires the action of the phospholipase PLDζ1. Mathematical modelling shows that impaired cellular polarity in roots of pldζ1 seedlings explains their different halotropism (salt avoidance) response.

The protocol was developed for observing salt-induced changes in Root System Architecture of Arabidopsis and other small plants. The seeds are germinated in control conditions, and exposed to stress 4 days after germination. As the agar plates are transparent, this system provides quick, easy and non-destructive method to observe development of roots. This protocol was used to produced the data in two manuscripts from our lab - Julkowska et al., 2014 Plant Physiology and Julkowska et al., 2017 Plant Cell.

Salinity is one of the most devastating abiotic stresses accounting for major crop losses yearly. Plant roots respond to high salt by several morphological adaptations, and strikingly can bend away from high salt patches. The latter process is termed halotropism, and occurs through redistribution of auxin in the root in response to a salt gradient. Halotropism presents an early and unique response to salt, that is specific to NaCl, and thus provides an excellent read-out to identify genetic loci that are required for the response of plants to salinity. Here, a natural variation screen for halotropic response of 333 Arabidopsis accessions revealed quantitative difference in the response within 24 hours. These data were successfully used to identify novel genetic components associated with this NaCl-specific response through GWAS. For three selected genes, the transcription factor WRKY25, a cation-proton exchanger CHX13, and a gene of unknown function DOB1 (Double Bending 1), follow-up characterisation of knockout mutants in Col-0 confirmed their role in halotropism. Also ion accumulation and shoot biomass under salt stress were affected in the mutants. Thus, our GWAS analysis has identified new genetic components contributing to main root halotropic responses, that provide new leads on the genetic architecture underlying plant responses to salt.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane., Plant cells maintain strict proton gradients over different membranes. Here, Dejonghe et al. show that several protonophores, including the known tyrosine kinase inhibitor TyrphostinA23, inhibit clathrin-mediated endocytosis by disturbing these gradients and causing cytoplasmic acidification.

Alterations of hydrogen peroxide (H2O2) levels have a profound impact on numerous signaling cascades orchestrating plant growth, development, and stress signaling, including programmed cell death. To expand the repertoire of known molecular mechanisms implicated in H2O2 signaling, we performed a forward chemical screen to identify small molecules that could alleviate the photorespiratory-induced cell death phenotype of Arabidopsisthaliana mutants lacking H2O2-scavenging capacity by peroxisomal catalase2. Here, we report the characterization of pakerine, an m-sulfamoyl benzamide from the sulfonamide family. Pakerine alleviates the cell death phenotype of cat2 mutants exposed to photorespiration-promoting conditions and delays dark-induced senescence in wild-type Arabidopsis leaves. By using a combination of transcriptomics, metabolomics, and affinity purification, we identified abnormal inflorescence meristem 1 (AIM1) as a putative protein target of pakerine. AIM1 is a 3-hydroxyacyl-CoA dehydrogenase involved in fatty acid &beta, oxidation that contributes to jasmonic acid (JA) and salicylic acid (SA) biosynthesis. Whereas intact JA biosynthesis was not required for pakerine bioactivity, our results point toward a role for &beta, oxidation-dependent SA production in the execution of H2O2-mediated cell death.

Freezing limits plant growth and crop productivity, and plant species in temperate zones have the capacity to develop freezing tolerance through complex modulation of gene expression affecting various aspects of metabolism and physiology. While many components of freezing tolerance have been identified in model species under controlled laboratory conditions, little is known about the mechanisms that impart freezing tolerance in natural populations of wild species. Here, we performed a quantitative trait locus (QTL) study of acclimated freezing tolerance in seedlings of Boechera stricta, a highly adapted relative of Arabidopsis (Arabidopsis thaliana) native to the Rocky Mountains. A single QTL was identified that contained the gene encoding ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (BstDGAT1), whose expression is highly cold responsive. The primary metabolic enzyme DGAT1 catalyzes the final step in assembly of triacylglycerol (TAG) by acyl transfer from acyl-CoA to diacylglycerol. Freezing tolerant plants showed higher DGAT1 expression during cold acclimation than more sensitive plants, and this resulted in increased accumulation of TAG in response to subsequent freezing. Levels of oligogalactolipids that are produced by SFR2 (SENSITIVE TO FREEZING2), an indispensable element of freezing tolerance in Arabidopsis, were also higher in freezing-tolerant plants. Furthermore, overexpression of AtDGAT1 led to increased freezing tolerance. We propose that DGAT1 confers freezing tolerance in plants by supporting SFR2-mediated remodeling of chloroplast membranes.

Standard protocol to examine lipid binding of the proteins isolated from the E.coli with the GST tag

SummaryGlobal food production is set to keep increasing despite a predicted decrease in total arable land [1]. To achieve higher production, denser planting will be required on increasingly degraded soils. When grown in dense stands, crops elongate and raise their leaves in an effort to reach sunlight, a process termed shade-avoidance [2]. Shade is perceived by a reduction in the ratio of red (R) to (FR) light and results in the stabilisation of a class of transcription factors known as PHYTOCHROME INTERACTING FACTORs (PIFs) [3,4]. PIFs activate the expression of auxin biosynthesis genes [4,5] and enhance auxin sensitivity [6], which promotes cell wall loosening and drives elongation growth. Despite our molecular understanding of shade-induced growth, little is known about how this developmental programme is integrated with other environmental factors.Here we demonstrate that low levels of NaCl in soil strongly impair the ability of plants to respond to shade. This block is dependent upon abscisic acid (ABA) signalling and the canonical ABA signalling pathway. Low R:FR light enhances the expression of a positive regulator of the brassinosteroid (BR) signalling pathway, BRASSINOSTEROID SIGNALLING KINASE 5 (BSK5). We found that ABA inhibits BSK5 up-regulation and interferes with GSK3-like kinase inactivation by the BR pathway, thus leading to a suppression of PIF function. By demonstrating a link between the ABA and BR-signalling pathways this study provides an important step forward in our understanding of how environmental cues are integrated into plant development.

In most abiotic stress conditions, including salinity and water deficit, the developmental plasticity of the plant root is regulated by the phytohormone auxin. Changes in auxin concentration are often attributed to changes in shoot-derived long-distance auxin flow. However, recent evidence suggests important contributions by short-distance auxin transport from local storage and local auxin biosynthesis, conjugation, and oxidation during abiotic stress. We discuss here current knowledge on long-distance auxin transport in stress responses, and subsequently debate how short-distance auxin transport and indole-3-acetic acid (IAA) metabolism play a role in influencing eventual auxin accumulation and signaling patterns. Our analysis stresses the importance of considering all these components together and highlights the use of mathematical modeling for predictions of plant physiological responses., Highlights Recent findings in active auxin transport over organelle membranes and the influence of apoplastic and cytosolic pH on passive auxin flow into the cell highlight the importance of short-distance auxin transport for local auxin maxima in roots exposed to stress. Based on previously published gene expression data, we have identified stress-induced expression patterns of IAA biosynthesis and conjugation genes in the root that could be relevant for IAA accumulation patterns. These results underscore the importance of incorporating IAA homeostasis into models predicting auxin levels and flow. Recent advances in mathematical models involving auxin-related processes highlight the value of combining in planta and in silico experiments when unraveling complex hormonal changes in plants.

Polarized cell growth in plants is maintained under the strict control and exquisitely choreographed balance of exocytic and endocytic membrane trafficking. The pollen tube has become a model system for rapid polar growth in which delivery of cell wall material and membrane recycling are controlled by membrane trafficking. Endocytosis plays an important role that is poorly understood. The plant AP180 N-Terminal Homolog (ANTH) proteins are putative homologs of Epsin 1 that recruits clathrin to phosphatidylinositol 4, 5-bisphosphate (PIP2) containing membranes to facilitate vesicle budding during endocytosis. Two Arabidopsis ANTH encoded by the genes AtAP180 and AtECA2 are highly expressed in pollen tubes. Pollen tubes from T-DNA inserted knockout mutant lines display significant morphological defects and unique pectin deposition. Fluorescent tagging reveals organization into dynamic foci located at the lateral flanks of the pollen tube. This precisely defined subapical domain coincides which clathrin-mediated endocytosis (CME) and PIP2 localization. Using a liposome-protein binding test, we showed that AtECA2 protein and ANTH domain recombinant proteins have strong affinity to PIP2 and phosphatidic acid containing liposomes in vitro. Taken together these data suggest that Arabidopsis ANTH proteins may play an important role in CME, proper cell wall assembly and morphogenesis.

Salinity is one of the major abiotic factors threatening food security worldwide. Recently, our understanding of early processes underlying salinity tolerance has expanded. In this review, early signaling events, such as phospholipid signaling, calcium ion (Ca(2+)) responses, and reactive oxygen species (ROS) production, together with salt stress-induced abscisic acid (ABA) accumulation, are brought into the context of long-term salt stress-specific responses and alteration of plant growth. Salt-induced quiescent and recovery growth phases rely on modification of cell cycle activity, cell expansion, and cell wall extensibility. The period of initial growth arrest varies among different organs, leading to altered plant morphology. Studying stress-induced changes in growth dynamics can be used for screening to discover novel genes contributing to salt stress tolerance in model species and crops.

Environmental stresses, such as shading of the shoot, drought, and soil salinity, threaten plant growth, yield, and survival. Plants can alleviate the impact of these stresses through various modes of phenotypic plasticity, such as shade avoidance and halotropism. Here, we review the current state of knowledge regarding the mechanisms that control plant developmental responses to shade, salt, and drought stress. We discuss plant hormones and cellular signaling pathways that control shoot branching and elongation responses to shade and root architecture modulation in response to drought and salinity. Because belowground stresses also result in aboveground changes and vice versa, we then outline how a wider palette of plant phenotypic traits is affected by the individual stresses. Consequently, we argue for a research agenda that integrates multiple plant organs, responses, and stresses. This will generate the scientific understanding needed for future crop improvement programs aiming at crops that can maintain yields under variable and suboptimal conditions.

International audience; Tropisms represent fascinating examples of how plants respond to environmental signals by adapting their growth and development. Here, a novel tropism is reported, halotropism, allowing plant seedlings to reduce their exposure to salinity by circumventing a saline environment. In response to a salt gradient, Arabidopsis, tomato, and sorghum roots were found to actively prioritize growth away from salinity above following the gravity axis. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by the PIN-FORMED 2 (PIN2) auxin efflux carrier. We show that salt-induced phospholipase D activity stimulates clathrin-mediated endocytosis of PIN2 at the side of the root facing the higher salt concentration. The intracellular relocalization of PIN2 allows for auxin redistribution and for the directional bending of the root away from the higher salt concentration. Our results thus identify a cellular pathway essential for the integration of environmental cues with auxin-regulated root growth that likely plays a key role in plant adaptative responses to salt stress.

Nutrient availability and salinity of the soil affect the growth and development of plant roots. Here, we describe how inorganic phosphate (Pi) availability affects the root system architecture (RSA) of Arabidopsis (Arabidopsis thaliana) and how Pi levels modulate responses of the root to salt stress. Pi starvation reduced main root length and increased the number of lateral roots of Arabidopsis Columbia-0 seedlings. In combination with salt, low Pi dampened the inhibiting effect of mild salt stress (75 mm) on all measured RSA components. At higher salt concentrations, the Pi deprivation response prevailed over the salt stress only for lateral root elongation. The Pi deprivation response of lateral roots appeared to be oppositely affected by abscisic acid signaling compared with the salt stress response. Natural variation in the response to the combination treatment of salt and Pi starvation within 330 Arabidopsis accessions could be grouped into four response patterns. When exposed to double stress, in general, lateral roots prioritized responses to salt, while the effect on main root traits was additive. Interestingly, these patterns were not identical for all accessions studied, and multiple strategies to integrate the signals from Pi deprivation and salinity were identified. By genome-wide association mapping, 12 genomic loci were identified as putative factors integrating responses to salt stress and Pi starvation. From our experiments, we conclude that Pi starvation interferes with salt responses mainly at the level of lateral roots and that large natural variation exists in the available genetic repertoire of accessions to handle the combination of stresses.

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues., Author summary Plants are constantly exposed to external stresses of biotic and abiotic nature, as well as internal stresses, resulting from growth and mechanical tension. Feedback information about the integrity of the cell wall can enable the plant to perceive such stresses, and respond adequately. Plants are known to perceive signals from their environment through receptor kinases at the plant cell surface. Here, we reveal that the Arabidopsis thaliana receptor kinase MIK2 regulates responses to cell wall perturbation. Moreover, we find that MIK2 controls root growth angle, modulates cell wall structure in the root tip, contributes to salt stress tolerance, and is required for resistance against a root-infecting pathogen. Our data suggest that MIK2 is involved in sensing cell wall perturbations in plants, whereby it allows the plant to cope with a diverse range of environmental stresses. These data provide an important step forward in our understanding of the mechanisms plants deploy to sense internal and external danger.

Highlight Study of natural variation of Arabidopsis reveals salinity tolerance to be linked to plant development and expression of novel candidate genes., Natural variation among Arabidopsis accessions is an important genetic resource to identify mechanisms underlying plant development and stress tolerance. To evaluate the natural variation in salinity stress tolerance, two large-scale experiments were performed on two populations consisting of 160 Arabidopsis accessions each. Multiple traits, including projected rosette area, and fresh and dry weight were collected as an estimate for salinity tolerance. Our results reveal a correlation between rosette size under salt stress conditions and developmental differences between the accessions grown in control conditions, suggesting that in general larger plants were more salt tolerant. This correlation was less pronounced when plants were grown under severe salt stress conditions. Subsequent genome wide association study (GWAS) revealed associations with novel candidate genes for salinity tolerance such as LRR-KISS (At4g08850), flowering locus KH-domain containing protein and a DUF1639-containing protein. Accessions with high LRR-KISS expression developed larger rosettes under salt stress conditions. Further characterization of allelic variation in candidate genes identified in this study will provide more insight into mechanisms of salt stress tolerance due to enhanced shoot growth.

International audience; Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLC. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca2+ ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

To face future challenges in crop production dictated by global climate changes, breeders and plant researchers collaborate to develop productive crops that are able to withstand a wide range of biotic and abiotic stresses. However, crop selection is often focused on shoot performance alone, as observation of root properties is more complex and asks for artificial and extensive phenotyping platforms. In addition, most root research focuses on development, while a direct link to the functionality of plasticity in root development for tolerance is often lacking. In this paper we review the currently known root system architecture (RSA) responses in Arabidopsis and a number of crop species to a range of abiotic stresses, including nutrient limitation, drought, salinity, flooding, and extreme temperatures. For each of these stresses, the key molecular and cellular mechanisms underlying the RSA response are highlighted. To explore the relevance for crop selection, we especially review and discuss studies linking root architectural responses to stress tolerance. This will provide a first step toward understanding the relevance of adaptive root development for a plant's response to its environment. We suggest that functional evidence on the role of root plasticity will support breeders in their efforts to include root properties in their current selection pipeline for abiotic stress tolerance, aimed to improve the robustness of crops.

The Sucrose non-fermenting-1 related protein kinase 2 (SnRK2) family represents a unique family of plant specific-protein kinases implicated in cellular signalling in response to osmotic stress. In our studies, we observed that two class 1 SnRK2 kinases, SnRK2.4 and SnRK2.10, are rapidly and transiently activated in Arabidopsis roots after exposure to salt. Under saline conditions, snrk2.4 knockout mutants had a reduced primary root length, while snrk2.10 mutants exhibited a reduction in the number of lateral roots. The reduced lateral root density was found to be a combinatory effect of a decrease in the amount of lateral root primordia and an increase in the amount of arrested lateral root primordia. The phenotypes were in agreement with the observed expression patterns of genomic YFP-fusions of SnRK2.10 and 2.4, under control of their native promoter sequences. SnRK2.10 was found to be expressed in the vascular tissue at the base of a developing lateral root, whereas SnRK2.4 was expressed throughout the root, with higher expression in the vascular system. Salt stress triggered a rapid re-localization of SnRK2.4-YFP from the cytosol to punctate structures in root epidermal cells. Differential centrifugation experiments of isolated Arabidopsis root proteins, confirmed recruitment of endogenous SnRK2.4/2.10 to membranes upon exposure to salt, supporting their observed binding affinity for the phospholipid phosphatidic acid. Together, our results reveal a role for SnRK2.4 and 2.10 in root growth and architecture in saline conditions. (c) 2012 The Authors. The Plant Journal (c) 2012 Blackwell Publishing Ltd.

PTEN (phosphatase and tensin homologue deleted on chromosome ten) proteins are dual phosphatases with both protein and phosphoinositide phosphatase activity. They modulate signalling pathways controlling growth, metabolism and apoptosis in animals and are implied in several human diseases. In the present paper we describe a novel class of PTEN pro-teins in plants, termed PTEN2, which comprises the AtPTEN (Arabidopsis PTEN) 2a and AtPTEN2b proteins in Arabidopsis. Both display low in vitro tyrosine phosphatase activity. In addition, AtPTEN2a actively dephosphorylates in vitro the 3′ phosphate group of PI3P (phosphatidylinositol 3-phosphate), PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) and PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate). In contrast with animal PTENs, PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) is a poor substrate. Site-directed mutagenesis of AtPTEN2a and molecular modelling of protein–phosphoinositide interactions indicated that substitutions at the PTEN2 core catalytic site of the Lys267 and Gly268 residues found in animals, which are critical for animal PTEN activity, by Met267 and Ala268 found in the eudicot PTEN2 are responsible for changes in substrate specificity. Remarkably, the AtPTEN2a protein also displays strong binding activity for PA (phosphatidic acid), a major lipid second messenger in plants. Promoter::GUS (β-glucuronidase) fusion, transcript and protein analyses further showed the transcriptional regulation of the ubiquitously expressed AtPTEN2a and AtPTEN2b by salt and osmotic stress. The results of the present study suggest a function for this novel class of plant PTEN proteins as an effector of lipid signalling in plants.

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is an enzyme playing a crucial role in photosynthesis of C4 plants. Here, we identify anionic phospholipids as novel regulators that inhibit C4 PEPC activity and provide evidence that the enzyme partially localizes to membranes.

Salinity is one of the major environmental factors limiting growth and productivity of rice plants. In this study, the effect of salt stress on phospholipid signaling responses inrice leaves was investigated. Leaf cuts were radiolabeled with 32 P-orthophosphate and the lipids extracted and analyzed by thin-layer chromatography, autoradiographyand phosphoimaging. Phospholipids were identifi ed by co-migration of known standards. Results showed that 32 P i was rapidly incorporated into the minor lipids, phosphatidylinositol bisphosphate (PIP 2 ) and phosphatidic acid (PA) and, interestingly, also into the structural lipids phosphatidylethanolamine (PE) and phosphatidylglycerol(PG), which normally label relatively slowly, like phosphatidylcholine (PC) and phosphatidylinositol (PI). Only very small amounts of PIP 2 were found. However, inresponse to salt stress (NaCl), PIP 2 levels rapidly (can be generated via the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK) or directly via phospholipase D (PLD). The latter can be measured invivo, using a transphosphatidylation assay. Interestingly, these measurements revealed that salt stress inhibited PLD activity, indicating that the salt stress-induced PAresponse was not due to PLD activity. Comparison of the 32 P-lipid responses in salt-tolerant and salt-sensitive cultivars revealed no signifi cant differences. Togetherthese results show that salt stress rapidly activates several lipid responses in rice leaves but that these responses do not explain the difference in salt tolerance between sensitive and tolerant cultivars.