Soil salinity presents a dual challenge for plants, involving both osmotic and ionic stress. In response, plants deploy distinct yet interconnected mechanisms to cope with these facets of salinity stress. In this investigation, we observed a substantial overlap in the salt (NaCl)-induced transcriptional responses of Arabidopsis roots with those triggered by osmotic stress or the plant stress hormone abscisic acid (ABA), as anticipated. Notably, a specific cluster of genes responded uniquely to sodium (Na+) ions and are not regulated by the known monovalent cation sensing mechanism MOCA1. Surprisingly, expression of sodium-induced genes exhibited a negative correlation with the ABA response and preceded the activation of genes induced by the osmotic stress component of salt. Elevated exogenous ABA levels resulted in the complete abolition of sodium-induced responses. Consistently, the ABA insensitive snrk2.2/2.3 double mutant displayed prolonged sodium-induced gene expression, coupled with increased root cell damage and root swelling under high salinity conditions. Moreover, ABA biosynthesis and signaling mutants were unable to redirect root growth to avoid high sodium concentrations and had increased sodium accumulation in the shoot. In summary, our findings unveil an unexpected and pivotal role for ABA signaling in mitigating cellular damage induced by salinity stress and modulating sodium-induced responses in plant roots.

Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploited a real-time salt-induced tilting assay. This assay follows root growth upon both gravitropic and salt challenges, revealing that root bending upon tilting is modulated by Na+ ions, but not by osmotic stress. Next, we measured this salt-specific response in 345 natural Arabidopsis (Arabidopsis thaliana) accessions and discovered a genetic locus, encoding the cell wall-modifying enzyme EXTENSIN ARABINOSE DEFICIENT TRANSFERASE (ExAD) that is associated with root bending in the presence of NaCl (hereafter salt). Extensins are a class of structural cell wall glycoproteins known as hydroxyproline (Hyp)-rich glycoproteins, which are posttranslationally modified by O-glycosylation, mostly involving Hyp-arabinosylation. We show that salt-induced ExAD-dependent Hyp-arabinosylation influences root bending responses and cell wall thickness. Roots of exad1 mutant seedlings, which lack Hyp-arabinosylation of extensin, displayed increased thickness of root epidermal cell walls and greater cell wall porosity. They also showed altered gravitropic root bending in salt conditions and a reduced salt-avoidance response. Our results suggest that extensin modification via Hyp-arabinosylation is a unique salt-specific cellular process required for the directional response of roots exposed to salinity.

Soil salinity presents a dual challenge for plants, involving both osmotic and ionic stress. In response, plants deploy distinct yet interconnected mechanisms to cope with these facets of salinity stress. In this investigation, we observed a substantial overlap in the salt (NaCl)-induced transcriptional responses of Arabidopsis roots with those triggered by osmotic stress or the plant stress hormone abscisic acid (ABA), as anticipated. Notably, a specific cluster of genes responded uniquely to sodium (Na+) ions. Surprisingly, expression of sodium-induced genes exhibited a negative correlation with the ABA response and preceded the activation of genes induced by the osmotic stress component of salt. Elevated exogenous ABA levels resulted in the complete abolition of sodium-induced responses. Consistently, ABA signalling mutants displayed prolonged sodium-induced gene expression, coupled with increased root cell damage under high salinity conditions. Moreover, ABA signalling mutants were unable to redirect root growth to avoid high sodium concentrations and failed to contain their root cell swelling in the presence of elevated salt levels.In summary, our findings unveil an unexpected and pivotal role for ABA signaling in mitigating cellular damage induced by salinity stress and modulating sodium-specific responses in plant roots.### Competing Interest StatementThe authors have declared no competing interest.

Lateral root placement, outgrowth and density are influenced by environmental changes, including salinity stress. CYP79B2 and B3 are two cytochrome P450 enzymes previously identified as required for root architecture remodeling in salt. They produce iAOx, a metabolite that can be converted into indole glucosinolates (IGs), camalexin and indole-3-acetic acid (IAA), a type of auxin. We report here that lateral root appearance, induced by an auxin maximum in the bending zone after gravistimulation, is delayed in the absence of CYP79B2/B3. This delay traces back to a decrease in early lateral root growth after emergence, taking place before lateral roots are macroscopically visible. We measured gene transcripts and abundance of metabolites in the iAOx pathway in root segments that are forming lateral roots. Genes involved in tryptophane and IG biosynthesis were upregulated in cyp79b2/b3 mutants, suggesting a transcriptional feedback-loop. Salt stress was found to increase the expression of genes involved in IAN biosynthesis, a precursor of both IAA and camalexin, in the root during lateral root formation. Moreover, salt increases the concentration of IAN in tissue forming lateral roots in a CYP79B2/B3 dependent manner, but these changes in IAN did not coincide with altered IAA levels. Both the reduction in lateral root density under salt and the delayed lateral root appearance in cyp79b2/b3 knock-out mutants can be complemented by exogenous application of IAN. Our results reveal a role for the iAOx pathway in regulating the timing of lateral root appearance, allowing the modulation of lateral root density under salt stress.

The root system architecture of a plant changes during salt stress exposure. Different accessions of Arabidopsis thaliana have adopted different strategies in remodelling 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 homoeostasis. 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 homoeostasis 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 modelling, 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.

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.

Soil salinity is an increasing worldwide problem for agriculture, affecting plant growth and yield. In our attempt 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 have observed 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. These cell wall modifications might be partly responsible for the activation of the signaling pathways required to activate salt stress responses. In fact, by adding calcium chloride or by chemically inhibiting pectin methyl esterase (PME) activity we observed reduced activation of the early signaling protein Mitogen Activated Protein Kinase 6 (MPK6) as well as a reduced amplitude in salt-induced marker gene induction. We show that MPK6 is required for the full induction of the salt-induced gene expression markers we tested. However, the sodium specific root halotropism response is likely regulated by a different branch of the pathway being independent of MPK6 or calcium application but influenced by the cell wall sensors FER/HERK1/THE1-4 and 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, likely being in part responsible for the response phenotypes observed in salt treated plants. Introduction In plants, cell walls act as a barrier that protects plants to environmental threats and functions as structural support for the whole plant body [1]. In young Arabidopsis plants, primary cell walls (CWs) are made of complex polysaccharide networks where the main load bearing component is cellulose, a linear polymer composed of amorphous and crystalized ß 1-4 linked glucose monomers. Cellulose fibrils are intimately connected with pectin, another major component of the cell wall, described to be essential for regulating cell expansion and elasticity (reviewed in [2]). Contrary to cellulose, which is synthetized at the plasma membrane level, pectins are synthetized in Golgi bodies and released in the apoplast in a highly methylated form [3], [4]. Cell walls are subjected to controlled modifications that allow the cell to extend, driving the direction of cell growth (reviewed in [5]). Cell wall modifications also happen in response to both biotic and abiotic stresses and these structural changes are perceived and sensed by plants. Receptors/sensors are responsible for triggering intracellular downstream signals in response to turgor pressure driven cell wall-dependent mechanical distortions, a process called cell wall integrity (CWI) maintenance [6], [7]. Several members of the Catharanthus roseus Receptor Like Kinase1 Like (CrRLK1L) family have been described to play a role in mechanisms related to cell wall remodeling (e.g. cellulose impairment, pathogen infections, fertility) [8]. CrRLK1Ls contain 2 malectin domains, proposed to selectively bind complex carbohydrates, and an intracellular kinase domain responsible for transmitting signals intracellularly [9]. In Arabidopsis, 17 genes encoding for CrRLK1Ls have been identified and in some cases their functional roles have extensively been studied. For example FERONIA (FER), appears to be vital in multiple aspects of plant life (e.g. fertility, root hair development, mechanical stress, immune responses) while THESEUS1 (THE1), HERKULES 1/2 (HERK1/2) and CURVY (CVY1) are essential for cell elongation and cell morphology related processes [8], [10], [11]. Even if CrRLK1Ls are extensively studied, the mechanism through which they work is not fully understood. It seems that CrRLK1Ls might form complexes through transient hetero-oligomerization as found for the newly identified LETUM1/LETUM2 modulating plant autoimmunity and HERK1/ANJEA or ANXUR (ANX)/BUDDHA’S PAPER SEAL (BUPS) that control pollen tube related mechanisms [12], [13]. CrRLK1Ls also act as receptors for small extracellular peptides called Rapid Alkalinization Factors (RALFs). In Arabidopsis, 34 different RALF genes have been identified and RALF22, 23, 33, 34 and RALF1 have been reported to function as FER ligands, with the latter being required for Arabidopsis ovule fertilization [12], [14]–[16]. On the other hand, THESEUS1 (THE1) seems to be the receptor of RALF34, and both are essential for regulating lateral root development in Arabidopsis [16]. Some CrRLK1Ls have been defined being responsible for sensing cell wall alterations and mechanical stress responses with THESEUS1 (THE1) being essential to fully activate at least one branch of the CWI signaling pathways [17], [18]. In Arabidopsis several other proteins have been characterized to play a role in CWI signaling, including stretch-activated plasma membrane-localized calcium channels (MCAs) and the Nitrate Reductase 1 and 2 (NIA1/2), the latter being recently described to be play an essential role in the cell wall damage dependent cell cycle regulation [2], [5], [17], [18]. Interestingly, many of the CWI players have been found to overlap with salt stress response regulators. This is the case for Leucin Rich Repeat Receptor Like Kinases MIK2/KISS (MALE DISCOVERER 1-INTERACTING), FEI1/2, FERONIA, but also for NIAs and MCAs [19], [20, p. 5], [21]–[23]. Intriguingly, while loss of function mutants for mik2, fei1/2, nia1/2 and mcas (mca1,2) were reported to be highly sensitive to salt application, their responses to cellulose induced cell wall damage varied, with fei1/2 being extremely sensitive and mik2, nia1/2 and mca1, being partly or almost completely insensitive to cellulose inhibition [17], [18], [20]–[23]. Yet how and why CWI and salt induced signaling overlap has been poorly investigated. For decades, researchers have tried to discover how plants sense salt. Salt application simultaneously induces ionic and osmotic stress, which both trigger subsequential general and specific signaling responses. Within seconds to minutes after salt treatment, early signaling molecules are induced, such as calcium spikes, reactive oxygen species (ROS) accumulation and mitogen protein kinase (MAPK) cascade phosphorylation (reviewed in [24], [25]). Only recently, the identification of MONOCATION-INDUCED [Ca2+]i INCREASES 1 (MOCA1) has proposed that salt is sensed at the plasma membrane. MOCA produces glycosyl inositol phosphorylceramide (GIPC) sphingolipids, able to bind to monovalent cations (such as Na2+), triggering the opening of unknown plasma membrane localized Ca2+ channels involved in calcium spikes burst [26]. The accumulation of intracellular calcium is necessary to activate the salt-overlysensitive (SOS) pathway, which consists of the Na+/H+ exchanger SOS1 and SOS2/3 signaling components, all required for limiting the damaging effects of salt on cells by increasing Na+ efflux [27]–[29]. On the other hand, the change in main root direction observed in high salt concentrations, called halotropism, was actually stronger in the sos mutants, and was shown to be dependent on auxin distribution [30], [31]. Interestingly, salt-stress induced calcium spikes depend on FER, as fer-4 loss of function mutants are unable to induce calcium bursts as wild type plants [19]. FERONIA seems to be required to maintain cell wall architecture in response to salt stress since loss of function fer-4 mutants display cell bursting and severe cell swelling in the root in the presence of high salt concentrations. It has been proposed that the salt oversensitive phenotypes observed in FER mutants depend on the enhanced cell wall softening due to the presence of cell wall localized sodium ions. These results are corroborated by supporting data showing that FER’s malectin domain can bind pectin macromolecules in vitro [19]. Exogenous calcium and boron application, can complement, to some extent, FER-dependent salt-induced phenotypes suggesting that salt application might affect pectin architecture [19]. No available information exists for a role of other CrRLK1L members in sensing salt induced cell wall damage. However, other cell wall localized proteins such as the Leucine-rich repeat extensins (LRX) 3/4/5 have been reported be involved in helping the physical association between RALF22/23 and FERONIA causing FER inactivation through its internalization upon salt stress [32]. Here we report that salt changes cell wall architecture by de-methyl esterification of pectin, which in turn is proposed to be responsible for a range of early cellular signaling, gene expression and phenotypic responses. In our attempt to expand our current knowledge on the role of other CrRLK1L in the abiotic field, we have analyzed a complete set of salt stress responses to shed some lights on the insight of early and late salt stress signaling events in previously characterized mutants. We also show that while loss of function mutants lacking THE1 or HERK1 alone show wild type like salt stress responses, plants lacking both HERK1 in combination with a truncated, gain of function allele version of THESEUS1 (herk1the1-4 mutants [33]) show severe salt sensitivity, similarly to that of fer-4 loss of function mutants. FER and HERK1/THE1-4 are required for dampening the salt-induced Mitogen Activated protein Kinase 6 (MPK6)

Crop loss due to soil salinization is an increasing threat to agriculture worldwide. This review provides an overview of cellular and physiological mechanisms in plant responses to salt. We place cellular responses in a time- and tissue-dependent context in order to link them to observed phases in growth rate that occur in response to stress. Recent advances in phenotyping can now functionally or genetically link cellular signaling responses, ion transport, water management, and gene expression to growth, development, and survival. Halophytes, which are naturally salt-tolerant plants, are highlighted as success stories to learn from. We emphasize that ( a) filling the major knowledge gaps in salt-induced signaling pathways, ( b) increasing the spatial and temporal resolution of our knowledge of salt stress responses, ( c) discovering and considering crop-specific responses, and ( d) including halophytes in our comparative studies are all essential in order to take our approaches to increasing crop yields in saline soils to the next level.

The current model for vesicular traffic to and from the plasma membrane is accepted, but the molecular requirements for this coordination are not well defined. We have identified the hot ABA-deficiency suppressor1 mutant, which has a stomatal function defect, as a clathrin heavy chain1 (CHC1) mutant allele and show that it has a decreased rate of endocytosis and growth defects that are shared with other chc1 mutant alleles. We used chc1 alleles and the related chc2 mutant as tools to investigate the effects that clathrin defects have on secretion pathways and plant growth. We show that secretion and endocytosis at the plasma membrane are sensitive to CHC1 and CHC2 function in seedling roots and that chc mutants have physiological defects in stomatal function and plant growth that have not been described previously. These findings suggest that clathrin supports specific functions in multiple cell types. Stomata movement and gas exchange are altered in chc mutants, indicating that clathrin is important for stomatal regulation. The aberrant function of chc mutant stomata is consistent with the growth phenotypes observed under different water and light conditions, which also are similar to those of the secretory SNARE mutant, syp121. The syp121 and chc mutants have impaired endocytosis and exocytosis compared with the wild type, indicating a link between SYP121-dependent secretion and clathrin-dependent endocytosis at the plasma membrane. Our findings provide evidence that clathrin and SYP121 functions are important for the coordination of endocytosis and exocytosis and have an impact on stomatal function, gas exchange, and vegetative growth in Arabidopsis (Arabidopsis thaliana).