Calcium Live Imaging at Multi-Scales from Cellular to Organ Level in Arabidopsis thaliana.

Plants must adapt to environmental constraints. For this, they are able to perceive several types of stress in isolation or in combination manner. At the cellular level, after the perception of stress, cell signaling is set up to allow the establishment of the specific response. The calcium ion is known to be one of the ubiquitous second messengers which is involved in most of the stresses perceived by the plant. Changes of free cytosolic calcium but also in other cellular compartments are able to activate or inactivate several mechanisms involved in the cell to cope with the changes of environmental conditions. Several calcium reporters have been intensively used to visualize calcium signals in different conditions. In this chapter, we will present only genetically encoded fluorescent reporters for calcium imaging in living plant tissues to measure variations in calcium at several scales. The FRET (fluorescence resonance energy transfer) YC3.60 and the intensiometric GCamP3 sensors will be used in this method chapter. The image analyses will be also detailed for fluorescence quantification of calcium variation.

AtDTX25, a member of the multidrug and toxic compound extrusion family, is a vacuolar ascorbate transporter that controls intracellular iron cycling in Arabidopsis.

Iron (Fe) is an essential element, its transport is regulated by the cell redox balance. In seeds, Fe enters the embryo as Fe2+ and is stored in vacuoles as Fe3+ . Through its ferric reduction activity, ascorbate plays a major role in Fe redox state and therefore Fe transport within the seed. We searched for ascorbate membrane transporters responsible for controlling Fe reduction by screening the yeast ferric reductase-deficient fre1 strain and isolated AtDTX25, a member of the Multidrug And Toxic compound Extrusion (MATE) family. AtDTX25 was shown to mediate ascorbate efflux when expressed in yeast and Xenopus oocytes, in a pH-dependent manner. In planta, AtDTX25 is highly expressed during germination and encodes a vacuolar membrane protein. Isolated vacuoles from AtDTX25-1 knockout mutant contained less ascorbate and more Fe than wild-type (WT), and mutant seedlings were highly sensitive to Fe deficiency. Iron imaging further showed that the remobilisation of Fe from vacuoles was highly impaired in mutant seedlings. Taken together, our results established AtDTX25 as a vacuolar ascorbate transporter, required during germination to promote the reduction of the pool of stored Fe3+ and its remobilisation to feed the developing seedling.

Manganese triggers phosphorylation-mediated endocytosis of the Arabidopsis metal transporter NRAMP1.

The NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 1 (NRAMP1) transporter guarantees plant survival of manganese (Mn) deficiency by mediating Mn entry into root cells. Unlike other high-affinity metal transporters, NRAMP1 is only slightly regulated at the transcriptional level. We show here that adequate Mn content in tissues is safeguarded through a tight control of the quantity of NRAMP1 present at the surface of root cells. Depending on Mn availability, an NRAMP1-GFP fusion protein cycles dynamically between the plasma membrane (PM) and endosomal compartments. This involves a clathrin-mediated endocytosis pathway, as disrupting this pathway in auxilin-overexpressor lines prevents NRAMP1 internalization. Mutation of the phosphorylated serine residues 20, 22 and 24 in the cytosol-exposed N terminus of NRAMP1 alters its membrane distribution. Indeed, a phospho-dead mutation stabilizes NRAMP1 at the PM, regardless of the Mn regime, and dramatically reduces plant tolerance to Mn toxicity. Conversely a phosphomimetic mutant is constitutively internalized into endosomes. Together, these data establish that phosphorylation of NRAMP1 is the trigger for its Mn-induced endocytosis and represents the main level of regulation of this transporter. Furthermore, the extent of Mn toxicity observed when interrupting NRAMP1 membrane cycling undermines the dogma that Mn is only marginally toxic to plants.

Functional analysis of TmHKT1;4-A2 promoter through deletion analysis provides new insight into the regulatory mechanism underlying abiotic stress adaptation.

MAIN CONCLUSION: Bioinformatic, molecular, and biochemical analysis were performed to get more insight into the regulatory mechanism by which TmHKT1;4-A2 is regulated. HKT transporters from different plant species have been shown to play important role in plant response to salt. In previous work, TmHKT1;4-A2 gene from Triticum monococcum has been characterized as a major gene for Nax1 QTL (Tounsi et al. Plant Cell Physiol 57:2047-2057, 2016). So far, little is known about its regulatory mechanism. In this study, the promoter region of TmHKT1;4-A2 (1400 bp) was isolated and considered as the full-length promoter (PA2-1400). In silico analysis revealed the presence of important cis-acting elements related to abiotic stresses and phytohormones. Interestingly, our real-time RT-PCR analysis provided evidence that TmHKT1;4-A2 is regulated not only by salt stress but also by osmotic, heavy metal, oxidative, and hormones stresses. In transgenic Arabidopsis plants, TmHKT1;4-A2 is strongly active in vascular tissues of roots and leaves. Through 5'-end deletion analysis, we showed that PA2-1400 promoter is able to drive strong GUS activity under normal conditions and in response to different stresses compared to PA2-824 and PA2-366 promoters. These findings provide new information on the regulatory mechanism of TmHKT1;4-A2 and shed more light on its role under different stresses.

A Plasma Membrane Nanodomain Ensures Signal Specificity during Osmotic Signaling in Plants.

In the course of their growth and development, plants have to constantly perceive and react to their environment. This is achieved in cells by the coordination of complex combinatorial signaling networks. However, how signal integration and specificity are achieved in this context is unknown. With a focus on the hyperosmotic stimulus, we use live super-resolution light imaging methods to demonstrate that a Rho GTPase, Rho-of-Plant 6 (ROP6), forms stimuli-dependent nanodomains within the plasma membrane (PM). These nanodomains are necessary and sufficient to transduce production of reactive oxygen species (ROS) that act as secondary messengers and trigger several plant adaptive responses to osmotic constraints. Furthermore, osmotic signal triggers interaction between ROP6 and two NADPH oxidases that subsequently generate ROS. ROP6 nanoclustering is also needed for cell surface auxin signaling, but short-time auxin treatment does not induce ROS accumulation. We show that auxin-induced ROP6 nanodomains, unlike osmotically driven ROP6 clusters, do not recruit the NADPH oxidase, RBOHD. Together, our results suggest that Rho GTPase nano-partitioning at the PM ensures signal specificity downstream of independent stimuli.

The Arabidopsis NRT1.1 transceptor coordinately controls auxin biosynthesis and transport to regulate root branching in response to nitrate.

In agricultural systems, nitrate is the main source of nitrogen available for plants. Besides its role as a nutrient, nitrate has been shown to act as a signal molecule in plant growth, development, and stress responses. In Arabidopsis, the NRT1.1 nitrate transceptor represses lateral root (LR) development at low nitrate availability by promoting auxin basipetal transport out of the LR primordia (LRPs). Here we show that NRT1.1 acts as a negative regulator of the TAR2 auxin biosynthetic gene in the root stele. This is expected to repress local auxin biosynthesis and thus to reduce acropetal auxin supply to the LRPs. Moreover, NRT1.1 also negatively affects expression of the LAX3 auxin influx carrier, thus preventing the cell wall remodeling required for overlying tissue separation during LRP emergence. NRT1.1-mediated repression of both TAR2 and LAX3 is suppressed at high nitrate availability, resulting in nitrate induction of the TAR2 and LAX3 expression that is required for optimal stimulation of LR development by nitrate. Altogether, our results indicate that the NRT1.1 transceptor coordinately controls several crucial auxin-associated processes required for LRP development, and as a consequence that NRT1.1 plays a much more integrated role than previously expected in regulating the nitrate response of root system architecture.

Paspalum urvillei and Setaria parviflora, two grasses naturally adapted to extreme iron-rich environments.

Paspalum urvillei and Setaria parviflora are two plant species naturally adapted to iron-rich environments such as around iron mines wastes. The aim of our work was to characterize how these two species cope with these extreme conditions by comparing them with related model species, Oryza sativa and Setaria viridis, that appeared to be much less tolerant to Fe excess. Both Paspalum urvillei and Setaria parviflora were able to limit the amount of Fe accumulated within roots and shoots, compared to the less tolerant species. Perls/DAB staining of Fe in root cross sections indicated that Paspalum urvillei and Setaria parviflora responded through the build-up of the iron plaque (IP), suggesting a role of this structure in the limitation of Fe uptake. Synchrotron µXRF analyses showed the presence of phosphorus, calcium, silicon and sulfur on IP of Paspalum urvillei roots and µXANES analyses identified Fe oxyhydroxide (ferrihydrite) as the main Fe form. Once within roots, high concentrations of Fe were localized in the cell walls and vacuoles of Paspalum urvillei, Setaria parviflora and O. sativa whereas Setaria viridis accumulated Fe in ferritins. The Fe forms translocated to the shoots of Setaria parviflora were identified as tri-iron complexes with citrate and malate. In leaves, all species accumulated Fe in the vacuoles of bundle sheath cells and as ferritin complexes in plastids. Taken together, our results strongly suggest that Paspalum urvillei and Setaria parviflora set up mechanisms of Fe exclusion in roots and shoots to limit the toxicity induced by Fe excess.

Highlight on the expression and the function of a novel MnSOD from diploid wheat (T. monococcum) in response to abiotic stress and heavy metal toxicity.

Superoxide dismutases (SODs) play a pivotal role in improving abiotic stress tolerance in plant cells. A novel manganese superoxide dismutase gene, denoted as TmMnSOD, was identified from Triticum monococcum. The encoded protein displayed high sequence identity with MnSOD family members and was highly homologous to TdMnSOD from durum wheat. Furthermore, the 3D structure analysis revealed that TmMnSOD displayed homotetramer subunit organization, incorporating four Mn2+ ions. Notably, TmMnSOD structure contains predominantly alpha helices with three beta sheets. On the other hand, under stress conditions, TmMnSOD transcript level was significantly up-regulated by salt, oxidative and heavy metal stresses. At the functional level, TmMnSOD imparts tolerance of yeast and E. coli cells under diverse stresses. Promoter analysis of TmMnSOD gene showed the presence of a great number of salt and pathogen-responsive cis-regulatory elements, highlighting the interest of this gene in breeding programs towards improved tolerance to salt stress in wheat.

The Rhizophagus irregularis Genome Encodes Two CTR Copper Transporters That Mediate Cu Import Into the Cytosol and a CTR-Like Protein Likely Involved in Copper Tolerance.

Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic conditions. In this study, we have characterized two Cu transporters of the CTR family (RiCTR1 and RiCTR2) and a CTR-like protein (RiCTR3A) of Rhizophagus irregularis. Functional analyses in yeast revealed that RiCTR1 encodes a plasma membrane Cu transporter, RiCTR2 a vacuolar Cu transporter and RiCTR3A a plasma membrane protein involved in Cu tolerance. RiCTR1 was more highly expressed in the extraradical mycelia (ERM) and RiCTR2 in the intraradical mycelia (IRM). In the ERM, RiCTR1 expression was up-regulated by Cu deficiency and down-regulated by Cu toxicity. RiCTR2 expression increased only in the ERM grown under severe Cu-deficient conditions. These data suggest that RiCTR1 is involved in Cu uptake by the ERM and RiCTR2 in mobilization of vacuolar Cu stores. Cu deficiency decreased mycorrhizal colonization and arbuscule frequency, but increased RiCTR1 and RiCTR2 expression in the IRM, which suggest that the IRM has a high Cu demand. The two alternatively spliced products of RiCTR3, RiCTR3A and RiCTR3B, were more highly expressed in the ERM. Up-regulation of RiCTR3A by Cu toxicity and the yeast complementation assays suggest that RiCTR3A might function as a Cu receptor involved in Cu tolerance.

Localization and expression analysis of a novel catalase from Triticum monococcum TmCAT1 involved in response to different environmental stresses.

Catalase proteins play a crucial role in detoxifying hydrogen peroxide, generated during plant growth, and in response to various environmental stresses. Despite their importance, little is known about their localization and expression in wheat. In this study, we identified and characterized a novel peroxisomal catalase gene from Triticum monococcum, designated as TmCAT1. Phylogenetic analysis revealed that TmCAT1 shared high identity with TdCAT1 and other plant catalases belonging to subfamily 1. We predicted the 3D structure model and the oligomerization arrangement of TmCAT1. Besides, we displayed an arrangement in asymmetric unit, which involved interactions including, mainly, residues from N-terminal domain. Interestingly, sequence analysis indicated that TmCAT1, like TdCAT1, had the peroxisomal targeting signal (PTS1) around its C-terminus. Transient expression of TmCAT1-GFP and TdCAT1-GFP in tobacco leaves revealed that the two fused proteins are targeted into peroxisomes. However, the truncated forms lacking the tripeptide QKL remained in the cytosol. Concerning the expression profile analysis, TmCAT1 is expressed especially in leaves in normal condition. On the other hand, it is up-regulated by different stress incorporating salt, osmotic, oxidative, heavy metal and hormones stresses. Functional analysis by heterologous expression in yeast cells showed that TmCAT1 improved tolerance to multiple abiotic stresses. The presence of important cis-regulatory elements in the promoter region of TmCAT1 strongly reinforces the interest of this gene in plant adaptation to various stresses.

Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus.

The split GFP technique is based on the auto-assembly of GFP when two polypeptides-GFP1-10 (residues 1-214; the detector) and GFP11 (residues 215-230; the tag)-both non-fluorescing on their own, associate spontaneously to form a fluorescent molecule. We evaluated this technique for its efficacy in contributing to the characterization of Cauliflower mosaic virus (CaMV) infection. A recombinant CaMV with GFP11 fused to the viral protein P6 (a key player in CaMV infection and major constituent of viral factory inclusions that arise during infection) was constructed and used to inoculate transgenic Arabidopsis thaliana expressing GFP1-10. The mutant virus (CaMV11P6) was infectious, aphid-transmissible and the insertion was stable over many passages. Symptoms on infected plants were delayed and milder. Viral protein accumulation, especially of recombinant 11P6, was greatly decreased, impeding its detection early in infection. Nonetheless, spread of infection from the inoculated leaf to other leaves was followed by whole plant imaging. Infected cells displayed in real time confocal laser scanning microscopy fluorescence in wild type-looking virus factories. Thus, it allowed for the first time to track a CaMV protein in vivo in the context of an authentic infection. 11P6 was immunoprecipitated with anti-GFP nanobodies, presenting a new application for the split GFP system in protein-protein interaction assays and proteomics. Taken together, split GFP can be an attractive alternative to using the entire GFP for protein tagging.

Osmotic Stress Activates Two Reactive Oxygen Species Pathways with Distinct Effects on Protein Nanodomains and Diffusion.

Physiological acclimation of plants to an everchanging environment is governed by complex combinatorial signaling networks that perceive and transduce various abiotic and biotic stimuli. Reactive oxygen species (ROS) serve as one of the second messengers in plant responses to hyperosmotic stress. The molecular bases of ROS production and the primary cellular processes that they target were investigated in the Arabidopsis (Arabidopsis thaliana) root. Combined pharmacological and genetic approaches showed that the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) pathway and an additional pathway involving apoplastic ascorbate and iron can account for ROS production upon hyperosmotic stimulation. The two pathways determine synergistically the rate of membrane internalization, within minutes after activation. Live superresolution microscopy revealed at single-molecule scale how ROS control specific diffusion and nano-organization of membrane cargo proteins. In particular, ROS generated by RBOHs initiated clustering of the PLASMA MEMBRANE INTRINSIC PROTEIN2;1 aquaporin and its removal from the plasma membrane. This process is contributed to by clathrin-mediated endocytosis, with a positive role of RBOH-dependent ROS, specifically under hyperosmotic stress.

Anatomical and hormonal description of rootlet primordium development along white lupin cluster root.

Cluster root (CR) is one of the most spectacular plant developmental adaptations to hostile environment. It can be found in a few species from a dozen botanical families, including white lupin (Lupinus albus) in the Fabaceae family. These amazing structures are produced in phosphate-deprived conditions and are made of hundreds of short roots also known as rootlets. White lupin is the only crop bearing CRs and is considered as the model species for CR studies. However, little information is available on CRs atypical development, including the molecular events that trigger their formation. To provide insights on CR formation, we performed an anatomical and cellular description of rootlet development in white lupin. Starting with a classic histological approach, we described rootlet primordium development and defined eight developmental stages from rootlet initiation to their emergence. Due to the major role of hormones in the developmental program of root system, we next focussed on auxin-related mechanisms. We observed the establishment of an auxin maximum through rootlet development in transgenic roots expressing the DR5:GUS auxin reporter. Expression analysis of the main auxin-related genes [TIR, Auxin Response Factor (ARF) and AUX/IAA] during a detailed time course revealed specific expression associated with the formation of the rootlet primordium. We showed that L. albus TRANSPORT INHIBITOR RESPONSE 1b is expressed during rootlet primordium formation and that L. albus AUXIN RESPONSE FACTOR 5 is expressed in the vasculature but absent in the primordium itself. Altogether, our results describe the very early cellular events leading to CR formation and reveal some of the auxin-related mechanisms.

Intracellular Distribution of Manganese by the Trans-Golgi Network Transporter NRAMP2 Is Critical for Photosynthesis and Cellular Redox Homeostasis.

Plants require trace levels of manganese (Mn) for survival, as it is an essential cofactor in oxygen metabolism, especially O2 production via photosynthesis and the disposal of superoxide radicals. These processes occur in specialized organelles, requiring membrane-bound intracellular transporters to partition Mn between cell compartments. We identified an Arabidopsis thaliana member of the NRAMP family of divalent metal transporters, NRAMP2, which functions in the intracellular distribution of Mn. Two knockdown alleles of NRAMP2 showed decreased activity of photosystem II and increased oxidative stress under Mn-deficient conditions, yet total Mn content remained unchanged. At the subcellular level, these phenotypes were associated with a loss of Mn content in vacuoles and chloroplasts. NRAMP2 was able to rescue the mitochondrial yeast mutant mtm1∆ In plants, NRAMP2 is a resident protein of the trans-Golgi network. NRAMP2 may act indirectly on downstream organelles by building up a cytosolic pool that is used to feed target compartments. Moreover, not only does the nramp2 mutant accumulate superoxide ions, but NRAMP2 can functionally replace cytosolic superoxide dismutase in yeast, indicating that the pool of Mn displaced by NRAMP2 is required for the detoxification of reactive oxygen species.

Distinct amino acids in the C-linker domain of the Arabidopsis K+ channel KAT2 determine its subcellular localization and activity at the plasma membrane.

Shaker K(+) channels form the major K(+) conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K(+) channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K(+) rectifying channel1 (AtKC1). These two subunits contribute to K(+) transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch 312TVRAASEFA320 that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K(+)) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.

In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

New insights into Fe localization in plant tissues.

Deciphering cellular iron (Fe) homeostasis requires having access to both quantitative and qualitative information on the subcellular pools of Fe in tissues and their dynamics within the cells. We have taken advantage of the Perls/DAB Fe staining procedure to perform a systematic analysis of Fe distribution in roots, leaves and reproductive organs of the model plant Arabidopsis thaliana, using wild-type and mutant genotypes affected in iron transport and storage. Roots of soil-grown plants accumulate iron in the apoplast of the central cylinder, a pattern that is strongly intensified when the citrate effluxer FRD3 is not functional, thus stressing the importance of citrate in the apoplastic movement of Fe. In leaves, Fe level is low and only detected in and around vascular tissues. In contrast, Fe staining in leaves of iron-treated plants extends in the surrounding mesophyll cells where Fe deposits, likely corresponding to Fe-ferritin complexes, accumulate in the chloroplasts. The loss of ferritins in the fer1,3,4 triple mutant provoked a massive accumulation of Fe in the apoplastic space, suggesting that in the absence of iron buffering in the chloroplast, cells activate iron efflux and/or repress iron influx to limit the amount of iron in the cell. In flowers, Perls/DAB staining has revealed a major sink for Fe in the anthers. In particular, developing pollen grains accumulate detectable amounts of Fe in small-size intracellular bodies that aggregate around the vegetative nucleus at the binuclear stage and that were identified as amyloplasts. In conclusion, using the Perls/DAB procedure combined to selected mutant genotypes, this study has established a reliable atlas of Fe distribution in the main Arabidopsis organs, proving and refining long-assumed intracellular locations and uncovering new ones. This "iron map" of Arabidopsis will serve as a basis for future studies of possible actors of iron movement in plant tissues and cell compartments.

AtKC1 is a general modulator of Arabidopsis inward Shaker channel activity.

A functional Shaker potassium channel requires assembly of four α-subunits encoded by a single gene or various genes from the Shaker family. In Arabidopsis thaliana, AtKC1, a Shaker α-subunit that is silent when expressed alone, has been shown to regulate the activity of AKT1 by forming heteromeric AtKC1-AKT1 channels. Here, we investigated whether AtKC1 is a general regulator of channel activity. Co-expression in Xenopus oocytes of a dominant negative (pore-mutated) AtKC1 subunit with the inward Shaker channel subunits KAT1, KAT2 or AKT2, or the outward subunits SKOR or GORK, revealed that the three inward subunits functionally interact with AtKC1 while the outward ones cannot. Localization experiments in plant protoplasts showed that KAT2 was able to re-locate AtKC1 fused to GFP from endomembranes to the plasma membrane, indicating that heteromeric AtKC1-KAT2 channels are efficiently targeted to the plasma membrane. Functional properties of heteromeric channels involving AtKC1 and KAT1, KAT2 or AKT2 were analysed by voltage clamp after co-expression of the respective subunits in Xenopus oocytes. AtKC1 behaved as a regulatory subunit within the heterotetrameric channel, reducing the macroscopic conductance and negatively shifting the channel activation potential. Expression studies showed that AtKC1 and its identified Shaker partners have overlapping expression patterns, supporting the hypothesis of a general regulation of inward channel activity by AtKC1 in planta. Lastly, AtKC1 disruption appeared to reduce plant biomass production, showing that AtKC1-mediated channel activity regulation is required for normal plant growth.