A phospho-switch constrains BTL2-mediated phytocytokine signaling in plant immunity.

Enabling and constraining immune activation is of fundamental importance in maintaining cellular homeostasis. Depleting BAK1 and SERK4, the co-receptors of multiple pattern recognition receptors (PRRs), abolishes pattern-triggered immunity but triggers intracellular NOD-like receptor (NLR)-mediated autoimmunity with an elusive mechanism. By deploying RNAi-based genetic screens in Arabidopsis, we identified BAK-TO-LIFE 2 (BTL2), an uncharacterized receptor kinase, sensing BAK1/SERK4 integrity. BTL2 induces autoimmunity through activating Ca2+ channel CNGC20 in a kinase-dependent manner when BAK1/SERK4 are perturbed. To compensate for BAK1 deficiency, BTL2 complexes with multiple phytocytokine receptors, leading to potent phytocytokine responses mediated by helper NLR ADR1 family immune receptors, suggesting phytocytokine signaling as a molecular link connecting PRR- and NLR-mediated immunity. Remarkably, BAK1 constrains BTL2 activation via specific phosphorylation to maintain cellular integrity. Thus, BTL2 serves as a surveillance rheostat sensing the perturbation of BAK1/SERK4 immune co-receptors in promoting NLR-mediated phytocytokine signaling to ensure plant immunity.

CmCNIH1 improves salt tolerance by influencing the trafficking of CmHKT1;1 in pumpkin.

Pumpkin is often used as a rootstock for other Cucurbitaceae crops due to its resistance to soil-borne diseases and abiotic stress. Pumpkin rootstocks use a sodium transporter (CmHKT1;1) to promote the transport of Na+ from the shoot to the root effectively and improve the salt tolerance of the scion. However, the molecular regulatory mechanisms that influence the activity of CmHKT1;1 during salt stress response remain unknown. In this study, CmCNIH1, a cornichon homolog, was identified as a potential cargo receptor for CmHKT1;1. Yeast two-hybrid, biomolecular fluorescence complementation and luciferase complementary assays demonstrated that CmCNIH1 and CmHKT1;1 could interact. CmCNIH1 was a key component of the cellular vesicle transport machinery located in the endoplasmic reticulum (ER), ER export site and Golgi apparatus. A CmCNIH1 knockout mutant was more sensitive to salt stress than the wild-type (WT). In addition, ion homeostasis was disrupted in cmcnih1 mutants, which had higher Na+ and lower K+ content in shoots and roots than the WT. Two-electrode voltage-clamp experiment displayed that CmCNIH1 could not influence the Na+ current that passed through the plasma membrane (PM) in CmHKT1;1-expressing Xenopus laevis oocytes. Data from co-localization assays indicated that intact CmCNIH1 protein could alter the subcellular localization of CmHKT1;1 in tobacco leaf, pumpkin root and yeast. In summary, CmCNIH1 may function as a cargo receptor that regulates the localization of CmHKT1;1 to the PM to improve salt tolerance in pumpkin.

Analysis of ion transport properties of Glycine max HKT transporters and identifying a regulation of GmHKT1;1 by the non-functional GmHKT1;4.

The HKT transporter plays an important role for plants in response to salt stress, but the transport property of the soybean HKT transporters at the molecular level is still unclear. Here, using Xenopus oocyte as a heterologous expression system and two-electrode voltage-clamp technique, we identified four HKT transporters, GmHKT1;1, GmHKT1;2, GmHKT1;3, and GmHKT1;4, which all belong to type I subfamily, but having distinct ion transport properties. While GmHKT1;1, GmHKT1;2 and GmHKT1;3 function as Na+ transporters, GmHKT1;1 is less selective against K+ than the two other transporters. Astonishingly, GmHKT1;4, which lacks transmembrane segments and has no ion permeability, is significantly expressed, and its gene expression pattern is different from the other three GmHKTs under salt stress. Interestingly, GmHKT1;4 reduced the Na+/K+ currents mediated by GmHKT1;1. Further study showed that the transport ability of GmHKT1;1 regulated by GmHKT1;4 was related to the structural differences in the first intracellular domain and the fourth repeat domain. Overall, we have identified one unique GmHKT member, GmHKT1;4, which modulates the Na+ and K+ transport ability of GmHKT1;1 via direct interaction. Thus, we have revealed a new type of HKTs interaction model for altering their ion transport properties.

Genetic variation of BnaA3.NIP5;1 expressing in the lateral root cap contributes to boron deficiency tolerance in Brassica napus.

Boron (B) is essential for vascular plants. Rapeseed (Brassica napus) is the second leading crop source for vegetable oil worldwide, but its production is critically dependent on B supplies. BnaA3.NIP5;1 was identified as a B-efficient candidate gene in B. napus in our previous QTL fine mapping. However, the molecular mechanism through which this gene improves low-B tolerance remains elusive. Here, we report genetic variation in BnaA3.NIP5;1 gene, which encodes a boric acid channel, is a key determinant of low-B tolerance in B. napus. Transgenic lines with increased BnaA3.NIP5;1 expression exhibited improved low-B tolerance in both the seedling and maturity stages. BnaA3.NIP5;1 is preferentially polar-localized in the distal plasma membrane of lateral root cap (LRC) cells and transports B into the root tips to promote root growth under B-deficiency conditions. Further analysis revealed that a CTTTC tandem repeat in the 5'UTR of BnaA3.NIP5;1 altered the expression level of the gene, which is tightly associated with plant growth and seed yield. Field tests with natural populations and near-isogenic lines (NILs) confirmed that the varieties carried BnaA3.NIP5;1Q allele significantly improved seed yield. Taken together, our results provide novel insights into the low-B tolerance of B. napus, and the elite allele of BnaA3.NIP5;1 could serve as a direct target for breeding low-B-tolerant cultivars.

The ATR-WEE1 kinase module inhibits the MAC complex to regulate replication stress response.

DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.

CsMYB96 confers resistance to water loss in citrus fruit by simultaneous regulation of water transport and wax biosynthesis.

A Citrus sinensis R2R3 MYB transcription factor (CsMYB96) has previously been shown to be strongly associated with the expression of many genes related to wax biosynthesis in the fruit. In this study, CsMYB96 was found to alleviate water loss by simultaneously regulating the expression of genes encoding plasma membrane intrinsic proteins (CsPIPs) and wax-related genes. Expression profiling indicated that CsPIP1;1 and CsPIP2;4 had high expression that was representative of other aquaporins, and they were down-regulated in the peel of post-harvest citrus fruit. CsPIP2;4 was further characterized as the predominant CsPIP, with high expression and high-water channel activity. Transient overexpression of CsPIP2;4 accelerated water loss in citrus fruit. In silico analysis further indicated that the expression of CsMYB96 had a significant negative correlation with that of CsPIPs. In vivo and in vitro experiments confirmed that CsMYB96 was able to directly repress the expression of CsPIPs. In addition, CsMYB96 was able to activate wax-related genes and promote wax biosynthesis for defense against water loss. Transient and stable overexpression of CsMYB96 reduced water loss from both citrus fruit and Arabidopsis.

Citrus NIP5;1 aquaporin regulates cell membrane water permeability and alters PIPs plasma membrane localization.

KEY MESSAGE: The ER or donut-like structures localized aquaporin NIP5;1, which interacts with PIPs and alters their localization from plasma membrane to donut-like structures, regulates water permeability. NOD26-like intrinsic proteins (NIPs) play important roles in nutrient uptake and response to various stresses. However, there have been few studies of their functions in water transportation in citrus. Here, we demonstrate the functions of a novel citrus NIP aquaporin (CsNIP5;1) via multiple physiological and biochemical experiments. CsNIP5;1 showed high water permeability when expressed in Xenopus laevis oocytes and yeast. However, subcellular localization assays showed that this protein was localized in the endoplasmic reticulum (ER) or donut-like structures in citrus callus and tobacco leaf. Meanwhile, overexpression of CsNIP5;1 led to a reduction in the water permeability of citrus callus. Protein-protein interaction experiments and subcellular localization assays further revealed that CsNIP5;1 physically interacted with PIPs (CsPIP1;1 and AtPIP2;1), which altered their subcellular localization from the plasma membrane to donut-like structures. Together, CsNIP5;1 was identified as a good water channel when expressed in oocytes and yeast. Meanwhile, CsNIP5;1 participated in the regulation of water permeability of citrus callus, which may be associated with CsNIP5;1-induced re-localization of water channels PIPs. In summary, these results provide new insights into the regulatory mechanism of AQPs-mediated water diffusion.

An amiRNA screen uncovers redundant CBF and ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses.

Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetic screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.

The BIG protein distinguishes the process of CO2 -induced stomatal closure from the inhibition of stomatal opening by CO2.

We conducted an infrared thermal imaging-based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO2 . This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO2 insensitive 1 (cis1). BIG mutants are compromised in elevated CO2 -induced stomatal closure and bicarbonate activation of S-type anion channel currents. In contrast with the wild-type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO2 . However, like the wild-type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO2 . BIG mutants also display wild-type stomatal aperture responses to the closure-inducing stimulus abscisic acid (ABA). Our results indicate that BIG is a signaling component involved in the elevated CO2 -mediated control of stomatal development. In the control of stomatal aperture by CO2 , BIG is only required in elevated CO2 -induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO2 -mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO2 -mediated responses.

Nitric oxide negatively regulates AKT1-mediated potassium uptake through modulating vitamin B6 homeostasis in Arabidopsis.

Nitric oxide (NO), an active signaling molecule in plants, is involved in numerous physiological processes and adaptive responses to environmental stresses. Under high-salt conditions, plants accumulate NO quickly, and reorganize Na(+) and K(+) contents. However, the molecular connection between NO and ion homeostasis is largely unknown. Here, we report that NO lowers K(+) channel AKT1-mediated plant K(+) uptake by modulating vitamin B6 biosynthesis. In a screen for Arabidopsis NO-hypersensitive mutants, we isolated sno1 (sensitive to nitric oxide 1), which is allelic to the previously noted mutant sos4 (salt overly sensitive 4) that has impaired Na(+) and K(+) contents and overproduces pyridoxal 5'-phosphate (PLP), an active form of vitamin B6. We showed that NO increased PLP and decreased K(+) levels in plant. NO induced SNO1 gene expression and enzyme activity, indicating that NO-triggered PLP accumulation mainly occurs through SNO1-mediated vitamin B6 salvage biosynthetic pathway. Furthermore, we demonstrated that PLP significantly repressed the activity of K(+) channel AKT1 in the Xenopus oocyte system and Arabidopsis root protoplasts. Together, our results suggest that NO decreases K(+) absorption by promoting the synthesis of vitamin B6 PLP, which further represses the activity of K(+) channel AKT1 in Arabidopsis. These findings reveal a previously unidentified pivotal role of NO in modulating the homeostasis of vitamin B6 and potassium nutrition in plants, and shed light on the mechanism of NO in plant acclimation to environmental changes.

Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell.

Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action.

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

PYR/RCAR receptors contribute to ozone-, reduced air humidity-, darkness-, and CO2-induced stomatal regulation.

Rapid stomatal closure induced by changes in the environment, such as elevation of CO2, reduction of air humidity, darkness, and pulses of the air pollutant ozone (O3), involves the SLOW ANION CHANNEL1 (SLAC1). SLAC1 is activated by OPEN STOMATA1 (OST1) and Ca(2+)-dependent protein kinases. OST1 activation is controlled through abscisic acid (ABA)-induced inhibition of type 2 protein phosphatases (PP2C) by PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR) receptor proteins. To address the role of signaling through PYR/RCARs for whole-plant steady-state stomatal conductance and stomatal closure induced by environmental factors, we used a set of Arabidopsis (Arabidopsis thaliana) mutants defective in ABA metabolism/signaling. The stomatal conductance values varied severalfold among the studied mutants, indicating that basal ABA signaling through PYR/RCAR receptors plays a fundamental role in controlling whole-plant water loss through stomata. PYR/RCAR-dependent inhibition of PP2Cs was clearly required for rapid stomatal regulation in response to darkness, reduced air humidity, and O3. Furthermore, PYR/RCAR proteins seem to function in a dose-dependent manner, and there is a functional diversity among them. Although a rapid stomatal response to elevated CO2 was evident in all but slac1 and ost1 mutants, the bicarbonate-induced activation of S-type anion channels was reduced in the dominant active PP2C mutants abi1-1 and abi2-1. Further experiments with a wider range of CO2 concentrations and analyses of stomatal response kinetics suggested that the ABA signalosome partially affects the CO2-induced stomatal response. Thus, we show that PYR/RCAR receptors play an important role for the whole-plant stomatal adjustments and responses to low humidity, darkness, and O3 and are involved in responses to elevated CO2.

Mechanisms of plant saline-alkaline tolerance.

Saline-alkaline soil affects crop growth and development, thereby suppressing the yields. Human activities and climate changes are putting arable land under the threat of saline-alkalization. To feed a growing global population in limited arable land, it is of great urgence to breed saline-alkaline tolerant crops to cope with food security. Plant salt-tolerance mechanisms have already been explored for decades. However, to date, the molecular mechanisms underlying plants responses to saline-alkaline stress have remained largely elusive. Here, we summarize recent advances in plant response to saline-alkaline stress and propose some points deserving of further exploration.

Transmembrane potential, an indicator in situ reporting cellular senescence and stress response in plant tissues.

BACKGROUND: Plant cells usually sustain a stable membrane potential due to influx and/or efflux of charged ions across plasma membrane. With the growth and development of plants, different tissues and cells undergo systemic or local programmed decline. Whether the membrane potential of plasma membrane could report senescence signal of plant tissues and cells is unclear. RESULTS: We applied a maneuverable transmembrane potential (TMP) detection method with patch-clamp setup to examine the senescence signal of leaf tissue cells in situ over the whole life cycle in Arabidopsis thaliana. The data showed that the TMPs of plant tissues and cells were varied at different growth stages, and the change of TMP was higher at the vegetative growth stage than at the reproductive stage of plant growth. The distinct change of TMP was detectable between the normal and the senescent tissues and cells in several plant species. Moreover, diverse abiotic stimuli, such as heat stress, hyperpolarized the TMP in a short time, followed by depolarized membrane potential with the senescence occurring. We further examined the TMP of plant chloroplasts, which also indicates the senescence signal in organelles. CONCLUSIONS: This convenient TMP detection method can report the senescence signal of plant tissues and cells, and can also indicate the potential of plant tolerance to environmental stress.

Differential sodium and potassium transport selectivities of the rice OsHKT2;1 and OsHKT2;2 transporters in plant cells.

Na(+) and K(+) homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na(+) transport or Na(+) and K(+) transport when expressed in Xenopus laevis oocytes and yeast. However, the Na(+)/K(+) selectivities of the K(+)-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5' untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K(+) permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na(+) uptake, but little Rb(+) uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na(+)-K(+) cotransport in plant cells such that extracellular K(+) stimulated OsHKT2;2-mediated Na(+) influx and vice versa. Furthermore, at millimolar Na(+) concentrations, OsHKT2;2 mediated Na(+) influx into plant cells without adding extracellular K(+). This study shows that the Na(+)/K(+) selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K(+) and Ca(2+) down-regulated OsHKT2;1-mediated Na(+) influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.

Interplay between hydrogen sulfide and other signaling molecules in the regulation of guard cell signaling and abiotic/biotic stress response.

Stomatal aperture controls the balance between transpirational water loss and photosynthetic carbon dioxide (CO2) uptake. Stomata are surrounded by pairs of guard cells that sense and transduce environmental or stress signals to induce diverse endogenous responses for adaptation to environmental changes. In a recent decade, hydrogen sulfide (H2S) has been recognized as a signaling molecule that regulates stomatal movement. In this review, we summarize recent progress in research on the regulatory role of H2S in stomatal movement, including the dynamic regulation of phytohormones, ion homeostasis, and cell structural components. We focus especially on the cross talk among H2S, nitric oxide (NO), and hydrogen peroxide (H2O2) in guard cells, as well as on H2S-mediated post-translational protein modification (cysteine thiol persulfidation). Finally, we summarize the mechanisms by which H2S interacts with other signaling molecules in plants under abiotic or biotic stress. Based on evidence and clues from existing research, we propose some issues that need to be addressed in the future.

Hydrogen sulfide (H2S) signaling in plant development and stress responses.

ABSTRACT: Hydrogen sulfide (H2S) was initially recognized as a toxic gas and its biological functions in mammalian cells have been gradually discovered during the past decades. In the latest decade, numerous studies have revealed that H2S has versatile functions in plants as well. In this review, we summarize H2S-mediated sulfur metabolic pathways, as well as the progress in the recognition of its biological functions in plant growth and development, particularly its physiological functions in biotic and abiotic stress responses. Besides direct chemical reactions, nitric oxide (NO) and hydrogen peroxide (H2O2) have complex relationships with H2S in plant signaling, both of which mediate protein post-translational modification (PTM) to attack the cysteine residues. We also discuss recent progress in the research on the three types of PTMs and their biological functions in plants. Finally, we propose the relevant issues that need to be addressed in the future research. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-021-00035-4.

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K channels in Arabidopsis guard cells.

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)-induced stomatal closure response in the [Ca2+]i-elevation-independent pathway? (ii) How do ABA-insensitive mutants affect the [Ca2+]i-elevation-independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward-rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA-induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA-insensitive mutants ost1-2, abi2-1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i-dependent ABA signaling. Interestingly, patch-clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S-type anion channels and down-regulate inward-rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms.

Identification of vacuolar phosphate efflux transporters in land plants.

Inorganic phosphate (Pi) is an essential component of all life forms. Land plants acquire Pi from the soil through roots and associated symbioses, and it is then transported throughout the plant. When sufficient, excess Pi is stored in vacuoles for remobilization following Pi deficiency. Although Pi release from the vacuoles to the cytoplasm serves as a critical mechanism for plants to adapt to low-Pi stress, the transporters responsible for vacuolar Pi efflux have not been identified. Here, we identified a pair of Oryza sativa vacuolar Pi efflux transporters (OsVPE1 and OsVPE2) that were more abundant in plants grown under Pi-deficient conditions. These OsVPE proteins can transport Pi into yeast cells and Xenopus laevis oocytes. Vacuolar Pi content was higher in the loss-of-function Osvpe1 Osvpe2 double mutant than in wild type, particularly under low-Pi stress. Overexpression of either OsVPE1 or OsVPE2 in transgenic plants reduced vacuolar Pi content, consistent with a role in vacuolar Pi efflux. We demonstrate that these VPE proteins evolved from an ancient plasma membrane glycerol-3-phosphate transporter protein. Together, these data indicate that this transporter was recruited to the vacuolar membrane to catalyse Pi efflux during the course of land plant evolution.