SummaryCalcium signalling involves sensor proteins that decode temporal and spatial changes in cellular Ca 2+ concentration. Calcineurin B-like proteins (CBLs) represent a unique family of plant calcium sensors that relay signals by interacting with a family of protein kinases, designated as CBL-interacting protein kinases (CIPKs). In a reverse genetic screen for altered drought tolerance, we identified a loss-of-function allele of CIPK23 as exhibiting a drought-tolerant phenotype. In the cipk23 mutant, reduced transpirational water loss from leaves coincides with enhanced ABA sensitivity of guard cells during opening as well as closing reactions, without noticeable alterations in ABA content in the plant. We identified the calcium sensors CBL1 and CBL9 as CIPK23-interacting proteins that targeted CIPK23 to the plasma membrane in vivo. Expression analysis of the CIPK23, CBL1 and CBL9 genes suggested that they may function together in diverse tissues, including guard cells and root hairs. In addition, expression of the CIPK23 gene was induced by low-potassium conditions, implicating a function of this gene product in potassium nutrition. Indeed, cipk23 mutants displayed severe growth impairment on media with low concentrations of potassium. This phenotype correlates with a reduced efficiency of K + uptake into the roots. In support of the conclusion that CBL1 and CBL9 interact with and synergistically serve as upstream regulators of CIPK23, the cbl1 cbl9 double mutant, but not the cbl1 or cbl9 single mutants, exhibit altered phenotypes for stomatal responses and low-potassium sensitivity. Together with the recent identification of the potassium channel AKT1 as a target of CIPK23, these results imply that plasma membrane-localized CBL1-and CBL9-CIPK23 complexes simultaneously regulate K + transport processes in roots and in stomatal guard cells.

Potassium (K ؉ ) is an essential nutrient for plant growth and development. Plants often adapt to low K ؉ conditions by increasing their K ؉ uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K ؉ transporter 1 (AKT1), responsible for K ؉ uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel.

). † These authors contributed equally to this work. SummaryCalcium serves as a critical messenger in many adaptation and developmental processes. Cellular calcium signals are detected and transmitted by sensor molecules such as calcium-binding proteins. In plants, the calcineurin B-like protein (CBL) family represents a unique group of calcium sensors and plays a key role in decoding calcium transients by specifically interacting with and regulating a family of protein kinases (CIPKs). We report here that the CBL protein CBL10 functions as a crucial regulator of salt tolerance in Arabidopsis. Cbl10 mutant plants exhibited significant growth defects and showed hypersensitive cell death in leaf tissues under high-salt conditions. Interestingly, the Na + content of the cbl10 mutant, unlike other salt-sensitive mutants identified thus far, was significantly lower than in the wild type under either normal or high-salt conditions, suggesting that CBL10 mediates a novel Ca 2+ -signaling pathway for salt tolerance. Indeed, the CBL10 protein physically interacts with the salt-tolerance factor CIPK24 (SOS2), and the CBL10-CIPK24 (SOS2) complex is associated with the vacuolar compartments that are responsible for salt storage and detoxification in plant cells. These findings suggest that CBL10 and CIPK24 (SOS2) constitute a novel salt-tolerance pathway that regulates the sequestration/compartmentalization of Na + in plant cells. Because CIPK24 (SOS2) also interacts with CBL4 (SOS3) and regulates salt export across the plasma membrane, our study identifies CIPK24 (SOS2) as a multi-functional protein kinase that regulates different aspects of salt tolerance by interacting with distinct CBL calcium sensors.

Nutrient sensing is critical for plant adaptation to the environment. Because of extensive farming and erosion, low content of mineral nutrients such as potassium (K ؉ ) in soils becomes a limiting factor for plant growth. In response to low-K conditions, plants enhance their capability of K ؉ uptake through an unknown signaling mechanism. Here we report the identification of a Ca 2؉ -dependent pathway for low-K response in Arabidopsis. We are not aware of any other example of a molecular pathway for a nutrient response in plants. Earlier genetic analyses revealed three genes encoding two Ca 2؉ sensors (CBL1 and CBL9) and their target protein kinase (CIPK23) to be critical for plant growth on low-K media and for stomatal regulation, indicating that these calcium signaling components participate in the low-K response and turgor regulation. In this study, we show that the protein kinase CIPK23 interacted with, and phosphorylated, a voltage-gated inward K ؉ channel (AKT1) required for K ؉ acquisition in Arabidopsis. In the Xenopus oocyte system, our studies showed that interacting calcium sensors (CBL1 and CBL9) together with target kinase CIPK23, but not either component alone, activated the AKT1 channel in a Ca 2؉ -dependent manner, connecting the Ca 2؉ signal to enhanced K ؉ uptake through activation of a K ؉ channel. Disruption of both CBL1 and CBL9 or CIPK23 gene in Arabidopsis reduced the AKT1 activity in the mutant roots, confirming that the Ca 2؉ -CBL-CIPK pathway functions to orchestrate transporting activities in planta according to external K ؉ availability.calcium signaling ͉ potassium channel ͉ nutrient sensing ͉ potassium uptake ͉ protein kinase P lants and microbes are able to adapt to widely varying environmental conditions, including the availability of inorganic nutrients. The response to K ϩ is especially relevant because it is the major inorganic osmoticum that contributes to turgor pressure and, hence, to cellular growth and development (1, 2). Both plants and fungi maintain internal K ϩ near 100 mM even when extracellular K ϩ varies between 1 M and 100 mM (i.e., over five orders of magnitude in free concentration). A large collection of transporters is required for this homeostatic mechanism (3-6), yet little is known about the mechanism underlying regulation and integration of the transport activities in concert with the extracellular K ϩ levels. Some studies suggest that plants enhance their capability of K ϩ uptake by activating some K ϩ transporters in response to K ϩ -deficient conditions (7-10). A signaling process exists for the plants to ''monitor'' external K ϩ concentration and ''respond'' to the low-K condition by enhancing the capability for K ϩ acquisition. This signaling pathway represents a typical nutrient sensing and response process in plants about which our knowledge is extremely limited. One recent study indicates that low-K status in the soil triggers elevated production of H 2 O 2 that may serve as a signaling molecule to alter the expression of certain genes (11). Becaus...

Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of seed dormancy and adaptation to abiotic stresses. Previous work identified OsPYL/RCARs as functional ABA receptors regulating ABA-dependent gene expression in Oryza sativa. OsPYL/RCARs thus are considered to be good candidate genes for improvement of abiotic stress tolerance in crops. This work demonstrates that the cytosolic ABA receptor OsPYL/RCAR5 in O. sativa functions as a positive regulator of abiotic stress-responsive gene expression. The constitutive expression of OsPYL/RCAR5 in rice driven by the Zea mays ubiquitin promoter induced the expression of many stress-responsive genes even under normal growth conditions and resulted in improved drought and salt stress tolerance in rice. However, it slightly reduced plant height under paddy field conditions and severely reduced total seed yield. This suggests that, although exogenous expression of OsPYL/RCAR5 is able to improve abiotic stress tolerance in rice, fine regulation of its expression will be required to avoid deleterious effects on agricultural traits.

Potassium is one of the major macro-nutrients essential for a number of cellular processes in plants. Low potassium level in the soil represents a limiting factor for crop production. Recent studies have identified potassium transporters that are involved in potassium acquisition, and some of them are critical for potassium nutrition under low potassium conditions. However, little is understood on the molecular components involved in low potassium signaling and responses. We report here the identification of a calcineurin B-like protein-interacting protein kinase (CIPK9) as a critical regulator of low potassium response in Arabidopsis. The CIPK9 gene was responsive to abiotic stress conditions, and its transcript was inducible in both roots and shoots by potassium deprivation. Disruption of CIPK9 function rendered the mutant plants hypersensitive to low potassium media. Further analysis indicated that K + uptake and content were not affected in the mutant plants, implying CIPK9 in the regulation of potassium utilization or sensing processes.

BackgroundFK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.ResultsFKBP and CYP proteins in rice (Oryza sativa cv. Japonica) were identified and classified, and given the appropriate name for each IMM, considering the ortholog-relation with Arabidopsis and Chlamydomonas or molecular weight of the proteins. 29 FKBP and 27 CYP genes can putatively be identified in rice; among them, a number of genes can be putatively classified as orthologs of Arabidopsis IMMs. However, some genes were novel, did not match with those of Arabidopsis and Chlamydomonas, and several genes were paralogs by genetic duplication. Among 56 IMMs in rice, a significant number are regulated by salt and/or desiccation stress. In addition, their expression levels responding to the water-stress have been analyzed in different tissues, and some subcellular IMMs located by means of tagging with GFP protein.ConclusionLike other green photosynthetic organisms such as Arabidopsis (23 FKBPs and 29 CYPs) and Chlamydomonas (23 FKBs and 26 CYNs), rice has the highest number of IMM genes among organisms reported so far, suggesting that the numbers relate closely to photosynthesis. Classification of the putative FKBPs and CYPs in rice provides the information about their evolutional/functional significance when comparisons are drawn with the relatively well studied genera, Arabidopsis and Chlamydomonas. In addition, many of the genes upregulated by water stress offer the possibility of manipulating the stress responses in rice.

Potato (Solanum tuberosum) is relatively vulnerable to abiotic stress conditions such as drought, but the tolerance mechanisms for such stresses in potato are largely unknown. To identify stress-related factors in potato, we previously carried out a genetic screen of potato plants exposed to abiotic environmental stress conditions using reverse northern-blot analysis. A cDNA encoding a putative R1-type MYB-like transcription factor (StMYB1R-1) was identified as a putative stress-response gene. Here, the transcript levels of StMYB1R-1 were enhanced in response to several environmental stresses in addition to drought but were unaffected by biotic stresses. The results of intracellular targeting and quadruple 9-mer protein-binding microarray analysis indicated that StMYB1R-1 localizes to the nucleus and binds to the DNA sequence G / A GATAA. Overexpression of a StMYB1R-1 transgene in potato plants improved plant tolerance to drought stress while having no significant effects on other agricultural traits. Transgenic plants exhibited reduced rates of water loss and more rapid stomatal closing than wild-type plants under drought stress conditions. In addition, overexpression of StMYB1R-1 enhanced the expression of droughtregulated genes such as AtHB-7, RD28, ALDH22a1, and ERD1-like. Thus, the expression of StMYB1R-1 in potato enhanced drought tolerance via regulation of water loss. These results indicated that StMYB1R-1 functions as a transcription factor involved in the activation of drought-related genes.