In the case of K+ sensing, we have shown here that not only the transporter proteins but also the components in the low-K+ response signaling pathways respond to K+ status by altering their protein abundance . In addition, the regulation of protein levels occurs at the post-translational level for all components, calling for future effort to identify the enzymes and regulators that connect nutrient availability to protein stability. In fact, our effort here indicates that one mechanism for protein stability control, at least for CBL calcium sensors, involves phosphorylation by their partner kinases . Although previous studies showed that the CBLs can be phosphorylated in vitro by their partner CIPKs, functional relevance of such phosphorylation is proposed to enhance interaction between CBLs and CIPKs in yeast and protoplast transient expression system or to play a role in activating AKT1 in Xenopus Oocytes. In this study, we identified specific CIPKs responsible for CBL phosphorylation in planta and provided a link between CBL phosphorylation and control of the protein stability in response to changing K+ status. Because the abundance of downstream kinases and transporters are also tightly associated with their phosphorylation status, we propose that the “CBL-CIPK-transporter” pathway represents a phosphorylation-dependent protein stabilization and activation cascade . Investigating the mechanism underlying regulated protein stability in response to K status and how different CIPK members may differentially regulate different CBLs will be a major direction to focus on in the future. For further understanding of phosphorylation-dependent protein stability control, we identified the kinases and phosphatases that contribute to the reversible phosphorylation of the CBL-type Ca2+sensors. It is particularly interesting to find that the CBLphosphorylating kinases are activated by the low-K+ stress and, in contrast, the PP2C phosphatases function in response to low- to high-K+ switch. Furthermore, it is significant to reveal that the CIPKs responsible for low-K+ response phosphorylated both the PM-CBLs and VM-CBLs ,macetero de 7 litros whereas the HAB1/ABI1/ABI2/PP2CA phosphatases involved in high-K+ response specifically act on the VM-CBLs . Such findings clearly indicate that highK+ -induced dephosphorylation are pathway-specific and does not simply represent a reverse mode of low-K+ -induced phosphorylation.

Taken together with previous finding that the high K+ -responsive PP2Cs have been shown to be critical regulators of ABA responses, we expect that K+ -nutrient sensing may crosstalk to ABA signaling through these and possibly other components. The finding that the addition of ABA to the high-K+ medium blocked the VM-CBL2/3 dephosphorylation and ABA deficient mutant aba2-1 showed a similar hypersensitive phenotype to cbl2/3 and cipk9/ 23 mutants supported this notion . Considering that these PP2C members can physically interact with both CIPKs and CBLs, and that HAB1/ABI1/ABI2/PP2CA repress the auto-phosphorylation of CIPK9 and CBL2/3 transphosphorylation , we proposed that HAB1/ABI1/ABI2/PP2CA phosphatases may control the phosphorylation levels of CBL2/3 by at least three possible mechanisms: by interacting and dephosphorylating CBL2/3 directly, by dephosphorylating and inhibiting CIPK9/23 kinase activity, and/or by sequestering CIPK9/23 proteins from binding to CBL2/3. Future work is thus expected to resolve these possibilities and to identify other early events in sensing K+ status in plants. Another significant finding in this work is the relationship between the dual CBL-CIPK pathways in response to low-K+ stress. Our recent study indicated that VM-CBL2/3-CIPK pathway is more critical than the PM-CBL1/9-CIPK pathway because cbl2cbl3 double mutant showed severe growth inhibition in a broad range of external K+ regimes whereas the cbl1cbl9 double mutant displayed much less defect under the same conditions. This is consistent with our results in this study that the activity of VM-CBL2/3-CIPK pathway is more sensitive to K+ deficiency and is activated earlier than PM-CBL1/9-CIPK pathway during high- to low-K+ transfer . These results further support the notion that VM-CBL2/3-CIPK pathway for K+ remobilization may serve as a primary mechanism for plants to respond andadapt to K+ -deficiency. Along this line, we also found that, although CBL2/3 and CBL1/9 are spatially separated in the cell, VM-CBL2/3 are essential for the stabilization of CBL1/9 proteins in response to low-K+ stress . Concerning the molecular link that enables such coordination between VM-CBL2/3-CIPK and PM-CBL1/9-CIPK pathways, we hypothesize that the partner kinases shared by CBL2/3 and CBL1/9, i.e., CIPK9/23, may serve as the “bridge” of the dual pathways. When plants face low-K+ stress, early signals, such as Ca2+ spikes, may first activate CBL2/3 that preferentially recruit CIPK9/23 kinases to the vacuolar membrane to phosphorylate and stabilize CBL2/3. Stable CBL-CIPK complexes phosphorylate and activate their target transporters such as TPKs to retrieve K+ from the vacuolar store. With prolonged K+ deficiency, the PM-CBL1/9 recruit the hyperactive CIPKs that may shuttle between VM and PM to form CBL-CIPK complexes at the plasma membrane where CIPKs phosphorylate CBL1/9 and K+ transporters, e.g., AKT1, boosting K+ uptake from K+ -limited environments.

Future work should be directed to monitoring the time course of low-K+ generated Ca2+ signature and its correlation with the sequential activation of VM-CBL-CIPK and PM-CBL-CIPK modules, as well as dissecting the possible “CIPK shuttling” mechanism between the VM and PM. Additionally, it would be interesting to explore the molecular toolkit for the production of Ca2+ signals that activate CBL2/ 3 and CBL1/9 in response to low-K+ stress.All seeds were surface sterilized with 10% bleach for 10 min, washed three times with water and sown on the growth medium solidified with 0.8% BD BBLTM select agar. The recipe of the growth medium was modified from MS medium with a reduced level of NH4 + unless indicated otherwise, which contained the following components: 3 mM Ca 2, 1.25 mM NH4H2PO4, 1.5 mM MgSO4, 1× Murashige and Skoog micronutrients , and 1% sucrose. The pH of the medium was adjusted to 5.8 using NaOH. The final K+ concentration in the medium was adjusted by adding KCl as the K+ source. For the germination phenotyping assay, seedings were germinated on modified MS medium shown above with different concentrations of K+ and incubated at 4 °C for 4 d for stratification, then were transferred to a growth chamber with 80 μmol m−2 s−1 light intensity with a 12 h light/ 12 h dark photoperiod for the indicated days. For the post-germination phenotyping assay, seeds were germinated on modified MS medium containing 20 mM K+ and grown for 4 days. The seedlings were then transferred onto various agarose-solidified modified MS medium 2, 1.25 mM NH4H2PO4, 1.5 mM MgSO4, 1× Murashige, and Skoog micro-nutrients, and 1% sucrose, pH5.8 supplemented with different concentrations of K+ for subsequent growth under 80 μmol m−2 s−1 light intensity with a 12 h light/12 h dark photoperiod. At the end of assay, the root length of seedlings was measured by Image J software. For phenotypic assay in the hydroponics, seeds were germinated on MS medium and grown for 7 days. The seedlings were then transferred to the liquid solution containing 1.4 mM Ca2, 0.1 mM Ca2, 0.125 mM MgSO4, 0.025 mM MgCl2, as well as 1/6 strength of MS minor salts and supplemented with different concentrations of KCl. All the hydroponic solutions for plant growth were replaced with fresh ones twice a week.Total RNA was extracted from plant samples using the TRIZOL reagent . After being treated with DNase I to remove DNA contamination, cDNA was synthesized using SuperScript II reverse transcriptase kit . The quantitative real-time PCR analysis was performed on the DNA Engine Opticon System using the SYBR Green Realtime PCR Master Mix .

All experiments were performed using three biological replicates,macetas 30 litros and actin served as an internal standard. The relative expression of each gene was calculated using ΔΔCT method. Each experiment was repeated with three different batches of samples and RT-PCR reactions were performed with three technical replicates for each sample. The primers used in quantitative real-time PCR are listed in Supplementary Table 1.For total protein extraction, Arabidopsis seedlings were grounded in the presence of liquid nitrogen to a fine powder and extracted with 2× SDS sample buffer . Aliquots of denatured total protein were separated by 12% SDS-PAGE and transferred to PVDF membrane. For the detection of phosphorylated CBL proteins, the total protein was separated by 10% SDS-PAGE with 15 μM Phos-tag and transferred to PVDF membrane. For immunoblot analyses, anti-CBL1, anti-CBL3, anti-β-tubulin , anti-GAPDH , anti-actin , anti-Flag were used as primary antibodies. The anti-CBL1 rabbit polyclonal antibody was made using recombinant CBL1 protein purified from E coli as antigen by Cocalico company . Each experiment was repeated at least three times, and one representative result was shown. Quantification of immunoblots was done using Image J software.Arabidopsis seedlings were grounded in the presence of liquid nitrogen to fine powder and extracted with buffer containing 50 mM HEPES , 150 mM NaCl, 50 mM β-glycerophosphate, 2 mM DTT, 1% Triton X-100 and 10% glycerol, with EDTA-free protease inhibitors . After centrifugation for 10 min at 20,000 g, the supernatant was isolated and used as protein samples for Phostag gel analysis. For dephosphorylation of CBL1/9 and CBL2/3, 50 μL supernatant was incubated with 1 μL λ-PPase and 5 μL 10 mM MnCl2 under 30 °C for the indicated times. For dephosphorylation of CIPK9-3Flag, CIPK23-3Flag, AKT1-3Flag proteins, the supernatant was incubated with 10 μL prewashed anti-Flag M2 agarose beads for 1 h at 4 °C on a roller shaker. The beads were then washed three times with the extraction buffer described above. The protein bound beads were incubated with 1 μL λ-PPase and 5 μL 10 mM MnCl2 under 30 °C for 30 min. The dephosphorylation reaction was stopped by adding 2× SDS loading buffer and boiled for 10 min.CIPK9, CBL2 and CBL3 were cloned in pGEX4T-1 vector and expressed in E.coli as a GST-tag fusion protein, ABI1, ABI2, PP2CA and HAB1 were cloned in pMAL-c2X vector as a MBP -tag fusion protein. All MBP- and GST-fused proteins were purified according to standard instructions. For in vitro phosphorylation, 0.5–2.0 mg of purified proteins was incubated in kinase reaction buffer containing 20 mM Tris , 2.5 mM MnCl2, 0.5 mM CaCl2, 1 mM DTT, 10 mM ATP and 2 μCi 32γP at 30 °C for 30 min and terminated by 5× SDS–PAGE loading buffer. The samples were subsequently analyzed using a 12% SDS-PAGE gel, followed by Coomassie staining and autoradiography. Coomassie staining was used to verify the quality of samples and loading consistency.To measure the K content, plant roots and shoots were harvested separately at the end of each phenotypic assay and surface-washed with double-distilled water for 30 s. The samples were then thoroughly dried in the oven at 99 °C. The dry tissues were grounded in a mortar, collected into a 15 ml tube, and dissolved with 1 ml ultrapure HNO3 . The tubes were incubated in a water bath at 99 °C for 4 h. Digested samples were diluted with double-distilled water and the K concentration in the solution were determined by inductively coupled plasma optical emission spectroscopy .In flowering plants, a primary role for boron is to form a diester cross-link between two monomers of rhamnogalacturonan-II , a pectic polysaccharide present in the cell walls of all vascular plants . Rhamnogalacturonan-II is a structurally complex domain of pectin , which comprises 12 different monosaccharides that are linked together by at least 20 different glycosidic linkages . Nevertheless, its structure is largely conserved in vascular plants . The majority of RG-II exists in the wall as a dimer that is generated by forming a borate diester between the D-apiose of side chain A of two RG-II molecules. The inability of RG-II to properly assemble into a dimer results in the formation of cell walls with abnormal biochemical and biomechanical properties and has a severe impact on plant productivity.Nevertheless, the mechanisms that drive the interactions between borate and RG-II are poorly understood . There is increasing evidence that alteration of RG-II structure and cross-linking have severe impacts on plant growth, development and viability. To date, the only characterized RG-II biosynthetic enzymes are the rhamnogalacturonan xylosyl transferases , which catalyze the transfer of xylose from UDP-xylose to fucose to form ɑ-xylose–fucose in vitro . Inactivation of RGXT1 and -2 has no discernible effect on plant growth or RG-II structure , implying redundancy of function, whereas mutations affecting RGXT4 lead to defects of root and pollen tube growth that are lethal . Mutations that prevent the synthesis of UDP-Api and CMP-Kdo are also lethal and provide further evidence for the essential role of RG-II in plant growth .