With respect to K+ uptake, two CBL proteins, CBL1 and CBL9, function together with CIPK1/9/23 at the plasma membrane where they activate K+ channels and carriers such as AKT1 and HAK5 through phosphorylation. In parallel, CBL2 and CBL3 recruit four CIPKs, CIPK3/9/23/26, to the vacuolar membrane where the CBL-CIPK modules initiate K+ remobilization by activating transporters including two-pore K + -channels. These findings demonstrate that the PM-CBL1/9- CIPK23 and VM-CBL2/3-CIPK3/9/23/26 signaling modules serve as response mechanisms that link the low-K+ stress to the activation of the transport activities to maintain K+ homeostasis. Although the dual CBL-CIPK pathways regulating K+ channels and transporters have been well established, it remains unknown how these CBL-CIPK proteins are modified in response to changes in K+ status in the environment. In this study, we monitored the behaviors of CBL1/9, CBL2/3, CIPK9/23, AKT1, and TPK1 proteins and revealed K+ dose- and time-dependent post translational modifications of these proteins in response to changing K+ nutrient status, establishing previously unanticipated mechanisms for nutrient sensing in plants. The abundance and phosphorylation of these proteins reflect the activity of the dual CBL-CIPK pathways and coincide with the nutrient availability: when K+ levels are low, CBL-CIPK-K+ channel becomes more abundant and more active to boost K+ uptake from soil and vacuole release into the cytosol; when K+ levels are high, CBL-CIPK-K+ channel activity is not required and thus dephosphorylated and degraded.
Interestingly,cultivo del arandano azul although CBL1/9 and CBL2/3 are localized to distinct subcellular compartments, CBL2/3 activates prior to and contributes to the accumulation of CBL1/9 proteins, revealing a unique mechanism for crosstalk between PM and VM pathways. Together with our previously described genetic analysis, these biochemical data further support the conclusion that VM-CBL2/3-CIPK pathway functions as a primary mechanism for plants to respond and adapt to low-K+ stress.When facing K+ deficiency, plants activate the CBL1/9-CIPK23 pathway to boost K+ -uptake capacity by activating K+ channels and transporters in roots. To address the molecular mechanisms underlying CBLCIPK activation in response to low-K+ , we examined the behavior of the CBL proteins to explore the possible processes that couple the external K+ status to the function of the calcium sensors. Using a polyclonal antibody against a recombinant CBL1 protein, we monitored the content of native CBL1 and CBL9 proteins in Arabidopsis seedlings. Due to high homology and functional redundancy between CBL1 and CBL9, the CBL1 polyclonal antibody reacted with both CBL1 and CBL9 proteins , as demonstrated by the presence of a protein band at the correct molecular weight in the wild type, cbl1 or cbl9 single mutant, but not in the cbl1/9 double mutant. To determine whether CBL1/9 protein level is regulated in response to the change of external K+ levels, we performed western blot analysis using total protein extracted from Arabidopsis seedlings grown on the modified MS medium with different K+ levels . Our results suggested that, with a decreasing level of external K+ , the primary roots of wild type seedlings shortened, and CBL1/9 protein abundance increased . In particular, a large boost of CBL1/9 protein occurred from 1 mM to 0.1 mM, physiological K+ levels associated with natural soils.
To further determine the kinetic changes of CBL1/9 levels in response to external K+ levels, we transferred seedlings from high- to low-K+ condition, or vice versa. Interestingly, while the increase in CBL1/9 abundance by high- to low-K+ transfer was not detectable until 5 days upon K+ deprivation, the decrease of CBL1/9 proteins happened much faster and reached the lowest level on the third day . We reasoned that during the pre-culture under high K+ condition, seedlings accumulated excess K+ , which supplied plants with sufficient K+ in the first 7 days upon low-K+ treatment, leading to a delayed K+ -starvation response. In contrast, low- to high-K+ transfer immediately allowed starved seedlings to acquire sufficient K+ , making the CBL1/9 signaling proteins obsolete, and resulting in a more rapid degradation response. These results revealed that the abundance of CBL1/9 proteins is fine-tuned by extracellular K+ levels. We also measured CBL1 and CBL9 mRNA levels under highand low-K+ and found no significant differences , indicating that a post translational modification is responsible for the fluctuation in CBL1/9 protein abundance. In addition to PM-CBL1/9-CIPK23 pathway, VM-CBL2/3-CIPK3/9/ 23/26 represents another indispensable pathway for plants to cope with K+ deficiency. Unlike PM-CBL1/9-CIPK modules which are mainly responsible for boosting K+ uptake, VM-CBL2/3-CIPK pathway mediates K+ remobilization from vacuoles into cytoplasm by activating tonoplast TPKs channels. Despite the different subcellular locations and functional processes they participate, the PM- and VM-CBL-CIPK pathways are both activated upon K+ -deficiency. We thus investigated whether the vacuolar CBL2/3 protein levels were, like PM-CBLs, governed by external K+ levels as well. CBL2 and CBL3 showed high homology and CBL3 antibody we generated recognized both CBL2 and CBL3 in Arabidopsis . We examined CBL2/3 protein levels in seedlings grown under various concentrations of K+ and found that CBL2/3 protein abundance was enriched without altering transcription level under low-K+ stress . Previous work showed that CBL1 and CBL9 proteins are phosphorylated in vitro. To monitor if this is the case in plants as a response to K+ shortage, we grew wild-type seedlings on sufficient-K+ medium for 4 days and then transferred them to K+ -de®- cient conditions and took samples at five different time points .
We confirmed that this low-K+ treatment was effective by examining the expression level of HAK5, a marker gene in Arabidopsis response to K+ deficiency. As shown in Supplementary Fig. 3, HAK5 transcript level began to increase after 1d of low-K+ transfer and showed a striking increase on day 3 and peaked on day 7 . Using Phos-tag mobility shift assay, we found that the level of CBL1/9 phosphorylation was strongly induced on the 3rd day after low-K+ transfer, followed by a further increase on day 7 and remained high on day 9 . The pattern observed on the up-regulation of HAK5 expression and CBL1/9 phosphorylation suggested that low-K responsive events consistently happened at both the transcriptional level and post translational level. Furthermore, low- to high-K+ transfer triggered the dephosphorylation of CBL1/9 , suggesting that external K+ level modulates the phosphorylation status of CBL1/9. Phosphatase treatment confirmed that the mobility shift of CBL1/9 proteins under low-K+ treatment resulted from phosphorylation . We also generated transgenic plants harboring UBQ10: CBL1-3flag or UBQ10: CBL9-3flag construct in the cbl1/9 double mutant background. In both cases, protein and phosphorylation levels of CBL1-3flag and CBL9-3flag were dramatically elevated by low-K+ treatment .We further examined the phosphorylation status of CBL2/3 under low-K+ treatment using the phos-tag gel assay and found that, like PMCBL1/9, the vacuolar CBL2/3 were also highly phosphorylated under low- K+ treatment . Furthermore, high- to lowK+ transfer enhanced the phosphorylation whereas low- to high-K+ transfer triggered the dephosphorylation of CBL2/3 , suggesting that external K+ level controls CBL2/3 phosphorylation status. In parallel with the change of phosphorylation, CBL2/3 protein level was also elevated during high- to low-K+ transfer and decreased during low- to high-K+ transfer . As the tonoplast CBL2/3-CIPK3/9/23/26 module also functions in Mg2+ homeostasis under high-Mg2+ stress, we next examined whether CBL2/3 are phosphorylated in response to high Mg2+. Interestingly, high-Mg2+ stress failed to enhance CBL2/3 phosphorylation . As a further confirmation, we transferred the seedlings from low-K+ to high-K+ or high-K+ plus high-Mg2+. Ifhigh-Mg2+ stress also enhances CBL2/3 phosphorylation as low-K+ does, we would expect to see the inhibitory effect of high-Mg2+ on high-K+ -triggered CBL2/3 dephosphorylation. However,arándanos azules cultivo this was not the case as the degree of CBL2/3 dephosphorylation was similar between transfer to high-K+ or high-K+ plus high-Mg2+ . These results indicated that, although CBL2/3 function in both low-K+ and high-Mg2+ responses, their phosphorylation is regulated in a low-K+ stress-specific manner. CBLs and CIPKs form CBL-CIPK complexes to regulate their downstream target proteins. We found that CBL1/9 and CBL2/ 3 respond to low-K+ stress by increasing protein abundance . For increased levels of CBLs to have functional relevance, we speculate that their partner kinases, CIPK9 and CIPK23, may also respond to K+ deficiency in the similar manner. Under low-K+ treatment, CIPK9 mRNA level was elevated dramatically in the wild-type plants , which is consistent with the previous study. The CIPK23 mRNA level did not change significantly under low-K+ stress condition used in this study , which differs from the previous findings that CIPK23 mRNA level is induced by low-K+ treatment. Such discrepancy may result from variations in experimental conditions and developmental stages of plant materials. To exclude the contribution of transcriptional control, we generated transgenic plants harboring UBQ10: CIPK9-3flag or UBQ10: CIPK23- 3flag construct in the cipk9/23 double mutant background.
In both cases, CIPK9-3flag and CIPK23-3flag were functionally equivalent to the native proteins because they rescued the low-K+ -sensitive phenotype in cipk9/23 double mutant . In western blot analyses using flag-antibody, we found that protein levels of CIPK9-3flag and CIPK23-3flag were dramatically elevated by low-K+ treatment . Moreover, both CIPK9-3flag and CIPK23-3flag proteins in seedlings grown under low-K+ treatment became up-shifted in the phos-tag gel, and the mobility shift was removed by phosphatase treatment , confirming that low-K+ stress enhances the phosphorylation of CIPK9 and CIPK23 in plants. Previous studies demonstrated that CIPK9/23, like many other kinases, can auto-phosphorylate in vitro. We examined whether low-K+ -induced CIPK9/23 phosphorylation was auto-catalyzed in planta. To this end, we introduced the kinase-dead version of CIPK9 or CIPK23 fused with 3x flag tag into cipk9/23 double mutant. To ensure correct CIPK9K48N−3flag and CIPK23K60N−3flag transgenes were introduced, we sequenced the genomic DNA from several transgenic lines and confirmed that all UBQ10: CIPK9K48N−3flag plants contained the K48N mutation and all UBQ10: CIPK23K60N−3flag plants contained the K60N mutation . To test if the kinasedead versions of CIPKs were indeed inactive in vivo, we analyzed CBL1/9 phosphorylation in the transgenic lines expressing similar level of wild type CIPK or kinase-dead CIPK. Our results showed that low-K+ -induced CBL1/9 phosphorylation was dramatically reduced in transgenic seedlings expressing the kinase-dead version of CIPK 9 as compared with UBQ10: CIPK9-3flag control . Similar result was observed when comparing the seedlings expressing wild type CIPK23-3flag versus kinase-dead CIPK23K60N−3flag . These results suggested that the kinase activity was ablated in the CIPK9K48N or CIPK23K60N mutants in plants. We next investigated whether low-K+ -induced phosphorylation of CIPK9/23 results from auto-activity of the kinases by comparing seedlings expressing wild type or kinase-dead version of CIPK9 or CIPK23. Interestingly, the phosphorylation levels of CIPK9K48N−3flag or CIPK23K60N−3flag, despite their lack of kinase activity, were phosphorylated at a similar level as their wild type controls in response to low-K+ treatment . These results suggested that low-K+ -induced phosphorylation of CIPK9 and CIPK23 was catalyzed by other kinase in plants. Although previous studies show that PM-CBL-CIPK pathway phosphorylates and activates AKT1 channel in Xenopus oocyte, it remains unknown if AKT1 protein is phosphorylated in planta as a response to low-K+ stress. Further, our finding of low-K+ -induced increase in protein abundance of both CBLs and CIPKs prompted us to examine the abundance of their target channels such as AKT1. As done with CBLs and CIPKs, we constructed UBQ10: AKT1-3flag construct and introduced it into akt1 mutant. The tagged version of AKT1 was functional as shown by its capability in complementing akt1 mutant phenotype under low-K+ stress . As shown by western blot, AKT1-3flag protein accumulated at a much higher level when seedlings were grown under low-K+ conditions . Using phos-tag assay, we found that AKT1-3flag proteins also became up-shifted in the gel when plants were grown under low-K+ conditions and the mobility shift was removed by phosphatase , confirming that low-K+ stress enhances the in planta phosphorylation of AKT1. As TPKs serve as the target channels of VM-CBL2/3-CIPKs module to activate K+ efflux from vacuole, we examined whether TPKs channels were, like AKT1, also phosphorylated by low-K+ stress using TPK1 as an example. We introduced 3x flag epitope-tagged TPK1 into Col background and found that K+ starvation clearly enhanced TPK1 phosphorylation status . Interestingly, abundance of TPK1 was not affected by low-K+ treatment , suggesting that the abundance of TPK1 may not be limiting and phosphorylation holds the key for activation of the channel.Given that CBL1/9 proteins interact with and are phosphorylated by CIPK23 and CIPK9 in vitro, and CIPK9 and CIPK23 play essential roles in regulating K+ uptake and homeostasis, we examined whether these CIPKs are responsible for low-K+ -enhanced CBL1/9 phosphorylation in vivo.