We first monitored the phosphorylation status of CBL1/9 in wild type, cipk23, and cipk9 single mutants but found no significant difference among seedlings of different genotypes . In cipk9/23 double mutant, however, low-K + -induced CBL1/9 phosphorylation was dramatically reduced , suggesting that CIPK23 and CIPK9 function redundantly in phosphorylating CBL1/9 proteins under low-K+ stress. Interestingly, CIPK9/23 appeared to contribute to CBL1/9 accumulation because CBL1/9 protein abundance was much lower in cipk9/ 23 double mutant than in the wild type under low-K+ condition . Furthermore, the kinase activity of CIPK9/23 appeared to be required for CBL1/9 accumulation under low-K+ stress, since low-K+ -triggered CBL1/9 protein accumulation was clearly reduced in lines expressing the kinase-dead version of CIPKs as compared to lines expressing wild-type versions . Considering that CBL2/3 are phosphorylated by CIPK9 in vitro and CIPK9/23 were required for CBL1/9 phosphorylation in plant response to low-K+ stress , we speculated that low-K+ -triggered CBL2/3 phosphorylation may also depend on CIPK9/23. Indeed, cipk9/ 23 double mutants showed a drastic reduction in CBL2/3 phosphorylation status as compared to wild-type seedlings under low-K+ conditions. By comparing the intensity of CBL2/3 phosphorylation in cipk9 and cipk23 single mutants, cipk9/23 double mutant, and wild type, we found that CIPK9 played a more dominant role over CIPK23 in phosphorylating CBL2/3 . This result is in line with the previous findings that CIPK9 is more important than CIPK23 in vacuolar K+ -remobilization and that CIPK9 preferentially phosphorylates CBL2/3 over CBL1/9 in vitro.

Additionally,maceta de 30 litros low-K+ -induced increase in CBL2/3 protein abundance showed a clear reduction in cipk9/23 double mutants as compared to the wild type , suggesting that CIPK9/23 not only mediates the phosphorylation but also facilitate the accumulation of CBL2/3 proteins under K+ -deficiency condition. Our data so far tightly linked CBLs phosphorylation to their stability: low-K+ -induced phosphorylation parallels with higher protein abundance and high-K+ -induced dephosphorylation correlates with protein degradation . Moreover, CIPKs kinase activity appeared to be indispensable for low-K+ -induced CBLs accumulation . To further confirm that phosphorylation contributes to the abundance of CBLs under low-K+ stress, we identified and mutated the phosphorylation sites in CBLs. Previous in vitro studies identified a conserved Ser residue within the C terminus of CBL1, CBL9, and CBL2 as the only site phosphorylated by their interacting CIPKs. To investigate whether the same site is phosphorylated in plants upon low-K+ stress, we used CBL1 as an example and introduced 3x flag epitope-tagged CBL1WT, CBL1S201A into cbl1/9 double mutant. Figure 2d showed that CBL1S201A mutation abolished low-K+ -mediated phosphorylation, confirming that Ser201 is the only phosphorylation site in CBL1 in Arabidopsis seedlings under low-K+ treatment. We next explored the relationship between CBL1 phosphorylation and protein abundance by analyzing the effect of S201A mutation on the CBL1 protein accumulation in response to K+ deprivation . Upon high- to low-K+ transfer, accumulation of CBL1S201A−3flag was reduced by more than half as compared to CBL1WT −3flag, confirming that the CBL1 phosphorylation contributes to its protein accumulation upon low-K+ stress.

Despite lack of phosphorylation, abundance of CBL1S201A−3flag was induced by low-K+ albeit to a lesser extent , implicating other mechanism, in addition to phosphorylation, in controlling CBL1 stability. We investigated whether phosphorylation of CBL1 at Ser-201 impacts its function in plants under low-K+ stress. Consistent with the previous study, cbl1/9 double mutant plants were stunted with chlorotic leaves when grown on low-K+ medium containing 20 mM NH4 + . Expression of CBL1WT−3flag, but not CBL1S201A−3flag, fully rescued the growth of cbl1/ 9 double mutant . This result indicated that S201 site phosphorylation is important for the function of CBL1 in plants under low-K+ stress.Previous studies show that, at the early stage of plant responses to K+ shortage, vacuolar K+ concentration drops dramatically to maintain steady cytoplasmic K+ levels. In addition, the vacuolar CBL-CIPK pathway is more important in response to short-term K+ -deficiency. We, therefore, hypothesized that, when facing low-K+ availability, activation of vacuolar K+ remobilization by VM-CBL2/3-CIPK pathway may serve as a primary mechanism to supplement cytoplasmic K+. As a result, VM-CBL-CIPK pathway may be activated first in response to low-K+ stress. To test this hypothesis, we grew wild-type seedlings on the high-K+ medium for 6 days and transferred them to low-K+ medium to monitor the time course of phosphorylation of CBL1/9 and CBL2/3 in parallel. As shown in Fig. 3a, b, the phosphorylation level of CBL2/3 proteins was elevated by 4.5-fold on the third day upon K+ starvation treatment, whereas CBL1/9 phosphorylation level did not show a significant increase until the fifth day, supporting the notion that VMCBL2/3 activation happens before PM-CBL1/9 and vacuolar K+ remobilization is the primary response to low-K+ stress.To further explore the relationship between the two CBL-CIPK pathways, we examined whether disruption of one pathway had positive or negative impact on the activation of the other pathway in response to low-K+ stress.

We found that low-K+ -induced increase in CBL2/3 protein abundance and phosphorylation remained the same in the wild type and cbl1/9 double mutant , indicating that PM-CBL1/9 proteins are not essential for the activation of VM-CBL2/3-CIPK pathway. In contrast, protein abundance of CBL1/9 was severely reduced in cbl2/3 double mutant as compared to the wild type , suggesting that CBL2/3 are indispensable for the upregulation of CBL1/9 protein accumulation under low K conditions. To further confirm this observation, we crossed UBQ10:CBL1-3flag transgenic line into the cbl2/3 background and compared the protein level of CBL1-3flag in cbl2/3 and wild-type backgrounds. Like the native CBL1, CBL1-3flag protein level remained very low in the cbl2/3 during a 9-day low-K+ treatment . Because a constitutive promoter was utilized to drive transcription of CBL1-3flag protein, changes in low-K+ -induced CBL1/9 protein accumulation, as regulated by CBL2/3, occurred at a post-translational level. Taken together, these results support the conclusion that VM-CBL2/3-CIPK pathway is the first responder to low-K+ stress and, further, the vacuolar pathway positively impacts the activation of the PM-CBL1/9-CIPK pathway.If the CBLs are phosphorylated by CIPKs and become more stable in response to low-K+ stress, they may be dephosphorylated and destabilized upon K+ replenishment. The such reversible regulatory mechanism may hold the key to enabling plant adaptation to the changes in K+ nutrient status. To address this mechanism further, we sought to identify the phosphatases required for the dephosphorylation of CBLs in response to high-K+ . We previously showed that multiple PP2C Group A members interact with CIPKs and CBLs in yeast. We hypothesized that CBLs, CIPKs, and PP2Cs may form alternate complexes in regulating the kinase activity of CIPKs and the phosphorylation levels of CBLs. To test this hypothesis, we performed in vitro kinase assay and tested the effect of four group A PP2Cs, HAB1/ABI1/ABI2/PP2CA, on the phosphorylation of CIPK9 and/or CBL2/3. As shown in Fig. 4a, b, the recombinant CIPK9, but not the dead kinase CIPK9K48N, showed autophosphorylation activity and phosphorylated CBL2 and CBL3. The kinase activity of CIPK9 was dramatically enhanced by CBL2 or CBL3, consistent with a previous study. When ABI1, ABI2, HAB1, or PP2CA was added to the reaction, bothautophosphorylation and CBL2/3 transphosphorylation were effectively abolished . When the phosphatase activity was disrupted by a mutation in these PP2Cs, however, they became ineffective in blocking the CIPK and CBL phosphorylation. For example, D177A mutation in ABI1 or D142A mutation in PP2CA,maceta plastico cuadrada which abrogates Mn2+ binding and impairs phosphatase activity, eliminated the inhibitory effect of ABI1 and PP2CA against CIPK9 and CBL2/3 phosphorylation in our assays . Interestingly, CIPK9 phosphorylated MBP-ABI2 and MBP-HAB1 proteins but not MBP-ABI1 and MBP-PP2CA, implying that CIPK9 may utilize ABI2 and HAB1 as substrates thereby regulating their phosphatase activities. To further investigate whether ABI1, ABI2, PP2CA and HAB1 dephosphorylate CBL2/3 in planta, we monitored CBL2/3 dephosphorylation in two triple mutants impaired in the above four phosphatases, hab1abi1pp2ca and hab1abi1abi2. In wild type seedlings, CBL2/3 was substantially dephosphorylated 3 days after transfer from low- to high-K+ . In striking contrast, CBL2/3 remained phosphorylated in hab1abi1pp2ca and hab1abi1abi2 triple mutants through 4–5 days after K+ -replenishment. Surprisingly, the PMlocalized CBL1/9 proteins underwent dephosphorylation to the same extent in the triple mutants as in the wild type . These observations identified HAB1/ABI1/ABI2/PP2CA as essential components required for the dephosphorylation of CBL2/3, but not for CBL1/9, in response to sufficient-K+ levels despite the fact that same CIPKs are responsible for phosphorylating both CBL1/9 and CBL2/3 .

Consistent with the phosphorylation status during low- to high-K+ transfer, CBL2/3 protein abundance was significantly reduced in the wild type but not in hab1abi1pp2ca and hab1abi1abi2 triple mutants , suggesting that these phosphatases are also involved in the regulation of degradation of CBL2/3 under high-K+ condition. Given that all of these four PP2C group A members act as key negative regulators of ABA responses, and that the biosynthesis of ABA in both leaves and roots showed an increase after K+ starvation, we hypothesized that high-K+ activates HAB1/ABI1/ABI2/ PP2CA phosphatases by down-regulating ABA level, leading to CBL2/3 dephosphorylation and degradation. This possibility was supported by the observation that high-K+ -induced dephosphorylation and degradation of CBL2/3 was substantially reduced by the addition of ABA to the medium .The HAB1/ABI1/ABI2/PP2CA phosphatases countered CBL2/3-CIPK9 phosphorylation , which may negatively regulate CBL-CIPK activity in response to low-K+ stress. We examined hab1abi1pp2ca and hab1abi1abi2 mutants under low-K+ stress and found that they displayed an opposite phenotype to the cbl2/3 double mutant. At 20 mM K+ concentration , there was no discernible difference in the seedlings of the mutants. Under a low-K+ condition , cbl2/3 double mutant showed strong growth retardation as exemplified by shorter roots during early seedling development , consistent with the finding in our previous study. In contrast, both hab1abi1pp2ca and hab1abi1abi2 triple mutants were significantly more tolerant to low-K+ condition, showing longer primary roots than both cbl2 cbl3 double mutant and the wild type . To further address the role of HAB1/ABI1/ABI2/PP2CA in low-K+ stress during an extended growth period, we cultured the seedlings hydroponically and examined their phenotypes for 4 weeks. As shown in Figure 5e, f, hab1abi1pp2ca and hab1abi1abi2 triple mutants as well as cbl2/3 double mutant did not show any significant difference from the wild-type plants under high-K+ condition. When grown in the low-K + solution , cbl2/3 mutant seedlings were severely stunted with leaf necrosis as previously reported. In contrast, hab1abi1pp2ca, hab1abi1abi2 mutants grew significantly better than wild-type plants , suggesting that HAB1/ABI1/ABI2/PP2CA phosphatases play a negative role in plant growth under low-K+ stress. To further assess the function of HAB1/ABI1/ABI2/PP2CA phosphatases in plant response to low-K+ stress, we evaluated the growth of the dominant gain-of-function mutant abi1-1c in which the mutated ABI1 becomes constitutively active. Our results showed that abi1-1c seedlings were hypersensitive to low-K+ condition as indicated by shorter roots, although the phenotype was not as strong as in the cipk9/23 double mutant . In the hydroponic solutions containing low-K+ , the fresh weight of abi1-1c plants was reduced to 60% of that of the wild type . In addition, ABA-deficient mutant aba2-1 was also more stunted than wild-type plants under low-K+ conditions . Moreover, abi1-1c and aba2-1 plants grown under low-K+ showed a significantly higher K content than the wild type, in line with the elevated K content in the cipk9/23 double mutant . Taken together, these data support the conclusion that HAB1/ABI1/ABI2/PP2CA phosphatases play a negative role in plant response and adaptation to low-K+ stress.To respond and adapt to low-K+ environments, plants have evolved an intricate mechanism involving dual CBL-CIPK calcium signaling pathways. The PM-CBL1/9-CIPK pathway activates the AKT1 channel and other transporters in the roots to boost K+ uptake, and the VM-CBL2/3- CIPK pathway enhances vacuolar K+ -remobilization to support cytoplasmic metabolism. Although the CBL-CIPK-K+ transport nexus has been well established, the early events that link K+ status to activation of CBL-CIPK proteins remains unclear. In this study, we identified post translational modifications of PM-CBL1/9-CIPKs-AKT1 and VM-CBL2/3- CIPK9/23-TPK1 proteins in response to changing external K+ levels, adding unique insights to the molecular processes responsible for nutrient sensing in plants. In the context of mineral nutrient transport in plants, studies have shown that abundance of transporter proteins can respond to substrate concentration. For example, phosphate uptake transporters of the PHT1 family are induced by low-P at the transcriptional level and degraded in response to P repletion.