In other words, CIPK24 probably functions as a major component downstream of the CBL4-mediated pathway, alternative or additional kinases other than CIPK24 are likely to be involved in the CBL10-mediated pathway. Among all the single and double mutants, sos1 was the most severely affected with similar phenotype to all the double mutants combining cbl10 and any SOS pathway mutant , suggesting that SOS1 may serve as a predominant determinant in plant salt tolerance or as a converging point for the CBL4 and CBL10-mediated pathways.Maintenance of the K+/Na+ homeostasis within plant cells is critical for plant salt tolerance. The SOS pathway components are believed to maintain a low Na+ content and normal K+ content in the cytosol by exporting excessive Na+ back to the external space [4,31]. In contrast, CBL10 is hypothesized to sequester Na+into the vacuole [16]. Since the whole-plant phenotype analysis did not provide a clear-cut answer regarding the relationship of the CBL10 and SOS pathway , we resorted to an analysis on the Na+/K+ homeostasis in the mutant plants to further delineate the relationship of the CBL10- and CBL4-directed pathways. Plants of various genotypes were grown ina normal hydroponic medium for three weeks and thentransferred to the same medium or a medium containing 5 mM or 50 mM NaCl and cultured for five more days.When grown in a medium without NaCl, no significant difference in the Na+ and K+ contents was found between the wild type and all the mutants in either roots or shoots. When grown in a medium containing 5 mM NaCl condition,hydroponic bucket the wild type and cbl10 plants had similar Na+ and K+ contents in roots and shoots as those plants grown under control conditions without the NaCl supplement.

In contrast, the SOS pathway mutants and all double mutants showed higher Na+ and lower K+ content in roots with no significant change in shoots. When grown under high salt conditions , all plants of different genotypes displayed significantly increased Na+ content and decreased K+ content in both roots and shoots, as compared to those grown under control conditions. The sos mutants accumulated much more Na+ and less K+ than the wild type in both roots and shoots , consistent with results in earlier studies. In contrast to the sos mutants, cbl10 contained less Na+ and more K+ in the roots than the wild type and approximately equal Na+ and K+ contents to the wild type in shoots, similar to results shown in a previous report. To our surprise, in 50 mM NaCl condition, all three cbl10-associated double mutants, including sos1 cbl10, cipk24 cbl10 and cbl4 cbl10, accumulated significantly less Na+ than the sos single mutants in the shoots albeit the Na+ content was still higher than that of the wild-type plants . These results strongly suggested that CBL10 should regulate a transport process that is independent from the SOS1-mediated Na+ efflux. Otherwise, the salt content in the mutants should follow the same pattern when either the CBL10 pathway or SOS pathway is disrupted. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 14 the SOS1-mediated Na+ efflux. Otherwise, the Tissue-specific expression pattern and subcellular locations of specific downstream partners including CIPKs underlies specificity for CBLs to mediate the stress response. We revisited the subcellular localization of CBL4 and CBL10 in different assay systems. In Arabidopsis mesophyll protoplasts, the empty green fluorescent protein alone was ubiquitously distributed in the cell . CBL4 is uniquely localized to the plasma membrane, while CBL10 is preferentially localized to the intracellular membranes including tonoplast . In the epidermal cells of N. benthamiana, CBL10 is also predominantly localized to the vacuolar membrane . Furthermore, when vacuoles were released from isolated mesophyll protoplasts of the transgenic plant, they showed a clear Venus, an enhanced yellow fluorescent protein, signal at the tonoplast . On the other hand, the CBL10-Venus signal largely overlapped with the two-pore K+ channel 1 -mCherry fusion protein , a tonoplast marker.

In addition, we generated transgenic Arabidopsis lines that constitutively expressed the CBL10-Venus fusion protein in the cbl10 mutant background. Importantly, the expression of CBL10-Venus complemented the salt-sensitive phenotype of cbl10, confirming the proper function of the fusion protein . As observed in transiently transformed N. benthamiana cells, the Venus fluorescence in these plants was found to be intracellularly targeted . Additionally, previous work in poplars suggested that the targeting of CBL10 to the tonoplast is required for salt stress adaptation. Based on these results, we speculate that CBL10 serves as a calcium sensor protein probably in the vacuolar membrane. However, we cannot exclude the possibility that CBL10 may also be associated with other types of membrane in some specific cell types or under certain physiological conditions. Future studies should be directed to alternative approaches, such as the transmission electron microscopy, to conclusively determine the subcellular localization of CBL10 with higher resolution in plant cells. High concentrations of Na+ in the cytoplasm disrupt the ionic balance and the uptake of essential mineral nutrients, such as K+, which in turn causes adverse effects on many metabolic pathways. To cope with salt stress, plants have evolved various tolerance mechanisms including two transport processes at the single cell level. Either exporting Na+ out of the cell, or compartmentalizing excessive Na+ into the vacuol. These two transport mechanisms act in a coordinated manner to maintain a low Na+ concentration in the cytoplasm. However, it remains unknown if they are regulated by the same or different signaling pathways. The SOS pathway is generally viewed as a signaling mechanism for the activation of the Na+ efflux through SOS1, a NHX-type Na+/H+ exchanger in the plasma membrane. The loss of function of SOS genes thus results in hypersensitivity to NaCl, coupled with the Na+ over-accumulation in the cytoplasm. On the other hand, some Na+/H+ exchangers are localized in the tonoplast and may be involved in transporting Na+ from the cytoplasm to the vacuole. However, the exact role of different NHX isoforms responsible for salt tolerance remains unclear. Interestingly, the two distinct but inter-connected salt transport processes appear to be both regulated by calcium signaling, in which calcineurin B-like proteins are thought to be the primary calcium sensors during salt stress adaptation. Among them, CBL4 and CBL10 display distinct tissue expression patterns and subcellular localizations.

The spatial specificity of these two calcium sensors may contribute to their functional diversification in salt stress adaptation. In order to understand how they work synergistically in the regulation of salt tolerance, we genetically analyzed the salt-sensitive phenotype of the cbl4 cbl10 double mutant in comparison with the single mutants. The cbl4 cbl10 double mutant was dramatically more sensitive to salt as compared to the cbl10 and cbl4 single mutants,stackable planters suggesting that CBL4 and CBL10 either functionally overlap or each directs an independent salt-tolerance pathway. If the two CBLs are functionally overlapping, they should regulate the same transport processes and then the double mutant should not only show more severe phenotype but also show more severe deviation in the Na+ and K+ contents as compared to the wild-type plants. However, that was not the case: cbl4 and cbl10 displayed generally opposite Na+ and K+ profiles. Although the cbl4 cbl10 double mutant plants showed Na+ over-accumulation compared to the wild type, but significantly lower Na+ content than the cbl4 single mutant . This suggests that CBL10 should not be involved in the CBL4-regulated Na+ extrusion process , although these two calcium sensors interact with a common downstream kinase CIPK24 . Instead, CBL10 should regulate a distinct Na+-transport process in response to high salt, probably the Na+ sequestration into the vacuole, as suggested by its tonoplast localization and the lower Na+ content in the cbl10 mutants. This is consistent with the general theme that the Na+ efflux or Na+ sequestration into the vacuole both contribute to salt tolerance and disrupting either may result in elevation of the Na level in the cytoplasm and thus leading to salt sensitivity. Certainly disrupting both transport processes would lead to more severe salt sensitivity, which match the more sensitive phenotype of cbl4 cbl10. Previous studies suggested that CIPK24 serves as the common downstream target of CBL4 and CBL10 by forming CBL4-CIPK24 or CBL10-CIPK24 complex at the plasma or vacuolar membrane separately.Although our findings in this study supported this hypothesis, they also suggested that other CIPKs, in addition to CIPK24, should be also involved in the CBL10-mediated pathway based on the genetic evidence that double mutants of cbl4 cbl10 and cipk24 cbl10 displayed a significant enhancement in Na+ sensitivity as compared to cipk24 . Indeed, screened by the yeast two-hybrid assay, we found that CBL10 did interact with other CIPKs in addition to CIPK24 . Various combinations of CBL10 with different CIPKs may target different target proteins and exhibit diverse functions. To examine whether SOS1 is a downstream component of CBL10 in the pathway, we also compared the salt sensitivity between sos1 cbl10 and sos1. In our test conditions, the salt sensitivity of cbl4 cbl10 and sos1 cbl10 was comparable to sos1 , suggesting that SOS1 may serve as a converging point for the two CBL pathways. However, the double mutants cbl4 cbl10 and sos1 cbl10 accumulated much lower Na+ content than the single mutants of cbl4 and sos1, respectively, under salt conditions , which implies that CBL10 and SOS1 functions in two different transport processes in regulating Na+ homeostasis.

For instance, in the sos single mutants in which the Na+ efflux is blocked, the CBL10 pathway functions to transport Na+ into the vacuole leading to the over-accumulation of Na+ in plant tissues. When the vacuole sequestration is defective in the cbl10-associated double mutants, the Na+ uptake is inhibited as a feedback of lacking storage space, leading to less accumulation and thus lower Na+ content in these double mutants as compared to the sos single mutants . Despite overall lower Na+ content in plant tissues, the double mutants showed similar salt sensitivity as sos1 because the majority of Na+ in these double mutants is in the cytoplasm effectively causing toxicity. Our results thus provide an example where a two-tier evaluation system must be implemented for dissecting salt tolerance mechanism in plants: First by whole-plant phenotyping and further by the analysis of Na+/K+ homeostasis . Concerning the target transporters for CBL10, all evidence so far supports the hypothesis that the CBL10-CIPK pathway may regulate Na-transporters in the tonoplast. Sequestration of Na+ into the vacuole is presumably fulfilled by an array of Na + transporters that include the vacuole-localized NHX-type Na+ /H+ transporters. However, recent genetic evidence indicates that vacuole-localized antiporters NHX1-4 have Na+-transport activities but may not contribute much to the vacuolar Na+ compartmentation, because the quadruple knockout mutant nhx1/2/3/4 is not more sensitive to NaCl than the wild type. Furthermore, vacuoles isolated from the quadruple mutant still retain the Na+ uptake that is independent to the pH gradient, implicating the presence of NHX-independent Na+ transporters in Arabidopsis vacuoles. We speculate that some of these unknown transporters may serve as CBL10-CIPK targets. On the other hand, endosomal compartments emerge as critical players that may be directly involved in controlling Na+ homeostasis. A possible but yet to be proved model is that the Na+ sequestration into the plant vacuole may actually be achieved, at least in part, through endosomal Na+ scavenging processes and subsequent fusion to the vacuole. NHX5 and NHX6 are localized to endosomal compartments and associated with protein trafficking from the Golgi/Trans-Golgi Network to vacuoles. Supporting this hypothesis is the finding that disruption of two endosomal NHXs in the nhx5 nhx6 double mutant showed increased sensitivity to salinity. Considering the fact that a proportion of the CBL10 protein was also localized to the dynamic endosomal compartments, NHX5/6 could also act as the candidate targets of the CBL10-CIPK complexes. In a recent work, translocon of the outer membrane of the chloroplasts 34 was identified as a novel interaction partner protein of CBL10 at the outer membrane of chloroplasts, clearly indicating that CBL10 can relay Ca2+ signals in more diverse ways than currently known. Identification of target transporter directly regulated by the CBL10-CIPK module is an important and challenging task for future research, which would also unravel the pathway through which Na+ is deposited into the plant vacuole.