The addition of Al slightly reduced the elongation of roots both with and without a cap-like structure, and the degree in the reduction was larger in structure-less roots . In the absence of Al, the removal of a cap-like structure hardly decreased the root elongation, although it slightly increased the occurrence of root bending . In roots without cap-like structures, this facilitation of root bending may result in a sight decrease in the Al-resistant root elongation. The present study revealed that the presence of a cap-like structure around root apices in A. mangium ameliorates an Al-facilitated bending of root growth direction. Root caps play major roles in root gravitropism through the localization of amyloplast granules and/or the basipetal flow of auxin . Our further analysis of the cap-like structure identified that a major attached position of a structure to the root was confined to columella root cap region, and the rigidity between this connection was enhanced during prolonged Al exposure periods . The undetached state of a cap-like structure may have a role in protecting inner root cap cell function from instant Al-caused damages. An apparent root bending induced by Al seems to require high concentrations, since no similar root bending in Al-sensitive plants has been reported. It is likely that a much lower concentration of Al may be sufficient for the complete arrest of root elongation until root cap cell dysfunction become recognizable as a root bending. The present study did not find any detrimental role of a floating,raspberry cultivation pot cap-like structure around the root in Al-resistant root elongation, similar to previous findings on root caps .

In the present study, however, we did not evaluate potential roles of undetached tissues on Al tolerance mechanisms. A cycle of detachment and formation of caplike structures imply that non-detached original tissues in the root surface area of root apex may play roles in the Al-resistant root elongation in A. mangium. Another study of A. mangiumrevealed that more than half of fibrillary tissue initials separated from the root surface were present beyond the entire root elongation region . In summary, we have found a novel cap-like structure of tissues for the protection of root cap cells at the root apex of A. mangium. Further characterization of the detachment pattern of the tissues from the root surface should provide better understanding of their roles in high Al tolerance mechanisms. Soil salinity imposes ion toxicity, hyperosmotic stress, and secondary stresses such as oxidative damage and nutritional disorders on plants. Plants have evolved several mechanisms to respond to the harsh environment and adjust their growth under high salt conditions. One critical mechanism involves calcium elevation and calcium-dependent signaling pathways in plant cells. The SOS pathway represents a calcium-dependent signaling pathway responsible for Na+ homeostasis and salt tolerance in Arabidopsis. The pathway starts from CBL4 , a calcium sensor protein that is supposed to respond to the specific Ca2+ signals triggered by excess Na+. The CBL4 protein interacts with a serine/threonine protein kinase that activates the SOS1, a Na+/H+ antiporter, leading to the Na+ efflux from the cytosol. In addition to interacting with SOS1 at the plasma membrane, CIPK24 is also reported to regulate the activity of several tonoplast localized transporters by interacting with them, such as the Ca2+/H+ antiporter, the vacuolar V-ATPase and the Na+/H+ exchanger. Additionally, CIPK24 may link flowering time and salt stress response as its activity was regulated by a photoperiodicity and circadian clock switch GIGANTEA.

CBL4 also plays a critical role in the development of lateral roots through the modulation of auxin gradients and maxima in roots under mild salt conditions. While CBL4 mainly functions in root tissues, CBL10 appears to be preferentially expressed in the shoots and plays a key role in salt stress tolerance as well. Similar to CBL4, CBL10 also physically interacted with CIPK24. However, in contrast to the CBL4-CIPK24 complex at the plasma membrane, the CBL10-CIPK24 interaction was primarily associated with vacuolar compartments. As CBL4 and CBL10 cannot replace each other’s functions, they must fulfill distinct regulatory functions in the salt stress response of Arabidopsis plants. Furthermore, a recent study has demonstrated that CBL10 is critical for reproductive development under salinity conditions and it functions independently from the SOS pathway. Studies so far have shown that the salt-tolerance function conferred by CBL10 is conserved in Arabidopsis, poplar and tomato. In poplar, two CBL10 homologs have been identified and they serve similar functions. In tomato, a CBL10 homologue has been shown to function in Na+/Ca2+ homeostasis under salt stress and reactive oxygen species signaling in plant immunity. The CBL-CIPK pathway is widely accepted as a major mechanism underlying plant response and adaptation to different external stresses that trigger Ca2+ signaling events . Although several other CBL-CIPK pairs have been implicated in salt tolerance, the CBL4- and CBL10-dependent pathways play dominant roles in salt tolerance. In this study, we examined the genetic interaction of the two major salt response pathways directed by CBL4 and CBL10 that mediate salt tolerance by regulating processes at the plasma membrane and intracellular membranes, respectively.

Detailed characterization of the cbl4 cbl10, cipk24 cbl10 and sos1 cbl10 double mutants under salt stress demonstrated that CBL4 and CBL10 fulfill distinct mechanisms of salt tolerance in plants. The original sos3 mutant is an ethyl methane sulfonate -mutagenized allele in which SOS3 lacks three amino acids. Although this mutated version of CBL4 is impaired in Ca2+ binding, it is probably not a null allele, which may still have a residual function. We thus isolated an independent T-DNA insertional cbl4 allele in which the full-length transcript became undetectable by reverse transcription-polymerase chain reaction . As expected, cbl4 is more sensitive than the original sos3 mutant in 50 mM and 75 mM NaCl conditions . To investigate the functional interaction between CBL10 and three components of the SOS pathway , a series of double mutants were created by genetic crossing and their genotypes were confirmed by RT-PCR . To test the salt sensitivity of the cbl4 cbl10 mutant, five-day-old mutant and wild-type plants were transferred to the Murashige and Skoog medium with a series of NaCl concentrations. We found that cbl4 cbl10 showed dramatically enhanced sensitivity to Na stresses than the single mutants in all tested concentrations of NaCl , and the growth difference between the double and single mutant plants was much more pronounced as the Na+ level was elevated . On the normal MS medium without NaCl, all mutants exhibited no differences from the wild type . On the MS medium with 5 mM NaCl, the cbl4 cbl10 double mutant already exhibited significant sensitivity with shorter root and smaller shoots as compared to the wild type, whereas the cbl4 and cbl10 single mutants did not show a significant difference from the wild-type plants . To determine whether the hypersensitivity of these mutants to NaCl is specifically attributable to Na+, we replaced 50 mM NaCl with 50 mM NaNO3 and 50 mM KCl and found that the mutant seedlings were sensitive to 50 mM NaNO3 but showed no difference in growth compared with the wild type on the medium containing 50 mM KCl . This result indicates that the mutants were specifically sensitive to Na+ but not to K+ or Cl−. To extend the phenotypic analysis of the cbl4 cbl10 mutants in mature plants,low round pots we also examined the salt sensitivity of the cbl4 cbl10 mutant plants in hydroponic solutions with defined levels of external Na+ . We found that cbl4 cbl10 grown in hydroponic conditions also showed dramatically enhanced sensitivity to Na+ stresses as compared to the single mutants. Under 15 mM Na+ conditions, the cbl4 cbl10 plants were much more stunted than single mutants, as revealed also by the shoot biomass . Taken together, these results demonstrated that CBL10 and CBL4 function additively in salt tolerance in Arabidopsis. After being grown in the hydroponic 1/6 MS medium supplemented with 15 mM NaCl for six days, cipk24 cbl10 showed more salt-sensitive phenotype than cipk24 or cbl10, as revealed by the shoot biomass . The growth difference between cipk24 cbl10 and the single mutant plants was much more pronounced after the plants were grown in the medium containing 5 mM NaCl for 25 days . As illustrated in Figure S3g, the cipk24 cbl10 leaves were chlorotic and could hardly bolt while the wild type and the single mutant plants successfully bolted and flowered. On the standard MS medium without NaCl, the wild type and all the mutant plants grow normally and exhibited no differences .

While the cipk24 cbl10 double mutant showed more severe phenotype than each of the single mutant , the cipk24 cbl4 double mutant showed similar phenotype to cipk24 . Therefore, the functional interaction between CBL10 and CIPK24, if any, was distinct from the relationship of CBL4 and CIPK24 that forms the linear pathway. 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. 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, 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. Tissue-specific expression pattern and subcellular locations of specific downstream partners including CIPKs underlies specificity for CBLs to mediate the stress response.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 otherhand, 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.