The accumulation of sodium in soil (saline conditions) negatively affects plant growth and development. The Salt Overly Sensitive (SOS) pathway in Arabidopsis (Arabidopsis thaliana) functions to remove sodium from the cytosol during vegetative development preventing its accumulation to toxic levels. In this pathway, the SOS3 and CALCINEURIN B-LIKE10 (CBL10) calcium sensors interact with the SOS2 protein kinase to activate sodium/proton exchange at the plasma membrane (SOS1) or vacuolar membrane. To determine if the same pathway functions during reproductive development in response to salt, fertility was analyzed in wild type and the SOS pathway mutants grown in saline conditions. In response to salt, CBL10 functions early in reproductive development before fertilization, while SOS1 functions mostly after fertilization when seed development begins. Neither SOS2 nor SOS3 function in reproductive development in response to salt. Loss of CBL10 function resulted in reduced anther dehiscence, shortened stamen filaments, and aborted pollen development. In addition, cbl10 mutant pistils could not sustain the growth of wild-type pollen tubes. These results suggest that CBL10 is critical for reproductive development in the presence of salt and that it functions in different pathways during vegetative and reproductive development.

The SALT-OVERLY-SENSITIVE (SOS) pathway in Arabidopsis (Arabidopsis thaliana) functions to prevent the toxic accumulation of sodium in the cytosol when plants are grown in salt-affected soils. In this pathway, the CALCINEURIN B-LIKE10 (AtCBL10) calcium sensor interacts with the AtSOS2 kinase to activate the AtSOS1 plasma membrane sodium/proton exchanger. CBL10 has been duplicated in Eutrema (Eutrema salsugineum), a salt-tolerant relative of Arabidopsis. Because Eutrema maintains growth in salt-affected soils that kill most crop plants, the duplication of CBL10 provides a unique opportunity to functionally test the outcome of gene duplication and its link to plant salt tolerance. In Eutrema, individual down-regulation of the duplicated CBL10 genes (EsCBL10a and EsCBL10b) decreased growth in the presence of salt and, in combination, led to an even greater decrease, suggesting that both genes function in response to salt and have distinct functions. Cross-species complementation assays demonstrated that EsCBL10b has an enhanced ability to activate the SOS pathway while EsCBL10a has a function not performed by AtCBL10 or EsCBL10b. Chimeric EsCBL10a/EsCBL10b proteins revealed that the specific functions of the EsCBL10 proteins resulted from changes in the amino terminus. The duplication of CBL10 increased calcium-mediated signaling capacity in Eutrema and conferred increased salt tolerance to salt-sensitive Arabidopsis.

The presence of varied numbers of CALCINEURIN B-LIKE10 (CBL10) calcium sensor genes in species across the Brassicaceae and the demonstrated role of CBL10 in salt tolerance in Arabidopsis thaliana and Eutrema salsugineum provided a unique opportunity to determine if CBL10 function is modified in different species and linked to salt tolerance. Salinity effects on species growth and cross-species complementation were used to determine the extent of conservation and divergence of CBL10 function in four species representing major lineages within the core Brassicaceae (A. thaliana, E. salsugineum, Schrenkiella parvula, and Sisymbrium irio) as well as the first diverging lineage (Aethionema arabicum). Evolutionary and functional analyses indicate that CBL10 duplicated within expanded lineage II of the Brassicaceae and that, while portions of CBL10 function are conserved across the family, there are species-specific variations in CBL10 function. Paralogous CBL10 genes within a species diverged in expression and function probably contributing to the maintenance of the duplicated gene pairs. Orthologous CBL10 genes diverged in function in a species-specific manner, suggesting that functions arose post-speciation. Multiple CBL10 genes and their functional divergence may have expanded calcium-mediated signaling responses and contributed to the ability of certain members of the Brassicaceae to maintain growth in salt-affected soils.