Calcium is a component of signal transduction pathways that allow plants to respond to numerous endogenous and environmental signals during growth and development. Calcium-mediated signaling involves multiple components including: 1) channels, pumps, and exchangers that act in concert to generate a change in cytosolic calcium, 2) calcium-binding proteins that sense the calcium change, and 3) downstream target proteins that modify enzyme activity and gene expression needed for the subsequent response. One method for achieving specificity during calcium signaling is through regulation of the calcium-binding proteins that perceive changes in cytosolic calcium. These proteins can be regulated through differences in expression in response to stimuli, localization within the cell or plant, affinity for calcium, and interaction with downstream target proteins; all of which can result in specific cellular responses. My projects have focused on the Arabidopsis thaliana (Arabidopsis) CALCINEURIN B-LIKE10 (CBL10) calcium-binding protein, and specifically on understanding: 1) how post-transcriptional regulation of the CBL10 gene is used to modulate seedling growth in saline conditions (salinity), and 2) CBL10’s function in the flower during growth in salinity. In addition, 3) I have examined the roles of two putative CBL10-interacting proteins in plant growth and development. CBL10 is alternatively spliced into two transcripts; CBL10 encoding the characterized, full-length protein and CBL10 LONG A (CBL10LA) encoding a putative truncated protein due to a pre-mature termination codon within a retained intron. When seedlings are grown in the absence of salinity, both alternatively spliced transcripts are detected; however, in response to salinity, levels of the CBL10LA transcript are reduced. My data suggest a model in which the relative abundance of the two transcripts regulates the SALT-OVERLY-SENSITIVE (SOS) pathway involved in maintaining cellular sodium ion homeostasis. The presence of CBL10LA in the absence of salinity ensures that the SOS pathway is inactive. The removal of CBL10LA in response to saline conditions results in CBL10 activation of the SOS pathway to prevent sodium ions from accumulating to toxic levels in the cytosol. Successful fertilization during flowering requires the coordinated development of stamens and pistils. Stamens must elongate and anthers dehisce to release pollen onto the stigma while the pistil prepares to receive the pollen and promote growth and targeting of the female gametophyte. When the cbl10 mutant is grown in salinity, flowers are sterile due to decreased stamen elongation, reduced anther dehiscence, and abnormal pistil development. My studies demonstrated that the SOS pathway is not involved in maintaining flower development in salinity and indicate that CBL10 functions in different pathways to regulate vegetative and reproductive development during growth in saline conditions. An in silico search for Arabidopsis proteins that might interact with CBL10 resulted in the identification of two components of the Mediator complex involved in the regulation of transcription in eukaryotes. While additional studies I carried out suggest that interaction with CBL10 is unlikely, I have shown that these proteins are important for plant growth in high levels of chloride and in maintenance of growth in short-day conditions.

The buildup of salt in soils is a major abiotic stress that affects agricultural productivity, limiting the growth and yield of most crop species which cannot tolerate even modest levels of salinity (glycophytes). Genetic variability for salt tolerance exists as some plants (halophytes) have adapted to environments with high levels of salt. Understanding how salt tolerance has been acquired in halophytic species will be an important part of strategies to improve the ability of crops to grow in saline soils. The CALCINEURIN B-LIKE10 (AtCBL10) calcium sensor was identified as a component of salt signaling in the glycophyte Arabidopsis thaliana (A. thaliana) based on hypersensitivity of the Atcbl10 mutant to salt. When A. thaliana is grown in the presence of salt, AtCBL10 interacts with the AtSOS2 protein kinase to activate the AtSOS1 sodium/proton exchanger, resulting in the removal of sodium ions from the cytosol. Eutrema salsugineum (E. salsugineum), a halophytic relative of A. thaliana, has two CBL10 genes (EsCBL10a and EsCBL10b). In this research, the duplication of CBL10 in E. salsugineum was characterized and the functions of EsCBL10a and EsCBL10b in salt tolerance were determined. My analyses indicate that the coding sequences of EsCBL10a and EsCBL10b are highly conserved, as they share 85% nucleotide identity. An analysis of transcript structure indicates transcripts from EsCBL10a and EsCBL10b loci are alternatively spliced, but in distinct ways. My results suggest that EsCBL10a and AtCBL10 likely share the ancestral genomic position, while EsCBL10b might have moved to a different genomic region, and that the duplication took place prior to the divergence of expanded Lineage II species. The expression patterns of EsCBL10a and EsCBL10b are different; EsCBL10b transcript is high in shoots and low in roots while EsCBL10a transcript is detectable in both tissues. Preliminary analysis of E. salsugineum lines with reduced expression of EsCBL10a and EsCBL10b suggest that both genes might play a role during growth in the presence of salt, but that these roles are distinct.