Arabidopsis Calmodulin-like Proteins CML13 and CML14 Interact with Calmodulin-Binding Transcriptional Activators and Function in Salinity Stress Response.

Eukaryotic cells use calcium ions (Ca2+) as second messengers, particularly in response to abiotic and biotic stresses. These signals are detected by Ca2+ sensor proteins, such as calmodulin (CaM), which regulate the downstream target proteins. Plants also possess many CaM-like proteins (CMLs), most of which remain unstudied. We recently demonstrated that Arabidopsis CML13 and CML14 interact with proteins containing isoleucine/glutamine (IQ) domains, including CaM-binding transcriptional activators (CAMTAs). Here, we show that CaM, CML13 and CML14 bind all six members of the Arabidopsis CAMTA family. Using a combination of in planta and in vitro protein-interaction assays, we tested 11 members of the CaM/CML family and demonstrated that only CaM, CML13 and CML14 bind to CAMTA IQ domains. CaM, CML13 and CML14 showed Ca2+-independent binding to the IQ region of CAMTA6 and CAMTA3, and CAMTA6 in vitro exhibited some specificity toward individual IQ domains within CAMTA6 in split-luciferase in planta assays. We show that cml13 mutants exhibited enhanced salinity tolerance during germination compared to wild-type plants, a phenotype similar to camta6 mutants. In contrast, plants overexpressing CML13-GFP or CML14-GFP in the wild-type background showed increased NaCl sensitivity. Under mannitol stress, cml13 mutants were more susceptible than camta6 mutants or wild-type plants. The phenotype of cml13 mutants could be rescued with the wild-type CML13 gene. Several salinity-marker genes under CAMTA6 control were similarly misregulated in both camta6 and cml13 mutants, further supporting a role for CML13 in CAMTA6 function. Collectively, our data suggest that CML13 and CML14 participate in abiotic stress signaling as CAMTA effectors.

In Vivo Phosphorylation of the Cytosolic Glucose-6-Phosphate Dehydrogenase Isozyme G6PD6 in Phosphate-Resupplied Arabidopsis thaliana Suspension Cells and Seedlings.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first committed step of the oxidative pentose phosphate pathway (OPPP). Our recent phosphoproteomics study revealed that the cytosolic G6PD6 isozyme became hyperphosphorylated at Ser12, Thr13 and Ser18, 48 h following phosphate (Pi) resupply to Pi-starved (-Pi) Arabidopsis thaliana cell cultures. The aim of the present study was to assess whether G6PD6 phosphorylation also occurs in shoots or roots following Pi resupply to -Pi Arabidopsis seedlings, and to investigate its relationship with G6PD activity. Interrogation of phosphoproteomic databases indicated that N-terminal, multi-site phosphorylation of G6PD6 and its orthologs is quite prevalent. However, the functions of these phosphorylation events remain unknown. Immunoblotting with an anti-(pSer18 phosphosite-specific G6PD6) antibody confirmed that G6PD6 from Pi-resupplied, but not -Pi, Arabidopsis cell cultures or seedlings (i.e., roots) was phosphorylated at Ser18; this correlated with a significant increase in extractable G6PD activity, and biomass accumulation. Peptide kinase assays of Pi-resupplied cell culture extracts indicated that G6PD6 phosphorylation at Ser18 is catalyzed by a Ca2+-dependent protein kinase (CDPK), which correlates with the 'CDPK-like' targeting motif that flanks Ser18. Our results support the hypothesis that N-terminal phosphorylation activates G6PD6 to enhance OPPP flux and thus the production of reducing power (i.e., NADPH) and C-skeletons needed to establish the rapid resumption of growth that ensures Pi-resupply to -Pi Arabidopsis.