Characterisation of a novel calciumsensor in Arabidopsis thaliana

Abstract

In dicotyledonous plants calcium is predominantly stored in the vacuoles of leaf mesophyll cells, a process in which the Arabidopsis thaliana tonoplast-localised Ca2+/H+ antiporter 1 (AtCAX1) was previously identified as having an essential role. Simultaneous loss-of-function of AtCAX1, and its close homolog AtCAX3, or an overexpression of a constitutively active form (sCAX1) can cause a number of physiological perturbations. The transcriptional profiles concurrent with these perturbations were examined in a set of Arabidopsis cax mutants (cax1, cax3, cax1/cax3 and cax1/sCAX1, and parental wildtype Col-0) as means to uncover novel Ca2+-signalling elements. A core set of misexpressed genes was examined, in a preliminary screen using putative loss-of-function Arabidopsis mutants, but no calcium-related phenotypes were identified. Instead, the most highly misexpressed gene in cax1 and cax1/cax3 lines was selected for further functional characterisation. Calmodulin-like 41 (CML41) was negatively correlated with CAX1 expression so it was hypothesised that it might behave as a transcriptional regulator of CAX1 or as a Ca2+ signalling element downstream of CAX1 function. During cloning it was discovered that CML41 was likely transcribed into two transcripts – a full-length CML41 (CML41FL), which is annotated in the NCBI database, and a novel shortersplicing transcript named CML41 Short (CML41S). The proteins encoded by CML41FL and CML41S were predicted to have 4 and 3 putative EF-hand calcium binding domains respectively, and both were demonstrated to have calcium-binding capacity in vitro, indicating that CML41FL and CML41S may act as Ca2+ sensors in planta. Both proteins have the same targeting signal peptide and share a similar subcellular localisation pattern being predominantly localised in the cytoplasm of young developing leaves, and roots under standard growth conditions, but are translocated to plasmodesmata (PD) in mature and old vegetative leaves. Furthermore, a TEOSINTE BRANCHED 1, cycloidea and proliferating cell factor (TCP) transcription factor 14 (TCP14) was demonstrated to interact with both CML41FL and CML41S, but the function of these interactions remains obscure. Misexpression (35S CMV driven amiRNA knockdown or overexpression) of either CML41FL or CML41S had no effect on CAX1 transcript abundance, so it is more likely that CML41 acts as a downstream Ca2+ signal element rather than in controlling CAX1 expression. In silico analysis of gene expression indicates that CML41 is highly up-regulated during biotic stress, senescence, in response to changes in photoperiod and calcium treatments, so the phenotypes of CML41 misexpressing plants were examined under these and related conditions. Both CML41FL and CML41S expression was induced in leaves infiltrated with flg22 – an elicitor of P. syringae inducing pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) signalling in plants. Knocking-down CML41FL expression significantly reduced the callose deposition at PD in leaves in response to flg22, whereas in normal conditions a constitutive overexpression of CML41FL failed to increased callose deposition. Together this implies that CML41FL (and/or CML41S) may function as a Ca2+ sensor downstream of the flg22-triggered immune response to modulate callose accumulation, and its activation may require an elevation of cytosolic Ca2+. The overexpression of CML41S and silencing of CML41FL both accelerated chlorophyll breakdown and senescence of individual leaves induced by dark, although their expression was not altered during the conditions imposed here. High calcium supplementation (50 mM) inhibited primary root growth of wild-type and CML41 overexpression lines whereas it was not affected in CML41-knocked-down amiRNA lines. At 12.5 mM calcium, as compared to 0.3 mM, primary root growth of wild-type and CML41-knocked-down amiRNA plants was stimulated but this was not observed in CML41FL- or CML41S-overexpression plants. In plants expressing CML41-GFP translational fusions, both CML41FL and CML41S were translocated from the cytoplasm to the PD at the root tip under high calcium conditions. These results suggest that a root-growth responses to high external calcium might involve the translocation of CML41 from the cytoplasm to the PD. Here, I demonstrate that a previously uncharacterised member of the CML family is likely to have key roles in biotic stress responses, in regulation of dark-induced leaf senescence and regulation of root sensitivity to environmental calcium levels. A number of experimental avenues are opened up by this work, especially in respect to the relative contributions of CML41FL and CML41S to the above phenotypes.