Isolation of a Calmodulin-binding Transcription Factor from Rice (Oryza sativa L.)

Abstract

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horseradish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca 2؉ -dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5-TWCG(C/T)GTKKKKTKCG-3 (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate ␤-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

Identification of a Calmodulin-binding NAC Protein as a Transcriptional Repressor in Arabidopsis

“…CaM also regulates the transcriptional activities of transcription factors. CaM actually binds transcription factors and activates or inhibits their DNA-binding properties (13)(14)(15)(16)32). In this report, we isolated a CaM-binding NAC transcription factor (CBNAC) that can function as a transcriptional repressor.…”

“…Full-length CBNAC cDNA was obtained by PCR using a 5Ј primer containing a BamHI site and a 3Ј primer containing an SmaI site. The PCR product was inserted into the pUC::smGFP expression vector (32). Plasmids were introduced by polyethylene glycol-mediated transformation (33) into Arabidopsis protoplasts prepared from leaf tissues.…”

“…In this study, we used the RBSS method to characterize the DNA-binding specificity of CBNAC. This method is convenient and rapid for investigating DNA sequences bound by an individual protein (32,55,56). Different approaches used to identify core binding sequences can produce different results with other NAC proteins (25).…”

“…To produce double-stranded, random binding DNA sequences 400 pmol of R1 and 800 pmol of TNR20 (or R2 and TNR16) were annealed, and second strand synthesis was catalyzed by Klenow fragment polymerase in the presence of dNTPs (final concentration, 0.25 mM each). The DNA binding assay was performed as described previously (32). Out of the five selection cycles (binding/mobility shift/elution/PCR amplification) conducted, the first three selections were performed using binding conditions of 50 mM KCl, whereas the latter two selections were carried out at 100 mM and 150 mM KCl, respectively.…”

Coping with Stresses: Roles of Calcium- and Calcium/Calmodulin-Regulated Gene Expression

“…Although this study provided evidence that CaM is involved in regulating the activity of a defense-associated TF, it did not link the pathogen-induced Ca 2+ signature to transcription. Choi et al (2005) studied the function of the rice CAMTAs (Os-CBTs) in Arabidopsis protoplasts using synthetic promoters and found that Ca 2+ /CaM suppressed the CAMTA-mediated transcription activation. Constitutive activation of plant defense responses in camta3 mutants is temperature dependent (Du et al, 2009).…”