Research article Expression of calmodulin genes in wild type and calmodulin mutants of Arabidopsis thaliana under heat stress Nisreen A. AL-Quraan 1 , Robert D. Locy, Narendra K. Singh * Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA article info Article history: Received 6 October 2009 Accepted 24 April 2010 Available online 11 May 2010 Keywords: Arabidopsis Calmodulin CAM expression Ca 2þ Heat stress RT-PCR abstract Calmodulin (CaM), a calcium-regulated protein, regulates the activity of a number of key enzymes and plays important roles in cellular responses to environmental changes. The Arabidopsis thaliana genome contains nine calmodulin (CAM) genes. To understand the role of specific CAM genes in heat stress, the steady-state level of mRNA for the nine CAM genes in root and shoot tissues of seedlings grown at normal growth temperature (25  C) and during heat stress at 42  C for 2 h was compared in T-DNA insertional mutant lines of 7 CAM genes and the wild type using gene specific primers and RT-PCR. Compared to growth at 25  C, the mRNA levels of all CAM genes were up-regulated in both root and shoot after heat treatment with the notable exception of CAM5 in root and shoot, and CAM1 in shoot where the mRNA levels were reduced. At 25  C all cam mutants showed varying levels of mRNA for corresponding CAM genes with the highest levels of CAM5 gene mRNA being found in cam5-1 and cam5-3. CAM5 gene mRNA was not observed in the cam5-4 allele which harbors a T-DNA insertion in exon II. The level of respective CAM gene mRNAs were reduced in all cam alleles compared to levels in wild type except for increased expression of CAM5 in roots and shoots of cam5-1 and cam5-3. Compared to wild type, the level of mRNA for all CAM genes varied in each cam mutant, but not in a systematic way. In general, any non-exonic T- DNA insertion produced a decrease in the mRNA levels of the CAM2 and CAM3 genes, and the levels of CAM gene mRNAs were the same as wild type or lower in the cam1 , cam4, cam5-2, and cam6-1 non- exonic mutant alleles. However, the level of mRNA for all genes except CAM2 and CAM3 genes was up- regulated in all cam2 and cam3 alleles and in the cam5-1 and cam5-3 alleles. During heat stress at 42  C the level of CAM gene mRNAs were also variable between insertional mutants, but the level of CAM1 and CAM5 gene mRNAs were consistently greater in response to heat stress in both root and shoot. These results suggest differential tissue-specific expression of CAM genes in root and shoot tissues, and specific regulation of CAM gene mRNA levels by heat. Each of the CAM genes appears to contain noncoding regions that play regulatory roles resulting in interaction between CAM genes leading to changes in specific CAM gene mRNA levels in Arabidopsis. Only exonic insertion in CAM5 gene resulted in a loss-of- function of CAM5 gene among the mutants we surveyed in this study. Published by Elsevier Masson SAS. 1. Introduction Calmodulins (CaM) are highly conserved and ubiquitous trans- ducers of Ca þ2 signals in all eukaryotes. The regulatory abilities of CaMs are revealed by their ability to modulate the activities of enzymes involved in various physiological responses including growth, gravitropism, phototropism, environmental stress, and biotic defense responses [18,22,23,32,44]. CaM harbors no intrinsic enzyme activity of its own. All calmodulin proteins are highly conserved with 4 repeating units called EF-hands. Each EF-hand binds a single Ca þ2 ion [5,33]. Upon Ca þ2 binding to the EF-hand domains, CaM alters its structure [38] by revealing hydrophobic surfaces that serve to interact with target proteins. In this way the Ca þ2 -dependent regulation of target proteins is mediated. The genomic database of Arabidopsis thaliana reveals 9 CAM gene loci. The first 7 calmodulin genes (CAM1eCAM7) produce proteins consisting of 149 amino acids each, while CAM8 and CAM9 are predicted to code for proteins that are 151 amino acids each Abbreviations: CAM, calmodulin gene; CaM, calmodulin protein; Ca þ2 /CaM, calcium/calmodulin complex; RT-PCR, reverse transcriptase-polymerase chain reaction; UTR, Un-translated region. * Corresponding author. Tel.: þ1 334 844 1667; fax: þ1 334 844 1645. E-mail address: singhna@auburn.edu (N.K. Singh). 1 Present address: Department of Biotechnology and Genetic Engineering, Jordan University of Science and Technology, Irbid 22110, Jordan. Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Published by Elsevier Masson SAS. doi:10.1016/j.plaphy.2010.04.011 Plant Physiology and Biochemistry 48 (2010) 697e702