Abstract
Calcium ion (Ca2+) is a universal second messenger that plays a critical role in plant responses to diverse physiological and environmental stimuli. The stimulus-specific signals are perceived and decoded by a series of Ca2+ binding proteins serving as Ca2+ sensors. The majority of Ca2+ sensors possess the EF-hand motif, a helix-loop-helix structure which forms a turn-loop structure. Although EF-hand proteins in model plant such as Arabidopsis have been well described, the identification, classification, and the physiological functions of EF-hand-containing proteins from soybean are not systemically reported. In this study, a total of at least 262 genes possibly encoding proteins containing one to six EF-hand motifs were identified in soybean genome. These genes include 6 calmodulins (CaMs), 144 calmodulin-like proteins (CMLs), 15 calcineurin B-like proteins, 50 calcium-dependent protein kinases (CDPKs), 13 CDPK-related protein kinases, 2 Ca2+- and CaM-dependent protein kinases, 17 respiratory burst oxidase homologs, and 15 unclassified EF-hand proteins. Most of these genes (87.8%) contain at least one kind of hormonal signaling- and/or stress response-related cis-elements in their -1500 bp promoter regions. Expression analyses by exploring the published microarray and Illumina transcriptome sequencing data revealed that the expression of these EF-hand genes were widely detected in different organs of soybean, and nearly half of the total EF-hand genes were responsive to various environmental or nutritional stresses. Quantitative RT-PCR was used to confirm their responsiveness to several stress treatments. To confirm the Ca2+-binding ability of these EF-hand proteins, four CMLs (CML1, CML13, CML39, and CML95) were randomly selected for SDS–PAGE mobility-shift assay in the presence and absence of Ca2+. Results showed that all of them have the ability to bind Ca2+. This study provided the first comprehensive analyses of genes encoding for EF-hand proteins in soybean. Information on the classification, phylogenetic relationships and expression profiles of soybean EF-hand genes in different tissues and under various environmental and nutritional stresses will be helpful for identifying candidates with potential roles in Ca2+ signal-mediated physiological processes including growth and development, plant-microbe interactions and responses to biotic and abiotic stresses.
Highlights
During their life cycles, plants encounter a variety of external stresses such as heat, cold, drought, flooding, salinity, nutrient deficiency, and attacks from insects and pathogens
“EF-hand,” “calmodulin,” “calmodulinlike,” “calcineurin B-like,” and “calcium-dependent protein kinase” were each used as a key word to perform homolog searches
There were at least 144 genes encoding for calmodulin-like proteins (CMLs) proteins; these CMLs share at least 12.8% overall amino acid identity with Arabidopsis thaliana CaM2 (AtCaM2), and have no other known functional domains except the EF-hands (Supplemental Table S2)
Summary
Plants encounter a variety of external stresses such as heat, cold, drought, flooding, salinity, nutrient deficiency, and attacks from insects and pathogens. A steep Ca2+ gradient across the plasma membrane as well as the inner membrane system enclosing the cellular Ca2+ storages is established under the controls of stimulus responsive Ca2+ channels, pumps and transporters (McAinsh and Pittman, 2009; Kudla et al, 2010) It has been well-documented that various external biotic or abiotic stimuli can quickly trigger specific and distinct spatial-temporal patterns of changes in cytosolic Ca2+ concentration, and “Ca2+ signatures” have been coined to describe the specificities of calcium signals triggered by the causative stimuli (Webb et al, 1996; McAinsh and Pittman, 2009). Upon Ca2+ binding, Ca2+ sensors undergo conformational changes that either promote their interactions with target proteins or alter their own enzymatic activities, and transmit these stimulus-specific signals into biochemical, molecular, cellular or physiological events, which help plants survive the unfavorable environmental conditions
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