Analysis of EF-Hand Proteins in Soybean Genome Suggests Their Potential Roles in Environmental and Nutritional Stress Signaling

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.

Arabidopsis Calmodulin-Like Proteins, CML15 and CML16 Possess Biochemical Properties Distinct from Calmodulin and Show Non-overlapping Tissue Expression Patterns

“…For Arabidopsis, transcriptome database mining predicted that CaMs are broadly expressed across tissues and developmental stages whereas CMLs cluster into five main expression groups that range from highly restrictive to widespread like the CaMs (McCormack et al, 2005 ). Likewise, transcriptome analysis for soybean CMLs indicates similar expression-based clustering with many displaying broad tissue distribution and at least 7 CMLs being floral specific (Zeng et al, 2017 ). Promoter-reporter analysis has corroborated or expanded upon these data for a few Arabidopsis CMLs .…”

“…A recent reorganization of the Arabidopsis phylogeny places CML15 and CML16 in subgroup II (Zhu et al, 2015 ). Orthologs of both these CMLs are found across plant taxa (Zhu et al, 2015 ; Zeng et al, 2017 ). Although CML15 and CML16 are predicted to possess 4 EF-hand motifs, variations within the Ca 2+ -binding loops and adjacent regions are present when compared to CaM2.…”

“…In the genetic model plant, Arabidopsis , there are about 250 EF-hand proteins encoded in the genome (Day et al, 2002 ). Similarly, 262 EF-hand proteins are predicted in soybean, suggesting such complexity is found throughout plant taxa (Zeng et al, 2017 ). In addition to CaMs, plants possess three additional large families of Ca 2+ sensors: Ca 2+ -dependent protein kinases (CPKs), calcineurin B-like proteins (CBLs), and CMLs (DeFalco et al, 2010 ; Ranty et al, 2016 ).…”

Hydrogel-based transparent soils for root phenotyping in vivo

“…Real-time PCR analysis was used to investigate whether the TS medium provides adequate access to nutrients and oxygen, and how it compares to hydroponics and field soil. We characterized the expression of five genes that have been associated with abiotic stress [N deficiency, Glyma.12g221500 (14); P, Fe, and Zn deficiency, Glyma.08g053500 (15); drought, Glyma.04G203300 (16); hypoxia, Glyma.11G121800 and Glyma.04G240800 (10, 17)] and three genes that have been associated with the development PLANT BIOLOGY ENGINEERING of lateral roots and root tips (Glyma.06G070500, Glyma.08G133800, Glyma.02G043400) in plants (root tissues of G. max, IA2102, 12 d old, n = 4) grown in hydroponics, TS, and field soil, using soil extract as a nutrient medium, as described for the phenotyping study. Relative expression was calculated by the 2 −ΔΔCt method by considering soil treatment as the calibrator (18).…”

Identification and functional analysis of soybean GmSBH1 gene promoter conferring high temperature- and humidity-induced expression

“…The cis-acting HSE was verified to have transcriptional activation function (Supplemental Figure 3) and interacted with 82 kinds of different proteins under HTH stress ( Figure 5; Supplemental Table 1). The function of these 82 proteins could be roughly divided into 8 categories of seed storage proteins, seed mature proteins (Shewry et al, 1995;Natarajan et al, 2006), defense proteins (Lee et al, 2016), ribosomal proteins (Mager, 1988), signal transduction proteins (Zeng et al, 2017), enzyme and protease inhibitors (Tormo et al, 2006), other functional proteins, and unknown functional proteins. These proteins can provide a basis for studying the regulatory mechanism of GmSBH1 under HTH stress.…”