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.

Keywords: EF-hand motif; Rboh (respiratory burst oxidase homolog); calcineurin B-like protein (CBL); calcium signal; calcium-dependent protein kinase (CDPK); calmodulin; calmodulin-like protein (CML); soybean (Glycine max).

FIGURE 1

The number of genes encoding proteins containing 1, 2, 3, 4, 5, or 6 EF-hand motifs. The number of EF-hand motifs in each protein was predicted using InterPro database (http://www.ebi.ac.uk/interpro/).

FIGURE 2

Graphical representation of locations for putative EF-hand protein-encoding genes on each soybean chromosome. CaM/CMLs are marked in red, CBLs are marked in green, CDPK/CRK/CCaMKs are marked in blue, respiratory burst oxidase homologs (Rbohs) are marked in orange, and the other unclassified EF-hand proteins (UEPs) are marked in purple. Genes within a light green box are putatively tandemly duplicated.

FIGURE 3

Unrooted phylogenetic tree of EF-hand proteins in soybean (Glycine max). The alignment for the phylogenetic tree was performed with ClustalW using full-length amino acid sequences. The phylogenetic tree was created with the MEGA6 software and the neighbor-joining method with 1,000 bootstrap replications. All the 150 CaM/CML proteins are marked with red circles, 15 CBL proteins are marked with green squares, 65 CDPK/CRK/CCaMK proteins are marked with purple triangles, 17 Rboh proteins are marked with pink inverted triangles, and 15 UEPs are marked with blue diamonds. The bar indicates the relative divergence of the sequences examined.

FIGURE 4

Schematic diagrams of representative soybean EF-hand proteins. EF-hands and other domains were predicted using InterPro database (http://www.ebi.ac.uk/interpro/).

FIGURE 5

Unrooted phylogenetic tree of the CaM and CML proteins from soybean (G. max) and Arabidopsis thaliana. The alignment for the phylogenetic tree was performed with ClustalW using full-length amino acid sequences. The phylogenetic tree was created with the MEGA6 software and the neighbor-joining method with 1,000 bootstrap replications. All the 150 CaM/CML proteins from soybean are marked with red circles, while 57 CaM/CML proteins from Arabidopsis are marked with blue triangles. Roman numerals designate the subfamilies. The bar indicates the relative divergence of the sequences examined and bootstrap values are displayed next to the branch.

FIGURE 6

Unrooted phylogenetic tree of the total CBL proteins from soybean (G. max) and A. thaliana. The alignment for the phylogenetic tree was performed with ClustalW using full-length amino acid sequences. The phylogenetic tree was created with the MEGA6 software and the neighbor-joining method with 1,000 bootstrap replications. The 15 CBLs from soybean are marked with green squares, while the 10 CBLs from Arabidopsis are not marked. Locus name of each gene is shown in brackets. The bar indicates the relative divergence of the sequences examined and bootstrap values are displayed next to the branch.

FIGURE 7

Unrooted phylogenetic tree of the GmCDPK, GmCRK, and GmCCaMK proteins. The alignment for the phylogenetic tree was performed with ClustalW using full-length amino acid sequences. The phylogenetic tree was created with the MEGA6 software and the neighbor-joining method with 1,000 bootstrap replications. All the 50 GmCDPKs are marked with triangles, 13 GmCRKs are marked with inverted triangles, and 2 GmCCaMKs are marked with diamonds, while proteins from Arabidopsis (34 CDPKs and 8 CRKs), and CCaMKs from Medicago truncatula (MtCCaMK) or Lotus japonicus (LjCCaMK) are not marked. Locus name of each gene is shown in brackets. The bar indicates the relative divergence of the sequences examined and bootstrap values are displayed next to the branch.

FIGURE 8

Unrooted phylogenetic tree of the Rboh proteins from soybean (G. max) and A. thaliana. The alignment for the phylogenetic tree was performed with ClustalW using full-length amino acid sequences. The phylogenetic tree was created with the MEGA6 software and the neighbor-joining method with 1,000 bootstrap replications. Seventeen GmRbohs are marked with pink inverted triangles, while 10 AtRboh proteins are not marked. Locus name of each gene is shown in brackets. The bar indicates the relative divergence of the sequences examined and bootstrap values are displayed next to the branch.

FIGURE 9

Heat map representation for tissue-specific expression patterns of the predicted GmCaM/CML genes according to Illumina transcriptome data. The Reads/Kb/Million (RPKM) normalized log2 transformed counts were visualized in the heat map. The red colors indicate expression intensity, gray color indicates no expression.

FIGURE 10

Heat map representation for tissue-specific expression patterns of predicted EF-hand protein-coding genes with the exception of GmCaM/CMLs according to Illumina transcriptome data. The RPKM normalized log2 transformed counts were visualized in the heat map. The red colors indicate expression intensity, gray color indicates no expression detected.

FIGURE 11

Heatmap representation for expression profiles of EF-hand protein-encoding genes in response to cold, drought, flooding, phosphorus deficiency, and Bradyrhizobium japonicum inoculation (24–48 HAI). The intensities of the color represent the relative magnitude of fold changes in log2 values according to microarray or high-throughput sequencing data. Red color indicates induction, green color indicates repression, gray color means there is no significant expressional change.

FIGURE 12

Heat map representation for expression profiles of some selected EF-hand genes in response to stresses. The expression of EF-hand genes were analyzed by qRT-PCR in roots of 12-day-old seedling, after treatment with 200 mM NaCl (salt), 10% PEG 6000, phosphate deficiency (-Pi), iron deficiency (-Fe), and zinc deficiency (-Zn) for 2, 8, and 24 h. The intensities of the color represent the relative magnitude of fold changes (treatment/control) in log2 values of three technical replicates. The asterisks indicate an absolute fold change ≥ 2 and P-value < 0.05 by Student’s t-test. Red color indicates induction, green color indicates repression.

FIGURE 13

Effect of Ca²⁺ on the electrophoretic mobility of GmCML1, GmCML13, GmCML39, and GmCML95 proteins. Samples (1–2 μg) of purified, recombinant AtCaM7 (positive control), GmCML1, GmCML13, GmCML39, and GmCML95 containing 1.0 mM CaCl2, 1.0 mM EGTA, or 1.0 mM MgCl₂ were separated on SDS–PAGE gels supplemented with 1.0 mM CaCl₂, 1.0 mM EGTA, or 1.0 mM MgCl₂, respectively, and stained with Coomassie Brilliant Blue. The positions of the molecular weight markers (kDa) are indicated on the left.