Ca2+/Cation Antiporters (CaCA): Identification, Characterization and Expression Profiling in Bread Wheat (Triticum aestivum L.)

Abstract

The Ca²⁺/cation antiporters (CaCA) superfamily proteins play vital function in Ca²⁺ ion homeostasis, which is an important event during development and defense response. Molecular characterization of these proteins has been performed in certain plants, but they are still not characterized in Triticum aestivum (bread wheat). Herein, we identified 34 TaCaCA superfamily proteins, which were classified into TaCAX, TaCCX, TaNCL, and TaMHX protein families based on their structural organization and evolutionary relation with earlier reported proteins. Since the T. aestivum comprises an allohexaploid genome, TaCaCA genes were derived from each A, B, and D subgenome and homeologous chromosome (HC), except chromosome-group 1. Majority of genes were derived from more than one HCs in each family that were considered as homeologous genes (HGs) due to their high similarity with each other. These HGs showed comparable gene and protein structures in terms of exon/intron organization and domain architecture. Majority of TaCaCA proteins comprised two Na_Ca_ex domains. However, TaNCLs consisted of an additional EF-hand domain with calcium binding motifs. Each TaCaCA protein family consisted of about 10 transmembrane and two α-repeat regions with specifically conserved signature motifs except TaNCL, which had single α-repeat. Variable expression of most of the TaCaCA genes during various developmental stages suggested their specified role in development. However, constitutively high expression of a few genes like TaCAX1-A and TaNCL1-B indicated their role throughout the plant growth and development. The modulated expression of certain genes during biotic (fungal infections) and abiotic stresses (heat, drought, salt) suggested their role in stress response. Majority of TaCCX and TaNCL family genes were found highly affected during various abiotic stresses. However, the role of individual gene needs to be established. The present study unfolded the opportunity for detail functional characterization of TaCaCA proteins and their utilization in future crop improvement programs.

Keywords: Ca2+/cation antiporters; TaCAX; TaCCX; TaMHX; TaNCL; Triticum aestivum; abiotic; biotic.

Figure 1

Distribution of the TaCaCA superfamily genes on A, B, and D subgenomes and chromosomes. Figure shows the frequency of TaCaCA genes on A, B, and D sub-genomes (A) and pictorial representation on various chromosomes (B). The figures of various chromosomes are adapted from the IWGSC website (www.wheatgenome.org). The TaCAX, TaCCX, TaNCL, and TaMHX genes are represented by red, green, blue and yellow bars, respectively. The homeologous genes in each family are shown by similar symbol.

Figure 2

Phylogenetic analysis of the CaCA superfamily proteins. The evolutionary relationship between CaCA proteins of T. aestivum, Arabidopsis, and rice was analyzed using conserved core domain (A) and α2-repeat motif (B). Figure shows tight clustering of the CAX, CCX, NCL, and MHX family proteins from various plants, which are shown with red, purple, green, and blue color lines and fonts, respectively. The CAX and CCX family proteins are further classified into two (Type 1A and Type 1B) and three (group 1, 2, and 3) categories, respectively. Orange and pink shaded regions show Type 1A and Type 1B CAX proteins, while yellow, blue and purple shaded regions represent group 1, 2, and 3 CCX proteins, respectively.

Figure 3

Gene and protein structure analysis. (A) Intron/exon configuration of TaCaCA superfamily genes. Exons and introns are shown as yellow boxes and black lines, respectively. Un-translated regions (UTRs) are shown as blue boxes. Various intron phases are represented as- 0; intron phase 0, 1; intron phase 1, 2; intron phase 2. (B) Figure shows domain architecture of TaCaCA proteins. The red, yellow and blue colored boxes represent Na_Ca_ex, Caca2 and EF-hand domains, respectively.

Figure 4

Multiple sequence alignment of CCX proteins. Figure shows alignment of CCX protein sequences of T. aestivum, Arabidopsis and rice. The group 1, 2, and 3 CCX sequences are separated by green lines. Conserved α1 and α2-repeat regions are shown in black colored boxes. The signature motifs “GNG(A/S)PD” in α1-repeat and “G(N/D)SxGD” in α2-repeat motif reported by Cai and Lytton (2004a) are shown in purple boxes. The predicted transmembrane (TM) spans are over-lined in blue color.

Figure 5

Multiple sequence alignments of NCL and MHX proteins. (A) Figure shows alignment of EF-hand domain and α-2 repeat region of NCL proteins from T. aestivum, Arabidopsis, and rice. Pink asterisks indicate calcium binding sites in EF-hand domain. (B) Alignment of α-1 and α-2 repeat regions of human NCXs (HsNCX1; NP_066920, HsNCX2; NP_055878 and HsNCX3; NP_891977) with MHX proteins from T. aestivum, Arabidopsis and rice. The signature motifs “GSSAPE” in α1-repeat and “GTSVPD” α2-repeat of human NCX protein are shown in pink coloured boxes. Residues involved in Na⁺/Ca²⁺ exchange in HsNCX1 are indicated by asterisks, while the red asterisks indicates the residues which are not conserved in the MHX proteins.

Figure 6

Relative expression profile of TaCaCA superfamily genes in various tissue developmental stages. Heat map shows relative expression profile of (A) TaCAX, (B) TaCCX, (C) TaMHX, and (D) TaNCL genes in three developmental stages of each root, leaf, stem, spike, and grain tissue. The developmental stages are shown in Zadoks scale.

Figure 7

Differential expression analysis of TaCaCA superfamily genes under biotic and abiotic stresses. The heat map shows differential expression pattern of (1) TaCAX genes under (A) biotic, (B) heat/drought, and (C) salt stress, (2) TaCCX genes under (D) biotic, (E) heat/drought and (F) salt stress (F), (3) TaNCL genes under (G) biotic, (H) heat/drought and (I) salt stress, and (4) TaMHX genes under (J) biotic, (K) heat/drought stress and (L) salt stress. The symbols shown in figure indicate-HS; heat stress, DS; drought stress, HD; combination of heat and drought stress, Bgt; after Blumeria graminis infection, Pst; after Puccinia striiformis inoculation.