Proton-pumping pyrophosphatase homeolog expression is a dynamic trait in bread wheat (Triticum aestivum)

Abstract

Proton-pumping pyrophosphatases (H⁺-PPases) have been shown to enhance biomass and yield. However, to date, there has been little work towards identify genes encoding H⁺-PPases in bread wheat (Triticum aestivum) (TaVPs) and limited knowledge on how the expression of these genes varies across different growth stages and tissue types. In this study, the IWGSC database was used to identify two novel TaVP genes, TaVP4 and TaVP5, and elucidate the complete homeolog sequences of the three known TaVP genes, bringing the total number of bread wheat TaVPs from 9 to 15. Gene expression levels of each TaVP homeolog were assessed using quantitative real-time PCR (qRT-PCR) in four diverse wheat varieties in terms of phenotypic traits related to high vacuolar pyrophosphatase expression. Homeolog expression was analyzed across multiple tissue types and developmental stages. Expression levels of the TaVP homeologs were found to vary significantly between varieties, tissues and plant developmental stages. During early development (Z10 and Z13), expressions of TaVP1 and TaVP2 homeologs were higher in shoot tissue than root tissue, with both shoot and root expression increasing in later developmental stages (Z22). TaVP2-D was expressed in all varieties and tissue types and was the most highly expressed homeolog at all developmental stages. Expression of the TaVP3 homeologs was restricted to developing grain (Z75), while TaVP4 homeolog expression was higher at Z22 than earlier developmental stages. Variation in TaVP4B was detected among varieties at Z22 and Z75, with Buck Atlantico (high biomass) and Scout (elite Australian cultivar) having the highest levels of expression. These findings offer a comprehensive overview of the bread wheat H⁺-PPase family and identify variation in TaVP homeolog expression that will be of use to improve the growth, yield, and abiotic stress tolerance of bread wheat.

Keywords: gene expression; gene identification; homeolog; synteny analysis; vacuolar pyrophosphatases.

FIGURE 1

Intron‐exon analysis and amino acid alignment of TaVP homeologs. (a) Intron‐exon structures of TaVP wheat homeologs. Exons and introns are represented by blue and gray lines, respectively. Scale bars are indicated for both, with sequences shown in a 5′ to 3′ direction. (b) Amino acid sequence alignment of a highly conserved region (amino acid residues 285–355) containing a pyrophosphatase binding domain and three residues involved in proton translocation (indicated by stars). Alignment contains the TaVP homeologs and type I orthologs from Vigna radiata (VrVP1), Arabidopsis thaliana (AtAVP1), Hordeum vulgare (HVP1, HVP3, HVP10), Zea mays (ZmVPP1), and Oryza sativa (OVP1–6), as well as type II orthologs of Arabidopsis thaliana (AtAVP2;1, AtVHP2;2) and Zea mays (ZmGPP). Shading indicates conservation level of amino acid residues where dark blue = 100%, light blue = 80–99%, purple = 60–79%, and no color = < 60%

FIGURE 2

Phylogenetic analysis of type I H^+‐PPases. Unrooted phylogenetic tree of type I H⁺‐PPase proteins from Vigna radiata (VrVP1), Arabidopsis thaliana (AVP1), Hordeum vulgare (HVP1, HVP10, HVP3), Zea mays (ZmVPP1), Oryza sativa (OVP1–6), and bread wheat homeologs (TaVP1–TaVP4). Phylogeny was created in MEGA6© via the maximum‐likelihood method and formatted with iTOL. Analysis was validated with 1,000 bootstrap replicates, and bootstrap support for each node is indicated. Scale bar represents 0.1 amino acid substitutions per residue. Dotted lines are for labeling purposes only and are not indicative of branch length

FIGURE 3

TaVP expression profile at seedling stage. Expression of TaVP homeologs in shoot (white columns) and roots (gray columns) of 6‐day‐old wheat seedlings (Z10). Expression data displayed as normalized relative quantity (NRQ) for four bread wheat varieties, Vigour18, Mocho de Espiga Branca, Scout, and Buck Atlantico. Values are means of three to five biological replicates and three technical replicates. Error bars represent standard error of the mean

FIGURE 4

TaVP expression profile at third leaf stage. Expression of TaVP homeologs in third leaf (white columns), third leaf sheath (dotted columns), and root (gray columns) tissue of plants with fully expanded 3rd leaves (Z13). Expression data displayed as normalized relative quantity (NRQ) for four bread wheat varieties, Vigour18, Mocho de Espiga Branca, Scout, and Buck Atlantico. Values are means of three to five biological replicates and three technical replicates. Error bars represent standard error of the mean

FIGURE 5

TaVP expression profile at second tiller stage. Expression of TaVP homeologs in third leaf (white columns), third leaf sheath (dotted columns), first leaf of the first tiller (black columns), first leaf of the second tiller (bricked columns), and root (gray columns) tissue of tillering plants (Z22). Expression data displayed as normalized relative quantity (NRQ) for four bread wheat varieties, Vigour18, Mocho de Espiga Branca, Scout, and Buck Atlantico. Values are means of three to five biological replicates and three technical replicates. Error bars represent standard error of the mean

FIGURE 6

TaVP expression profile at grain development stage. Expression of TaVP homeologs in the developing grain of field grown plants (Z75). Expression data displayed as normalized relative quantity (NRQ) for four bread wheat varieties, Vigour18, Mocho de Espiga Branca, Scout, and Buck Atlantico. Values are means of three to five biological replicates and three technical replicates. Error bars represent standard error of the mean

FIGURE 7

Heat map of TaVP gene expression through time. Each column represents an individual TaVP homeolog, with developmental stages (Z10, Z13, Z22, and Z75), varieties (Vigour18, Mocho de Espiga Branca [Mocho], Scout, and Buck Atlantico) and tissue types (third leaf, sheath, first tiller leaf, second tiller leaf, root, and grain) displayed along the y axis. Colors indicate TaVP expression levels, with white and dark blue corresponding to the lowest and highest expression levels, respectively. Gray shading indicates samples for which no qRT‐PCR expression data were obtained