Systematic analysis of sequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize

Abstract

Background: Members of the homeodomain-leucine zipper (HD-Zip) gene family encode transcription factors that are unique to plants and have diverse functions in plant growth and development such as various stress responses, organ formation and vascular development. Although systematic characterization of this family has been carried out in Arabidopsis and rice, little is known about HD-Zip genes in maize (Zea mays L.).

Methods and findings: In this study, we described the identification and structural characterization of HD-Zip genes in the maize genome. A complete set of 55 HD-Zip genes (Zmhdz1-55) were identified in the maize genome using Blast search tools and categorized into four classes (HD-Zip I-IV) based on phylogeny. Chromosomal location of these genes revealed that they are distributed unevenly across all 10 chromosomes. Segmental duplication contributed largely to the expansion of the maize HD-ZIP gene family, while tandem duplication was only responsible for the amplification of the HD-Zip II genes. Furthermore, most of the maize HD-Zip I genes were found to contain an overabundance of stress-related cis-elements in their promoter sequences. The expression levels of the 17 HD-Zip I genes under drought stress were also investigated by quantitative real-time PCR (qRT-PCR). All of the 17 maize HD-ZIP I genes were found to be regulated by drought stress, and the duplicated genes within a sister pair exhibited the similar expression patterns, suggesting their conserved functions during the process of evolution.

Conclusions: Our results reveal a comprehensive overview of the maize HD-Zip gene family and provide the first step towards the selection of Zmhdz genes for cloning and functional research to uncover their roles in maize growth and development.

Figure 1. Phylogenetic relationship and gene structure of the 55 predicted maize HD-Zip proteins.

NJ tree (the part of left side): The unrooted tree was generated with MEGA4.0 program using the full-length amino acid sequences of the 55 maize HD-Zip proteins. The bootstrap values are indicated at the branches in black numbers, and the proteins were named according to their gene codes (see Table S1). Gene structure (the part of right side): Exons and introns are indicated by white boxes and single lines, respectively. Thick gray lines represent the untranslated regions (UTRs). The length of each HD-Zip gene can be estimated using the scale at the bottom.

Figure 2. Phylogenetic relationships of maize, rice and Arabidopsis HD-Zip proteins.

The tree was generated with MEGA4.0 program using the NJ method. Bootstrap values are indicated at the branches in black numbers. The unrooted tree in the lower-left corner was constructed using the same method.

Figure 3. Distribution of 20 putative conserved motifs in maize HD-Zip proteins.

Motifs of HD-Zip proteins were identified by MEME web server. Note that the length of each box in the proteins does not represent the actual motif size, and the colored boxes were ordered manually according to the results of the MEME analysis. The conserved amino acid sequences and length of each motif are shown in table 2.

Figure 4. Chromosomal locations of maize HD-Zip genes on all 10 chromosomes.

Markers next to the gene names represent the classes to which each HD-Zip gene belongs (HD-Zip I, ▴; HD-Zip II, ★; HD-Zip III, ▾; HD-Zip IV, ). Chromosome numbers are shown at the top of each vertical gray bar, and the names on the left side of each chromosome correspond to the approximate locations of each HD-Zip gene. Genes involved in segmental duplication are joined by dashed lines, and the red rectangle indicates the gene cluster on each chromosome. The scale is in megabases (Mb).

Figure 5. Distribution of major stress-related cis-elements in the promoter sequences of the 17 Zmhdz I genes.

Putative ABRE, LTRE and DRE core sequences are represented by different symbols as indicated. The cis-elements distributed on the sense strand and reverse strand are indicated above and below the black lines, respectively.

Figure 6. Expression patterns of the 17 Zmhdz I genes under drought stress.

Relative expressions of Zmhdz I genes were examined by qRT-PCR and normalized by Actin 1 expression. The Y axis is the scale of the relative expression level. The X axis is the time course of drought stress treatment. CK, normal plant, D1 (slight stress), leaves sampled at 3 h after treatment, D2 (moderate stress), leaves sampled at 6 h after treatment and D3 (severe stress), leaves sampled at 12 h after treatment. Date represent mean ± SD.