Genome-Wide Identification of the Eucalyptus urophylla GATA Gene Family and Its Diverse Roles in Chlorophyll Biosynthesis

Abstract

GATA transcription factors have been demonstrated to play key regulatory roles in plant growth, development, and hormonal response. However, the knowledge concerning the evolution of GATA genes in Eucalyptus urophylla and their trans-regulatory interaction is indistinct. Phylogenetic analysis and study of conserved motifs, exon structures, and expression patterns resolved the evolutionary relationships of these GATA proteins. Phylogenetic analysis showed that EgrGATAs are broadly distributed in four subfamilies. Cis-element analysis of promoters revealed that EgrGATA genes respond to light and are influenced by multiple hormones and abiotic stresses. Transcriptome analysis revealed distinct temporal and spatial expression patterns of EgrGATA genes in various tissues of E. urophylla S.T.Blake, which was confirmed by real-time quantitative PCR (RT-qPCR). Further research revealed that EurGNC and EurCGA1 were localized in the nucleus, and EurGNC directly binds to the cis-element of the EurGUN5 promoter, implying its potential roles in the regulation of chlorophyll synthesis. This comprehensive study provides new insights into the evolution of GATAs and could help to improve the photosynthetic assimilation and vegetative growth of E. urophylla at the genetic level.

Keywords: Eucalyptus; GATA; evolution; photosynthesis; transcription factors.

Figure 1

Phylogenetic relationships between GATA proteins from Arabidopsis, poplar, rice, and Eucalyptus. The GATA members of Eucalyptus grandis were classified into four subfamilies together with the homologous genes of other plants. A, B, C, and D represent the four subfamilies, respectively.

Figure 2

Predicted Eucalyptus grandis GATA (EgrGATA) protein phylogeny, conserved amino acid motifs, and gene structures. (A) Unrooted tree of EgrGATA proteins based on maximum-likelihood method. (B) Position of exons and UTRs in the EgrGATA gene models (yellow line represents exons, and blue line represents UTRs). (C) Composition and distribution of overrepresented amino acid motifs (sequences of conserved motifs are given in Figure S1).

Figure 3

Analysis of cis-elements in the promoters of EgrGATA genes. (A) Overview of the types and numbers of cis-elements of the four subfamilies identified from the PLANTCARE database. (B) The number of light-responsive, hormone-responsive, stress-, and plant-growth-related elements present in the promoter region of each gene.

Figure 4

Distribution of EgrGATA genes on the chromosomes of Eucalyptus grandis and synteny analysis of GATA genes. (A) Chromosomal distributions of EgrGATA genes. Color gradient from blue to red on the chromosomes indicates low to high gene density, respectively. (B) Synteny analyses of GATA genes between Eucalyptus grandis and other module plant species (A. thaliana and P. trichocarpa). Green lines indicate the collinear blocks within Eucalyptus and other plant genomes.

Figure 5

Tissue-specific gene expression patterns of 22 EgrGATA genes. The expression patterns of genes in terminal bud (TB), juvenile leaves (JL), transition leaves (TL), mature leaves (ML), senescent leaves (SL), young stem (S1), transition stem (S2), mature stem (S3), bark (BA), developing secondary xylem (DSX), and mature xylem (MX). The red and blue colors indicate the high and low transcript abundance, respectively. Expressions normalized by log2.

Figure 6

The qRT-PCR analyses of EgrGATA genes in leaves and stems. The qRT-PCR analyses of eight genes expressed in terminal bud (TB), spire leaves (SL), transition leaves (TL), mature leaves (ML), senescent leaves (SL), and developing secondary xylem (DSX). Photosystem I light-harvesting complex gene 3 (LHCA3). The different lowercase letters show statistically significant differences by the Tukey-Kramer multiple comparison test at p < 0.05.

Figure 7

EurGNC is a nuclear-localized transcriptional regulator. (A) Subcellular localization of EurGNC and EurCGA1 proteins. Fluorescence signals of GFP were detected in tobacco leaf epidermal cells. Left panel, DAPI and GFP image; middle panel, bright field; and right panel, merging of GFP and bright field. Bar, 25 µm. (B) Yeast one-hybrid assay to detect whether EurGNC regulates downstream genes (EurGUN5 and EurLHCB3). AbA (100 ng/L) was used to repress the autoactivation. Empty pGADT7 (AD) and AbAi were used as negative controls, while pGADT7-p53 and pAbAi-53 were used as positive controls.