Genome-Wide Comprehensive Analysis of the GASA Gene Family in Populus

Abstract

Gibberellic acid-stimulated Arabidopsis (GASA) proteins, as cysteine-rich peptides (CRPs), play roles in development and reproduction and biotic and abiotic stresses. Although the GASA gene family has been identified in plants, the knowledge about GASAs in Populus euphratica, the woody model plant for studying abiotic stress, remains limited. Here, we referenced the well-sequenced Populus trichocarpa genome, and identified the GASAs in the whole genome of P. euphratica and P. trichocarpa. 21 candidate genes in P. trichocarpa and 19 candidate genes in P. euphratica were identified and categorized into three subfamilies by phylogenetic analysis. Most GASAs with signal peptides were located extracellularly. The GASA genes in Populus have experienced multiple gene duplication events, especially in the subfamily A. The evolution of the subfamily A, with the largest number of members, can be attributed to whole-genome duplication (WGD) and tandem duplication (TD). Collinearity analysis showed that WGD genes played a leading role in the evolution of GASA genes subfamily B. The expression patterns of P. trichocarpa and P. euphratica were investigated using the PlantGenIE database and the real-time quantitative PCR (qRT-PCR), respectively. GASA genes in P. trichocarpa and P. euphratica were mainly expressed in young tissues and organs, and almost rarely expressed in mature leaves. GASA genes in P. euphratica leaves were also widely involved in hormone responses and drought stress responses. GUS activity assay showed that PeuGASA15 was widely present in various organs of the plant, especially in vascular bundles, and was induced by auxin and inhibited by mannitol dramatically. In summary, this present study provides a theoretical foundation for further research on the function of GASA genes in P. euphratica.

Keywords: GASA genes; Populus; bioinformatics; growth and development; phytohormone.

Figure 1

Positions of PtrGASA and PeuGASA genes on P. trichocarpa chromosomes. The P. trichocarpa chromosomes where the PtrGASA genes are located are shown in orange. The reference P. trichocarpa chromosomes where the PeuGASA genes are located are shown in blue. The positions of genes and the size of chromosomes can be estimated using the scale on the left of the picture.

Figure 2

Neighbor-Joining phylogenetic tree of GASA domains. The 97 conserved GASA domains of GASAs in sequenced plants, Selaginella moellendorffii, Picea abies, Oryza sativa ssp. japonica, Arabidopsis thaliana, Populus trichocarpa, and Populous euphratica were analyzed. The tree was divided into three phylogenetic subgroups, which were shown in different colors.

Figure 3

Analysis of the evolutionary relationship among GASA genes. (a) Evolution analysis of GASA gene family in P. trichocarpa; (b) Collinear analysis of GASA gene family between the Arabidopsis and P. trichocarpa. Different sizes of fan rings represented different sizes of chromosomes. The colored lines represented collinear GASA gene pairs.

Figure 4

Gene structures and motif analysis of GASA gene family in P. euphratica and P. trichocarpa. (a) Phylogenetic tree of PtrGASAs and PeuGASAs. Subfamily A/B/C were represented by green, red and blue respectively; (b) Gene structures of PtrGASAs and PeuGASAs. Upstream or downstream non-coding regions were represented by green rounded rectangles, exons by orange rounded rectangles, and introns by gray lines; (c) Conserved motifs of PtrGASAs and PeuGASAs. Conservative motifs were presented in different colored boxes. The length of each nucleotide sequence or protein sequence can be estimated using the scale below the picture.

Figure 5

Subcellular localization of PeuGASA proteins. The Super::GFP and Super::PeuGASA-GFP fusion proteins were transiently expressed in tobacco.

Figure 6

Expression patterns of GASA genes in different P. euphratica tissues. Data were presented as mean ± standard error (SE). Subsequent multiple comparisons were evaluated based on the least significant difference (LSD) test to calculate p-values (Asterisks denoted significant differences: * p ≤ 0.05; *** p ≤ 0.01).

Figure 7

Expression analysis of PeuGASA genes in response to SA, ABA, MeJA, EBR, GA₃ and IAA treatments. Numbers are the time (h) of treatment. The heat map was based on the calculation of three replicates of qRT-PCR data and the transformation of log2 algorithm. Multiple comparisons were evaluated based on the least significant difference (LSD) test to calculate p-values (Asterisks denoted significant differences: * p ≤ 0.05; ** p ≤ 0.01).

Figure 8

Expression analysis of PeuGASA genes in response to drought stress. W-A, W-B, W-C, and W-D respectively represented four drought treatments with different soil volumetric water content (soil-VWC): control (43 ± 1% soil-VWC), mild drought (33 ± 1% soil-VWC), moderate drought (23 ± 1% soil-VWC) and severe drought (13 ± 1% soil-VWC). Data were presented as mean ± standard error (SE). Subsequent multiple comparisons were evaluated based on the least significant difference (LSD) test to calculate p-values (Asterisks denoted significant differences: * p ≤ 0.05; *** p ≤ 0.01).

Figure 9

The cis-acting elements of PeuGASA genes. (a) Numbers and gradient red colors indicate the number of cis-acting elements; (b) Color-coded histograms indicate the number of cis-acting elements of genes in each category; (c) Pie charts show the proportion of different cis-acting elements in each category.

Figure 10

GUS activity of the PeuGASA15 promoter. (a) 3-day-old plant; (b) 4-day-old plant; (c) 7-day-old plant; (d,e) Cotyledons and young leaves of 7-day-old plant; (f) 14d-day-old plant; (g) inflorescence; (h–k) Expanding flowers; (l) Fading flowers; (m) Expanded flower; (n) Petal; (o) Stamen; (p,q) Silique.

Figure 11

Three-week-old ProGASA15::GUS transgenic Arabidopsis seedlings growing on 1/2 MS AGAR plates were transplanted into medium without or containing 300 mM mannitol, 150 mM NaCl, 20 μM ABA, 20 mg/L IAA, respectively.