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. 2021 Jul 6:12:686852.
doi: 10.3389/fgene.2021.686852. eCollection 2021.

Genome-Wide Identification of the Early Flowering 4 (ELF4) Gene Family in Cotton and Silent GhELF4-1 and GhEFL3-6 Decreased Cotton Stress Resistance

Miaomiao Tian  1   2 , Aimin Wu  2 , Meng Zhang  2 , Jingjing Zhang  2 , Hengling Wei  2 , Xu Yang  2 , Liang Ma  2 , Jianhua Lu  2 , Xiaokang Fu  2 , Hantao Wang  2 , Shuxun Yu  1   2
Affiliations

Genome-Wide Identification of the Early Flowering 4 (ELF4) Gene Family in Cotton and Silent GhELF4-1 and GhEFL3-6 Decreased Cotton Stress Resistance

Miaomiao Tian et al. Front Genet. .

Abstract

The early flowering 4 (ELF4) family members play multiple roles in the physiological development of plants. ELF4s participated in the plant biological clock's regulation process, photoperiod, hypocotyl elongation, and flowering time. However, the function in the ELF4s gene is barely known. In this study, 11, 12, 21, and 22 ELF4 genes were identified from the genomes of Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. There ELF4s genes were classified into four subfamilies, and members from the same subfamily show relatively conservative gene structures. The results of gene chromosome location and gene duplication revealed that segmental duplication promotes gene expansion, and the Ka/Ks indicated that the ELF4 gene family has undergone purification selection during long-term evolution. Spatio-temporal expression patterns and qRT-PCR showed that GhELF4 genes were mainly related to flower, leaf, and fiber development. Cis-acting elements analysis and qRT-PCR showed that GhELF4 genes might be involved in the regulation of abscisic acid (ABA) or light pathways. Silencing of GhELF4-1 and GhEFL3-6 significantly affected the height of cotton seedlings and reduced the resistance of cotton. The identification and functional analysis of ELF4 genes in upland cotton provide more candidate genes for genetic modification.

Keywords: ELF4; cotton; expression patterns; fiber; gene silencing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Chromosomal distribution of ELF4s in Gossypium raimondii, G. barbadense, G. arboreum, and G. hirsutum. The scale represents megabases (Mb). The chromosome numbers are indicated on the left of each vertical bar.
FIGURE 2
FIGURE 2
Phylogenetic tree of ELF4s. The 95 ELF4 protein sequences of G. raimondii, G. arboreum, G. hirsutum, G. barbadense, A. thaliana, Populus trichocarpa, Oryza sativa, Theobroma cacao, and Glycine max were aligned with ClustalX 2.0, and the phylogenetic tree was generated.
FIGURE 3
FIGURE 3
The gene pairs of segment duplication in GrELF4s, GaELF4s, and GhELF4s. The chromosomes of G. raimondii, G. arboretum, and G. hirsutum are filled with green, blue, and yellow, respectively. Gene pairs involved in segment duplication are linked by a line. The scale on the circle is in megabases (Mb).
FIGURE 4
FIGURE 4
The gene structure and conserved protein motif of four cotton species ELF4. (A) The phylogenetic tree analysis of the ELF4 gene. (B) Analysis of exons and intron in ELF4 Genes. (C) The predicted ELF4 genes motif.
FIGURE 5
FIGURE 5
The cis-acting elements of stress-related and plant hormone response in the GhELF4 genes promoter regions. The proportion of each type of related cis-acting elements in 21 GhELF4 genes.
FIGURE 6
FIGURE 6
Expression prediction of GhELF4s in different tissues. 0 to 25 DPA indicates 0, 5, 10, 15, 20, and 25 days after anthesis. The scale bars in the upper right corner represent log2-transformed FPKM values.
FIGURE 7
FIGURE 7
Relative expression levels of GhELF4s in different tissues. (A) Expression analysis of GhELF4 in roots, stems, leaves and flowers. (B) Expression analysis of GhELF4s in fiber developmental stages. 0, 5, 10, 15, 20, and 25 represent 0, 5, 10, 15, 20, and 25 DPA fibers. Error bars showed the standard deviation of three biological replicates.
FIGURE 8
FIGURE 8
Expression analysis of GhELF4s under ABA and light treatment via qRT-PCR. (A) Expression analysis of GhELF4s under ABA treatment via qRT-PCR. The TM-1 cotton seedlings were sprayed with water and 100 μM ABA during the two-leaf flattening period, and cotton cotyledon samples were taken at 3, 6, 9, 12, and 24 h. Error bars show the standard deviation of three biological replicates. (B) qRT-PCR results of GhELF4s gene response to light at different times. Error bars show the standard deviation of three biological replicates.
FIGURE 9
FIGURE 9
Silencing of GhELF4-1 and GhEFL3-6 decreased resistance to salt stress in cotton. (A) Comparison of pYL156-GhELF4-1 plant phenotype and pYL-156-cal1, pYL-156; (B) Comparison of pYL156-GhEFL3-6 plant phenotype and pYL-156-cal1, pYL-156; (C) The relative expression levels of pYL-156 and pYL-156-GhELF4-1; (D) The relative expression levels of pYL-156 and pYL-156-GhEFL3-6; (E) Plant height statistics of pYL-156, pYL-156-GhELF4-1 and pYL-156-GhEFL3-6; (F) VIGS plant Peroxidase (POD) activity assay; (G) VIGS plant catalase (Silva et al., 2020) activity assay; **P < 0.01, *P < 0.05.
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