GhSOC1s Evolve to Respond Differently to the Environmental Cues and Promote Flowering in Partially Independent Ways

Abstract

Gossypium hirsutum is most broadly cultivated in the world due to its broader adaptation to the environment and successful breeding of early maturity varieties. However, how cotton responds to environmental cues to adjust flowering time to achieve reproductive success is largely unknown. SOC1 functions as an essential integrator for the endogenous and exogenous signals to maximize reproduction. Thus we identified six SOC1-like genes in Gossypium that clustered into two groups. GhSOC1-1 contained a large intron and clustered with monocot SOC1s, while GhSOC1-2/3 were close to dicot SOC1s. GhSOC1s expression gradually increased during seedling development suggesting their conserved function in promoting flowering, which was supported by the early flowering phenotype of 35S:GhSOC1-1 Arabidopsis lines and the delayed flowering of cotton silencing lines. Furthermore, GhSOC1-1 responded to short-day and high temperature conditions, while GhSOC1-2 responded to long-day conditions. GhSOC1-3 might function to promote flowering in response to low temperature and cold. Taken together, our results demonstrate that GhSOC1s respond differently to light and temperature and act cooperatively to activate GhLFY expression to promote floral transition and enlighten us in cotton adaptation to environment that is helpful in improvement of cotton maturity.

Keywords: Gossypium hirsutum L.; SOC1-like gene; environmental response; evolution; flowering time control.

FIGURE 1

Sequence analysis of GhSOC1s. (A) Gene structure of GhSOC1s. (B) Sequence conservation of the M, I, K, C domain of GhSOC1s. (C) Structure prediction of GhSOC1s. The interface of the MADS domain with DNA groove are marked in red. The orange color highlights the important residues for dimerization and the green color highlights the important residues for tetramerization. Triangles mark the varied Leucine residues. (D) 3-D structure of the K domain. The varied Leucine residues of GhSOC1-1 are highlighted in red. Close-up display the varied interacting residues depicted as sticks.

FIGURE 2

Phylogenetic analysis of SOC1-like genes in cotton and other plants. SsSOC1a and SsSOC1b in Saccharum spontaneum was download from the article (Fatima et al., 2020). Other SOC1 sequences were downloaded from NCBI and their accession numbers are given in Supplementary Table 2.

FIGURE 3

Prediction of cis-acting elements in GhSOC1s genome. The coding region and non-coding region are presented in gray and black lines.

FIGURE 4

Expression analysis of GhSOC1s. (A) Tissue specific expression of GhSOC1s in G. hirsutum. (B) Expression pattern of GhSOC1s in a flower. Pi, pistil; St, stamen; Pe, petal; Ca; calyx; To, torus. (C) Expression changes of GhSOC1s during seedling development. 3TLS, 4TLS, and 5TLS demonstrates the third, fourth and fifth true leaf stages, respectively. The expression is normalized to GhHIS and the maximum expression is set as 100%. Error bars represent the standard deviations of three biological replicates. (D) Subcellular localization of GhSOC1-1 in tobacco leaves. GFP, GFP fluorescence; DAPI, fluorescence of 40,6-diamino-2-phenylindole; BF, Bright field; Merge, merge of GFP, DAPI, and BF images.

FIGURE 5

Overexpression of GhSOC1-1 promotes flowering of Arabidopsis. (A) Genotype verification of positive plants in T1 generation. M, DNA molecular weight marker DL 2000; A-C, water control, WT control and positive control; 1-4, PCR detection of positive plants. (B) Phenotype of T3 generation line. (C) Relative expression of GhSOC1-1 and statistics of flowering time in transgenic lines and wild-type Arabidopsis. Asterisks indicate statistically significant differences according to Student’s t-test (**P < 0.01). (D) Expression of AtLFY, AtAP1, and AtFT in transgenic and wild-type Arabidopsis. The expression is normalized to AtTUB2 and the maximum expression is set as 100%. Error bars represent the standard deviations of three biological replicates.

FIGURE 6

VIGS of GhSOC1s in G. hirsutum delays flowering time. (A) Albino phenotype of TRV:CLA1 as a control. (B) Delayed flowering phenotype of VIGS plants. Red triangles point the flower buds displayed in the left. CK, empty vector control; VI, silence plants. (C) Detection of the efficiency of gene silencing. The expression was normalized to GhHIS and the maximum expression is set as 100%. Error bars represent the standard deviations of three biological replicates. (D,E) Maturity traits of VIGS plants in branch of first square (D) and days from sowing until square appears (E). Asterisks indicate statistically significant differences among 15 individuals according to Student’s t-test (**P < 0.01). (F) Expression of GhLFY, GhAP1, and GhFT in Silencing and control plants. The expression is normalized to GhHIS and the maximum expression is set as 100%. Error bars represent the standard deviations of three biological replicates.

FIGURE 7

Responses of GhSOC1s to light and temperature. (A) Quick response of GhSOC1s to day length shift from long-day to short-day for 48 h under 28°C. (B) Quick response of GhSOC1s to temperature shift for 48 h examined in seedlings grown under 28°C and long-day condition. (C) GhSOC1s expression is examined under continues long-day and short-day conditions under 28°C at 3TLS and 5TLS. (D) GhSOC1s expression is examined under different temperature conditions under long-day at 3TLS and 5TLS. The expression is normalized to GhHIS. Error bars represent the standard deviations of three biological replicates. Different letters suggest significant differences calculated by Student’s t-test (P < 0.01).

FIGURE 8

Proposed regulatory model of GhSOC1s. HAT, high ambient temperature; LAT, low ambient temperature; LD, long-day condition; SD, short-day condition.