SiNF-YC2 Regulates Early Maturity and Salt Tolerance in Setaria italica

Abstract

Early maturity is an important agronomic trait in most crops, because it can solve the problem of planting in stubble for multiple cropping as well as make full use of light and temperature resources in alpine regions, thereby avoiding damage from low temperatures in the early growth period and early frost damage in the late growth period to improve crop yield and quality. The expression of genes that determine flowering affects flowering time, which directly affects crop maturity and indirectly affects crop yield and quality. Therefore, it is important to analyze the regulatory network of flowering for the cultivation of early-maturing varieties. Foxtail millet (Setaria italica) is a reserve crop for future extreme weather and is also a model crop for functional gene research in C4 crops. However, there are few reports on the molecular mechanism regulating flowering in foxtail millet. A putative candidate gene, SiNF-YC2, was isolated based on quantitative trait loci (QTL) mapping analysis. Bioinformatics analysis showed that SiNF-YC2 has a conserved HAP5 domain, which indicates that it is a member of the NF-YC transcription factor family. The promoter of SiNF-YC2 contains light-response-, hormone-, and stress-resistance-related elements. The expression of SiNF-YC2 was sensitive to the photoperiod and was related to the regulation of biological rhythm. Expression also varied in different tissues and in response to drought and salt stress. In a yeast two-hybrid assay, SiNF-YC2 interacted with SiCO in the nucleus. Functional analysis suggested that SiNF-YC2 promotes flowering and improves resistance to salt stress.

Keywords: QTL mapping; Setaria italica; SiNF-YC2; abiotic stress; early maturity; photoperiod sensitivity.

Figure 1

QTL mapping and candidate gene screening for early-maturity traits in foxtail millet. (a) Heading phenotype of parent plants. (b) Location of SiNF-YC2 on the chromosome.

Figure 2

Bioinformatics analysis of SiNF-YC2. (a) Conserved domain prediction. (b) Protein secondary structure analysis. (c) cis-Acting elements in SiNF-YC2 promoter. (d) Evolutionary analysis of SiNF-YC2 (At: Arabidopsis thaliana; Zm: Zea mays; Si: Setaria italica; Os: Oryza sativa). The asterisk indicates SiNF-YC2. (e) Protein tertiary structure prediction. (f) Cloning of SiNF-YC2 (M: 2 kb DNA marker).

Figure 3

Expression analysis of SiNF-YC2. (a) Short-day (SD) photoperiod. (b) Long-day (LD) photoperiod. (c) Relative expression of SiNF-YC2 in different tissues of foxtail millet. Expression of SiNF-YC2 under (d) 20% PEG6000, (e) 200 mM NaCl, and (f) 100 μM ABA treatments. Different letters on columns indicate significant differences at the level of 0.05. p < 0.05 indicates significant differences between statistical data, p > 0.05 indicates insignificant differences between statistical data, and p < 0.01 indicates extremely significant differences between statistical data. Different lowercase letters indicate differences between the control and treatment at the same time point; different capital letters indicated differences in SiNF-YC2 expression in millet exposed to stress at different time points.

Figure 4

SiNF-YC2 and SiCO interact in the nucleus. (a) Subcellular localization of SiNF-YC2 protein. (b) Interaction of SiNF-YC2 and SiCO. DDO: two-deficient SD/-Leu-Trp medium; QDO: four-deficient SD/-Ade-His-Leu-Trp medium.

Figure 5

SiNF-YC2 positively regulates flowering time. (a) PCR detection of transgenic Arabidopsis (M: 2 kb DNA marker; +: positive control; −: negative control; WT: wild-type Arabidopsis; 1–10: transgenic Arabidopsis OE-1 to OE-10). (b) Quantitative screening of SiNF-YC2. (c) Flowering phenotypes of wild-type and transgenic Arabidopsis. (d) Flowering time statistics. (e) Rosette leaf phenotypes of wild-type and transgenic Arabidopsis. Different letters on columns indicate significant differences at the level of 0.05. p < 0.05 indicates significant differences between statistical data, p > 0.05 indicates insignificant differences between statistical data, and p < 0.01 indicates extremely significant differences between statistical data. Different lowercase letters indicate differences between different strains.

Figure 6

Comparison of transgenic Arabidopsis germination under different stress treatments. Response of transgenic Arabidopsis treated with (a–c) mannitol, (d–f) ABA, and (g–k) salt. (a,d,g) Germination rate, (b,e,h) root length, (c,f,i) root length data analysis, (j) salt stress at seedling stage, and (k) survival rate. WT: wild type. OE: SiNF-YC2 transgenic Arabidopsis. Bar = 1.5 cm. Different letters on columns indicate significant differences at the level of 0.05. p < 0.05 indicates significant differences between statistical data, p > 0.05 indicates insignificant differences between statistical data, and p < 0.01 indicates extremely significant differences between statistical data. Different lowercase letters indicate differences between different strains.