Identification of the NF-Y Gene Family and Functional Analysis of PmNF-YA8 in Broomcorn Millet

Nuclear factor Y (NF-Y) transcription factors regulate the expression of target genes by specifically binding to the CCAAT element in the promoter region of target genes, and play a role in plant responses to abiotic stresses such as drought and salinity. Broomcorn millet exhibits strong tolerance to drought and barrenness, serving as an indispensable crop for saline-alkali and marginal lands. Systematic elucidation of the functions of the NF-Y gene family in broomcorn millet will provide a theoretical basis for enriching the genetic theory of crop stress tolerance and promoting drought-resistant breeding practices.

Genome data of broomcorn millet and bioinformatics approaches were employed to identify NF-Y family members, physicochemical properties, gene structures, construct phylogenetic trees, and predict conserved domains as well as cis-acting elements in promoters. Reverse Transcription Quantitative PCR (RT-qPCR) was used to determine the expression of these genes in different plant tissues, and the expression characteristics of PmNF-YA8 under drought stress were verified through transgenic functional validation in Arabidopsis thaliana.

Based on the proso millet reference genome, 33 PmNF-Y genes (including 11 PmNF-YA, 12 PmNF-YB, and 10 PmNF-YC) were identified, encoding proteins with lengths of 122-571 amino acids, isoelectric points of 4.74-10.19, and molecular weights of 13.58-59.17 kDa. Chromosomal localization analysis showed that these 33 genes (PmNF-YA1-PmNF-YA11, PmNF-YB1-PmNF-YB12, and PmNF-YC1-PmNF-YC10) were distributed across 15 chromosomes. Two types of drought stress-responsive elements were predicted in the promoter regions of PmNF-Y family members. A total of 3, 47, and 59 NF-Y orthologous gene pairs were identified between broomcorn millet and three other plant species (Arabidopsis thaliana, rice, and maize), respectively. RT-qPCR analysis revealed that PmNF-Y genes were widely expressed in tissues such as roots, stems, and leaves. The expression level of PmNF-YA8 was up-regulated in both XHS and NM9 varieties after drought stress. Transgenic Arabidopsis thaliana lines overexpressing PmNF-YA8 were generated, and phenotypic and physiological analyses showed that under 300 mmol·L^-1 mannitol stress, the average root length of transgenic plants (14.03 mm) was longer than that of wild-type (WT) plants (9.07 mm). Additionally, transgenic plants had a lower MDA increase range (55.23%-57.12%) compared with WT plants (100.78%), with SOD and POD activities increased by 24.28%-27.43% and 163.57%-341.33%, respectively, and Pro content (151.31-175.14 μg·g^-1) higher than that of WT plants (143.78 μg·g^-1).

A total of 33 NF-Y family members were identified in broomcorn millet. The PmNF-YA8 gene was cloned from broomcorn millet and heterologously transformed into Arabidopsis thaliana. Its heterologous expression enhanced the drought stress tolerance of Arabidopsis thaliana plants by regulating the expression of stress-related genes, thus making PmNF-YA8 a key candidate gene for modulating drought tolerance in broomcorn millet.