Genome-wide identification of BcGRF genes in flowering Chinese cabbage and preliminary functional analysis of BcGRF8 in nitrogen metabolism

Abstract

Growth-regulating factors (GRFs) are a unique family of transcription factors with well-characterized functions in plant growth and development. However, few studies have evaluated their roles in the absorption and assimilation of nitrate. In this study, we characterized the GRF family genes of flowering Chinese cabbage (Brassica campestris), an important vegetable crop in South China. Using bioinformatics methods, we identified BcGRF genes and analyzed their evolutionary relationships, conserved motifs, and sequence characteristics. Through genome-wide analysis, we identified 17 BcGRF genes distributed on seven chromosomes. A phylogenetic analysis revealed that the BcGRF genes could be categorized into five subfamilies. RT-qPCR analysis showed that BcGRF1, 8, 10, and 17 expression clearly increased in response to nitrogen (N) deficiency, particularly at 8 h after treatment. BcGRF8 expression was the most sensitive to N deficiency and was significantly correlated with the expression patterns of most key genes related to N metabolism. Using yeast one-hybrid and dual-luciferase assays, we discovered that BcGRF8 strongly enhances the driving activity of the BcNRT1.1 gene promoter. Next, we investigated the molecular mechanism by which BcGRF8 participates in nitrate assimilation and N signaling pathways by expressing it in Arabidopsis. BcGRF8 was localized in the cell nucleus and BcGRF8 overexpression significantly increased the shoot and root fresh weights, seedling root length, and lateral root number in Arabidopsis. In addition, BcGRF8 overexpression considerably reduced the nitrate contents under both nitrate-poor and -rich conditions in Arabidopsis. Finally, we found that BcGRF8 broadly regulates genes related to N uptake, utilization, and signaling. Our results demonstrate that BcGRF8 substantially accelerates plant growth and nitrate assimilation under both nitrate-poor and -rich conditions by increasing the number of lateral roots and the expression of genes involved in N uptake and assimilation, providing a basis for crop improvement.

Genome-Wide Identification and Expression Analysis of Growth-Regulating Factors in Eucommia ulmoides Oliver (Du-Zhong)

“…For instance, in A. thaliana, AtGRF5 and AtGRF6 do not exhibit complementarity to the mature miR396s sequences [1,2,21]. Similarly, homologs of AtGRF5/6 in Brassica campestris and Brassica rapa also lack the conserved "RSRK-VE" motif in their coding sequences and do not have the target binding sites for miR396s [7,14]. Interestingly, OsGRF11 and its homologs ZmGRF4/10 belong to distinct subgroup members of AtGRF5/6 and do not possess the conserved "RSRK-VE" motif either [13,48].…”

“…The WRC domain is considered to function in the DNA-binding process due to the containing of a putative nuclear localization signal and a C3H-type zinc (Zn) finger motif [3,5]. Moreover, the WRC domains in most plant GRFs contain a conserved "RSRK-VE" motif at its terminus, which corresponds to a nucleotide sequence that complements the mature miR396 sequences [6,7]. Numerous studies have unequivocally demonstrated that miR396 can bind to its target sites on GRF to negatively regulate the expression of the GRF gene [8][9][10][11].…”

“…The first plant GRF, OsGRF1, was previously identified in deepwater rice with high expression levels in internode intercalary meristem in response to GA 3 [3]. Currently, members of GRFs have also been identified in several other plant species: 9 in A. thaliana, 12 in O. sativa, 17 in Brassica rapa, 15 in Lactuca sativa, and 19 in Populus alba × P. glandulosa [5][6][7][13][14][15][16].…”