Transcription factors ZmNF-YA1 and ZmNF-YB16 regulate plant growth and drought tolerance in maize

Abstract

The identification of drought stress regulatory genes is crucial for the genetic improvement of maize (Zea mays L.) yield. Nuclear factors Y (NF-Ys) are important transcription factors, but their roles in the drought stress tolerance of plants and underlying molecular mechanisms are largely unknown. In this work, we used yeast two-hybrid screening to identify potential interactors of ZmNF-YB16 and confirmed the interaction between ZmNF-YA1 and ZmNF-YB16-YC17 and between ZmNF-YA7 and ZmNF-YB16-YC17. ZmNF-YB16 interacted with ZmNF-YC17 via its histone fold domain to form a heterodimer in the cytoplasm and then entered the nucleus to form a heterotrimer with ZmNF-YA1 or ZmNF-YA7 under osmotic stress. Overexpression of ZmNF-YA1 improved drought and salt stress tolerance and root development of maize, whereas zmnf-ya1 mutants exhibited drought and salt stress sensitivity. ZmNF-YA1-mediated transcriptional regulation, especially in JA signaling, histone modification, and chromatin remodeling, could underlie the altered stress tolerance of zmnf-ya1 mutant plants. ZmNF-YA1 bound to promoter CCAAT motifs and directly regulated the expression of multiple genes that play important roles in stress responses and plant development. Comparison of ZmNF-YB16- and ZmNF-YA1-regulated genes showed that ZmNF-YA1 and ZmNF-YB16 have similar biological functions in stress responses but varied functions in other biological processes. Taken together, ZmNF-YA1 is a positive regulator of plant drought and salt stress responses and is involved in the root development of maize, and ZmNF-Y complexes with different subunits may have discrepant functions.

Figure 1

ZmNF-YB16 interact with ZmNF-YC17 in vitro and in vivo. A, Transcriptional activation analysis of ZmNF-YB16. Numbers at the top indicate the position of AAs. The N-terminal 21–90 AA represents the histone-like domain. Different lengths of proteins were cloned and fused to the GAL4 DNA binding (BD) vector. Transformants were screened on SD/-Leu and SD/-His, and β-galactosidase (β-gal) activities were validated by X-Gal staining and quantified by the ONPG assay. B, Growth of yeast cells harboring ZmNF-YB16 and ZmNF-YC17 fused to the GAL4 BD domain (pGBKT7-BD) and GAL4 activity domain (pGADT7-AD). SD/-Leu/-Trp (DDO) is the nonselective medium, and SD/-Leu/-Trp/-Ade/-His (QDO) is the selective medium. C, In vitro pull-down assay of ZmNF-YC17-GST protein and ZmNF-YB16-His protein. The fractions pulled down were detected with an His antibody. D, Verification of the in vivo interaction between ZmNF-YB16 and ZmNF-YC17 by the BiFC system in N. benthamiana. Expression of ZmNF-YB16-cYFP and ZmNF-YC17-nYFP was observed in leaves. The YFP fluorescence signal was detected in the group co-transformed of ZmNF-YB16-cYFP and ZmNF-YC17-nYFP (lower row), but not in the control groups (Rows 1–3). bar = 20 µm. E, Co-IP assay to test the direct interaction between ZmNF-YB16 and ZmNF-YC17. IP: antibody, antibody used for immunoprecipitation, IB: antibody, antibody used for western blot.

Figure 2

ZmNF-YB16/ZmNF-YC17/ZmNF-YA1/7 form heterotrimers in maize. A, Transcriptional activation analysis of ZmNF-YC17. Numbers at the top indicate the position of AAs. The N-terminal 91–160 AA represents the histone-like domain region. The different lengths of proteins were cloned and fused to the GAL4 BD vector. Transformants screen, β-gal activities, X-Gal staining, and ONPG quantification were performed as that in Figure 1A. B, Growth of yeast cells harboring various truncated forms of ZmNF-YC17 fused to the GAL4 BD vector. ZmNF-YB16 was fused to the GAL4 AD vector. Transformants screen, X-Gal staining were performed as that in Figure 1B. C, Subcellular localization analysis of ZmNF-YB16 and ZmNF-YC17 under normal condition. bar = 10 µm. D, Subcellular colocalization analysis of ZmNF-YB16 and ZmNF-YC17 under osmotic stress conditions. ZmNF-YB16 was fused to the pUC18-P35S-GFP vector, and ZmNF-YC17 was fused to the pUC18-P35S-mCherrry vector. bar = 10 µm. E, Confirmation of subcellular location changes by cytosol and nuclear fraction and western-blot. Relative expression levels were quantified using Image J. F, Yeast three-hybrid assay to identify candidate trimers containing ZmNF-YB16 and ZmNF-YC17. The growth of yeast cells harboring NF-YA family proteins fused to the GAL4 AD, ZmNF-YB16 fused to the GAL4 BD, and ZmNF-YC17 fused to a NLS on selective medium. Transformants screen, X-Gal staining were performed as that in Figure 1B. G, Subcellular localization of ZmNF-YA1 and ZmNF-YA7 under normal conditions. ZmNF-YA1 and ZmNF-YA7 were fused to the pUC18-P35S-GFP vector, respectively. bar = 10 µm.

Figure 3

Loss of function of ZmNF-YA1 suppresses plant growth and root development in maize. A, Root phenotypes of W22 and zmnf-ya1 mutant plants hydroponically cultured for 10 d. B and C, Total axial root length and lateral root numbers of W22 and zmnf-ya1 mutant plants hydroponically cultured for 10 d. D, Plant height of WT and mutant plants hydroponically cultured for 5, 8, 10, and 12 d. E, Eleven-day-old hydroponically cultured maize plants under normal conditions (Normal Control) and treated with 12% PEG₆₀₀₀, 120 mM NaCl, or 15 mM LiCl for 10 d. F–H, Shoot dry weight, root dry weight and axial root length of WT and ZmNF-YA1 mutants under different conditions and the loss led by stresses. The images of panel A and E were digitally extracted for comparison. All values are means ± SD of three independent experiments (each with five seedlings per line). bar = 5 cm. The asterisks indicate a significant difference at *0.01 < P ≤ 0.05, **P ≤ 0.01 by a t test.

Figure 4

OE of ZmNF-YA1 improves abiotic stress tolerance in maize. A and B, DH4866 (transgene donor inbred line) and ZmNF-YA1 OE lines hydroponically cultured for 6 d and 10 d. C, Root phenotypes of DH4866 and ZmNF-YA1 OE lines hydroponically cultured for 10 d. D and E, Total axial root length and lateral root numbers of DH4866 and ZmNF-YA1 OE lines hydroponically cultured for 10 d. F, Eleven-day-old hydroponically cultured maize plants of DH4866 and OE lines under normal conditions (Normal Control) and treated with 12% PEG₆₀₀₀, 120 mM NaCl, or 15 mM LiCl for 10 d. The images of panels A–C, and F were digitally extracted for comparison. G–I, Shoot dry weight, root dry weight, and axial root length of DH4866 and ZmNF-YA1 OE lines under different conditions and the loss led by stresses. All values are means ± SD of three independent experiments (each with five seedlings per line). bar = 5 cm. The asterisks indicate a significant difference at *0.01<P≤0.05, **P≤0.01 by a t test.

Figure 5

Transcriptome analysis of the zmnf-ya1 mutant and WT under normal and drought stress. A–C, Significantly enriched GO terms of the Clades 1–3 DEGs repressed by ZmNF-YA1 identified in Supplemental Figure S10D. DEGs in Clades 1–3 were upregulated DEGs in the zmnf-ya1 mutant (i.e. repressed by ZmNF-YA1) and they could be further grouped into Clades 1–3 based on their response to drought stress. D–F, Significantly enriched GO terms of the Clades 4–6 DEGs induced by ZmNF-YA1 identified in Supplemental Figure S10E. DEGs in Clades 4–6 were downregulated DEGs in the zmnf-ya1 mutant (i.e. induced by ZmNF-YA1) and they could be further grouped into Clades 4–6 based on their response to drought stress. Values indicate the fold enriched and the FDR values for the corresponding GO terms. The color intensity indicates the enrichment degree. All the GO enrichment analysis results of the six clades DEGs identified in Supplemental Figures S10D and S10E were list in Supplemental Table S4.

Figure 6

Validation of candidate downstream genes. A Chip-qPCR analysis of the seven candidate downstream genes. B, Distribution of the CCAAT motif on the promoters of candidate downstream genes. C, EMSA showing that ZmNF-YA1 binds to the CCAAT motif at −1,457∼−1,387 bp of the ZmLOX5 promoter. D, EMSA showing that ZmNF-YA1 binds to the CCAAT motif at −68∼−109 bp of the ZmLOX5 promoter. E, Yeast one-hybrid analysis of seven candidate downstream genes. C, Control; A, co-transformed with ZmNF-YA1; ABC, co-transformed with ZmNF-YA1, ZmNF-YB16 and ZmNF-YC17. F, Relative expression level boxplot of the seven candidate downstream genes. The blue boxes indicate the expression levels in W22 and the orange boxes indicate the value from the zmnf-ya1. In each box, different lines represent the minimum, maximum, median, first quartile, and three quartiles of the dataset.

Figure 7

Proposed models of the possible roles of the ZmNF-Y complex in maize development and the stress response. ZmNF-YB16 interacts with ZmNF-YC17 in the cytoplasm forming a dimer, and upon exposure to abiotic stress, the dimer enters the nucleus and binds to ZmNF-YA1 or ZmNF-YA7, forming the ZmNF-YA1-YB16-YC17 or ZmNF-YA7-YB16-YC17 heterotrimer. The expression of ZmNF-YB16 and ZmNF-YA1 is induced by stress treatment, whereas ZmNF-YC17 expression is not. OE of ZmNF-YA1 and ZmNF-YB16 significantly improves abiotic stress tolerance and modifies plant growth and development. The downstream targets of the ZmNF-YA1-YB16-YC17 and other ZmNF-YA1-related transcriptional regulation complexes contribute to changed abiotic stress tolerance and plant growth and development in maize. These processes include but are not limited to transcriptional regulation of the ZmNF-YA1 complex through both the CCAAT-box and chromatin remodeling. The target genes coding for TFs, stress response genes, growth and development regulation and translation regulation genes are changed and further contribute to the stress tolerance and growth regulation in maize. Some of these were selected and confirmed as direct targets of the ZmNF-YA1 complex such as ZmbHLH116, ZmPOD64, ZmAMY, ZmLOX5, ZmCNR9, ZmMBF1c, and ZmLSD1. ZmNF-YA7 is suggested to interact with other members and play important roles in flowering regulation and stress response.