Family-wide survey of miR169s and NF-YAs and their expression profiles response to abiotic stress in maize roots

Abstract

Previous studies have identified miR169/NF-YA modules are important regulators of plant development and stress responses. Currently, reported genome sequence data offers an opportunity for global characterization of miR169 and NF-YA genes, which may provide insights into the molecular mechanisms of the miR169/NF-YA modules in maize. In our study, fourteen NF-YA transcription factors with conserved domains were identified based on maize genome loci. The miR169 gene family has 18 members that generate 10 mature products, and 8 of these mature miR169 members could target 7 of 14 ZmNF-YA genes in maize. The seven ZmNF-YA proteins were localized to the nucleus while lacked transcriptional activity. We investigated the expression patterns of the zma-miR169 members and their targeted ZmNF-YA genes in maize roots treated by drought stress (polyethylene glycol, PEG), hormone stress (abscisic acid, ABA), and salt stress (NaCl). The zma-miR169 family members were downregulated in short term (0 ∼ 48 h) and generally upregulated over the long term (15 days) in response to the three abiotic stress conditions. Most of the targeted ZmNF-YA genes exhibited a reverse correlation with zma-miR169 gene expression over both the short term and long term. Maize root elongation was promoted by PEG and ABA but repressed by NaCl over the long term. Apparently, ZmNF-YA14 expression perfectly matched the zma-miR169 expression and corresponded to root growth reversely.

Figure 1. Nuclear acid sequence alignments of mature zma-miR169 (A) and zma-miR169 target sites in ZmNF-YA family members (B).

Zma-miR169d and zma-miR169e are deleted at sequence 11.

Figure 2. Amino acid alignment of NF-YA core domains from Sc, Saccharomyces cerevisiae; At, Arabidopsis; Hs, Homo sapiens; Zm, Zea mays.

Figure 3. Phylogenetic analyses of NF-YA proteins.

(A) Phylogenetic analysis of ZmNF-YA family members. (B) Phylogenetic analysis of NF-YA proteins from maize, rice, and Arabidopsis. (C) Syntenic relationships of NF-YA genes between maize and rice. Each line represents an orthologous gene. The loci of NF-YA genes involved are shown in Table S3. Phylogenetic trees were constructed by neighbor joining with complete deletions as implemented by Molecular Evolutionary Genetics Analysis software, version 5.0 (MEGA5) . Reliability values at each branch represent bootstrap samples (1000 replicates).

Figure 4. Mapping of Zm-NFYA cleavage sites generated by zma-miR169s.

Cleavage sites are indicated by arrows. 5′ termini of mRNA fragments isolated from maize were determined from cloned 5′RACE products (Figure S4). The frequencies of cleavage site usage are indicated by fractional numbers.

Figure 5. Expression profiles of seven maize ZmNF-YA genes and localization and transcriptional activity of ZmNF-YA proteins.

(A) ZmNF-YA expression patterns in maize tissues. Values represent the means of normalized signal reads. Error bars represent standard errors for three independent normalized signal values. (B) Localization of ZmNF-YA:GFP fusion proteins in maize mesophyll cells. Blue: nucleus stained with Hoechst33342; Green: ZmNF-YA:GFP fluorescence. Cambridge blue: merged images. (C) Transcriptional activation by ZmNF-YAs in a yeast two-hybrid system. Negative control: pAS2 vector. Positive control: ZmNF-YB.

Figure 6. Expression profiles of mature zma-miR169s and ZmNF-YAs in roots in response to PEG (A), ABA (B), and salt (C).

Expression levels of U6 and Tub5 were used as internal references for expression of zma-miR169s and ZmNF-YAs, respectively. Values represent the means and the error bars represent standard errors for three independent experiments.

Figure 7. Maize root growth and zma-miR169/ZmNF-YA14 module expression in response to treatment with NaCl, ABA, or PEG.

(A) Root phenotypes in response to treatment with NaCl, ABA, or PEG for 15 days. (B) Root lengths of seedlings treated with NaCl, ABA, or PEG. (C) Relative expression levels of zma-miR169 genes in seedlings treated with NaCl, ABA, or PEG. (D) Relative expression levels of ZmNF-YA14 in seedlings treated with NaCl, ABA, or PEG.

Figure 8. Putative model of zma-miR169/ZmNF-YA14 module response to stress in maize root.