Annotation, phylogeny and expression analysis of the nuclear factor Y gene families in common bean (Phaseolus vulgaris)

Abstract

In the past decade, plant nuclear factor Y (NF-Y) genes have gained major interest due to their roles in many biological processes in plant development or adaptation to environmental conditions, particularly in the root nodule symbiosis established between legume plants and nitrogen fixing bacteria. NF-Ys are heterotrimeric transcriptional complexes composed of three subunits, NF-YA, NF-YB, and NF-YC, which bind with high affinity and specificity to the CCAAT box, a cis element present in many eukaryotic promoters. In plants, NF-Y subunits consist of gene families with about 10 members each. In this study, we have identified and characterized the NF-Y gene families of common bean (Phaseolus vulgaris), a grain legume of worldwide economical importance and the main source of dietary protein of developing countries. Expression analysis showed that some members of each family are up-regulated at early or late stages of the nitrogen fixing symbiotic interaction with its partner Rhizobium etli. We also showed that some genes are differentially accumulated in response to inoculation with high or less efficient R. etli strains, constituting excellent candidates to participate in the strain-specific response during symbiosis. Genes of the NF-YA family exhibit a highly structured intron-exon organization. Moreover, this family is characterized by the presence of upstream ORFs when introns in the 5' UTR are retained and miRNA target sites in their 3' UTR, suggesting that these genes might be subjected to a complex post-transcriptional regulation. Multiple protein alignments indicated the presence of highly conserved domains in each of the NF-Y families, presumably involved in subunit interactions and DNA binding. The analysis presented here constitutes a starting point to understand the regulation and biological function of individual members of the NF-Y families in different developmental processes in this grain legume.

Keywords: CCAAT box; gene regulation; legumes; nodulation; symbiosis; transcription factors.

Figure 1

Gene structure of PvNF-Y subunits family members in P. vulgaris. Scheme of intron/exon organization of gene members of PvNF-YA (A), PvNF-YB (B), and PvNF-YC (C) families predicted from the gene models and RNA sequencing studies available at the P. vulgaris genome V1.0 database. Lines represent introns and boxes represent exons. Gray boxes correspond to 5′ and 3′ untranslated regions (UTR) and black boxes correspond to coding sequence (CDS) regions. The sizes of intron and exon can be estimated using the reference scale bar of 500 bp. Note that PvNF-YB12 does not have a 5′ UTR annotated in the common bean genome. Similarly, that PvNF-YC5 and -YC7 do not have either 5′ or 3′ UTRs annotated in the common bean genome.

Figure 2

Multiple alignments of conserved regions of PvNF-Y families. Sequence alignments among the highly conserved domains of NF-YA (A), NF-YB (B), and NF-YC (C) proteins of common bean (Pv) and Mus musculus (Mouse). Multiple protein sequence alignments were generated by ClustalW (Thompson et al., 1994). Conserved regions were shading using BOXSHADE 3.21 to produce a graphic representation of the alignment. Identical amino acids are highlighted in black boxes, and similar residues are highlighted in gray boxes. The DNA-binding domain and the domains required for the interaction with the other subunits previously defined in yeast and mammals are indicated.

Figure 3

Phylogenic trees for the PvNF-Y families. Phylogenetic trees for the NF-YA (A), NF-YB (B), and NF-YC (C) families were constructed using the neighbor-joining method based on the multiple sequence alignment analysis of the full-length proteins obtained from the common bean genome V1.0 database, including the mouse NF-Y amino acid sequences as root (rooted tree). All trees were determined and constructed using MEGA 5 from a ClustalW analysis. Reliability values at each branch represent bootstrap samples (10,000 replicates).

Figure 4

Expression analysis of NF-Y family members in different organs. Relative transcript levels obtained by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) of PvNF-YA (A), PvNF-YB (B), and PvNF-YC (C) gene family members in different organs: leaves, stems, roots, and nodules of 7 or 14 days post-inoculation (dpi) with Rhizobium etli SC15 (Nod SC15 7dpi) or 55N1 (Nod 55N1 7dpi). Expression levels were normalized to elongation factor 1α (eEF1α) values and are presented as relative to the root sample. Error bars represent standard deviation (SD) of at least three technical replicates. Results are representative of two independent biological experiments. Single, double, and triple asterisks indicate that values are significantly different from the control value in an unpaired two-tailed t-test with p < 0.05, 0.01, and 0.001, respectively. Note that in (B,C) the Y axis was segmented to accommodate the expression values of genes that are expressed at much higher levels n shoot than in root tissue.

Figure 5

Expression analysis of PvNF-Y family members in common bean roots upon inoculation with R. etli strains SC15 or 55N1. Relative transcript levels of PvNF-YA (A), PvNF-YB, (B), and PvNF-YC (C) in roots 24 h post-inoculation with either strain SC15 or 55N1 of R. etli or with the medium used to growth the bacteria (YEM) as control, as determined by RT-qPCR. Expression data were normalized to eEF1α and presented as relative to roots inoculated with YEM. Error bars represent SD of at least three technical replicates. Results are representative of two independent biological experiments. Single, double, and triple asterisks indicate that values are significantly different from the control value in an unpaired two-tailed t-test with p < 0.05, 0.01, and 0.001, respectively.