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Identification and characterization of NF-Y transcription factor families in Canola (Brassica napus L.)

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Abstract

NF-Y (NUCLEAR FACTOR-Y), a heterotrimeric transcription factor, is composed of NF-YA, NF-YB, and NF-YC proteins in yeast, animal, and plant systems. In plants, each of the NF-YA/B/C subunit forms a multi-member family. NF-Ys are key regulators with important roles in many physiological processes, such as drought tolerance, flowering time, and seed development. In this study, we identified, annotated, and further characterized 14 NF-YA, 14 NF-YB, and 5 NF-YC proteins in Brassica napus (canola). Phylogenetic analysis revealed that the NF-YA/B/C subunits were more closely clustered with the Arabidopsis thaliana (Arabidopsis) homologs than with rice OsHAP2/3/5 subunits. Analyses of the conserved domain indicated that the BnNF-YA/B/C subfamilies, respectively, shared the same conserved domains with those in other organisms, including Homo sapiens, Saccharomyces cerevisiae, Arabidopsis, and Oryza sativa (rice). An examination of exon/intron structures revealed that most gene structures of BnNF-Y were similar to their homologs in Arabidopsis, a model dicot plant, but different from those in the model monocot plant rice, suggesting that plant NF-Ys diverged before monocot and dicot plants differentiated. Spatial-tempo expression patterns, as determined by qRT-PCR, showed that most BnNF-Ys were widely expressed in different tissues throughout the canola life cycle and that several closely related BnNF-Y subunits had similar expression profiles. Based on these findings, we predict that BnNF-Y proteins have functions that are conserved in the homologous proteins in other plants. This study provides the first extensive evaluation of the BnNF-Y family, and provides a useful foundation for dissecting the functions of BnNF-Y.

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Abbreviations

NF-Y:

NUCLEAR FACTOR-Y

HAP:

Heme Activator Protein

CBF:

CCAAT-binding Factor

LEC1:

LEAFY COTYLEDON 1

L1L:

LEAFY COTYLEDON1-LIKE

LEC2:

LEAFY COTYLEDON 2

EST:

Expressed sequence

ORF:

Open reading frame

CDS:

Coding sequence

UTR:

Untranslated region

bZIP:

The basic domain-leucine zipper

FT:

FLOWERING LOCUS T

SOC1:

SUPPRESSOR OF OVEREXPRESSION OF CO 1

Ehd1:

Early heading date1

Hd3a:

Heading date 3a

RFT1:

RICE FLOWERING LOCUS 1

LHCB:

Light-harvesting Chlorophyll a/b-binding protein

qRT-PCR:

Quantitative real-time PCR

DAP:

Days after planting

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Acknowledgments

We thank Prof. Zhaopu Liu from Nanjing Agricultural University for kindly supplying the canola (Nanyanyou 1) seeds. We are grateful to Dr. Zengrong Huang and Ms Jin Wang from Nanjing Agricultural University for advice for qRT-PCR. We also thank Kathleen Farquharson for valuable comments on the manuscript revision. This research was supported by grants from the Natural Science Foundation of Jiangsu province (BK2011635), the State Key Laboratory of Crop Genetics and Germplasm Enhancement (ZW2010004), a China Postdoctoral Science Foundation-funded project (20110491441), a Jiangsu Postdoctoral Science Foundation-funded project (1101013B), Fundamental Research Funds for the Central Universities (KYZ201206) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (RAPD program (809001), and the Technological Innovation Foundation for Young Scientists of Nanjing Agricultural University (Y201058).

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The authors declare that they have no competing interests.

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Correspondence to Mingxiang Liang.

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X. Yin, Z. Lin, and Q. Zheng contributed equally to this work.

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Supplementary Fig. S1 Phylogenetic trees of canola and Arabidopsis NF-Y families based on the full-length proteins. The phylogenetic trees were constructed using the full-length proteins by the neighbor-joining method implemented by MEGA software, version 4.1. The numbers at each branch point represent the bootstrap scores (1,000 replicates). A branch with a bootstrap score below 50 was usually considered unreliable. Sequences of the mouse NF-Y subunits were used to root the trees, respectively. a Phylogenetic tree of canola and Arabidopsis NF-YA families based on the full-length proteins. b phylogenetic tree of canola and Arabidopsis NF-YB families based on the full-length proteins. c phylogenetic tree of canola and Arabidopsis NF-YC families based on the full-length proteins

Supplementary Fig. S2 Phylogenetic trees for BnNF-Y, AtNF-Y and OsHAP families based on the conserved domains and the full-length proteins. a Phylogenetic tree of BnNF-YA, AtNF-YA and OsHAP2 families for the conserved domains. b phylogenetic tree of BnNF-YA, AtNF-YA and OsHAP2 families for the full-length proteins. c phylogenetic tree of BnNF-YB, AtNF-YB and OsHAP3 families for the conserved domains. d phylogenetic tree of BnNF-YB, AtNF-YB and OsHAP3 families for the full-length proteins. e phylogenetic tree of BnNF-YC, AtNF-YC and OsHAP5 families for the conserved domains. f phylogenetic tree of BnNF-YC, AtNF-YC and OsHAP5 families for the full-length proteins. The phylogenetic trees were constructed using full-length proteins by neighbor-joining method implemented by Molecular Evolutionary Genetics Analysis (MEGA) software, version 4.1. The numbers at each branch point represent the bootstrap scores (1,000 replicates). A branch with a bootstrap score below 50 was usually considered unreliable. Sequences of the mouse NF-Y subunits were used to root the trees, respectively

Supplementary Fig. S3 Amino acid alignment of conserved domains of NF-Y proteins from different organisms. Hs, Homo sapiens; Mm, Mus musculus; Sc, Saccharomyces cerevisiae; Bn, Brassica napus; At, Arabidopsis thaliana; and Os, Oryza sativa. Numbers in parentheses correspond to the actual positions of the first amino acids of the conserved domains within each protein; numbers above the alignment were used as reference points in the text; and numbers to the right correspond to the amount of each amino acid presented here. In the consensus line, uppercase letters represent identity in more than 50 % of sequences and X represents less than 50 % identity. a NF-YAs alignment. To eliminate some gaps resulting from nonhomologous sequences, the Q residues were removed from BnNF-YA13 and BnNF-YA14 between the K and P residues at position 31/32, respectively. The amino acid sequence VLD between the A and Q residues at 22/23 and the F between the K and R residues at position 29/30 from BnNF-YA3 were removed. The NF-YA conserved regions were composed of two alpha-helices: A1 mediates the NF-YB–NF-YC interaction and A2 is responsible for CCAAT binding (Xing et al. 1993; Mantovani et al. 1994; Xing et al. 1994). b NF-YBs alignment. The secondary structures, alpha-helices (solid blue rectangles) and coils (black lines), are represented on the top of the alignment, based on (Romier et al. 2003). The DNA-binding and subunit-binding domains are represented as black and colored bars, respectively (Romier et al. 2003; Sinha et al. 1996). The NF-YC interaction domain extends across two independent regions and partly overlaps with the DNA-binding and NF-YA interaction domains. c NF-YCs alignment. To eliminate gaps of nonhomology, the amino acid sequence DTLTRS was removed from AtNF-YC7 between the S and D residues at position 57/58, and YVNFQK was removed from AtNF-YC12 between the paired I residues at 18/19. Furthermore, the amino acid sequences RSA and VDGGGGGGGGA were removed from OsHAP5G between the K and G residues at position 11/12 and between the V and R residues at position 16/17, respectively. The secondary structures, alpha-helices (solid blue rectangles) and coils (black lines), are represented on the top of the alignment, based on (Romier et al. 2003). The DNA-binding and subunit-binding domains are represented by black and colored bars, respectively (McNabb et al. 1997; Kim et al. 1996; Romier et al. 2003). The NF-YA interaction domain extends across two separate regions. The DNA-binding domain in NF-YC consists of two amino acids, AR

Supplementary Fig. S4 Exon/Intron structures of OsHAPs. Black boxes denote exons within coding regions and the lines connecting them represent introns. The length of boxes and lines represents the size (bp) of the corresponding exon and intron, respectively, as do the numbers in black boxes or above the lines. The gene structures without the untranslated regions (UTRs) were constructed using the Gene Structure Display Serve tool (http://gsds.cbi.pku.edu.cn/)

Supplementary Fig. S5 Semi-quantitive RT-PCR analysis of BnNF-Ys for 75-DAP leaves at two given cycles. L-1-1, L-1-2, and L-1-3 are the three biological samples of 75-DAP leaves

Supplementary Table S1 The accession numbers (TC# or GB#) of the sequences from the oilseed rape database

Supplementary Table S2 All primers for genomic DNA, cDNA and qRT-PCR

Supplementary Table S3 Putative NF-Ys in Brassica rapa

Supplementary Table S4 The putative functions of BnNF-Y

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Liang, M., Yin, X., Lin, Z. et al. Identification and characterization of NF-Y transcription factor families in Canola (Brassica napus L.). Planta 239, 107–126 (2014). https://doi.org/10.1007/s00425-013-1964-3

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