Genome-Wide Survey and Expression Analysis of the Plant-Specific NAC Transcription Factor Family in Soybean During Development and Dehydration Stress

DNA RESEARCH 18, 263–276, (2011) Advance Access Publication on 18 June 2011 doi:10.1093/dnares/dsr015 Genome-Wide Survey and Expression Analysis of the Plant-Specific NAC Transcription Factor Family in Soybean During Development and Dehydration Stress DUNG TIEN Le 1,2, RIE Nishiyama 1, YASUKO Watanabe 1, KEIICHI Mochida 3, KAZUKO Yamaguchi-Shinozaki 4, KAZUO Shinozaki 3, and L AM-SON PHAN Tran 1, * Signaling Pathway Research Unit, Plant Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan1; Agricultural Genetics Institute, Vietnamese Academy of Agricultural Science, Pham-Van-Dong Str., Hanoi, Vietnam2; Gene Discovery Research Group, Plant Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan3 and Japan International Center of Agricultural Sciences, Ibaraki 305-8686, Japan4 *To whom correspondence should be addressed. Tel. þ81 45-503-9593. Fax. þ81 45-503-9591. E-mail: Edited by Satoshi Tabata (Received 24 April 2011; accepted 24 May 2011) Abstract Plant-specific NAC transcription factors (TFs) play important roles in regulating diverse biological processes, including development, senescence, growth, cell division and responses to environmental stress stimuli. Within the soybean genome, we identified 152 full-length GmNAC TFs, including 11 membrane-bound members. In silico analysis of the GmNACs, together with their Arabidopsis and rice counterparts, revealed similar NAC architecture. Next, we explored the soybean Affymetrix array and Illumina transcriptome sequence data to analyse tissue-specific expression profiles of GmNAC genes. Phylogenetic analysis using stress-related NAC TFs from Arabidopsis and rice as seeding sequences identified 58 of the 152 GmNACs as putative stress-responsive genes, including eight previously reported dehydration-responsive GmNACs. We could design gene-specific primers for quantitative real-time PCR verification of 38 out of 50 newly predicted stress-related genes. Twenty-five and six GmNACs were found to be induced and repressed 2-fold or more, respectively, in soybean roots and/or shoots in response to dehydration. GmNAC085, whose amino acid sequence was 39%; identical to that of wellknown SNAC1/ONAC2, was the most induced gene upon dehydration, showing 390-fold and 20-fold induction in shoots and roots, respectively. Our systematic analysis has identified excellent tissue-specific and/or dehydration-responsive candidate GmNAC genes for in-depth characterization and future development of improved drought-tolerant transgenic soybeans. Key words: soybean; NAC transcription factors; dehydration; sequence analysis; expression analysis 1. Introduction Cultivated soybean (Glycine max L.), which provides an abundant source of oil and protein-rich food for human consumption and animal feed, is one of the major and most important legume crops native to East Asia. Soybean growth, productivity and seed quality are adversely affected by a wide range of environmental stresses.1,2 Among the adverse environmental factors, drought is considered the most devastating abiotic stress. Drought stress affects all stages of plant growth and development, resulting in significant yield loss by 40%; as well as severely impacting seed quality.2 In response to drought # The Author 2011. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 264 Characterization of Soybean GmNAC Family stress, plants activate a number of defence mechanisms that function to increase tolerance to water deficit.3,4 The early events of the adaptation of plants to drought stress includes the perception of stress signals and subsequent signal transduction, leading to the activation of various physiological and metabolic responses.3,5,6 Within the signal transduction networks that are involved in the conversion of stress signal perception to stress-responsive gene expression, various transcription factors (TFs) and cis-acting elements contained in stress-responsive promoters function not only as molecular switches for gene expression, but also as terminal points of signal transduction in the signalling processes. The identification and molecular tailoring of novel TFs have the potential to overcome a number of important limitations involved in the generation of transgenic crop plants with superior yield under stress conditions. Within higher plants, 7%; of their genomes encodes for putative TFs.7 Typically, the TFs contain a distinct type of DNA-binding domain and transcriptional regulation region (TRR) and are capable of activating or repressing the transcription of multiple target genes.3,5,8,9 The NAC TFs contain a highly conserved N-terminal DNA-binding NAC domain and a variable C-terminal TRR.10 Research in Arabidopsis has indicated that there are at least five different target DNA-binding sites for the NAC TFs. These include the drought-responsive NAC recognition sequence (NACRS) containing the CACG core motif; the iron deficiency-responsive IDE2 motif containing the core sequence CA(A/C)G(T/C)(T/C/A)(T/ C/A); the CBNACBS-binding site of the Arabidopsis calmodulin-binding CBNAC protein having the GCTT as core-binding motif; the secondary wall NAC-binding element (SNBE) with 19-bp consensus sequence (T/A)NN(C/T) (T/C/G)TNNNNNNNA(A/C)GN(A/C/ T) (A/T); and the 21-bp segment of the 35S promoter (283 to 263) containing two core sequences CGTA and CGTC.11 – 17 In addition to DNA binding, the NAC domain also possesses the capacity for mediating protein:protein interactions.13,18 The highly variable C-terminal TRRs of NAC TFs can act as either a transcriptional activator or a repressor.11,14,19 Interestingly, the C-terminal domains of numerous NAC TFs also exhibit protein-binding activity.14 On the other hand, the C-terminal regions of some NAC TFs also contain transmembrane motifs (TMs) which are responsible for the anchoring to the plasma membrane. These NAC TFs are classified as membraneassociated and are designated as NTL (NTM1-Like or ‘NAC with Transmembrane Motif 1’-Like) TFs.20,21 NAC TFs have been shown to regulate a number of biological processes, including those which protect plants under water stress conditions.10,22,23 There [Vol. 18, are at least 105 ANAC and 140 ONAC members in Arabidopsis and rice (Oryza sativa), respectively.24 – 26 The first evidence demonstrating the involvement of NAC TFs in the improvement of drought tolerance in plants was reported in Arabidopsis by the identification and functional analyses of the ANAC019, ANAC055 and ANAC072 genes. Following this work, a number of studies on abiotic stress-related functions of NAC TFs (...truncated)