Identification and functional prediction of stress responsive AP2/ERF transcription factors in Brassica napus by genome-wide analysis

Highlights

• By implementing genome-wide analysis, 321 AP2/ERF transcription factors were identified in Brassica napus.

• BnAP2/ERFs were formed through duplication events, occurring before Brassica napus formation.

• The BnERF039 and BnERF245 are candidates for improving salt-tolerant Brassica napus.

• BnERF227, BnERF228, BnERF234, BnERF134, BnERF132, BnERF176, and BnERF235 are candidates for improving Leptosphaeria-tolerant Brassica napus.

Abstract

Using homology and domain authentication, 321 putative AP2/ERF transcription factors were identified in Brassica napus, called BnAP2/ERF TFs. BnAP2/ERF TFs were classified into five major subfamilies, including DREB, ERF, AP2, RAV, and BnSoloist. This classification is based on phylogenetic analysis, motif identification, gene structure analysis, and physiochemical characterization. These TFs were annotated based on phylogenetic relationship with Brassica rapa. BnAP2/ERF TFs were located on 19 chromosomes of B. napus. Orthologs and paralogs were identified using synteny-based methods Ks calculation within B. napus genome and between B. napus with other species such as B. rapa, Brassica oleracea, and Arabidopsis thaliana indicated that BnAP2/ERF TFs were formed through duplication events occurred before B. napus formation. Kn/Ks values were between 0 and 1, suggesting the purifying selection among BnAP2/ERF TFs. Gene ontology annotation, cis-regulatory elements and functional interaction networks suggested that BnAP2/ERF TFs participate in response to stressors, including drought, high salinity, heat and cold as well as developmental processes particularly organ specification and embryogenesis. The identified cis-regulatory elements in the upstream of BnAP2/ERF TFs were responsive to abscisic acid. Analysis of the expression data derived from Illumina Hiseq 2000 RNA sequencing revealed that BnAP2/ERF genes were highly expressed in the roots comparing to flower buds, leaves, and stems. Also, the ERF subfamily was over-expressed under salt and fungal treatments. BnERF039 and BnERF245 are candidates for salt-tolerant B. napus. BnERF253-256 and BnERF260-277 are potential cytokinin response factors. BnERF227, BnERF228, BnERF234, BnERF134, BnERF132, BnERF176, and BnERF235 were suggested for resistance against Leptosphaeria maculan and Leptosphaeria biglobosa.

Introduction

APETALA2/Ethylene Responsive Factors (AP2/ERF) represent a large family of transcription factors (TFs) in plants. AP2/ERF TFs are identified by the presence of an AP2 DNA-binding domain consisting 60–70 highly conserved amino acids (aa) (Licausi et al., 2013, Sakuma et al., 2002). The AP2 domain was initially identified in Arabidopsis and tobacco AP2 and ERF proteins, respectively (Jofuku et al., 1994, Ohme-Takagi and Shinshi, 1995). This domain consists of 3 stranded-anti-parallel β-sheets and an α-helix parallel to β-sheets (Allen et al., 1998). Two regions designated as YRG and RAYD element, at the N- and C-terminal of the AP2 domain, play an important role in the DNA-binding activity. YRG element contains almost 20 residues that facilitates DNA-binding through the basic and hydrophilic moieties. RAYD element with about 40 residues interacts with the major groove of DNA through amphipathic α-helix formation (Okamuro et al., 1997). Although the AP2 domain had been initially considered as a plant specific domain, it was later found in viral and bacterial HNH endonuclease (A class of homing endonucleases). Hence, the gain of AP2 domain in plants was as a result of horizontal gene transfer (Magnani et al., 2004).

AP2/ERF superfamily has been divided into four major subfamilies according to DNA-binding domain and sequence similarity. These four subfamilies are Dehydration Responsive Element Binding (DREB) Factor, ERF, AP2, and a subfamily called Related to ABI3 and VP1 (RAV) (Riechmann et al., 2000, Sakuma et al., 2002). A fifth subfamily could be found in most of the higher plants nominated as the Soloist. The Soloist subfamily has a single AP2 domain with low conservation (Song et al., 2013). Within this manuscript, DREB, ERF, AP2, and RAV have been called subfamilies and their clusters have been defined as groups. The DREB and ERF subfamilies, both containing a single AP2 domain, are distinguished from each other based on their differences in the conserved residues of DNA-binding domain. Two classification methods have been suggested for DREB and ERF subfamilies. According to Sakuma et al., DREB and ERF subfamilies are subdivided into A1-A6 and B1-B6 groups, respectively, based on the DNA-binding domain (Sakuma et al., 2002). Nakano clusters ERF and DREB subfamilies together into 12 groups in A. thaliana and 15 groups in rice based on other conserved motifs outside of the AP2 domain and gene structure (Nakano et al., 2006). The majority of AP2 subfamily members have two AP2 domains. The RAV subfamily is identified by the presence of one extra DNA-binding domain, called B3 domain (Licausi et al., 2010a, Nakano et al., 2006).

AP2/ERF TFs are generally known to activate gene expression through Dehydration Responsive Element (DRE)/C-repeat (CRT) and Low Temperature Responsive Element (LTRE). However, there are some other regulatory elements attributed to abscisic acid (ABA)-dependent and independent pathways. The ERF subfamily binds to GCC box (AGCCGCC), while the DREB subfamily interacts with (A/GCCGAC) (Mizoi et al., 2012, Ohme-Takagi and Shinshi, 1995, Yamaguchi-Shinozaki and Shinozaki, 2005). The AP2 subfamily binds to a completely different sequence, called GCAC(A/G)N(A/T)TCCC(A/G)ANG(C/T) in spite of having the AP2 domain (Gong et al., 2008, Nole-Wilson and Krizek, 2000). The RAV subfamily generally trans-activates the CAACA or CACCTG elements (Sohn et al., 2006).

AP2/ERF superfamily could be considered as an activator or repressor. Generally, repressors in the AP2/ERF superfamily consist of two major motifs, including the ERF-associated amphiphilic repression (EAR) motif (DLNxxP or LxLxLx) in the C-terminal (Hiratsu et al., 2003, Ohta et al., 2001) and B3 repression domain (BRD) motif (RLFGV) in the RAV subfamily. Activators usually do not have a specific motif and merely have regions rich in acidic amino acids, such as Gln, Ser, Pro, and Thr (Liu et al., 1999). However, the EDLL motif has been recognized as a potent plant transcription activation within the AP2/ERF TFs (Tiwari et al., 2012).

AP2/ERF TFs have important functions in biological processes, including development, reproduction, primary and secondary metabolite biosynthesis, and adaptation to biotic and abiotic stresses (Licausi et al., 2013, Mizoi et al., 2012). They are mainly activated in response to stresses such as drought (Golldack et al., 2011, Yamaguchi-Shinozaki and Shinozaki, 2006), heat (Sakuma et al., 2006b), water logging (Hinz et al., 2010), high salinity (Abogadallah et al., 2011), freezing (Yang et al., 2005), osmotic stress (Zhu et al., 2010) and pathogen stimuli (Gutterson and Reuber, 2004). For example, the DREB subfamily is mainly induced in response to abiotic stresses (Mizoi et al., 2012). DREB A1/Cold Binding Factor (CBF) group is considered as a master regulator of cold-stress. Several genes being assigned to cold stress, including Late Embryogenesis Abundant (LEA) genes, fatty acid biosynthesis genes, and sugar metabolism genes are highly activated in transgenic plants, over-expressing DREB1/CBF TFs (Fowler and Thomashow, 2002, Maruyama et al., 2009). The increased numbers of CBF TFs in Eucalyptus grandis might be associated with well acclimation of Eucalyptus to different climate regions (Cao et al., 2015). However, DREB1/CBF TFs are not only induced in response to cold stress, but also to circadian clock, high salinity, dehydration, and growth (Mizoi et al., 2012). DREB A2 group (equal to group IV) plays a dual function in drought and heat shock adaptive pathways through activating genes encoding LEA proteins and heat-shock proteins (Sakuma et al., 2006b). DREB A6 group (equal to group I) is responsible for the biosynthesis of lipid and cell-wall components (Mizoi et al., 2012). Some other roles, including ABA and sugar signaling (Arenas-Huertero et al., 2000, Huijser et al., 2000, Song et al., 2005), increasing drought tolerance through changing architecture of roots and leaves (Abogadallah et al., 2011; Wilson et al., 1996c), regulation of DREB A1/A2 in a negative feedback (Mizoi et al., 2012) and binding to the promoters of genes, involved in the biosynthesis of antioxidants, have been attributed to the other members of DREB (Shaikhali et al., 2008). Group V is involved in the wax biosynthesis and secondary wall formation (Aharoni et al., 2004, Broun et al., 2004, Lasserre et al., 2008). Group V as a part of ERF B6 includes groups V, VI-L, and Xb-L according to Nakano’s classification method. Members of the ERF B1 group (equal to group VII) were activated under oxygen limitation, and conferred submergence tolerance (Licausi et al., 2010b, Xu et al., 2006). The ERF B3 group (equal to group IX) was responsible for secondary metabolite biosynthesis (Mizoi et al., 2012, Shen et al., 2016). The ERF subfamily is also associated with developmental processes (Vernié et al., 2008) and pathogen defensive procedures (Fischer and Dröge-Laser, 2004) along with drought and high salinity (Fujimoto et al., 2000, Park et al., 2001). The AP2 subfamily mostly carry out developmental roles entailing floral development (Elliott et al., 1996, Horstman et al., 2014), fruit size (Chialva et al., 2016), leaf shape and development (Luo et al., 2012, van der Graaff et al., 2000), embryogenesis (El Ouakfaoui et al., 2010, Heidmann et al., 2011), and plant height (Luo et al., 2012). The RAV subfamily is poorly studied. It has been shown that the RAV TFs are involved in postponing floral induction (Castillejo and Pelaz, 2008), growth inhibition (Zhao et al., 2008), bud outgrowth (Moreno-Cortés et al., 2012), leaf senescence (Woo et al., 2010) as well as tolerance to cold, osmotic stress (Zhuang et al., 2011) and pathogen infection (Sohn et al., 2006).

Brassica napus is the second oilseed crop in the world (Fareeha Zafar et al., 2015). It is considered as one of the crops of choice for biodiesel and biofuel production (Blackshaw et al., 2011, Hemmati et al., 2017).

B. napus (genome AnAnCnCn) is an allopolyploid formed by hybridization between B. oleracea (genome CoCo) and B. rapa (genome ArAr) as ancestors (Allender and King, 2010). Moreover, B. napus shares more than 86% similarity in protein coding sequence with the model plant A. thaliana (Cavell et al., 1998). As a consequence, having the knowledge of B. napus closest relatives allows us to investigate its genome.

Cultivation of B. napus is jeopardized by a number of biotic stresses, including fungal and viral pathogens, insects, and weeds. The fungal genus Leptosphaeria, including Leptosphaeria maculan and Leptosphaeria biglobosa are economically important fungal plant pathogens (Howlett, 2004, Van de Wouw et al., 2008). A number of abiotic stresses adversely affect B. napus growth and productivity, of which cold (Meza-Basso et al., 1986), heat (Yu et al., 2014), salinity, flooding, and drought are of special notice.

Since, AP2/ERF TFs are ideal candidates for crop improvement, elucidation of this superfamily in B. napus would have drastic effects on the crop yield and resistance toward biotic and abiotic stresses. In parallel with our study, another paper describing some aspects of genome wide analysis of AP2/ERF TFs in B. napus has been published by Song (Song et al., 2016). The major approach of the mentioned study was finding out the origination, expansion, and evolutionary relation of AP2/ERF TFs within the main land plants. However, in this manuscript, our main purpose is the elucidation of AP2/ERF functions based on various bioinformatic analyses which are quite different than the ones in the paper of Song et al. We report the identification of 321 AP2/ERF TFs in B. napus genome. Furthermore, cluster analysis of the identified proteins, motif-distribution recognition, gene structure analysis in conjunction with gene ontology annotation, evolutionary analysis, and protein characterization were carried out. Expression profile in different tissues and expression profile under biotic and abiotic stresses were monitored via digital RNA-seq data. Results from this study provide the basis for functional experiments associated with genetic engineering of B. napus.