Transcription factors (TFs) regulate expression of genes that mediate growth processes and environmental response and are employed as a principal source of the diversity and change that underlie evolution (Riechmann and Ratcliffe 2000). GATA TFs are transcriptional regulatory proteins that contain a characteristic type-IV zinc finger (Cys-X2-Cys-X17 − 20-Cys-X2-Cys) and a DNA-binding domain recognize the conserved GATA motif in the promoter sequence of target genes (Scazzocchio 2000; Lowry and Atchley 2000). Fungal GATA TFs are mainly involved in the relation of nitrogen metabolism (Michielse et al. 2014; Pfannmüller et al. 2017), light responses (Purschwitz et al. 2008; Fuller et al. 2013), siderophore biosynthesis and mating-type switching (Jung and Kronstad 2011). Few GATA TFs in fungus also take part in response to the abiotic stresses, such as the SreA, SreB, LreA, LreB, GLN3 and GAT1 (Chung et al. 2020; Crespo et al. 2001; Purschwitz et al. 2008; Fuller et al. 2013; Marty et al. 2015). The number of the GATA TFs is conserved among A. clavatus, A. flavus, A. fumigatus, A. nidulans, A. niger and A. oryaze that possess six GATA TFs, suggesting that filamentous fungi share an identical composition of GATA TFs with each other (Kobayashi et al. 2007). However, in this study, we identified seven A. oryzae GATA TFs from the A. oryzae 3.042 genome using an HMM model. Six known A. oryzae GATA TFs, consistent with the report of Kobayashi et al. (2007), were classified into six functional subgroups based on the number of ZnF_GATA domains and zinc finger motif of GATA domain sequences with other Aspergillus GATA TFs from FTFD, while the novel AoSnf5 encoding GATA TF also clustered into NSDD subgroups together with AoNsdD (Fig. 2). Conserved motifs demonstrated that GATA TF menbers had similar motif compositions could be clustered into one subgroup (Fig. 3), which suggests they may have similar genetic functions within the same subgroups. In addition, the motif distribution further confirms the accuracy of the phylogenetic relationship of Aspergillus GATA TFs. The analyses of phylogenetic tree and conserved motifs demonstrated that the evolution of GATA TFs among different Aspergillus was very conservative which might have the same evolutionary events and perform similar function among the Aspergillus GATA proteins within the same subgroups.
Although most GATA domains harbor a class-IV zinc-finger motif, this structure differs among kingdoms (Lowry and Atchley 2000). In plants, most GATA domains have a single Cys-X2-Cys-X18-Cys-X2-Cys motif, but some harbor more than two zinc-finger motifs or 20-residue within zinc-finger loops (Reyes et a. 2004; Behringer and Schwechheimer 2015). In animals, the GATA domain harbors two zinc-finger motifs with Cys-X2-Cys-X17-Cys-X2-Cys, but only the C-terminal finger is associated with DNA binding (Patient and Mcghee 2002). Fungal GATA TFs are combination of both animal and plant GATA TFs in terms of the amino acid residues present in the zinc-finger loop (Teakle and Gilmartin 1998). The majority of fungal GATA TFs contain a single zinc-finger domain and fall into two different categories: animal-like with 17-residue loops(Cys-X2-Cys-X17-Cys-X2-Cys), and plant-like with 18-residue loops (Cys-X2-Cys-X18-Cys-X2-Cys) (Teakle and Gilmartin 1998; Scazzocchio 2000; Patient and Mcghee 2002). Nineteen- and 20-residue zinc-finger loops (Cys-X2-Cys-X19 − 20-Cys-X2-Cys) are also found in fungi, albeit rarely (Scazzocchio 2000; Maxon and Herskowitz 2001). Except for the 17- and 18-residue zinc-finger loops in A. oryzae GATA TFs, the novel AoSnf5 contains 20-residue in the zinc-finger loops (Cys-X2-Cys-X20-Cys-X2-Cys), which are rarely found in fungi (Table 1 and Fig. 1). To our knowledge, GATA TF with 20-residue zinc-finger loops was found in Aspergillus for the first time. In addition, AoSreA harbors two ZnF-GATA domains with the form of Cys-X2-Cys-X17-Cys-X2-Cys, which is the typical GATA characteristic in animals (. Lowry and Atchley 2000; Patient and Mcghee 2002). Therefore, the features of A. oryzae GATA TFs strongly demonstrate that A. oryzae GATA TFs might be the combination of both plant and animal GATA TFs, which is consistent with the report that fungal GATA TFs are combination of both plant and animal GATA TFs in terms of the numbers of ZnF-GATA domains and amino acid residues present in the zinc-finger loop.
TFs are one of the key transcriptional regulators governing gene regulation and exhibit different expression profiles under distinct physiological and environmental conditions and act as synchronizing elements between stimuli and response. Many studies have revealed the GATA TFs are involved in the regulation of various abiotic stress responses in plants (Peng et al. 2015; Gupta et al. 2017; Nutan et al. 2019) and few fungi (Crespo et al. 2001; Fulle et al. 2013; Marty et al. 2015; Chung et al. 2020). The temperature and salt concentration are two of the most important environmental factors affecting the growth of A. oryzae during fermentation process (Machida et al. 2008; Chen et al. 2011; Bechman et al. 2012; Wang et al. 2013). AreA and AreB function as positive and negative transcriptional regulators participating in regulating nitrogen metabolism and carbon metabolism in Fusarium fujikuroi and Aspergillus nidulans (Michielse et al.2014; Pfannmüller et al. 2017; Chudzicka-Ormaniec et al. 2019). The expression level of AoAreA and AoAreB showed opposite trends at high temperature (42 ℃) compared with CK (30 ℃) in A. oryzae (Fig. 5A), which indicated AoAreA and AoAreB might also act respectively as negative and positive transcriptional regulators under high-temperature stresses. The AoNsdD and AoSnf5, clustering into NSDD subgroup in the NJ_tree (Fig. 2), were strongly induced under high salt stresses. NsdD has been reported as a key repressor affecting the quantity of asexual spores in Aspergillus (Lee et al. 2014; 2016), but there is lack of research on NsdD in response to adversity stress in Aspergillus. Apart from the regulation of siderophore biosynthesis and iron metabolism, SreA is also related with the maintenance of cell wall integrity and negatively impacts resistance as ΔsreA increases resistance to H2O2, calcofluor white, and Congo red (Chung et al. 2020).The expression level of AoSreA was significantly downregulated under 42 ℃ and high salt stresses, which indicates AoSreA might negatively impact high-temperature and high salt resistance. In contrast, AoSreA was significantly upregulated at 22 ℃, and there is a report that the SreB strongly expresses and contributes to filamentous growth at 22 ℃ via lipid metabolism in Blastomyces dermatitidis (Marty et al. 2015). AoSreA and SreB shared the same conserved ZnF_GATA domain (Figure S2), which demonstrates that overexpression AoSreA in A. oryzae might also enhance the growth of mycelium at 22 ℃. Moreover, AoCreA, interacting with AoSreA protein within the PPI network, has the same expression patterns as AoSreA, which indicates AoSreA might positively regulate the AoCreA under temperature and high salt stresses. Curiously, the expression level of AoCreA was inhibited under high salt stresses in A. oryzae, which conflicted with the previous study that ΔcreA mutants of Fusarium graminearum are sensitive to salt stress (Hou and Wang 2018). However, the results provide insights into the critical role of SreA in resistance to different temperatures and high salt stresses in A. oryzae.
LreA and LreB, is the GATA TFs of WC-1 and WC-2 subgroups involve in the regulation of blue- and red-light responses (Purschwitz et al. 2008; Fuller et al.2013). AoLreA and AoLreB, dividing respectively into WC-1 and WC-2 subgroups in NJ_tree (Fig. 2), acts as a dimer and contain typical PAS dimerization domains that display in Table 1 and Fig. 1B. Previous studies have demonstrated that the PAS domain also functions in sensing environmental or physiological signals including oxidative and heat stress (Nan et al. 2011; Corrada et al. 2016). Therefore, except for the regulation of blue- and red-light responses, the PAS domains presenting in AoLreA and AoLreB may facilitate the environmental response analysis of A. oryzae GATA TFs. Additionally, LreA and LreB is a regulatory complex of the global regulator VeA, while VeA plays a critical role in environmental stress responses in A. cristatus, and the ΔveA mutants are more sensitive to high salt, osmotic pressure, and temperature stress (Calvo 2008; Tan et al. 2018). In our study, AoLreA and AoLreB was increased under high-temperature (42 ℃) stresses, and AoLreA was significantly induced expression under 5.0 and 10.0 g/100 mL NaCl stresses. The results demonstrated that AoLreA and AoLreB might act as a regulatory complex of the global regulator VeA in response to temperature and high salt stresse in A.oryzae. Hence, the expression patterns of these A. oryzae GATA TFs under distinct environmental conditions provided useful information for the further analysis of A. oryzae GATA TFs in regulation of various abiotic stress responses in Aspergillus.