Time-specific quick salt response modules in the roots of bermudagrass
Previous transcriptome analysis of plants under salt stress reveals differential response strategy at different stages of stress [32, 33]. For instance, plants response to the initial osmotic stress by increasing the intracellular concentrations of osmolytes . After NaCl exposure for 24 to 72 h, alleviating Na+ toxicity raises to a more urgent task [23, 24]. To investigate the transcriptome adjustments of bermudagrass roots to the salt shock in the early phase, 1 h was firstly chosen to study the immediate salt response. We next chose 6 h as a treat time point to investigate the immediate following reaction after the earliest response to salt (1 h) based on the previous study showing that soybean faced to an initial osmotic stress stage in 1 h to 4 h after salt treatment . Moreover, 24 h was still chosen to investigate if the salt response strategy begins to change in bermudagrass because 24 h might be a turning point at which the salt response strategy might begin to change in some plants [23, 24].
In bermudagrass, about 2.4 and 6 times more specific salt-responsive genes were differentially regulated in the roots exposed to salt for 1 h compared to those exposed to salt for 6 h or 24 h respectively, suggesting that more genes and categories responded quickly after salt exposure (Fig. 3). For example, several signal receptors like kinases (e.g. LRR, thaumatin-like, RLK1, DUF26, LLD, LRK10 like, PERK, and WAK) were detected immediately and exclusively up-regulated at 1 h (Fig. S4a). These signal receptors kinases always response at earlier time point to function in protein phosphorylation and modification, which is an important step in initiating salt response signalling pathways and ultimately leading to a transcriptional regulation [34-38]. Moreover, the salt signal could also immediately trigger the downstream hormones pathways, which are known to be involved in stress responses in a wide range [19, 50]. In this study, genes involved with ABA biosynthesis and signal transduction sub-bins (17.1.1, 17.1.2, 17.1.3) were consistently up-enriched at all three-time points (e.g. NCED; PP2C and ABFs) (Fig.5a), suggesting the established role to salt response . However, we also noticed that transcripts involved in the metabolism of biosynthesis and signal transduction of ETH (e.g. ACC synthase; ACC oxidase and ERF) and JA (e.g. AOS1 and AOC4), were exclusively over-represented at 1 h of salt exposure (Fig. 4a; Table S2), indicating that these salt responsive hormones metabolism pathways might participate in the quick response to salt stress in the roots of bermudagrass [33, 39, 40]. In addition, the induction of transcripts involved in CTK and GA degradation were noticed (Fig. 4a; Table S2). Transcripts encoding gibberellin-degrading enzyme gibberellin 2-oxidase (homologs of At4g21200 and At1g75450 respectively) suggested the cell growth were partly inhibited to survive under salt stress. The expression of at least 9 transcripts of AtCKX6 (At1g75450) homologs were regulated (Table S2), which encoding a cytokinin oxidase/dehydrogenase that participate in catalysing the degradation of cytokines [41-42]. These results suggested that hormone signaling does not work alone while mediating salt response but might function in multifarious crosstalk network with other hormones.
Intracellular phosphorylation events are downstream of secondary messengers, such as CDPKs [9-14] and MAPK cascades [43-45], which are reported to be essential sensor-transducers in plants. In this study, some gene members involved in calcium signaling responded immediately after salt exposure for 1 h (e.g. CDPK11, CAM3, CPK5 and CML43) (Fig. S4a; Table S2). Some calcium-transporting ATPase encoding genes were specifically over-represented at 1 h, which could further promote the transmembrane transport of Ca2+ (Table S2). A MAPK2 gene (cluster-342212.26954), which is a homolog to At2g43790 was also up-regulated exclusively at 1 h (Fig. S2b) and might interplay with ROS and hormone in salt response [46, 47]. The immediate up-regulation of these protein kinases encoding genes might further trigger downstream transcriptome reconfiguration to cope with the stressful salt condition .
In this study, we also identified more than ten transcription factor families, which were significantly induced at one or more time points after salt exposure (Fig. S5). The induced TFs number was much more at 1 h than latter time points. Among those TFs, AP2, WRKY, bHLH and HB families accounted for a large ratio of the total number of salt-induced TFs identified and the three families (MYB, HB, bZip) were significantly induced at all three time points (Fig. 5b; Table S2). One HSF transcription factor was investigated as a hub gene of brown4 co-expression network in this study (Fig. 6d). The expression of this HSF transcription factor showed up-regulated by salt at all three time points and it could be a good target for future studies (Fig. 7f; S7). Consistent with the previous studies that WRKY TFs could positively or negatively participate in salt tolerance , we observed that transcripts for 20 of the 23 WRKY TFs detected significantly induced in response to 1 h salt treatment in the roots (Fig. S5; Table S2). The AP2/EREBP family was also reported to include some stress-responsive TFs . We also observed that 16 of 17 AP2 transcripts were up-regulated after 1 h salt treatment (Table S2). Under salt stress, another most affected TF family in the roots was bHLH, with 24 of 28 transcripts being induced at 1 h and 10 of 19 were increased at 6 h by salt stress (Table S2). Among these induced bHLH TFs, some important members which have been reported to positively participate in salt stress response such as bHLH92 . The Aux/IAA families were significantly enriched in salt-responsive transcripts especially at 1 h with all 12 transcripts all up-regulated by salt stress (e.g. IAA5, 12, 20, 24, 18, 23) (Table S2). These salt response Aux/IAA genes have a central role in auxin response and might act to integrate environmental inputs into the auxin gene regulatory network . Therefore, here, we noticed that some molecular processes, such as signal transduction, hormone metabolism and regulation of TFs were induced at the earlier time point and might form a cascade to active the downstream response factors.
Common and distinctive positive salt response mechanisms in the roots of bermudagrass
Plants have evolved diverse gene families for the detoxification of ROS caused by harsh environments such as salt [19, 20]. In our study, the POD activity was significantly higher in the roots of 1 h and 6 h salt-treated plants compared to that in their respective control roots (Fig. 1b). However, the SOD activity of 1 h and 6 h salt-treated roots showed an upward trend, but the increase was not significant compared to their respective control plants (Fig. 1c). Accordingly, a few members of POD encoding genes were up-regulated but SOD encoding genes not up-regulated in our transcriptome data (Fig. 4d; Table S2). Because oxidative stress is a consequence of the deterioration of lipid peroxidation (indicated by MDA) brought about by ROS, we also measured the MDA content in the roots. However, the roots MDA content displayed a higher value than control plants until exposed to salt for 24 h (Fig. 1a), suggesting a progressive accumulation with the increased treatment time. Other members of gene families encoding oxidases-copper, glutathione S transferases, beta 1,3 glucan hydrolases, UDP glucosyl and glucoronyl transferases, plastocyanin-like proteins (Fig. 4d; Table S2) were also up-regulated at one or more time points to cope with the salt stress. For example, UDP glucosyl transferases UGT79B2/B3 in Arabidopsis was reported to contribute to cold, salt and drought stress tolerance via modulating anthocyanin accumulation and enhancing ROS scavenging . Consistent with the previous studies in plants, some bioactive secondary metabolites in the roots of bermudagrass (e.g. carotenoids, tocopherols and flavonoids) [55-57] were also over-represented under salt and might also serve as ROS scavengers (Fig. 4b; Table S2). As expected, the genes regulating levels of osmoprotectants were also highly upregulated in this study. They included genes encoding galactinol synthases, raffinose synthase, trehalose, callose and galactose (Fig. S4d), which were reported to be the first stress-inducible genes under salt stress [23-26].
The plant cell wall consists of cellulose, hemicellulose, lignin, pectin and many glycoproteins [58, 59] and is considered to be an important factor involved in sensing of and response to salt stress. We also noticed that genes involved in cellulose synthase (10.2), hemicellulose synthesis (10.3) and lignin synthesis (16.2.1) were over-represented in the salt-treated roots of bermudagrass (Fig. 4f). The expression of glycoside hydrolase (GH17) family genes was significantly induced under 1 h of salt stress (Fig. 4d; Table S2), suggesting it may participate in the post-translational modifications of cell wall proteins and lead to the alteration of cell wall flexibility [60, 61]. In addition, a limited number of other cell-wall related gene families which function in cell wall extensibility were also showed differential regulation in salt responsive transcripts. For example, the expression of MUR4 was found up-regulated in the roots of bermudagrass (Fig. 4f), and was reported to be involved in the biosynthesis of UDP-arabinose. Mutation in MUR4 affects cell wall integrity and leads to reduced root elongation and defective cell-cell adhesion under high salinity . Moreover, several AGPs (arabinogalactan proteins) encoding genes were found up-regulated by salt at the transcript level in our study (Fig.4f). The AGPs on cell walls or plasma membranes are also reported to be associated with cell growth [63, 64] and one AGP (SOS5) was known to contribute to salt tolerance in Arabdiopsis . We further noticed an earlier response of lipid metabolism in the roots of bermudagrass. In particular, the expression of genes involved in FA synthesis and elongation were down-regulated while genes involved in FA desaturation and lipid degradation were significantly up-regulated immediately when exposed to salt for 1 h (Fig. S4b). Studies have shown that FA desaturases play an important role in the maintenance of the biological function of membranes in plant cells under different conditions including salt stress [66, 67]. Here, salt stress markedly changed the expression of genes encoding ω-3 FA desaturases which might lead to an alteration of FA composition (Fig. S4b, Table S2). The immediate regulation of genes coding for a recombination of lipid composition can provide novel insights for the improvement of salt tolerance in bermudagrass.
Other than secondary metabolisms-related genes which significantly participated in cell wall modification (Fig. 4f), some important secondary metabolism pathways were significantly induced in a prolonged time point, suggesting slightly slower reactions that may involve metabolic adjustment [68, 69]. For example, the polyamine synthesis sub-bin was over-represented only after 6 h and 24 h salt treatment. Some sub-bins included in secondary metabolism such as simple phenol, glucosinolates, isoflavones and tocopherol biosynthesis were specifically over-represented at 24 h (Fig. 4b; Table S2). These secondary metabolisms were previously reported to be involved in plants oxidative response in some species [68, 69]. For example, the expression of laccase encoding genes was found up-regulated especially when exposed to salt for 24 h, which might participate in the oxidation and reduction of simple phenols in the roots of bermudagrass and alleviate the oxidize stress caused by salt stress [70, 71].
Categories down-regulated by salt stress in the roots of bermudagrass
In this study, down-regulated genes were more abundant at all three time points respectively (Fig. 2c), suggesting an impact of the huge negative regulation of transcription on plant metabolism and functioning. Actually, important enriched categories such as hormone metabolism, transcription factors, misc and secondary metabolism also contained large number of down-regulated genes (Table S2). For instance, genes involved in brassinosteroid synthesis or degradation (e.g. CYP450 family members) and signal transduction (e.g. BRI) showed significantly down-regulated by salt stress (Table S2), suggesting an interaction of hormones to participate in salt response in bermudagrass . Although a series of TF families showed up-regulated, other TF families such as C2H2 and HAP had large number down-regulated genes at one or more time points (Table S2). HAP transcription factor AtHAP3b and C2H2 protein Zat7 were previously reported to play key roles in primary root elongation to promote drought tolerance and in salt resistance in Arabidopsis, respectively [73, 74].
In previous proteomic studies, NaCl treatment decreased protein translation, which is consistent with the downregulation of most transcripts for almost all ribosomal proteins in this study (Fig. S3; Table S2) [27, 75]. We also noticed that the number of DEGs after 1 h salt treatment was relatively more than the number after 6 h and 24 h salt treated (Fig. 2c, d). More ribosomal proteins 40S and 60S subunits and protein targeting categories also showed significant over-represented in down-regulated genes immediately after exposed to salt for 1 h, suggesting that more genes involved in protein or amino acid metabolism were quickly and negatively regulated. Genes involved in protein translational modification such as kinase and ubiquitination pathways were up-regulated (Fig. S3). Notably, the E3 RING and E3 SCF proteins were significantly enriched in the salt-induced genes (Fig. S3; Table S2), suggesting that these enzymes may function in ways that might be independent on the 26S proteasome during salt response . The inhibited protein synthesis and enhanced protein degradation might hike the concentration of free amino acid, especially proline, which can act as an osmotic protective substance. In this study, the proline content in the roots of bermudagrass showed significantly induced after NaCl exposure (Fig. 1d). These free amino acids could further initiate synthesis of dehydrin or polyamine, which might function in the maintenance of the structure of the protein and cell membrane under salt . However, proline synthetic related category showed not significantly over-represented, suggesting that genes involving in proline metabolism might not receive significant transcriptional regulation at salt all treat time points in this study.
Moreover, salt stress downregulated the tricarboxylic acid cycle (TCA)-related genes, which is the main respiratory pathway were generally down-regulated by salt stress (Fig. S1a; Table S2). For example, genes encoding pyruvate dehydrogenases, which function in the conversion of pyruvate to acetyl-CoA and thereby links the glycolytic pathway to the TCA cycle, were enriched among down-regulated profiles (Fig. S1a; Table S2). Also, genes encoding components of the mitochondrial electron transport chain such as NAD(P)H dehydrogenases and F1-ATPase were also exclusively enriched among down-regulated profiles (Fig. S1b; Table S2). This suggested that the mitochondria might be damaged by oxidative stress. Also, we noticed that genes involved in DNA synthesis and cell organization were down-regulated especially at 1 h and 6 h (Fig. S1c, 1d). These categories might function together to save energies and materials to maintain plants growth and development under salt stress. A proposed model of key categories positively and negatively affected by salt stress in the roots of bermudagrass was provided (Fig. 8). Generally, signal perception and transduction categories such as signaling receptor kinase, hormone and signal pathways immediately when exposed to salt. The transcription factors response at earlier time point to further positively or negatively regulate the downstream response genes. In this salt response categories, some categories such as lipid metabolism and protein synthesis response much earlier while other categories involved in secondary metabolite biosynthesis response at latter time point [26, 77].