Time-specific quick salt response modules in the roots of bermudagrass
Previous transcriptome analysis of plants under salt stress reveals differential response at early and late stages of stress [36, 37]. The temporal dynamic gene expression changes could give us a more comprehensive analysis to evaluate a plant’s response to a stress factor. Through comprehensive transcriptional analysis of the salt-responsive DEGs in the roots of bermudagrass which were exposed to salt for multi-time points, our results provided a list of key response genes and categories showing dynamic expression change pattern. Generally, compared with their respective control roots, 44,847 (4760 up- and 40,087 down-regulated), 18,793 (3730 up- and 15,063 down-regulated), 7362 (1915 up- and 5447 down-regulated) DEGs were specifically regulated in 1 h, 6 h or 24 h salt-treated bermudagrass, respectively (Fig. 2c). The diversity of temporal expression patterns of DEGs further detected by STEM also indicated a time-specific response and a highly complex regulatory network underlying the response to salt stress in the roots (Fig. 3). These data also showed that about 2.4 and 6 times more specific salt-responsive genes were differentially regulated in the roots exposed to salt for 1 h than those exposed to salt for 6 h or 24 h respectively, suggesting that more genes showed a quick response when subjected to salt (Fig. 3). As a consequence, more salt response categories were enriched at 1 h compared to later time points (Fig. S6; Table S3).
For example, after perceiving abiotic stimuli, signal receptors kinases always response at earlier time point to function in protein phosphorylation and modification, which is an important mechanism in initiating salt response signalling pathways and leading to transcription regulation [38-42]. In the roots of bermudagrass, several signal receptors like kinases (e.g. LRR, thaumatin-like, RLK1, DUF26, LLD, LRK10 like, PERK, WAK) were found immediately and specifically up-regulated at 1 h compared to the latter time points (Fig. S4a). Moreover, the salt signal could also immediately trigger the downstream hormones pathways, which are known to be involved stress responses in a wide range [22, 23, 24, 60]. In this study, genes involved in ABA synthesis and signal transduction sub-bins (17.1.1, 17.1.2, 17.1.3) showed consistently up-enriched at all three time points (e.g. NCED, PP2C, ABFs) (Fig.5a), suggesting the established role to salt response [23, 24]. However, we also noticed that transcripts involved in the metabolism of biosynthesis and signal transduction of ETH and JA were specifically over-represented when the roots were exposed to salt for 1 h (Fig. 4a; Table S3), indicating that these salt responsive hormones metabolism pathways might participate in the quick response to salt stress in the roots of bermudagrass [37, 43, 44]. In addition, the induced of transcripts involved in CTK and GA degradation were noticed (Fig. 4a; Table S3). 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 showed up-regulated (Table S3), which encoding a cytokinin oxidase/dehydrogenase participated in catalysing the degradation of cytokines [45-46]. These results suggested that hormone signaling does not work alone while mediating salt response and might function in multifarious crosstalk network with other hormones.
Intracellular phosphorylation events are downstream of secondary messengers (Ca2+, ROS or hormones), such as CDPKs [11-17] and MAPK cascades [47-49], which are reported to be essential sensor-transducers in plants. In this study, some gene members involved in calcium signaling also showed a quick response immediately after the roots were exposed to salt for 1 h (e.g. CDPK11, CAM3, CPK5, CML43) (Fig. S4a; Table S3). Some calcium-transporting ATPase encoding genes were specifically over-represented at 1 h, which could further promote the transmembrane transport of Ca2+ (Table S3). A mitogen-activated protein kinase MAPK2 (cluster-342212.26954), which is a homolog to At2g43790 was also up-regulated specifically at 1 h (Fig. S2b), might interplay with ROS and hormone in salt response [50, 51]. The immediately up-regulation of these protein kinases encoding genes might further trigger downstream transcriptome reconfiguration to cope with the stressful salt condition [52].
In this study, we also identified more than ten transcription factor families, which were significantly induced at one or more time points after the roots were exposed to salt condition (Fig. S5) and the induced TFs number was much more at 1 h than that at 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 three families (MYB, HB, bZip) were significantly induced at all three time points (Fig. 5b; Table S5). Through WGCNA, one HSF transcription factor was investigated as hub gene. This HSF transcription factor showing up-regulated by salt at all three time points could be a good target to study in the future (Fig. 7f). Consistent with the previous studies that WRKY TFs could positively or negatively participate in salt tolerance [50-59], we observed that transcripts for 20 of the 23 WRKY TFs detected significantly induced in response to 1 h salt treatment in the roots of bermudagrass (Fig. S5; Table S5). The AP2/EREBP family were reported to include some stress responsive transcription factors [61, 62]. In this study, we also observed 16 of 17 AP2 transcripts were up-regulated after 1 h salt treatment. When exposed to salt, another most affected TF family in the roots of bermudagrass was bHLH, with 24 of 28 transcripts were induced at 1 h and 10 of 19 were increased at 6 h by salt stress (Table S5). Among these induced bHLH transcription factors, some important members which have been reported positively participated in salt stress response such as bHLH92 [63]. 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 S5). 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 [64]. In addition, a series of transcription factors (e.g. ARF, ARR, C3H, NAC, Trihelix, AS2, JUMONJI, PHOR1, Psudo ARR) showed specifically up-enriched under 1 h salt treatment compared to the later time points, implying that these TFs might be specifically involved in quick salt response in the roots of bermudagrass (Fig. S5; Table S5). Therefore, in this study, we noticed that some molecular processes, such as signal transduction, hormone metabolism and regulation of transcription factors were induced at 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
It is well known that plants have evolved large gene families for detoxification of ROS caused by harsh environments such as salt [20, 21, 64, 65]. In our study, the POD activity was significantly higher in the roots of 1 h and 6 h salt-treated seedlings compared to that in their respective control roots (Fig. 1b) and a few members of POD encoding genes were up-regulated (Fig. 4d; Table S3). 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 S3) also showed 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 [66]. Consistent with the previous studies, some bioactive secondary metabolities in the roots of bermudagrass (e.g. carotenoids, tocopherols and flavonoids) [67-69] were also over-represented under salt and might also serve as ROS scavengers (Fig. 4b; Table S3). As expected, genes regulating levels of osmoprotectants also showed highly stress responsive in salt-treated roots of bermudagrass, such as 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 [28-31].
The plant cell wall is considered an important factor involved in sensing of and response to salt stress, which mainly consists of cellulose, hemicellulose, lignin, pectins and many glycoproteins [70, 71]. In this study, 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 when exposed to salt for 1 h (Fig. 4d; Table S3), which might be participated in the post-translational modifications of cell wall proteins and lead to the alteration of cell wall flexibility [72-75]. 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 of bermuagrass. For example, the expression of MUR4 was found up-regulated in the roots of bermudagrass (Fig. 4f), which 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 [76]. Moreover, a number of 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 [77, 78] and one AGP (SOS5) was known to contribute to salt tolerance in Arabdiopsis [79]. We further noticed that the lipid metabolism showed a quick response in the roots of bermudagrass. 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 [80-82]. In our study, salt stress markedly changed the expression of genes encoding ω-3 FA desaturases and might lead to an alteration of FA composition (Fig. S4b, Table S3). The immediately regulated expression of genes coding for recombination of lipid composition can provide novel insights for the improvement of salt tolerance in bermudagrass.
Except for secondary metabolisms related genes which significantly participated in cell wall modification (Fig. 4f), some important secondary metabolism pathways were significantly induced in the later phase, suggesting longer-term reactions that may involve metabolic adjustment [83, 84]. For example, the polyamine synthesis sub-bin showed over-represented only after 6 h and 24 h salt treatment. Some sub-bins included in secondary metabolism were specifically over-represented at 24 h, such as simple phenol, glucosinolates, isoflavones and tocopherol biosynthesis (Fig. 4b; Table S3). These secondary metabolisms were previously reported to be involved in plants oxidative response in some species [83, 84]. 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 oxidise stress caused by salt stress [85-87].
Categories downregulated by salt stress in the roots of bermudagrass
In previous proteomic studies, protein translation always showed a decrease following NaCl treatment, which is consistent with this study that the majority of transcripts for almost all ribosomal proteins were down-regulated (Fig. S3; Table S3) [31, 88]. The inhibited protein synthesis and enhanced protein degradation might increase the concentration of free amino acid, especially proline. Proline can be used as osmotic protective substance under osmotic stress and free amino acid could further used for the synthesis of dehydrin or polyamine, which might function in the maintenance of the structure of protein and cell membrane under salt. However, genes involved in protein translational modification such as kinase and ubiquitination pathway related genes were up-regulated (Fig. S3). Notably, E3 RING and E3 SCF proteins were significantly enriched in salt induced genes (Fig. S3; Table S3), suggesting that these enzymes function in ways that might be independent of the 26S proteasome in salt response [89, 90]. Moreover, genes required for the tricarboxylic acid cycle (TCA) , which is the main respiratory pathway were generally down-regulated by salt stress (Fig. S1a; Table S3). 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 S3). Genes encoding components of the mitochondrial elecron transport chain such as NAD(P)H dehydrogenases and F1-ATPase were also especially enriched among down-regulated profiles (Fig. S1b; Table S3), suggesting that the mitochondria might be damaged by oxidative stress. In addition, we also noticed that genes involved in DNA synthesis and cell organization also showed down-regulated (Fig. S1c, d). These down-regulation pathways might function together in conserving energy to maintain plants growth and development[31, 91].