Potential candidate genes conferring wheat ER stress response
In this study, we analyzed the responses of wheat seedlings to ER stress from four biological levels: morphological, physiological, cytological and molecular, and we identified the genes likely involved in the regulation of stress.
Genes related to protein processing in ER
In the process of protein folding, molecular chaperones are indispensable. The aforementioned molecular chaperones, such as Bip, CNX/CRT, GRP94, and PDIs, play critical roles in protein folding, particularly when cells are subjected to malfolded proteins and unassembled complexes. Under numerous stresses, Bip expression is significantly up-regulated, including ER stress agents such as TM and DTT [4, 21, 25], drought [50], cold [51], Cd2+ [5] and insect and pathogen attack [1]. Overexpression of Bip confers drought tolerance in soybean (Glycine max) [52] and tobacco (Nicotiana tabacum) [52, 53] and increases the tolerance to Cd2+ in tobacco [5]. In this study, we found the number of chaperones accounted for more than half of the 66 DEGs in the “protein processing in endoplasmic reticulum” pathway within the three treatment groups (Additional file 14: Table S9). Among these DEGs, Bip genes were significantly up-regulated under DTT, and the expression was even higher after co-treatment with TUDCA; additionally, we detected the dynamic changes of Bip genes and similar results were obtained (Additional file 14: Table S9 and Additional file 15: Figures S6A-B), implying a role in the regulation of wheat response to ER stress.
The expression of SHD, the only ortholog of GRP94 in Arabidopsis, was up-regulated after 2 and 4 h of treatment with ER stress agents TM or DTT [22, 27]. However, in the present study, compared with the control, we found GRP94 expression was down-regulated under DTT treatment in wheat at 48 h and was up-regulated after TUDCA co-treatment (Additional file 14: Table S9 and Additional file 15: Figure S6C). Therefore, we further studied the expression of GRP94 under different time points, and we found GRP94 was up-regulated under DTT treatment at 4 h and 24 h, and then down-regulated at 48 h and 96 h. However, under DTT + TUDCA co-treatment, GRP94 was up-regulated at 4 h, but down-regulated at 24 h, and then up-regulated at 48 h and 96 h (Additional file 15: Figure S6C).
CNX and CRT are ER chaperone proteins, bind calcium ions and participate in protein folding. Rice CNX (Os-CNX) is induced by various abiotic stresses, and overexpression of Os-CNX in tobacco confers drought tolerance [54]. Furthermore, wheat CRT (Ta-CRT) is induced by drought, and overexpression of Ta-CRT in tobacco plants increases drought resistance [55]. In our study, Ta-CRT was not induced by DTT treatment at 48 h, but the homologous ER-lumenal protein, Ta-CNX, was significantly up-regulated under DTT and further up-regulated after co-treatment with TUDCA (Additional file 14: Table S9 and Additional file 16: Figure S7A).
PDIs are molecular chaperones that catalyze the formation of disulfide bonds between unfolded proteins. In our study, eight genes related to PDI were obtained in wheat. Except for Traes_6AS_5896DC565, the other seven genes exhibited the same trend: PDIs were markedly up-regulated under DTT and further up-regulated under TUDCA co-treatment (Additional file 14: Table S9, Additional file 15: Figure S6D and Additional file 16: Figures S7B-C).
Other chaperones, such as DnaJ and those in Hsp70 and Hsp20 families, participate in ERAD (Fig. 7). Except for chaperones, other genes were related to ubiquitin-ligase complexes, including two E3 ubiquitin-protein ligase RNF5 genes (RMA1) and two ubiquitin-conjugating enzyme E2 D/E genes (UbcH5). Compared with the control, RMA1s (Novel07430 and Novel07785) were down-regulated under DTT and up-regulated after co-treatment with TUDCA, whereas UbcH5s (Traes_6BS_72F59E261 and Traes_6AS_E011BC5BB) showed the opposite trend (Additional file 14: Table S9). These results indicate that molecular chaperones actively participate in the regulation of plant response to ER stress.
Photosynthesis-related genes
A total of 158 photosynthesis-related genes were identified by RNA-seq. In addition to the aforementioned genes identified in the “photosynthesis” pathway, 65 DEGs were involved in “photosynthesis-antenna proteins” pathway. In this pathway, the DEGs were chlorophyll a/b-binding proteins, which primarily collect and transfer light energy to photosynthetic reaction centers and are down-regulated when plants are subjected to environmental stresses [56]. In this study, chlorophyll a/b-binding proteins were down-regulated under DTT but up-regulated after co-treatment with TUDCA (Additional file 16: Figures S7D-F). Correspondingly, chlorophyll a and b contents also showed a marked decrease under DTT treatment, whereas the effects were alleviated by TUDCA co-treatment (Figs. 2A, B). Furthermore, chlorophyll is the most important pigment in plant photosynthesis for the absorption and transmission of light energy, and the pathway of chlorophyll biosynthesis is completed by a series of enzymatic reactions. We identified 49 DEGs involved in “porphyrin and chlorophyll metabolism” pathway, and they were key enzyme genes in chlorophyll biosynthesis and might play critical roles in maintaining plant growth under stress conditions.
Antioxidant enzyme genes
One of the common responses when plants are subjected to a wide range of biotic and abiotic stresses is the generation of ROS [57], which cause oxidative damage to plants [58]. Fortunately, plants developed an antioxidant defense system, which primarily consists of antioxidant enzymes to scavenge ROS and protect cells from oxidative injury [58]. The over-accumulation of ROS can result in cytomembrane damage and even cell death [31, 58]. In this study, a total of 42 DEGs were identified that were related to antioxidant enzymes, including PODs (39 genes), SODs (2 genes) and CAT (1 gene). The relative expression of SODs was down-regulated under DTT treatment and up-regulated after DTT + TUDCA co-treatment. We monitored the dynamic changes of one SOD gene (Traes_7AL_E14A72218), and similar results were obtained over time (Additional file 15: Figure S6E). CAT expression exhibited a trend of continuous down-regulation. For PODs, the expression patterns were different under the three treatments (Additional file 16: Figures S7G-I). Correspondingly, the activity of antioxidant enzymes SOD and CAT increased under DTT treatment compared with that of the control, whereas activity level was eased by TUDCA co-treatment (Figs. 2E, F), indicating the ROS levels were reduced by TUDCA. Thus, these antioxidant enzyme genes may play critical roles in the process of wheat response to stress.
Plant hormone-related genes
A total of 318 genes were identified with involvement in plant hormone biosynthesis and signal transduction. Among these genes, 110 genes were involved in the signal transduction of plant hormones, with those related to ABA signaling pathway predominantly induced, including protein phosphatase 2Cs (PP2Cs), serine/threonine-protein kinase SRK2s (SnRK2s), ABA-responsive element binding factors (ABFs), auxin-responsive protein IAAs (AUX/IAAs), and abscisic acid receptor PYR/PYL families (PYR/PYLs). Researchers identify these genes as involved in stress responses. For example, TaPP2C expression is induced by water stress, and TaPP2C may be an early signal molecule [59]. ABFs act as bZIP TFs and play important roles in responding to environmental stresses. Similarly, OsABF1 is induced by abiotic stress and increases stress signaling in rice [60], and SnRK2 is involved in dehydration stress signaling in Arabidopsis [61]. In this study, compared with the control, the expression of almost all TaPP2Cs, ABFs, SnRK2s was up-regulated under DTT, whereas almost all were down-regulated after TUDCA co-treatment.
The other 208 genes were related to plant hormone biosynthesis. Among these genes, the most abundant genes were involved in SA, ET, auxin and JA biosynthesis. For example, JA is an important signaling molecule and is an endogenous regulator in plant defense against environmental stresses. In rice, the 12-oxo-phytodienoic acid reductase gene (OsOPR1) encodes 12-oxo-phytodienoate reductase, which is involved in the biosynthesis of JA, and OsOPR1 may play a regulatory role in rice defense, stress response and reproductive development [62]. In foxtail millet, SiOPR1 encodes a putative 12-oxophytodienoic acid reductase 1, which plays an important role in response to drought stress [63]. In this study, 9 JA biosynthesis-related genes (12-oxophytodienoate reductase 1, OPR1) were identified that participated in “alpha-linolenic acid metabolism” pathway. Furthermore, the 9 OPR1 genes exhibited different expression patterns. For example, the relative expression of Traes_2AS_A9F768C2B and Traes_2DS_A886F6C92 was down-regulated under DTT treatment but was up-regulated under DTT + TUDCA co-treatment. Additionally, the expression of Traes_6DL_94DCF0B70 exhibited a trend of continuous up-regulation (Additional file 16: Figures S7J-L). The results showed TUDCA increased the expression of OPR1, which was followed by an increase in resistance of wheat to ER stress. Therefore, plant hormone-related genes might play important roles in wheat response to ER stress and could act as signal molecules.
Transcription factors
Based on the RNA-seq data in this study, we found the top five most abundant TF families were MYB, NAC, orphans, bHLH, and bZIP. MYB families play an important role in regulatory networks that control metabolism, development and response to environmental stresses [64]. For example, Arabidopsis MYB112 promotes anthocyanin formation under salinity and high light stress [65]. The orthologous gene of AtMYB112, Traes_1AS_36AF74187, was up-regulated under DTT and further up-regulated after TUDCA co-treatment, and we detected the dynamic changes of this gene and similar results were obtained over time (Additional file 15: Figure S6F); however, another MYB-related family gene, Novel12259, exhibited a different expression pattern under DTT treatment (Additional file 16: Figure S7M). Additionally, MYBs often combine with bHLHs in plant gene regulation under stress [66]. In plants, bHLHs regulate abiotic stress response and tolerance, and TabHLH39 improves tolerance to drought, salt and cold stress in transgenic Arabidopsis [67]. In our study, compared with the control, the expression of TabHLH39 (Novel07753) was down-regulated under DTT, whereas expression was up-regulated after TUDCA co-treatment (Additional file 16: Figure S7N).
In plants, NAC TFs are a large family of regulators and play vital roles in plant development and in response to environmental stresses [68]. In Arabidopsis, NAC062, NAC089 and NAC103 are identified as ER stress-related MTFs, playing important roles in responding to ER stress. In this study, the aforementioned MTFs were not detected, but other important NACs were identified, such as ANAC102, TaNAC6, TaNAC8 and TaNAC29. ANAC102 affects viability of Arabidopsis seeds after low-oxygen treatment [69], and GmNAC6 (Glycine max NAC6) is induced by ER stress and osmotic stress and participates in the NRP-mediated cell-death signaling pathway induced by ER stress and osmotic stress [70]. TaNAC8 functions as a transcriptional activator and is involved in resisting abiotic and biotic stresses in wheat [71], and overexpression of TaNAC29 in plants increases tolerance to high salinity and dehydration [72]. Therefore, these NAC genes may play important roles in wheat response to ER stress. Compared with the control, almost all of these TF genes were similarly up-regulated under DTT and down-regulated after TUDCA co-treatment.
bZIP TFs regulate processes that include pathogen defense, light and stress signaling, seed maturation and flower development [73]. In Arabidopsis, bZIP60, bZIP28 and bZIP17 are also MTFs. For example, bZIP60 plays an important role in ER stress responses in Arabidopsis through the up-regulation of genes encoding factors that aid in protein folding and degradation [24, 25]. In our study, compared with the control, the orthologous genes of AtbZIP60 (Traes_7AL_25850F96F, Traes_7BL_625F55A12 and Traes_7DL_3CE000E38) were all up-regulated under DTT and were further up-regulated after TUDCA co-treatment.
WRKYs are also widely involved in biotic and abiotic stress responses [74]. For example, WRKY33 is a TF that plays an important role in plant defense against environmental stresses. In Arabidopsis, WRKY33 is vital for plant resistance to necrotrophic pathogens [75], and WRKY33 is also an autophagy regulatory gene, which is up-regulated by ER stress [28]. WRKY33 participates in heat tolerance in Arabidopsis [76], and overexpression of AtWRKY33 increases salt stress tolerance in Arabidopsis [77]. In our study, compared with the control, the expression of Novel13869 (AtWRKY33/TaWRKY27) was down-regulated under DTT but up-regulated by TUDCA co-treatment (Additional file 16: Figure S7O).
Other-related genes
In addition to the potential candidate genes mentioned above, we screened another 10 genes with fold changes greater than 4, such as MLO, TPP, P5CS and SRG1 (Additional file 17: Table S10).
For example, MLO protein, which is a calmodulin-binding protein (CBP), is involved in biotic and abiotic stress responses of plants [78]. Mlo is a key gene for resistance to powdery mildew in barley, and the wild-type gene has a negative regulatory function in plant defense, whereas mlo mutants show greatly increased resistance [78, 79]. Additionally, mlo mutants exhibited spontaneous mesophyll cell death, indicating Mlo likely has a functional role in cell death protection during environmental stresses [78]. We speculate that the MLO protein may play a part in inhibiting the progress of cell death; thus, the cell death ratio was reduced.
Trehalose 6-phosphate phosphatase (TPP) is involved in trehalose biosynthesis during chilling stress in rice [80], and overexpression of OsTPP1 confers stress tolerance in rice [81]. Moreover, yeast TPP expressed in tobacco results in drought tolerance [82].
Delta-1-pyrroline-5-carboxylate synthetase (P5CS) is a bifunctional enzyme involved in proline biosynthesis [83]. Proline is accumulated by overexpressed P5CS, which confers salt tolerance in transgenic potato [84] and water and salt tolerance in transgenic rice [85].
Additionally, the other genes may also play indispensable roles in ER stress response in wheat. For example, plant SRG1 is a senescence-related gene 1 and a member of the Fe (II) / ascorbate oxidase superfamily [86], which plays an important role in anti-oxidative stress [87], and pectin lyase-like is a superfamily protein that is related to cell wall degradation and fruit softening [88].
Comprehensive analysis of possible mechanism of ER stress regulation in wheat
To further understand ER stress regulation in wheat, other two wheat genotypes with contrasting tolerance to PEG stress, Hanxuan10 and Zhengyin1 which were used in our previous study [89], were utilized to observe the changes of their morphological, physiological and molecular index. The treatments were performed on Hanxuan10 and Zhengyin1 seedlings as that on Yunong211. And the gene expressions were monitored by qRT-PCR at 4 h, 1 d, 2 d and 4 d after treatments. Similar results were observed in Hanxuan10 and Zhengyin1 as that in Yunong211 under DTT treatment and DTT + TUDCA co-treatment (Additional files 18, 19, 20: Figures S8-S10).
Although the chemical chaperone TUDCA has been widely employed to ease ER stress in mammals and Arabidopsis, we conducted a set of experiments to evaluate the effects of TUDCA on wheat normal growth. We found no obvious side effects of a single TUDCA treatment, based on molecular, cellular, physiological and morphological changes (Additional files 21, 22, 23, 24: Figures S11–14). We also studied the effects of TUDCA on seedling growth of Hanxuan10 and Zhengyin1 and we observed a similar result as that of Yunong211 (Additional file 20: Figures S10 and Additional file 25-26: Figures S15-S16). In our previous study, we have reported TUDCA could alleviate osmotic stress induced cell death in which ER stress related genes were involved [37]. And we also revealed that foliar spraying 100 μg/mL of TUDCA solution had no obvious effect on seeding growth except for slightly improving the physiological characteristics of wheat leaves and enhancing the expression of TabZIP60 under normal growing conditions. And in this study, we didn’t observe obvious side effects of TUDCA on seedling growth either except for slightly reducing root length (at 2 day), and increasing fresh weight (at 3 d and 4 d) and dry weight (at 2 d, 3 d and 4 d). Interestingly, after analyzing the RNA-seq and qPCR data using samples collected from 4 groups (Control, single DTT treated, single TUDCA treated and DTT and TUDCA co-treated), we did find several genes responded to single TUDCA treatment. We speculated the function of these genes might be a possible reason to explain the slight changes of seedling when they were treated with TUDCA only but the solid evidence is lack at present. Therefore, we need to further study these genes to understand their functions on regulation plant growth under ER stress.
As discussed earlier, we analyzed the potential candidate genes conferring wheat ER stress resistance. To confirm whether these genes really respond to ER stress, we randomly chose several genes to detect their expression under various treatments (control, 20% PEG, 20% PEG+TUDCA, 42 °C heat, 42 °C heat+TUDCA, TUDCA), time courses (4 h, 1 d, 2 d and 4 d) and wheat cultivars (Yunong211, Hanxuan10 and Zhengyin1) (Additional file 27: Figure S17). Combined the changes of these genes under DTT treated and DTT + TUDCA co-treated, chlorophyll a/b-binding protein, SOD and bHLH39 are conserved across species. Here, based on the response of potential candidate genes to ER stress, we develop a hypothetical model to elucidate a possible mechanism of wheat response to ER stress (Fig. 14).
In this study, we used DTT and TUDCA to induce or suppress ER stress, respectively. When plants are subjected to ER stress, ROS are induced. Under ER stress, calcium homeostasis is imbalanced and calcium ions are released from the ER into mitochondria. Subsequently, calcium accumulation in the mitochondria leads to the release of Cyt C and the decrease of Cyt C oxidase activity, and finally, ROS accumulate and cell death is induced [18, 32]. To protect against the damage of ROS, plant cells and its organelles such as mitochondria and chloroplasts employ antioxidant defense systems to scavenge the ROS. The cells also initiate UPR and regulate ER stress responsive genes, such as molecular chaperones, to help correct folding of proteins in the ER. In Arabidopsis, the cells regulate genes related to survival, such as bZIP28 [21,22,23], bZIP60 [24, 25], NAC103 [26], NAC062 [27], AGB1 [90] and BI-1 [10] in response to ER stress. However, the ability of plants to suppress ER stress is limited. Therefore, when ER stress is too severe or chronic, the cells will initiate genes associated with death, such as NAC089 [28], BAG6 [91] and Metacaspase [92], with subsequent induction of cell death. Here, based on aforementioned analyses, we provide some new insights about potential candidate genes that are involved in ER stress responses for cell survival in wheat. We conclude that genes related to molecular chaperones, photosynthesis, antioxidant enzymes, plant hormones, TFs, and others may play vital roles in responding to ER stress and promoting cell survival.