Changes of phenotype and photosynthesis parameters of two varieties under moderate drought
Under moderate drought stress, the net photosynthetic rate (PN) and stomatal conductance (gs) were significantly decreased compared to control plants, while the intercellular CO2 concentration (Ci) were increased. As shown in (Fig. 1b, c, d), the changes of each parameters in SD902 were higher than SD609. Compared with control, the intercellular CO2 concentration in SD609 and SD902 increased by 16.3% and 20.9%, respectively.
Drought stress significantly induced different phenotypic responses between the two varieties. Under moderate drought stress, the leaf tips of SD902 curled more severely than SD609, and the seedlings of SD609 appeared greener than those of SD902 (Fig. 2a). Moreover, the chlorophyll content (SPAD) in SD609 was higher than 902 in both control and water deficit conditions (Fig. 1a), which was consistent with the visual observation.
Changes of photosynthetic characteristics and protective enzymes of two varieties under moderate drought
Drought stress also significantly changed the photosynthetic efficiency, which can be reflected by energy conversion in PSII and PSI. As shown in Fig. 3a, b, the value of Y(II) and Y(I) under drought stress were lower than under control conditions in two varieties, especially in SD902. The decline of Y(II) was accompanied by the increase of Y(NPQ) and Y(NO) in both varieties, of which Y(NO) increased significantly in SD902. Similarly, the increase in Y(I) was accompanied by the changes in Y(NA) and Y(ND). The changes in Y(NA) and Y(ND) were higher in SD902 than that in SD609 (Fig. 3a, b). The values of Y(CEF) of two varieties under moderate drought stress was examined by estimation of ETRI and ETRII (Fig. 3c, d). When exposed to moderate drought stress, values for ETRI and ETRII significantly decreased compared to control conditions. There is a clear difference in Y(CEF) between the two varieties Fig. 3e. Compared with control, Y(CEF) was increased in SD609 under drought stress whereas decreased in SD902.
The ability of the antioxidant enzymes, including SOD, POD and GR in two varieties leaves were influenced by moderate drought (Fig. 2b, c, d, e)). Nevertheless, the degree of elevation of these antioxidant enzymes was relatively higher in SD609 than in SD902. We found that the activities of SOD, POD and GR in SD609 increased by 26.4%, 20% and 40.1%, respectively. However, the activities of SOD, POD and GR in SD902 increased by 8.1%, 15.6% and 13.9%, respectively.
Comprehensive proteome analysis of SD609 and SD902
To compare the differences in protein expression levels between the control- and drought-treated maize seedlings of each genotype, we used the iTRAQ approach and performed quantitative analysis on SD609 and SD902. The present study identified 5488 proteins with a 1% FDR from 15079 distinct peptides derived from 126307 spectra. A search for proteins with fold-changes >1.5 or <0.67 (P < 0.05) resulted in the identification of 2559 proteins between the control and drought stress in SD609 and SD902. We further screened the specific proteins between two varieties based on their fold-changes in expression level. As shown in Fig. 4a, a total of 198 (108 increased and 90 decreased) and 102 (66 increased and 36 decreased) proteins exhibited significant changes (P < 0.05) in SD609 and SD902, respectively. Only 39 proteins overlapped between the two varieties, of which 26 were up-regulated and 13 down-regulated. Moreover, 159 drought responsive proteins were unique in drought-tolerant variety SD609, while 63 specific protein in drought-sensitive SD902 (Additional Table 1).
Next, the WoLFPSORT database was used to predict subcellular localization (Fig. 4b, c). Of these DEPs, the subcellular localization showed most proteins were located in chloroplasts in SD609 (Fig. 4b) and SD902 (Fig. 4c), indicating that moderate drought stress mobilized many chloroplast proteins in SD609 and SD902. We further performed gene ontology (GO) and KEGG analysis to annotate their functions (Fig. 5). Among them, Fig. 5a and Fig. 5b respectively represented the GO terms of specific DEPs in SD609 and SD902 induced by drought stress. The GO terms of shared DEPs in both two varieties were shown in Fig. 5c. The GO data revealed that the specific in SD609 (Fig. 5a) were highly enriched in photosynthesis, whereas the specific DEPs in SD902 (Fig. 5b) were mainly included response to stress. The biological process of shared DEPs in SD609 and SD902 involved photosynthesis and protein-chromophore linkage (Fig. 5c). KEGG pathway analysis was used to identify the metabolic pathways of the differentially expressed proteins in SD609 and SD902 (Fig. 5d, e). The upregulated DEPs in SD609 (Fig. 5d) under drought stress were frequently associated with photosynthesis, while downregulated DEPs were associated with photosynthesis-antenna protein and pyruvate metabolism etc. The KEGG pathways protein processing endoplasmic reticulum and photosynthesis-antenna proteins were more frequent in SD902 enriched proteins, whereas porphyrin and chlorophyll metabolism were more frequent in suppressed DEPs (Fig. 5e). Collectively, GO and KEGG enrichment results provided an overview showing that moderate drought leads to the accumulation and suppression of distinct sets of proteins in SD609 and SD902.
Photosynthesis-related DEPs observed in tolerant variety SD609
Photosynthesis proteins in tolerant variety SD609
Photosynthesis is the main physiological process of plants and responds quickly to stress. A total of 36 DEPs in SD609 (10 up-regulated and 24 down-regulated) were related to electron transport (Additional Table 1). Among these proteins, five proteins associated with PSII were increased under drought stress, including PSII repair protein PSB27-H1, Oxygen-evolving enhancer (OEE) protein 1-1, OEE1, OEE2-1 and PSII 11kD protein. The protein level of 14 light-harvesting Chl a/b binding protein complexes (LHCs) (such as LHCII, LHCP) were decreased by 0.22- to 0.55-fold. The protein Plastoquinol-plastocyanin reductase and plastocyanin in Cytochrome b6/f complex were also up-accumulated by 1.7-fold relative to control. Nine proteins (4 up-regulated and 5 down-regulated) annotated in PSI were also changed under drought stress, of which the Ferredoxin 2 (FDX2), FDX5, two PSI reaction center subunit IV A proteins were increased, other proteins such as PSI subunit O, PSI-G, PSI-K as well as PSI-B were decreased. Moreover, four chlorophyll biosynthesis proteins were down-regulated induced by drought. In the dark (Calvin cycle) reactions of photosynthesis, nine phosphoenolpyruvate carboxylase (PEPC) proteins were decreased by 0.5- to 0.66-fold. The expression profiles of the photosynthesis proteins suggest that the system structure and the ability of electron transport is enhanced in the drought-tolerant seedling leaves but reduced in the drought-sensitive seedling leaves under the same drought stress.
Reactive Oxygen Species (ROS) scavenging proteins in tolerant variety SD609
Plants have evolved the antioxidant defense system including enzymatic and non-enzymatic mechanisms to scavenge the increase of ROS under water deficit conditions. In our study, 16 proteins in ROS scavenging were observed in SD609 (Additional Table 1). The expression of SOD in SD609 was up-regulated by 2.4-fold, and five peroxidase proteins was enhanced under drought stress. Moderate drought stress also activated proteins associated with the ascorbate-glutathione (AsA-GSH) cycle and thioredoxin-peroxiredoxin (Trx-Prx) pathway, such as glutathione reductase (GR), together with 2-Cys peroxiredoxin BAS1, thioredoxin M (TrxM).
Energy metabolism-related proteins in tolerant variety SD609
Consistent with enriched GO biological process terms, mitochondrial electron chain and ATP synthesis-related proteins linked with energy metabolism were changed in SD609 under drought stress (Additional Table 1). Here, electron transport proteins such as cytochrome c oxidase subunit and NADH-ubiquinone oxidoreductase B18 subunit were increased. Furthermore, two ATP synthase subunit proteins were also induced by drought. Their changes imply that SD609 enhanced energy production to cope with moderate drought stress.
Photosynthesis-related DEPs observed in sensitive variety SD902
The photosynthesis-antenna proteins pathway was significantly enriched in SD902 under water deficit conditions (Fig. 5e). However, the number of photosynthesis proteins detected in SD609 is higher than those in SD902 (Additional Table 1). Eight DEPs (LHCs) involved in photoreaction were up-regulated by approximately 1.7-fold. Besides, 17 DEPs participating in photosynthesis were down-regulated. These 17 DEPs were grouped into three types: (i) five proteins related to Chl biosynthesis (for example, uroporphyrinogen decarboxylase and NADPH-protochlorophyllide oxidoreductase) were detected under drought stress. (ii) four Ferredoxin-NADP(H) oxidoreductase (FNR) protein, which catalyzes the electron transfer between NADP(H) and ferredoxin (Fd), were decreased in abundance. (iii) eight pyruvate, phosphate dikinase (PPDK) protein involved in carbon fixation were significantly down-regulated in response to moderate drought stress. The down-regulation of most photosynthetic proteins may explain the changes in photosynthetic parameters of SD902 under drought stress. Besides, six sucrose synthase proteins involved in starch and sucrose metabolism were up-accumulated in SD902 under drought stress.
Common DEPs between two maize varieties
In our results, 39 proteins shared in SD609 and SD902 were evident (Additional Table 1), including GO enrichment (Fig. 5c) of both datasets for: photosynthesis and generation of precursor metabolites and energy. The most abundant photosynthesis-related proteins were LHCs and NADPH-protochlorophyllide oxidoreductase, in which the membrane proteins of LHCs bind chlorophyll and transfer energy to the reaction centers for photosynthesis. Among the 35 identified DEPs, most protein were increased in two varieties. As shown in Additional Table 1, the heat shock protein (HSP26) and other small heat proteins (sHSP), glutathione reductase, were up-regulated in both varieties under drought stress. By contrast, the LHCs was increased in SD902, but decreased in SD609.
Protein interaction network in tolerant variety under moderate drought
To predict the protein interactions and functional relations among DEPs, protein-protein interaction network analysis was performed with confidence scores > 0.5 to identify the interactions of specific DEPs in SD609 (Fig. 6). Four main groups of interacting protein species were identified in the network. The functions of the proteins in these four clusters are generally focused on photosynthesis, ROS scavenging, protein folding and energy metabolism. Most proteins in these clusters were increased, which displays the pivotal response of these proteins under drought conditions. It is found that plastocyanin (103629356) interacts with 24 other proteins, and OEE1 (100272890) is also linked with 24 other proteins, such as PSI-K, Cytochrome b6/f complex proteins and TRM1. Furthermore, ATP synthase subunit (100281924) interacts with 21 other proteins. These results indicate proteins involved in different metabolic pathways responded to drought stress by interacting.
Expression levels of genes encoding DAPs in response to moderate drought
Next, the transcriptional expression in thirteen selected proteins were measured by real quantitative real-time PCR (qRT-PCR). qRT-PCR results showed that the expression patterns of half of DEPs were coincided well with their corresponding coding genes. Of these 13 proteins, seven genes expression (Fig. 7a, d, e, h, i, j, k) were consistent with proteomic results. The expression patterns of other six genes showed opposite trends with their homologous proteins. These results may be due to a time delay between mRNA and proteins or posttranscriptional and transcriptional regulatory mechanisms. Thus, most of the qRT-PCR results confirmed our proteomic results.