Heterosis is ubiquitous and has attracted much scientific attention. Many methods, like genomics and transcriptomics, have been used in heterosis studies , and now proteomics is also being used. ZD808 is the main cultivated variety in southwestern China, which has a complicated eco-environment with barren soil and frequent drought and disease occurrences, while ZD909 is the main cultivated variety in the Huang-Hui-Hai area of China. ZD808 can adapt to adverse environmental conditions and exhibits excellent resistance levels to stresses, such as drought , while ZD909 has a strong photosynthetic capacity . So that maize hybrids ZD808 and ZD909 have excellent yield traits compared with those of their parents [30, 33] (Table 1). Here, proteomic techniques were used to analyze the two hybrids and their parental lines to provide new clues regarding the heterosis mechanisms.
Heterosis is affected, to some extent, by many factors [3, 5], and NAPs may be a major source of heterosis. By using label-free quantitative proteomics, 2,222 and 2,486 proteins were quantified from seedling leaves of ZD808 group and ZD909 group, respectively, means that there is a difference in the analysis results between groups. The Pearson’s correlation analysis revealed that the correlations of the quantitative proteins of three independent biological replicates were relatively high, and the two hybrids were more similar to their female parents (Fig. 2A, B). Venn diagrams independently illustrated the relationships of DAPs and NAPs between two hybrids. Some DAPs of ZD808 and ZD909 were simultaneously identified as up-regulated proteins and/or down-regulated proteins when compared with any parent, but there were no significant differences between the numbers of simultaneously identified DAPs in most intersections of the two data sets (Fig. 2C). The comparative analysis of common DAPs indicated that there were more common up-regulated DAPs than common down-regulated DAPs in both ZD808 and ZD909 (Fig. 2D, E). However, the NAP analysis showed that their patterns were significantly different in ZD808 and ZD909 (Fig. 2F). The Cluster analysis showed a difference distribution of NAPs between two hybrids, but similarity of two hybrids and their female parents is higher than hybrids and their male parents, it indicated female parent had more influence on hybrids NAPs.
All the identified proteins were classified into 33 pathways as well as a group in which proteins were not annotated (Fig. 3). The pathway enrichment analysis showed that most of the up-regulated DAPs in ZD808 and down-regulated DAPs in ZD909, when compared with any parent, were enriched in many pathways. In ZD808, DAPs up-regulated by at least a twofold change in the hybrids when compared with both parents were enriched in almost all of the pathways, especially stress-related pathways such as amino acid metabolism, signaling, lipid metabolism, secondary metabolism, stress, redox, and hormone metabolism. In ZD909, DAPs up-regulated by at least a twofold change in the hybrids when compared with both parents were mainly enriched in cell, stress, and mitochondrial electron transport/ATP synthesis and transport. Thus, the functions of the DAPs in ZD808 and ZD909 appeared to be different. However, in ZD808, most of the ‘++’ and ‘+’ NAPs were only enriched in stress-related pathways, while ‘− −’ and ‘−’ NAPs were only obviously enriched in PS pathways. However, in ZD909, the ‘++’ and ‘+’ NAPs were mainly enriched in PS pathways, while ‘− −’ and ‘−’ NAPs were enrichment in RNA, amino acid metabolism, secondary metabolism, and redox. Thus, stress-related pathways were enhanced in ZD808, and PS-related pathways were enhanced in ZD909.
Because non-additively expressed proteins were predicted to play important roles in heterosis [3, 16, 35], all the identified NAPs were further analyzed using MapMan (Table S3, S4). Most of the NAPs identified from the two hybrids could be divided into four classes of pathways, stress responses, PS, amino acid metabolism and protein. We believe that if the majority of the NAPs in a pathway were up- or down-regulated, then the pathway was enhanced or impeded, respectively. Stress responses play crucial roles when plants are subjected to abiotic or biotic stresses. The MapMan analysis showed that some up-regulated NAPs of ZD808 were enriched in pathways involved in stress responses (Fig. 4). Among these proteins, 70-kDa heat shock proteins (Hsp70s) are a widely expressed family with similar structures that exist in almost all organisms. Hsp70s are important components of the cell’s machinery for protein folding, and they help to protect cells from cold, heat, and drought stresses [36-38]. Other proteins shown in Figure 4 are also related to basal metabolism and stress responses. The basal metabolism and stress responses were enhanced in hybrid ZD808 compared with its parents. Some studies reported the relationship between heterosis and stress tolerance, but a great difference exist on different species and different stress . Some evidence showed that there is negative relationship between expression of defense response genes and growth heterosis , there are also some evidences of a positive or no relationship between heterosis and stress tolerance [40, 41]. In this study, stress responses were enhanced at three-leaf stage, and yield increased at maturity stage, it suggested that there had a positive relationship between heterosis and anti-stress in ZD808.
Increased vigor or yield is usually the focus of heterosis research. PS is a key process in promoting plant growth and development. The main PS-related pathways enriched with NAPs of ZD808 and ZD909 are shown in Figure 4. Half of the NAPs of ZD808 involved in PS were classified as ‘− −’ or ‘−’, with only three being classified as ‘+’ or ‘++’. This indicated that the PS action in hybrid ZD808 was weaker than in its parents. Unlike in ZD808, most of the NAPs related to PS in ZD909 were classified as ‘+’ or ‘++’. Thus, there is a great difference in the PS activity between the two hybrids at the three-leaf stage. The enhanced of PS-related pathways at three-leaf stage could be the bases on heterosis of yield at maturity stage.
Amino acid metabolism also plays important roles in the growth processes of plants. A total of 14 NAPs in ZD808 and ZD909 were enriched in amino acid metabolism-related pathways. Among them, chloroplastic 3-dehydroquinate synthase, shikimate dehydrogenase1, acetolactate synthase 1, ketol-acid reductoisomerase, chloroplastic 2-isopropylmalate synthase 1, and cysteine synthase were identified as ‘++’ or ‘+’ in ZD808, with only D-3-phosphoglycerate dehydrogenase belonging to ‘− −’. However, in ZD909, all of these NAPs were classified as ‘− −’ or ‘−’. This indicates that amino acid metabolism was enhanced in hybrid ZD808 and impeded in ZD909. Moreover, protein pathways were greatly affected by hybridization, including amino acid activation, protein degradation (serine protease and ubiquitin), protein folding, protein synthesis (elongation, initiation and ribosomal protein), protein targeting (in the chloroplast and nucleus), assembly and cofactor ligation, and posttranslational modification. In ZD808, most of the NAPs related to protein synthesis, folding, amino acid activation, and targeting were classified as ‘+’ or ‘++’. However, in ZD909, most of the NAPs in protein-related pathways were classified as ‘− −’ or ‘−’. This indicated that the effects of protein synthesis and other protein-related pathways in the two hybrids were very different.
In the PRM verification of some key proteins, the abundance levels of 12 NAPs related to PS and stress were analyzed (Fig. 6). The PRM results in parents and hybrids were similar to the label-free quantitative proteomics results. Four stress response-related proteins, chaperone protein ClpB3, D-3-phosphoglycerate dehydrogenase (PGDH), glutathione S-transferase 6 (GST6), and membrane steroid-binding protein 1 (MSBP1), were verified by PRM. The direct interaction of ClpB3 with Hsp70 may eventually result in the refolding, and hence the reactivation, of deoxyxylulose 5-phosphate synthase, which promotes the repair of the methylerythritol 4-phosphate pathway [42-44]. This pathway synthesizes the metabolic precursors of isoprenoids, such as carotenoids, and the prenyl chains of chlorophylls, tocopherols, or plastoquinone. PRM verification classified ClpB3 as ‘++’ in ZD808, while it was classified as ‘+’ in label-free quantitative proteomics. The high abundance level of ClpB3 in hybrid ZD808 indicates that its stress-response capability is greater than that of its parents. PGDH, which originates from glycolysis and the Calvin cycle, participates in the biosynthesis of L-serine . Arabidopsis thaliana PGDH1 is also essential for plant adaptation to high CO2, as well as for embryogenesis and pollen development . Recently, a study showed that PGDH activity increased in sugar beet after exposure to salt stress . PGDH activity is increased in sugar beet after exposure to salt stress. PGDH was down-regulated in hybrid ZD808 and classified as ‘− −’ NAPs by both label-free quantitative proteomics and PRM. Even though PGDH was down-regulated in ZD808, the response to salt stress-related PGDH in ZD808 may be greater than the responses of its parents. GST is a multifunctional enzyme that participates in reducing the formation of reactive oxygen species and cell toxicity [48, 49]. GST6 is up-regulated under salt-stress conditions . GST6 was classified as a ‘−’ NAP in ZD808, and PRM verification classified it as ‘− −’. Thus, the response to salt stress-related GST6 may be high in ZD808. MSBP1 inhibits cell elongation  and negatively regulates brassinosteroid signaling . MSBP1 expression is also closely related to salinity tolerance . Like many stress-related NAPs in ZD909, this protein was down-regulated, which was verified by PRM.
Eight proteins related to PS, fructose-bisphosphate aldolase 7 (FBA7), ferredoxin-NADP reductase (FNR), photosystem II subunit PsbS1 (PsbS1), post-illumination chlorophyll fluorescence increase (PIFI), chlorophyll a-b binding proteins (CAB1 and CAB2), photosystem I P700 chlorophyll a apoprotein A2 (psaB), photosynthetic NDH subunit of lumenal location 1 (PNSL1), were also subjected to PRM verification (Fig. 6). These proteins had important roles in PS. PIFI, CAB1 and 2, psaB, and PNSL1 were identified in ZD909 as ‘+’ NAPs, and most of them were classified as ‘+’or ‘++’ in the PRM verification. PIFI is essential for NDH-mediated nonphotochemical reduction of the plastoquinone pool in chlororespiratory electron transport . CABs are a class of important proteins in thylakoid membranes of higher plants, and take part in both photosystems I and II. In addition, CABs may be involved in abiotic stress responses . It was also found that CABs were highly expressed in the F1 hybrid of Chinese cabbage at two days after sowing, means that early developmental events in the germinating seedling of the hybrid may be important for later developmental vigor and yield advantage . PsaB a primary electron donor of photosystem I. PNSL1 is a lumen subcomplex L subunit of chloroplast NDH. Unlike in ZD909, most proteins related to PS identified by label-free quantitative proteomics were down-regulated, and FBA7, FNR, and PsbS1 classifications were verified by the PRM. FBAs participate in carbon ﬁxation, glycolysis, and Calvin cycle pathways. FNR catalyzes reversible electron transport between ferredoxin and NADP. PsbS1 is a subunit of photosystem II. These roles indicated that PS was decreased in ZD808 compared with its parents. Based on the label-free quantitative proteomics and PRM analyses of NAPs, the heterosis of maize hybrids ZD808 and ZD909 may be mainly related to PS and/or stress resistance.
In addition to the above-mentioned proteins which related to stress responses and photosynthesis, a protein expressed one of reported genes associated with heterosis, ERBB-3 binding protein 1 (EBP1), was up-regulated in ZD909 (Table S2). EBP1 regulates organ size through cell growth and proliferation . And more, some DAPs/NAPs in ZD808 and ZD909 are not yet functionally annotated (Table S1, S2). And even though analysis of DAPs and NAPs in the germinating seedling of hybrids ZD808 and ZD909 has given us some useful information in understanding the reason of valuable agronomic traits, proteomics research throughout the growth stage is still lacking. All these results indicate that the mechanism of maize heterosis needs further study.