Heterosis, characterized by superior traits in hybrid progeny compared to both parents, is crucial in crop production. Achieving heterosis involves labor-intensive field tests, prompting the need to understand its molecular mechanisms. Various methods, including physiological, genetic, and molecular approaches, have been employed. Transcriptomic and proteomic studies, particularly the analysis of differentially accumulated proteins (DAPs) and non-additive proteins (NAPs), have gained prominence in heterosis research. Despite progress in heterosis-related gene identification, comprehending the underlying causes and outcomes remains challenging.
Case 1 Decoding Heterosis: Proteomic Insights from Maize Seedling Leaves of ZD808 and ZD909 Hybrids.
Sample
The study focuses on maize, a globally significant cereal crop, and specifically on hybrids ZD808 and ZD909. These hybrids exhibit strong heterotic phenotypes, including increased seedling vigor, larger ears, and enhanced stress resistance. ZD808, derived from the parents CL11 and NG5, is primarily cultivated in southwestern China, while ZD909, bred from Z58 and HD568, is mainly planted in the Huang-Huai-Hai region. Both hybrids cover extensive cropland in China and demonstrate remarkable agronomic traits compared to their respective parents. The analysis concentrates on seedling leaves, aiming to uncover proteomic characteristics associated with the observed heterosis in these maize hybrids.
LC-MS Quantitative Identification of Proteins: Statistical Analysis
Quantitative identification of proteins was conducted using liquid chromatography-mass spectrometry (LC-MS) based on non-labeled proteomic techniques, with three biological replicates from six different varieties. A total of 2222 proteins were quantitatively identified in ZD808 and its parents, while 2486 proteins were identified in ZD909 and its parents.
To discern differentially accumulated proteins (DAPs), a 2-fold change was chosen as the empirical threshold. Comparative analysis revealed that, in ZD808, there were 833 upregulated and 459 downregulated proteins compared to the parent strains CL11 or NG5. In comparison, ZD909 exhibited 537 upregulated and 1167 downregulated proteins. Among the DAPs, ZD808 had 325 commonly upregulated and 54 commonly downregulated proteins compared to the parents, while ZD909 had 309 commonly upregulated and 143 commonly downregulated proteins. Non-additive proteins (NAPs) were found to be potentially associated with hybrid vigor. In the seedlings of ZD808 and ZD909, 188 and 263 NAPs were identified, respectively. Among all identified NAPs from ZD808 and ZD909, only four were commonly identified in both hybrids. Clustering NAPs from the two hybrids highlighted significant differences in distribution, with ZD808 showing greater differences between the parent strains than between the hybrid and one parent.
Comparisons of DAPs between ZD808 and ZD909 indicated a higher number of commonly upregulated DAPs than commonly downregulated ones. In NAPs, ZD808 exhibited a higher abundance of both high-abundance and low-abundance NAPs compared to its parents, while ZD909 showed the opposite pattern. The comparison of DAPs and NAPs between ZD808 and ZD909 suggested that, in ZD808, the quantity of commonly upregulated proteins was higher than the quantity of NAPs classified as "++" and "+", aligning with the common trend of downregulated proteins in both ZD808 and ZD909.
Pathway Enrichment Analysis of DAPs and NAPs
Enrichment analysis of pathways associated with differentially accumulated proteins (DAPs) and non-additive proteins (NAPs) identified from ZD808 and ZD909 was conducted using MapMan software, resulting in the categorization of 34 classes. In comparison to any parent, the hybrids exhibited at least a 2-fold change in various pathways, including protein, RNA, amino acid metabolism, signal transduction, lipid metabolism, secondary metabolism, photosynthesis (PS), stress response, oxidation-reduction, hormone metabolism, and development. Conversely, in comparison to both parents, the hybrids showed at least a 2-fold change in downregulated DAPs associated with protein pathways.
NAPs classified as "++" and "+" in ZD808 were associated with pathways such as protein, RNA, miscellaneous, secondary metabolism, amino acid metabolism, oxidation-reduction, signal transduction, and tricarboxylic acid cycle (TCA)/organ transformation. Conversely, ZD909 NAPs classified as "++" and "+" were primarily associated with PS pathways. The categorization of ZD808's NAPs as "--" and "-" was related to protein, PS, and other pathways. Similarly, ZD909's NAPs categorized as "--" and "-" were associated with protein, RNA, amino acid metabolism, transport, secondary metabolism, pyrroloquinoline synthesis, and oxidation-reduction pathways.
Enhanced Stress-Related Pathways in ZD808
A more in-depth analysis revealed the enrichment of stress response-related pathways in both ZD808 and ZD909, with a total of 76 NAPs identified. These pathways included secondary metabolism, protein hydrolysis, tricarboxylic acid (TCA) cycle, abiotic stress, oxidation-reduction status, and signal transduction. Among these, 44 NAPs were identified in ZD808 and its parents, with 32 NAPs upregulated in ZD808. In contrast, 36 NAPs were identified in ZD909 and its parents, with only 11 showing upregulation.
Enhancement of Photosynthesis (PS)-Related Pathways in ZD909:
A total of 27 non-additive proteins (NAPs) from both ZD808 and ZD909 were found to be enriched in PS-related pathways, including light reactions, Calvin cycle, and photophosphorylation. The majority of NAPs associated with PS were identified in ZD909 and its parents, with all except two NAPs in ZD909 showing upregulation, primarily participating in light reactions. Among these, 10 NAPs were identified in ZD808 and its parents, but only 3 NAPs were upregulated, mainly enriching in light reactions and Calvin cycle. The results indicated that, in ZD808, approximately half of the NAPs related to PS were classified as "--" or "-", whereas, in contrast, the majority of PS-related NAPs in ZD909 were classified as "+" or "++".
Parallel Reaction Monitoring (PRM) Validation:
PRM measurements were employed to validate a subset of NAPs obtained from non-labeled proteomic analysis. Given that the characteristic peptides of target proteins must exhibit uniqueness, only proteins with distinct peptide sequences were selected for PRM analysis. Some proteins related to PS and stress pathways were chosen for PRM validation. The validation results demonstrated that the intensity of most PS-related proteins in hybrid ZD808 was lower than that of its parents, while in ZD909, the trend was opposite.
Reference
- Wang, Daoping, et al. "Comparative proteomic analysis reveals that the Heterosis of two maize hybrids is related to enhancement of stress response and photosynthesis respectively." BMC Plant Biology 21 (2021): 1-15.
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