iTRAQ-based quantitative proteomic analysis provides insight into the water stress response of corn seedlings

Primary data analysis and protein identification

First, we tested the repeatability of two samples at the same time, demonstrating that our results have good repeatability and confirming the credibility of the data (Fig. 1A). Integrating all the data, we identified a total of 3063 proteins from the 12 samples and annotated them using different databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for understanding the high-level functions and utilities of the biological system. The Clusters of Orthologous Groups of Proteins (COG) database contains the phylogenetic classification of proteins encoded in complete genomes. The Gene Ontology (GO) database is the largest source of information on gene functions. The InterPro provides functional analysis of proteins by classifying them into families and predicting domains and sites (Fig. 1B). We then analyzed the function of these proteins using KEGG annotation, revealing that these identified proteins are involved in various metabolic processes. The most enriched pathways were carbohydrate metabolism, amino acid metabolism, energy metabolism, and mRNA translation (Fig. 1C). These data reveal metabolic changes in plant cells in response to water-limited growing conditions. Considering the important role of transcription factors in regulating various vital activities, we categorized the transcription factors detected, revealing that the most abundant categories included bHLH, MYB, NAC, and G2-like proteins (Fig. .1D).

Figure 1

Basic iTRAQ output details. (A) Scatter plots showing the correlation between two replicates for different conditions. (B) Venn diagrams of peptides identified using different annotation techniques. (VS) The KEGG annotation for the identified peptides. (D) Frequency of each class of transcription factors in the identified peptides.

Analysis of drought-sensitive DAPs after a 3-day drought

To explore factors with important effects on drought response, we first analyzed samples after 3 days of drought treatment. We identified a total of 362 DAPs between control and experimental plants subjected to a 3-day drought, including 214 upregulated proteins and 148 downregulated proteins (Fig. 2A, Tables S1 and S2). Gene ontology (GO) annotations were used to assign and analyze pathway enrichment of DAPs, which were classified into 35 GO terms (Fig. 2B). The most common terms in cell components are organelles and cell parts. The most enriched terms in the biological processes category are metabolic processes, cellular processes, responses to stimuli, and biological regulation, while the most enriched terms in the molecular functions category are catalytic activity and binding. . (Fig. 2B). Moreover, further analysis of the enriched GO terms (p-value

Figure 2
Figure 2

DAPs involved in drought tolerance pathways. (A) Map of the volcano showing the DAPs identified following the treatment of the drought in 3 days. (B) GO annotation for all DAPs. (VS) GO enrichment analysis of upregulated proteins after 3-day dryness.

Flavonoids are a large class of low-weight phenols, which play vital roles in various biochemical and physiological processes in plants, such as UV protection, defense against pathogens and water stress.17.18. We showed that some of the DAPs (eg, shikimate O-hydroxycinnamoyl transferase and flavonoid 3′-monooxygenase) are involved in flavonoid biosynthesis and oxidative phosphorylation pathways (Figure S1B). These data suggest that drought stimuli may affect flavonoid biosynthesis and the oxidative phosphorylation pathway.

Comparison of DAP between plants after 3 days and 6 days of drought

We further compared the DAPs of plants subjected to 3- and 6-day droughts to the corresponding control, respectively. A total of 20 and 41 DAPs were generally downregulated and upregulated, respectively, in plants subjected to 3- and 6-day droughts (Fig. 3A,B and Table S3). These overlapping DAPs may therefore be important for drought response, so we further analyzed the function of the overlapping proteins. Some of these up-regulated DAPs have been annotated as metabolite, chaperone, and transferase interconverting enzymes (Fig. 3C). Additionally, some of the down-regulated DAPs are annotated as transporter, scaffold protein, and DNA metabolism proteins (Figure S1C).

picture 3
picture 3

Comparison of DAP after a drought of 3 days and 6 days. (A) Venn diagram illustrating the overlap between down-regulated proteins in plants subjected to 3-d and 6-d drought treatments. (B) Venn diagram illustrating the overlap between upregulated proteins in plants subjected to 3-d and 6-d drought treatments. (VS) Categorization of the 41 upregulated proteins in 3-d and 6-d dryness treatments.

Drought-sensitive proteins such as dehydrins and heat shock proteins (HSPs) are produced to protect the intracellular metabolic machinery. COR410 dehydrin localizes to the plasma membrane and accumulates under water stress in wheat (Triticum aestivum)19. Here we found that COR410 was up-regulated in plants after 3 days of drought, suggesting that it also participates in the drought response in maize. HSPs participate in various stress responses, not just heat stress. Six of the 41 HSP20 family members were found to be upregulated in the dried plants in the present study, indicating that the HSP20 family may play a crucial role in the water stress response in maize. The abundance of proteins related to biogenesis and cell wall degradation, such as glucan endo-1,3-beta-glucosidase 7, was also significantly increased in dried samples. Some of the other DAPs were also involved in this metabolic pathway, such as the X2 isoform of glutamate decarboxylase and the protein GDP-fucose O-fucosyltransferase. Some DAPs have a currently unknown function and may represent novel regulatory targets for further drought research.

DAP analysis after rehydration

To further explore whether DAPs returned to normal levels when water supply returned, we analyzed protein levels of samples rehydrated for 1 day after a 6-day drought compared to control samples. Corn was still alive after 6 days of drought treatment, but some leaves appeared to be wilted. We found a total of 231 DAPs, of which 127 were upregulated and 104 were downregulated (Fig. 4A). Some of the DAPs under drought treatment returned to the same abundance as the control when water supply was restored (Fig. 4B, Table S5), suggesting that the expression of these proteins is most sensitive to stimulation by drought. GO analyzed indicated that some of these DAPs belong to binding proteins and have ATP-dependent activity, suggested that these proteins might participate in signal transduction (Fig. 4C and D). On the other hand, some of the DAPs have not returned to their normal state. levels after the water supply resumes.

Figure 4
number 4

The change in drought response proteins after rewatering. (A) Map of the volcano showing the DAPs identified following the rehydration treatment. (B) Scatter plots showing change in abundance of DAPs from drought treatments after rewatering. Red indicates up-regulation after re-watering, while blue represents down-regulation. The circle represents an upregulated protein in dried plants, while a triangle represents a downregulated protein. (VS) GO annotation for recovery proteins. (D) Functional analysis of salvage proteins.

Pooled analysis of DAP change trend during drought and rewatering

We performed cluster analysis on the trend of change of DAPs in different samples and found that some proteins were up-regulated under dryness stimulation (Fig. 5A). Through time-series clustering, we found that DAP expression levels showed four main trends during drought treatment and re-watering: there was no obvious rule in Group 1 DAPs; Group 2 DAPs gradually increased throughout the dryness and rehydration period; Group 4 DAPs peaked after 3 days of dryness treatment and then remained high; while group 3 DAPs show a wave-like change, increasing after 3 days of drought, decreasing after 6 days of drought, and then finally increasing again after rewatering (Fig. 5B).

Figure 5
number 5

Pooled analysis of DAPs during drought treatments. (A) Heat map illustrating DAP expression profiles at three time points (3-day drought, 6-day drought, and rewatering). (B) Four enriched clusters of gene expression profiles determined using the K-means algorithm. (VS) Randomly selected DAPs were analyzed by RT-qPCR. (D) GO group 3 protein enrichment result. Red color represents BP (biological process), green color represents MF (molecular function), and blue color represents CC (cellular component). (E) GO cluster 4 protein enrichment result. Red color represents BP (biological process), green color represents MF (molecular function), and blue color represents CC (cellular component).

We detected transcriptional changes in genes encoding DAPs using RT-qPCR. For the 16 randomly selected DAPs, mRNA changes were consistent with the trend of protein changes for 8 DAPs, indicating that maize regulated expression changes of these proteins at the transcriptional level under drought stimuli ( Fig. 5C). In contrast, there are still mRNA changes inconsistent with protein changes for 8 other DAPs (data not shown), indicating that regulation of the abundance of these proteins may occur at the level of translation or stability. proteins.

To explore the function of these different cluster proteins, we use GO Analyzer to annotate each cluster. There were no enrichment terms for groups 1 and 2. Some of the group 3 proteins participate in catabolic process and metabolic process, and group 4 proteins are involved in redox process and the metabolic process (Fig. 5D and E).

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