Label-free quantitative proteomic analysis of alfalfa in response to microRNA156 under high temperature

Abstract

Background: Abiotic stress, including heat, is one of the major factors that affect alfalfa growth and forage yield. The small RNA, microRNA156 (miR156), regulates multiple traits in alfalfa during abiotic stress. The aim of this study was to explore the role of miR156 in regulating heat response in alfalfa at the protein level.

Results: In this study, we compared an empty vector control and miR156 overexpressing (miR156OE) alfalfa plants after exposing them to heat stress (40 °C) for 24 h. We measured physiological parameters of control and miR156OE plants under heat stress, and collected leaf samples for protein analysis. A higher proline and antioxidant contents were detected in miR156OE plants than in controls under heat stress. Protein samples were analyzed by label-free quantification proteomics. Across all samples, a total of 1878 protein groups were detected. Under heat stress, 45 protein groups in the empty vector plants were significantly altered (P < 0.05; |log₂FC| > 2). Conversely, 105 protein groups were significantly altered when miR156OE alfalfa was subjected to heat stress, of which 91 were unique to miR156OE plants. The identified protein groups unique to miR156OE plants were related to diverse functions including metabolism, photosynthesis, stress-response and plant defenses. Furthermore, we identified transcription factors in miR156OE plants, which belonged to squamosa promoter binding-like protein, MYB, ethylene responsive factors, AP2 domain, ABA response element binding factor and bZIP families of transcription factors.

Conclusions: These results suggest a positive role for miR156 in heat stress response in alfalfa. They reveal a miR156-regulated network of mechanisms at the protein level to modulate heat responses in alfalfa.

Keywords: Alfalfa; Heat stress; LC-MS/MS; Proteomic; miR156.

Fig. 1

MiR156 alters the physiological responses of alfalfa to heat stress. Levels of a antioxidants, and b proline in EV control and A8. Asterisk (*) shows statistical significance at p < 0.05 where n = 3–4 (t-test)

Fig. 2

Comparison of protein with differential abundance in EV and A8. Venn diagram shows the number of significantly downregulated (a) and upregulated (b) proteins in EV and A8 under heat stress relative to corresponding non-stress controls

Fig. 3

Gene Ontology (GO) enrichment analysis, a Fractional distribution of GO terms based on molecular function, cellular component and biological process. Tree maps of b biological process, c cellular component and d molecular function of identified proteins with differentially altered abundance in EV controls under heat stress

Fig. 4

Gene Ontology (GO) enrichment analysis, a Fractional distribution of GO terms based on molecular function, cellular component and biological process. Tree maps of b biological process, c cellular component and d molecular function of identified proteins with differentially altered abundance in miR156 overexpressing genotype (A8) under heat stress

Fig. 5

A proposed model of miR156-regulated heat stress response in alfalfa