# Transcriptome Sequencing Analysis Reveals the Mechanisms of Poly-γ-Glutamic Acid Enhanced the Chilling and Freezing Tolerance in Wheat

## TL;DR

This study shows that γ-PGA improves wheat's cold tolerance by boosting osmoprotectants and activating key genes involved in cold response pathways.

## Contribution

The study identifies a core cold-responsive gene network regulated by γ-PGA and reveals specific genes involved in cold tolerance mechanisms in wheat.

## Key findings

- γ-PGA-treated wheat seedlings showed a 128.81% higher survival rate after freezing stress.
- γ-PGA upregulates key genes in phenylpropanoid-flavonoid, glutathione, and lipid metabolism pathways.
- Common cold-responsive genes were identified, forming a core network enriched in carbon and cell wall organization pathways.

## Abstract

γ-PGA significantly enhances the cold resistance of wheat seedlings under both chilling (4 °C) and freezing (−18 °C) stress. Physiologically, γ-PGA promoted the accumulation of osmoregulatory substances (proline and soluble sugars) and activated the antioxidant enzyme system. Transcriptome analysis revealed that γ-PGA regulates a core cold-responsive gene network, particularly upregulating key genes involved in phenylpropanoid-flavonoid metabolism, glutathione metabolism, and lipid metabolism.

Low-temperature stress significantly limits wheat growth and productivity. Poly-γ-glutamic acid (γ-PGA) is an environmentally friendly green molecular material that plays an important role in plant growth and regulation; however, its protective mechanisms against cold stress in wheat remain poorly understood. In this study, the effect of γ-PGA on both chilling (4 °C) and freezing (−18 °C) resistance in wheat seedlings and its underlying mechanisms were comparatively studied. The results showed that the γ-PGA-treated seedlings exhibited a 128.81% higher survival rate after freezing stress and maintained significantly greater biomass accumulation under both stress conditions (62.44% and 26.56% higher dry weight under chilling and freezing stress, respectively). A physiological analysis revealed that γ-PGA enhanced osmoprotectant (proline and soluble sugars) accumulation and activated key antioxidant enzymes (SOD, POD, and APX). Then, an RNA-seq analysis identified 11,401 and 7721 differentially expressed genes under chilling and freezing stress, respectively, with 3598 common genes constituting a core cold-response network. KEGG and GO analyses demonstrated significant enrichment in pathways related to carbon metabolism, glutathione metabolism, phenylpropanoid–flavonoid biosynthesis, fatty acid metabolism, and cell wall organization. Notably, γ-PGA strongly upregulated key genes in phenylpropanoid–flavonoid metabolism (TraesCS2B02G615000 and TraesCS2B02G624400), glutathione metabolism (TraesCS1B02G127900), and lipid metabolism (TraesCS1B02G018700). These results provide comprehensive molecular insights into γ-PGA-mediated cold tolerance and support its potential application in sustainable wheat production under low-temperature stress conditions.