Mechanistic insights into the delayed yellowing of broccoli by the combination of ectoine and lactide peptide

Abstract

Preventing the postharvest yellowing of broccoli florets is a significant challenge. Our study investigated the effect of a combined treatment of ectoine and lactide peptide (ELP, soak treatment for 10 min) on the postharvest physiology and yellowing of broccoli. The treatment effectively reduced yellowing of the broccoli and inhibited the loss of nutrients. Transcriptomic and metabolomic analyses showed that the treatment with ectoine and lactide peptides reduced the accumulation of linoleic acid by downregulating the expression of LOX2 and EPHX2. Up-regulated the expression of HIS4 and down-regulated the expression of P4HA and PAO3, thereby synergistically promoting the accumulation of histidine and proline. Regarding flavonoid biosynthesis, CHS was upregulated and caffeoylquinic acid exhibited increased abundance. Results of the study enhance our understanding of the molecular mechanisms underlying the regulation of broccoli yellowing and demonstrate the potential use of a combined ectoine and lactide peptide treatment for the postharvest preservation of vegetables.

Introduction

Broccoli (Brassica oleracea L.) is highly valued for its nutritional profile, rich in vitamins, minerals, and bioactive compounds. However, its postharvest life is remarkably short, often limited by rapid senescence. This process is most visibly marked by the degradation of chlorophyll, leading to yellowing, alongside a decline in nutritional quality and increased susceptibility to decay.

To combat these issues, researchers are increasingly looking toward "green" bioactive substances. This study explores a novel synergistic treatment using ectoine, a natural osmoprotectant produced by extremophilic bacteria, and lactide peptides, bioproducts derived from corn syrup known to enhance stress tolerance in plants. The combination, referred to as ELP, aims to provide a sustainable solution for extending the shelf life of this high-value vegetable.

Methodology

To evaluate the efficacy of the ELP treatment, broccoli florets were subjected to a 10-minute soak in an ELP solution (2 mL/L ectoine and 6 mL/L lactide peptide complex). Following treatment, samples were stored at 20°C to simulate typical retail shelf conditions.

The study utilized a multi-omics approach to dissect the physiological and molecular responses:

• Physiological Profiling: Standard quality markers were monitored, including weight loss, color coordinates ($L^*, a^*, b^*$), respiration rate, ethylene production, and electrolyte leakage.
• Biochemical Assays: Antioxidant enzyme activities (CAT, POD, APX) and nutrient levels (Vitamin C, total phenols) were measured.
• Transcriptomics and Metabolomics: RNA-seq was performed to identify Differentially Expressed Genes (DEGs), while UPLC-QTOF-MS non-targeted metabolomics identified Differentially Abundant Metabolites (DAMs). Data were mapped against the Brassica oleracea reference genome (GCA_900416815.2) and the METLIN database.

Results and Discussion

Physiological and Visual Preservation

The ELP treatment demonstrated a clear ability to delay the onset of senescence. Visually, treated broccoli maintained its green color for approximately one day longer than the control group. By the fourth day of storage, chlorophyll retention was significantly higher in the ELP group (47.4% remaining) compared to the control group (only 21.9% remaining).

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Furthermore, ELP treatment effectively slowed the "metabolic clock" of the florets. This was evidenced by a significant reduction in respiration rates and ethylene generation—the primary hormone driving senescence. The treatment also preserved cellular integrity, as indicated by lower levels of malonaldehyde (MDA) and reduced electrolyte leakage compared to untreated samples.

Molecular Mechanisms of Action

The multi-omics analysis revealed that ELP treatment triggers a complex metabolic "re-wiring" that protects the plant tissue:

1. Amino Acid Homeostasis: The treatment upregulated HIS4 and downregulated P4HA and PAO3, leading to the accumulation of histidine and proline. These amino acids serve as critical non-enzymatic antioxidants and osmoprotectants, stabilizing cellular structures.
2. Lipid Membrane Protection: Senescence is often driven by lipid peroxidation. ELP treatment downregulated LOX2 and EPHX2, genes responsible for breaking down linoleic acid. By maintaining higher linoleic acid levels and reducing its conversion into harmful oxylipins, the treatment preserved membrane integrity.
3. Enhanced Flavonoid Biosynthesis: The upregulation of CHS (Chalcone Synthase) led to an increase in caffeoylquinic acid, boosting the broccoli's natural antioxidant capacity to neutralize reactive oxygen species (ROS).

Conclusion

The combination of ectoine and lactide peptide represents a promising, eco-friendly postharvest treatment for broccoli. By simultaneously suppressing ethylene production, enhancing antioxidant defenses, and preserving lipid membranes, ELP treatment significantly delays yellowing and nutrient loss. While further research is needed to evaluate industrial scalability and performance within the cold chain, these findings provide a robust molecular foundation for using ELP as a sustainable preservative in the vegetable industry.

@inproceedings{wu2025mechanistic,
  author = {Ke Wu and Xiaozhen Yue and Song Zhang and Chunjie Zhang and Jieyu Lv and Bangjin Yan and Mina Wang and Shuzhi Yuan and Xiaodi Xu and Jinhua Zuo and Aili Jiang and Qing Chen and Qing Wang},
  title = {{Mechanistic insights into the delayed yellowing of broccoli by the combination of ectoine and lactide peptide}},
  booktitle = {Npj},
  year = {2025},
  doi = {10.1038/s41538-025-00580-y},
}