HrβVPE1 Regulates Seed Germination in Hippophae rhamnoides by Controlling Degradation of Outer Endosperm Cells

doi:10.13560/j.cnki.biotech.bull.1985.2025-1299

Abstract

Abstract:

Objective This study aimed to investigate the effects of the Hippophae rhamnoides vacuolar processing enzyme gene HrβVPE1 on programmed cell death (PCD) and starch degradation in outer endosperm cells during seed germination, thereby providing a theoretical basis for clarifying its molecular regulatory mechanism in seed germination. Method The HrβVPE1 gene was cloned from Hippophae rhamnoides seeds. Bioinformatics approaches were adopted to analyze the nucleotide and amino acid sequences of HrβVPE1, followed by multiple sequence alignment and phylogenetic tree construction. Quantitative real-time PCR (RT-qPCR) was employed to detect the expression patterns of HrβVPE1 at different seed germination stages. Agrobacterium-mediated transient transformation was conducted on Nicotiana benthamiana leaves to determine the subcellular localization of HrβVPE1. Virus-induced gene silencing (VIGS) was used to downregulate HrβVPE1 expression in H. rhamnoides seeds. Iodine-potassium iodide (I₂-KI) staining, DAPI (4′,6-diamidino-2-phenylindole) staining, and double-probe staining with FDA (fluorescein diacetate) and FM4-64 were performed to observe the impacts of HrβVPE1 silencing on starch degradation, vacuole dynamics, nuclear morphology, plasma membrane integrity, and cell viability of outer endosperm cells. Additionally, an HrβVPE1 overexpression vector was constructed and transformed into Arabidopsis thaliana. The seed germination rate of transgenic A. thaliana was calculated, and RT-qPCR was used to detect the expression levels of multiple PCD-related genes in overexpressing plants. Result The full-length cDNA of HrβVPE1 was 1 482 bp, encoding a polypeptide of 493 amino acids. Phylogenetic analysis revealed that HrβVPE1 had a close genetic relationship with vacuolar processing enzymes from woody plants such as Populus and Salix species. Subcellular localization results indicated that HrβVPE1 was localized to the vacuolar membrane. RT-qPCR analysis showed that HrβVPE1 exhibited high expression levels in the middle and late stages of seed germination. Silencing of HrβVPE1 significantly delayed starch degradation, inhibited the fusion of protein storage vacuoles (PSVs) and the degradation of cell nuclei in outer endosperm cells, blocked the PCD process, and prolonged cell viability. Overexpression of HrβVPE1 in A. thaliana promoted seed germination, upregulated the expression of positive PCD regulatory genes (AtCEP1, AtXCP2, AtMC9), and downregulated the expression of negative PCD regulatory genes (AtMOD1, AtDAD2). Conclusion HrβVPE1 plays a critical role in H. rhamnoides seed germination by regulating PCD processes in outer endosperm cells and thereby mediating starch degradation.

Key words: Hippophae rhamnoides, HrβVPE1, programmed cell death (PCD), outer endosperm cell, starch degradation, seed germination, virus-induced gene silencing (VIGS), vacuole fusion

CHENG Zi-yi, WANG Pei-rong, ZHONG Zi-ning, WEI Ming, WU Guo-qiang. HrβVPE1 Regulates Seed Germination in Hippophae rhamnoides by Controlling Degradation of Outer Endosperm Cells[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1299.

Figures/Tables 9

Fig. 1 Phylogenetic tree of HrβVPE1

Fig. 2 Expression analysis of HrβVPE1A: Relative expression levels of HrβVPE1 in H. rhamnoides seeds during 0-10 d of germination. B: Subcellular localization observation results of HrβVPE1, GFP-K (GFP protein channel), VPE (HrβVPE1-GFP fusion protein channel), γ-TIP-mcherry (tonoplast marker channel), CHI (chloroplast marker fluorescence channel), DIC (differential interference contrast bright-field image), and Merge (merged image of all fluorescence channels and bright field). The scale is 20 μm. Different lowercase letters indicate significant differences (P<0.05), the same below

Fig. 3 Effects of HrβVPE1 silencing on gene expression and phenotype during seed germination in H. rhamnoidesA: Relative expression of HrβVPE1 mRNA. EV: Empty vector control group; VIGS: HrβVPE1 gene silencing treatment group. B: Seed germination rates of the EV group and VIGS group. **P<0.01

Fig. 4 Changes in starch degradation and starch content of seeds in different treatment groups during 1-6 d of germinationA: Starch degradation dynamics of seeds in different treatment groups during 1-6 d of germination. Each row shows seed samples from three groups at 1-6 d of germination, with unstained seeds on the left and I2-KI stained seeds on the right for each sample. The scale is 2 mm. CK: normally germinating seed group; EV: empty vector control group; VIGS: HrβVPE1 gene-silenced group; the same applies hereinafter. B: Changes in starch content of seeds in different treatment groups during 1-6 d of germination

Fig. 5 Morphological characteristics of vacuoles and nuclei in outer endosperm cells in seeds from different treatment groups during 1-6 d of germinationRow a shows the bright-field observation, and row b shows nuclear fluorescence observation results after DAPI staining. The scale bar is 50 μm. The area indicated by the arrow shows an enlargement of the red box at ×2.5 magnification

Fig. 6 Cell death status of outer endosperm cells in seeds from different treatment groups during 1-6 d of germinationRow a: Bright-field observation. Row b: FDA-stained fluorescence images. Row c: FM4-64-stained fluorescence images. Row d: Merged fluorescence images of FDA and FM4-64 staining. The scale bar for row a is 50 μm, and that for rows b, c, and d is 100 μm

Fig. 7 Analysis of gene expression (A), enzyme activity (B) and seed germination characteristics (C) of A. thaliana overexpression HrβVPE1WT: Wild-type A. thaliana. 2-1, 5-2: HrβVPE1 overexpression lines

Fig. 8 Relative expressions of PCD-related genes in overexpression HrβVPE1 A. thaliana

Fig. 9 Schematic diagram of the mechanism of HrβVPE1 regulating PCD and seed germination in outer endosperm cells of H. rhamnoides seeds

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