Integrated physiological and proteomic analysis of embryo and endosperm reveals central salt stress response proteins during seed germination of winter wheat cultivar Zhengmai 366

Abstract

Background: Salinity is a major abiotic stressor that affects seed germination, plant growth, and crop production. Seed germination represents the beginning of plant growth and is closely linked with subsequent crop development and ultimate yield formation. This study attempted to extend findings regarding the potential proteomic dynamics during wheat seed germination under salt stress and to explore the mechanism of crop salt response.

Results: Salt stress significantly affected seed physiological activities during the germination process, resulting in significant decreases in phytohormone and α-amylase activity and significant increases in soluble sugar, starch, and ADP glucose pyrophosphorylase activity. A comparative proteomics approach was applied to analyze the dynamic proteome changes of embryo and endosperm during seed germination in Chinese winter wheat cultivar Zhengmai 366 under salt stress. Two-dimensional electrophoresis identified 92 and 61 differentially accumulated proteins (DAPs) in response to salt stress in embryo and endosperm, respectively. Both organs contained a high proportion of DAPs involved in stress defense, energy metabolism, and protein/amino acid metabolism. The endosperm had more DAPs related to storage proteins and starch metabolism than the embryo, and 2% of DAPs participating in lipid and sterol metabolism were specifically detected in the embryo.

Conclusions: Seed physiological activities were significantly affected during the germination process when subjected to salt stress. The DAPs involved in stress defense and energy metabolism were upregulated whereas those related to reserve substance degradation and protein/amino acid metabolism were significantly downregulated, leading to delayed seed germination under salt stress. Our proteomic results revealed synergistic regulation of the response to salt stress during seed germination.

Keywords: Bread wheat; Embryo; Endosperm; Proteome; Salt stress; Seed germination.

Fig. 1

Morphological and ultrastructural changes during seed germination in elite Chinese bread wheat cultivar Zhengmai 366 under salt stress. a Seed morphology. CK: water soaked seeds; S: 180 mM salt solution soaked seeds. b SEM images of seed germination from 5 periods in CK and salt treatment group. The scale bar is 50 μm. The A and B starch granules are marked with blue arrows and red arrows

Fig. 2

Physiological parameter changes at different seed germination stages in Zhengmai 366 under salt stress. a Seed germination rate; b Relative water content; c Soluble sugar; d Starch; e GA content; f IAA content; g ZR content; h ABA content; i α-Amylase; j AGPase; k Sucrose synthase; l Protein. Error bars indicate standard errors of three biological replicates. Statistically significant differences compared to the control were calculated based on an independent Student’s t-tests: *p < 0.05; **p < 0.01. CK: control group. S: salt treatment group

Fig. 3

Principal component analysis (a) and principle component regression analysis (b) of 11 physiological parameters at different seed germination stages in Zhengmai 366. CK0h, CK6h, CK12h, CK18h, CK24h represent five different germination stages: 0,6, 12, 18 and 24 h in CK group, respectively; S6 h, S12 h, S18 h and S24 h represent five different germination stages: 6, 12, 18 and 24 h in salt treatment group (180 mM salt solution soaked seeds), respectively. 1: ZR content; 2: ABA content; 3: GA content; 4: IAA content; 5: Starch content; 6: Soluble sugar content; 7: α-amylase activity; 8: Relative water content (RWC); 9: Sucrose synthase activity; 10: AGPase activity; 11: Protein content

Fig. 4

2DE images of embryo and endosperm during seed germination in Zhengmai 366. Numbered spots indicate differentially accumulated protein (DAP) spots identified by MALDI-TOF/TOF-MS with significant accumulation changes under salt stress. a Embryo gel, samples were extracted from the embryo and electro focused on an 18 cm pH 3–10 linear IPG strip. b Endosperm gel, samples were extracted from the endosperm and electro focused on an 18 cm pH 3–10 linear IPG strip. The different colours of protein spots are corresponding to protein expression patterns shown in Fig. 7. The red spots: Cluster I; the orange spots: Cluster II; the yellow spots: Cluster III; the green spots: Cluster IV; the black spots: Cluster V; the blue spots: Cluster VI; the purple spots: Cluster VII; the light blue spots: Cluster VIII

Fig. 5

Functional classification, subcellular localization and Venn diagram of the identified DAPs in response to salt stress from wheat embryo and endosperm during seed germination. a Functional classification of DAP spots in embryo. b Functional classification of DAP spots in endosperm. c Venn diagram analyses of DAPs in embryo and endosperm. d Subcellular localization. Labels: Pero, Peroxisome; Nucl, Nuclear; Vaco, Vacuole; Mito, Mitochondria; Extr, Extracellular; Chlo, Chloroplast; Cyto, Cytoplasm

Fig. 6

Subcellular localization of the identified three DAPs in Arabidopsis protoplasts. GFP: GFP fluorescence signal (green); Chlorophyll: chlorophyll autofluorescence signal (red); DAPI: fluorescent images of nucleic stained by DAPI (blue); Bright light: field of bright light; Merged: emergence of the GFP fluorescence signal, chlorophyll autofluorescence signal, and bright light field. Simultaneous detection of two fluorescence signals (green and blue) demonstrates co-localization (cyan). Scale bar 5 μm. LEA, late embryogenesis abundant protein Lea14-A; AR, aldose reductase; cyMDH, cytosolic malate dehydrogenase

Fig. 7

Accumulation patterns of the DAPs responsive to salt stress from 2-DE maps of embryo and endosperm during seed germination. a Hierarchical clustering of DAPs from embryo; b Hierarchical clustering of DAPs from endosperm. Each column represents samples from control and salt treatment groups. Each row displays the change of a DAP spot using color-coding based on the relative ratio. 0 h, CK6h, CK12h, CK18h, CK24h represent five different germination stages: 0,6, 12, 18 and 24 h in CK group, respectively; 0 h, S6 h, S12 h, S18 h and S24 h represent five different germination stages: 0,6, 12, 18 and 24 h in salt treatment group, respectively

Fig. 8

RT-qPCR analysis of the representative DAP genes in embryo (a) and endosperm (b) under control and salt treatment group of Zhengmai 366. Statistically significant differences compared to the control were calculated based on an independent Student’s t-tests: *p < 0.05; **p < 0.01

Fig. 9

Metabolic changes of the key proteins in embryo and endosperm under salt stress. Circles represent proteins expressed in the embryo, squares represent proteins expressed in the endosperm. The red color represents upregulation and green represents downregulation under salt stress. ROSS, ROS Scavenging; Osm, Osmotic homoeostasis; Fer, Fermentation metabolism. 3PG, Glyceraldehyde 3-phosphate; 2PG, 2-Phosphoglycerate; APX, Ascorbate peroxidase; AR, Aldose reductase; CATI, Catalase isozyme 1; EC, Enolase; EF-1β, Elongation factor 1-beta; eIF 5A3, eukaryotic translation initiation factor 5A3; F-1,6-2P, Fructose 1,6 di/bis phosphate; FBA, Fructose-bisphosphate aldolase cytoplasmic isozyme; G-1P, Glucose-1- phosphate; G-6P, Glucose-6- phosphate; G3P, glyceraldehyde-3-phosphate; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; LEA, Late embryogenesis abundant protein Lea14-A; PEP, Phosphoenolpyruvate; PER1, 1-Cys peroxiredoxin; PGAM, Phosphoglycerate mutase; PGM, Phosphoglucomutase; PGK, 3-phosphoglycerate kinase; PMS, Putative methionine synthase; SOD, Superoxide dismutase; TIF IIB, Transcription initiation factor IIB. The orange arrow represents the stress of suffering; the blue arrow represents the order that the protein of different functional groups responding stress