Hydro-Electro Hybrid Priming Promotes Carrot (Daucus carota L.) Seed Germination by Activating Lipid Utilization and Respiratory Metabolism

Abstract

Carrot (Daucus carota L.) is widely cultivated as one of the most important root crops, and developing an effective presowing treatment method can promote the development of modern mechanized precision sowing. In the present study, a novel seed priming technology, named hydro-electro hybrid priming (HEHP), was used to promote the germination of carrot seeds. Seed germination experiments showed that HEHP was able to increase the germination index (GI) and vigor index (VI) by 3.1-fold and 6.8-fold, respectively, and the effect was significantly superior to that of hydro-priming (HYD) and electrostatic field treatment (EF). The consumption and utilization rate of seed storage reserves were also greatly improved. Meanwhile, both glyoxysomes and mitochondria were found to appear ahead of time in the endosperm cells of HEHP through observations of the subcellular structure of the endosperm. Activities of isocitrate lyase (ICL), NAD-dependent malate dehydrogenase (MDH), pyruvate kinase (PK), and alcohol dehydrogenase (ADH) were significantly increased by HEHP. From transcriptome results, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the glyoxylate cycle, glycolysis, gluconeogenesis, and the citrate cycle were significantly enriched and real-time quantitative PCR (qRT-PCR) analysis confirmed the expression pattern of 15 critical differentially expressed genes (DEGs) in these pathways. All DEGs encoding MDH, phosphoenolpyruvate carboxykinase (PEPCK), and PK were upregulated in HEHP; thus, it is reasonable to infer that the transformation of malate, oxalacetate, phosphoenolpyruvate, and pyruvate in the cytoplasm may be pivotal for the energy supply during early germination. The results suggest that the optimal effect of HEHP is achieved by initiating stored lipid utilization and respiratory metabolism pathways related to germination.

Keywords: carrot; hydro-electro hybrid priming; lipid utilization; respiratory metabolism; seed germination.

Figure 1

Seed germination and seedling growth status of carrot seeds after different priming treatments. (CK, the control; EF, high-voltage electrostatic field treatment; HYD, hydro-priming treatment; HEHP, hydro-electro hybrid priming treatment) (A) Photograph of seed germination experiment at 120 h after sowing; (B) Seedlings randomly selected from each treatment at 120 h after sowing; (C) Change trend of germination percentage with time after sowing.

Figure 2

Changes of the consumption and utilization rate of carrot seed storage reserves after different priming treatments. Values represent the means ± SE from three biological replications. The different lowercase letters above the bars indicate significant differences in 95% probability level (p < 0.05, Duncan test was performed after ANOVA analysis).

Figure 3

Changes of the subcellular structure of endosperm of carrot seeds after different priming treatments. (A1,A2) Two images from a CK seed endosperm before sowing; (B1,B2) Two images from a CK seed endosperm 20 h after sowing; (C1,C2) Two images from a HYD seed endosperm before sowing; (D1,D2) Two images from a HYD seed endosperm 20 h after sowing; (E1,E2) Two images from a HEHP seed endosperm before sowing; (F1,F2) Two images from a HEHP seed endosperm 20 h after sowing. (A1–F1) 4300×. (A2–F2) 22,000×. L: Oleosome; DL: Oleosome in the process of degradation; CW: Cell wall; DCW: Cell wall in the process of degradation; CM: Cell membrane; Mi: Mitochondrion; Gl: Glyoxysome; V: Vesicle; N: Nucleus; Nu: Nucleolus; BG: High electron dense substances.

Figure 4

Changes of the key enzyme activities after different priming treatments. ICL, isocitrate lyase; MDH, NAD-dependent malate dehydrogenase; PK, pyruvate kinase; ADH, alcohol dehydrogenase. Values represent the means ± SE from three biological replications. The different lowercase letters above the bars indicate significant differences in 95% probability level (p < 0.05, Duncan test was performed after ANOVA analysis).

Figure 5

Venn diagram showing specific and overlapping identified genes among all the treatments.

Figure 6

Gene ontology (GO) enrichment analysis of DEGs among CK, HYD and HEHP. (A) Top 20 enriched GO terms of the DEGs identified in CK vs. HEHP; (B) Top 20 enriched GO terms of the DEGs identified in CK vs. HYD; (C) Top 20 enriched GO terms of the DEGs identified in HYD vs. HEHP.

Figure 7

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs among CK, HYD, and HEHP. (A) The top 20 enriched KEGG pathways of the DEGs identified in CK vs. HEHP; (B) The top 20 enriched KEGG pathways of the DEGs identified in CK vs. HYD; (C) The top 20 enriched KEGG pathways of the DEGs identified in HYD vs. HEHP.

Figure 8

DEGs among CK, HYD and HEHP involved in the glyoxylate cycle and respiratory metabolism. ICL, isocitrate lyase; FH, fumarate hydratase; SDH, succinate dehydrogenase; MDH, NAD-malate dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase; PDC, pyruvate decarboxylase; ADH, alcohol dehydrogenase.

Figure 9

Real-time quantitative PCR (qRT-PCR) analysis of 15 selected DEGs at S0 and S20. (A) Expression patterns of 15 selected DEGs from qRT-PCR; (B) Correlation analysis between the results of qRT-PCR and transcriptome (Spearman correlation coefficient was used. * means significant at the level of p < 0.05. ** means significant at the level of p < 0.01). The corresponding relationship between gene ID and its encoded enzyme are as follows: LOC108215541, ICL; LOC108224240, SDH iron-sulfur subunit; LOC108204633, FH; LOC108210669, LOC108223687, LOC108199904 and LOC108193312, MDH; LOC108206052 and LOC108205258, PEPCK; LOC108219027 and LOC108208954, PK; LOC108223320 and LOC108209732, PDC; LOC108199900 and LOC108214136, ADH. The different lowercase letters above the bars indicate significant differences in 95% probability level (p < 0.05, Duncan test was per-formed after ANOVA analysis).

Figure 10

Statistics of DEGs among CK, HYD and HEHP encoding MYB, bZIP and HB-other family TFs.