DA-6 promotes germination and seedling establishment from aged soybean seeds by mediating fatty acid metabolism and glycometabolism

Abstract

Soybean seeds contain higher concentrations of oil (triacylglycerol) and fatty acids than do cereal crop seeds, and the oxidation of these biomolecules during seed storage significantly shortens seed longevity and decreases germination ability. Here, we report that diethyl aminoethyl hexanoate (DA-6), a plant growth regulator, increases germination and seedling establishment from aged soybean seeds by increasing fatty acid metabolism and glycometabolism. Phenotypic analysis showed that DA-6 treatment markedly promoted germination and seedling establishment from naturally and artificially aged soybean seeds. Further analysis revealed that DA-6 increased the concentrations of soluble sugars during imbibition of aged soybean seeds. Consistently, the concentrations of several different fatty acids in DA-6-treated aged seeds were higher than those in untreated aged seeds. Subsequently, quantitative PCR analysis indicated that DA-6 induced the transcription of several key genes involved in the hydrolysis of triacylglycerol to sugars in aged soybean seeds. Furthermore, the activity of invertase in aged seeds, which catalyzes the hydrolysis of sucrose to form fructose and glucose, increased following DA-6 treatment. Taken together, DA-6 promotes germination and seedling establishment from aged soybean seeds by enhancing the hydrolysis of triacylglycerol and the conversion of fatty acids to sugars.

Fig. 1.

Natural aging significantly decreases soybean seed germination and seedling establishment abilities. (A–D) Representative photographs of naturally aged soybean seeds during the imbibition process (60 h after sowing). Soybean seeds were stored for 5, 10, 22, or 34 months after harvest and then subjected to analysis. Scale bar=10 mm. (E) The quantitative analysis of final germination rates of different samples (A–D) are shown (72 h after sowing). (F) The early seedling establishment phenotype of different samples (A–D) immediately following germination (2 weeks after sowing). Scale bar=100 mm. (G) The quantitative analysis of the seedling establishment rates of different samples are shown (2 weeks after sowing). Percentages are the average of three repeats ±SE. The germination experiments were performed under 25 °C and 60% relative humidity conditions, while the seedlings were grown under 25 °C and 16 h light with 8 h dark conditions. The asterisk (*) indicates a significant difference at P<0.0 by Student’s t-test analysis.

Fig. 2.

DA-6 promotes germination and seedling establishment from naturally aged soybean seeds. (A) Representative photographs of naturally aged soybean seeds (stored for 12 months) during the imbibition process (60 h after sowing), with or without DA-6 treatment. Scale bar=10 mm. (B) The quantitative analysis of final germination rates of seeds stored for 12 months in the absence or presence of DA-6 treatment. (C) The early seedling establishment phenotype of seeds stored for 12 months with or without DA-6 application. Scale bar=100 mm. (D) The statistical data of early seedling establishment for (C) are shown (15 d after sowing). Percentages are the average of three repeats ±SE. The germination experiments were performed under 25 °C and 60% relative humidity conditions, while the seedlings were grown under 25 °C and 16 h light with 8 h dark conditions. The asterisk (*) indicates a significant difference at P<0.05 by Student’s t-test analysis. A 200 μM concentration of exogenous DA-6 was used.

Fig. 3.

DA-6 enhances the germination ability of artificially aged soybean seeds. (A–C) Representative photographs of different types of soybean seeds (healthy seeds without CDT, CDT seeds with H₂O, and CDT seeds with DA-6 treatment) during imbibition (48 h after sowing). Scale bar=10 mm. (D) The quantitative analysis of final germination rates of different samples (A–C) is shown. (E and F) The radical length (E) and fresh weight (F) of germinated soybean seeds were analyzed (48 h after sowing). The germination experiments were performed under 25 °C and 60% relative humidity with dark conditions; the germination rates under dark conditions were recorded using a green safety light, according to a previous assay (Barrero et al., 2014). The average percentages of three repeats ±SE are shown. The asterisk (*) indicates a significant difference at P<0.05 by Student’s t-test analysis. A 200 μM concentration of exogenous DA-6 was used.

Fig. 4.

DA-6 positively regulates the seedling establishment of artificially aged soybean seeds. (A) The early seedling establishment phenotype of different types of soybean seeds (healthy seeds without CDT, CDT seeds with H₂O, and CDT seeds with DA-6 treatment). Scale bar=100 mm. (B) The statistical data of early seedling establishment for (A) are shown (15 d after sowing). (C) Representative images of the height of soybean seedlings. Scale bar=100 mm. (D) The quantitative analysis of plant height for (C). Percentages are the average of three repeats ±SE. The seedlings were grown under 25 °C and 16 h light with 8 h dark conditions. The asterisk (*) indicates a significant difference at P<0.05 by Student’s t-test analysis. A 200 μM concentration of exogenous DA-6 was employed.

Fig. 5.

DA-6 treatment increases the concentration of soluble sugars during imbibition by aged soybean seeds. Different types of soybean seeds (healthy seeds without CDT, CDT seeds with H₂O, and CDT seeds with DA-6 treatment) were employed. (A) Soluble sugar quantification analysis. (B) Fructose concentration quantification. (C) Sucrose concentration analysis. The average percentages of four repeats ±SE are shown. Asterisks (*) and (**) indicate a significant difference at P<0.05 and P<0.01, respectively, by Student’s t-test analysis. A 200 μM concentration of exogenous DA-6 was used.

Fig. 6.

DA-6 treatment increases the concentration of total fatty acids in aged soybean seeds during imbibition. Different types of soybean seeds (healthy seeds without CDT, CDT seeds with H₂O, and CDT seeds with DA-6 treatment) were employed. (A) The heat map analysis of the contents of several types of fatty acids during the soybean seed imbibition process with the time-course. The heat map was created by the Illustrator software. The fatty acid level from low (L) to high (H) indicates the minimum and maximum in the entire database. (B) The total fatty acid concentration in different types of soybean seeds during imbibition. (C) The unsaturated fatty acid concentration in soybean seeds during imbibition. (D) The saturated fatty acid concentration in soybean seeds during imbibition. The average percentages of four repeats ±SE are shown. Asterisks (*) and (**) indicate a significant difference at P<0.05 and P<0.01, respectively, by Student’s t-test analysis. A 200 μM concentration of exogenous DA-6 was employed.

Fig. 7.

Positive effect of DA-6 on the transcription of several key genes which are involved in hydrolysis of triacylglycerol in aged soybean seeds during imbibition. Gene expression was investigated by qPCR assay during the course of the imbibition process. Different types of soybean seeds (CDT seeds with H₂O and CDT seeds with DA-6 treatment) were employed. These different types of soybean seeds were employed for mRNA extraction, and three replications were performed. GmSDP6 and GmSDP1 encode FAD-G3P dehydrogenase and patatin-like domain-containing triacylglycerol lipase, and GmACX2 and GmPCK1 encode acyl-CoA oxidase and phosphoenolpyruvate carboxykinase, respectively. Those genes are all involved in the pathways by which the triacylglycerol was transferred to fatty acids and sugars during imbibition. (A) GmSDP1; (B) GmSDP6; (C) GmACX2; (D) GmPCK1; (E) GmMFP2; (F) GmMDAR4; (G) GmCOMATOSE. Asterisks (*) and (**) indicate a significant difference at P<0.05 and P<0.01, respectively, by Student’s t-test analysis. A 200 μM concentration of exogenous DA-6 was used.

Fig. 8.

DA-6 treatment increases the invertase activity in aged soybean seeds during imbibition. Different types of soybean seeds (healthy seeds without CDT, CDT seeds with H₂O, and CDT seeds with DA-6 treatment) were employed. The invertase enzyme activity in these distinct types of seeds was analyzed during imbibition. The average percentages of three repeats ±SE are shown. Asterisks (**) indicate a significant difference at P<0.01 by Student’s t-test analysis. A 200 μM concentration of exogenous DA-6 was used.

Fig. 9.

The proposed working model through which DA-6 promotes germination and seedling establishment from aged soybean seeds. In the aged soybean seeds, the transition from triacylglycerol to fatty acid and glycerol and then to the soluble sugars was blocked in the absence of DA-6. The application of exogenous DA-6 treatment in aged soybean seeds promoted the transcription of several key genes and elevated the activity of invertase which is involved in this pathway. Taken together, this model revealed that DA-6 promotes the germination and seedling establishment from aged soybean seeds by increasing the hydrolysis of triacylglycerol and the conversion of fatty acids and glycerol to sugars.