Enhanced seed viability and lipid compositional changes during natural ageing by suppressing phospholipase Dα in soybean seed

Abstract

Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols (TAG), i.e. the storage lipids. Phospholipase D (PLD) catalyses the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild-type (WT) seeds stored for 33 months were non-viable, 30%-50% of PLDα-knockdown (PLD-KD) soybean seeds stored for 33 months germinated. WT and PLD-KD seeds increased in lysophospholipid levels and in TAG fatty acid unsaturation during ageing, but the levels of lysophospholipids increased more in WT than in PLD-KD seeds. The loss of viability of WT seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural ageing, PLDα affects the soybean phospholipid profile and the TAG profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long-term storage.

Figure 1

pSPLDi PCR, western blotting for PLDα, and a PLDα enzyme activity in immature cotyledons from, (a) and (b), heterozygous T₂ SW1 and, (c) and (d), a T₅ SW line. (a) and (c) The presence of the pSPLDi transgene was confirmed with GOI PCR. + indicates PCR-positive for pSPLDi, and − indicates PCR-negative. Immunoblot analysis of PLDα was performed with Arabidopsis PLDα1 antibody, and PLDα proteins were detected at the 92 kDa position, based on protein markers. (b) and (d) PLDα enzyme assay of T₂ SW1 and T₅ SW seeds. PLDα enzyme activity was expressed as nmol of PC hydrolyzed min⁻¹ mg⁻¹total protein. Error bars indicate ± SE from three independent measurements .

Figure 2

Effect of PLDα suppression on seed germination of soybean seeds stored for 33 months. T₂ SW1-3 and SW1-7 seeds were tested for germination, the presence of the pSPLDi transgene, and electrolyte leakage. (a) Fresh and aged soybean seeds were placed on a wet paper roll for 10 days, and soybean sprouts were counted to calculate germination (%, number of germinated seeds/total number of seeds × 100). Values are mean ± SD (n = 30 in three replications). (b) PCR analysis for the presence of pSPLDi transgene in soybean sprout from 33 month-old soybean seeds. (c) Effect of PLDα suppression on electrolyte leakage of 33 month-old soybean seeds. Wild type (Fayette), SW1-3 transgene PCR negative (−), SW1-3 transgene PCR positive (+), SW1-7 transgene PCR negative (−), and SW1-7 transgene PCR positive (+) seeds were tested for ion leakage by conductivity measurements. * indicates a significant difference at the p < 0.05 level in transgenic (PCR positive) or null-transgenic (PCR negative) compared to the wild type genotype (n = 3).

Figure 3

Ultrastructure of cotyledon tissues andcell wall complex from fresh wild type [(a) and (d)], aged wild type [(b) and (e)], and aged PLD-KD [(c) and (f)] cotyledon tissues. In (b) and (e), black arrows indicate signs of partial plasmolysis. Bars in a, b, and e = 10 μm. Bars in d, e, and f= 0.5 μm. ML, middle lamella; PW, primary walls; PD, plasmodesmata.

Figure 4

Lipid molecular species of wild type and PLD-KD fresh and aged seeds. (a) The black bars and the hatched bars represent wild type fresh and aged seeds, respectively. (b) The black bars and the hatched bars represent PLD-KD fresh and aged seeds, respectively. The values are the mean ± SD (n = 5). An H or L in the hatched bars indicates that the value is significantly higher or lower than that of fresh seeds at p < 0.05, respectively.

Figure 5

Changes in molecular species of lysoPC, lysoPE and lysoPG caused by natural aging of (a) wild type and (b) PLD-KD seeds, as revealed by ESI-MS/MS. The black bars represent fresh seeds and the hatched bars represent old seeds. The values are the mean ± SD (n = 5). An H or L indicates that the value is significantly higher or lower than that of fresh seeds at p < 0.05.

Figure 6

Changes in triacylglycerol molecular species resulted by natural aging in (a) wild type and (b) PLD-KD seeds measured by ESI-MS/MS with standards. The black bars and the hatched bars represent fresh and aged respectively. The value are the mean ± SD (n = 5). An H or L in the aged seed indicates that the value is significantly higher or lower than that of fresh seed of each cultivar at p < 0.05.

Figure 7

Indexes of PL and TAG species in wild type and PLD-KD soybean seeds during natural aging. Unsaturation index of PL and TAG from fresh wild type (WT) seeds, fresh PLD-KD seeds, aged wild type seeds, and aged PLD-KD seeds. All values are mean ± SD of five biological replicates, and the significance data is shown belowat p <0.05 level.