Evidence for proteomic and metabolic adaptations associated with alterations of seed yield and quality in sulfur-limited Brassica napus L

Abstract

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Fig. 1.

Effects of control, LS70, LS53, and LS32 treatments on seed morphology and viability. A, morphology of mature seeds harvested from plants grown under control, LS70, LS53, and LS32 conditions. LS70, LS53, and LS32 treatments corresponded to sulfur limitation applied when the first petals fell (GS70), when the main inflorescence emerged (GS53), and at the beginning of the bolting stage (GS32). B, Brassica napus embryos isolated from control, LS70, LS53, and LS32 seeds and subjected to the tetrazolium assay. White arrows indicate unviable seeds in control, LS70, and LS53 seeds. For details about the method used, see the “Experimental Procedures” section.

Fig. 2.

Fatty acid composition of mature seeds harvested from plants grown under control, LS70, LS53, and LS32 conditions. The values are shown as the mean in mg g⁻¹ DW (n = 6 for control; n = 4 for LS70, LS53, and LS32). The ω6/ω3 ratios are indicated above the pie charts. *Significant differences from the control value (p < 0.05). Means ± S.E. are indicated in supplemental Table S1.

Fig. 3.

Two-dimensional electrophoresis gels of total proteins from control, LS70, LS53, and LS32 mature Brassica napus seeds. For each treatment, the induced and repressed protein spots are circled in red and green, respectively, on the corresponding gel image. These spots were identified by LC-MS/MS and are listed in Tables IV through VII. Unidentified spots or spots that were not associated with a functional group are listed in supplemental Table S2. M_r, molecular weight; pI, isoelectric point.

Fig. 4.

Effects of control, LS70, LS53, and LS32 treatments on mature seed proteome. A, distribution of the 208 proteins presenting a significant variation of abundance in LS70, LS53, and LS32 seeds relative to the control. B, number and distribution (%) in the functional groups of proteins changed in LS70, LS53, and LS32 seeds relative to the control.

Fig. 5.

Adaptation of seed storage protein (SSP) accumulation in response to LS70, LS53, and LS32 treatments. A, clustering of expression profiles of significantly modulated SSPs identified by mass spectrometry in LS70, LS53, and LS32 mature seeds relative to the control (n = 6 for control; n = 4 for LS70, LS53, and LS32). Proteins up- and down-regulated in the whole seed are marked in red and green, respectively (color code at the top of the figure). B, relative abundance of the different classes of SSPs and other proteins in the total protein content of Brassica napus mature seeds grown under control, LS70, LS53, and LS32 conditions. The values correspond to the mean ± S.E. (n = 6 for control; n = 4 for LS70, LS53, and LS32). Significant differences from the control were at *p < 0.05 or at **p < 0.01.

Fig. 6.

Effect of control, LS70, LS53, and LS32 treatments on theoretical and experimentally measured sulfur contents of mature seed proteins. A, theoretical S amino acid proportion calculated as the sum of relative abundances multiplied by the S amino acids (cysteine and methionine) proportion of the corresponding protein, given next to the protein name in brackets. B, S-proteic content of mature seeds grown under control, LS70, LS53, or LS32 conditions. For each treatment, the S-proteic/S ratio is shown directly above the corresponding bar. In both panels the values correspond to the mean ± S.E. (n = 6 for control; n = 4 for LS70, LS53, and LS32). *Significant difference from the control value (p < 0.05).

Fig. 7.

Schematic representation of changes in accumulation of proteins of carbon metabolism and associated proteins in mature LS70, LS53, and LS32 seeds compared with control seeds. Significantly up- and down-regulated proteins are shown in red and green, respectively. *Thiamine-pyrophosphate-dependent reaction. A, UTP-glucose-1-phosphate uridylyltransferase. B, β-glucosidase. C, cytosolic phosphoglucose isomerase. D, fructose-bisphosphate aldolase. E, glyceraldehyde-3-phosphate dehydrogenase. F, phosphoglycerate kinase. G, enolase. H, cytosolic malate dehydrogenase. I, glyoxalase I. J, NADP-malic enzyme. K, ATP synthase CF1 β subunit. L, citrate synthase. M, aconitase. N, isocitrate dehydrogenase. O, succinate dehydrogenase. P, alcohol dehydrogenase. Q, glyoxysomal malate synthase. DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; GSH, reduced form of glutathione; OAA, oxaloacetate.