Metabolism of sugars in the endosperm of developing seeds of oilseed rape

Abstract

The sugars in the endosperm of a developing seed have many potential roles, including the supply of carbon to the developing embryo and controlling gene expression in it. Our understanding of their metabolism is, however, fragmentary and is confined to a very few species (especially Vicia spp.). To develop a quantitative understanding of the regulation of sugars in seeds of oilseed rape (Brassica napus), we measured relevant enzyme activities, the sizes of the pools of sugars in the liquid endosperm, and the flux of sugars from the endosperm into the embryo. The concentrations of hexose sugars in the liquid endosperm decreased, and sucrose (Suc) increased through development. The overall osmotic potential also fell. The timing of the changes was not precise enough to determine whether they signaled the onset of rapid accumulation of storage products. Changes in endosperm invertase activity were complex and quantitatively do not explain the changes in sugars. The embryo can metabolize hexose sugars in addition to Suc, and possibly at higher rates. Therefore, in addition to invertase, the growing embryo itself has a potential to influence the balance of sugars in the endosperm. The activity of Suc synthase in the embryo was greater than that of invertase during development. This observation and a higher activity of fructokinase than glucokinase in the embryo are both consistent with the embryo using Suc as a carbon source.

Figure 1

Schematic view of sugar movements within a developing seed of oilseed rape.

Figure 2

Sugars in the liquid endosperm of developing seeds of oilseed rape. The weight of the embryo is used as a developmental scale (x axis), but the data in A and B relate only to the endosperm. A, Suc (□), Glc (○), and Fru (▴) are plotted as percentages of the total hexose moieties found in all three. B, The summed concentration of Suc, Glc, and Fru. Note that in A, which is intended to display the balance of forms carbohydrate, Suc counts as two hexose moieties. In B, which is concerned with the osmotic potential of endosperm solutes, Suc is counted as a single molecule. In both panels, each point represents a single silique. We harvested a range of siliques on 4 different d, and we have pooled the four data sets in this figure. C, The oil content of developing embryos, taken from Eastmond and Rawsthorne (2000). Each data point represents a value obtained from a batch of five embryos. Very similar developmental profiles have been measured previously within our laboratory (Murphy and Cummins, 1989; Kang et al., 1994; da Silva et al., 1997). Note the relative timings of the onset of rapid oil synthesis and the change in sugar content (A), taking into account the x axis scaling.

Figure 3

Acid invertase in the seed coat (A; cellular endosperm and testa) and the liquid endosperm (B) of oilseed rape seeds. Invertase was measured at pH 4.5 as moles of Suc consumed. Each point represents a single silique. Invertase was measured in different siliques on 3 different d, but the trends were similar in all three experiments. Therefore, this figure shows the combined data of all experiments. Data for liquid endosperm were initially measured per microliter of endosperm and were then converted to a seed basis using a separate curve of endosperm volume versus embryo weight. Embryo weight is used as a developmental scale. The lines have no significance except to emphasize the trend in the data and are best fit three-parameter exponential decay curves.

Figure 4

The pH dependence of invertases in developing seeds of oilseed rape. Invertases were extracted from young (▴) and old (●) seed coats and from embryos (○). Young seed coats came from seeds whose embryos weighed less than 0.5 mg. Old seed coats were from seeds with embryos weighing approximately 3 mg. Note that young seed coats have a higher activity of invertase than old ones, regardless of pH. Also note that neither the (acid) invertase of the old seed coat, nor the (alkaline) invertase of the embryo has an activity at pH 6, yet young seed coats do have an isoform of invertase active at this neutral pH. Data are plotted as mean ± se of three measurements from a single experiment.

Figure 5

The rate of uptake of Fru (▴), Glc (○), and Suc (□) by young (approximately 0.5 mg fresh weight) embryos of oilseed rape. These symbols are the same as used in Figure 2A. Note that uptake is approximately linear for 2 h. This figure shows a single experiment in which each point is the mean ± se of three measurements, each made on a single silique. Corresponding data in the text are means of these data and two other, similar experiments, not shown here.

Figure 6

Sugar-metabolizing enzymes in developing embryos of oilseed rape. A, Suc synthase (●) and alkaline invertase (○); B, fructokinase (▴) and glucokinase (○). Each point represents a single silique, and data are combined from three separate experiments. The lines merely indicate a trend and are best fit cubic curves.

Figure 7

An arithmetic model of the system described in Figure 1, using values similar to those measured in vivo where possible. A, The concentrations of Suc (double-chain line) and the hexoses (superimposed, solid line) that would be expected assuming the V_max of invertase follows that of the seed-coat acid invertase (dashed line). Note the delay before the concentrations of Glc and Fru fall and Suc increases. B, As in A, but assuming the V_max of invertase follows that of the acid invertase of the liquid endosperm. For the critical, early period of development, this invertase has a higher activity. Note that higher activities of invertase lead to a more pronounced plateau before the change in sugar concentrations. C, As in A, but with the V_max values of uptake of hexose adjusted, Fru 5% upward, Glc 5% downward. Note that this very modest change has large consequences for the concentrations of Glc (dashed line) and Fru (solid line), which no longer superimpose. Full details of the method of calculation are found in “Materials and Methods.”