In vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

Abstract

Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

Keywords: affinity; barley; biochemical characterization; expression levels; kinetics; stability; starch synthases; substrate specificity.

Figure 1

Substrate specificity of SSs. Relative activity for all SS enzymes with different MOS and polysaccharides as acceptors, assayed at 37°C with 1 mM ADP-Glc, 10 mM acceptor for MOS (eight blue bars, from darker for DP = 1 to lighter for DP = 8) and 1 mg/mL acceptor for branched polysaccharides (four green bars). All specific activities are normalized to the values with maltotetraose for SSI (orange bar), which corresponds to 0.302 μmol·min⁻¹·mg⁻¹. Two values that would require the y-axis scale to be doubled are shown numerically with broken bars. The asterisk denotes a value which was not determined.

Figure 2

Reversibility and long-range elongation profile of SSI. (A) Structure of the fluorescently labeled acceptor and proof of the absence of any reaction in the absence of both ADP and ADP-Glc, but in the presence of enzyme and fluorescent acceptor. (B) UPLC reaction profile over time after addition of 10 mM ADP. (C) UPLC reaction profile over time after addition of 5 mM ADP-Glc. (D) UPLC reaction profiles with a truncated version of HvSSI after addition of variable amounts of ADP-Glc, measured after all donor was depleted. Only for panels (D–F) in this figure are the data originating from the truncated version of HvSSI. (E) Molar fraction of each MOS species after integration of peak areas from panel (D). (F) Models of expected MOS species distributions with different kinetic models, compared with the molar fractions from panel e (“Experimental” corresponds to the red bars in panel E).

Figure 3

Observed and predicted reaction course for HvGBSSI action on maltotriose. (A) UPLC traces of reaction of GBSSI with unlabeled maltotriose. DPs of product and time points are indicated. Labeling was done after reaction termination. The DP = 3 peak is truncated in all traces up to 2 h for improved visibility of the other peaks. Boxes contain the traces for the larger peaks with a zoomed in vertical scale to illustrate the relative areas of peaks up to DP = 21, some peaks with even larger DPs are visible. (B) Observed (green bars) and predicted (blue bars) product distributions corresponding to the reaction progress after 6 h, before DP = 7 started to accumulate. The predicted product distribution was calculated assuming a purely distributive reaction mechanism and the individual reaction velocities for each acceptor measured at 37°C.

Figure 4

Temperature stability of the SS enzymes. Residual activity of SS samples after incubation at the indicated temperatures. Solid lines are sigmoidal fits to the experimental data.

Figure 5

Variation of SS activity with temperature. (A) Compared activity of SSI with different acceptors at different temperatures. The first seven are linear MOS, the last two branched polysaccharides, assayed at 0.1 mg/mL. All data presented as relative values to maltotetroase at 37°C. (B) The same for SSIIa, glycogen at 1 mg/mL concentration. (C) The same for SSIV. The assay temperatures are different from panel to panel, as are the gaps between temperatures, but the color scheme has been kept consistent.

Figure 6

Affinity of SSs for different substrates from glycan arrays. Carbohydrate microarray binding profile, which shows binding of SSs to various poly- and oligosaccharides. In addition, positive controls CBM20 (binding to starch), LM11 (binding to (1,4)-β-D-xylan/arabinoxylan) and LM6 (binding to (1,5)-α-L-arabinan) were included. The mean spot signals obtained from four experiments are presented in a heat map in which color intensity is correlated to signal. The highest signal in each data set was set to 100, and all other values were normalized accordingly as indicated by the color scale bar.

Figure 7

Temporal profile of protein abundance in barley endosperm. Barley grains were harvested between 0 and 24 DAF every 2 days and proteins from barley endosperm extracted. Equal amounts of protein were loaded onto SDS-PAGE and probed with the indicated primary antibodies. The horizontal blue lines show the alignment of the molecular weight markers around the relevant area. (Top) Immunoblots using the indicated antibodies; protein ladders are included in the left of each immunoblot and corresponding molecular weights are indicated to the left of the group. The rightmost lane in each blot shows recombinant enzyme controls added to each gel, of the same type that the antibodies were raised against. The molecular weights of the bands being integrated are: HvGBSSI, 59.0 kDa (61.3 kDa for the recombinant version); HvSSI, 67.5 kDa (69.8 kDa for the recombinant version); HvSSIIa, 81.6 kDa (84.6 kDa for the recombinant version); HvSSIV, 98.3 kDa (99.1 kDa for the recombinant version). The region used for integration in each case is illustrated with open green brackets; it is larger for SSIIa because the multiple bands were interpreted as being fragments of SSIIa, since their presence and pattern were occasionally detected also in SDS-PAGE of recombinant protein preparations; in any case, the band at 81.6 kDa dominates the overall intensity. (Bottom) Semi-quantitative analysis of protein abundance, ordered as for the immunoblots. The x-axis shows time as DAF, the y-axis shows protein quantity as calculated from immunoblot as percentage of total protein content.