Figure 1.
The mechanism of actions of three core enzymes in starch biosynthetic considered in the model.
Starch synthases (SSs) transfer ADP-glucose (red hexagon) to the non-reducing end of a pre-existing α-(1⟶4)-linked glucan. Starch branching enzymes (SBEs) transfer the cleaved chain onto a random position–which may include the original branch–via an α-(1⟶6) link. The length of the transferred portion by SBEs has to be ≥ Xmin and that of the remaining stub must be ≥ X0. Debranching enzymes (DBEs) hydrolyze α-(1⟶6) linkages, thereby removing the whole chain. These enzymatic schemes are focused on the form of glucans in terms of the CLD rather than the actual products. Together, one of each of SS, SBE and DBE, regardless of the actual isoforms, make up an enzyme set.
Figure 2.
Rice (cv Nipponbare) amylopectin CLD fitted with the substrate-competing model.
The amylopectin CLD was obtained by FACE [32]. X is the number of glucose residues on glucans released by unrestricted debranching of the starch. The black- and gray-filled circles are X = 6 and 32, respectively. The fitting to the overall experimental CLD (yellow filled circles) is given as green filled circles. The range of fitting X up to ≈ 60–70 is considered. Fitting procedures are given the Model section. Crosses mark the features of the CLD.
Figure 3.
Components of the CLD, given in Figure 2, fitted with the model.
(A) Fitting (red filled circles) to single-lamella (SL) chains with the substrate-competing model. X = 32 marks the apparent length of chain needed for the chains to enter the contiguous amorphous lamella (Figure 5). Where the slope changes significantly in Feature F gives the range of presumed three-lamellae-spanning chains. (B) Fitting (blue filled circles) to the type-2 TL chains with the substarte-competing model, which is the difference between the experimental and the calculated SL CLD in (A) and displaced (details in the Model section). The actual X is shown on the upper abscissa of (B).
Figure 4.
Fitted parameters for six replicates of the CLD described in Figure 2.
(A) Averages and ± standard deviations (thin bars) of β (the relative branching activity from an enzyme set to that of the total propagation from set (i) and (ii) in the SL range, or (iii) and (iv) in the TL range) from different enzyme sets; h(iii/i) is the ratio of the maximum of type-2 TL CLD to that of the SL CLD. (B) The modes (highest repeating values) of X0 and Xmin (which are discrete variables) from different enzyme sets. Where the thin bars are not seen means the standard deviation is small. The red and blue colors correspond to the calculated SL and type-2 TL CLD in Figure 2.
Figure 5.
A proposed mechanism for the formation of the arrays of semi-crystalline lamellae in amylopectin.
C and A indicate the crystalline and amorphous lamellae. The purple shaded regions indicate the nascent SL space where non-lamellar amylopectin chains, amorphous in nature, are being formed. Types of chains: (1) single-lamella (SL) chains that are (mostly) confined in crystalline lamellae (grey lines); (2) type-1 TL chains, crystalline-lamella-connecting chains, which span more than one crystalline lamellae (red lines); (3) type-2 TL chains, non-lamella-connecting chains, which protrude the SL space but remain in the amorphous lamellae (blue lines). The longest chain length confined to a crystalline lamella, predicted from Figure 2, is X ∼ 31 (i.e. chains ≥32 enters the contiguous amorphous lamella), while longer SL chains (red lines) with X ∼ 60–70 are sufficiently long to participate in crystalline formation in the subsequent crystalline lamella.
Figure 6.
The steady-state surface, which describes the restrictions on the parameters for amylopectin CLD to form.
Surface generated for X0(i), Xmin(i), X0(ii) and Xmin(ii) = 6, 7, 9 and 14 respectively. These values are the fitted parameters for Figure 2. The thick red line is an approximation to the steady-state line in our previous development (Figure 4 in [24]), where the exact steady-state line is when β(ii) (as defined in Figure 4) = 0. γ(i,ii) is the sum of relative debranching activities from set (i) and (ii) to that of propagation from set (i) and (ii).