Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming

Abstract

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.

Keywords: endosperm; high-amylose rice; metabolomics; starch biosynthesis; transcriptomics.

Fig. 1.

Structural comparison of wild-type OsSBEIIb and the mutated version in line E15. The protein is represented as a ribbon, whereas all of the labeled residues are represented as scaled ball and stick models. (A) Wild-type OsSBEIIb showing the position of Arg⁴⁷¹. The Inset box shows all of the key amino acids in the central catalytic domain (Asp⁴⁰², His⁴⁰⁷, Arg⁴⁷¹ [yellow], Asp⁴⁷³, Glu⁵²⁸, His⁵⁹⁵, and Asp⁵⁹⁶). Dashed lines show the intramolecular interactions (orange, salt bridge; green, hydrogen bond; purple, π-interactions). (B) Mutant OsSBEIIb in line E15 showing the position of His⁴⁰⁷ and Asp⁴⁰². The Inset box shows that His⁴⁰⁷ has the same interactions with His³⁶⁸ and Ser⁴⁰⁶ as shown for the wild-type protein, but Asp⁴⁰² no longer interacts with Arg⁴⁷¹ but retains the normal interactions with lle³⁵³ and Gln³⁵². Amino acids that interact with His⁴⁰⁷ and Asp⁴⁰² are colored yellow. Dashed lines show the intramolecular interactions (green, hydrogen bond; purple, π-interactions).

Fig. 2.

Starch properties and grain morphology of wild-type rice, Tos17 insertion line NE9005, and mutant line E15. (A) Properties of the starch in all three genotypes. Samples annotated with different letters are significantly different from one another as determined by ANOVA (P < 0.01). (B) Gross morphology (i–iii) and transverse sections (iv–vi) of the seeds, and scanning electron microscopy images (vii–ix) of starch grains in the endosperm. (Scale bars, 1 mm in i–vi; 5 µm in vii–ix.)

Fig. 3.

Expression analysis of 26 genes representing the starch biosynthesis pathway. (A) Heat map summarizing the transcriptional reprogramming of the starch biosynthesis pathway in the T2 leaves and T3 seeds of line E15. Fold changes are shown in red (increase) or blue (decrease) compared to wild type. (B) Expression level of all 26 genes relative to OsActin 15 DAF in the leaves (Top) and seeds (Bottom). Each value is the mean SD of at least three independent measurements with SEs, and significant differences were determined using Student’s t test (*P < 0.05, **P < 0.01).

Fig. 4.

Heat map of polar and nonpolar metabolites identified in rice endosperm. S1 and S2 are two biological replicates of line E15 and WT is a wild-type segregant (azygous control). Individual columns represent technical replicates of WT, S1, and S2 (Bottom of the plot). Fold changes are indicated by varying shades of blue (decrease) and red (increase) compared to wild-type seeds. N.C., not confirmed.

Fig. 5.

Metabolic pathway highlighting significant changes in the endosperm of line E15 compared to wild-type seeds. Significant changes are shown as gradients of red (higher levels in E15), blue (lower levels in E15), and green (detected only in E15). Metabolites with the same abundance in both genotypes are shown in gray and those undetected in either genotype are shown in white. The average of six technical replicates was used for all calculations. N.C., not confirmed.