Relationships between starch synthase I and branching enzyme isozymes determined using double mutant rice lines

Abstract

Background: Starch is the most important carbohydrate in plant storage tissues. Multiple isozymes in at least four enzyme classes are involved in starch biosynthesis. Some of these isozymes are thought to interact and form complexes for efficient starch biosynthesis. Of these enzyme classes, starch synthases (SSs) and branching enzymes (BEs) play particularly central roles.

Results: We generated double mutant lines (ss1/be1 and ss1L/be2b) between SSI (the largest component of total soluble SS activity) and BEI or BEIIb (major BEs in developing rice endosperm) to explore the relationships among these isozymes. The seed weight of ss1/be1 was comparable to that of wild type, although most ss1/be2b seeds were sterile and no double recessive plants were obtained. The seed weight of the double recessive mutant line ss1L/be2b, derived from the leaky ss1 mutant (ss1L) and be2b, was higher than that of the single be2b mutant. Analyses of the chain-length distribution of amylopectin in ss1/be1 endosperm revealed additive effects of SSI and BEI on amylopectin structure. Chain-length analysis indicated that the BEIIb deficiency significantly reduced the ratio of short chains in amylopectin of ss1L/be2b. The amylose content of endosperm starch of ss1/be1 and ss1L/be2b was almost the same as that of wild type, whereas the endosperm starch of be2b contained more amylose than did that of wild type. SSI, BEI, and BEIIb deficiency also affected the extent of binding of other isozymes to starch granules.

Conclusions: Analysis of the chain-length distribution in amylopectin of the double mutant lines showed that SSI and BEI or BEIIb primarily function independently, and branching by BEIIb is followed by SSI chain elongation. The increased amylose content in be2b was because of reduced amylopectin biosynthesis; however, the lower SSI activity in this background may have enhanced amylopectin biosynthesis as a result of a correction of imbalance between the branching and elongation found in the single mutant. The fact that a deficiency of SSI, BEI, or BEIIb affected the affinity of other starch biosynthetic isozymes for the starch granule implies that there is a close interaction among SSI, BEI and BEIIb during amylopectin biosynthesis in rice endosperm.

Figure 1

Pedigree and seed morphologies of double mutant lines. Morphology of dehulled rice seeds (upper panels) and seed cross-sections (lower panel). Seeds were obtained from double mutant lines derived from SSI and BEI or BEIIb mutants and their parent lines, and were observed under a stereo-microscope. Symbols in parentheses show genotypes of lines.

Figure 2

Enzyme activities on zymograms. Native-PAGE/activity staining of starch synthases (SSs; Panel A), branching enzymes (BEs; Panel B), and debranching enzymes (DBEs; Panel C) in developing endosperm of double mutant, parental mutant, and wild-type lines. Arrowheads indicate SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA (isoamylase), PUL (pullulanase), and PHO (phosphorylase) activity bands. Soluble protein extracts were prepared from developing endosperm. Amount of soluble protein is per mg fresh weight. Volumes of crude extract applied to the native gels in section ‘a’ are 2-fold and 4-fold greater than those applied in sections ‘b’ and ‘c’, respectively. Kin, Kinmaze; Nip, Nipponbare; T65, Taichung 65; WT, wild type.

Figure 3

Distributions of enzymes in total protein extract and three protein fractions. Immunoblotting of total protein extract (Total), soluble protein fraction (SP), loosely-bound protein fraction (LBP), and tightly-bound protein fraction (TBP) from three developing rice endosperms at 12–15 days after flowering (DAF). Samples are from double mutant, parental mutant, and wild-type lines. Immunoblotting analyses were conducted using antisera raised against rice SSI, GBSSI, BEI, and BEIIb. GBSSI from mature endosperms (mature GBSSI) were also analyzed. Amounts of protein loaded onto the gels were as follows: 1/25 of extracts in SP, 1/15 of extracts in LBP and 1/4 of extracts in TBP. Amount of protein bands in ‘Total’ was standardized against density of total protein bands stained with Coomassie Brilliant Blue (data not shown). Kin, Kinmaze; Nip, Nipponbare; T65, Taichung 65; WT, wild type.

Figure 4

Molecular structure analysis of amylopectin by capillary electrophoresis. Chain-length distribution patterns of endosperm amylopectin in mature endosperm (A, D). Differential plots between single mutant and wild-type lines (B, E). Differential plots between double mutant and wild type and the calculated profiles made by adding the profiles of single mutant lines (C, F). Numbers on plots represent DP values. Each figure shows one representative data set (one of at least three replicates prepared from different rice seeds from homogenous plants). Relative standard error of molar% of each chain length from DP5–60 was less than 2.5%. Kin, Kinmaze; Nip, Nipponbare; T65, Taichung 65; WT, wild type.

Figure 5

Size separation of debranched endosperm starch and purified amylopectin by gel-filtration. Size separation of debranched endosperm starch and purified amylopectin from double mutant (E and I), parental mutant (C, D, G and H), and wild-type lines (A, B and F) by gel filtration chromatography through three Toyopearl HW55S-HW50S columns. Graphs show elution profiles of isoamylase-debranched starch (blue lines) and purified amylopectin (red lines). Different X-axis scales reflect different flow rates among experiments. Fractions (Fr. I, II, and III) were divided at troughs of carbohydrate content curve, as detected by refractive index detectors (left Y-axis). Grey line in wild type (Nipponbare) indicates λ_max values of starch-iodine complexes (right Y-axis). Figures show one typical dataset (one of at least three replicates prepared from starch and purified amylopectin).