STRUCTURE AND FUNCTION OF ENDOSPERM STARCH FROM MAIZE MUTANTS DEFICIENT IN ONE OR MORE STARCH-BRANCHING ENZYME ISOFORM ACTIVITIES

Open Access
- Author:
- Xia, Huan
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 12, 2009
- Committee Members:
- Donald B Thompson, Dissertation Advisor/Co-Advisor
Donald B Thompson, Committee Chair/Co-Chair
Mark Guiltinan, Committee Chair/Co-Chair
John Neil Coupland, Committee Member
Devin G Peterson, Committee Member
Ming Tien, Committee Member - Keywords:
- starch branching enzyme
maize endosperm starch
starch structure
starch digestibility
maize mutant
maize kernel germination - Abstract:
- Maize starch-branching enzyme (SBE) isoforms are important in endosperm starch biosynthesis and consequently are expected to influence starch structure and function. The ae mutant, which is deficient in SBEIIb, has a profound effect on endosperm starch structure, leading to an increased amylose content and a reduced amylopectin branching. The study of the ae mutant suggests an important function of SBEIIb on starch structure. Although no effect of the sbe1a mutant, which is deficient in SBEI, on starch structure has been noted previously, for starch deficient in SBEIIb, a further deficiency of SBEI increased branching (Yao et al. 2004). A related study showed that for starch deficient in SBEIIb, a further deficiency of SBEIIa also increased branching (Yao et al. 2003). Taken together, these studies suggest a reciprocal inhibition between SBEI and SBEIIa. It can be reasoned from these studies that the effect of individual SBE isoform on starch structure may not be independent. The overall goal of this continuing line of SBE research is to understand the role of each of the SBE isoform in starch biosynthesis. The goal of this thesis is to understand the effects of deficiency of maize SBE isoform activities on endosperm starch molecular and granular structure and starch digestion, with emphasis on the effect of SBEI deficiency. A preliminary test of endosperm starch digestibility for the sbe1a mutant showed that deficiency of SBEI caused a decreased susceptibility of starch granules to pancreatic á-amylase. This was the first indication of an effect of deficiency of SBEI alone in synthesis of endosperm starch and justified further investigation of the function of SBEI isoform. Consequently, this thesis describes the study of the structure and function of sbe1a mutant endosperm starch compared to non-mutant (wild-type, Wt) starch. Starch from Wt and sbe1a mutant endosperm was subjected to in vitro pancreatic á-amylase digestion for 16 hr by the AOAC procedure for resistant starch (RS), to determine the proportion of RS. The digestion kinetics were analyzed using a double-exponential decay fit. sbe1a starch had a higher RS value (13.2%) compared to Wt (1.6%). Kinetic analysis showed that sbe1a starch had a lower proportion of a rapidly-digested component than Wt. Chain length profile was examined for the non-granular starch and starch fractions from Wt and sbe1a, as well as for the RS from Wt and sbe1a. Although chain length profiles of native starch molecules appeared very similar for Wt and sbe1a, some small differences between the two genotypes were observed in the chain length profile of their RS, as well as in the comparison of the chain length profile of their RS to native starch. Compared to Wt, fewer amylose-like chains were digested in RS from sbe1a. Iodine binding analysis of starch fractions showed that sbe1a samples had higher ëmax than Wt samples. Amylopectin fractionated from Wt and sbe1a starch was subjected to in vitro â-amylase hydrolysis over 24 hr. Hydrolysis of sbe1a chains was less complete as compared to Wt. The less complete hydrolysis is consistent with a higher proportion of closely associated branch points in amylopectin from sbe1a. The amylose fraction from Wt and sbe1a starch was subjected to exhaustive â-amylolysis. sbe1a starch had more long residual chains, suggesting that the distribution of branch points on these long amylose chains may be closer to the non-reducing ends. Debranching of â-limit dextrin from amylose by isoamylase was less complete in sbe1a, suggesting that closely associated branch points may be more prevalent in amylose from sbe1a. Granule structure for native starches and residual granule structure for the respective RS were examined by microscopy. Scanning and transmission electron micrographs show that the RS from sbe1a mutant retained more of the granule integrity. A resistant peripheral layer by microscopy observed for the sbe1a RS may help understand the decreased digestibility of sbe1a starch. Sbe1a is more strongly expressed in the later stage of endosperm development (Gao et al. 1996). Thus, if most of the later-synthesized starch is deposited in the peripheral region of the growing granule, starch synthesized in the sbe1a mutant would be expected to be affected predominantly in the peripheral region. A reasonable speculation is that the branching pattern might differ between the RS and the digested portion of sbe1a starch, and this difference might also account for the decreased digestibility of the peripheral region in sbe1a granule. Starch utilization and coleoptile growth of Wt and sbe1a mutant kernels were measured during kernel germination. After Day 6 germinating sbe1a kernels exhibited a slower rate of coleoptile growth and an accordingly decreased rate of starch hydrolysis as compared to Wt kernels, suggesting compromised starch utilization in germinating sbe1a kernels. Comparisons of RS values among two sets of mutant combinations both show a main effect of sbe1a and a significant difference between sbe1a and Wt. This consistent result substantially confirms our preliminary indication of an effect of sbe1a alone. For the first set of mutant combinations, sbe1a and sbe2a, the effects of sbe1a and sbe2a on both RS values and amylopectin branching are not independent, as a significant interaction is observed. In contrast, for the second set of mutant combinations, sbe1a and ae, the effects of sbe1a and ae on RS values are independent, but the effects of sbe1a and ae on amylose content are not independent. The significant statistical interaction terms suggest possible physical interactions among SBEs. These interactions might be mediated by other starch synthetic enzymes. The evidence obtained in this thesis, coupled with previous research, leads to several hypotheses about the specific functions of the three maize SBE isoforms in endosperm starch biosynthesis: 1) The SBEIIb protein is the dominant form of SBE, and is responsible for synthesizing branch points that are clustered. 2) When SBEIIb is present, SBEI is responsible for modulating the branching pattern by synthesizing branch points that are less locally clustered, and SBEIIa is responsible for modulating the branching pattern by synthesizing branch points that are more locally clustered. 3) When SBEIIb is absent, SBEI and SBEIIa may have a reciprocal inhibitory function on synthesis of branch points. This thesis also for the first time reports that a lack of SBEI activity resulted in an observable effect, which was seen on both starch molecular structure and starch function. Structural and functional analysis of endosperm starch deficient in SBEI activity strongly supports the hypothesis that SBEI is required to synthesize starch granules for normal kernel development, allowing efficient hydrolysis and utilization. Evidence from this thesis reveals a unique and essential function of SBEI in normal plant development, consistent with the evolutionary conservation of SBEI in all higher plants. The new knowledge generated in this thesis will contribute to our understanding of the function and evolution of the maize SBEs, and of their roles in the biosynthesis, hydrolysis and utilization of starch granules. Moreover, the novel sbe1a starch might have application as a food ingredient with nutritional benefit.