GENETIC AND BIOCHEMICAL ANALYSIS OF THE FUNCTIONS OF STARCH BRANCHING ENYMES IN Zea mays.

Open Access
Author:
Blackman, Jennifer Lynne
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Mark Guiltinan, Thesis Supervisor
  • Daniel J Cosgrove, Honors Advisor
Keywords:
  • Starch Branching Enzymes
  • Starch
  • Pollen
  • Maize
Abstract:
Zea mays L. (maize) contains starch branching enzymes (SBEs), which are glycosyl transferases that determine the structure of the starch within the plant by adding branches to amylose to form amylopectin. There are three forms of starch branching enzymes, SBEI, SBEIIa, and SBEIIb. Since Sbe2a and Ae (Sbe2b) are expressed both in the kernel endosperm and within mature pollen, these are thought to play specific roles in the synthesis of starch during plant development. Although the role of SBE has been well characterized in maize endosperm, the role of these genes in the other parts of the plant such as leaves and pollen is less well studied. To examine the role of SBEs in maize, two main approaches were taken. In the first chapter of the thesis, the proteins were studied in vivo through the use of a reverse genetics approach. In the second chapter of the thesis, purified SBE proteins were prepared to investigate in vitro the regulation of their biochemical activities. To assess the role(s) of SBEs in pollen development and successful pollination, we first determined the frequency of genetic transmission of the sbe2a-Mu mutation, a knock out allele generated by a Mutator transposon insertion (herein referred to as sbe2a-Mu), and ae alleles in an outcross of Sbe2a/sbe2a-Mu; ae/ae pollen to a plant wildtype for Sbe2a and Ae. In contrast to the expectation that 50% of offspring should contain the sbe2a-Mu allele, only 9% transmission of the allele was observed in the ae background. This suggests that pollen germination and/or growth requires the function of at least one of the SBEII class genes for proper function. Secondly, we tested the sbe2a-Mu; ae double mutant’s effect on pollen tube germination and/or growth. To address this, the lengths of double mutant pollen tubes were compared to single ae mutant tubes growing within wildtype silks and on synthetic media plates. In vitro, the lengths of the sbe2a-Mu; ae double mutant ii pollen tubes were shorter than ae single mutant types. Observations of pollen germination and growth made in vivo were consistent with this result. Fewer of the sbe2a-Mu; ae double mutant tubes were observed within wildtype pollinated silks as compared to the ae single mutant pollen. Together, these results indicate that the decrease in transmission rate of the sbe2a-Mu and ae mutant alleles to progeny could be due to the inability of the double mutant pollen to either germinate or grow as fast as the ae single mutant or wild type pollen. It is likely that the slow rate of growth observed in sbe2a-Mu; ae double mutant pollen results in its low rate of pollination and allele transmission. This suggested the hypothesis that wildtype and sbe single mutants are able to out-compete the sbe2a-Mu; ae double mutant pollen grains and successfully pollinate egg cells at a significantly higher rate. As part of a study of the biochemical regulation of the SBE enzymes, recombinantly expressed starch branching enzymes were expressed in E. coli, purified and their biochemical activities examined to investigate their regulation by redox conditions. Mutants were obtained with both single and multiple mutations in order to study the possible residues that are pertinent to redox regulation of these enzymes.