Functions Of Key Enzymes:AGPase And Isoamylase Involved In Maize Starch Biosynthesis

Starch is the major component in plant seeds and is the most carbon reserve. It provides more than80%calories in the world, and has become an essential commodity that is widely used in food, feed, fuel and industry. About70%of the maize endosperm is starch, which can affect the yield and quality of maize directly. So it is very important to do further research on starch metabolism.The core of starch biosynthesis pathway is orchestrated by four types of enzymes:ADP-glucose pyrophosphorylase (AGPase), starch synthase (SSs), starch branching enzymes (SBEs), and starch-debranchingenzymes (DBEs). Generally, there are multiple isoforms for each enzyme. The major enzymes involved in the process of starch degradation are as follows: glucan-water-dikinase, phosphogulucan-water-dikinase, isoamylase,β-amylase, a-amylase. Although the functions of most of these enzymes are very clear, the synergies of them are still remaining exclusive. We still have much work to do to answer the question that how the enzymes complexes work.In this work, we analyzed the functions of AGPase in maize tissues comprehensively, and the functional interaction between isoamylase-DBE and SSIII. Given this complexity, the precise genetic mechanisms responsible for AGPase activity in endosperm, leaf, embryo, and other tissues of maize are not fully understood. Four different transcripts potentially encoding AGPase SS have been detected by RT-PCR in maize endosperm.Three of these transcripts, namely BT2b, LEAFS, and EMBS potentially could encode plastidial forms of the protein. The distribution of transcripts encoding plastidial AGPase LS in maize tissues has not been reported. Two kinds of new double mutants of SSIII and ISA were identified: isa2-339/dull-Ref and isa2-339/dull-4059, which were good materials for the determination of functional interactions between enzyme proteins. The main results are as follows:1. The expression profiling of AGPase genes is tissue-specific and development stage specific; Recombinant forms of all seven proteins were expressed in E. coli, and which showed that the possible isoforms of AGPase in maize were:SH2/BT2a, SH2/LEAFS、SH2/EMBRYOS、EMBRYOL/EMBRYOS and LEAFL/LEAFS.2. We analyzed the properties of kinetics and heat resist of EMBRYOL+LEAFS isoforms:in contrast to the wild-type AGPase (SH2+BT2a), the Km value of EMBRYOL+LEAFS AGPase is smaller, so it has great affinity ability to bind the substrates, and the EMBRYOL+LEAFS AGPase exhibited greater heat stability. These properties are very interesting and important, because it will help us find the AGPases which have good properties of allosteric and heat stability, and develope new transgenetic plants.3. We screened and indentified more than20kinds of transponson mutants of AGPase genes, but just got two new effective mutants:leafL and embryos, which have good phenotype. The starch content of leafL-DsEx3/leafL-DsEx3homozygous mutant leaves was approximately23%of the amount in congenic wild type inbred r1-m. The embS-MuEx2mutation conditioned a decrease in embryo starch content to approximately46%of the wild type level. In both instances the difference between the mutant and wild type value was highly statistically significant (p value<0.0004). The leafL and embryos genes were shown to condition production of approximately6%and6.4%of the total amount of starch that accumulates in endosperm tissue, respectively.4. Two new double mutants were identified containing the null mutation isa2-339and either du1-Ref, encoding a truncated SSIII protein lacking the catalytic domain, or the null allele du1-R4059. In contrast to the single mutant parents, the double mutations of both SSIII and ISA2lead to starch deficient and accumulated phytoglycogen, and can change the structure of starch or phytoglycogen.The main innovation points in present work are as follows:(1)It is the first time to analyze all AGPase genes comprehensively, and clear the gene expression profiling and AGPase isoforms in maize tissues. (2) It is the first time to get mutant homozygotes of two new AGPase genes: leafL and embryos, and provide strong evidence that how important of the plastidial AGPase in controlling the carbon flux.(3) We find a new AGPase isoforms in maize that has very good properties of kinetics and heat stability: greater affinity ability to bind the substrates and greater heat stability than the most wild-type AGPase (SH2+BT2a).(4) It is the first time to get the double mutants of isa2-339/dul-Ref and isa2-339/dull-4059, which are important materials for the research on functional interaction between enzyme proteins, because they have special phenotype in contrast to the single mutants.