Effect Of The Activities Of Key Enzymes Involved In Starch Biosynthesis And Expression Profiles Of Genes Which Encoded Starch Synthase In Developing Wheat Grain

Two different wheat varieties were used to study the dynamic changes of theaccumulations of amylose and amylopectin, starch accumulation rate, the activity changes ofkey enzymes involved in starch biosynthesis, the accumulations of different amylopectinchain length and the expression profiles of genes which encoded starch synthase in the kernelduring grain filling. The results observed were:1. The changes of amylose and amylopectin accumulating rates of both waxy andnon-waxy wheat varieties showed a single peak curve. The accumulation courses of bothamylopectin and amylose were well fitted to the logistic equation by relating amylopectin andamylose contents against days post anthesis. The simulation parameters revealed that thehigher contents of amylopectin and amylose resulted from greater accumulation rate andmean accumulation rate, but accumulation duration probably played a less important role forthem.2. The activity changes of the adenosine diphosphorate glucose pyrophrylase (AGPP),soluble starch synthase (SSS), Granule-bound starch synthase (GBSS) and starch branchingenzyme (SBE) were all in the pattern of a single-peak curve, and peaked at20~25days afteranthesis.3. The accumulation rate of amylose was significantly or highly significantly correlatedwith the activities of AGPP, SSS, GBSS and SBE the accumulation rate of amylopectin wassignificantly correlated with the activity of SSS, and the accumulation rate of starch wassignificantly or highly significantly correlated with the activities of AGPP and SSS.4. Amylopectin were de-branched with isoamylase and named by different chains A andshort B (DP<29), mid-length B (29<DP<65.8) and long B (>65.8). Different amylopectinchain contents changed continually during grain development and varied differently betweenthe two types of wheat. Moreover, the AGPP, SSS, GBSS, and SBE activities exhibited aclose correlation with the different chains of amylopectin. 5. A comprehensive analysis of the transcript levels of genes which encodestarch-synthesis enzymes was fundamental for the assessment of the function of each enzymeand the regulatory mechanism for starch biosynthesis in sink organs. Using quantitativereal-time RT-PCR, an examination was made of the expression profiles of9wheat genesencoding three classes of enzymes, i.e. ADPglucose pyrophosphorylase, starch synthase, andstarch branching enzyme in developing grain, which were AGPP-L and AGPP-S encoded thelarge and small subunit of AGPP, GBSSI was encoded GBSS, SSI, SSII and SSIII wereencoded SS, SBEI, SBEIIa and SBEIIb were encoded SBE. The results showed that theexpression profiles of AGPL, AGPP-S, SSI, SSII, SSIII, SBEI, SBEIIa and SBEIIb weresimilar between non-waxy and waxy types of wheat, except GBSSI. Four patterns ofexpression in the seed were identified: group1gene, SSII, which was highly expressedthroughout granule development; group2gene, SBEIIa, which was expressed very early ingrain formation and are presumed to be involved in the construction of fundamental cellmachineries, de novo synthesis of glucan primers, and initiation of starch granules; group3genes, AGPP-L、AGPP-S, GBSSI, SSIII and SBEIIb, which are highly expressed throughoutendosperm development; group4genes, SSI and SBEI which have transcripts that are low atthe onset but which rise steeply at the start of starch synthesis in the endosperm and arethought to play essential roles in endosperm starch synthesis, Thus, The modes of geneexpression were tissueand developmental stage-specific.6. During the whole stage of gain development, GBSSI and SSIII genes higher expressedin non-waxy while SBEI and SBEIIb were lower expressed in non-waxy wheat.7. During the whole stage of grain development, SSI was generally expressed over themid-late grain development, while SSII was higher expressed over the early-mid graindevelopment, SSIII was higher expressed at the middle of grain development, and the GBSSIwas higher expressed during whole stage of grain development; SBEIIa was expressed overthe early-mid grain development, SBEIIb was higher expressen at the middle of graindevelopment, and SBEI was higher expressed over mid-late grain development.8. The results showed that the beginning biosynthesis of amylopectin was earlier thanamylose, based on the results as below:(1) The amylopectin appeared at5days after anthesis in non-waxy type of wheat;meanwhile, we did not found the amylose at5days after anthesis. The amylose content wasdetected about10days after anthesis, and the accumulation of amylopectin was increasedrapidly at this time. Moreover, we did not found amylose in the waxy type of wheat. (2) At5days after anthesis, we detected SSS activity which mainly was involved inamylopectin synthesis, but GBSS activity which mainly was involved in amylose synthesiswas not detected.(3) The amylopectin which were obtained at5days after anthesis were de-branched withisoamylase, we found that the different chains of amylopectin were synthsized at this time.(4) The expression level of genes which encoded starch snthase told us that the SSII andSBEIIa, which encoded the starch synthase maily involved in amylopectin synthesis, werehigher expressed at the initiation of grain development. Even though the GBSSI wereexpressed at this time, it probably belonged to post-transcriptional control gene. According tothe espression levele of GBSSI in waxy type of wheat, we speculated that the expression levelwere too lower to amylose synthesis.