Difference Of Gene Expression Profiling In The Endosperm Of Ae/wx And Sh1 Mutants And Starch Biosynthesis In Maize

Maize (Zea mays L. ssp. Mays) provides us food and feed for many years due to its starch content and quality. To study the molecular mechanisms of starch biosynthesis in maize, we employed 18K Affymetrix? maize Genomic Genechip? to reveal the differences of gene expression profiling of the 15 days endosperm after pollination (DAP) in ae/wx and sh1 mutants compared to B73. We found the relationship among starch metabolism, metabolisms of other components and gene expressing profiling due to the accumulation of sugar. Then, genes expression patterns of starch biosynthesis in leaves and developing endosperms of 5 maize inbreds were studied for the contribution of different genes to starch biosynthesis in endosperm. In addition, RNA interference was employed to knock down ZmSBEâ…¡b or ZmSBEâ… &ZmSBEâ…¡b for the requirement of high-amylose starch in industry. The main results were as follows:1. DNA microarrayThe 15 DAP endosperms of B73,sh1,ae/wx were employed to investigate the differenc of their gene expression profiling. Compared to B73, total 2706 and 1966 probe sets were different significantly (FDR<0.1%; q value<0.01) in sh1 and aw/wx mutants (total 17,622 probe sets detecting 13,495 genes in chip). To reduce the effect of gene expression in different genetic backgrounds, differentially expressed genes were analyzed in endosperm of B73 and Mo17 at 13 DAP and 19 DAP, from which 609 and 889 differentially expressed genes were identified (FDR<5%) as the ones caused by different backgrounds. After deducting 351 probe sets from ae/wx vs.B73 and 390 probe sets from sh1 vs.B73, there were 1429 (named Dae/wx) and 1557 (named Dsh1) probe sets surplus for further analysis, among which 835 probe sets kept the same tendency in both mutants. 90.6% (29/32) differentially expressed genes were confirmed by qRT-PCR.2. GO analysis of differentially expressed genesAccording to p-value, lipid transport (6/12, which means 6 differentially expressed genes of 12 members on chip, and the same as follows), pyruvate decarboxylase activity (4/6), sugar:hydrogen symporter activity (4/7) and structural constituent of cell wall (3/6) were revealed in Dae/wx, suggesting that carbohydrate metabolism and lipid transportation were affected significanty. Whereas, sugar:hydrogen symporter activity (6/7), Golgi apparatus (4/5), sucrose synthase activity (4/6), cysteine protease inhibitor activity (5/9), lipid transport (5/12), cell wall (6/18) and pyruvate decarboxylase activity (3/6) were displayed in Dsh1, suggesting that except for carbohydrate metabolism and lipid transportation, down-regulated expression of cysteine protease inhibitor suggests changes in stress responses and in cell biology.3. The functional annotation and classification of differentially expressed genesAbout 72% differentially expressed genes were annotated and classified into 14 classes, among which carbohydrate and energy metabolism, transcription and posttranscriptional processing, protein synthesis, posttranslational processing, stress/defence/senescence and transportation were affected significantly. All together, the genes in these processes accounted for 46.5% of the same tendent differences, 53.7% of the genes changed only in Dae/wx (Oae/wx) and 47.1% of the genes changed only in Dsh1 (Osh1). Further analysis suggests that there were a few genes in starch biosynthesis but more genes in sugar and energy metabolism expressed differentially. The down-regulation of trehalose-phosphate synthase (TPS) suggests the down-regulated activity of AGPase after posttranslational redox activation between Sh2 subunits. The analysis of promoters of sugar metabolism genes indicates several sugar response elements in most promoters. Pul and TPS may be repressed directly by high-level of sugar in which SREATMSD (TTATCC), TATCCAOSAMY (TATCCA) and TATCCAY MOTIFOSRAMY3D (TATCCAY) in their promoters should be the sugar repressive elements due to their down-regulated expressions.The differentially expressed genes reflect the difference of the starch accumulation and cell structure in two mutants. Due to the accumulation of sugar, signal transductions, especially sugar signal and stress signal, were changed significantly, which might alter the expression of hormone-related genes, cell organizational proteins genes, and cell cycle-related genes finally. In addition, there were maternally expressed genes (MEG) and seed maturation proteins up-regulated and embryo-specific proteins down-regulated.4. Expression patterns of genes differentially expressed in starch metabolism and cell cycle during endosperm development7 DAP kernals, 15 DAP endosperms and 25 DAP endosperms of B73, ae/wx and sh1 were used to study the expression patterns of 18 differentially expressed genes in sugar metabolism and 9 differentially expressed genes in cell cycle. We found that the expression patterns of Pul, SBEâ…¡a, UGPase, SUS3, SSâ…¡a in sh1 and Sh2-1, SUS3, SSâ…¡a in ae/wx were altered, revealing that these genes may interact with each other. In contrast, only the expression level of genes in sugar transport and signal were altered, suggesting that these genes are regulated indirectly by sugar level, cell development. Similarly, although the expression levels of the genes in cell cycle were changed significantly, the expreeion patterns were not changed distinctly, suggesting that the cell cycle in endosperm cells is not disturbed the cell division. As a result, endosperm cell development is delayed. Compared to B73, layered structure was not found in 7 DAP endosperm of ae/wx and sh1 mutants, and bigger nucleoli in ae/wx mutants and smaller nucleoli and more cell number in sh1 mutants were found, suggesting the significant alternation of the cytoarchitecture in the endosperms of ae/wx and sh1 mutants.5. Different contribution of the genes to starch biosynthesisLeaves and developing endosperms of Q319, C7-2, q404, ZN-A and S8 were used to study the expression patterns of 44 genes participating in starch synthesis. We found that ZmGBSSâ… ,ZmSSâ…¢,ZmSBEâ…¡b,ZmBT2-2,ZmSh2-2,ZmSh2-3,ZmBT1 were expressed exclusively in the metaphase and anaphase of developing endosperm, and the expression patterns of ZmSUS1,ZmSus1L,ZmSUS3,ZmSSâ… ,ZmSSâ…¡a,ZmSBEâ… ,ZmISO1,ZmPul were consistent with the process of starch synthesis in the endosperm, while ZmUGP3 and ZmSUT2 were highly-expressed in all samples, suggesting that these genes perhaps devoted to starch synthesis in endosperm. Although their expression patterns were down-regulated during the endosperm development, ZmBT2-1 and ZmSh2-1 were still expressed. ZmSUS1, ZmSUS1L,ZmBT2-1,ZmSH2-1,ZmBT1,ZmSSs,ZmSBEs,ZmISO1,ZmISO2,ZmPUL,ZmGPTs and ZmPPTs were localized to plastids, ZmSUS2,ZmSUS3,ZmUGPs,ZmBT2-2,ZmSH2-2,ZmSH2-3,ZmPGIå’ŒZmPPMs were localized to cytoplasm. Whereas, ZmSUTs were localized on cytoplasm membrane. These data provides more evidence about the different contribution of the genes to starch biosynthesis.6. High-amylose maize inbreds engineered by RNA interference (RNAi)To meet the requirement of high-amylose starch in industry, based on the expression profiles of starch biosynthesis-related genes and published data, SBEâ…¡b and SBEâ… &SBEâ…¡b were selected to knock down in maize by RNA interference approach, to create new high-amylose maize inbreds. Sh2 was cloned and modified by insertion of a tyrosine and a Serine between 495 and 496 amino acids, to enhance the activity of AGPase and to increase the starch biosynthesis. Transgenic plants were recovered via biolistic and Agrobacterium-mediated transformations.