ABSTRACT: Despite much knowledge is well-known on the transcriptional and enzymological regulation of many genes in starch synthesis, including SBEⅡb, Waxy, and SUS-SH1 genes, less information is available on their expression profiles as a consequence of their dysfunction. Hence, transcriptional adjustments in response to mutations were required for a better understanding of starch biosynthesis pathway, others metabolism and cell response. We used microarrays to detail the transcriptional adjustments in the endosperms of ae wx and sh1 at 15 days after pollination (DAP) compared to B73. Maize endosperms at 15 DAP were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homologic endosperms of middle ears in order to increase the temporal resolution of expression profiles.
Project description:Despite much knowledge is well-known on the transcriptional and enzymological regulation of many genes in starch synthesis, including SBEⅡb, Waxy, and SUS-SH1 genes, less information is available on their expression profiles as a consequence of their dysfunction. Hence, transcriptional adjustments in response to mutations were required for a better understanding of starch biosynthesis pathway, others metabolism and cell response. We used microarrays to detail the transcriptional adjustments in the endosperms of ae wx and sh1 at 15 days after pollination (DAP) compared to B73.
Project description:In angiosperms, endosperm plays a crucial role in coordinating seed development through genetic balance and molecular interaction, and is the primary tissue where genomic imprinting occurs. To identify small interfering RNA (siRNA) “imprintome” and its paternal transcriptome activation in early developing maize endosperms, we performed high-throughput small RNA sequencing of whole kernels at 0, 3 and 5 days after pollination (DAP) and endosperms at 7, 10 and 15 DAP, using B73 by Mo17 reciprocal crosses. We observed gradually increased expression of paternal siRNAs in 3- and 5-DAP kernels and balanced contribution of parental siRNA transcriptome in 7-DAP endosperm, followed by identification 460 imprinted siRNA loci with majority (456/460, 99.1%) being maternally expressed that occurred at 10 DAP. Genome-wide scanning found 13 imprinted genes harboring imprinted siRNA loci within their 2-Kb flanking regions, which was significantly different from random probability based on simulation analysis. Finally, gene ontology categories of “response to auxin stimulus”, “response to brassinosteroid stimulus” and “regulation of gene expression” for genes harboring 10-DAP specific siRNAs and “nutrient reservoir activity”, “protein localization to vacuole” and “secondary metabolite biosynthetic process” for genes harboring 15-DAP specific siRNAs indicated that siRNAs could be involved in influencing specific cellular or biochemical processes that are essential for endosperm development, e.g. nutrient uptake and allocation. Although the mechanism of how these siRNAs regulating endosperm key events remains unclear, this study provided us an alternative perspective of siRNA function in plant. The unpollinated kernels (0 DAP), the kernels of 3, 5 DAP and endosperms of 7 10, 15 DAP from the B73 and Mo17 reciprocal crosses were used to perform high-throughput sequencing using the Illumina HiSeq2000 platform
Project description:In angiosperms, the endosperm provides nutrients for embryogenesis or seed germination and is the primary tissue where gene imprinting occurs. To map the imprintome of the early developing endosperm in maize, we performed high-throughput transcriptome sequencing of the kernels at 0, 3, 5 days after pollination (DAP) and the endosperms at 7, 10, and 15 DAP produced from the B73 and Mo17 reciprocal crosses. We observed a gradual increase of paternal gene mRNAs in the 3- and 5-DAP kernels. In the 7-DAP endosperm, the majority of the tested genes reached a ratio of 2m:1p suggesting that paternal genes were nearly fully activated by 7 DAP. A total of 116, 234 and 63 imprinted genes exhibiting parent-specific expression were identified in the 7-, 10-, and 15-DAP endosperms, respectively. The highest amount of paternally expressed genes (PEGs) was found at 7 DAP mainly due to the significantly deviated parental allele expression ratio of these genes at this stage, while nearly 80% of the maternally expressed genes (MEGs) were specific to 10 DAP which were primarily attributed to the sharply increased expression levels compared to the other stages. GO enrichment analysis of the imprinted genes indicated the 10-DAP-specific MEGs were involved in the nutrient uptake and allocation through auxin signaling pathway in the maize endosperm coincident with the endosperm developmental stage associated with the beginning of starch and storage protein accumulation The unpollinated kernels (0 DAP), the kernels of 3, 5 DAP and endosperms of 7 10, 15 DAP from the B73 and Mo17 reciprocal crosses were used to perform high-throughput sequencing using the Illumina HiSeq2000 platform
Project description:In angiosperms, the endosperm provides nutrients for embryogenesis or seed germination and is the primary tissue where gene imprinting occurs. To map the imprintome of the early developing endosperm in maize, we performed high-throughput transcriptome sequencing of the kernels at 0, 3, 5 days after pollination (DAP) and the endosperms at 7, 10, and 15 DAP produced from the B73 and Mo17 reciprocal crosses. We observed a gradual increase of paternal gene mRNAs in the 3- and 5-DAP kernels. In the 7-DAP endosperm, the majority of the tested genes reached a ratio of 2m:1p suggesting that paternal genes were nearly fully activated by 7 DAP. A total of 116, 234 and 63 imprinted genes exhibiting parent-specific expression were identified in the 7-, 10-, and 15-DAP endosperms, respectively. The highest amount of paternally expressed genes (PEGs) was found at 7 DAP mainly due to the significantly deviated parental allele expression ratio of these genes at this stage, while nearly 80% of the maternally expressed genes (MEGs) were specific to 10 DAP which were primarily attributed to the sharply increased expression levels compared to the other stages. GO enrichment analysis of the imprinted genes indicated the 10-DAP-specific MEGs were involved in the nutrient uptake and allocation through auxin signaling pathway in the maize endosperm coincident with the endosperm developmental stage associated with the beginning of starch and storage protein accumulation
Project description:We performed a molecular characterization of the maize defective kernel mutant that is a seed-lethal mutant with developmental deficiency phenotypes, transcriptomic analysis revealed numerous differentially expressed genes related to storage proteins and starch biosynthesis. The developmental difference between the WT and mutant might be caused by differences in gene expression. To explain the developmental difference between them at the genomic level, RNA-Seq analysis was performed with total RNA isolated from WT and mutant endosperms at 10 DAP
Project description:Starch synthesis is an essential feature for crops, but its regulatory mechanism has been largely restricted to transcription factors. Starch synthesis parallels the endosperm development in maize. we obtained transcriptome, small RNAome and DNA methylome data from the multiple developing stages of endosperms, as well as kernel and leaf, via applying RNA-seq, sRNA-seq and BS-seq technologies. We demonstrated that genome-wide gene expression makes a sharp transition at the point when starch accumulation speeds between DAP 10-13, while the enriched up-regulation of starch and sucrose metabolism genes occurs earlier at DAP 9-10. Expression pattern analysis establishes a comprehensive network between starch synthesis genes and transcription factors. MiRNAs negatively regulating TF expression were extensively downregulated during maize endosperm development. We showed that TF ZmMYB33 and ZmMYB65 promotes the transcription of the GUS reporter under the control of Wx and Sbe2b/Bt2 gene promoters containing their binding motifs, respectively. We further showed that the abundantly expressed miR159k-3p negatively regulate the expression of these two transcription factors, suggesting that miRNAs are important regulators of starch synthesis. In addition, we showed that both starch synthesis genes and miRNAs are globally repressed by DNA methylation, while transcription factors are not. Taken together, the presented results establish the regulatory functions of miRNAs and DNA methylation in starch synthesis, and indicates DNA methylation as the master switch.
Project description:Starch synthesis is an essential feature for crops, but its regulatory mechanism has been largely restricted to transcription factors. Starch synthesis parallels the endosperm development in maize. we obtained transcriptome, small RNAome and DNA methylome data from the multiple developing stages of endosperms, as well as kernel and leaf, via applying RNA-seq, sRNA-seq and BS-seq technologies. We demonstrated that genome-wide gene expression makes a sharp transition at the point when starch accumulation speeds between DAP 10-13, while the enriched up-regulation of starch and sucrose metabolism genes occurs earlier at DAP 9-10. Expression pattern analysis establishes a comprehensive network between starch synthesis genes and transcription factors. MiRNAs negatively regulating TF expression were extensively downregulated during maize endosperm development. We showed that TF ZmMYB33 and ZmMYB65 promotes the transcription of the GUS reporter under the control of Wx and Sbe2b/Bt2 gene promoters containing their binding motifs, respectively. We further showed that the abundantly expressed miR159k-3p negatively regulate the expression of these two transcription factors, suggesting that miRNAs are important regulators of starch synthesis. In addition, we showed that both starch synthesis genes and miRNAs are globally repressed by DNA methylation, while transcription factors are not. Taken together, the presented results establish the regulatory functions of miRNAs and DNA methylation in starch synthesis, and indicates DNA methylation as the master switch.
Project description:Starch synthesis is an essential feature for crops, but its regulatory mechanism has been largely restricted to transcription factors. Starch synthesis parallels the endosperm development in maize. we obtained transcriptome, small RNAome and DNA methylome data from the multiple developing stages of endosperms, as well as kernel and leaf, via applying RNA-seq, sRNA-seq and BS-seq technologies. We demonstrated that genome-wide gene expression makes a sharp transition at the point when starch accumulation speeds between DAP 10-13, while the enriched up-regulation of starch and sucrose metabolism genes occurs earlier at DAP 9-10. Expression pattern analysis establishes a comprehensive network between starch synthesis genes and transcription factors. MiRNAs negatively regulating TF expression were extensively downregulated during maize endosperm development. We showed that TF ZmMYB33 and ZmMYB65 promotes the transcription of the GUS reporter under the control of Wx and Sbe2b/Bt2 gene promoters containing their binding motifs, respectively. We further showed that the abundantly expressed miR159k-3p negatively regulate the expression of these two transcription factors, suggesting that miRNAs are important regulators of starch synthesis. In addition, we showed that both starch synthesis genes and miRNAs are globally repressed by DNA methylation, while transcription factors are not. Taken together, the presented results establish the regulatory functions of miRNAs and DNA methylation in starch synthesis, and indicates DNA methylation as the master switch.
Project description:Waxy starch has an important influence on bread dough and the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. The waxy and non-waxy wheats had similar starch granule morphological features and developmental patterns, and similar amylopectin quality in the grain. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Dynamic transcriptional expression profiling of starch biosynthesis-related genes indicated similar transcriptional expression profiles in both cultivars. Our results revealed that phosphorylation modifications played critical roles in amylose and amylopectin biosynthesis, but the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.
Project description:Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.