## 2 Results and Discussion
### MapMan Analysis of Koshihikari Rice Seed Differentially Expressed Genes in Grades 2 and 3  
In order to visualize the DEGs that are involved in key pathways, a total of 42,560 Rice Japonica genes were plotted against the pathway maps of the Mapman tool. Among all the genes, DEGs with high fold changes were selected for the analysis, as discussed in methods for Mapman analysis. From the fold change cut-off threshold, a total of 161 and 490 highly regulated differentially expressed (HRDE) genes for each Y2 and Y3 were identified. Among these, 92 genes are common for both grades. A total of 68 and 398 genes are unique for Y2 and Y3, respectively resulting in a total of 560 HRDEs for both. An overview of these HRDEs in Y1-Y2 and Y1-Y3, and the related 36 pathway bins, are shown in (Figure 3A,B). Results clearly indicate that the highest fraction of the gene regulation occurred in 16, 17, 20, 26, 27, 28, 29, 30, 33, and 34 Mapman bins. The number of genes associated with each bin, specific to each pathway, is listed in supplementary Table S1. These bins are related to secondary metabolism, hormone metabolism, stress response, miscellaneous enzyme families, RNA, DNA, protein, signaling, development, and transport. In addition, bin 35 has the most differentially expressed genes; however, these genes are without any functional annotation. Figure 3: Mapman Bins for functional categorization of high temperature-responsive genes in (**A**) Y2: grade 2 Koshihikari rice seed and (**B**) Y3: grade 3 Koshihikari rice seed. Non-redundant 640 genes which expression are changed over 2-fold in the seeds of both grades 2 (Y2) and 3 (Y3) were functionally categorized into MapMan bins as described in Materials and Methods. The heat map with grid boxes shows the genes (blue, upregulated; red, downregulated) in each BIN for each grade seeds. Number of genes associated to each bin is listed in supplementary Table S1. The 36 BINS abbreviations: PS, photosynthesis; maCHO, major carbohydrate metabolism; miCHO, minor carbohydrate metabolism;G, glycolysis; FM, fermentation; GL/GC, gluconeogenesis/glyoxalate cycle; OPP, oxidative pentose phosphate; TCA/OT, tricarboxylic acid/organic acid transformations; MET/ATPs, mitochondrial electron transport/adenosine triphosphate; CW, cell wall; L, lipid metabolism; N-, nitrogen metabolism; AA, amino acid metabolism; S-A, sulfur assimilation; MH, metal handling; S, secondary metabolism; H, hormone metabolism; Co-F/V, co-factor and vitamin metabolism; TS, tetrapyrrole synthesis; ST, stress; RR, redox regulation; P, polyamine metabolism; N, nucleotide metabolism; BioDX, biodegradation of xenobiotics; C1, C1-metabolism; MISC, miscellaneous; RNA, ribonucleacid; DNA, deoxyribonucleic acid; PR, protein; SIG, signaling; C, cell; mRNA, messenger RNA; D, development; T, transport; NA, not assigned; MN, mineral nutrition. The overall expression profile indicates that stress response genes (bin 20) are strongly regulated in both Y2 and Y3. Y3 exhibited a larger number of strongly upregulated genes in comparison to Y2. Further, the differential gene expression patterns of Y2 and Y3 in three major processes, namely cell function, metabolism, and abiotic-biotic stress (Figures 4-6) were explored. Figure 4: A cell function map of highly regulated differentially expressed (HRDE) genes that are categorized based on various functions, generated using Mapman. (**A**) Map for HRDE genes related to Koshihikari rice grade Y2. (**B**) Map for HRDE genes related to Koshihikari rice grade Y3. Blue and red colored data points indicated highly up or downregulated genes. Numbering for each data point on the figure was given in left to right order to identify relevant gene information from supplementary Table S2. These numbers represent serial numbers for the genes in the supplementary table accordingly. Figure 5: A metabolism overview map of highly regulated differentially expressed (HRDE) genes that are categorized into various metabolic pathways generated using Mapman. (**A**) Map for HRDE genes related to Koshihikari rice grade Y2.