**6. Expression profiling unravels the complex molecular machinery involved in grain filling**

Quality of rice grains, the major human calorie provider is very significant in the present scenario of ensuring global food security. Quality and quantity of grain production is majorly dependent on the synthesis and storage of various macromolecules and minerals during the grain filling stage. In rice, grain filling happens in the endosperm tissue and is regulated by highly coordinated and synchronous pathways [4]. Endosperm acts as the nutrient reservoir for the developing embryo initially and to the germinating embryo over the course of time. Endosperm functions in the supply of nutrients to the growing embryo right from its syncytial state. Growth and expansion of the endosperm cells are limited by programmed cell death in mature seeds. Thus, the accumulation of storage reserve is dependent on the life span of endosperm cells [56]. Understanding the intricate machineries involved in grain filling is imperative in the identification and manipulation of the key regulatory pathways aimed at improving the quality and productivity of the crop varieties available. Expression analyses serve as a promising tool facilitating the identification of candidate genes regulating grain filling process in rice.

Major reserves accumulating in seeds include carbohydrates, storage proteins and lipid compounds. Biosynthesis of these storage macromolecules are coordinately controlled by different TFs and other TRs. Expression profiling has shown the co-expression of different TF genes including *bZIP*, *Dof* and *MYB* with many grain filling genes in rice [57]. Members belonging to these protein families have shown to play significant roles in the regulation of storage protein and starch biosynthesis [57–59]. Additionally, genes involved in the biosynthesis of macromolecules, various transporters for amino acid, sugar, phosphate, peptide, nitrate and ABC transporters show enrichment in the grain filling stage [57]. Transporter genes are essential for the uptake of nutrient and precursor molecules from the source tissues. Expression profiling also gives information regarding the genes involved in specific pathways. Furthermore, a detailed analysis can be useful in the identification of *cis*-elements enriched in specific process. Many of the grain filling genes contain a conserved *cis*-element 'AACA' in their promoters, suggesting its importance in the process. AACA element is essential for conferring the expression in rice seed [57, 60]. Transcript profiling has also shown that the milling yield and eating quality of rice grains depends on the proportion of starch and proteins in the grain. High quality rice grains contain a high composition of starch and protein biosynthetic transcripts. Massively parallel signature sequencing (MPSS) and sequencing by synthesis (SBS) have shown a higher level of alternative splicing and antisense transcripts for different metabolic genes in the high milling yield and eating quality varieties. These transcripts belong to starch, aspartate amino acid, storage protein and allergenic protein metabolism genes, indicating the complex transcriptional cascade involved in the regulation of rice grain quality [61]. Thus, expression profiling has not only improved our understanding in the grain filling process but also identified different transcripts essential for the process. Many of these genes can also serve as potential markers for the identification of superior rice varieties.

Grain chalkiness is another important agronomic trait influencing the market value of rice. It negatively affects the consumer preference and culinary quality. Analyses have shown the differential expression of a large repertoire of genes involved in signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis in a high chalky rice variety. Out of the different metabolic genes, starch metabolism genes can be considered as the major reason for grain chalkiness because of their opposite expression pattern in varieties showing varying levels of chalkiness. The sucrose and starch biosynthetic genes show up regulation in the chalky variety. Moreover, the nonstarchy polysaccharide transcripts show significant down regulation. Thus, the expression profiling suggests a positive correlation of the starchy polysaccharide transcripts with the chalky phenotype in rice grains. Additionally, the genes involved in oxido-reductive homeostasis also show significant up regulation in the chalky rice variety [62]. Thus, transcript profiling has the potential for the identification of candidate genes underlying a phenotype, including grain chalkiness.

A delay in the expression of various genes involved in the transformation of sucrose to starch has been identified as the major reason for poor grain filling in the inferior spikelets located on the lower secondary panicle branches. RNA-Seq analysis shows the lower expression of these genes in the inferior spikelets at an early stage of grain filling in comparison with the superior spikelets. However, it was reversed during the later stages of grain filling process. Low capacity of the sink tissue and the associated limited carbohydrate supply at the later stages of grain filling has been proposed as the probable reason for the poor filling of grains [63]. Thus, profiling of seed transcripts has greatly deepened our understanding of the molecular machinery involved in seed filling and panicle branching. This has served to identify the cascade of TRs involved in the process. It will definitely pave way for the identification of candidate genes and their introgression for the production of improved variety with better consumer preference.

storage protein and starch biosynthesis [57–59]. Additionally, genes involved in the biosynthesis of macromolecules, various transporters for amino acid, sugar, phosphate, peptide, nitrate and ABC transporters show enrichment in the grain filling stage [57]. Transporter genes are essential for the uptake of nutrient and precursor molecules from the source tissues. Expression profiling also gives information regarding the genes involved in specific pathways. Furthermore, a detailed analysis can be useful in the identification of *cis*-elements enriched in specific process. Many of the grain filling genes contain a conserved *cis*-element 'AACA' in their promoters, suggesting its importance in the process. AACA element is essential for conferring the expression in rice seed [57, 60]. Transcript profiling has also shown that the milling yield and eating quality of rice grains depends on the proportion of starch and proteins in the grain. High quality rice grains contain a high composition of starch and protein biosynthetic transcripts. Massively parallel signature sequencing (MPSS) and sequencing by synthesis (SBS) have shown a higher level of alternative splicing and antisense transcripts for different metabolic genes in the high milling yield and eating quality varieties. These transcripts belong to starch, aspartate amino acid, storage protein and allergenic protein metabolism genes, indicating the complex transcriptional cascade involved in the regulation of rice grain quality [61]. Thus, expression profiling has not only improved our understanding in the grain filling process but also identified different transcripts essential for the process. Many of these genes can also serve as potential markers for

Grain chalkiness is another important agronomic trait influencing the market value of rice. It negatively affects the consumer preference and culinary quality. Analyses have shown the differential expression of a large repertoire of genes involved in signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis in a high chalky rice variety. Out of the different metabolic genes, starch metabolism genes can be considered as the major reason for grain chalkiness because of their opposite expression pattern in varieties showing varying levels of chalkiness. The sucrose and starch biosynthetic genes show up regulation in the chalky variety. Moreover, the nonstarchy polysaccharide transcripts show significant down regulation. Thus, the expression profiling suggests a positive correlation of the starchy polysaccharide transcripts with the chalky phenotype in rice grains. Additionally, the genes involved in oxido-reductive homeostasis also show significant up regulation in the chalky rice variety [62]. Thus, transcript profiling has the potential for the identification of candidate genes underlying a phenotype,

A delay in the expression of various genes involved in the transformation of sucrose to starch has been identified as the major reason for poor grain filling in the inferior spikelets located on the lower secondary panicle branches. RNA-Seq analysis shows the lower expression of these genes in the inferior spikelets at an early stage of grain filling in comparison with the superior spikelets. However, it was reversed during the later stages of grain filling process. Low capacity of the sink tissue and the associated limited carbohydrate supply at the later stages of grain filling has been proposed as the probable reason for the poor filling of grains [63]. Thus,

the identification of superior rice varieties.

36 Advances in Seed Biology

including grain chalkiness.
