**7. Dissecting out the effect of temperature stress on seed filling at the molecular level using expression profiling**

Plants are sessile organisms and show ideal growth and development only when they are grown under optimal conditions. Temperature is one of the most important environmental factors which has a great influence on the growth and development of various plant species. It has got much attention because of the recent global warming. Rice grain weight and yield have been shown to be greatly reduced by the environmental temperature. Grain filling process is more affected by the increase in night temperature than by the day temperature, particularly. High temperature reduces grain yield by impairing the filling process. Transcriptome profiling has greatly helped in revealing the underlying mechanism of yield loss associated with high temperature stress.

An increase in the temperature affects the grain development process right from the opening of the glumes till the grain filling stage. At high temperature, the glumes remain unclosed which affects the germination rate as well as commercial quality of the seeds. Genes involved in cell wall metabolism and response to water and carbohydrate metabolism are up regulated following high temperatures stress [64]. A large number of genes show differential expression in heat sensitive and tolerant cultivars in the early milky stage of the rice seed. These genes are majorly involved in oxidation-reduction, metabolic, transport, transcript regulation, defense response and photosynthetic processes. It has been shown that high temperature disrupts the mitochondrial electron transport system. This further induces a higher concentration of hydrogen ions in the matrix, which affects the functional state of different enzymes involved in TCA cycle and other metabolic pathways [65]. Further, the elevated temperature has been shown to negatively affect the grain filling process. Impairment in grain filling has been shown to result from the shortage of storage material. RNA profiling shows the down regulation of different genes involved in sucrose import and starch biosynthesis. Concomitantly, many of the genes involved in starch degradation show up regulation in the heat sensitive variety. Further, high temperature also results in the inhibition of the respiratory chain. This leads to the inefficient production of energy/ATP, eventually leading to the poor filling in rice grains [66]. An elevation in temperature can negatively affect the development of rice grains in different ways as non-closure of glumes or poor grain filling. Sequence profiling show the down regulation of different genes involved in carbohydrate synthesis, sucrose transport and ETC genes, altogether resulting in poor energy production in the system. Thus, profiling studies have not only helped in understanding the mechanisms associated with poor grain filling and elevated temperature but also serve as a promising tool for the identification of candidates for improving the heat tolerant character in rice.

### **8. Summary and concluding remarks**

Transcriptome studies have gained momentum in the last decade by the advancements in sequencing techniques, bioinformatics tools and functional genomics. This has strengthened our knowledge of the seed development program by boosting data generation and interpretation. The outlook from these high-throughput studies is predominantly determined by the tissues under investigation. Seed development process can be studied in entirety through expression atlases. They provide information about the dynamics and specificity of transcriptomes across a range of tissues/organs associated with the growth and development of rice seed. Larger proportion of endosperm-specific genes obtained in comparative studies between vegetative and seed stages indicate the amount of reprogramming happening at the molecular level. The extent of involvement of various tissues in seed development as well as their molecular relatedness can also be assessed by means of transcript profiling of tissues in groups. This helps in visualizing the molecular changes occurring during progression of development in rice seed through subsequent stages by highlighting the factors that undergo change or remain constant. Several TFs and hormone-related genes show specific or preferential expression in seed tissues indicating their active involvement with the process. Molecular interactions between tissues, such as cross talk between embryo and endosperm, can also be revealed by transcriptome atlases. However, for obtaining detailed information about the developmental changes related to a particular tissue, it will be beneficial to study that tissue in isolation, as it will be cost effective and less time consuming. Transcriptome studies of rice embryo and endosperm have identified the genes and pathways that control various phases of their development. Several MTases, including MET1, genes associated with DNA/chromatin remodeling and homeobox TFs are up regulated in the initial stages of embryo development indicating induction of genes associated with zygotic development and organ formation. Polarized expression of various TFs, TRs and phytohormones, including auxin, GA and cytokinin, suggests their role in establishing apical-basal polarity in the embryo. In the early and middle stages of embryo development, pathways related to amino acid metabolism, lipid and energy metabolism, nucleic acid replication/processing and signal transduction are up regulated, while in the later stages, pathways related to starch biosynthesis and protein modification are up regulated. Also, up regulation of ABA biosynthesis and down regulation of ethylene biosynthesis in later stages suggests their antagonistic role in embryo maturation. Endosperm development starts little later than embryo in seed development. Higher expression of cell cycle related genes in the initial stages suggest that early endosperm development is mostly concerned with cell division and expansion. Variable expression pattern of TFs, such as MADS, AP2-EREBP, NACs and CCAAT, throughout different stages of endosperm development emphasizes their specific roles throughout the process. Up regulation of PCD related genes, starch and storage protein synthesis genes and carbohydrate and energy metabolism genes in the middle and later stages suggests that these stages of endosperm development are active in accumulation of storage reserves and programmed cell death.

Expression profiling studies performed till now have provided information about important genes and pathways, interrelatedness of various processes and cross talks between the key players. Nevertheless, obtaining a fully functional knowledge of this complex development process would require the enormous task of consolidation of the data generated by various studies in a single platform uniting all the pathways and regulatory networks controlling different aspects of seed development and the functional validation of these genes.
