## 2 Results and Discussion
### High Temperature-Triggered Regulatory Events in Koshiiukari Rice Seeds of Grades 2 and 3
#### 2.4.4 Secondary Metabolites  
Secondary metabolites are produced by plants through several metabolic pathways to support their survival in the environment. Some of the most commonly studied secondary metabolites are alkaloids, flavonoids, and terpenoids. Most plants regulate synthesis of these compounds for their survival based on the environmental conditions. Among most environmental factors, temperature is known to influence plant secondary metabolite production significantly [69]. In the current study, upregulation of some genes related to ent-kaurene synthase, anthocyanins, flavonoid biosynthesis, lignin biosynthesis, and lacases was observed. Three genes related to ent-kaurene synthase were found to be strongly upregulated in Y3 (Os04909900.1, Os02936140, and Os02936210), while only one gene (Os01914630) was mildly upregulated in Y2. These enzymes are known for their role in gibberell phytohormone biosynthesis and serve as intermediates in specific di-terpenoid metabolism [70]. Additionally, specific genes belonging to the anthocyanins and flavonoid biosynthesis pathway in Y3 were found to be upregulated. Some of these are chalcone synthase (Os11g32650.1), leucoanthocyanidin reductase (Os06g44170.1) and dihydroflavonol-4-reductase (DFR) (Os03g08624.1). On the contrary, in Y2, these genes were not found. However, the isoflavvon reductase related gene with moderate expression was found to be downregulated in both Y2 (Os01g01660.1) and Y3 (Os02g56460.1). This class of flavonoids was identified to exhibit antioxidative functions in plants like tomato [71]. These are also responsible for fruit and flower color in several plants. In _Arabidopsis_, the mutants with DFR deficient genes resulted in decreased levels of proanthocyanidin brown tannins on their seed coats [72; 73]. Another important metabolite that is very essential for plants is lignin. It provides structural support for the plants to grow and also protect the plant from pathogens. Its accumulation could be considered as a repair mechanism to prevent cell wall damage during heat stress. Genes related (Os04g01470.1, Os04g09680.1, and Os11g42200.1) to lignin biosynthesis were also found to be upregulated in both Y2 and Y3. Os04g01470.1 and Os04g09680.1 are the caffe acid 3-O-methyltransferase transferase (COMT) genes. These are involved in converting caffe acid to ferulic acid and 5-hydroxyferulic acid to sinapic acid [74]. Both the genes were observed to be upregulated in Y3, while the former was only observed in Y2. In addition, upregulation of lacases (Os11g42200.1) was observed in both Y2 and Y3. Higher lactose activity indicates induced catalysis of oxidative coupling between phenylpropane units to form lignin [75]. Heat stress in plants could also lead to osmotic stress due to disruption of osmotic hemostasis. In order to prevent osmotic stress plants produce carotenoids [74]. Here in Y2, a gene (Os01g14630.1) geranyl diphosphate synthase (GPPs) related to osmotic stress and carotenoid synthesis was strongly downregulated compared to Y3. Upregulation of GGPS during stress conditions leading to enhanced osmotic stress tolerance was found in _Arabidopsis thaliana_[76; 77]. The current result is quite opposite to previous reports. It could be due to abundance of GPPs in leaves compared to seed and differential expression of specific genes in specific tissues.