Flavonoids are one of the most important secondary metabolites in plants, and phenylalanine ammonia-lyase (PAL) is the first rate-limiting enzyme for their biosynthesis. However, detailed information on the regulation of PAL in plants is still little. In this study, PAL in E. ferox was identified and functionally analyzed, and its upstream regulatory network was investigated. Through genome-wide identification, we obtained 12 putative PAL genes from E. ferox. Phylogenetic tree and synteny analysis revealed that PAL in E. ferox was expanded and mostly preserved. Subsequently, enzyme activity assays demonstrated that EfPAL1 and EfPAL2 both catalyzed the production of cinnamic acid from phenylalanine only, with EfPAL2 exhibiting a superior enzyme activity. Overexpression of EfPAL1 and EfPAL2 in Arabidopsis thaliana, respectively, both enhanced the biosynthesis of flavonoids. Furthermore, two transcription factors, EfZAT11 and EfHY5, were identified by yeast one-hybrid library assays as binding to the promoter of EfPAL2, and further luciferase (LUC) activity analysis indicated that EfZAT11 promoted the expression of EfPAL2, while EfHY5 repressed the expression of EfPAL2. These results suggested that EfZAT11 and EfHY5 positively and negatively regulate flavonoid biosynthesis, respectively. Subcellular localization revealed that EfZAT11 and EfHY5 were localized in the nucleus. Our findings clarified the key EfPAL1 and EfPAL2 of flavonoid biosynthesis in E. ferox and established the upstream regulatory network of EfPAL2, which would provide novel information for the study of flavonoid biosynthesis mechanism.

Nelumbo nucifera Gaertn. is an important perennial aquatic herb that has high ornamental, edible, medicinal, and economic value, being widely distributed and used in China. The NAC superfamily (NAM, ATAF1/2, CUC2) plays critical roles in plant growth, development, and response to abiotic and biotic stresses. Though there have been a few reports about NAC genes in lotus, systematic analysis is still relatively lacking. The present study aimed to characterize all the NAC genes in the lotus and obtain better insights on the NnNACs in response to salt stress by depending on ABA signaling. Here, 97 NAC genes were identified by searching the whole lotus genome based on the raw HMM models of the conserved NAM domain and NAC domain. They were characterized by bioinformatics analysis and divided into 18 subgroups based on the phylogenetic tree. Cis-element analysis demonstrated that NAC genes are responsive to biotic and abiotic stresses, light, low temperature, and plant hormones. Meanwhile, NAC genes had tissue expression specificity. qRT-PCR analysis indicated that NAC genes could be upregulated or downregulated by NaCl treatment, ABA, and fluoridone. In addition, NAC016, NAC025, and NAC070, whose encoding genes were significantly induced by NaCl and ABA, were located in the nucleus. Further analysis showed the three NAC proteins had transcriptional activation capabilities. The co-expression network analysis reflected that NAC proteins may form complexes with other proteins to play a role together. Our study provides a theoretical basis for further research to be conducted on the regulatory mechanisms of salinity resistance in the lotus.

Optimal nitrogen (N) supply significantly increases the starch content, components, and yield of Nelumbo nucifera. However, the underlying transcriptional mechanism and starch accumulation under dose-dependent nitrogen fertilizer are poorly understood. In this study, we found that the optimal nitrogen fertilizer (N2, 30 kg/667 m²) was more beneficial to improve the stomatal conductance (Gs), leaf intercellular CO₂ concentration (Ci), transpiration rate (Tr), net photosynthetic rates (Pn), chlorophyll content, starch content, and plot yield. What is more, N2-fertilizer treatment induced a higher number of starch granule, AP2 content, and RVA curve peaks. Then, the transcriptomic analyses performed in control (CK) and N2-fertilizer treatment (N2) showed that the expressions of many differentially expressed genes (DEGs) were significantly induced by N2. KEGG and GO enrichment analysis showed that these DEGs were significantly enriched in biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, carbon metabolism, carbon fixation in photosynthetic organisms, plant hormone signal transduction, and starch and sucrose metabolisms, suggesting that nitrogen fertilizer induced alterations of photosynthesis- and starch accumulation-related gene expression profiles. Finally, six photosynthesis-related genes and fourteen starch synthesis-related genes were confirmed to be required for starch accumulation in the Nelumbo nucifera development. qPCR analysis of six starch accumulation-related genes demonstrated the accuracy of the transcriptome. Hence, our study provides valuable resource for future studies on molecular mechanisms underlying starch accumulation in Nelumbo nucifera rhizome under N-fertilizer treatment.

Starch is the final product of photosynthesis and the main storage form in plants. Studies have shown that there is a close synergistic regulatory relationship between ABA signal transduction and starch biosynthesis. In this study, we employed RNA sequencing (RNA-Seq) to investigate transcriptomic changes of the Euryale ferox seeds treated by exogenous ABA. The differentially expressed genes engaged in the “Starch and sucrose” and “TCA cycle” pathway. Furthermore, the key transcription factor EfABI4 in ABA signaling pathway and the key genes of starch biosynthesis (EfDBE1, EfSBE2, EfSS1, EfSS2, EfSS3, EfSS4 and EfGBSS1) were significantly up-regulated. Further, the Euryale ferox plant was treated with ABA, it was found that the total starch content of Euryale ferox seeds at different development stages was significantly higher than that of the control, and the key genes of starch synthesis in Euryale ferox seeds were also significantly up-regulated. Finally, yeast one-hybrid and dual luciferase assay proved that EfABI4 can promote the expression of EfSS1 by directly binding to its promoter. Subcellular localization results showed that EfABI4 protein was located at the nucleus and EfSS1 protein was located in the cytomembrane. These findings revealed that ABA promotes starch synthesis and accumulation by mediating EfABI4 to directly promote EfSS1 gene expression, which is helpful for understanding starch synthesis in seeds.

Slow-release fertilizer is an environmentally friendly fertilizer that is widely used in crop cultivation instead of traditional nitrogen fertilizer. However, the optimal application time of slow-release fertilizer and its effect on starch accumulation and rhizome quality of lotus remains unclear. In this study, two slow-release fertilizer applications (sulfur-coated compound fertilizer, SCU, and resin-coated urea, RCU) were fertilized under three fertilization periods (the erect leaf stage, SCU1 and RCU1; the erect leaf completely covering the water stage, SCU2 and RCU2; and the swelling stage of lotus rhizomes, SCU3 and RCU3) to study the effects of different application periods. Compared with CK (0 kg∙ha⁻¹ nitrogen fertilizer), leaf relative chlorophyll content (SPAD) and net photosynthetic rate (Pn) remained at higher levels under SCU1 and RCU1. Further studies showed that SCU1 and RCU1 increased yield, amylose content, amylopectin and total starch, and the number of starch particles in lotus, and also significantly reduced peak viscosity, final viscosity and setback viscosity of lotus rhizome starch. To account for these changes, we measured the activity of key enzymes in starch synthesis and the relative expression levels of related genes. Through analysis, we found that these parameters increased significantly under SCU and RCU treatment, especially under SCU1 and RCU1 treatment. The results of this study showed that the one-time application at the erect leaf stage (SCU1 and RCU1) could improve the physicochemical properties of starch by regulating the key enzymes and related genes of starch synthesis, thus improving the nutritional quality of lotus rhizome. These results provide a technical choice for the one-time application of slow-release fertilizer in lotus rhizome production and cultivation.