As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis and photoprotectors for plants to adapt to high light stress. Here, we report a small subunit (SSU) of geranylgeranyl diphosphate synthase (GGPPS) in Nicotiana tabacum, NtSSU II, which takes part in the regulation carotenoid biosynthesis by forming multiple enzymatic components with NtGGPPS1 and downstream phytoene synthase (NtPSY1). NtSSU II transcript is widely distributed in various tissues and stimulated by low light and high light treatments. The confocal image revealed that NtSSU II was localized in the chloroplast. Bimolecular fluorescence complementation (BiFC) indicated that NtSSU II and NtGGPPS1 formed heterodimers, which were able to interact with phytoene synthase (NtPSY1) to channel GGPP into the carotenoid production. CRISPR/Cas9-induced ntssu II mutant exhibited decreased leaf area and biomass, along with a decline in carotenoid and chlorophyll accumulation. Moreover, the genes involved in carotenoid biosynthesis were also downregulated in transgenic plants of ntssu II mutant. Taken together, the newly identified NtSSU II could form multiple enzymatic components with NtGGPPS1 and NtPSY1 to regulate carotenoid biosynthesis in N. tabacum, in addition to the co-expression of genes in carotenoids biosynthetic pathways.

As the largest group of structurally diverse metabolites, terpenoids are versatile natural compounds that act as metabolism mediators, plant volatiles, and ecological communicators. However, few terpenoid compounds have been identified in plant parts of sacred lotus (Nelumbo nucifera Gaertn.). To elucidate the molecular genetic basis of the terpene biosynthetic pathway, terpenes from different parts of the plant, including seeds (S), young leaves (YL), mature leaves (ML), white flowers (WF), yellow flowers (YF), and red flowers (RF), were identified by LC-MS/MS and the relative contents of the same terpenes in different parts were compared. The results indicate that all plant parts primarily consist of triterpenes, with only minor quantities of sesquiterpenes and diterpenes, and there were differences in the terpene content detected in different plant parts. To illustrate the biosynthesis of various terpenoids, RNA sequencing was performed to profile the transcriptomes of various plant parts, which generated a total of 126.95 GB clean data and assembled into 29,630 unigenes. Among these unigenes, 105 candidate unigenes are involved in the mevalonate (MVA) pathway, methyl-erythritol phosphate (MEP) pathway, terpenoid backbone biosynthesis pathway, and terpenoid synthases pathway. Moreover, the co-expression network between terpene synthase (TPS) and WRKY transcription factors provides new information for the terpene biosynthesis pathway.

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis as well as photoprotectors for plants to adapt to high light stress. Phytoene synthase (PSY) is the entry enzyme and also the major rate-limiting enzyme in the carotenoid pathway. Here, we report a dehydration-responsive element-binding protein (DREB) transcription factor member in Nicotiana tabacum K326, NtDREB-1BL1, which regulates carotenoids biosynthesis by binding to the NtPSY promoter. The NtDREB-1BL1 transcript was widely distributed in leaves by Real-time PCR. Confocal image revealed that NtDREB-1BL1 was localized in the nucleus. The chromatin immunoprecipitation (ChIP) with the qPCR technique indicated that NtDREB-1BL1 could anchor the promoter region of NtPSY. Overexpression (NtDREB-1BL1 OE) and RNA interference (NtDREB-1BL1 RNAi) of NtDREB-1BL1 were performed to evaluate its biological function in N. tabacum. Both carotenoid and chlorophyll contents increased in transgenic plants of NtDREB-1BL1 OE compared with wild-type (WT) plants, with the augment of the genes involved in carotenoid biosynthesis. In contrast, the contents of carotenoid and chlorophyll significantly decreased in transgenic plants of NtDREB-1BL1 RNAi compared to WT, along with the decline in the expression of genes related to carotenoid biosynthesis. Moreover, transgenic plants of NtDREB-1BL1 OE exhibited enhanced tolerance under drought stress, with the weakened tolerance of drought stress in transgenic plants of NtDREB-1BL1 RNAi. In conclusion, our results illustrated the new role of transcription factor NtDREB-1BL1 in improving carotenoid biosynthesis through regulating NtPSY expression.

The normal growth of Nelumbo nucifera, a widely planted aquatic crop in Asia, was severely ruined by replant disease. The mechanism of replant disease was still unknown in aquatic crops. Complementary transcriptomic and proteomic analyses were performed by comparing seedings of first-year planting (FP) and consecutive planting (CP). 9810 differentially expressed genes (DEGs) were identified between FP and CP. Additionally, 975 differentially expressed proteins (DEPs) were obtained. The correlation of proteome and transcriptome illustrated phenylpropanoid biosynthesis, flavonoid biosynthesis, metabolic pathways, and MAPK signaling pathways were significantly activated. Peroxidase, determined as one of the key proteins in replant disease of N. nucifera, was phylogenetically analyzed. A new depiction of the molecular mechanism causing replant disease in N. nucifera was illustrated. A consecutive monoculture stimulated the generation of reactive oxygen species (ROS) and ethylene, altered the metabolic balance of lignin and flavonoid, and attenuated the activity of antioxidant enzymes through DNA methylation. Therefore, the accumulation of autotoxic allelochemicals and the deficiency of antioxidant enzymes unavoidably suppressed the normal growth and development of replanted N. nucifera.