Time-course analysis and transcriptomic identification of key response strategies to complete submergence in Nelumbo nucifera.

Water submergence is an environmental stress with detrimental effects on plant growth and survival. As a wetland plant species, lotus (Nelumbo nucifera) is widely cultivated in flood-prone lowlands throughout Asian countries, but little is known about its endurance and acclimation mechanisms to complete submergence. Here, we combined a time-course submergence experiment and an RNA-sequencing transcriptome analysis on two lotus varieties of "Qiuxing" and "China Antique". Both varieties showed a low submergence tolerance, with a median lethal time of around 10 days. Differentially expressed gene (DEG) analysis and weighted gene co-expression network analysis (WGCNA) identified a number of key genes putatively involved in lotus submergence responses. Lotus plants under complete submergence developed thinned leaves and elongated petioles containing high density of aerenchyma. All four lotus submergence responsive ERF-VII genes and gene sets corresponding to the low oxygen "escape" strategy (LOES) were elevated. In addition, a number of lotus innate immunity genes were rapidly induced by submergence, likely to confer resistance to possible pathogen infections. Our data also reveals the likely involvement of jasmonic acid in modulating lotus submergence responses, but to a lesser extent than the gaseous ethylene hormone. These results suggest that lotus plants primarily take the LOES strategy in coping with submergence-induced complex stresses, and will be valuable for people understanding the molecular basis underlying the plant submergence acclimations.

Comprehensive analysis of AGPase genes uncovers their potential roles in starch biosynthesis in lotus seed.

BACKGROUND: Starch in the lotus seed contains a high proportion of amylose, which endows lotus seed a promising property in the development of hypoglycemic and low-glycemic index functional food. Currently, improving starch content is one of the major goals for seed-lotus breeding. ADP-glucose pyrophosphorylase (AGPase) plays an essential role in regulating starch biosynthesis in plants, but little is known about its characterization in lotus. RESULTS: We describe the nutritional compositions of lotus seed among 30 varieties with starch as a major component. Comparative transcriptome analysis showed that AGPase genes were differentially expressed in two varieties (CA and JX) with significant different starch content. Seven putative AGPase genes were identified in the lotus genome (Nelumbo nucifera Gaertn.), which could be grouped into two subfamilies. Selective pressure analysis indicated that purifying selection acted as a vital force in the evolution of AGPase genes. Expression analysis revealed that lotus AGPase genes have varying expression patterns, with NnAGPL2a and NnAGPS1a as the most predominantly expressed, especially in seed and rhizome. NnAGPL2a and NnAGPS1a were co-expressed with a number of starch and sucrose metabolism pathway related genes, and their expressions were accompanied by increased AGPase activity and starch content in lotus seed. CONCLUSIONS: Seven AGPase genes were characterized in lotus, with NnAGPL2a and NnAGPS1a, as the key genes involved in starch biosynthesis in lotus seed. These results considerably extend our understanding on lotus AGPase genes and provide theoretical basis for breeding new lotus varieties with high-starch content.

The Establishment of an Efficient Callus Induction System for Lotus (Nelumbo nucifera).

The lotus (Nelumbo nucifera) is one of the most popular aquatic plants in Asia, and has emerged as a novel model for studying flower and rhizome development, and primary and secondary metabolite accumulation. Here, we developed a highly efficient callus induction system for the lotus by optimizing a series of key factors that affect callus formation. The highest efficient callus production was induced on immature cotyledon and embryo explants grown on Murashige and Skoog (MS) basal medium containing an optimized combination of 3 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L 6-benzylaminopurine (6-BA). In addition, lotus callus induction was proven to be influenced by lotus genotypes, light conditions, the developmental stages of explants and the time of explant sampling. Collecting immature cotyledons from seeds of the genotype "Shilihe 1", at 9 days post pollination, and to culture the explants in darkness, are proposed as the optimum conditions for lotus callus induction. Interestingly, highly efficient callus induction was also observed in explants of immature embryo derived aseptic seedlings; and a small amount of lotus benzylisoquinoline alkaloid (BIA) and obvious expression of BIA biosynthetic genes were detected in lotus callus.

Investigation of benzylisoquinoline alkaloid biosynthetic pathway and its transcriptional regulation in lotus.

Lotus predominantly accumulates benzylisoquinoline alkaloids (BIAs), but their biosynthesis and regulation remain unclear. Here, we investigated structural and regulatory genes involved in BIA accumulation in lotus. Two clustered CYP80 genes were identified to be responsible for the biosynthesis of bis-BIAs and aporphine-type BIAs, respectively, and their tissue-specific expression causes divergence in alkaloid component between leaf and embryo. In contrast with the common (S)-reticuline precursor for most BIAs, aporphine alkaloids in lotus leaf may result from the (S)-N-methylcoclaurine precursor. Structural diversity of BIA alkaloids in the leaf is attributed to enzymatic modifications, including intramolecular C-C phenol coupling on ring A and methylation and demethylation at certain positions. Additionally, most BIA biosynthetic pathway genes show higher levels of expression in the leaf of high-BIA cultivar compared with low-BIA cultivar, suggesting transcriptional regulation of BIA accumulation in lotus. Five transcription factors, including three MYBs, one ethylene-responsive factor, and one basic helix-loop-helix (bHLH), were identified to be candidate regulators of BIA biosynthesis in lotus. Our study reveals a BIA biosynthetic pathway and its transcriptional regulation in lotus, which will enable a deeper understanding of BIA biosynthesis in plants.