The WRKY family is one of the largest transcription factor (TF) families in plants and plays central roles in modulating plant stress responses and developmental processes, as well as secondary metabolic regulations. Lotus (Nelumbo nucifera) is an aquatic crop that has significant food, ornamental and pharmacological values. Here, we performed an overview analysis of WRKY TF family members in lotus, and studied their functions in environmental adaptation and regulation of lotus benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 65 WRKY genes were identified in the lotus genome and they were well clustered in a similar pattern with their Arabidopsis homologs in seven groups (designated I, IIa-IIe, and III), although no lotus WRKY was clustered in the group IIIa. Most lotus WRKYs were functionally paired, which was attributed to the recently occurred whole genome duplication in lotus. In addition, lotus WRKYs were regulated dramatically by salicilic acid (SA), jasmonic acid (JA), and submergence treatments, and two lotus WRKYs, NnWRKY40a and NnWRKY40b, were significantly induced by JA and promoted lotus BIA biosynthesis through activating BIA biosynthetic genes. The investigation of WRKY TFs for this basal eudicot reveals new insights into the evolution of the WRKY family, and provides fundamental information for their functional studies and lotus breeding.

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 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.

Lotus (Nelumbo spp.) is an important aquatic ornamental genus in the family Nelumbonaceae comprising only 2 species: Nelumbo lutea with yellow flowers and Nelumbo nucifera with red or white flowers. The petal color variations between these 2 species have previously been associated with the potential activities of FLAVONOL SYNTHASE (FLS) and MYB5. However, the underlying genetic mechanisms of flower color divergence within the N. nucifera species remain unclear. Here, quantitative trait locus mapping led to the identification of MYB5, a candidate gene controlling petal color in N. nucifera. Genotyping of 213 natural lotus accessions revealed an 80 kb presence/absence variant (PAV) of the NnMYB5 gene that is associated with petal color variation. Transcriptome analysis, dual-luciferase, and yeast 1-hybrid assays showed that NnMYB5 could directly activate the anthocyanin transporter gene GLUTATHIONE S-TRANSFERASE2 (NnGST2). Heterologous expression of NnGST2 in Arabidopsis (Arabidopsis thaliana) and its overexpression in lotus petals induced anthocyanin accumulation. Deletion of the 80 kb PAV within NnMYB5 inactivated NnGST2 expression and blocked anthocyanin accumulation in white N. nucifera petals. In contrast, the anthocyanin deficiency of N. lutea occurred due to pseudogenized NlMYB5 alleles. Our results establish a regulatory link between NnMYB5 and NnGST2 in petal anthocyanin accumulation and demonstrate the independent mechanisms controlling flower coloration in Nelumbo.

Lotus, an economically significant aquatic crop within the genus Nelumbo (family Nelumbonaceae), includes the two extant species Nelumbo nucifera and Nelumbo lutea. Renowned for its ornamental, culinary, and medicinal value, lotus has seen notable advancements in genomics and molecular biology, particularly in reference genome sequencing and assembly, bioactive compound biosynthetic pathways, and molecular mechanisms underlying key traits. Critical genes linked to horticultural characteristics, edible quality, medicinal properties, and stress resilience have been identified and functionally validated. This review presents a thorough overview of recent molecular developments in lotus research, emphasizing genome sequencing and functional gene analysis. Additionally, it addresses prevailing challenges and future directions, providing valuable insights for foundational research and genetic enhancement of lotus.