NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.

FLOWERING LOCUS T genes control floral induction in lotus.

The transition to flowering is a vital process in the lotus life cycle that significantly impacts its ornamental value and seed production. However, the molecular basis of floral transition in lotus remains largely unknown. Here, eight homologous FLOWERING LOCUS T (FT) genes were initially characterized in lotus, which were designated as NnFT1-NnFT8. All of these genes were found to possess the conserved PEBP domain and exhibited high transcript levels in both lotus leaves and floral organs. The proNnFT:ß-glucuronidase (GUS) assay exhibited GUS staining in the vascular tissues of leaves. Furthermore, subcellular localization revealed that NnFT proteins were present in various cellular organelles, including the nucleus, cytoplasm, and endoplasmic reticulum. Overexpression of two NnFT homologs, NnFT2 and NnFT3, rescued the late flowering phenotype in the Arabidopsis ft-10 mutant, indicating the stimulative roles of NnFTs in floral induction. Moreover, NnFTs demonstrated interactions with a bZIP transcription factor, FLOWERING LOCUS D (NnFD), both in vitro and in vivo. These findings will not only deepen our understanding of the regulatory mechanism underlying lotus floral transition, but also provide valuable genetic resources for creating new lotus varieties with extended blooming periods using molecular strategies in the future.

Metabolite profiling and screening of callus browning-related genes in lotus (Nelumbo nucifera).

Callus browning is a major drawback to lotus callus proliferation and regeneration. However, the underlying mechanism of its formation remains largely unknown. Herein, we aimed to explore the metabolic and molecular basis of lotus callus browning by combining histological staining, high-throughput metabolomics, and transcriptomic assays for lotus callus at three browning stages. Histological stained brown callus cross sections displayed severe cell death symptoms, accompanied by an obvious accumulation of polyphenols and lignified materials. Widely targeted metabolomics revealed extensively decreased accumulation of most detected flavonoids and benzylisoquinoline alkaloids (BIAs), as well as a few phenolic acids, amino acids and their derivatives in callus with browning symptoms. Conversely, the contents of most detected tannins were significantly increased. Subsequent comparative transcriptomics identified a set of differentially expressed genes (DEGs) associated with the biosynthesis and regulation of flavonoids and BIAs in lotus. Notably, callus browning was coupled with significantly up-regulated expression of two polyphenol oxidase (PPO) and 17 peroxidase (POD) encoding genes, while the expression of ethylene associated genes remained at marginal levels. These results suggest that lotus callus browning is primarily controlled at the level of metabolism, wherein the oxidation of flavonoids and BIAs is crucially decisive.

Transcription factor NnMYB5 controls petal color by regulating GLUTATHIONE S-TRANSFERASE2 in Nelumbo nucifera.

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.

Comprehensive genome-wide identification and functional characterization of MAPK cascade gene families in Nelumbo.

Mitogen-activated protein kinase (MAPK) cascade signaling pathway plays pivotal roles in various plant biological processes. However, systematic study of MAPK cascade gene families is yet to be conducted in lotus. Herein, 198 putative MAPK genes, including 152 MAP3Ks, 15 MKKs, and 31 MPKs genes were identified in Nelumbo. Segmental duplication was identified as the predominant factor driving MAPK cascade gene family expansion in lotus. MAPK cascade genes in N. nucifera and N. lutea shared high degree of sequence homologies, with 84, 9, and 19 homologous MAP3K, MKK, and MPK gene pairs being detected between the two species, respectively, with most genes predominantly undergoing purifying selection. Gene expression profiling indicated that NnMAPK cascade genes were extensively involved in plant development and submergence stress response. Co-expression analysis revealed potential interaction between transcription factors (TFs) and NnMAPK cascade genes in various biological processes. NnMKK showed predicted interactions with multiple NnMAP3K or NnMPK proteins, which suggested that functional diversity of MAPK cascade genes could be as a result of their complex protein interaction mechanisms. This first systematic analysis of MAPK cascade families in lotus provides deeper insights into their evolutionary dynamics and functional properties, which potentially could be crucial for lotus genetic improvement.

Genome-wide study and functional characterization elucidates the potential association of late embryogenesis abundant (LEA) genes with lotus seed development.

Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins imperatively associated with plant growth and development, as well as cell protection from abiotic stress. However, the genome-wide characterization of LEA gene family remains limited, especially in aquatic species such as lotus (Nelumbo spp.). Here, 57 putative LEA genes, including 28 NnLEAs and 29 NlLEAs were identified in the N.nucifera and N.lutea genomes, respectively. A total of 27 homologous LEA gene pairs were identified, indicating high degree of sequence homologies between the two Nelumbo species. Secondary structure prediction indicated high prevalence of alpha (α) helix structure among LEA proteins in the LEA_1, LEA_4, and SMP groups. Screening of putative promoter cis-elements revealed that NnLEA genes were involved in diverse biological processes. Most NnLEA genes were predominantly expressed in the late cotyledons and plumules development stages, suggesting their potential vital roles in lotus seed maturation. In addition, genes co-expressed with NnLEAs were involved in ABA signaling, seed maturation, and development processes. Overall, this study provides new insights for the in-depth understanding of the functions of NnLEA proteins in lotus seed development, and could act as a useful reference for the molecular breeding of seeds with prolonged lifespan.

Genome-Wide Characterization and Comprehensive Analysis of NAC Transcription Factor Family in Nelumbo nucifera.

NAC (NAM, ATAF, and CUC) is a ubiquitously expressed plant-specific transcription factor (TF) family which is involved in the regulation of various biological processes. However, a systematic characterization of NAC gene family is yet to be reported in lotus. Here, 82 NnNAC genes which included five predicted membrane-bound NAC proteins were identified in the lotus genome. Phylogenetic analysis revealed seven-subfamily clusters (I-VII) of NnNAC proteins, with homologous gene pairs displaying similar conserved motifs and gene structure characteristics. Transactivation assay of NnNAC proteins revealed an extensive transcriptional activation capacity which is mediated by the highly divergent C-terminal activation domain (AD). Expression analysis of NnNAC genes in lotus tissues showed high transcript levels in root, stamen, petal and seed coat. In addition, 30 and 29 differentially expressed NnNAC candidate genes putatively involved in lotus seed development and response to complete submergence stress, respectively, were identified. Overall, our study provides potentially useful candidate gene resources for future molecular breeding of lotus varieties with novel agronomic traits.

Identification of QTLs and a putative candidate gene involved in rhizome enlargement of Asian lotus (Nelumbo nucifera).

KEY MESSAGE: QTL mapping studies identified three reliable QTLs of rhizome enlargement in lotus. NnBEL6 located within the confidence interval of the major QTL cqREI-LG2 is a key candidate gene enhancing rhizome enlargement. Lotus (Nelumbo) is perennial aquatic plant with nutritional, pharmacological, and ornamental significance. Rhizome is an underground lotus stem that acts as a storage organ and as a reproductive tissue for asexual production. The enlargement of lotus rhizome is an important adaptive strategy for surviving the cold winter. The aims of this study were to identify quantitative trait loci (QTLs) for rhizome enlargement traits including rhizome enlargement index (REI) and number of enlarged rhizome (NER), and to uncover their associated candidate genes. A high-density genetic linkage map was constructed, consisting of 2935 markers binned from 236,840 SNPs. A total of 14 significant QTLs were detected for REI and NER, which explained 6.7-22.3% of trait variance. Three QTL regions were repeatedly identified in at least 2 years, and a major QTL, designated cqREI-LG2, with a rhizome-enlargement effect and about 20% of the phenotypic contribution was identified across the 3 climatic years. A candidate NnBEL6 gene located within the confidence interval of cqREI-LG2 was considered to be putatively involved in lotus rhizome enlargement. The expression of NnBEL6 was exclusively induced by rhizome swelling. Sequence comparison of NnBEL6 among lotus cultivars revealed a functional Indel site in its promoter that likely initiates the rhizome enlargement process. Transgenic potato assay was used to confirm the role of NnBEL6 in inducing tuberization. The successful identification QTLs and functional validation of NnBEL6 gene reported in this study will enrich our knowledge on the genetic basis of rhizome enlargement in lotus.

Transcriptome-Wide Characterization of Alkaloids and Chlorophyll Biosynthesis in Lotus Plumule.

Lotus plumule is a green tissue in the middle of seeds that predominantly accumulates bisbenzylisoquinoline alkaloids (bis-BIAs) and chlorophyll (Chl). However, the biosynthetic mechanisms of these two metabolites remain largely unknown in lotus. This study used physiological and RNA sequencing (RNA-Seq) approaches to characterize the development and molecular mechanisms of bis-BIAs and Chl biosynthesis in lotus plumule. Physiological analysis revealed that exponential plumule growth occurred between 9 and 15 days after pollination (DAP), which coincided with the onset of bis-BIAs biosynthesis and its subsequent rapid accumulation. Transcriptome analysis of lotus plumule identified a total of 8,725 differentially expressed genes (DEGs), representing ~27.7% of all transcripts in the lotus genome. Sixteen structural DEGs, potentially associated with bis-BIAs biosynthesis, were identified. Of these, 12 encoded O-methyltransferases (OMTs) are likely involved in the methylation and bis-BIAs diversity in lotus. In addition, functionally divergent paralogous and redundant homologous gene members of the BIAs biosynthesis pathway, as well as transcription factors co-expressed with bis-BIAs and Chl biosynthesis genes, were identified. Twenty-two genes encoding 16 conserved enzymes of the Chl biosynthesis pathway were identified, with the majority being significantly upregulated by Chl biosynthesis. Photosynthesis and Chl biosynthesis pathways were simultaneously activated during lotus plumule development. Moreover, our results showed that light-driven Pchlide reduction is essential for Chl biosynthesis in the lotus plumule. These results will be useful for enhancing our understanding of alkaloids and Chl biosynthesis in plants.

Comparative analyses of American and Asian lotus genomes reveal insights into petal color, carpel thermogenesis and domestication.

Nelumbo lutea (American lotus), which differs from Nelumbo nucifera (Asian lotus) morphologically, is one of the two remaining species in the basal eudicot family Nelumbonaceae. Here, we assembled the 843-Mb genome of American lotus into eight pseudochromosomes containing 31 382 protein-coding genes. Comparative analyses revealed conserved synteny without large chromosomal rearrangements between the genomes of American and Asian lotus and identified 29 533 structural variants (SVs). Carotenoid and anthocyanin pigments determine the yellow and red petal colors of American and Asian lotus, respectively. The structural genes encoding enzymes of the carotenoid and anthocyanin biosynthesis pathways were conserved between two species but differed in expression. We detected SVs caused by repetitive sequence expansion or contraction among the anthocyanin biosynthesis regulatory MYB genes. Further transient overexpression of candidate NnMYB5 induced anthocyanin accumulation in lotus petals. Alternative oxidase (AOX), uncoupling proteins (UCPs), and sugar metabolism and transportation contributed to carpel thermogenesis. Carpels produce heat with sugars transported from leaves as the main substrates, because there was weak tonoplast sugar transporter (TST) activity, and with SWEETs were highly expressed during thermogenesis. Cell proliferation-related activities were particularly enhanced in the warmer carpels compared with stamens during the cold night before blooming, which suggested that thermogenesis plays an important role in flower protogyny. Population genomic analyses revealed deep divergence between American and Asian lotus, and independent domestication affecting seed, rhizome, and flower traits. Our findings provide a high-quality reference genome of American lotus for exploring the genetic divergence and variation between two species and revealed possible genomic bases for petal color, carpel thermogenesis and domestication in lotus.

Color fading in lotus (Nelumbo nucifera) petals is manipulated both by anthocyanin biosynthesis reduction and active degradation.

Flower color is a key trait that determines the ornamental quality of aquatic lotus (Nelumbo nucifera). Color fading significantly decreases the ornamental value of lotus flowers. However, the molecular mechanism underlying lotus petal discoloration remains largely unknown. Here, the anthocyanin content and global transcriptional profiling of lotus petals of cultivar 'Qiusanse' in four developmental stages were analyzed. Five anthocyanin components were detected, and the total anthocyanin content decreased as the petal color changed from red to nearly white. Moreover, the malondialdehyde (MDA) content and peroxidase (POD) activity increased during color fading. RNA-seq analysis revealed a total of 4,092 differentially expressed genes (DEGs) between petal developmental stages. Notably, oxidoreductase and hydrolase activity related genes were overrepresented in DEGs. The expression pattern of key anthocyanin biosynthesis genes including, CHS, F3H, ANS, UFGT, and transcription factor regulators, including MYBs, WRKYs and bHLHs were correlated with anthocyanin accumulation. Interestingly, DEGs associated with anthocyanin degradation and vacuolar pH regulation, including peroxidase, proton pumps regulators such as WRKY3 and MYB5-like, were significantly upregulated during the late stages of flowering. This study reveals for the first time the transcriptional dynamics during lotus petal discoloration. Our results suggest the involvement of anthocyanin biosynthesis repressors and degrading genes as well as pH regulators in controlling color fading of lotus petals. The study also provides valuable information and candidate genes for improving the lotus flower color.

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.