The CYP80A and CYP80G Are Involved in the Biosynthesis of Benzylisoquinoline Alkaloids in the Sacred Lotus (Nelumbo nucifera).

Bisbenzylisoquinoline and aporphine alkaloids are the two main pharmacological compounds in the ancient sacred lotus (Nelumbo nucifera). The biosynthesis of bisbenzylisoquinoline and aporphine alkaloids has attracted extensive attention because bisbenzylisoquinoline alkaloids have been reported as potential therapeutic agents for COVID-19. Our study showed that NnCYP80A can catalyze C-O coupling in both (R)-N-methylcoclaurine and (S)-N-methylcoclaurine to produce bisbenzylisoquinoline alkaloids with three different linkages. In addition, NnCYP80G catalyzed C-C coupling in aporphine alkaloids with extensive substrate selectivity, specifically using (R)-N-methylcoclaurine, (S)-N-methylcoclaurine, coclaurine and reticuline as substrates, but the synthesis of C-ring alkaloids without hydroxyl groups in the lotus remains to be elucidated. The key residues of NnCYP80G were also studied using the 3D structure of the protein predicted using Alphafold 2, and six key amino acids (G39, G69, A211, P288, R425 and C427) were identified. The R425A mutation significantly decreased the catalysis of (R)-N-methylcoclaurine and coclaurine inactivation, which might play important role in the biosynthesis of alkaloids with new configurations.

NnWOX1-1, NnWOX4-3, and NnWOX5-1 of lotus (Nelumbo nucifera Gaertn)promote root formation and enhance stress tolerance in transgenic Arabidopsis thaliana.

BACKGROUND: Adventitious roots (ARs) represent an important organ system for water and nutrient uptake in lotus plants because of degeneration of the principal root. The WUSCHEL-related homeobox (WOX) gene regulates plant development and growth by affecting the expression of several other genes. In this study, three WOX genes, NnWOX1-1, NnWOX4-3, and NnWOX5-1, were isolated and their functions were assessed in Arabidopsis plants. RESULTS: The full lengths of NnWOX1-1, NnWOX4-3, and NnWOX5-1 were 1038, 645, and 558 bp, encoding 362, 214, and 185 amino acid residues, respectively. Phylogenetic analysis classified NnWOX1-1 and NnWOX4-3 encoding proteins into one group, and NnWOX5-1 and MnWOX5 encoding proteins exhibited strong genetic relationships. The three genes were induced by sucrose and indoleacetic acid (IAA) and exhibited organ-specific expression characteristics. In addition to improving root growth and salt tolerance, NnWOX1-1 and NnWOX4-3 promoted stem development in transgenic Arabidopsis plants. A total of 751, 594, and 541 genes, including 19, 19, and 13 respective genes related to ethylene and IAA metabolism and responses, were enhanced in NnWOX1-1, NnWOX4-3, and NnWOX5-1 transgenic plants, respectively. Further analysis showed that ethylene production rates in transgenic plants increased, whereas IAA, peroxidase, and lignin content did not significantly change. Exogenous application of ethephon on lotus seedlings promoted AR formation and dramatically increased the fresh and dry weights of the plants. CONCLUSIONS: NnWOX1-1, NnWOX4-3, and NnWOX5-1 influence root formation, stem development, and stress adaptation in transgenic Arabidopsis plants by affecting the transcription of multiple genes. Among these, changes in gene expression involving ethylene metabolism and responses likely critically affect the development of Arabidopsis plants. In addition, ethylene may represent an important factor affecting AR formation in lotus seedlings.

Combined analysis of microRNA and mRNA profiles provides insights into the pathogenic resistant mechanisms of the lotus rhizome rot.

Lotus rhizome rot caused by Fusarium oxysporum is a common vascular fungal disease in plants that significantly impacts the yield. However, only a few studies have studied the mechanism of Nelumbo nucifera responding to lotus rhizome rot. Here, we investigated the pathogenic genes and miRNAs in lotus rhizome rot to uncover the pathogenic resistant mechanisms by transcriptome and small RNA sequencing of lotus roots after inoculation with Fusarium oxysporum. GO and KEGG functional enrichment analysis showed that differential miRNAs were mostly enriched in starch and sucrose metabolism, biosynthesis of secondary metabolites, glutathione metabolism, brassinosteroid biosynthesis and flavonoid biosynthesis pathways. Twenty-seven upregulated miRNAs, 19 downregulated miRNAs and their target genes were identified. Correlation analysis found that miRNAs negatively regulate target genes, which were also enriched in starch and sucrose metabolism and glutathione metabolism pathways. Their expression was measured by reverse transcription quantitative PCR (qRT-PCR), and the results were consistent with the transcriptome analysis, thus verifying the reliability of transcriptome data. We selected three miRNAs (miRNA858-y, miRNA171-z and a novel miRNA novel-m0005-5p) to test the relationship between miRNAs and their target genes. The activity of the GUS testing assay indicated that miRNA could decrease the GUS activity by inhibiting the expression of their target genes. Collectively, this study provides a comprehensive analysis of transcriptome and small RNA sequencing of lotus root after inoculation with Fusarium oxysporum, and we identified candidate miRNAs and their target genes for breeding strategies of Nelumbo nucifera.

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.

Genome-Wide Analysis of WUSCHEL-Related Homeobox Gene Family in Sacred Lotus (Nelumbo nucifera).

WUSCHEL-related homeobox (WOX) is a plant-specific transcription factor (TF), which plays an essential role in the regulation of plant growth, development, and abiotic stress responses. However, little information is available on the specific roles of WOX TFs in sacred lotus (Nelumbo nucifera), which is a perennial aquatic plant with important edible, ornamental, and medicinal values. We identified 15 WOX TFs distributing on six chromosomes in the genome of N. nucifera. A total of 72 WOX genes from five species were divided into three clades and nine subclades based on the phylogenetic tree. NnWOXs in the same subclades had similar gene structures and conserved motifs. Cis-acting element analysis of the promoter regions of NnWOXs found many elements enriched in hormone induction, stress responses, and light responses, indicating their roles in growth and development. The Ka/Ks analysis showed that the WOX gene family had been intensely purified and selected in N. nucifera. The expression pattern analysis suggested that NnWOXs were involved in organ development and differentiation of N. nucifera. Furthermore, the protein-protein interaction analysis showed that NnWOXs might participate in the growth, development, and metabolic regulation of N. nucifera. Taken together, these findings laid a foundation for further analysis of NnWOX functions.

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.

Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera).

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs markedly from that of opium poppy and other members of the Ranunculales, most notably in an abundance of BIAs possessing the (R)-stereochemical configuration and the absence of reticuline, a major branchpoint intermediate in most BIA producers. Owing to these unique metabolic features and the pharmacological potential of lotus, we set out to elucidate the BIA biosynthesis network in N. nucifera. Here we show that lotus CYP80G (NnCYP80G) and a superior ortholog from Peruvian nutmeg (Laurelia sempervirens; LsCYP80G) stereospecifically convert (R)-N-methylcoclaurine to the proaporphine alkaloid glaziovine, which is subsequently methylated to pronuciferine, the presumed precursor to nuciferine. While sacred lotus employs a dedicated (R)-route to aporphine alkaloids from (R)-norcoclaurine, we implemented an artificial stereochemical inversion approach to flip the stereochemistry of the core BIA pathway. Exploiting the unique substrate specificity of dehydroreticuline synthase from common poppy (Papaver rhoeas) and pairing it with dehydroreticuline reductase enabled de novo synthesis of (R)-N-methylcoclaurine from (S)-norcoclaurine and its subsequent conversion to pronuciferine. We leveraged our stereochemical inversion approach to also elucidate the role of NnCYP80A in sacred lotus metabolism, which we show catalyzes the stereospecific formation of the bis-BIA nelumboferine. Screening our collection of 66 plant O-methyltransferases enabled conversion of nelumboferine to liensinine, a potential anti-cancer bis-BIA from sacred lotus. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.

Proteomic and metabolomic analyses showing the differentially accumulation of NnUFGT2 is involved in the petal red-white bicolor pigmentation in lotus (Nelumbo nucifera).

Bicolor flower lotus is rare with high ornamental value. During the long history of breeding and artificial selection, a very famous lotus cultivar 'Da Sajin' with red and white picotee bicolor petals were obtained. In order to reveal the mechanism underlying the formation of its picotee bicolor pattern in the petal, an integrative metabolomics and proteomics analyses were conducted between red and white parts of its petals. The results showed that the defect of anthocyanidin 3-O-glucosyltransferases (UFGTs) accumulation resulted in the failure of the glycosylation of anthocyanidin, the last step of anthocyanin biosynthesis in white part of the petals. And proteomic data and biochemical analysis showed that the defect of UFGTs accumulation is not related to their transcription, but because of their degradation. Function of one differentially accumulated NnUFGT were proven being involved in anthocyanin biosynthesis through both in-vitro enzyme assay and in-vivo transgenic analyses. This regulation on the protein accumulation of structural genes in anthocyanin biosynthesis was not explored in any other plants, and hence supposed to be a novel mechanism for the formation of picotee bicolor pattern flower. The results not only provide some new insights into the understanding of lotus flower coloration, but also might assist the breeding of flower lotus.

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.

Integration of metabolomics and transcriptomics analyses investigates the accumulation of secondary metabolites in maturing seed plumule of sacred lotus (Nelumbo nucifera).

Lotus seed plumule (LP) is rich in a variety of antioxidant and anti-inflammatory secondary metabolites, making it a traditional food and medicine widely used in China. Physiological and histological evidences indicated that LP mainly accumulated metabolites in 15-24 days after pollination (DAP) during their development. To systematically investigate the dynamic accumulation of major secondary metabolites, the UPLC-HRMS-based widely targeted metabolomics analyses were performed on maturing LP at 15, 18, 21, and 24 DAP. In total, 767 metabolites were identified, including many secondary metabolites, e.g., 27 % flavonoids and 8 % alkaloids. Among them, 591 were identified as differentially accumulated metabolites (DAMs). The majority of secondary metabolites showed great accumulation after 18 DAP even at the late stage of LP maturation, such as hesperidin, neohesperidin, orobol, serotonin, and lotus special O-nornuciferine, endowing mature LP with effective pharmaceutical properties. The paralleled transcriptomic analysis identified 11,019 differentially expressed genes (DEGs). Based on the comprehensive data, several systematical metabolic regulation maps were established for different secondary metabolites, and 18 DAP was found as a switching point for LP maturing from active primary metabolism to massive secondary metabolites deposition. This study provides valuable information for understanding the mechanism of secondary metabolite accumulation in maturing LP and facilitates its pharmaceutical application.

TMT-based quantitative proteomic and physiological analyses on lotus plumule of artificially aged seed in long-living sacred lotus Nelumbo nucifera.

Seed longevity is important for the maintenance of seed nutritional quality, vigor, and germination potential during storage. Sacred lotus is known as one of the longest living seeds in the world and their ability to maintain longevity has been widely investigated. In this study, a suitable controlled deterioration treatment (CDT) method was first established to evaluate the vigor loss of lotus plumule (LP), and then the Tandem Mass Tags (TMT)-based proteomic analysis was performed on LP from the CDT-treated seed to quantitatively and qualitatively analyze the protein profile dynamic. In total, 4002 proteins were successfully quantified, of them, 558 differently accumulated proteins (DAPs) were identified. Protein processing and RNA-related proteins were found more easily to be affected by CDT, which may directly result in seed vigor loss. Meanwhile, CDT resulted in remarkable up-regulation of numerous proteins related to antioxidation, photosynthesis, RNA and DNA stability, starch and sucrose mobilization, and cell membrane and wall stability, which potentially played key roles in maintaining the lotus seed vigor under CDT. Histological and physiological analyses were also performed to verify some proteome results. This study provided both fundamental data and new insights to further uncover the secret of lotus seed longevity. SIGNIFICANCE: Seed aging affects the seed quality and can result in direct economic losses. The exceptional longevity of sacred lotus seed has attracted extensive attention. In this study, an optimized CDT method was used to mimic the natural aging process of sacred lotus seed, and based on TMT-based quantitative proteomic analysis on the LP profile of CDT-treated seeds, a series of differentially accumulation of specific proteins (DEPs) were revealed related to CDT resistance. Correspondingly, the physiological state and histological structure of the LP along with the CDT were detected to verify the proteome data. This study provided comprehensive information for the molecular basis of lotus seed aging analysis and facilitate to screen seed longevity related proteins for other plant species.

Prokaryotic communities adapted to microhabitats on the Indian lotus (Nelumbo nucifera) growing in the high-altitude urban Dal Lake.

Indian lotus (Nelumbo nucifera) is one of the dominant aquatic plants cultivated in Dal Lake, situated at 1586 m above mean sea level (MSL) in the northeast of Srinagar, Kashmir. Despite their economic and ecological role, the microbial communities associated with the lotus plant are still unexplored. In this study, we investigated the prokaryotic communities on surfaces of different lotus microhabitats (roots, rhizome, leaves, flowers, and fruits), lake water, and sediments using 16S rRNA gene amplicon sequencing. Overall, prokaryotic diversity decreased significantly on the surface of lotus microhabitats in comparison to the lake water and sediments. Among the microhabitats of lotus, roots and leaves harbored more diverse communities in comparison to rhizomes, fruits, and flowers. A total of 98 genera were shared by lotus and the Dal Lake sediments and water. However, significant differences were found in their relative abundance; for example, Pseudomonas was the most dominant genus on the majority of lotus microhabitats. On the other hand, Flavobacterium was highly abundant in the lake water, while a higher abundance of Acinetobacter was recorded in sediments. Additionally, we also noted the presence of potential human pathogenic genera including Escherichia-Shigella, Enterobacter, Pantoea, Raoultella, Serratia, and Sphingomonas on the lotus microhabitats. Predicted functions of prokaryotic communities revealed a higher abundance of genes associated with nutrient uptake in the microhabitats of the lotus. This study offered first-hand information on the prokaryotic communities harbored by lotus plants and water and sediments of the Dal Lake and demonstrated the adaptation of diverse communities to microhabitats of lotus.

Integrated metabolite profiling and transcriptome analysis reveal candidate genes involved in the formation of yellow Nelumbo nucifera.

As a worldwide major ornamental flower and a edible plant, lotus (Nelumbo nucifera) is also used as medicine and tea beverage. Here, transcriptome and metabolites of yellow (MLQS) and white (YGB) lotus cultivars during five key flower coloration stages were profiled. 2014 differentially expressed genes were detected with 11 carotenoids in lotus were identified for the first time. Then, regulatory networks between and within functional modules was reconstructed, and the correlation between module-metabolites and gene-metabolites was conducted within 3 core modules. 18 candidate genes related to the formation of yellow flower were screened out and a gene regulatory model for the flower color difference between MLQS and YGB were speculated as follows: The substrate competition between F3'H and F3'5'H and substrate specificity of FLS, together with differential expression of CCD4a and CCD4b were contribute to the differences in flavonoids and carotenoids. Besides, UGT73C2, UGT91C1-2 and SGTase, and regulation of UGTs by transcription factors PLATZ, MADS, NAC031, and MYB308 may also play a role in the upstream regulation. The following verification results indicated that functional differences existed in the coding sequences of NnCCD4b and promoters of NnCCD4a of MLQS and YGB. In all, this study preliminarily reveals the mechanism of yellow flower coloration in lotus and provides new ideas for the study of complex ornamental characters of other plants.

Identification of the NAC Transcription Factors and Their Function in ABA and Salinity Response in Nelumbo nucifera.

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.

Evolution of isoform-level gene expression patterns across tissues during lotus species divergence.

Both gene duplication and alternative splicing (AS) drive the functional diversity of gene products in plants, yet the relative contributions of the two key mechanisms to the evolution of gene function are largely unclear. Here, we studied AS in two closely related lotus plants, Nelumbo lutea and Nelumbo nucifera, and the outgroup Arabidopsis thaliana, for both single-copy and duplicated genes. We show that most splicing events evolved rapidly between orthologs and that the origin of lineage-specific splice variants or isoforms contributed to gene functional changes during species divergence within Nelumbo. Single-copy genes contain more isoforms, have more AS events conserved across species, and show more complex tissue-dependent expression patterns than their duplicated counterparts. This suggests that expression divergence through isoforms is a mechanism to extend the expression breadth of genes with low copy numbers. As compared to isoforms of local, small-scale duplicates, isoforms of whole-genome duplicates are less conserved and display a less conserved tissue bias, pointing towards their contribution to subfunctionalization. Through comparative analysis of isoform expression networks, we identified orthologous genes of which the expression of at least some of their isoforms displays a conserved tissue bias across species, indicating a strong selection pressure for maintaining a stable expression pattern of these isoforms. Overall, our study shows that both AS and gene duplication contributed to the diversity of gene function during the evolution of lotus.

Integrated Transcriptome and Targeted Metabolite Analysis Reveal miRNA-mRNA Networks in Low-Light-Induced Lotus Flower Bud Abortion.

Most Nelumbo nucifera (lotus) flower buds were aborted during the growing season, notably in low-light environments. How lotus produces so many aborted flower buds is largely unknown. An integrated transcriptome and targeted metabolite analysis was performed to reveal the genetic regulatory networks underlying lotus flower bud abortion. A total of 233 miRNAs and 25,351 genes were identified in lotus flower buds, including 68 novel miRNAs and 1108 novel genes. Further enrichment analysis indicated that sugar signaling plays a potential central role in regulating lotus flower bud abortion. Targeted metabolite analysis showed that trehalose levels declined the most in the aborting flower buds. A potential regulatory network centered on miR156 governs lotus flower bud abortion, involving multiple miRNA-mRNA pairs related to cell integrity, cell proliferation and expansion, and DNA repair. Genetic analysis showed that miRNA156-5p-overexpressing lotus showed aggravated flower bud abortion phenotypes. Trehalose-6-P synthase 1 (TPS1), which is required for trehalose synthase, had a negative regulatory effect on miR156 expression. TPS1-overexpression lotus showed significantly decreased flower bud abortion rates both in normal-light and low-light environments. Our study establishes a possible genetic basis for how lotus produces so many aborted flower buds, facilitating genetic improvement of lotus' shade tolerance.

Differential expression involved in starch synthesis pathway genes reveal various starch characteristics of seed and rhizome in lotus (Nelumbo Nucifera).

Lotus (Nelumbo nucifera) is one of the main aquatic vegetables in China. Its seed and rhizome are main edible parts which are rich in starch. Preliminary experiments of starch contents revealed that seed and rhizome expressed great differences in amylose and amylopectin contents. The rhizomes have higher amylopectin content, while the seeds have higher amylose content. In this study, we have estimated 16 varieties of lotus seeds and found that the amylose content of lotus seeds ranged from 30% to 50%, with an average amylose content of 43%, which showed high-amylose content characteristics. Morphological analysis of lotus seed shown that starch rapid accumulated in 20 DAF (day after fertilization) ∼ 26 DAF. Transcriptome of lotus seeds indicated that starch genes played an important role in seed development. Especially in 22 DAF, the genes which controlled amylose synthesis significantly increased, in contrast, the expression of amylopectin-related genes was stable and might limit the synthesis of amylopectin. We further analyzed the expression patterns of 11 key related genes between lotus seeds and rhizomes, and found that the expression of amylose-related genes to were higher in lotus seed, while the expression of genes related to amylopectin synthesis were higher in rhizome. This study provided a comprehensive research of molecular basis for starch in lotus seed and rhizome. Different expression among key genes during starch accumulation might be the principal cause of the differences in starch properties between seed and rhizome. PRACTICAL APPLICATION: Differential expression involved in starch synthesis pathway genes is the main reason for various starch characteristics of seed and rhizome in lotus. High amylose content in lotus seed is a valuable trait for developing functional food.

Distinct composition and amplification dynamics of transposable elements in sacred lotus (Nelumbo nucifera Gaertn.).

Sacred lotus (Nelumbo nucifera Gaertn.) is a basal eudicot plant with a unique lifestyle, physiological features, and evolutionary characteristics. Here we report the unique profile of transposable elements (TEs) in the genome, using a manually curated repeat library. TEs account for 59% of the genome, and hAT (Ac/Ds) elements alone represent 8%, more than in any other known plant genome. About 18% of the lotus genome is comprised of Copia LTR retrotransposons, and over 25% of them are associated with non-canonical termini (non-TGCA). Such high abundance of non-canonical LTR retrotransposons has not been reported for any other organism. TEs are very abundant in genic regions, with retrotransposons enriched in introns and DNA transposons primarily in flanking regions of genes. The recent insertion of TEs in introns has led to significant intron size expansion, with a total of 200 Mb in the 28 455 genes. This is accompanied by declining TE activity in intergenic regions, suggesting distinct control efficacy of TE amplification in different genomic compartments. Despite the prevalence of TEs in genic regions, some genes are associated with fewer TEs, such as those involved in fruit ripening and stress responses. Other genes are enriched with TEs, and genes in epigenetic pathways are the most associated with TEs in introns, indicating a dynamic interaction between TEs and the host surveillance machinery. The dramatic differential abundance of TEs with genes involved in different biological processes as well as the variation of target preference of different TEs suggests the composition and activity of TEs influence the path of evolution.

Integrated mRNA and Small RNA Sequencing Reveals a microRNA Regulatory Network Associated with Starch Biosynthesis in Lotus (Nelumbo nucifera Gaertn.) Rhizomes.

Internode starch biosynthesis is one of the most important traits in lotus rhizome because of its relation to crop productivity. Understanding the microRNA (miRNA) and mRNA expression profiles related to lotus internode starch biosynthesis would help develop molecular improvement strategies, but they are not yet well-investigated. To identify genes and miRNAs involved in internode starch biosynthesis, the cDNA and small RNA libraries of Z6-1, Z6-2, and Z6-3 were sequenced, and their expression were further studied. Through combined analyses of transcriptome data and small RNA sequencing data, a complex co-expression regulatory network was constructed, in which 20 miRNAs could modulate starch biosynthesis in different internodes by tuning the expression of 10 target genes. QRT-PCR analysis, transient co-expression experiment and dual luciferase assay comprehensively confirmed that NnumiR396a down-regulated the expression of NnSS2 and ultimately prevents the synthesis of amylopectin, and NnumiR396b down-regulated the expression of NnPGM2 and ultimately prevents the synthesis of total starch. Our results suggest that miRNAs play a critical role in starch biosynthesis in lotus rhizome, and that miRNA-mediated networks could modulate starch biosynthesis in this tissue. These results have provided important insights into the molecular mechanism of starch biosynthesis in developing lotus rhizome.