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.

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.

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.

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.

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.

Comprehensive Analysis and Functional Studies of WRKY Transcription Factors in Nelumbo nucifera.

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.

Two polyketide synthases are necessary for 4-hydroxy-5-methylcoumarin biosynthesis in Gerbera hybrida.

Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4-hydroxy-5-methylcoumarin (HMC). Unlike most coumarins, 5-methylcoumarins have been suggested to be derived through the acetate-malonate pathway. All of these polyketide-derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2-pyrone synthases (G2PS1-3). The enzymes are chalcone synthase-like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7-dihydroxy-5-methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site-directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.

Assessment of calcium and zinc accumulation in cultivated and wild apples.

BACKGROUND: Apple is one of the staple fruits worldwide which are a good source of mineral nutrients. However, little is known about genetic variation for mineral nutrition in apple germplasm. In this study, the calcium and zinc contents in mature fruits of 378 apple cultivars and 39 wild relatives were assessed. Mineral concentrations were quantified using flame atomic absorption spectroscopy (FAAS). RESULTS: Both calcium and zinc accumulation showed great variation among accessions tested. Overall, wild fruits were significantly richer in zinc than cultivated fruits, while the average concentration of calcium was similar between cultivated and wild fruits. The difference in zinc concentration between wild and cultivated fruits may be an indirect result of artificial selection on fruit characteristics during apple domestication. Moreover, calcium concentration in fruit showed a decreasing trend throughout fruit development of apple, while zinc concentration in fruit displayed a complex variation pattern in the late stages of fruit development. CONCLUSION: The finding of a wild genetic variation for fruit calcium and zinc accumulation in apple germplasm could be helpful for future research on genetic dissection and improvement of calcium and zinc accumulation in apple fruit. © 2017 Society of Chemical Industry.

Simple and fast electrochemical detection of sequence-specific DNA via click chemistry-mediated labeling of hairpin DNA probes with ethynylferrocene.

A universal and straightforward electrochemical biosensing strategy for the detection and identification of sequence-specific DNA via click chemistry-mediated labeling of hairpin DNA probes (hairpins) with ethynylferrocene was reported. In the target-unbound form, the immobilized hairpins were kept in the folded stem-loop configuration with their azido terminals held in close proximity of the electrode surface, making them difficult to be labeled with ethynylferrocene due to the remarkable steric hindrance of the densely packed hairpins. Upon hybridization, they were unfolded and underwent a large conformational change, thus enabling the azido terminals to become available for its subsequent conjugation with ethynylferrocene via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). After that, the quantitatively labeled ethynylferrocene could be exploited as the electroactive probes to monitor the DNA hybridization. As the unfolded hairpins were labeled in a stoichiometric ratio of 1 : 1, the electrochemical measurement based on differential pulse voltammetry enabled a reliable quantification of sequence-specific DNA. Under optimal conditions, the strategy could detect target single-stranded DNA (ssDNA) down to 0.296 pM with a good linear response over the range from 1 pM to 1 nM, and had excellent specificity in the genotyping of single-nucleotide polymorphisms. Furthermore, it also exhibited good detection reliability in serum samples and required no complicated protocols. More importantly, the simplicity of this strategy together with its compatibility with microfluidic chips makes it show great potential in clinical applications, where simple procedures are generally preferred.

Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida.

• Chalcone synthase (CHS) is the key enzyme in the first committed step of the flavonoid biosynthetic pathway and catalyzes the stepwise condensation of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone. In plants, CHS is often encoded by a small family of genes that are temporally and spatially regulated. Our earlier studies have shown that GCHS4 is highly activated by ectopic expression of an MYB-type regulator GMYB10 in gerbera (Gerbera hybrida). • The tissue- and development-specific expression patterns of three gerbera CHS genes were examined. Virus-induced gene silencing (VIGS) was used to knock down GCHS1 and GCHS4 separately in gerbera inflorescences. • Our data show that GCHS4 is the only CHS encoding gene that is expressed in the cyanidin-pigmented vegetative tissues of gerbera cv Terraregina. GCHS3 expression is pronounced in the pappus bristles of the flowers. Expression of both GCHS1 and GCHS4 is high in the epidermal cells of gerbera petals, but only GCHS1 is contributing to flavonoid biosynthesis. • Gerbera contains a family of three CHS encoding genes showing different spatial and temporal regulation. GCHS4 expression in gerbera petals is regulated post-transcriptionally, at the level of either translation elongation or protein stability.

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.

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.

Modification of Tobacco rattle virus RNA1 to serve as a VIGS vector reveals that the 29K movement protein is an RNA silencing suppressor of the virus.

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.

Virus-induced gene silencing for Asteraceae--a reverse genetics approach for functional genomics in Gerbera hybrida.

Virus-induced gene silencing (VIGS) is a natural defence mechanism in plants which leads to sequence-specific degradation of viral RNA. For identifying gene functions, Tobacco rattle virus (TRV)-based VIGS has been applied for silencing of endogenous genes in many plant species. Gerbera hybrida (Asteraceae) has emerged as a novel model for studies in flower development and secondary metabolism. For this highly heterozygous species, functional studies have been conducted through reverse genetic methods by producing stable transgenic lines, which, however, is labour-intensive and time-consuming. For the development of TRV-based VIGS system for gerbera, and for the first time for an Asteraceaeous species, we screened several gerbera cultivars and optimized the agroinfiltration methods for efficient silencing. Gene fragments for gerbera phytoene desaturase (GPDS) and Mg-chelatase subunits (GChl-H and GChl-I), expressed from a TRV vector, induced silencing phenotypes in leaves, scapes, and involucral bracts indicating their feasibility as markers for green tissues. In addition, robust silencing symptoms were achieved in gerbera floral tissues by silencing the anthocyanin pathway gene for chalcone synthase (GCHS1) and a gerbera B-type MADS-box gene globosa (GGLO1), confirming the phenotypes previously observed in stable transgenic lines. Unexpectedly, photobleaching induced by GPDS and GChl-H or GChl-I silencing, or by the herbicide norflurazon, resulted in silencing of the polyketide synthase gene G2PS1, which has no apparent connections to carotenoid or chlorophyll biosynthesis. We have shown feasibility of VIGS for functional studies in gerbera, but our results also show that selection of the marker gene for silencing must be critically evaluated.

Short-term exposure to silver nano-particles alters the physiology and induces stress-related gene expression in Nelumbo nucifera.

Lotus (Nelumbo nucifera) was used as model plant in this study to explore its physiology and molecular response upon short-term exposure to silver nano-particles (AgNPs). Accumulation patterns demonstrated a potential uptake of AgNPs by roots and transport to the leaves as a likely key translocation route in lotus. AgNPs exposure was negatively correlated with lotus growth, including germination rate and petiole length in a concentration-dependent manner. Synthesis of chloroplast pigments in lotus leaves was enhanced by low AgNPs concentration, but were inhibited at high concentration. Hydrogen peroxide (H2O2) was detected in lotus leaves after AgNPs treatment. Proline accumulation in lotus leaves was induced with the increase in AgNPs concentration and exposure time. Antioxidant enzyme activities of superoxide dismutase (SOD), peroxidase (POD) as well as catalase (CAT) were enhanced after the first day of AgNPs exposure, but declined with increased exposure time, indicating a time-dependent toxicity of AgNPs. In addition, real-time PCR revealed that two detoxification-related genes, GSH1 and GST, could be activated on the first day of AgNPs exposure, but down-regulated with prolonged AgNPs treatment. Photosynthesis-related RbcS gene was up-regulated, however, no obvious difference in the expression of RbcL was observed after the first day of AgNPs exposure. Moreover, WRKY70a and WRKY70b transcription factors exhibited similar expression patterns, with the highest induction after a 5 mg/L AgNPs exposure on the first day, which decreased with prolonged exposure time. This study provides useful references for further evaluation of the toxic mechanism of AgNPs and their bio-effects on aquatic plants and ecosystems.

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.

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.

Jasmonate-Responsive Transcription Factors NnWRKY70a and NnWRKY70b Positively Regulate Benzylisoquinoline Alkaloid Biosynthesis in Lotus (Nelumbo nucifera).

Lotus (Nelumbo nucifera) is a large aquatic plant that accumulates pharmacologically significant benzylisoquinoline alkaloids (BIAs). However, little is known about their biosynthesis and regulation. Here, we show that the two group III WRKY transcription factors (TFs), NnWRKY70a and NnWRKY70b, positively regulate the BIA biosynthesis in lotus. Both NnWRKY70s are jasmonic acid (JA) responsive, with their expression profiles highly correlated to the BIA concentration and BIA pathway gene expression. A dual-luciferase assay showed that NnWRKY70a could transactivate the NnTYDC promoter, whereas NnWRKY70b could activate promoters of the three BIA structural genes, including NnTYDC, NnCYP80G, and Nn7OMT. In addition, the transient overexpression of NnWRKY70a and NnWRKY70b in lotus petals significantly elevated the BIA alkaloid concentrations. Notably, NnWRKY70b seems to be a stronger BIA biosynthesis regulator, because it dramatically induced more BIA structural gene expressions and BIA accumulation than NnWRKY70a. A yeast two-hybrid assay further revealed that NnWRKY70b physically interacted with NnJAZ1 and two other group III WRKY TFs (NnWRKY53b and NnWRKY70a), suggesting that it may cooperate with the other group III WRKYs to adjust the lotus BIA biosynthesis via the JA-signaling pathway. To illustrate the mechanism underlying NnWRKY70b-mediated BIA regulation in the lotus, a simplified model is proposed. Our study provides useful insights into the regulatory roles of WRKY TFs in the biosynthesis of secondary metabolites.

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.

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.