The water lily genome and the early evolution of flowering plants.

Water lilies belong to the angiosperm order Nymphaeales. Amborellales, Nymphaeales and Austrobaileyales together form the so-called ANA-grade of angiosperms, which are extant representatives of lineages that diverged the earliest from the lineage leading to the extant mesangiosperms1-3. Here we report the 409-megabase genome sequence of the blue-petal water lily (Nymphaea colorata). Our phylogenomic analyses support Amborellales and Nymphaeales as successive sister lineages to all other extant angiosperms. The N. colorata genome and 19 other water lily transcriptomes reveal a Nymphaealean whole-genome duplication event, which is shared by Nymphaeaceae and possibly Cabombaceae. Among the genes retained from this whole-genome duplication are homologues of genes that regulate flowering transition and flower development. The broad expression of homologues of floral ABCE genes in N. colorata might support a similarly broadly active ancestral ABCE model of floral organ determination in early angiosperms. Water lilies have evolved attractive floral scents and colours, which are features shared with mesangiosperms, and we identified their putative biosynthetic genes in N. colorata. The chemical compounds and biosynthetic genes behind floral scents suggest that they have evolved in parallel to those in mesangiosperms. Because of its unique phylogenetic position, the N. colorata genome sheds light on the early evolution of angiosperms.

Diverse O-methyltransferases catalyze the biosynthesis of floral benzenoids that repel aphids from the flowers of waterlily Nymphaea prolifera.

Nymphaea is a key genus of the ANA grade (Amborellales, Nymphaeales, and Austrobaileyales) of basal flowering plants, which serve as a key model to study the early evolution of floral traits. In this study, we comprehensively investigated the emission, biosynthesis, and biological function of the floral scent in a night-blossoming waterlily Nymphaea prolifera. The headspace volatile collection combined with GC-MS analysis showed that the floral scent of N. prolifera is predominately comprised by methylated benzenoids including anisole, veratrole, guaiacol, and methoxyanisole. Moreover, the emission of these floral benzenoids in N. prolifera exhibited temporal and spatial pattern with circadian rhythm and tissue specificity. By creating and mining transcriptomes of N. prolifera flowers, 12 oxygen methyltransferases (NpOMTs) were functionally identified. By in vitro enzymatic assay, NpOMT3, 6, and 7 could produce anisole and NpOMT5, 7, 9, produce guaiacol, whereas NpOMT3, 6, 9, 11 catalyzed the formation of veratrole. Methoxyanisole was identified as the universal product of all NpOMTs. Expression patterns of NpOMTs provided implication for their roles in the production of the respective benzenoids. Phylogenetic analysis of OMTs suggested a Nymphaea-specific expansion of the OMT family, indicating the evolution of lineage-specific functions. In bioassays, anisole, veratrole, and guaiacol in the floral benzenoids were revealed to play the critical role in repelling waterlily aphids. Overall, this study indicates that the basal flowering plant N. prolifera has evolved a diversity and complexity of OMT genes for the biosynthesis of methylated benzenoids that can repel insects from feeding the flowers. These findings provide new insights into the evolutional mechanism and ecological significance of the floral scent from early-diverged flowering plants.

Biosynthesis and emission of methyl hexanoate, the major constituent of floral scent of a night-blooming water lily Victoriacruziana.

Among the factors that have made flowering plants the most species-rich lineage of land plants is the interaction between flower and insect pollinators, for which floral scent plays a pivotal role. Water lilies belong to the ANA (Amborellales, Nymphaeales, and Austrobaileyales) grade of basal flowering plants. In this study, Victoria cruziana was investigated as a model night-blooming water lily for floral scent biosynthesis. Four volatile compounds, including three benzenoids and one fatty acid methyl ester methyl hexanoate, were detected from the flowers of V. cruziana during their first bloom, with methyl hexanoate accounting for 45 % of total floral volatile emission. Emission rates were largely constant before significant drop starting at the end of second bloom. To understand the molecular basis of floral scent biosynthesis in V. cruziana, particularly methyl hexanoate, a transcriptome from the whole flowers at the full-bloom stage was created and analyzed. Methyl hexanoate was hypothesized to be biosynthesized by SABATH methyltransferases. From the transcriptome, three full-length SABATH genes designated VcSABATH1-3 were identified. A full-length cDNA for each of the three VcSABATH genes was expressed in Escherichia coli to produce recombinant proteins. When tested in in vitro methyltransferase enzyme assays with different fatty acids, both VcSABATH1 and VcSABATH3 exhibited highest levels of activity with hexanoic acid to produce methyl hexanoate, with the specific activity of VcSABATH1 being about 15 % of that for VcSABATH3. VcSABATH1 and VcSABATH3 showed the highest levels of expression in stamen and pistil, respectively. In phylogenetic analysis, three VcSABATH genes clustered with other water lily SABATH methyltransferase genes including the one known for making other fatty acid methyl esters, implying both a common evolutionary origin and functional divergence. Fatty acid methyl esters are not frequent constituents of floral scents of mesangiosperms, pointing to the importance for the evolution of novel fatty acid methyltransferase for making fatty acid methyl esters in the pollination biology of water lilies.

Molecular breeding of water lily: engineering cold stress tolerance into tropical water lily.

Water lilies (order Nymphaeales) are rich in both economic and cultural values. They grow into aquatic herbs, and are divided into two ecological types: tropical and hardy. Although tropical water lilies have more ornamental and medicinal values compared to the hardy water lily, the study and utilization of tropical water lilies in both landscaping and pharmaceutical use is greatly hindered due to their limited planting area. Tropical water lilies cannot survive the winter in areas beyond 24.3°N latitude. Here, the transgenic pipeline through the pollen-tube pathway was generated for water lily for the first time. To improve cold stress tolerance of tropical water lilies, a gene encoding choline oxidase (CodA) driven by a cold stress-inducible promoter was transformed into a tropical water lily through the pollen-tube transformation. Six independent transgenic lines were tested for survival rate during two winter seasons from 2015 to 2017 in Hangzhou (30.3°N latitude). PCR and southern blot detection revealed that the CodA gene had been integrated into the genome. Reverse transcription PCR showed that CodA gene was induced after cold stress treatment, and further quantitative real-time PCR revealed different expressions among six 4 lines and line 3 had the highest expression. Multiple physiological experiments showed that after cold stress treatment, both the conductivity and malondialdehyde (MDA) levels from transgenic plants were significantly lower than those of non-transgenic plants, whereas the content of betaine and the activity of superoxide dismutase, catalase, and peroxidase were higher than those from non-transgenic plants. These results suggest that expression of exogenous CodA gene significantly improved the cold stress tolerance of tropical water lilies through a wide range of physiological alterations. Our results currently expanded a six-latitude cultivating area of the tropical water lilies. These results not only illuminate the bright future for water lily breeding but will also facilitate the functional genomic studies.

Water lilies as emerging models for Darwin's abominable mystery.

Water lilies are not only highly favored aquatic ornamental plants with cultural and economic importance but they also occupy a critical evolutionary space that is crucial for understanding the origin and early evolutionary trajectory of flowering plants. The birth and rapid radiation of flowering plants has interested many scientists and was considered 'an abominable mystery' by Charles Darwin. In searching for the angiosperm evolutionary origin and its underlying mechanisms, the genome of Amborella has shed some light on the molecular features of one of the basal angiosperm lineages; however, little is known regarding the genetics and genomics of another basal angiosperm lineage, namely, the water lily. In this study, we reviewed current molecular research and note that water lily research has entered the genomic era. We propose that the genome of the water lily is critical for studying the contentious relationship of basal angiosperms and Darwin's 'abominable mystery'. Four pantropical water lilies, especially the recently sequenced Nymphaea colorata, have characteristics such as small size, rapid growth rate and numerous seeds and can act as the best model for understanding the origin of angiosperms. The water lily genome is also valuable for revealing the genetics of ornamental traits and will largely accelerate the molecular breeding of water lilies.