taxonID	type	description	language	source
03EBF734A03016240125F9B0FD84F8AD.taxon	description	Combining headspace collection with gas chromatography – mass spectrometry (GC-MS) analysis, four volatile compounds were detected from the fully-opened flowers of V. cruziana during their first bloom (Fig. 1 A). These four compounds were identified to be methyl hexanoate, benzyl alcohol, benzyl 2 - methylbutanoate and benzyl tiglate. Except benzyl 2 - methylbutanoate, the chemical identities of the other three compounds were verified by comparing their chromatograms (Fig. 1 B) and mass spectra (Fig. 1 C) with those of authentic standards. Among these compounds, methyl hexanoate was the most abundant constituent accounting for 45.5 % of the total emission. Benzyl alcohol, benzyl 2 - methylbutanoate and benzyl tiglate accounted for 37.8 %, 11.3 %, and 5.4 % of the total emission, respectively. 2.2. Emission of floral volatiles from different parts of V. cruziana flowers To determine the contribution of different parts of V. cruziana flowers to floral scent profile, the flowers of V. cruziana were divided into sepals, petals, stamen and pistils. These four parts were subjected to headspace collection, separately. The four floral volatiles, i. e. methyl hexanoate, benzyl alcohol, benzyl 2 - methylbutanoate and benzyl tiglate, were detected from each of the four parts. (Fig. 2). Of the four parts, stamen showed the highest rates of total emission (36.42 ± 7.6 nmol / g / h). Methyl hexanoate exhibited the highest rates of emission from stamens at 36.31 ± 7.5 nmol / g / h (Fig. 2), followed by pistils (12.11 ± 3.8 nmol / g / h), petals (2.09 ± 1.6 nmol / g / h) and sepals (1.31 ± 1.24 nmol / g / h). In contrast, the three benzenoids exhibited the highest rates of emission from the petals with the emission rate of 10.99 ± 3.3 nmol / g / h (Fig. 2). Their emission rates from sepals, stamens and pistils were 3.64 ± 1.9 nmol / g / h, 0.11 ± 0.01 nmol / g / h and pistils (0.01 ± 0.001 nmol / g / h), respectively. 2.3. Emission dynamics of floral volatiles from V. cruziana In our observation, the flowers of V. cruziana opened fully at 19: 00 on the first day, began to close in the following morning and became completely closed between 12: 00 and 13: 00. The flowers started to reopen between 16: 00 and 17: 00 on the same day. The representative flowers at the four stages were shown in Fig. 3 A. To examine the emission dynamics, floral volatiles of V. cruziana were continuously collected (1 - h collection followed by a 3 - h interval) for two days (12 / 12 h light / dark) and analyzed by GC-MS. The emission rates of total volatiles remained constant before a significant drop towards the end of second bloom (Fig. 3 B). The emission rates of benzenoids and methyl hexanoate were also analyzed separately. The emission patterns of benzenoids were very similar to those of total volatiles (Fig. 3 C). In contrast, there was a reduction in mission rates of methyl hexanoate towards the end of first closure before increasing during the second bloom (Fig. 3 D). Like total volatiles, the emission of both benzenoids (Fig. 3 C) and methyl hexanoate (Fig. 3 D) dropped significantly at the of the end of second bloom and after that. 2.4. Identification of candidate genes for the biosynthesis of methyl hexanoate To understand the molecular basis of floral volatile biosynthesis in V. cruziana, an RNA-Seq library using the RNA samples of whole flowers of V. cruziana at full blooming stage was constructed and sequenced. High quality sequencing data was obtained, and the clean bases of flower and leaf were 6.11 G (Table S 1). The de novo assembly yielded 64,472 unigenes (Table S 1). The unigenes longer than 1 k bp were 34483 that accounted for 53.49 % of the total unigenes and the N 50 was 2476 bp (Table S 1). More than 63.51 % unigenes were annotated in at least one database, and 16 % unigenes were annotated in all seven bioinformatics databases (Table S 1). The annotated unigenes were clustered in three GO function classification (biological process, cellular component, molecular function, Fig. S 1). Next, the transcriptome was specifically searched for candidate biosynthetic genes. This study focused on the biosynthesis of methyl hexanoate. Prior to this study, the biosynthesis of fatty acid methyl esters has been studied in another water lily N. colorata, in which methyl decanoate and methyl octanoate are biosynthesized by methyltransferases that belong to the SABATH family (Zhang et al., 2019). It was our hypothesis that the formation of methyl hexanoate in V. cruziana is also catalyzed by SABATH enzymes. As such, the flower transcriptome of V. cruziana was created (Fig. S 1) and searched for SABATH genes. Three putative full-length SABATH genes were identified, which were designated as VcSABATH 1, VcSABATH 2 and VcSABATH 3 with GenBank accession number of MZ 541994, MZ 541995 and MZ 541996, respectively. The proteins they encode are 364 (VcSABATH 1), 367 (VcSABATH 2), and 362 (VcSABATH 3) amino acids in length. Multiple sequence alignment revealed that all three VcSABATH proteins contain conserved residues for interacting with the carboxyl moiety of the substrate and the methyl donor S - adenosyl-L-methionine (Fig. 4). 2.5. Biochemical assay of VcSABATHs Next, in vitro methyltransferase enzyme assays were performed to determine which of the three VcSABATH genes is responsible for the biosynthesis of methyl hexanoate in V. cruziana flowers. A full-length cDNA for each of the three VcSABATH genes was synthesized and cloned into a protein expression vector (pET 32 a) and expressed in E. coil to produce recombinant enzymes. Individual recombinant VcSABATH proteins were tested in methyltransferase enzyme assays using hexanoic acid as substrate. While VcSABATH 2 was inactive, both VcSABATH 1 and VcSABATH 3 catalyzed the formation of methyl hexanoate (Fig. 5). Next, E. coli - expressed recombinant VcSABATH 1 and VcSABATH 3 were purified (Fig. S 2) and the purified proteins used to measure their respective specific activities. The specific activity of VcSABATH 1 and VcSABATH 3 using hexanoic acid as substrate was determined to be 23.2 ± 2.41 pkat / mg protein and 150.4 ± 3.6 pkat / mg protein, respectively. To determine substrate specificity of VcSABATH 1 and VcSABATH 3, they were also tested using three other fatty acids as substrate. The relative activity of VcSABATH 1 using octanoic acid, decanoic acid or dodecanoic acid as substrate was 39.4 %, 33.6 % and 16.9 % of that with hexanoic acid respectively. Similarly, the relative activity of VcSABATH 3 using octanoic acid, decanoic acid or dodecanoic acid as substrate was 33.5 %, 38.8 % and 17.3 % of that with hexanoic acid respectively. 2.6. Expression of VcSABATH 1 and VcSABATH 3 in different flower organs With VcSABATH 3 and VcSABATH 1 demonstrated to encode hexanoic acid methyltransferase, their expression in different parts of V. cruziana flowers was measured using reverse transcriptionquantitative PCR (RT-qPCR). The highest level of expression of VcSABATH 1 was detected in stamens (Fig. 6 A), whereas VcSABATH 3 showed the highest level of expression in pistils (Fig. 6 B).	en	Jiang, Yifan, Liu, Guanhua, Zhang, Wanbo, Zhang, Chi, Chen, Xinlu, Chen, Yuchu, Yu, Cuiwei, Yu, Dongbei, Fu, Jianyu, Chen, Feng (2021): Biosynthesis and emission of methyl hexanoate, the major constituent of floral scent of a night-blooming water lily Victoria cruziana. Phytochemistry (112899) 191: 1-10, DOI: 10.1016/j.phytochem.2021.112899, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112899
03EBF734A03016240125F9B0FD84F8AD.taxon	discussion	2.7. Phylogenetic analysis Prior to this study, the SABATH family of methyltransferases in plants has been relatively well studied with a number of members characterized (D’ Auria et al., 2003). These include salicylic acid MT (Ross et al., 1999; Chen et al., 2003, Zhao et al., 2010), jasmonic acid MT (Seo et al., 2001; Zhao et al., 2013), indole- 3 acetic acid MT (Qin et al., 2005; Zhao et al., 2007, 2008), gibberellic acid MT (Varbanova et al., 2007; Zhang et al., 2020). To understand the evolutionary relatedness of the two fatty acid methyltransferases from V. cruziana (VcSABATH 1 and VcSABATH 3) with known SABATH proteins, phylogenetic analyses were performed. Three VcSABATHs form a water lily-specific cluster with 6 full-length SABATH members from N. colorata except the putative IAMT (Nc 8 G 0124910 - 1) (Fig. 7). VcSABATH 1 was most closely related to three NcSABATHs including Nc 11 G 0120830 - 1 which was characterized to be decanoic acid methyltransferase (Zhang et al., 2019). VcSABATH 2 and VcSABATH 3 were most closely related to each other and together with two NcSABATHs of unknown functions they form a subcluster in water lily-specific cluster (Fig. 7).	en	Jiang, Yifan, Liu, Guanhua, Zhang, Wanbo, Zhang, Chi, Chen, Xinlu, Chen, Yuchu, Yu, Cuiwei, Yu, Dongbei, Fu, Jianyu, Chen, Feng (2021): Biosynthesis and emission of methyl hexanoate, the major constituent of floral scent of a night-blooming water lily Victoria cruziana. Phytochemistry (112899) 191: 1-10, DOI: 10.1016/j.phytochem.2021.112899, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112899
03EBF734A03416270273F9E5FB65F8DF.taxon	materials_examined	V. cruziana Orbign. (Nymphaeaceae) plants were grown in square ponds of 80 cm in depth in Hangzhou (N 30 ◦ 6 ′ 54 ′′, E 120 ◦ 6 ′ 36 ′′), China. Plants were applied with constant conditions of fertilization, irrigation, disease prevention, and pesticide application. The flowers were collected on the first day of full blooming.	en	Jiang, Yifan, Liu, Guanhua, Zhang, Wanbo, Zhang, Chi, Chen, Xinlu, Chen, Yuchu, Yu, Cuiwei, Yu, Dongbei, Fu, Jianyu, Chen, Feng (2021): Biosynthesis and emission of methyl hexanoate, the major constituent of floral scent of a night-blooming water lily Victoria cruziana. Phytochemistry (112899) 191: 1-10, DOI: 10.1016/j.phytochem.2021.112899, URL: http://dx.doi.org/10.1016/j.phytochem.2021.112899
