Volatile 1-octanol of tea (Camellia sinensis L.) fuels cell division and indole-3-acetic acid production in phylloplane isolate Pseudomonas sp. NEEL19

Tea leaves possess numerous volatile organic compounds (VOC) that contribute to tea’s characteristic aroma. Some components of tea VOC were known to exhibit antimicrobial activity; however, their impact on bacteria remains elusive. Here, we showed that the VOC of fresh aqueous tea leaf extract, recovered through hydrodistillation, promoted cell division and tryptophan-dependent indole-3-acetic acid (IAA) production in Pseudomonas sp. NEEL19, a solvent-tolerant isolate of the tea phylloplane. 1-octanol was identified as one of the responsible volatiles stimulating cell division, metabolic change, swimming motility, putative pili/nanowire formation and IAA production, through gas chromatography-mass spectrometry, microscopy and partition petri dish culture analyses. The bacterial metabolic responses including IAA production increased under 1-octanol vapor in a dose-dependent manner, whereas direct-contact in liquid culture failed to elicit such response. Thus, volatile 1-octanol emitting from tea leaves is a potential modulator of cell division, colonization and phytohormone production in NEEL19, possibly influencing the tea aroma.

Tea is one of the most popular beverages in the world, ranked second among non-alcoholic drinks. The nonvolatile and volatile compounds respectively determine the taste and aroma of tea 1 . Extensive research has been done characterizing volatile and non-volatile compounds, which together govern the flavor and quality of tea. Organic acids, sugars, free amino acids have been commonly identified in the non-volatile pool, whereas aldehydes, alcohols, ketones, sesquiterpenes and furans were found in the volatile fraction of tea [1][2][3][4][5][6][7][8][9] . Volatile organic compounds (VOC) released by plants can shape plant-associated microbial communities by exerting growth-inhibiting and/or promoting attributes 10,11 . Plant VOC usually exhibit antimicrobial properties and thought to act as defense agents 10,[12][13][14][15][16] . Plant VOC can repel herbivores or attract herbivore predators 17 . VOC such as methanol and monoterpenes were known to serve as the carbon source for plant-associated bacteria 10,[18][19][20] .
The interaction between plant VOC and phyllosphere microbes are bidirectional, and hence deserves more attention 10 . VOC composition of plants can be manipulated by plant-associated microbes and herbivorous insects 11,21 . To date, around 600 volatile compounds have been reported from tea 1 . VOC of tea phyllosphere exhibited insect attractant 22,23 and antimicrobial attributes 24 . However, the impact of tea VOC on bacteria that colonize the phylloplane remains unknown.
Some strains of Pseudomonas tolerate organic solvents in liquid cultures [25][26][27][28][29] . Representatives of Pseudomonas are one of the most abundant taxa colonizing the plant leaves 30 . Indole-3-acetic acid (IAA), a phytohormone capable of controlling several aspects of plant growth and development, can be synthesized by some Pseudomonas 31,32 . IAA modulates secondary metabolism and adventitious root formation in plants 33 . It mediates plant-microbe relationship and influences bacterial physiology 32,34 . A better understanding of chemical crosstalk between plant VOC and bacterial IAA producer may allow enhancement of tea plant growth and physiology, leading to improved flavor.
Here, we hypothesized that the solvent-tolerant strain associated with tea leaves can sense VOC and selfmodulate the biofilm and phytohormone formation. The hypothesis was tested by using an alcohol-tolerant phylloplane isolate Pseudomonas sp. NEEL19 and crude aqueous tea extract (TeaAq) that emit volatiles.

Results
Molecular identification and phylogenetic characterization. NEEL19 shared 100% 16S rRNA gene sequence similarity with Pseudomonas juntendi BML3 T , a bacterium of clinical origin 35 . It also shared ≥ 99.0% sequence similarities with sixteen Pseudomonas species and several genome-sequenced isolates (Table S1). However, in the neighbor-joining tree, NEEL19 established a distinct and strong phyletic lineage with P. alloputida VKh7 T (99.8% similarity; 74% bootstrap support), isolated from the bean rhizosphere 36 ( Figure S1). Phylogenetic linkages were also seen establishing with a clinical isolate P. mosselii CIP 105259 T37 , and solvent-tolerant P. putida strains BIRD-1 25 , S12 27,38 and KT2440 39 . Thus, NEEL19 was identified as a Pseudomonad, but a specieslevel distinction was not possible due to the limited variation in inter-species and intra-species 16S rRNA gene sequences in this diverse genus.
Chemical characterization of crude aqueous extract of fresh tea leaves (TeaAq). Clevenger apparatus ( Figure S5) was used to extract TeaAq through hydrodistillation from tea leaves. TeaAq appeared pale yellow with a distinct odor indicating the presence of VOC. The fourier transform infrared spectrometry (FT-IR) data revealed signatory infrared vibrational bands of aromatics and alcohols in TeaAq (Fig. 2a,b). Gas chromatography-mass spectrometry (GC-MS) detected several phytochemicals including 1-octanol in TeaAq (Fig. 2c, Table S3), whereas no traces of ethanol and 1-propanol were found. The presence of 1-octanol was verified through the retention time and mass profile of standard ( Fig. 2d-e).
Impact of OcV on cell motility. The colony diameter of NEEL19 reached ~ 2 cm within 36 h at 0.3% (w/v) agar, indicating rapid swimming motility on full-strength nutrient media ( Figure S7a). However, swarming and twitching motilities were absent as determined at 0.5% and 1.0% agar (w/v), respectively ( Figure S7b-c).

Discussion
Pseudomonas sp. NEEL19 isolated from tea phylloplane shared a high 16S rRNA gene sequence similarity and phylogenetic association with Pseudomonads of plant 36 and clinical 35,37 origins. Therefore, experiments were performed on NEEL19 to identify possible plant beneficial features and drug resistance. The co-occurrence of plant growth-promotive traits and antibiotic resistance detected in NEEL19 was in line with our earlier report on a plant-associated bacterium Burkholderia sp. LS-044 41 . The ability to utilize diverse compounds as sole carbon and energy sources suggested a remarkable nutritional versatility of NEEL19. Genome sequencing may shed more light on the poorly resolved taxonomic status and genetic make-up of NEEL19.
NEEL19 also exhibited phylogenetic proximity to solvent tolerant Pseudomonas strains such as S12 27,38 , BIRD-1 25 , KT2440 39 , VLB120 40 and IH-2000 28 . Distinguishing growth trends of NEEL19 recorded at 0.5-5% (v/v) ethanol and 1-octanol treatments suggested distinct impacts of these two alcohols on cell growth. Cells were short with a rough surface when treated with 1-octanol, whereas elongated with a smooth surface when exposed to ethanol, in liquid cultures. Ethanol-driven increment in cell size indicated solvent tolerance in NEEL19 since enhancement in cell size was reported to be an adaptive feature of solvent-tolerant strains 26 . In contrast, reduction in cell size presumably provides an increased surface area-to-volume ratio for the transport of long C-chain (≥ 3 C) alcohols. www.nature.com/scientificreports/ The tea leaves have been studied extensively for their VOC using various extraction and analytical techniques [1][2][3][4][5][6][7][8][9]42 . Hydrodistillation in the Clevenger apparatus facilitated the entrapment of tea VOC in aqueous form. While FT-IR provided a preliminary indication of functional groups, GC-MS facilitated specific identification of 1-octanol in TeaAq. Detection of 1-octanol was in line with earlier reports on tea leaves [2][3][4][5]9 .
Solvent-tolerant strains have been studied earlier by directly incorporating target solvents into liquid cultures [25][26][27][28][29][38][39][40] . In contrast, we carried out a detailed investigation on the impact of vapors of volatile compounds on a solvent-tolerant strain besides performing the solvent-emended liquid cultures. AB dye reduction assay, which involves a redox metabolic indicator resazurin 43 , was employed to probe bacterial cell viability, whereas phenol red assay was used to monitor media pH. 1-octanol emitting from TeaAq was identified as one of the responsible molecules modulating bacterial replication and metabolism (including IAA formation) through cell culture and colorimetric assays. Bacteria may produce IAA in Trp-dependent or independent manner 32,34 . Trp boosted the IAA formation in NEEL19 while concurrently suppressing the cell division under OcV and TeaV treatments. Thus, NEEL19 most likely to invest volatile 1-octanol emitting from tea leaves on IAA production than on cell replication in the presence of Trp.
Alcohols promote the biofilm formation in Pseudomonas presumably by modifying the cell surface 29 . OcV and TeaV exposures had a similar impact on bacterial cell size, surface features and putative intercellular pili/nanowire formation, indicating a definite role played by 1-octanol in the transformation. Furthermore, OcV was found to promote swimming motility, which influences bacterial colonization and biofilm formation. Bacterial pili act as nanowires promoting cell-cell aggregation and electroactive biofilm formation in Geobacter 44 . However, pili of P. aeruginosa reported to lack conductance 45 , possibly reflecting their main involvement in motility and establishment of biofilm than electron transport. IAA was reported to induce filamentation in Saccharomyces cerevisiae 46 and promote biofilm formation in Escherichia coli 47 . Therefore, the impact of OcV-driven IAA formation on the development of pili/nanowire and biofilm in NEEL19 warrants further investigation.
The impact of the vapors of 1-octanol on NEEL19 was assessed in detail to further ascertain its bioactivity. Cell count, viability, media alkalization and IAA formation were increased in NEEL19 when exposed to OcV in a dose-dependent manner. Thus, for a given amount of volatile 1-octanol emission, solvent-tolerant Pseudomonas would produce more biomass and IAA than solvent-sensitive microbiota, possibly manipulating the tea plant physiology and secondary metabolism.
OcAq failed to trigger bacterial Trp-dependent IAA production, whereas TeaAq boosted the formation of IAA. Results indicated the prerequisite of 1-octanol in vapor form for IAA production. On the other hand, multiple phytochemicals available in TeaAq most likely contributed to IAA formation in liquid cultures. The study highlighted the significance of the volatile micro-niche of tea phylloplane, where alcohol-phytohormone exchange occurs between the host and its inhabitant (Fig. 7).

Materials and methods
Reagents and chemicals. High purity (> 99.0%) methanol, ethanol, 1-propanol, 1-butanol and 1-octanol were obtained from Fisher Scientific (Leicestershire, UK). IAA and phenol red were purchased from Sigma Aldrich. AB dye was obtained from Invitrogen.
Extraction of tea leaf aqueous extract. Fresh and healthy tea leaves were collected from a tea plantation of Nantou County, Yuchi township, Taiwan (23°52′47"N, 120°54′46"E) on 19th November 2018. Leaves (100 g) Figure 7. Schematic representation of alcohol-phytohormone exchange occurring between tea phylloplane and its bacterial inhabitant discovered in this study. Images of tea plantation, scanning electron microscopy of NEEL19 and sample vials were from this study. www.nature.com/scientificreports/ were introduced into 500 ml deionized water and subjected to conventional hydrodistillation in a Clevenger apparatus ( Figure S5) for 2 h according to European Pharmacopoeia method 48 . Aqueous tea extract (TeaAq, 15 ml) was isolated, aliquoted and preserved at − 20 °C. Plant growth-promotive traits and biochemical features. N 2 fixation and IAA production were determined by acetylene reduction assay 51,52 and colorimetry 53 , respectively. Siderophore production was tested on CAS agar 54 . DNase activity was assessed using DNase test agar (Himedia). Catalase and oxidase activities, and hydrolysis of starch (0.2%, w/v) were determined as described elsewhere 55 . Biochemical and enzymatic analyses were performed using API 20 NE, API 20 E and API ZYM (bioMérieux) strips. Antibiotic resistance was tested using ATB staph strip (bioMérieux). Carbon source utilization was determined using GN2 MicroPlate (Biolog). Kit-based tests were done following the manufacturers' protocol. Bacterial growth, viability, media acidity/alkalinity and IAA production under the supply of carbon sources in vapor form. Culture microfuges exposed to vapors were retrieved from PPD after 30 h of incubation. Cell suspensions were transferred to a 96-well microplate for OD 600 measurements in a microplate reader (Biochrom Asys UVM 340). Cells grown on NA (Himedia) were counted (CFU ml −1 ). AB dye reduction (%) was estimated according to the manufacturer's protocol by introducing 10% (v/v) AB to culture suspension in a microplate, followed by reading the plates at 570 and 600 nm. Media acidity/alkalinity and IAA production were determined in cell-free culture supernatants using microplates. Acidity and alkalinity were probed by adding 10% (v/v) phenol red to the supernatant, and reading the plates at 415 and 560 nm, respectively. IAA production was assessed colorimetrically 53 with the following modifications: One-fold supernatant was mixed with four-fold Salkowski reagent in a 96-well microplate, incubated for 15 min at room temperature and read at 530 nm. IAA was quantified using a standard curve plotted for the IAA standard.

FT-IR and GC-MS analysis.
Motility assay. Preliminary experiments were carried on full-strength NB (Himedia) supplemented with 0.3, 0.5 and 1.0% (w/v) agar for swimming, swarming and twitching motilities 56,57 , respectively. Subsequently, motility was assessed on PPD pre-casted with 0.3% (w/v) agar-supplemented DSMZ 125* at the cell-compartment. 1-octanol (75 µl) was placed in the volatile carbon-compartment and plates were inoculated and sealed tightly with an insulation tape. Plates were incubated for 72 h at 30 °C and colony diameters were measured. Inoculated 1-octanol-free plates were used as control.
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