Connecting moss lipid droplets to patchoulol biosynthesis

Anantha Peramuna; Hansol Bae; Carmen Quiñonero López; Arvid Fromberg; Bent Petersen; Henrik Toft Simonsen

doi:10.1371/journal.pone.0243620

. 2020 Dec 7;15(12):e0243620. doi: 10.1371/journal.pone.0243620

Connecting moss lipid droplets to patchoulol biosynthesis

Anantha Peramuna ¹, Hansol Bae ^1,², Carmen Quiñonero López ¹, Arvid Fromberg ³, Bent Petersen ^4,⁵, Henrik Toft Simonsen ^1,^2,^*

Editor: Björn Hamberger⁶

PMCID: PMC7721168 PMID: 33284858

Abstract

Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. Here, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Three different LD-proteins: Oleosin (PpOLE1), Lipid Droplet Associated Protein (AtLDAP1) and Seipin (PpSeipin325) were tested as anchors. Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis. The expression of PTS physically linked to Seipin increased the LD size and the retention of patchoulol in the cell. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants.

Introduction

Plant-derived specialized metabolites such as terpenoids are exploited for their value in fragrances, flavors, and pharmaceutics industries [1]. The natural productions of several of these fragrant terpenoids are unsustainable due to agricultural and environmental factors, which is reflected in excessive use of water and the wholesale price. Patchouli oil, where the major component is patchoulol from Pogostemon cablin has a fluctuating market price of 30–200 US dollars/Kg. Production of terpenoids in a heterologous expression system (yeast, algae or moss) has had some successes [2–6]. However, high yields and efficient extraction methods comparable to the native plants are still a major challenge. Native plants have evolved to produce and store volatile terpenoids in specialized cells [7, 8]. Heterologous expression systems lack the cellular specialty needed to contain and produce high amounts of these compounds without being toxic to the cell [9, 10]. Thus, an alternative way to produce and store compounds inside expression platforms was investigated.

We explored a novel approach and targeted terpenoids into plant lipid droplets (LDs) that was recently tested by transient expression in Nicotiana benthamiana. However, an algae lipid droplet protein was used, and the results were not conclusive possibly due to the change in carbon flow [11]. Plant cytosolic LDs are dynamic organelles that originate from the endoplasmic reticulum (ER) [12–14]. LDs consist of a hydrophobic core surrounded by a mono-lipid-layer membrane and structural proteins that maintain the membrane stability of LDs [15]. P. patens LDs are found in the vegetative gametophytes and are abundant in the spores. LDs store triacylglycerol (TAG) and steryl esters (SE) in a liquid form as an energy source [16]. The lipophilic environment attracts small hydrophobic compounds produced in its proximity. Since LDs are naturally buoyant, they will upon lysis of the cell separate from the rest by centrifugation, a property that lowers the downstream purification cost. In this work, we overexpressed patchoulol synthase (PTS), which is a sesquiterpene synthase that catalyze the production of patchoulol from farnesyl diphosphate in the cytosol. PTS and patchoulol originates from the scrub Pogostemon cablin [2]. We also overexpressed PTS physically attached to three LD related proteins, which are a part of the LD biogenesis (oleosin, seipin and lipid droplet associated protein also known as small rubber particle protein) to produce the sesquiterpenoid patchoulol close to LDs. These three groups of proteins were selected based on their diverse structure and LD binding properties.

Oleosins have been found to be the most abundant protein in the P. patens LD proteome [16]. They are attached to the cytosolic side of the ER and here support a hairpin loop on the membrane surface that leads to the emerging LDs [16, 17]. The support of the hairpin loop supports sequestration of neutral lipids from the ER to form the LD [18]. Following this sequestration oleosins together with other proteins supports the detachment of the LDs from the ER and release into the cytosol [18]. Here oleosins supports the structure of the LDs and hinder a formation of large LDs [14].

Seipin proteins are known to play a structural role in LD biogenesis in yeast and Arabidopsis [19, 20]. Seipins have multiple membrane-spanning helices that localize the protein to the ER membrane. Although the function of seipins is unclear due to conflicting results among different organisms [21], seipins have shown to effect the accumulation of TAG in LDs of Arabidopsis [19]. Currently, no work has been performed for seipins in non-vascular plants.

Unlike oleosin and seipin, Lipid Droplet Associated Protein (LDAP) (also known as small rubber particle protein (SRPs)) does not contain any transmembrane domain. LDAPs are mainly folded as a helical protein with self-polymerization properties that is capable of covering the LD surfaces without the disruption of the LD membrane structure [22, 23]. LDAPs are stress-related and required for proper growth in most plants and are found in cells both with and without LDs [14]. Previous works in Arabidopsis and tobacco have shown that AtLDAP1 is localized to the LDs by covering the membrane surface of the LD [24–26].

Here, we also verified the localization of oleosin and LDAP1 to the LDs and seipins to the ER tubules in P. patens. Furthermore, by utilizing these LD-associated proteins as an anchor, we showed that the expression of LD protein-attached patchoulol synthase (PTS) led to a higher content of patchoulol in the LD as compared to the expression of free PTS.

Materials and methods

Plant material, growth conditions, and transformation

Wild type Physcomitrium patens (syn. Physcomitrella patens, Gransden ecotype) was obtained from the International Moss Stock Center at the University of Freiburg (http://www.moss-stock-center.org/). Growth conditions and transformation processes are similar to those previously published [27].

Strain construction

Transformation of P. patens was performed according to the protocol previously published [27–29] Approximately, 1.5 g (fresh weight) P. patens cells cultivated for five days were digested with 20 mL of 0.5% DriselaseR enzyme in 8.5% D-mannitol for 45 minutes with gentle shaking. Digested cells, filtered through a 100 μm pored mesh, were pelleted by centrifugation for 4 minutes at 150 x g. The pelleted protoplasts were washed and pelleted twice with a wash solution (8.5% D-mannitol, 10 mM CaCl₂). Washed protoplasts were concentrated to 1.6 × 10⁶ protoplasts/mL and suspended in MMM solution (9.1% D-mannitol, 10% MES, and 15 mM MgCl₂). Protoplasts were transformed by mixing 300 μL of protoplast with an equal amount of PEG and 5μg of total DNA. The mixture was incubated for 5 minutes at 45°C followed by another 5 minutes at room temperature. PEG in the mixture was diluted by adding 300 μL of 8.5% D-mannitol five times with 1 minute intervals in between, followed by adding 1 mL of 8.5% D-mannitol five more times. Transformed protoplasts were pelleted by centrifugation and the resulting pellet was resuspended in 500 μL of 8.5% D-mannitol and 2.5 mL of PRMT. The cells were spread on multiple PRMB solid media plates with a cellophane overlay. The cells were incubated overnight in a dark chamber and moved to continuous light for 5–7 days at 23°C. The regenerated protoplasts were transferred to PhyB media containing the appropriate selection marker for 2- weeks. After two weeks of selection, the cellophane was transferred for two more weeks on PhyB media and back on to selection for two weeks. Stable transformants were genotyped by PCR and transferred into PhyB media. A detailed description of this protocol is published in [28, 30]. Above transformation method yielded the following stable moss mutants for this experiment (for sequences see below): ZmUbi:PpOle1, ZmUbi:PpOle1-Venus, ZmUbi:PpOle1-PTS, ZmUbi:PpOle1-LP4/2A-PTS, ZmUbi:PpSeipin325, ZmUbi:PpSeipin325-Venus, ZmUbi:PpSeipin325-PTS, ZmUbi:AtLDAP1, ZmUbi:AtLDAP1-Venus, ZmUbi:PpLDAP1-PTS, ZmUbi:PTS (also see S1 Table in S1 File). Coding sequences of all the DNA fragments were inserted into the P. patens neutral 108 loci, with G418 selection cassette, under the Maize Ubiquitin 1 promoter and OCS terminator (S1 Fig in S1 File). The primers used are listed in the S2 Table in S1 File.

DNA fragments and genes

PpSeipin325 (Pp1S325_14V6.1) and PpOleosin1 (Pp1S84_138V6.1) (named PpOle1) was amplified from P. patens cDNA and chosen based on previous work on expression data and presence in all tissues of development [16]. The coding sequence of Arabidopsis thaliana lipid droplet associated protein 1 (AtLDAP1, At1g67360) and oleosin attached to PTS via LP4/2A linker was obtained using Thermofisher DNA synthesis services. The patchoulol synthase (PTS, AAS86323) was amplified using the pUNI33 PTS plasmid [2], and LP4/2A was obtained from DNA fragments previously shown to be functional in P. patens [30]. The G418 selection cassette and Maize Ubiquitin 1 promoter was amplified from the pMP1355 vector [30].

Microscopy

LDs were stained using either 0.5 μg/ml of BODIPY 505/515 (Invitrogen Molecular Probes) or LipidTOX™ Red Neutral Lipid Stain 577/609 (ThermoFisher). Images of BODIPY stained LDs were obtained with a Leica Las AF confocal laser microscope using a 488 nm laser excitation. A window of 510–530 nm was used for LD visualization and a window of 650–700 nm window was used to visualize the auto-fluorescence of chloroplasts. For co-localization studies, the yellow fluorescent protein Venus was excited with a 514-laser line and visualized with a window of 520–550. The LD staining dye HCS LipidTOX™ Red Neutral Lipid Stain was excited with the 594-laser line and visualized with a window of 605–630 nm. For the visualization of the ER, ER-Tracker™ Red (BODIPY™ TR Glibenclamide) dye was used. The emission and excitation parameters for ER-Tracker™ Red were similar to HCS LipidTOX™ Red Neutral Lipid Stain. Z-stacks were performed on all images with a line average of four. All the Z-stacks were combined using the Leica maximum projection function and Imaris software and processed using Imaris 9.1 (Oxford Instruments) and ImageJ [31] data analysis software. The size of the LDs was quantified by imaging with at least ten random images of multiple cells from biological triplicates. See Figs 1–3 for examples on how the pictures was zoomed in for the measurement, all pictures for LD size measurements is given in S3 File. The diameter of each non-overlapping LDs was measured using ImageJ and the size of the LDs was calculated using the volume of a sphere (V = 4/3 πr³).

Fig 1 — (A) Microscopy images of WT protonema cells grown in for 10 days (i) and WT gametophytes grown for 6 weeks (ii). Stained with BODIPY (green) and overlaid with chloroplast autofluorescence (red), showing the presence of cytosolic LDs. (B) (i) Image depicts 3D projection of surface rendering Z-stack image of a portion of a cell stained with BODIPY and ER-Tracker™ Red dye was used. and overlaid with red chloroplast autofluorescence (ii) BODIPY stained LDs in green overlaid with red chloroplast autofluorescence after removing the ER tracker stained Z-stack image. The right-hand side shows a model for the localization of LD-associated proteins. Seipin oligomers localize in the ER at the ER-LD contact site. Lipid droplet associated proteins mainly fold as a helical protein and covers the LD surface without disturbing the LD membrane. Oleosin proteins penetrate the LD membrane into the LD matrix with a long conserved hydrophobic hairpin structure.

Fig 3 — A) Processed confocal Z-stacks of Venus tagged AtLDAP1 (yellow), LipidTOX red-stained LD (green) and subsequent overlap of the two micrographs (scale = 10 μm). B) 3D projection of surface rendering Z-stack image of the stained LDs (green) with chloroplast auto-fluorescent (red) and subsequent overlap of Venus localization of AtLDAP1.

Metabolite extraction and GC-MS analysis

Biological triplicates of P. patens lines were grown for three weeks in 20 mL liquid PhyB (BCDAT) media under standard conditions, after which the tissues were washed, isolated and ground for patchoulol quantification inside the cells. For patchoulol quantification in the P. patens lines were extracted with an equal amount of 100% ethyl acetate by sonication for one hour. For patchoulol quantification outside of the cells, 10 mL of media was extracted with 2mL of 100% ethyl acetate after rapid shaking for 10 minutes. After phase separation 200 μL of the organic phase was loaded into GC vials and 1 μL was injected into the GC-MS. Patchoulol was quantified by using a standard curve (see S2 Fig in S1 File) of the authentic patchoulol standard, and the final concentration of patchoulol per dry weight was calculated. GC-MS analysis was performed on a GC-MS equipped with a ZB-5ms column (30 m x 0.25 mm x 0.25 μm) as previously described [2]. The samples were injected on a non-split mode using the following program: The initial temperature of the oven was held for 1 minute at 50°C. Then, the temperature was increased to 320°C at 15°C/min and held at 320°C for 5 minutes. The total run took 24 minutes. The injector temperature was set at 250°C, a temperature that can lead to rapid degradation with sesquiterpenoids [32]. This was however not the case and the downstream analysis was not affected [32]. The ion source temperature of the mass spectrometer was at 230°C. The obtained data obtained were compared with the standard for verification and quantification purposes.

PTS expression analysis by RT-PCR

RNA transcription level for patchoulol synthase was measured by extracting the total RNA from 7-day-old cells, using Spectrum™ Plant Total RNA Kit (Sigma, STRN250). A total of 500 ng of each RNA sample was reverse transcribed to cDNA with the iScript^TM cDNA Synthesis kit (BIO-RAD). RNA extraction was performed on biological triplicates for each P. patens mutant line. RT-PCR was performed using QuantiFast® SYBR® Green PCR (Qiagen) according to the manufacturer’s protocol. The following amplification program was used: 95°C 5 min, 40 cycles at 95°C 10 s followed by 60°C 30 s. Samples were amplified in duplicates from the same RNA isolation. RT-PCRs were performed using three biological replicates with qPCR primers designed for PTS, actin, and tubulin (S2 Table in S1 File). RT-PCR efficiency, E, was estimated for each gene by generating standard curves by plotting quantification cycle (Cq) values (y) against the log of a serial cDNA dilution (x). For this, a cDNA sample was used as template in a range of 25, 5, 2.5, 1.2 and 0.6 ng. The RT-PCR efficiencies were calculated from the slope of the linear regression equations of the standard curves, along with the regression coefficient (R2). The equation used was: E = 10(–1/a), E values in a range of 1.90–2.10 (PCR efficiency between 90% and 110%) with a regression coefficient below 0.02 are acceptable. All PCR efficiencies displayed between 100% and 115%.

Growth measurement

Plant height was measured on biological triplicates of wild type (WT), and the ZmUbi:AtLDAP1-PTS and ZmUbi:PTS lines. These were grown for 20 days in BCD liquid media. Cell lines were imaged using 5X bright field microscopy by attaining at least 15 random images of each cell line. The plant height of all the gametophytes with a minimum of two nodes in the image was calculated using ImageJ software by drawing a segmented line along the length of the stem manually. Measuring tool is freely available in the Image J software.

Lipid droplet extraction

Protoplasts of biological triplicates were isolated according to a previously published protocol [28] from cells that were cultivated in PhyB liquid media for three weeks. Washed protoplasts were lysed through osmotic pressure by adding 10 mL of deionized water. Lysed cells were pelleted at 5,800 x g for 10 minutes and the supernatant was mixed with sucrose to a final concentration of 0.3 M. The mixture was loaded into an ultracentrifuge tube with a 2 mL 150 mM KCl layer on top and centrifuged for 40 minutes at 135,000 x g in a swinging bucket rotor. The top 1.5 ml of the KCl layer containing the LDs were isolated using a glass tube and transferred into another ultracentrifuge tube for further purification. The isolated 1.5 mL of 150 mM KCl with LDs was carefully overlaid with a 10.5 mL of 10 mM KCl followed by 2 mL of diH₂O. This mixture was further centrifuged by ultracentrifugation using the same settings as above. Purified LDs on the top of the water layer were confirmed by BODIPY staining. The purified LDs were extracted with ethyl acetate and the extracts were analyzed using GC-MS. The GC-MS analysis was performed on a Thermo Scientific TSQ 8000 Evo with a TriPlus RSH Autosampler equipped with agitation oven and needle bake out option. The injection port on the GC was set at 200°C, with a splitless time of 3 min. The flow through the column was 1.0 mL/min using Helium as carrier gas. The column used was a 30m DB-5MS column, 0.250 mm i.d., 0.25 μm film thickness. The oven temperature program was set to 60°C for 3 min, raised at 10°C/min to 300°C and a hold time of 3 min. The MS Ion source temperature was 200°C, transfer line temperature was 250°C, and the scan from m/z 50 to m/z 300 using 70 eV electrical ionization with a 5 min. solvent delay. Data and spectra were analyzed using Thermo Scientific Tracefinder software version 4.1 and the NIST library database version NIST 14 and compared to previously published data [2].

RNA extract for sequencing

The total RNA was extracted using the Spectrum™ Plant Total RNA Kit (Sigma, STRN250) from cells grown in PhyB media for 7 days after blending as described for RT-PCR. This yielded 300 μg total RNA as determined by nano-drop. RNA integrity was initially confirmed by agarose gel electrophoresis and the visualization of intact ribosomal RNA bands. Subsequent RNA quality control was carried out on a 2100 Bioanalyzer (Agilent Technologies, Hørsholm, Denmark) and each sample received an RNA integrity numbers (RIN) of greater than 8.5.

The total RNA of biological triplicates was pooled to make one technical sample, and total RNA samples were submitted to Macrogen (Seoul, Korea) for stranded mRNA library preparation using an Illumina Truseq Stranded mRNA library prep kit. The sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, "tagmentation" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments were then PCR amplified and gel purified. For cluster generation, the library was loaded into a flow cell where fragments were captured on a lawn of surface-bound oligos complementary to the library adapters. Each fragment was then amplified into distinct, clonal clusters through bridge amplification. When cluster generation was complete, the templates were ready for sequencing. The sequencing was performed using Novaseq 150bp paired-end sequencing providing 30–40 million reads per sample.

Illumina SBS technology utilizes a proprietary reversible terminator-based method that detects single bases as they are incorporated into DNA template strands. As all 4 reversible, terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias and greatly reduces raw error rates compared to other technologies.

Sequencing data was converted into raw data for the analysis. The raw reads were uploaded to the Sequence Read Archive (SRA) at NCBI with the Bioproject accession number PRJNA603435.

Trinity assembly and transcriptome annotation

A de novo transcriptome was assembled as a reference for read mapping and Differential Expression (DE) analysis using Trinity v2.4.0 [33]. As recommended in the Trinity protocol, one single Trinity assembly was generated by combining all reads across samples as input to ease following downstream analysis. Quality trimming and adapter removal was performed using trimmomatic [34] with default parameters. Transcript abundance was estimated using the alignment-based quantification method RSEM that uses Bowtie2 [35] as an alignment method. Transcript and gene expression matrices were generated, and the numbers of expressed genes were calculated. Finally, differential expression analysis was performed at the gene level using edgeR [36] with a dispersion rate of 0.1. Extractions and clustering of differentially expressed genes were performed with combinations of P-value cutoff for FDR of 1e-3 and fold-change values of 2, 4, 16, 64 and 256. Functional annotation of the transcriptome was performed using the annotation suite Trinotate. The functional annotation includes homology searches to BLAST, SwissProt, PFAM and various annotation databases such as eggNOG/GO/Kegg.

Results

Lipid droplet formation in wild type P. patens

To understand the LD biogenesis during default condition, we observed the formation of LDs in two different developmental stages in P. patens wild type. When compared to the protonema cells where LDs were sparsely scattered, we could observe a substantial increase in the numbers of LD in mature leafy gametophytes (Fig 1A). This is in line with previous studies that showed the relationship between LD biogenesis and senescence [37, 38]. The processed confocal images of BODIPY and ER-tracker stained tissue suggested that the LDs in P. patens originated from the ER (Fig 1B), as previously observed [16]. In the combined images of chloroplasts and LDs (average size of 3.12 E^-11 ± 6.21E^-16 mm³ (Table 1, S4 Fig in S1 File), it was observed that LDs seems to be positioned on the outer edges of the ER adjacent to the chloroplasts (Fig 1A and 1B). This indicate that LD formation in P. patens is similar to that of higher plants. The abundance of LD scaffolds in mature cells of P. patens showed the feasibility of targeting PTS to LDs for stable storage of patchoulol in vivo. For this purpose, we selected three LD-associated proteins due to their diverse way of association to LDs (Fig 1C).

Table 1. LD size of different P. patens lines (based on biological triplicates).

Cell type	LD Diameter (mm)	Volume (mm³)	Fold increase of volume
WT	4.73E-04 ± 1.28E-05	3.12E-11 ± 6.21E-16	-
PTS-only	5.65E-04 ± 1.30E-05	5.30E-11 ± 6.49E-16	1.70 *
PpOle1-LP4/2A-PTS	5.49E-04 ± 1.13E-05	4.88E-11 ± 4.28E-16	1.56 *
PpSeipin325 OE	6.60E-04 ± 1.49E-05	8.45E-11 ± 9.65E-16	2.71 ^**
PpSeipin325-Venus	5.99E-04 ± 1.30E-05	6.33E-11 ± 6.40E-16	2.03 **
PpSeipin325-PTS	6.74E-04 ± 1.32E-05	9.01E-11 ± 6.77E-16	2.88 **

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The size of the LDs was assessed by a combination of random confocal images and ImageJ analysis of the LDs of biological triplicates. More than 10 different random micrographs and at least 200 LDs were analyzed per cell type. The significance of the data was determined by one-way ANOVA test comparing the P. patens lines with the WT; P < 0.05 = *, P < 0.005 = **.

Localization of oleosin, LDAP and Seipin in P. patens leafy gametophyte tissues

To test if the LD related proteins, PpOLE1, PpSeipin325, AtLDAP1 are localized to the LDs of P. patens, the proteins were tagged with the reporter protein Venus using a flexible linker to minimize the steric effects of the two proteins. Confocal images of the leafy gametophyte tissues showed that PpOLE1 is specifically localized to the LDs (Fig 2), which confirms previous findings of the localization of oleosin proteins to the LD in P. patens gametophyte [16].

Fig 2 — (A) PpOLE1 was visualized by the fluorescence of the C-terminal tagged Venus and LDs were visualized by LipidTOX Red stain (artificial yellow color). (B) 3D projection of surface rendering Z-stacks of LipidTOX Red stained LDs (artificial yellow color) were merged with Venus for the depiction of LD co-localization)).

LDAP is one of the most abundant proteins in Arabidopsis leaf-LD proteome and shown to control the LD biogenesis in leaves [39]. The Arabidopsis genome contains three LDAP genes, but P. patens only contains one LDAP homolog (PpLDAP). Here AtLDAP1 was investigated as this was already characterized and had shown to be involved in LD stability [39]. According to the Affymetrix GeneChip data, PpLDAP is expressed in gametophytes and showed an increased expression in reproductive stages like the other LD-proteins (S5 Fig in S1 File). As seen in the confocal micrographs, AtLDAP1 is localized to the LDs of P. patens (Fig 3), this confirms the possible association of LDAP to LDs in P. patens. The result suggests that LD protein interaction is highly conserved in the evolutionary lineage, from P. patens to higher plants. Compared to the WT, the ZmUbi:AtLDAP1 line was observed to produce LDs in excess. Due to the frequent overlapping of the LDs in the processed confocal Z-stack images, the average LD number per cell was not obtained. Further studies are required to conclude the effect and the function of LDAPs on LD biogenesis in P. patens.

The localization of seipins was confirmed by examination of PpSeipin325 (one of two seipins in P. patens, Fig 4, S6 Fig in S1 File). Composite confocal images of the ZmUbi:PpSeipin325-Venus line showed that the majority of PpSeipin325 follows the ER pattern, which confirms previous findings in other organisms [19, 20]. Further image processing, using Imaris software showed that LDs emerge from a tiny round clearing of the seipin expression pattern (Fig 3).

Patchoulol accumulation in P. patens with PTS and oleosin overexpression

Previous work has demonstrated that 0.2–0.8 mg/g DW patchoulol could be produced in P. patens by overexpressing PTS, with higher concentration by plastid targeting and overexpression of FPP synthase [2]. In the current work, the ZmUbi:PTS line produced 32.89 μg/g of patchoulol, retaining 10.07 ± 0.42 μg/g DW inside the cell and 22.82 ± 1.18 μg/g DW excreted to the media (Table 2). Although this is lower than the previously published result, in this work PTS synthase was targeted to the P. patens neutral 108 locus with a single copy integration, unlike the random and multiple integrations of PTS in the previously published work [2].

Table 2. Patchoulol production of three weeks old modified P. patens cell lines (biological triplicates).

Cell Type	Inside the cell (μg/g)	Outside the cell (μg/g)	Total Production (μg/g)	% Inside
PpOle1-PTS	1.86 ± 0.30	2.43 ± 0.17	4.3	43 *
PpOle1-LP4/2A-PTS	4.67 ± 0.30	12.63 ± 2.26	17.3	27
PpSeipin325-PTS	1.81 ± 0.34	3.13 ± 0.72	4.95	37
AtLDAP1-PTS	5.14 ± 0.79	7.41 ± 0.89	12.54	41 *
PTS-Only	10.07 ± 0.42	22.82 ± 1.18	32.9	31

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Patchoulol was extracted from cells (inside) and the media (outside) using ethyl acetate. Production was calculated via GC/MS analysis of the samples. The amount of patchoulol inside the cell was divided by the total amount to calculate the percentage of patchoulol contained in the cells. The significance of the data was determined by student T-test comparing the retained patchoulol in P. patens lines with the PTS alone line; P < 0.05 = *.

To address the question of the effect of LD-anchoring on patchoulol production, PTS was linked to PpOLE1 by two different linkers: Flexible Gly-Ser (GS) linker for physical attachment and translationally cleaved LP4/2A linker [27, 40]. Unlike the GS linker, the LP4/2A linker facilitates the release of PTS from PpOLE1 upon cleavage by protease, this at least at 80% cleavage efficiency [40, 41], allowing us to directly compare the retention of patchoulol inside the cells with or without the LD anchor.

As seen in Table 2, the physically linked PTS (ZmUbi:PpOle1-PTS) retained the most patchoulol inside the cell, amounting to 43% of the total patchoulol production, where the translationally cleaved ZmUbi:PpOle-LP42A-PTS retained only 27% and the PTS only ZmUbi:PTS retaining 31% (Table 2). Taken together, the proximity of PTS to PpOLE1 aided the retention of patchoulol inside the cells. We also confirmed the occurrence of patchoulol and the two minor biosynthesis products, seychelene and patchoulene, in the LDs by the GC-MS analysis of the purified LDs of ZmUbi:PpOle1-PTS line (S2 Fig in S1 File).

Gentle osmolytic lysis of protoplast and a thorough wash step was attempted to prevent any ER membrane residues or carry-over patchoulol from the cytoplasm, to the LD fraction. Other bryophyte species, such as Marchantia polymorpha L, can accumulate sesquiterpenoids in LDs along with neutral lipids as previously observed [42, 43]. Following the cell lysis, only the free LDs were isolated, and not the LDs still physically attached to the ER. As shown in Table 1 the isolated free-LDs in the ZmUbi:PTS line was larger than in WT, where biosynthesis of patchoulol in the cell could have had an effect on the increased LD size. Over-expression of PTS and oleosin (ZmUbi:PpOle-LP4/2A-PTS) as translationally cleaved proteins produced smaller LD compared to ZmUbi:PTS line. Overexpression of oleosin proteins has previously been shown to decrease the size of LDs in seeds [44]. Thus, the LD volume reduction in ZmUbi:PpOle-LP4/2A-PTS compared to ZmUbi:PTS could be partially explained by the functional properties of the oleosin protein that lead to more but smaller LDs.

The occurrence of LDs in each of the ZmUbi:PTS, ZmUbi:PpOle-PTS and ZmUbi:PpOle-LP4/2A-PTS lines were comparably higher than in WT cells (Fig 5). The increased number of LDs in ZmUbi:PpOle-PTS and ZmUbi:PpOle-LP4/2A-PTS lines were expected due to the known function of oleosin. However, the increased number of LDs in ZmUbi:PTS line was unexpected. This observation of an increased number of LDs was also documented in artemisinin P. patens mutants produced in a previous work [45].

Patchoulol accumulation and growth effects in PTS linked AtLDAP1

To gain insight into the effects of LDAP linked PTS, ZmUbi:AtLDAP1-PTS was stably transfected to P. patens. After 3 weeks, the ZmUbi:AtLDAP1-PTS line produced a total of 12.5 μg/g DW patchoulol (Table 2). The ZmUbi:AtLDAP1-PTS line produced the highest amount of patchoulol compared to any LD targeted PTS line. The high patchoulol production of the ZmUbi:AtLDAP1-PTS line could be attributed to the structural association of AtLDAP1 to the LDs. The unlikely presence of any transmembrane domains in the LDAP protein structure possibly increased the folding efficacy of attached PTS, to yield a high production of patchoulol. Overall, the retention of patchoulol inside the cells of the ZmUbi:AtLDAP1-PTS line was comparable to the translationally cleaved ZmUbi:Oleosin-LP4-2A-PTS line. The ZmUbi:AtLDAP1-PTS line retained 41% patchoulol inside the cell as compared to 28% of the ZmUbi:Oleosin-LP4/2A-PTS line (Table 2). The ZmUbi:AtLDAP1-PTS line also harbored a large number of LDs similar to that of ZmUbi:Oleosin-LP4/2A-PTS (Fig 5). The size of the LDs in the ZmUbi:AtLDAP1-PTS was not measured as the LDs clumped together and was impossible to seperate. This subset of data re-validates the significance of having the PTS in close proximity to LD for storage purposes and depicts the importance of the structural properties of the LD-anchor for patchoulol biosynthesis.

Patchoulol production in PpSeipin325-PTS mutants and size of LD

PTS was also attached to the PpSeipin325, due to the unique association of seipin proteins to the LD expansion on the ER (Fig 1C). In principle, the ZmUbi:PpSeipin325-PTS line could function from the outside of the emerging LD, aiding the expansion of the LD with neutral lipids and patchoulol. The engineered ZmUbi:PpSeipin325-PTS, produced a total of 4.95 μg/g patchoulol in 3 weeks (Table 2). The very low production of patchoulol in PpSeipin325-PTS line could partially be explained by a very low expression of PTS in the ZmUbi:PpSeipin325-PTS line. This could be due to the large size of PpSeipin325, which subsequently leads to lower expression and likely alters the folding and properties of the PTS and thereby the kinetics (S3 Fig in S1 File).

To further demonstrate the transfer and storage of patchoulol to LDs, we examined the expansion of the LD size in the ZmUbi:PpSeipin325-PTS cell line relative to that of ZmUbi:PpSeipin325-Venus. The outside association of seipin protein made the ZmUbi:PpSeipin325-PTS cell line an ideal candidate to study the LD expansion in relation to patchoulol production. The ZmUbi:PpSeipin325-Venus cell line was selected as the control to have similar biological conditions of having an attached protein to seipin. The ZmUbi:PpSeipin325-Venus line produced LDs of 6.33E-11 ± 6.4E-16 mm³ in size as compared to the ZmUbi:PpSeipin325-PTS that produced a LD of 9.01E-11± 6.77E-16 mm³. On average, LDs of ZmUbi:PpSeipin325-PTS line expanded by 188% to the WT and 42% to the ZmUbi:PpSeipin-Venus control (Table 1, S4 Fig in S1 File).

Our confocal micrographs suggest that P. patens seipins are localized mainly to the ER and around the base of the LDs. Work done on Arabidopsis seipins has shown that AtSeipin1 is capable of producing bulky LDs [19]. Interestingly, larger LDs were observed in the ZmUbi:PpSeipin325 line in 3-week-old leaf tissues when compared to WT. On average, the LDs in the ZmUbi:PpSeipin325 line was 2.71 fold larger than wild type LDs (Table 1, S4 Fig in S1 File). This suggests that similar to AtSeipin1, PpSeipin325 is involved in the expansion of the LDs.

Effect on plant growth

P. patens expressing only PTS had 26% lower growth in plant height when compared to WT after 20 days, measured in millimeter of the gametophyte growth. However, producing patchoulol with PTS attached to LD-associated proteins had no negative effect on the growth but provided an even faster biomass development than WT. This was observed for the ZmUbi:AtLDAP1-PTS when the plant heights were compared to WT, and ZmUbi:PTS lines. The average WT gametophyte grown for 20 days, grew to 0.32 ± 0.01 mm in height, followed by 0.24 ± 0.01mm for ZmUbi:PTS and 0.65 ± 0.03 mm for ZmUbi:AtLDAP1-PTS cell lines. The ZmUbi:AtLDAP1-PTS line had a 2-fold plant-height increase over the WT.

Transcriptomic sequencing data

Due to the unexpected increased number of LDs in ZmUbi:PTS line, transcriptome analysis for each mutant was performed. The transcriptomic data obtained showed that among the genes upregulated 256-fold or more, two P. patens genes were present in all our samples (Fig 6, Table 3, summary in S2 File). This was GDSL esterase/lipase At4g16230-like (XP_024368984) and non-specific lipid-transfer protein 3-like (XP_024363543). A third lipase, the GDSL esterase/lipase At5g03820-like (XP_024382012), is represented in all but the PpSeipin325 overexpression line. More lipases are also upregulated, and genes related to photosynthesis is downregulated along with other genes in all overexpression lines. In order to confirm the involvement of these genes in LD biogenesis further studies are needed.

Fig 6 — For some this also show that there is little to no change in one or more P. *patens* lines, but a 256-fold change in just one P. *patens* line. The annotation of the contig numbers is seen in Table 3.

Table 3. List of contigs shown in Fig 3.

Contig number in sequencing assembly and general regulation (U/D/N)	P. patens NCBI #	Description from P. patens annotation
DN00929 (U)	XP_024395254	chalcone synthase 6-4-like
DN02000 (U)	PNR47760	hypothetical protein
DN02561 (U)	XP_024385936	pectinesterase 15 -like
DN05532 (U)	XP_024380033	alpha carbonic anhydrase 7-like
DN05993 (D)	N/A	uncharacterized protein
DN06081 (U)	XP_024395980	peroxidase 21-like isoform X1
DN06295 (D)	XP_024375887	chlorophyll a-b binding protein of LHCII type 1-like
DN06494 (U)	XP_024382012	GDSL esterase/lipase At5g03820-like
DN11934 (U)	XP_024368003	uncharacterized protein
DN12351 (D)	XP_024367311	copper transporter 5.1-like
DN12927 (D)	XP_024362229	germin-like protein 9–3
DN16400 (U)	XP_024395390	receptor-like protein 51
DN17926 (U)	XP_024368032	alpha carbonic anhydrase 5-like
DN18350 (D)	XP_024397347	enolase 1-like
DN20064 (D)	XP_024392911	protein LURP-one-related 15-like
DN20134 (D)	XP_024403288	polyphenol oxidase-like
DN20262 (D)	PNR36153	hypothetical protein
DN23122 (N)	XP_024378832	dirigent protein 19-like
DN23815 (D)	XP_024360335	1-Cys peroxiredoxin-like
DN24061 (U)	XP_024368984	GDSL esterase/lipase At4g16230-like
DN24155 (D)	PNR55678	hypothetical protein
DN26229 (D)	XP_024400099	copper chaperone for superoxide dismutase
DN26375 (U)	XP_024382195	uncharacterized protein
DN27466 (D)	XP_024399688	phospho-2-dehydro-3-deoxyheptonate aldolase 2
DN29028 (N)	N/A	uncharacterized protein
DN29463 (D)	NP_904206.1	PSII 44 kD protein
DN30687 (D)	N/A	uncharacterized protein
DN32215 (U)	PNR26297	UDP-glycosyltransferase 83A1-like
DN32629 (N)	N/A	uncharacterized protein
DN32661 (D)	PNR52172	hypothetical protein
DN32681 (U)	PNR53501	hypothetical protein
DN32998 (U)	XP_024363339	abscisic acid 8'-hydroxylase 3-like
DN33997 (D)	N/A	uncharacterized protein
DN34516 (N)	XP_024359073	chlorophyll a-b binding protein
DN34803 (U)	XP_024369838	uncharacterized protein
DN38215 (D)	N/A	uncharacterized protein
DN38263 (U)	XP_024381396	uncharacterized protein
DN38313 (D)	XP_024360503	GDSL esterase/lipase At4g01130-like
DN38368 (D)	PNR36232	hypothetical protein PHYPA_022083
DN38568 (U)	XP_024393797	uncharacterized protein
DN38832 (U)	PNR53501	hypothetical protein
DN39595 (D)	XP_024358907	formin-1-like
DN45734 (D)	PNR34789	hypothetical protein
DN46340 (D)	PNR36032	hypothetical protein
DN49887 (U)	XP_024393312	aldehyde oxidase GLOX-like
DN50002 (D)	NP_904175.1	PSII 47kDa protein
DN50057 (D)	XP_024367800	ferric reduction oxidase 6-like
DN50091 (U)	PNR39672	hypothetical protein
DN50465 (U)	XP_024363543	non-specific lipid-transfer protein 3-like
DN55646 (U)	XP_024375379	non-specific lipid-transfer protein 2G-like
DN55842 (U)	XP_024377855	probable xyloglucan endotransglucosylase/hydrolase
DN61444 (U)	XP_024388184	pathogen-related protein-like
DN61594 (U)	XP_024393924	germin-like protein 1–1

Open in a new tab

The up (U) or down (D) regulation or no change (N) is given for the majority of P. patens lines as compared to the expression in the wild type. The annotation provided is based on a BLAST search towards the P. patens genome v. 3.3, and the NCBI numbering is included.

Discussion

Our results from Table 1 demonstrate the relationship between LD expansion and expression of PTS in the cells. While the results are not conclusive for routing of patchoulol into LDs, they do demonstrate that increasing the size and numbers of LDs in the cell will retain more patchoulol inside the cells and the LDs. The level of patchoulol biosynthesis is not as high in the PTS-LD linked mutants as the two mutants where it is not linked. This also fits with what has previously been observed in N. benthamiana [11] and likely arise from a lower enzyme activity of PTS when the enzyme is attached to the LD’s. The difference between the PTS alone and PpOle1-LP4/2A-PTS is probably due to change in transcription/translation of the longer construct; however, this is not examined.

We suggest that newly synthesized patchoulol in the PpSeipin325-PTS, AtLDAP1-PTS and PpOle1-PTS lines could enter the LD via two routes: passive diffusion the LD through the membrane in the cytoplasm or across the ER membrane into the ER tubule and then enter the LD through the ER-LD contact site. The latter is particularly interesting for the PpSeipin325-PTS, whereas for LDAP and Ole it is more likely that it is passive diffusion directly into LD. It is unknown if one route is preferred over the other, though both routes requires a passive diffusion over a lipid membrane.

Microscopic analysis verified the localization of PpOLE1 and AtLDAP1 to the cytosolic LDs. Over-expression of both PpOle1 and AtLDAP1 increased the number of cytosolic LDs compared to the WT. Localization of PpSeipin325 was restricted to the reticular ER pattern and over-expression of PpSeipin325 increased the size of the LDs by 170%. Thus, we hypothesized that PpSeipin325, similar to AtSeipin1, is involved in the expansion of LDs. Large groups of clustering LDs were observed in the leaf tissue of three weeks old mutant line (Fig 5). LD structural proteins such as oleosins and LDAPs have the ability to coat the LDs and prevent the LDs from aggregation. The rapid expansion of the LDs in the ZmUbi:PpSeipin325-PTS line could hinder the natural localization process of LD structural proteins, leading to clustered LDs. Overall, an increase in LD numbers seems to provide a new pool for storage of sesquiterpenoids. Future work could focus on co-expressing PpSeipin325 with PpOLE1 or AtLDAP1 to attain a homogeneous pool of LDs to store patchoulol with expression of PTS.

As previously shown, the heterologous expression of terpenoids leads to growth deficiencies in cells [9, 10, 46]. It was observed that the production of terpenoids in P. patens led to a 48% lower biomass formation during 18 days of cultivation [46]. Here, P. patens expressing only PTS had 26% lower growth in plant height when compared to WT after 20 days. This stress response is probably also related to the enhanced LD production, which are formed to lower the stress burden to the cells. This stress response hypothesis is also supported by the enhanced growth in mutants where PTS is attached to the LDs thus ensuring that patchoulol is captured inside the LDs. A 2-fold increase in plant height of the ZmUbi:AtLDAP1-PTS is in line with previous observations where the over-expression of AtLDAP1 in Arabidopsis increased the height by 5-fold [24]. How the LDAP proteins are involved in tissue growth in Arabidopsis and P. patens, is yet to be investigated, but the increase in growth and biomass overall lead to higher production of the final product. From the transcriptomic data in Table 3, it is clear that several lipases and non-specific lipid transporter proteins (LTPs) are upregulated in all the mutant lines. This indicates that the increased lipid droplet formation also initiates a higher production and transport of lipids. Our data supports that there is tight link between neutral lipid levels, LD formation and terpenoid production capacity. This link is supported by the recent study that showed the biogenesis of LD in chloroplast with modified oleosin remarkably increased the terpene production [47]. Previously published work in some bryophytes also suggest that LTPS are involved in the trafficking of fatty acids and larger lipids [48–50]. Thus, the increased lipid biosynthesis and lipid content in the cell could provide better storage capacity for terpenoids. It requires further studies to prove this possibility, but this holds the potential for novel biotechnological developments.

Conclusion

We observed 26% less growth in plant height when patchoulol is biosynthesized in P. patens, just by over-expressing PTS. This finding was in line with similarly published works, however, in the LD targeted PTS line, ZmUbi:AtLDAP1-PTS we observed 2-fold increased growth in plant height compared to WT. This shows that we can biosynthesize terpenoids in P. patens, increase growth, and potentially device a new extraction method, which is very useful for future industrial production.

All the LD protein-PTS attached lines retained more patchoulol in the cell, over none-attached ZmUbi:PTS and ZmUbi:PpOle1-LP4/2A-PTS lines. The size and number of LDs in patchoulol biosynthesis lines were increased; the size was increased with up to 188% and about 25% more patchoulol was retained inside the cell, though the production was significantly reduced. Thus, there is a potential to use the LDs for storage of small molecules during production in bioreactors. However, it requires further work to be established this for production of any small molecule.

Supporting information

S1 File. The file contains the following data.

(PDF)

Click here for additional data file.^{(607.2KB, pdf)}

S2 File. Differential expressed genes in lipid bodies.

Genes 256-fold differentially expressed between the mutants are shown in tables of the individual mutants.

(PDF)

Click here for additional data file.^{(304.5KB, pdf)}

S3 File. Images used for measurement of LD size, and the measurements in excel sheets.

(ZIP)

Click here for additional data file.^{(75.9MB, zip)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

AP, HB, and HTS were supported by The Danish Council for Independent Research (#4005-00158B). CQ was supported by funding from the People Program (Marie Curie Actions) of the European 517 Union's Seventh Framework Program FP7/2007-2013/ under REA grant agreement n° [607011]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Mosspiration Biotech provided support in the form of part of the salaries for authors [HB and HTS], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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PLoS One. doi: 10.1371/journal.pone.0243620.r001

Decision Letter 0

Björn Hamberger

13 Oct 2020

PONE-D-20-29050

Connecting moss lipid droplets to patchoulol biosynthesis

PLOS ONE

Dear Dr. Simonsen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Three scientists, who are each experts in specific aspects of the manuscript and at different academic stages, have kindly provided specific input. All three were very supportive, an opinion which I can only second. No additional experiments were suggested and the data was considered scientifically sound. However, there are suggestions and comments regarding the text, figures, minor details, and in particular one part, which appears to have been accidentally copied from another paper. Overall, I hope this will allow you to make a swift revision.

==============================

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[AP, HB, and HTS were supported by The Danish Council for Independent Research (#4005-00158B).

516 CQ was supported by funding from the People Program (Marie Curie Actions) of the European

517 Union's Seventh Framework Program FP7/2007-2013/ under REA grant agreement n° [607011].]

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review Connecting moss lipid droplets to patchoulol biosynthesis

In this manuscript, the authors have targeted a patchoulol synthase to lipid droplets (LD) or the ER by fusing it to different LD/ER proteins. The targeting appears to be successful. The goal to increase the production of patchoulol in comparison to previous studies where patchuolol synthase was not targeted to LDs was not reached, as the levels that could be obtained were much lower than previously described. The targeting in this study had however a benefit to reach higher levels. Furthermore, the LD size was slightly increased by the overexpression. While the authors did not get very far in a biotechnological sense, I like the idea of using LDs as sites for the synthesis and storage of hydrophobic compounds.

Comments

1. In the abstract the authors write:

“Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis.”

However, there could also be an effect on LD degradation. Also, the number of LDs was not quantified. It is furthermore strange that in Figure 1 the number of LDs in the wild type is very high and then in Figure 5 much lower. Where all the images taken at the same time of the day or the plants fixed? LDs undergo a circadian rhythm.

2. It would be interesting to see examples, how the LD size was determined. I don’t think that LD size can be reliably measured by confocal microscopy as they are too small. I mean again it would be great to see the original pictures they took, but I would assume that the LDs are only a few pixels in diameter. Furthermore, often LDs seem to be clustered together and single LDs cannot be resolved.

Calculating volumes from this data to enlarge the differences is not necessary. The data should be presented in a box plot or better a swarm plot so that individual sizes can be seen.

3. In the lines 51-53 reviews on plant LDs should be cited instead or at least additionally. Paper number 14 for example does not mention plants at all.

4. The authors should at least briefly describe the reaction catalyzed by PTS and where it is normally located and what the substrate is.

5. Line 62 “Oleosins are the most abundant protein in the P. patens LD proteome”. On what publication is this based on? Was it really quantified? Is this true for spores only or in all moss tissues?

6. Lines 64-66. “Oleosins sequestrate neutral lipids from the ER bilayer and this way extract the LDs from the ER. This extraction of LD to the cytosol is controlled solely by the innate properties of oleosin, and modified oleosin can redirect the LD to the ER lumen and then to the vacuoles”.

While these sentences are a 1:1 plagiate from Anthony Huangs review, I feel that this statement is also misleading. I don’t think the role of oleosins in LD formation is so clear. They are definitely not needed in plants and also the absence of proteins including VAP27-1, Seipin, LDAP and LDIP have a strong influence on the proper formation of LDs indicating that it is not oleosin alone that does the job in vivo.

7. The authors should discuss that the accumulation of LDs could also just be a stress symptom as the plants are clearly stressed by PTS overexpression as they grow smaller. I don’t think that the small amount of patchoulol produced can really substantially contribute to LD formation. If the authors want to make this claim or indicate this possibility they must measure the amount of TAG and sterol esters to show that these do not increase.

Reviewer #2: The present study by Peramuna et al investigates synthetic biology approaches to co-produce high value sesquiterpene (patchoulol) together with three different Lipid droplet proteins Oleosin (PpOLE1), Lipid Droplet (LD) Associated Protein or Seipin (PpSeipin325) for redirecting the patchoulol production in Physcomitrella patens. The study involves overexpressing patchoulol synthase (PTS) physically attached to three proteins to produce the sesquiterpenoid patchoulol close to LDs. The authors also verified the localization of PpOLE1 and AtLDAP1 to the LDs and PpSeipin325 to the ER tubules in P. patens. By utilizing these LD-associated proteins as an anchor, the authors showed that the expression of LD protein-attached patchoulol synthase (PTS) led to a higher content of patchoulol in the LD as compared to the expression of free PTS.

The study in principle is sound. The experiments are well done, and the data generated by the authors appears to be of good quality. The study lays a groundwork for better targeted metabolic engineering of P. patens for terpene production in future. The manuscript is a great fit for the journal and hence is recommended for acceptance once the following comments and queries are addressed:

Comments

Line 40-41. Please add the reference for ….. “fluctuating market price of 30-200 US dollars/Kg.”

Line Line 48-51. The statement needs further refinement. The use of term “novel” and “the results were not conclusive” is somewhat misleading as the use of both LDAPs and Oleosins for terpene compartmentalization has been well demonstrated previously [ref 11 and 47]

Results:

P. patens has three oleosin genes, OLE1-OLE3, and OLE1 and OLE2 has two splice variants each [Ref. 15]. The authors do not explain why OLE1 was used for the present study and also which splice variant of OLE1 was used?

Line 291- 294: the supplementary figure mentions PpSRP instead of PpLDAP. Please correct this.

Line 257: Please check and correct the order in which supplementary figures are mentioned in the text.

Line 288-296: Although it does not matter from the synthetic biology point of view to use AtLDAP1 instead of PpLDAP, it would have been more useful to test the biological function of PpLDAP and compare it with AtLDAP1. I do not suggest the authors to do it for the current manuscript, but it is surely something they can do for future work, given the robustness of their moss transgenic system and the expertise in the field.

Line 301-305: The authors do not explain why PpSeipin325 was chosen for the study, since there are two of them present in P. patens? Were these already characterized in the past? If these were, please mention that in the text. If not, the rationale for not using PpLDAP does not hold that well. The authors are advised to strengthen this section of the manuscript.

Line 327: The authors do not explain why they chose a neutral locus Pp108 instead of random and multiple integration locus, when the production was demonstrated to be higher in their previous research?

Line 335: What is the difference between using a free PTS and one tagged to Oleosin via LP4/2A linker, because principally PTS will be free in the cytosol in both the cases? Also, why was not LP4/2A linker tested with AtLDAP1 and Seipin325 constructs?

Line 407: replace “tested” with “observed”

Line 423-431: The authors talk about the effect of the different combinations of lipid droplets/PTS on the P. patens growth. But there are no figures/pictures to support the findings. I think this is very important observation and some representative images/figure in the main text would be quite valuable.

Line 433: Although the transcriptome data seems to be a detailed study on its own and could have been very useful in understanding the reasons for unexpected increase of LDs in some of the lines, it does not seem to be the case here. I would suggest the authors to put more details in this section. The authors can use some of the bioinformatics tools to provide a chart or a diagram of major metabolic pathways that are affected in these lines.

Conclusion: The conclusion sections needs some refinement. It just seems like an extension of results/ discussion.

Figures:

1. Please label the subcellular organelles in the microscopy images.

2. If possible, add the corresponding phase contrast images of the microscopy images.

3. Add a comparative plant growth figure of wild and transgenic lines.

Legends:

Line 444-448: Please add a description of the transgenic lines in the legend section.

Reviewer #3: This review is being written from the perspective of a senior PhD candidate with background in Physcomitrella patens, terpene biosynthesis, and molecular biology and expertise in bioinformatics, genetics, and genomics. Peremuna et al. clearly express their teams attempts to increase yield of patchoulol and localize PTS production to the ER and more specifically lipid droplets. When compared to previously published papers their work appeared to have mixed successes, with lower patchoulol productions then in other lines, which they account for by the location of gene insertion being in a neutral locus. Additionally, lines targeted to lipid droplets showed less patchoulol production but better retention within the cell and healthier plant growth when compared to non-localized mutants.

I think the logic and science in this paper is sound and I very much so appreciated the transparency with the mixed success of patchoulol production and PTS integration into the lipid droplets. I also think this paper does a great job of bringing attention to the questions that arose throughout their scientific process that remained unanswered, posing future directions not only to themselves but the readers as well. Their work and the publishing of this work will likely save others time and energy in the future from having to repeat similar or the same experiments tested here.

I think this paper requires no major edits but could be improved by including more background on why a heterologous system is necessary in the first place. Also, be aware of the use of acronyms and abbreviations as they were not always as clear as they could be (ie. Line 29 – LD/ER refers to Lipid Droplet/endoplasmic reticulum but ER is not defined previously; Line 72 - PpOLE1/PpSeipin325/AtLDAP1 not being formally highlighted in the introduction; Table 2 - Possible typo stating PpLDAP1-PTS, instead of AtLDAP1-PTS (check between lines 382 and 684); Line 490 – Adding another acronym (LTPs) near the end of the paper and is only used one other time seems unnecessary). With a paper containing many acronyms and frequently referring to each of them in different orders in each section it is important to make sure the information is conveyed clearly.

Lastly, some constructive advice to the authors for their future work I have some suggestions regarding my expertise. (Line 235) In the future you may want to refer to the published genome and trancriptome of P. patens instead of recreating a de novo transcriptome with Trinity, even though these lines have had gene transformations, the universal gene names of P. patens would likely be helpful. Additionally, the version of trinity used is a few years old and many updates have occurred since this version (Version 2.11.0 is currently available). (Line 433) As you investigate more into the differential gene expression make sure to consider genes that are less than a 256-fold change in expression as well. 256-fold is a good place to start but there is probably a lot of useful information at less extremes as well. (Line 298) You mention in a few places that there was an observed increase in LD number per cell but you weren’t able to get a calculation on the actual number of droplets that changed. You may be able to use your average lipid droplet volume and average total lipid production to get an idea of how many droplets there were using the following equation and solving for DropletNumber LDVolume*NumberOfDroplets=TotalLipidContent?

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Reviewer #1: No

Reviewer #2: Yes: Wajid Waheed Bhat

Reviewer #3: Yes: Davis T. Mathieu

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PLoS One. 2020 Dec 7;15(12):e0243620. doi: 10.1371/journal.pone.0243620.r002

Author response to Decision Letter 0

15 Oct 2020

Comments to the reviewers:

Reviewer 1

1. In the abstract the authors write:

“Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis.”

Answer: In figure 1 the LD’s was taken at day 10 and at 6 weeks. Figure 5 is after 3 weeks of growth. We did not observe that LD’s was influenced by circadian rhythms, as we the plants was grown un 24h light. Thus Figure 5 is best compared with Figure 1.Ai. Figure text for figure 5 is updated.

Answer: We would like to refer the reviewer to see Figs 1, 2 and 3 for examples on how the pictures was zoomed in for the measurement. It is quite clear that some are bundles of LD’s where as others are clearly single LDs. This is also described in M&M, the text is updated and strengthened here.

Calculating volumes from this data to enlarge the differences is not necessary. The data should be presented in a box plot or better a swarm plot so that individual sizes can be seen.

Answer: We are not sure this would provide further evidence and support. And as such will keep the current representation.

3. In the lines 51-53 reviews on plant LDs should be cited instead or at least additionally. Paper number 14 for example does not mention plants at all.

Answer: the references are updated.

4. The authors should at least briefly describe the reaction catalyzed by PTS and where it is normally located and what the substrate is.

Answer: sure, this is added to the text.

And yes it was quantified and it is not only in spores. We would like to refer the reviewer to this publication.

Answer: The text was updated to reflect more recent knowledge and the comment from the reviewers.

Answer: This is added to the discussion.

Reviewer 2:

Line 40-41. Please add the reference for ….. “fluctuating market price of 30-200 US dollars/Kg.”

Answer: Please see reference 2, where these numbers are given.

Answer: the sentence was rephrased.

Results:

Answer: The full native sequence of PpOleosin1 (Pp1S84_138V6.1) was used as given in M&M and chosen based on expression in all tissues. M&M is updated to reflect this.

Line 291- 294: the supplementary figure mentions PpSRP instead of PpLDAP. Please correct this.

Answer: The text is updated accordingly.

Line 257: Please check and correct the order in which supplementary figures are mentioned in the text.

Answer: The text is updated accordingly.

Answer: We fully agree, and hope to start this work soon.

Answer: The full native sequence of PpSeipin325 (Pp1S325_14V6.1) was used as given in M&M and chosen based on expression in all tissues. M&M is updated to reflect this.

Answer: Previous research have not examined how many copies was inserted into the genome. It is much likely that 5-8 copies of PTS were inserted previously. With a controlled 108 integration one know precisely what was done. There is not need to explain this.

Answer: The discussion is updated to reflect this.

Line 407: replace “tested” with “observed”

Answer: The text is updated accordingly.

Answer: this data is represented in the tables. A figure cannot be produced.

Answer: The current discussion of the transcriptome data reflects what we find must valuable at the current state. Further analysis of these data would require further in vivo studies as well to cooperate the findings. Thus, we have chosen to represent that here, and share these data, without a too detailed discussion and conclusions that we find it hard to support.

Conclusion: The conclusion sections needs some refinement. It just seems like an extension of results/ discussion.

Answer: The text is updated accordingly.

Figures:

1. Please label the subcellular organelles in the microscopy images.

Answer: The text is updated accordingly and updated legend.

2. If possible, add the corresponding phase contrast images of the microscopy images.

Answer: we do not have this image.

3. Add a comparative plant growth figure of wild and transgenic lines.

Answer see comment above on the same issue.

Legends:

Line 444-448: Please add a description of the transgenic lines in the legend section.

Answer: The text is updated accordingly.

Reviewer 3

Table 2 - Possible typo stating PpLDAP1-PTS, instead of AtLDAP1-PTS (check between lines 382 and 684);

Line 490 – Adding another acronym (LTPs) near the end of the paper and is only used one other time seems unnecessary). With a paper containing many acronyms and frequently referring to each of them in different orders in each section it is important to make sure the information is conveyed clearly.

Answer: The text is updated accordingly.

Answer: we investigated the suggested methods and idea and will use in future studies. We tried to use the average volume but the data we had and the measurements was not really sufficient at this stage. Thus we have chosen to stick with what we have. But we are very glad for the advises. As mentioned above, a further analysis of the transcriptome data is for sure needed, however this is currently not possible for us at this stage, and would require further in vivo studies.

PLoS One. doi: 10.1371/journal.pone.0243620.r003

Decision Letter 1

Björn Hamberger

12 Nov 2020

PONE-D-20-29050R1

Connecting moss lipid droplets to patchoulol biosynthesis

PLOS ONE

Dear Dr. Simonsen,

Thank you for re-submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has significantly improved but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Next to minor text edits, which are all specified below, one remaining comment addresses the data availability for the quantification of the lipid droplets, which should be addressed.

==============================

Please submit your revised manuscript by Dec 27 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Björn Hamberger

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: 1. I still believe that if a statement is made about the number of LDs these should be quantified with sufficient numbers of biological replicates and images.

Please provide all the images taken for measurement of number and LD size in the supplements according to the PLoS ONE policy. Also please give all the individual datapoints of all experiments in supplemental table as outlined in the PLOS data policy.

https://journals.plos.org/plosone/s/data-availability

Answer: This is based on Huang C-Y, Chung C-I, Lin Y-C, Hsing Y-IC, Huang AHC. Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution. Plant Physiol. 2009;150: 1192–1203.And yes it was quantified and it is not only in spores. We would like to refer the reviewer to this publication

Reply: Thank you for pointing out this publication. It should be cited right after this statement not only after the next statement. Furthermore, in this paper only one tissue was investigated. Therefore, the statement should be refined and made more specific. Also the statement itself is based on a blue gel, where one band was further investigated by MALDI-MS. A quantification is something different to me. But ok.

6. Thank you for amending the statement but the new statement

“Following this sequestration Oleosins together with other proteins supports the detachment of the LDs from the ER and release into the cytosol.”

Could use a citation for example a recent review or the new Vap27 paper by the chapman group.

Reviewer #2: The revised version of the manuscript by Peramuna et al does answer most of the comments/queries raised by the reviewers, and with some minor revisions, the manuscript is deemed fit for acceptance in PLOS One. The comments are as under:

1. Line 70: change “Oleosins to “oleosins”.

2. Line 354: Please clarify the statement “Gentle lysis of protoplast with osmosis and….”

3. Line 408: replace “properly” with “probably”

4. Line 460: replace “with” with “between”

5. Line 463-464: Rephrase the sentence “The amount of production is not as high in the……..”.

6. Line 5465, 466 and 489: “properly” with “probably”

7. Line 490-491: please rephrase the sentence “This is supported by that attaching………….”

8. Line 501-502: rephrase “Previously published works also suggest that some bryophyte LTPs are involved….” to “Previously published work in some bryophytes also suggest that LTPS are involved….”

9. Line 502-503: Please rephrase the sentence “ Thus, with increased lipid biosynthesis and more lipids in the cell could provide…….”.

Reviewer #3: All comments I made from first review were adequately answered. Upon reviewing second submission, the paper has made excellent revisions in the introduction and results section, adding to clarity and necessity of research. No Further major edits necessary.

Minor edits to consider after my second reading:

Line 60 - Sentence regarding the fragrance of patchoulol is unnecessary for the understanding of the paper as a whole. Authors choice to leave it in or not

Line 280 - typo (show --> shows)

Line 396 - "The size of the LDs in the ZmUbi:AtLDAP1-PTS was not measured due to their direct binding to the LDs.": This sentence was unclear to me. Is "their" referring to the protein? Did you not measure lipid droplet size because of direct binding of AtLDAP1-PTS to LDs? Wouldn't a larger lipid droplet support your argument that it was correctly targeted to the LDs.

Line 464 - "This also fits with what was observed in N. benthamiana [11] and properly arise from a change in the kinetics for the PTS to yield lower turnover numbers when this attached to the LD's": Consider rewording this sentence, Current wording is confusing and/or grammatically incorrect(?)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Wajid Waheed Bhat

Reviewer #3: Yes: Davis T. Mathieu

PLoS One. 2020 Dec 7;15(12):e0243620. doi: 10.1371/journal.pone.0243620.r004

Author response to Decision Letter 1

17 Nov 2020

Reviewer #1: 1. I still believe that if a statement is made about the number of LDs these should be quantified with sufficient numbers of biological replicates and images.

https://journals.plos.org/plosone/s/data-availability

comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. I still believe that if a statement is made about the number of LDs these should be quantified with sufficient numbers of biological replicates and images.

https://journals.plos.org/plosone/s/data-availability

Answer: The data is provided as extra supplementary files.

Answer: The reference was added and the text was slightly altered.

6. Thank you for amending the statement but the new statement

“Following this sequestration Oleosins together with other proteins supports the detachment of the LDs from the ER and release into the cytosol.”

Could use a citation for example a recent review or the new Vap27 paper by the chapman group.

The reference was added and the text was slightly altered.

1. Line 70: change “Oleosins to “oleosins”.

2. Line 354: Please clarify the statement “Gentle lysis of protoplast with osmosis and….”

3. Line 408: replace “properly” with “probably”

4. Line 460: replace “with” with “between”

5. Line 463-464: Rephrase the sentence “The amount of production is not as high in the……..”.

6. Line 5465, 466 and 489: “properly” with “probably”

7. Line 490-491: please rephrase the sentence “This is supported by that attaching………….”

9. Line 502-503: Please rephrase the sentence “ Thus, with increased lipid biosynthesis and more lipids in the cell could provide…….”.

Answer: All the corrections has been made in the manuscript.

Minor edits to consider after my second reading:

Line 60 - Sentence regarding the fragrance of patchoulol is unnecessary for the understanding of the paper as a whole. Authors choice to leave it in or not

Line 280 - typo (show --> shows)

Answer: All the corrections have been incorporated and the text has been clarified.

PLoS One. doi: 10.1371/journal.pone.0243620.r005

Decision Letter 2

Björn Hamberger

25 Nov 2020

Connecting moss lipid droplets to patchoulol biosynthesis

PONE-D-20-29050R2

Dear Dr. Simonsen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Björn Hamberger

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I am excited to see this improved manuscript accepted and look forward seeing this solid study published.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0243620.r006

Acceptance letter

Björn Hamberger

27 Nov 2020

PONE-D-20-29050R2

Connecting moss lipid droplets to patchoulol biosynthesis

Dear Dr. Simonsen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Björn Hamberger

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. The file contains the following data.

(PDF)

Click here for additional data file.^{(607.2KB, pdf)}

S2 File. Differential expressed genes in lipid bodies.

Genes 256-fold differentially expressed between the mutants are shown in tables of the individual mutants.

(PDF)

Click here for additional data file.^{(304.5KB, pdf)}

S3 File. Images used for measurement of LD size, and the measurements in excel sheets.

(ZIP)

Click here for additional data file.^{(75.9MB, zip)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Connecting moss lipid droplets to patchoulol biosynthesis

Anantha Peramuna

Hansol Bae

Carmen Quiñonero López

Arvid Fromberg

Bent Petersen

Henrik Toft Simonsen

Roles

Abstract

Introduction

Materials and methods

Plant material, growth conditions, and transformation

Strain construction

DNA fragments and genes

Microscopy

Fig 1. Wild type LD staining.

Fig 3. Co-localization of AtLDAP1 to P. patens LDs.

Metabolite extraction and GC-MS analysis

PTS expression analysis by RT-PCR

Growth measurement

Lipid droplet extraction

RNA extract for sequencing

Trinity assembly and transcriptome annotation

Results

Lipid droplet formation in wild type P. patens

Table 1. LD size of different P. patens lines (based on biological triplicates).

Localization of oleosin, LDAP and Seipin in P. patens leafy gametophyte tissues

Fig 2. Co-localization of PpOLE1 to LDs of P. patens.

Fig 4. Localization of PpSeipin325 in P. patens.

Patchoulol accumulation in P. patens with PTS and oleosin overexpression

Table 2. Patchoulol production of three weeks old modified P. patens cell lines (biological triplicates).

Fig 5. Ectopic expression of LD-associated proteins and PTS promoted LD occurrence.

Patchoulol accumulation and growth effects in PTS linked AtLDAP1

Patchoulol production in PpSeipin325-PTS mutants and size of LD

Effect on plant growth

Transcriptomic sequencing data

Fig 6. Log (x+) normalized heatmap of the different P. patens lines and the wildtype P. patens with a 256-fold up (red) or down (blue) regulation of gene expression in one or more P. patens lines compared to the wild type.

Table 3. List of contigs shown in Fig 3.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Björn Hamberger

Roles

Author response to Decision Letter 0

Decision Letter 1

Björn Hamberger

Roles

Author response to Decision Letter 1

Decision Letter 2

Björn Hamberger

Roles

Acceptance letter

Björn Hamberger

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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