Our wild-type/control Arabidopsis was Arabidopsis thaliana ecotype Columbia-0 (Col-0). Knockout mutants of pdat1 are homozygotes of T-DNA insertion mutant lines SALK_065334 (pdat1 KO1) and SALK_032261 (pdat1 KO2), which were obtained from the Arabidopsis Biological Resource Center (The Ohio State University). PCR screening for pdat1 homozygotes was performed using primers detailed in Supplementary Table 1. Genomic DNA for the PCR was isolated from 4-week old leaves by phenol extraction method (Sambrook et al., 1989) and the reaction was performed using Taq DNA Polymerase (Thermo Fisher Scientific).

AtPDAT1 (At5g13640) overexpression lines were kindly provided by Anders S. Carlsson in pART27 vector, with AtPDAT1 being expressed under the 35S Cauliflower Mosaic Virus promoter (CaMV; Ståhl et al., 2004; Banaś et al., 2014).

Lifecycle events of pdat1 knockout mutant, PDAT1-overexpressing, and wild-type lines were recorded simultaneously during plant development. All of the studied lines were grown together in a growth chamber at 23°C. The applied photoperiod consisted of 16 h of light (120 μmol photons m^–2 s^–1) and 8 h of dark. The relative humidity was at 60%. Lifecycle events were recorded on 6 or more different plants for each line.

The seeds of tested Arabidopsis lines were surface-sterilized by immersion in ethanol (70%, 2 min) followed by 1 min wash in distilled water and immersion in calcium hypochlorite (4%, 10 min). After that, the seeds were washed with distilled water (4 times) and planted on plates containing: 1% agar, 0.33× Murashige-Skoog medium (MS) and 1% sucrose. The seeds were sown on agar on a straight line in an upper part of the plate (up to 10 seeds per plate) and put in 4°C for 48 h vernalization. After that the plates were placed in the growth chamber vertically. After the needed amount of time (depending on the experiment) the rosette leaves and roots were separated and weighed. Under all temperature conditions the plants were grown with 16 h light (120 μmol photons m^–2 s^–1)/8 h dark photoperiod. For optimal growth condition measurements, the plates were placed for 4 weeks in 23°C. For heat stress measurements, the plates where placed for 3 weeks in 23°C, then for 2 h in 40°C (in MLR-352H Climate Chamber, Panasonic; humidity set at 60%). Now, depending on the experimental destination they were either placed for 2 h back into 23°C (for relative expression measurements) or placed for an additional week back into 23°C (for fresh weight measurements, lipid extraction). For cold stress measurements, the plants were put into growth chamber with 23°C for 2 weeks and then for additional 2 weeks into cold chamber (4°C), after which the fresh weight was measured.

Three 4-week-old rosettes of in vitro cultivated Arabidopsis were weighed and subjected to a Bligh and Dyer lipid extraction (Bligh and Dyer, 1959). One-fifth of the chloroform extract was transmethylated in 2 ml solution of 2% H₂SO₄ dissolved in dry methanol (45 min, 90°C), extracted to heptane with 50 nmol of heptadecanoic acid (internal standard) and then directly analyzed with GC-FID (Shimadzu GC-2010) utilizing 60 m × 0,25 mm BPX70 column (SGE Analytical Science). The rest of the chloroform extract was separated on TLC plate (silica gel 60; Merck) in heptane:diethyl ether:acetic acid (70:30:1) mobile phase. TAG spots were visualized under UV light after staining by 0.05% primuline solution and later scraped off. Silica gel samples containing TAG lipid group were from then on processed as described above for the one-fifth of the chloroform extract.

To obtain microsomal fraction from roots and rosettes, the seeds of tested Arabidopsis lines were sterilized and placed on MS plates as described above (only spread throughout the plate and not in a straight line). After 2 weeks of cultivation in 23°C, they were transferred into flasks containing 0.5× MS medium and 1% sucrose and grown with gentle shaking (80 rpm) for three more weeks. For all 5 weeks in vitro Arabidopsis lines were cultivated under the same light conditions as pot-cultivated plants (16 h light/8 h dark). The roots from approximately 20 plants were then carefully separated from leaves and homogenized separately in a glass homogenizers with homogenization buffer (0.1 M potassium phosphate buffer with 7.2 pH, 0.33 M sucrose, 1 mg/ml BSA and catalase 1000 units/ml) in a cold room (4°C) in ice. After homogenates were ready they were filtered through two-layer Miracloth and centrifuged at 20 000 × g for 12 min. The supernatants were filtered through two-layer Miracloth and centrifuged again, this time at 100 000 × g for 100 min. The supernatants were disposed of and the obtained pellets (containing microsomal fraction) were suspended in 0.1 M potassium phosphate buffer (pH 7.2). Aliquots of each suspension were used to measure microsomal protein content via Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific) and analyze phosphatidylcholine (PC) content as previously described by Klińska et al. (2019). PC content is equivalent to membrane concentrations in microsomal fractions. We show how both values relate to each other in Supplementary Table 4.

For conducting enzyme assays we have utilized substrates both employed by the enzymes and with proven in vitro activity (Ståhl et al., 2004; Banaś et al., 2014; Jasieniecka-Gazarkiewicz et al., 2017). For LPCAT activity tests, following optimization, 5 nmol of exogenous sn-1-18:1-lysophosphatidylcholine and 5 nmol of exogenous [¹⁴C]18:2-CoA were used with aliquots of microsomal fractions equivalent to 0.05 nmol (both roots and rosettes) of endogenous PC (0.022 μg of microsomal protein). The reaction components were diluted in 100 μl of 100 mM potassium buffer (pH 7.2). Addition of microsomal fraction was treated as the beginning of the reaction, which lasted for 30 min, and was conducted in 30°C. The reaction was terminated by addition of 375 μl of chloroform:methanol (1:2; v:v), 5 μl of glacial acetic acid, 125 μl of chloroform and 125 μl of water. Chloroform fraction was separated by centrifugation and transferred to a new tube. Extracts were then separated on silica gel 60 plates (Merck) utilizing thin-layer chromatography (TLC) principles in a glass chamber containing chloroform:methanol:acetic acid:water (90:15:10:2,5; v:v:v:v). The product of the reaction ([¹⁴C]-PC was visualized and quantified with electronic autoradiography (Instant Imager, Packard Instrument Co.).

AcylCoA:lysophosphatidylethanolamine acyltransferase activity tests were conducted and assessed in similar fashion, only instead of sn-1-18:1-lysophosphatidylcholine, sn-1-18:1-lysophosphatidylethanolamine was used, and microsomal fraction concentration went up to 0.5 nmol (0.22 μg of microsomal protein for both roots and rosettes) of endogenous PC per assay. The desired product was not [¹⁴C]-PC, but [¹⁴C]-PE.

Phospholipid:diacylglycerol acyltransferase activity assays were conducted in a slightly different manner. 18 h before the planned activity assays aliquots of 12 nmol of microsomal fractions were lyophilized (Heto PowerDry LL3000 Freeze Dryer, Thermo Electron Corporation). 5 nmol of sn-1-18:1-sn-2-[¹⁴C]18:2-PE and 5 nmol of sn-1,2-18:1-diacylglycerol for each assay were diluted in benzene and added to lyophilized microsomal fraction. The benzene was then evaporated under nitrogen, and 100 μl of 100 mM potassium buffer (pH 7.2) was added (beginning of the reaction). The reaction lasted for 2 h in 30°C. The reaction was terminated as described above for LPCAT activity. Reagent-containing chloroform extracts were separated by TLC on silica gel 60 plates with solvent system of hexane:diethyl ether:acetic acid (70:30:1; v:v:v:v). The product, which was [¹⁴C]-TAG, was visualized and quantified with electronic autoradiography.

Before the experiments, primers for qPCR were designed (Supplementary Table 2) for the following genes: ACT2 (At3g18780), PP2A (At1g69960), LPCAT1 (At1g12640), LPCAT2 (At1g63050), LPEAT1 (At1g80950), LPEAT2 (At2g45670), PDAT1 (At5g13640), ATG8a (At4g16520). Heat-stressed or non-stressed leaves of 3-week-old Arabidopsis grown in vitro were flash-frozen in liquid nitrogen. Total RNA was extracted from samples with GeneMatrix Universal RNA Purification Kit (EurX). RNA was then incubated with dsDNase (Thermo Fisher Scientific) to remove genomic DNA and then cDNA was synthesized with Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific). Maxima SYBR Green/ROX qPCR Master Mix (2×; Thermo Fisher Scientific) was used for qPCR analysis. qPCR measurements were conducted in QuantStudio^TM 3 Real-Time PCR System (Applied Biosystems). All procedures were performed according to the manufacturers’ instructions. The acquired results were analyzed employing the 2^–ΔΔCT algorithm (Winer et al., 1999).

We began our journey into investigating AtPDAT1 by studying phenotypic differences between selected pdat1 knockout mutants and PDAT1-overexpressing lines, as well as wild-type Arabidopsis thaliana. After 48 h vernalization, the seeds were planted into soil and observed while growing under assigned standard conditions (23°C, 16 h light/8 h dark photoperiod, 60% humidity). Key plant lifecycle events which were examined under utmost scrutiny were: appearance of the first flower buds (known as 0 DAF – day after flowering), opening of the first flower, yellowing of the first rosette leaf, yellowing of the last leaf in the rosette and cessation of flowering (all flowers of the plant becoming siliques). The obtained results are presented in Figure 1 and Supplementary Table 3.

Contrary to our expectations, there was no drastic difference between wild-type and pdat1 knockout lines generative lifecycle events. Wild-type and mutant lines went through the main milestones in flower development at similar points in time (they occurred at similar days after sowing), from first appearance of flower buds, through first flower opening to cessation of flowering, understood as all flowers on a single plant becoming siliques. PDAT1-overexpressing lines, on the other hand, began flowering significantly earlier than control, but ceased to produce flowers around the same time as wild-type and mutant lines. Their generative lifecycle was thus effectively longer. The wild-type plants were also visibly smaller than PDAT1-overexpressing plants (Figure 2).

Both the knocking-out and overexpression of PDAT1, influenced the advancement of Arabidopsis leaves yellowing (Figure 1). Lines with knockout of pdat1, entered senescence earlier than wild-type and had an all-yellow leaves rosette sooner. Overexpression of the gene, on the other hand, retarded plant aging and resulted in PDAT1-overexpressing plants going through the appearance of the first yellow leaf in the rosette at average 5 or 6 days later than wild-type (depending on the PDAT1-overexpressing line) and exhibiting an all-yellow leaves rosette 6 or 8 days after wild-type, on average. Therefore, PDAT1 expression level acts on Arabidopsis senescence and lifespan.

In order to check the effect, which both PDAT1 overexpression and pdat1 knockout would have on plant size (weight), we cultivated A. thaliana lines in vitro in assigned standard conditions, described in the previous chapter. Subsequently, we measured the fresh weight of their rosettes and roots after 4 weeks had passed (Supplementary Figure 1 contains photos of the cultivation at 3 weeks). The rosettes of PDAT1-overexpressing Arabidopsis lines weighed significantly more than wild-type control (126 and 123% of the control weight; Figure 3A). Clear difference was also observed in pdat1 mutants, which weighed 67 and 74% of control weight, depending on the mutant line. The differences were also significant in roots (Figure 3B), especially between pdat1 knockout plants and control, since the pdat1 mutant Arabidopsis had significantly lower root weight at 56 and 61% of the weight of control’s roots. No trends in regard to total fatty acid composition were observed (Supplementary Figure 2). In TAG there was a slight decrease in molar proportion of 18:3 in pdat1 knockout lines (Supplementary Figure 3), which would be in accordance with PDAT1’s specificity toward unsaturated fatty acids.

Because of the previous experiment’s outcome we decided to test the investigated Arabidopsis lines in stress conditions, to see, if PDAT1 overexpression or knockout were influencing plant’s resilience to hostile environmental conditions. The first series of experiments we designed focused on short-term heat stress. In our experiment 3-week-old Arabidopsis seedlings grown in vitro in optimal temperature (23°C) were exposed to heat for 2 h (40°C, 60% humidity). After the shock treatment, plants were placed back into optimal temperature, where they recovered for 7 days (Supplementary Figure 4). Then we proceeded to measure the fresh weight of all investigated plants’ rosettes (Figure 4A) and roots (Figure 4B).

Rosettes of wild-type (Figure 4A) experienced a small boost in comparison with 4-week-old seedlings, grown only in optimal temperature (Figure 3A). Heat-shocked knockout lines’ rosettes also weighed more than those grown in optimal temperature, but their weight in relation to the stressed control mirrored optimal conditions. However, that was not true for root measurements (Figure 4B). The weight of pdat1 mutant roots constituted more percentage of the control than their unstressed counterparts in previous experiment – 78 and 75% of control’s root weight in comparison to 56 and 61% in optimal conditions. The biggest change comparing to unstressed Arabidopsis in vitro growth occurred in PDAT1-overexpressing plants’ roots. The difference between them and control grew 40% in case of both overexpressing lines in comparison to optimal conditions. The weight divergence between wild-type and PDAT1-overexpressing plants increased also in case of rosettes (by 18% and 9% for particular lines). Total fatty acid composition was not affected by changes observed in the studied lines (Supplementary Figure 5). What distinguished the heat-shocked plants from the non-stressed ones was the TAG content, which increased in all of the studied lines (Figure 5). However, we could not discern any trends between the wild-type, the overexpressing lines and the mutant lines. Similarly, to non-stressed Arabidopsis, TAG of knockout lines contained lower mole percent of 18:3 in comparison to other studied plants (Supplementary Figure 6).

After short-term heat stress, we decided to subject our Arabidopsis lines to prolonged cold exposure. We selected 4°C as our testing cold temperature. Knowing, the plants would not germinate in 4°C, we first cultivated them in optimal temperature for 2 weeks (in vitro) and then placed them in the cold chamber for the next 2 weeks. Both wild-type control and pdat1 knockout reacted extremely to the stress factor, experiencing arrested growth and loss of pigment (Figures 6A–C). PDAT1-overexpressing lines were the most resilient, continuing to grow, with most or all of the rosette leaves preserving the green color. The measured fresh weight of rosette leaves and roots reflected that (Figures 6D,E). While both rosettes and roots of the pdat1 mutant lines weighed significantly less than control’s rosettes and roots, the PDAT1-overexpressing lines’ rosettes weighed on average 3.75 and 4.2 times more and roots average weight was 5.41 and 7.06 times more than their wild-type counterparts.

Some of the Arabidopsis plants, which were subjected to short-term heat stress were not given 1 week to recover, but their rosettes were instead harvested 2 h after the high-temperature treatment and used for relative expression measurements. For comparison, relative expression was also measured in rosettes of 3-week-old seedlings, which were grown with heat-stressed plants, but not exposed to 2 h in 40°C.

Taking into the account just wild-type lines, it turns out, that PDAT1 is expressed more than three times more in rosettes of seedlings, which were cultivated in 40°C for 2 h (Figures 7A,B) in comparison to non-stressed wild-type. It means, that enduring a short period of heat exposure triggers tripling of PDAT1 expression in Arabidopsis plants.

Investigated PDAT1-overexpressing lines exhibited a monumental increase in PDAT1 relative expression in comparison to wild-type (Figures 7C,D), just in optimal conditions. The increase of PDAT1 expression between PDAT1-overexpressing lines in optimal conditions versus the same lines after heat stress was noticeable and statistically significant in one PDAT1-overexpressing line in reference to ACT2 and in both lines in reference to PP2A (Figures 7A,B).

Since plant stress response stimulates autophagocytic process, we decided to measure relative expression levels of ATG8a gene, which encodes a protein regarded as essential in autophagy (Bu et al., 2020). ATG8a expression levels were measured in wild-type plants, PDAT1-overexpressing plants and pdat1 knockout mutants, both those cultivated only in optimal temperature for 3 weeks and those exposed to 2 h heat stress with 2 additional hours after shock treatment.

ATG8a relative expression was on the same level and not statistically different in any of the lines cultivated without heat-shock treatment (Figure 8). In comparison, all ATG8a expression levels in every Arabidopsis line subjected to high temperature were significantly multiple times higher than optimal-temperature-only wild-type control. The highest ATG8a relative expression in heat-exposed plants was found in PDAT1-overexpressing line 2 followed by expression in pdat1 knockout mutant line 2, then wild-type, then pdat1 knockout line 1 with the lowest ATG8a expression after heat exposure presented by PDAT1-overexpressing line 1. Expression in all plants subjected to heat-shock was on similar level. Based on those results, it seems that PDAT1 overexpression or knockout in A. thaliana does not correlate with higher or lower expression of autophagy-related ATG8a gene, neither in standard growth conditions nor after 2 h following the exposure to high-temperature stress.

Phospholipid:diacylglycerol acyltransferase enzymes are intertwined with LPLAT enzymes, since products of forward reactions of LPLAT enzymes are substrates for PDAT in DAG acylation and one of the products of PDAT action (LPL) is a substrate of LPLAT enzymes. For that reason, we decided to conduct in vitro assays using microsomal fractions from rosettes and roots of wild-type and PDAT1-overexpressing Arabidopsis cultures. First, we measured PDAT endogenous activity in root microsomal fractions from both wild-type control and PDAT1-overexpressing lines to confirm that those lines showed higher enzyme activity in vitro. PDAT1 activity in roots (Figure 9A; Supplementary Figure 7A) correlated with PDAT1 relative expression measurements in rosettes, even though plants for both experiments were grown in different conditions (Figures 7C,D). PDAT1 overexpression in roots was confirmed in its endogenous enzyme activity, which was 6.7 and 3.1-fold higher in PDAT1-overexpressing lines than in wild-type control.

With higher activity in PDAT1 OE confirmed, we advanced to LPLAT activity measurements. Since phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in plants, we concentrated on LPCAT and LPEAT activity of the microsomal fractions. We measured the analyzed enzyme’s activity as amount of product (an appropriate phospholipid) de novo synthesized in an in vitro reaction utilizing the amount of microsomal fraction containing 1 mg of microsomal protein during 1 h. Of the two LPLAT, LPCAT turned out to be the more active enzyme in microsomal fractions from both wild-type and PDAT1-overexpressing line (after optimizing reactions we used aliquots of root microsomal fraction equivalent to 0.05 nmol of endogenous PC for LPCAT assays and equivalent to 0.5 nmol of endogenous PC for LPEAT assays). LPCAT was significantly more active in PDAT1-overexpressing line 1 roots (7.7 times) and rosettes (by 27%) and PDAT1-overexpressing line 2 (6.3 times) roots and rosettes (by 52%) (Figures 9B,D; Supplementary Figures 7B,D). Our preliminary studies of microsomal LPLAT activity in wild-type and PDAT1-overexpressing line 2 (Supplementary Figure 8) showed higher LPCAT activity in rosettes of PDAT1-overexpressing line 2 and lower LPCAT activity in the same line in roots. However, LPCAT activity was always higher in comparison to wild-type control.

The investigated LPEAT activity was also higher in microsomal fraction from PDAT1-overexpressing lines in both rosettes and roots (Figures 9C,E; Supplementary Figures 7C,E). The increase in LPEAT activity in rosettes was more pronounced reaching twice the activity of control in both PDAT1-overexpressing lines.

Knowing how PDAT1 overexpression affects LPLAT activity in vitro, we proceeded to analyze the relative expression of two LPCAT and two LPEAT isoenzymes (LPCAT1, LPCAT2, LPEAT1 and LPEAT2) in rosettes of 3-week-old plants cultivated in optimal growth conditions in vitro, as well as in rosettes of 3-week-old plants harvested after 2 h heat stress, followed by 2 h in optimal conditions.

We first measured relative expression of LPCAT1 in both optimal and heat-shock conditions throughout all lines investigated in this study (Figure 10). The expression of this gene did not change in any lines in non-stressed plants except for it being lower than control in PDAT1-overexpressing line 1 with PP2A as a reference. In stress conditions, a significant difference was found between PDAT1-overexpressing line 1 and WT with one of the reference genes (ACT2). Those differences, however, were not much lower than in WT and they were not found to be significant with the other reference gene nor were they confirmed by expression measured in PDAT1-overexpressing line 2. The second LPCAT isoenzyme’s expression turned out to be much more diverse (Figure 11). In optimal conditions LPCAT2 expression was more than two times higher than control in both studied PDAT1-overexpressing lines, and with both reference genes. It was also significantly lower in pdat1 mutant line 1 with both ACT2 and PP2A. Expression of LPCAT2 grew in all the lines in stress conditions, and was still significantly higher in PDAT1-overexpressing lines than in wild-type. LPEAT1 relative expression (Figure 12) showed no difference between the lines in neither plants subjected to non-stress and stress conditions. The enzyme’s expression increased, however, between plants grown in optimal conditions and plants that endured heat-shock treatment, in all the studied lines. Its isoenzyme, LPEAT2, did not have increased expression in PDAT1-overexpressing lines neither (Figure 13). In stress conditions LPEAT2 expression was slightly (yet significantly with PP2A) lower after heat-shock than in optimal conditions. Non-stressed pdat1 mutant lines were exhibiting marginal increase in LPEAT2 expression.