Plant materials and growth conditions
All Arabidopsis (A. thaliana) lines used in this study were in the Columbia(Col-0) ecotype background. The ati1 ati2 mutant and ATI1-GFP transgenic line were described previously24. These lines were identified by genomic PCR using the primers listed in Supplemental Table 1. After surface sterilization, Arabidopsis seeds were germinated on half-strength Murashige and Skoog (MS) agar with 1% (w/v) sucrose and 1% (w/v) agar, and the seedlings were grown in a greenhouse with a day/night regimen of 16/8 h (23°C/21°C), an average light intensity of 160 mmol m−2 s−1, and a relative humidity of 70–80%. Seven-day-old seedlings were transplanted to a soil/vermiculite mixture (1:1) and were watered with Hoagland’s nutrient solution.
RNA extraction, RT-qPCR, and plasmid construction
Total RNA was isolated from Arabidopsis shoots grown in soil for 4 weeks, and cDNA was isolated using the PrimeScript™ II First-Strand cDNA Synthesis Kit (TaKaRa). LPATs were constructed into pCAMBIA1307-GFP, and mCherry-LPAT2, LDAP1-mCherry, and GFP-ATI2 into pCAMBIA130121. To suppress the expression of LPAT2, Pro35S:amiLPAT2 was generated using WEB microRNA designer (http://wmd3.weigelworld.org). The endogenous miRNA precursor MIR319a was cloned from RS30037and constructed into 1300-Super vector with the LPAT2 target gene. All constructs were confirmed by sequencing. Primers are listed in Supplementary Table 1.
The indicated constructs were introduced into Arabidopsis Col-0 plants by Agrobacterium tumefaciens (GV3101) using the floral dipping method. For transient expression in tobacco leaves, the GV3101 strains were infiltrated into tobacco leaves, stored under dark conditions overnight, and exposed to light for 48 h. For dark treatment, tobacco leaves were incubated under dark conditions for 3 d.
GST pull-down assay
The fragments of LPAT1 (448-657) and LPAT2 (100-918) were cloned into pGEX-4T-1 (GE Healthcare), and the N-terminal ATI2 (630 bp) was constructed into pCOLD1. Fusion proteins were expressed in Escherichia coli BL21 (DE3; Promega) according to the manufacturer’s instructions. GST-LPAT-bound beads were incubated with total protein extracted from cells expressing His-ATI2 at 4℃ for 3 h with gentle rotation. The beads were collected, washed three times with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O, 2 mM KH2PO4), boiled in 1× SDS loading buffer, and proteins were separated by SDS-PAGE.
Y2H, BiFC, and LCI assays
Y2H assays were performed according to the instructions of the Matchmaker Gold Yeast Two-Hybrid System (TaKaRa, Shiga, Japan). LPAT1/2 and ATI1/2 were cloned into pGBKT7 (BD) and pGADT7 (AD), respectively, and co-transformed into the yeast strain Y2H Gold. Colonies were photographed after 3 d at 30°C on –Leu–Trp dropout (DDO) and –Leu–Trp–His–Ade dropout (QDO) agar. BD and AD empty vectors were used as the negative controls.
For BiFC assays,the full-length coding sequences of LPATs and ATI2 were cloned into the binary nYFP and cYFP vectors, respectively, using the gene-specific primers listed in Supplementary Table 1. Transformed tobacco leaves were incubated for 72 h in the dark at room temperature before confocal microscopy analysis. The YFP signal was imaged at an excitation wavelength of 512 nm, and the emission signal was recovered at 525 to 565 nm. LCI assays were performed as described previously38. After infiltration at 28°C for 48 h, LUC activity was detected using a live plant imager (Tanon-5200).
Coimmunoprecipitation assay (co-IP)
LPAT2-GFP and ATI2-Flag were co-expressed in N. benthamiana leaves for co-IP assay. Total proteins were extracted in ice-cold immunoprecipitation buffer (50 mM Tris-MES, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 1.0% NP‐40, 1 mM DTT, 1 mM PMSF and 1´ protease inhibitor cocktail). Extracted proteins were incubated with anti‐GFP agarose beads (Shenzhen KT Life Technology) for 3 h at 4°C. After washing five times with ice‐cold immunoprecipitation buffer, the beads were boiled in 1× SDS loading buffer to release proteins, which were analyzed by immunoblotting using anti‐Flag (TransGen) or anti‐GFP antibodies (Abcam). This experiment was repeated at least three times.
FRET-FLIM analysis and fluorescence recovery after photobleaching (FRAP)
Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM) analysis was performed to detect the in vivo interaction between mCherry-LPAT2 and GFP-ATI2. The FRET pairs GFP and mCherry were used as the donor and acceptor fluorophores. The mCherry-LPAT2 and GFP-ATI2 plasmids were transiently transfected into tobacco cells. The scanning confocal microscope SP8 FALCON (Leica Microsystems) with the 20× dry objective lens was used for fluorescence confocal imaging and fluorescence lifetime imaging. A white-light laser system was used to excite GFP (488 nm), and hybrid photon detectors were used to collect emission at 495–545 nm from tobacco epidermal cells. The region of interest (ROI) was selected and lifetime values were calculated from at least 10 individual epidermal cells. All data manipulations were performed and fitted using LAS X ver. 3.5.2 software (Leica).
FRAP analysis was performed using FRAP Wizard implemented in LAS software (Leica). Two frames were scanned before bleaching, and a circular ROI covering the lipid droplet with 100% laser intensity for five frames was performed. Fluorescence recovery was measured at 15 s intervals until the fluorescence plateaued. Fluorescence in the ROI was quantified using LAS software (Leica).
LD isolation and lipid profile analysis
LDs were isolated as described previously40. Briefly, 6-week-old Arabidopsis rosette leaves were collected and ground into homogenates in extraction buffer (0.6 M sucrose, 10 mM NaH2PO4, pH 7.5, 150 mM NaCl, 0.1% [v/v] Tween-20, 1 mM PMSF, and 1´ protease inhibitor cocktail). Floating buffer 1 (0.4 M sucrose, 10 mM NaH2PO4, pH 7.5, 150 mM NaCl, 0.1% [v/v] Tween-20, 1 mM PMSF, and 1´ protease inhibitor cocktail) was slowly added to the tube as the upper layer, and centrifuged at 4°C 100,000 g for 90 min. Next, the accumulated LDs in the top layer were transferred to a new tube, and floating buffer 2 (0.2 M sucrose, 10 mM NaH2PO4, pH 7.5, 150 mM NaCl, 0.1% [v/v] Tween-20, 1 mM PMSF, and 1´ protease inhibitor cocktail) was added to the tube, followed by centrifugation at 100,000 g for 90 min. The upper-layer LD component was used for LD lipid extraction and analysis of lipid molecular species.
TEM observation, confocal microscopy, and quantitative analysis
For TEM observation, Arabidopsis seeds were fixed, embedded in Spur’s resin, sectioned, and contrasted as described previously39. Images were obtained using a H-7650 transmission electron microscope (Hitachi).
Confocal fluorescence images were obtained using a SP8 confocal laser scanning microscope (Leica). BODIPY 493/503 (Invitrogen) and MDH were used to stain LDs as described previously4,41. In brief, LDs were stained with 2 µg mL–1 BODIPY (4 mg/mL stock in DMSO) in 50 mM PIPES buffer (pH 7.0) and excited using a 488 nm laser with emission at 500–540 nm. For MDH staining, 0.1 mM MDH (0.1 Mstock in DMSO) in 50 mM Tris-HCl buffer (pH 7.0) was excited using a 405 nm laser with emission at 420–480 nm. LDs were stained for 15 min washed thrice in 50 mM Tris-HCl buffer and observed by confocal microscopy.
Phylogenetic analysis
The protein sequences for phylogenetic analysis were obtained from the NCBI database. The amino acid sequences were aligned using ClustalW and adjusted manually. Phylogenetic trees were mapped using MEGA version 7 software by the neighbor-joining method. Trees were drawn to scale, with branch lengths identical to the units used to infer evolutionary distances from phylogenetic trees.
Drug and salt treatment
Two-week-old Arabidopsis seedings were treated as follows. For OA treatment, the transgenic lines were incubated with OA (200 µM) for the indicated times. For inhibitor treatments, plants were transferred to half-strength MS medium containing DMSO, 1-But (0.1%, v/v), R59949 (12.5 μM), or CI-976 (50 μM) for 12 h. For salt treatment, five-day-old seedlings were transferred to half-strength MS agar with or without 200 mM NaCl. After 10 d of treatment, new growth of true leaves was defined as survival.
Statistical analysis
Statistical significance was analyzed by two-sided Student’s t-test (P < 0.05 and P < 0.01). Data are means ± SEM unless stated otherwise. Graphs were generated using GraphPad Prism ver. 8 for Windows or Microsoft Excel for Windows.