Transcriptomic and lipidomic profiles of glycerolipids during Arabidopsis flower development

Transcriptomic and lipidomic profiles of glycerolipids duringArabidopsis flower development

Yuki Nakamura1,2,3,4, Norman Z. W. Teo3,4,5, Guanghou Shui3,6, Christine H. L. Chua4, Wei-Fun Cheong3,

Sriram Parameswaran4, Ryota Koizumi7, Hiroyuki Ohta8,9, Markus R. Wenk3,5,10 and Toshiro Ito4,5

1Institute of Plant and Microbial Biology, Academia Sinica, 128 sec.2 Academia Rd, Nankang, Taipei 11529, Taiwan; 2PRESTO, Japan Science and Technology Agency, A-1-8 Honcho

Kawaguchi, Saitama, Japan; 3Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 28 Medical Drive, Singapore city 117456, Singapore; 4Temasek

Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore city 117604, Singapore; 5Department of Biological Sciences, National University of Singapore,

Singapore city, 117543 Singapore; 6State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101,

China; 7Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan; 8Center for Biological Resources and

Informatics, Tokyo Institute of Technology, 4259-B-65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan; 9Earth-Life Science Institute, Tokyo Institute of Technology, Ookayama,

Meguro-ku, Tokyo 152-8551, Japan; 10Swiss Tropical and Public Health Institute, University of Basel, Socinstrasse 57, PO Box 4002, Basel, Switzerland

Authors for correspondence:Yuki Nakamura

Tel: +886 2 27871130Email: [email protected]

Toshiro Ito

Tel: +65 68727016Email: [email protected]

Received: 10 November 2013

Accepted: 19 February 2014

New Phytologist (2014)doi: 10.1111/nph.12774

Key words: Arabidopsis thaliana, flowerdevelopment, glycerolipids, lipidomics, syn-chronized flower development.

Summary

� Flower glycerolipids are the yet-to-be discovered frontier of the lipidome. Although ample

evidence suggests important roles for glycerolipids in flower development, stage-specific lipid

profiling in tiny Arabidopsis flowers is challenging. Here, we utilized a transgenic system to

synchronize flower development in Arabidopsis.� The transgenic plant PAP1::AP1-GR ap1-1 cal-5 showed synchronized flower development

upon dexamethasone treatment, which enabled massive harvesting of floral samples of

homogenous developmental stages for glycerolipid profiling.� Glycerolipid profiling revealed a decrease in concentrations of phospholipids involved in sig-

naling during the early development stages, such as phosphatidic acid and phosphatidylinosi-

tol, and a marked increase in concentrations of nonphosphorous galactolipids during the late

stage. Moreover, in the midstage, phosphatidylinositol 4,5-bisphosphate concentration was

increased transiently, which suggests the stimulation of the phosphoinositide metabolism.

Accompanying transcriptomic profiling of relevant glycerolipid metabolic genes revealed

simultaneous induction of multiple phosphoinositide biosynthetic genes associated with the

increased phosphatidylinositol 4,5-bisphosphate concentration, with a high degree of differ-

ential expression patterns for genes encoding other glycerolipid-metabolic genes. The phos-

phatidic acid phosphatase mutant pah1 pah2 showed flower developmental defect,

suggesting a role for phosphatidic acid in flower development.� Our concurrent profiling of glycerolipids and relevant metabolic gene expression revealed

distinct metabolic pathways stimulated at different stages of flower development in Arabidopsis.

Introduction

Flower development is a highly coordinated event throughoutthe life cycle of seed plants with the aim of successful fertilizationand propagation of the subsequent generation. In Arabidopsisand other model plants, genetic study has contributed signifi-cantly to unraveling how the dramatic event of flower develop-ment is finely regulated (Causier et al., 2010). To furtherunderstand the molecular basis of the developmental process, asystems approach demands -omics study.

Because the Arabidopsis inflorescence consists of tiny flowersin different developmental stages, harvesting a sufficient amountof flower samples in a homogenous floral stage is challengingeven for RNA/DNA extraction. In transcriptomic studies, efforts

have involved synchronizing flower development by means oftransgenic technology (Gomez-Mena et al., 2005). In Arabidop-sis, double knockout of APETALA1 and CAULIFLOWER (ap1cal ) causes arrest of flower development at the initial step, thusleading to an inflorescence with a cluster of overproliferatednascent floral buds (Kempin et al., 1995). Expressing a gene forthe initial step of flower development can be a trigger to releasethis developmental arrest, which leads to a synchronized develop-ment of the ‘cauliflower’ flower. Gomez-Mena et al. (2005) usedAGAMOUS (AG) as a trigger to create a transgenic line, 35S::AG-GR ap1-1 cal-1, showing successful release of the arrest toreveal a transcriptional network controlled by AG (Gomez-Menaet al., 2005). To further study the initial stage of flower develop-ment that 35S::AG-GR ap1-1 cal-1 skips, Wellmer et al. (2006)

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created 35S::AP1-GR ap1-1 cal-1 to demonstrate a high degree ofdifferential gene expression during the early stages of flowerdevelopment. In line with the systems understanding, we still lackinformation on downstream events associated with the revealedgene expression pattern, that is, metabolomic or proteomic studywith the synchronized flower system following the transcriptomicstudies highlighted earlier (Bellaire et al., 2014).

Polar glycerolipids (hereafter glycerolipids) consist of a distinctset of metabolites that serve as membrane constituents (e.g. majorphospholipids, galactolipids and a sulfolipid) and signaling mole-cules (e.g. phosphatidic acid (PA), phosphatidylinositol (PI) andphosphoinositides). Distinct biochemical features of glycerolipidmetabolism are shown in Petunia flowers. Digalactosyldiacylglyc-erol (DGDG) is the major glycolipid in flowers, and metabolismin diacylglycerol (DAG) production reveals floral organ-specificity (Nakamura et al., 2003; Nakamura & Ohta, 2007). InArabidopsis, knockout studies have provided ample evidence ofglycerolipid biosynthesis involved in flower development. Forexample, a knockout mutant of glycerol 3-phosphate acyltrans-ferase 1 (GPAT1) that catalyzes the first step of the extraplasti-dic Kennedy pathway results in aberrant pollen structure(Zheng et al., 2003). Knockout of plastidic lysophosphatidateacyltransferase 1 (LPAT1) induces embryonic lethality (Kim &Huang, 2004; Yu et al., 2004). A leaky mutant of CTP:phos-phorylethanolamine cytidylyltransferase 1 (PECT1) affectsflower development, fertility and embryonic development (Mi-zoi et al., 2006). Knockout of phosphatidylserine synthase 1(PSS1) confers a male gametophytic defect (Yamaoka et al.,2011). This evidence suggests the critical roles of glycerolipidsin multiple stages of flower development.

Here, to understand glycerolipid profiles during flower devel-opment, we developed an Arabidopsis transgenic line with syn-chronized flower development. With this system, glycerolipidprofiling by a lipidomics platform revealed a marked decrease inPA and PI concentrations in the early developmental stages and asubsequent increase in galactolipid concentrations, mainly mo-nogalactosyldiacylglycerol (MGDG), in the later stages. Togetherwith the concomitant transcriptomic profiling of glycerolipidmetabolic genes by quantitative reverse transcription polymerasechain reaction (qRT-PCR), our analyses reveal distinct patternsof glycerolipid metabolism at different stages of flower develop-ment in Arabidopsis.

Materials and Methods

Plant growth and treatment conditions

Arabidopsis thaliana (L.) Heynh plants were grown under contin-uous light (150 lmol m�2 s�1). The pah1 pah2 mutant plantswere grown at 22°C, and pAP1::AP1-GR ap1-1 cal-5 plants weregrown at 18°C to avoid precocious initiation of flower develop-ment. Dexamethasone (DEX) was applied to the inflorescencesof pAP1::AP1-GR ap1-1 cal-5 by dipping in an aqueous solution(pH 7.0) containing 1 lM DEX and 0.015% (v/v) Silwet L-77.Construction of pAP1::AP1-GR ap1-1 cal-5 was described previ-ously (Bellaire et al., 2013).

Lipid extraction

Inflorescences of pAP1::AP1-GR ap1-1 cal-5 were harvested,immediately frozen in liquid nitrogen, and kept at �80°C untillipid extraction. Before lipid extraction, frozen tissues were incu-bated in hot (75°C) isopropanol containing 0.05% (v/v) butyl-ated hydroxytoluene (cat. no. B1378; Sigma-Aldrich) for 15 min.Total lipid was extracted from c. 500 ll (c. 10 mg DW) frozentissue as previously described (Bligh & Dyer, 1959).

Lipid analysis by high-performance liquid chromatography(HPLC)/mass spectrometry

An Agilent HPLC system coupled with an Applied BiosystemsTriple Quadrupole/Ion Trap mass spectrometer (4000Qtrap;Applied Biosystems, Foster City, CA, USA) was used to quantifyindividual polar lipids (phospholipids). Polar phospholipid spe-cies were quantified using targeted lipidomic approaches as previ-ously described (Fei et al., 2008). Briefly, from product andprecursor ion analysis of head groups, multiple reaction monitor-ing transitions were set up to quantify various polar lipids. Indi-vidual lipid concentrations were quantified by normalizing to thecorresponding spiked internal standards. Dimyristoyl phosphati-dylcholine (28:0-PC) was used to quantify endogenous PC,dimyristoyl phosphatidylethanolamine (28:0-PE) was used forendogenous PE, dimyristoyl phosphatidylserine (28:0-PS) wasused for endogenous PS, dimyristoyl phosphatidylglycerol (28:0-PG) was used for endogenous PG, and dimyristoyl phosphatidicacid (28:0-PA) was used for endogenous PA (all of these stan-dards were from Avanti Polar Lipids (Alabaster, AL, USA)).Dioctanoyl PI (16:0-PI; Echelon Biosciences, Salt Lake City,UT, USA) was used to quantify PI species.

Galactolipids, MGDG and DGDG were analyzed using anAgilent 1100 HPLC system coupled with an Applied Biosystems4000 QTrap mass spectrometer (W.-F. Cheong et al., unpub-lished). The HPLC system is made up of an Agilent 1100 binarypump, an Agilent 1100 thermo sampler and an Agilent 1100 col-umn oven. A Kinetex 2.6 u C18 100A column (i.d.4.69 100 mm; Phenomenex, Torrance, CA, USA) was used toperform lipid separation with a mobile phase containing chloro-form : methanol : 2% 50 mM sodium acetate at flow rate of180 ll min�1 for 25 min. The MS instrument was operated inpositive electronspray ionization (ESI) mode with a capillary volt-age of 5000 V, capillary temperature of 300°C and the collisionenergy ranged from 70 to 75 V. Product ion scan was performedusing this approach to generate specific multiple reactionmonitoring (MRM) transitions for the individual species ofMGDG and DGDG lipids. Individual lipid concentrations werequantified according to spiked purified standards, MGDG andDGDG, from Matreya LLC (Pleasant Gap, PA, USA).

Quantification of phosphatidylinositol 4-phosphate (PI4P)and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)

Phosphatidylinositol 4-phosphate (PI4P) and PI(4,5)P2 werequantitatively analyzed as previously described (Nasuhoglu et al.,

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2002) with a Dionex Ion Chromatography 3000 system (Dionex,Sunnyvale, CA, USA). Lipid extracts were deacylated by incuba-tion with 0.5 ml methylamine reagent (MeOH : 40% methylaminein water : 1-butanol : water (47 : 36 : 9 : 8, v/v)) at 50°C for45min. The aqueous phase was dried, resuspended in 0.5 ml of 1-butanol : petroleum ether : ethyl formate (20 : 40 : 1, v/v), andextracted twice with an equal volume of water. Aqueous extractswere dried, resuspended in water, and subjected to anion-exchange HPLC on an Ionpac AS11-HC column (Dionex). Neg-atively charged glycerol head groups were eluted with a 1.5–86 mM KOH gradient and detected online by suppressed con-ductivity 75 in a Dionex ion chromatography system equippedwith an ASRS-ultra II self-regenerating suppressor (Dionex).Individual peaks of head groups were identified by matchingtheir standard retention times and peak areas were calculatedusing Chromeleon software (Dionex). Lipid concentrations werecalculated with deacylated anionic phospholipids such asdipalmitoyl PI4P and dipalmitoyl PI(4,5)P2 (both from EchelonBiosciences) used as standards.

RNA extraction, cDNA synthesis and qRT-PCR

Total RNA was extracted from the floral samples with theRNeasy Plant Mini Kit (Qiagen), and cDNA was synthesizedwith the SuperScript III First-Strand reverse transcriptase kit(Invitrogen). Specific primers were designed as listed in Support-ing Information Table S1 and confirmed to be specific to the tar-get gene by dissociation analysis. qRT-PCR involved the 7900HT Fast Real Time PCR System (Applied Biosystems) withPower SYBR Green PCR Master Mix. For each qRT-PCR run,triple technical replicates were prepared and results were

averaged. Data in figures were further averaged by three biologi-cal replicates of samples. Error bars are indicated for genes with> twofold change in expression. For other genes, SD values were< 10%. The phosphoinositide-biosynthetic gene nomenclature isas defined previously (Mueller-Roeber & Pical, 2002). PIPK10(At4g01190) and PIPK11 (At1g01460) are according to Pereraet al. (2005) and PIPLC6 (At2g40116), PIPLC8 (At3g47220)and PIPLC9 (At3g47290) are according to Hunt et al. (2004).

Results and Discussion

Observation of a transgenic line that synchronizes flowerdevelopment

The pAP1::AP1-GR ap1-1 cal-5 transgenic plants were modi-fied from a previously used design (35S::AP1-GR ap1-1 cal-1)by using the native promoter of AP1 (pAP1) to reflect itsendogenous spatiotemporal expression properties. We trans-formed it in the ap1-1 cal-5 genetic background, because cal-5and ap1-1 are both in the Ler ecotype background while cal-1is in the WS ecotype background (Kempin et al., 1995; Ferran-diz et al., 2000). AP1-GR is an AP1 gene with a carboxytermi-nal fusion to the steroid-binding domain of the ratglucocorticoid receptor GR (Lloyd et al., 1994). Under normalconditions, the AP1-GR fusion protein is bound to Hsp90 andremains in the cytoplasm, where a nuclear protein AP1 is inac-tive. Upon treatment with DEX, AP1-GR is released fromHsp90 and translocates at the nucleus to induce expression ofits target genes (Fig. S1). With a single DEX application(1 lM), our pAP1::AP1-GR ap1-1 cal-5 line showed highlysynchronized flower development until anthesis (Fig. 1a). When

(a)

(b)

Fig. 1 Developmental stage-specificglycerolipid profiling in Arabidopsis thaliana

flowers. (a) Synchronized flowerdevelopment of dexamethasone (DEX)-treated pAP1::AP1-GR ap1-1 cal-5 flowers.Photographs indicate time (d) after DEXtreatment (e.g. D2, 2 d after DEX treatment).Bars, 5 mm. (b) Profiling of glycerolipidclasses during flower development. pAP1::AP1-GR ap1-1 cal-5 flowers were harvestedat different times after DEX treatment, andtotal lipids were extracted and analyzed asdescribed in the Materials and Methodssection. Data are means� SD (n = 4).Asterisks indicate significance (P < 0.01) fromthe previous time point. PI,phosphatidylinositol; PE,phosphatidylethanolamine; PA, phosphatidicacid; PG, phosphatidylglycerol; PS,phosphatidylserine; SQDG,sulfoquinovosyldiacylglycerol; PC,phosphatidylcholine; MGDG,monogalactosyldiacylglycerol; DGDG,digalactosyldiacylglycerol.


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plants were grown under optimal conditions (18°C,150 lmol m�2 s�1 continuous light), inflorescences reached thefully opened flower stage (stage 13; Smyth et al., 1990) at 14 dafter DEX treatment. Lipid composition was not affected byDEX treatment in the flowers of ap1-1 cal-5 in the absence ofpAP1::AP1-GR (Fig. S2).

Glycerolipid profiles during flower development

We performed developmental stage-specific glycerolipid profilingusing this Arabidopsis pAP1::AP1-GR ap1-1 cal-5 system. Themost dramatic change was in the profile of PA, which showed atransient increase (12–16 mol%) at day 2 after DEX treatment(hereafter D2), when ectopic inflorescence meristems were con-verted to floral meristems, followed by a rapid decrease (from 16to 4 mol%; Figs 1b, 2). Concomitantly, the concentration of PCmoderately increased at the midstage and that of MGDGincreased steeply at the late stages. Other glycerolipid profileswere fairly stable, although some changes occurred at D2. Thetemporary concomitant PA decrease and MGDG increasesuggests that PA is probably dephosphorylated by PA phospha-tase to yield DAG, which is then converted to MGDG byMGDG synthase (MGD).

Acyl profile of polar glycerolpid classes during flowerdevelopment

To further detail the glycerolipid profiles, we analyzed differentmolecular species of phospholipid and nonphosphorous glycer-olipid classes during flower development (Fig. 2). Here, we dis-cuss each profile by lipid class.

PA All of the five major species (34:3, 34:2, 36:5, 36:4 and36:3) found in flowers showed a transient increase at D2, fol-lowed by a rapid decrease in subsequent developmental stages.The extent of the decrease differed between C34 and C36 species;the level of 36:4 and 36:3 rapidly decreased at D4-6, and that of34:3 and 34:2 was high before decreasing after D6. This tempo-rary high level of C16 fatty acid-containing species may causetransient compositional changes in the PA pool at D2–4, whichmay affect PA signaling. In particular, the profiles for 36:4 and36:3 at D0–4 coincided well with those of PC and PE, so thetransient increase of these PA species may be derived from that ofPC or PE without remodeling acyl moieties, that is either byphospholipase D (PLD) or nonspecific phospholipase C (NPC)together with diacylglycerol kinase (DGK). It should be notedthat all phospholipid classes increased 36:3 and 36:4 composi-tion, whereas 36:6 in galactolipids decreased. This suggests highrecycling of phospholipids via PA but limited synthesis of galac-tolipids at this time.

PC Levels of two C34 species (34:3 and 34:2) remained stable,but that of C36 species increased gradually at the later stages.Because the PC class contains mostly C36 species, at the laterstages, it is enriched with polyunsaturated C18-containing fattyacid.

PE In contrast to PC, the PE class exhibits mostly C34 species.The profile of the major species resembled that of the PC classafter D6, with a difference found at earlier stages: C34 speciesretained similar levels from D0 to D2, but 36:4 and 36:3 showeda transient increase at D2, as did the corresponding species in thePC class. This transient increase coincided with the profile of PA,which suggests that the PLD pathway may contribute to the PAincrease at D2.

PG We found a differential profile among the five molecularspecies of PG analyzed (32:1, 32:0, 34:4, 34:3 and 34:2): aconcomitant decrease in 32:0 and increase in 34:4. Because32:0 represents the de novo synthesis of PG and 34:4 corre-sponds to 18:3/t16:1-PG, a representative plastidic species, thisprofile suggests compositional changes of PG within the plast-ids by the action of plastidic fatty acid desaturases (FAD4, 6,7 and 8) or decreased concentration of extraplastidic PG (e.g.mitochondrial PG), which is abundant in 16:0 (Li-Beissonet al., 2010).

PI Two major PI species (34:3 and 34:2) decreased duringArabidopsis flower development. This decrease can be associ-ated with repression of PI biosynthesis or stimulation of phos-phoinositide biosynthesis that uses PI as an initial substrate.During flower development, both PIS1 and PIS2 showed sta-ble transcript abundances (Fig. 3d); the genes are responsiblefor PI biosynthesis (Collin et al., 1999; Xue et al., 2000; Justinet al., 2003; Lofke et al., 2008). The finding suggests theutilization of PI for phosphoinositide metabolism (describedfurther in Figs 4, 5).

PS In agreement with the previous analysis that flower PS con-tains very long-chain fatty acids (VLCFAs) ranging from C20 toC24 (Yamaoka et al., 2011), we detected C38 and C40 species inaddition to C34 and C36 species. Although the total PS concen-tration was stable during flower development, we observed aslight decrease for species containing VLCFAs. Consistent withPC and PE, 36:4 and 36:3 showed a transient increase at D2,which suggests an involvement of these PS species in PAmetabolism.

MGDG Monogalactosyldiacylglycerol content was markedlyincreased at the later stages of flower development (Fig. 1b). Ofthe two major species, 34:6, representing prokaryotic MGDG(18:3/16:3-MGDG), showed a significant increase starting atD6–8 (Fig. 2), while the increase in 36:6 species (18:3/18:3-MGDG) was not obvious. Because MGD1 catalyzes the prokary-otic pathway of MGDG biosynthesis and contributes critically tothe MGDG concentrations (Kobayashi et al., 2007), it suggeststhat MGD1 mainly contributes to the increase in MGDG at thisstage.

DGDG We observed only a slight increase in the level ofDGDG species (Fig. 2), despite the marked increase in MGDG.This finding suggests that DGDG biosynthesis is not much influ-enced by increased MGDG during flower development.



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Sulfoquinovosyldiacylglycerol (SQDG) Major SQDG species(34:3, 34:2 and 36:6) showed a transient decrease during themidstage of flower development. The later increase in 34:3 spe-cies corresponds with increases in 34:6-MGDG and 34:4-PGspecies in stimulation of plastidic lipid biosynthesis. A high levelof SQDG at the initial stage of flower development is intriguing,with no further supportive data for the increase in SQDG in thisdevelopmental stage.

Transcriptional profile of genes involved in glycerolipidmetabolism

To correlate the glycerolipid profiles analyzed with the expressionof relevant metabolic genes, we profiled the transcript

abundances of such genes by qRT-PCR using the specific primersin Table S1. We compared our qRT-PCR data with microarraydataset publicly available for the later stages of wildtype Arabid-opsis flower development (Schmid et al., 2005). We found fairlygood agreement between them, suggesting that our data for ear-lier stages of flower development also reflects that of wildtypeflowers. Here, we grouped genes by the metabolic pathways oflipid class to which they contribute or probably contribute.

PS biosynthesis Phosphatidylserine is synthesized from PE byPSS1 and metabolized to PE by PS decarboxylase (PSD). PSS1 isthe predominant isogene in charge of PS biosynthesis becausegene knockout leaves no detectable PS and confers severe growthdefect and microspore development (Yamaoka et al., 2011).

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Fig. 2 Profiling of molecular species of glycerolipid classes of Arabidopsis thaliana flowers in Fig. 1. pAP1::AP1-GR ap1-1 cal-5 flowers were harvested atdifferent times after dexamethasone (DEX) treatment (e.g. D2, 2 d after DEX treatment), and total lipid was extracted and analyzed as described in theMaterials and Methods section. Data are means� SD (n = 4). Asterisks indicate significance (P < 0.01) from the previous time point. See Fig. 1(a) fordevelopmental stages of each time after DEX treatment. PI, phosphatidylinositol; PE, phosphatidylethanolamine; PA, phosphatidic acid; PG,phosphatidylglycerol; PS, phosphatidylserine; SQDG, sulfoquinovosyldiacylglycerol; PC, phosphatidylcholine; MGDG, monogalactosyldiacylglycerol;DGDG, digalactosyldiacylglycerol.





A study of three isoforms of PSD, PSD1, PSD2 and PSD3,showed that triple knockout resulted in no detectable PSD activ-ity and reduced PE content in mitochondria, although the maincontributors to the conversion of PS to PE in other organellesremain elusive (Nerlich et al., 2007). The transcriptional profileof these genes showed rather stable expression during flowerdevelopment, but a decrease in PSD1 level at later stages(Fig. 3a). This finding agrees with the microarray data of wild-type flowers in which PSD1 expression is higher at stage 9 than atlater stages, and increase in the level of PS-containing 34:2 and36:4 species in later flower development (Fig. 2). The decrease inlevel of PS-containing VLCFA species may not be explained byPSD, although an increase in level of VLCFA species in psd1 psd2psd3 flowers (Nerlich et al., 2007) suggests that PSD activity maybe involved in the metabolic fate of VLCFA-containing PS.

PC and PE biosynthesis Among the genes analyzed for PC andPE biosynthesis, phosphorylethanolamine N-methyltransferase2(NMT2) showed a transient three-fold increase in level at D6(Fig. 3b) and choline kinase1 (CK1) showed a threefold increase inlevel at later stages of flower development (Fig. 3c). NMT2 cata-lyzes the second and third steps of phosphor-base methylation(BeGora et al., 2010), and its knockout specifically affects the 34:3species of PE. NASCArray data show a marked increase in NMT2expression between stages 9 and 12 of flower development. Becauseonly this species showed a transient increase in level at D6 withinPE molecular species, NMT2 may be involved in this temporarychange in PE quality at the midstage of flower development. How-ever, this change is not critical for plant viability because the nmt2mutant showed no reproductive defect (BeGora et al., 2010). Func-tional study of CK1 has not been reported; however, the increasein its level coincides with the increase in level of C36 species of PC(Fig. 2), for a possible involvement of CK1 in this change.

Anionic phospholipid biosynthesis Genes involved in PG andPI biosynthesis were analyzed (Fig. 3d). Cytidine diphosphatediacylglycerol (CDP-DAG) synthase (CDS) catalyzes the conver-sion of PA to CDP-DAG; phosphatidylglycerol synthase (PGPS)catalyzes the conversion of CDP-DAG to PG phosphate (PGP),which is subsequently converted to PG; and PI synthase (PIS)converts CDP-DAG to PI. Among these genes, only CDS3showed an increase in level, by nine-fold, at the midstage andlater flower development stages (Fig. 3d). Among the five CDSgenes, the activity of CDS3 was lowest in yeast, although knock-out study has not been reported (Haselier et al., 2010). AlthoughCDS4 and CDS5 are localized at chloroplasts and contribute toplastidic lipid biosynthesis (Haselier et al., 2010), an associationof increased abundance of plastidic species of PG (34:4; Fig. 2)and CDS3 transcripts suggests an involvement in PG biosynthe-sis. PIS1 and PIS2 showed stable transcript abundances, althoughPI decreased during flower development (Figs 1b, 2). This find-ing suggests utilization of PI for phosphoinositide metabolism.

Nonphosphorous glycerolipid biosynthesis Biosynthesis ofMGDG, DGDG and SQDG occurs exclusively at plastids. Atransient decrease in transcripts of SQD1 and SQD2 (Fig. 3e)

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Fig. 3 Gene expression profiles of characterized or putative glycerolipidbiosynthetic genes during flower development of Arabidopsis thaliana.Data are means� SD fold change of transcript abundances relative to thatat D0, averaged by triple biological replicates. Asterisks indicatesignificance (P < 0.01) from the previous time point. (a) Phosphatidylserine(PS) biosynthetic genes; (b) phosphatidylcholine (PC) andphosphatidylethanolamine (PE) biosynthetic genes except choline kinases;(c) choline kinases; (d) phosphatidylglycerol (PG) and phosphatidylinositol(PI) biosynthetic genes; (e) nonphosphorous glycerolipid biosyntheticgenes; (f) glycerol 3-phosphate acyltransferase (GPAT) genes exceptGPAT2 and GPAT5, (g) GPAT2 and GPAT5 genes; (h) lysophosphatidicacid acyltransferase (LPAT) genes; (i) phosphatidic acid phosphatase genesincluding lipid phosphate phosphatase (LPP) and phosphatidatephosphohydrolase (PAH); (j) nonspecific phospholipase C (NPC) genes; (k)phospholipase D (PLD) genes; (l) DGK4 and DGK6 genes. Note that they-axis is logarithmic in (g) and (l). DGK, diacylglycerol kinase.



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NewPhytologist6

agrees well with SQDG profiles at the early stages of flower devel-opment (Fig. 2), which suggests that this profile is probably con-trolled transcriptionally. DGD1 and DGD2 showed a stableexpression pattern, which agrees well with DGDG profiles.MGD2 showed a sixfold increase in level at later stages, whichagrees with previous studies showing flower-specific expression ofMGD2 and strong promoter MGD2-GUS staining at stamensand developing pollen tubes (Awai et al., 2001; Kobayashi et al.,2004). However, knockout of MGD2 did not affect flower devel-opment, although the MGDG concentration in the mgd2mutantflower has not been reported (Kobayashi et al., 2009). Becausethe concentrations of prokaryotic MGDG were increased exclu-sively during development (Fig. 2), this profile indicates thatMGD1 mainly contributed to this increase. Contribution ofMGD1 to the reproductive process remains elusive, because theknockout mutant of MGD1 cannot reach this developmentalstage owing to a severe growth defect at germination (Kobayashiet al., 2007). Active expression of MGD1 at stamens was shownby GUS staining (Kobayashi et al., 2004).

Acyltransferases in the Kennedy pathway Glycerol 3-phosphateacyltransferase catalyzes the initial step of de novo glycerolipidbiosynthesis, and hence serves as a committed step of glycerolipidbiosynthesis. The sole isoform of plastidic GPAT (ACT1)showed a stable expression pattern during flower development,but several extraplastidic GPATs showed increased levels at mid-and/or late stages (Fig. 3f). GPAT2 and 7 showed transientincreases in level at D6–8, by 20-fold and threefold, respectively(Fig. 3f,g). In the later stages, GPAT1, 3, 5 and 6 showed markedincreases in level, by five-, four-, eight- and 140-fold, respectively(Fig. 3f,g). An increase in GPAT1 transcripts at the later stageagrees with previous findings that GPAT1 is important in tape-tum and anther development (Zheng et al., 2003; Yang et al.,2012). Which isoforms are the main extraplastidic GPATs is stillunclear (Yang et al., 2012). Moreover, some are involved in sur-face lipid biosynthesis. For example, GPAT5 is involved insuberin biosynthesis and GPAT6 is a bifunctional enzyme codingfor phosphatase activity to produce monoacylglycerol (Beissonet al., 2007; Li-Beisson et al., 2009). Surface lipid biosynthesismay be stimulated in the later stages, but we did not profile thesein this study.

Lysophosphatidic acid acyltransferase catalyzes the second acyl-ation to lysophosphatidic acid (LPA) to produce PA. The onlyplastidic isoform, LPAT1, showed stable expression, as did ACT1(Fig. 3f,h). Among four extraplastidic LPAT-coding genes,LPAT3 showed a transient increase in level at mid- and laterstages (Fig. 3h). This profile agrees with the initiation and matu-ration of pollen development, which is consistent with the studyof LPAT3 suggesting roles in pollen development (Kim et al.,2005). The profile of PA showed a continuous decrease at thisstage (Fig. 2), which suggests that PA synthesized by LPAT3 canbe rapidly converted to other glycerolipids or that LPAT3 specifi-cally produces PA in pollen that cannot be detected by harvestingentire flowers for lipid extraction.

PA phosphatases (PAPs) Arabidopsis has two types of PAPs, amembrane-bound type named lipid phosphate phosphatase(LPP) and a soluble type termed phosphatidate phosphohydro-lase (PAH; Nakamura et al., 2007, 2009b). Despite a continuousdecrease in PA during flower development (Fig. 2), most PAPgenes showed stable expression profiles (Fig. 3i). LPPa4 showedan 18-fold increase in level at D6, which suggests involvement inreproductive organ development. However, knockout of LPPc,which showed a stable expression pattern, has a lethal effect,probably the result of a male gametophytic defect (Nakamuraet al., 2007); the knockout phenotype of LPPa4 is not known.Overall, decrease in PA during the flower development is unlikelyto be controlled by the transcriptional level of PAP genes.

NPC Nonspecific phospholipase C hydrolyzes PC and othermajor membrane phospholipids to produce DAG and the corre-sponding polar head group (Nakamura et al., 2005). Among sixisoforms, NPC4 and NPC5 levels were increased eight- and 55-fold, respectively, at D6 (Fig. 3j). These NPCs are involved inphospholipid hydrolysis for galactolipid biosynthesis during

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Fig. 4 Profiling of phosphatidylinositol 4-phosphate (PI4P) andphosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) during flowerdevelopment of Arabidopsis thaliana. pAP1::AP1-GR ap1-1 cal-5 flowerswere harvested at different times after dexamethasone (DEX) treatment(e.g. D2, 2 d after DEX treatment), and total lipid was extracted andanalyzed as described in the Materials and Methods section. Data aremeans� SD (n = 4). Asterisks indicate significance (P < 0.01) from theprevious time point. See Fig. 1 for developmental stages of each time afterDEX treatment.





phosphate starvation (Nakamura et al., 2005; Gaude et al.,2008). Fig. 2 shows an increase in galactolipid (particularlyMGDG) from D6 onward, so these NPCs may contribute to thisincrease. Indeed, some phospholipid species showed a decrease inlevel when these NPCs were induced.

PLD Phosopholipase D hydrolyzes primary phospholipids toproduce PA. Of the 12 PLD isoforms in Arabidopsis, we couldnot design a specific primer set for PLDc3 because of highhomology with PLDc2 and PLDc1. As shown in Fig. 3(k),PLDf2 was induced transiently at D8 by 4.5-fold, and PLDa2and PLDb1 were induced at later stages by four- to fivefold.PLDf2 is induced by phosphate starvation and functions in par-allel with NPC4 and NPC5 in galactolipid biosynthesis (Cruz-Ramirez et al., 2006). Although induction of PLDf2 was slightlylater than that of NPC4 and NPC5, these enzymes may cooperatein the increase in galactolipid biosynthesis in the later stages offlower development. Although PLDb1 is involved in the defenseresponse (Zhao et al., 2013), the roles of PLDa2 and PLDb1 inflowers are unknown. These PLDs may contribute to galactolipid

synthesis at later stages or function in local PA signaling for floralmaturation.

DGK Diacylglycerol kinase phosphorylates DAG to producePA (Katagiri et al., 1996). Among seven DGK isoforms in Ara-bidopsis, five showed stable expression (Fig. S3), although DGK4and DGK6 showed an increase, by 38- and 800-fold, respectively,at D6 (Fig. 3l). Induction of DGK anticipates PA production,although we found no increase in PA at this developmental stage(Figs 2, 3). Neither DGK4 nor DGK6 have been functionallycharacterized. Because high functional redundancy is reportedamong DGK isoforms, these DGKs may work together for PAsignaling in addition to the PLD above in the midstage of flowerdevelopment.

Phosphoinositide profiles during flower development

The lipidomic profiling of PI showed a continuous decreasein expression throughout Arabidopsis flower development(Figs 1b, 2), although genes involved in PI synthesis (PIS1 and

(a) (b)

(c)

(e)

(d)

Fig. 5 Gene expression profiles ofcharacterized or putative phosphoinositidekinases and phosphoinositide-specificphospholipase C (PIPLC) genes during flowerdevelopment of Arabidopsis thaliana. Dataare means� SD fold change of transcriptabundance relative to that at D0, averagedby triple biological replicates. Asterisksindicate significance (P < 0.01) from theprevious time point. (a) Type III PI 4-kinase(PI4K) genes; (b) type II PI4K genes; (c) PI4P5-kinase (PIP5K) genes and PI4P kinase(PIPK) genes; (d) PIPLC genes exceptPIPLC6; (e) PIPLC6 gene. See Fig. 1(a) fordevelopmental stages of each time afterdexamethasone (DEX) treatment.



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NewPhytologist8

PIS2) showed stable expression (Fig. 3d). Phosphoinositidemetabolism involving PI as an initial substrate may be stimulatedduring flower development. The major pathway of phosphoinosi-tide metabolism is phosphorylation of PI by PI 4-kinase (PI4K)to produce PI4 phosphate (PI4P), which is further phosphory-lated by PI phosphates 5-kinase (PIP5K) to produce PI(4,5)P2.Eventually, this bis-phosphorylated substrate is hydrolyzed byphosphoinositide-specific phospholipase C (PIPLC) to produceinositol 1,4,5-trisphosphate (IP3) and DAG (Boss & Im, 2012).IP3 and its phosphorylated derivatives trigger intracellular signal-ing such as Ca2+ efflux (Im et al., 2010), whereas DAG isconverted to PA by DGK for a distinct signaling function (Ariszet al., 2009). In this context, we profiled two phosphoinositides,PI4P and PI(4,5)P2, according to the stages shown in Fig. 1(a).PI4P level showed a slight transient increase at D4–6, and PI(4,5)P2 showed a clear transient increase at D10 (Fig. 4). Thus,phosphoinositide metabolism may be induced at the midstage offlower development, and a decrease in PI concentration is likelyfor stimulation of phosphoinositide metabolism.

Gene expression profiles of phosphoinositide metabolicgenes during flower development

To investigate whether the transient increase in levels of PI4Pand PI(4,5)P2 coincides with induced gene expression involvedin the metabolism of these phosphoinositides, we used qRT-PCRanalysis for transcript profiling of 13 PI4K, nine PIP5K, twoPIPK and nine PIPLC isoforms. We detected no transcript ofPI4Kc10 in any of the floral samples analyzed, so it was excludedfrom the study. Interestingly, some of the genes were inducedconcomitantly at D8 (Fig. 5), almost when PI(4,5)P2 levelincreases significantly during flower development. These genesinclude PI4Ka2, PI4Kc1, PI4Kc7, PI4Kc8 and PI4Kc9 forPI4K-coding genes; PIP5K3 and PIPK11 for PIPK-coding genes;and PIPLC1, PIPLC3, PIPLC5, PIPLC6 and PIPLC7 for PIP-LC-coding genes. Involvement of kinases in flower developmentis partially known. For example, PIPK11 is one of the 150 genesspecifically expressed in pollen, and is important in pollen tubefunction (Becker et al., 2003; Ischebeck et al., 2011). It is intrigu-ing that PIP5K3 expression was found to be transiently increasedduring flower development, as this enzyme has previously beenreported to be only expressed in roots, specifically root hairs(Kusano et al., 2008; Stenzel et al., 2008). This suggests a pos-sible function of PIP5K3 in pollen tubes, which are highlysimilar to root hairs in structure. Involvement of PI4Kc sub-family in phosphoinositide metabolism remains elusive as theseenzymes have not been shown to harbor PI4K activity, butrather seem to be protein kinases (Galvao et al., 2008; Liuet al., 2013). By contrast, involvement of specific PIPLC iso-forms in flower development is still unknown in Arabidopsis,although these isoforms encode functional enzymes (Hirayamaet al., 1995; Sanchez & Chua, 2001; Xu et al., 2004). Thesimultaneous increase in the level of multiple genes at D8 sug-gests a coordinated transcriptional control that achieves a tran-sient increase in PI(4,5)P2 level for a signaling functionpossibly required in flower development.

Flower developmental defect found in the pah1 pah2mutant

To demonstrate the function of galactolipids, which changes dur-ing flower development, we observed the flower phenotype oflipid biosynthetic gene mutants. As shown in Figs 1(b) and 2, PArapidly decreased in the early stages of flower development.Because the pah1 pah2mutant was previously reported to be defi-cient in major PA phosphatase activity and to retain higher PAconcentrations (Nakamura et al., 2009b), we observed flowerphenotype. We noted that pah1 pah2 flowers showed weak termi-nal flower phenotypes (Fig. 6a), which is caused by precocioustermination of inflorescence meristem activity (Shannon &Meeks-Wagner, 1991; Alvarez et al., 1992). In addition, fusionof the floral organ was occasionally observed. Fig. 6(b) showed achimeric organ between petals and stamens in addition to fourpetals. A magnified observation by scanning electron microscopyrevealed that petal-like tissue was attached to the anther (Fig. 6c).Fused pistils (Fig. 6d) or fused stamens (Fig. 6e) were often foundas well. Furthermore, abnormal phyllotaxis was observed in theinflorescence (Fig. 6f). We found significantly increased PA con-centrations in flowers of pah1 pah2 (Fig. 6g). Organ fusion andabnormal phyllotaxis are commonly found in mutants defectivein organ boundary formation (Aida & Tasaka, 2006). Togetherwith terminal flower formation, our observation suggests thatrapid decrease of PA in the early stage of flower developmentmay facilitate proper organogenesis through meristem mainte-nance and organ boundary formation.

Distinct glycerolipid metabolism stimulated at differentstages of flower development

By using a transgenic tool to synchronize flower development,our concurrent profiling of glycerolipids and relevant metabolicgene expression during flower development has highlighted thestimulation of distinct metabolic pathways at different develop-mental stages. Here, to summarize our findings, we separated the2 wk of flower development into three stages: early stage (D0–4),midstage (D6–8) and late stage (D10–14; Fig. 7).

Early stage The highlight of the early stage was the high concen-tration of PA: a transient increase at D2 (by 17 mol%) of totalpolar glycerolipids, followed by a rapid decrease (Fig. 1b). Thisunusually high concentrations of PA were previously reported inpistils of Petunia flowers (Nakamura & Ohta, 2007). Given thatPA concentrations in vegetative tissues are minimal (e.g. 7-wk-old leaves contain 0.7 mol% PA; Welti et al., 2002), this high PAcontent is probably established during the floral transition.Indeed, a small increase in transcripts of GPAT2 and LPAT3,encoding activity for de novo glycerolipid biosynthesis (Fig. 3f–h),suggests slight transcriptional induction of the Kennedy pathwayat the early stage. We still do not know why PA concentration isso high in flowers (Nakamura & Ohta, 2007). This PA poolcould be used for active signaling; however, it could also be apool for galactolipid biosynthesis given the quantitatively equiva-lent changes in mol%. Here, PAP activity to hydrolyze PA may





be stimulated post-transcriptionally because no PAP isoformsshowed induced gene expression at this stage (Fig. 3i). PAH1 andPAH2 play major roles in PA hydrolysis (Nakamura et al.,2009b). The Saccharomyces cerevisiae ortholog of PAH1 andPAH2, Pah1p, requires phosphorylation/dephosphorylation inregulating enzyme activity and controls expression of phospho-lipid biosynthetic genes (O’Hara et al., 2006). Likewise, floral PAproduced by PAH1 and PAH2 may be involved in generegulations.

Midstage The profile becomes more complex at this stage. Theincrease in PC can be achieved by stimulation of the PC biosyn-thetic pathway, as indicated by the induction of NMT2 expres-sion (Fig. 3b). However, the induction of acyltransferaseexpression (GPAT2, GPAT7 and LPAT3; Fig. 3f,g) implies stim-ulation of de novo glycerolipid biosynthesis. The induction ofPLDf2, NPC4, NPC5, LPPa4 and DGK4 (Fig. 3i–l) stimulatesturnover of the PC–PA–DAG triangle, for fine-tuning the

expression of these metabolites. The transient increase in levels ofPI4P and PI(4,5)P2 (Fig. 5), another highlight of the midstage, israther straightforward, being controlled transcriptionally, whichis followed by simultaneous induction of a set of kinases andphospholipases. Phosphoinositide signaling may be required atthe midstage, probably to coordinate the onset of reproductiveorgan development.

Late stage Multiple genes are expressed at this stage, but anobvious lipid profile is the increase in prokaryotic species ofMGDG (Fig. 2). Apparently, this increase involves MGD1, asknocking out of MGD1, but not MGD2 and MGD3, affects theprokaryotic species of MGDG (Kobayashi et al., 2007, 2009).Stimulation of galactolipid biosynthesis during flower develop-ment was found previously in Petunia (Nakamura et al., 2003).However, in Petunia, the increase in DGDG exceeds that ofMGDG, for DGDG is the major glycolipid (Nakamura et al.,2003). The increase in MGDG concentration may be explained

(a)

(c)

(e) (f) (g)

(d)

(b)

Fig. 6 Flower developmental defect in pah1

pah2mutant of Arabidopsis thaliana. (a) Aterminal flower in the pah1 pah2mutant.Bar, 5 mm. (b) A flower producing a chimericorgan between petals and stamens (indicatedby red arrow) in addition to four petals. Bar,1 mm. (c) Scanning electron microscopyimage of a chimeric organ between petalsand stamens found in the pah1 pah2 flower.An, anther; fl, filament; pe, petal; po, pollen.Bar, 100 lm. (d) Fused pistils in pah1 pah2.Bar, 1 mm. (e) A pah1 pah2 flowercontaining fused stamen filaments indicatedby red arrow. Bar, 500 lm. (f) Abnormalflower phyllotaxis found in the inflorescenceof pah1 pah2. Unusual positioning ofpedicels is indicated by red arrows. Bar,10mm. (g) Increased phosphatidic acid (PA)concentration in floral buds of pah1 pah2.Data are means� SD (n = 4). WT, wildtype.



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NewPhytologist10

in part by the development of sepals that contain chloroplasts.However, because pistils in Petunia have higher galactolipid bio-synthetic activity than do leaves, developing carpels in Arabidop-sis flower may produce a large amount of MGDG. Alternatively,galactolipid accumulation in the developing pollen tube mayexplain the finding (Nakamura et al., 2009a; Botte et al., 2011).

Our concurrent profiling of glycerolipids and relevant meta-bolic gene expression revealed distinct metabolic pathways stimu-lated at different stages of flower development in Arabidopsis.The most dramatic change observed in Fig. 1(b) – a transientincrease and subsequent rapid decrease in PA in the early stages; amoderate increase in PC at the midstage; and a steep increase inMGDG in the later stages – suggests that PA could be

dephosphorylated to DAG by PA phosphatase, and that this isutilized as a substrate for PC and MGDG at later stages (Nakam-ura et al., 2009b; Eastmond et al., 2010). At the midstage, repro-ductive organs are massively developed, and thus thesemembrane lipids may be required as a constituent of such organs.It will be interesting to understand why MGDG is increased atlater stages. MGDG is an essential chloroplast membrane lipidfor photosynthetic function; however, its role in the later stagesof flower development is unknown. Previously, MGDG biosyn-thetic activity in the pistils of Petunia flowers was shown to behigher than that in leaves (Nakamura et al., 2003). Pistils maycontain some chloroplasts, as judged from the light green color ofthe organ; however, the higher MGDG biosynthetic activity inpistils suggests that MGDG is required for pistil developmentthat is not associated with known photosynthetic function. Apartfrom these major glycerolipids, we found dynamic changes in theprofiles of minor glycerolipids, such as phosphoinositides, evenin the earlier stages. Direct binding of phosphoinositides to keyproteins in meristem maintenance is known. For example, POL-TERGEIST (POL) and PLL1, which are components of theCLAVATA3 (CLV3)/WUSCHEL (WUS) pathway and areessential for maintenance of meristems, bind to some phospho-inositide species for stimulation (Gagne & Clark, 2010). In addi-tion, the Arabidopsis homolog of trithorax (ATX1), whichregulates AGAMOUS (AG) required for the initiation of repro-ductive organ development, binds phosphatidylinositol 5-phos-phate (Alvarez-Venegas et al., 2006). It is possible that thedynamic change in phosphoinositide profile is related to regula-tory function in meristem fate determination. A number of func-tional studies are anticipated on the basis of our current profiling.

Acknowledgements

The authors thank Siou-Ting Gan for technical assistance,Temasek Life Sciences Laboratory for resources, facilities, andtechnical assistance, and Arabidopsis TAIR (http://arabidopsis.org) for information and materials. This work was supportedby research grants to T.I. from the Temasek Life Sciences Lab-oratory, the National Research Foundation of Singapore,under its Competitive Research Programme (CRP AwardNRFCRP001-108); a grant to Y.N. and T.I. from PRESTO,Japan Science and Technology Agency, 4-1-8 Honcho Kawag-uchi, Saitama, Japan; a grant to Y.N. from the Institute ofPlant and Microbial Biology, Academia Sinica, Taipei; grantsto M.R.W. from the Singapore National Research Foundationunder CRP Award No. 2007-04, the Biomedical ResearchCouncil of Singapore (R-183-000-211-305), the NationalMedical Research Council (R-183-000-224-213), and the Sys-temsX.ch RTD project LipidX. Y.N. was supported by theJapanese Society for the Promotion of Science (JSPS) Postdoc-toral Fellowship for Research Abroad.

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Fig. 7 Glycerolipid metabolism suggested to be stimulated at the earlystage, midstage and late stage of flower development in Arabidopsis

thaliana. Increased glycerolipid classes at each stage are highlighted withan orange background. Genes with expression induced at the respectivestages are shown in blue at the corresponding metabolic steps at whichthese gene products catalyze or are likely to catalyze. CDP-DAG, cytidindiphospho-diacylglycerol; CDS, cytidin diphospho-diacylglycerol synthase;Cho, choline; CK, choline kinase; DAG, sn-1,2-diacylglycerol; DGK,diacylglycerol kinase; G3P, glycerol 3-phosphate; GPAT, glycerol 3-phosphate acyltransferase; LPA, lysophosphatidic acid; LPAT,lysophosphatidic acid acyltransferase; LPP, lipid phosphate phosphatase;MGD, monogalactosyldiacylglycerol synthase; MGDG,monogalactosyldiacylglycerol; NMT, phosphor-base N-methyltransferase;NPC, nonspecific phospholipase C; P-Etn, phosphorylethanolamine; P-Cho, phosphorylcholine; PA, phosphatidic acid; PAH, phosphatidic acidphosphohydrolase; PC, phosphatidylcholine; PI, phosphatidylinositol;PI4K, phosphatidylinositol 4-kinase; PI4P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PIPK,phosphatidylinositol 4-phosphate 5-kinase; PIPLC, phosphoinositide-specific phospholipase C; PLD, phospholipase D.





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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 A schematic illustration of the GR system.

Fig. S2 Effect of DEX treatment on lipid composition.

Fig. S3 Gene expression profiles of DGK1, 2, 3, 5 and 7.

Table S1 List of oligonucleotide primers used in this study

Please note: Wiley Blackwell are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.





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Transcriptomic and lipidomic profiles of glycerolipids during Arabidopsis flower development