Seed Fatty Acid Reducer acts downstream of gibberellin signalling pathway to lower seed fatty acid storage in Arabidopsis

Seed Fatty Acid Reducer acts downstream of gibberellinsignalling pathway to lower seed fatty acid storagein Arabidopsispce_2546 2155..2169

MINGXUN CHEN1, XUE DU1, YANG ZHU1, ZHONG WANG1, SHUIJIAN HUA3, ZHILAN LI1,4, WANGLI GUO1,GUOPING ZHANG1, JINRONG PENG2 & LIXI JIANG1

1Key Laboratory of Crop Germplasm Resource of Zhejiang Province, College of Agriculture and Biotechnology, 2College ofAnimal Sciences, Zhejiang University, Hangzhou 310058, 3Zhejiang Academy of Agricultural Sciences, Hangzhou 310021,and 4Zhejiang Provincial Natural Science Foundation of China, Hangzhou 310012, China

ABSTRACT

Previous studies based on microarray analysis have foundthat DELLAs down-regulate several GDSL genes inunopened flowers and/or imbibed seeds. This suggests therole of DELLAs in seed fatty acid (FA) metabolism. In thepresent study, enhancement of gibberellin (GA) signallingthrough DELLA mutation or exogenous gibberellin acidA3 (GA3) resulted in the up-regulated expression of tran-scription factors for embryogenesis and seed development,genes involved in the FA biosynthesis pathway, and fiveGDSL-type Seed Fatty Acid Reducer (SFAR) genes. SFARoverexpression reduced the total seed FA content and ledto a particular pattern of seed FA composition. This ‘SFARfootprint’ can also be found in plants with enhanced GA3

signalling. By contrast, the loss of SFAR function dramati-cally increases the seed FA content. The transgenic linesthat overexpress SFAR were less sensitive to stressful envi-ronments, reflected by a higher germination rate and betterseedling establishment compared with the wild type (WT)plants. The GDSL-type hydrolyzer is a family of proteinslargely uncharacterized in Arabidopsis. Their biologicalfunction remains poorly understood. SFAR reduces seedFA storage and acts downstream of the GA signallingpathway. We provide the first evidence that some GDSLproteins are somehow involved in FA degradation inArabidopsis seeds.

Key-words: fatty acid; GA signalling pathway; GDSL-typeSFAR genes.

INTRODUCTION

Gibberellins (GAs) are a large group of tetracyclic diterpe-noids that are essential for many aspects of plant growthand development, such as seed germination, stem elonga-tion, leaf expansion, trichome development, and flower and

fruit development (Swain, Reid & Kamiya 1997; King,Moritz & Harberd 2001; Sasaki et al. 2003; Cheng et al. 2004;Sun et al. 2004; Fleet & Sun 2005).The GA signal is receivedand transduced by the GID1 GA receptor/DELLA repres-sor pathway (Ueguchi-Tanaka et al. 2007). In Arabidopsis,the DELLA proteins, namely, GA INSENSITIVE (GAI),REPRESSOR OF ga1–3 (RGA), RGA-LIKE1 (RGL1),RGA-LIKE2 (RGL2) and RGA-LIKE3 (RGL3), consti-tute the nuclear negative regulators in the GA signallingpathway (Peng & Harberd 1997; Silverstone, Ciampaglio &Sun 1998; Yu et al. 2004). These five DELLA proteins haveboth unique and overlapping functions (Silverstone et al.2001; Lee et al. 2002; Jiang & Fu 2007). Genetic studiesindicate that GAI and RGA function in stem elongation asGA-sensitive repressors (Peng & Harberd 1997; Silverstoneet al. 1998; Dill & Sun 2001). The loss of function of GAIand RGA completely restores the dwarf phenotype, and thecombination of RGA, RGL1 and RGL2 loss-of-functionmutations represses petal, stamen filament and antherdevelopment in ga1–3 mutants (Cheng et al. 2004; Yu et al.2004). The RGL2 gene encodes the predominant repressorof seed germination in Arabidopsis, and its function isenhanced by the other DELLA proteins GAI, RGA andRGL1 (Tyler et al. 2004; Cao et al. 2005).

Previous studies have indicated that GA regulatesembryogenesis during the torpedo and early cotyledonstages when cells in the embryonic axis elongate (Hays,Yeung & Pharis 2002). More recently, Singh et al. (2010)reported that overexpression of a GA inactivation genecauses seed abortion, demonstrating that active GAs in theendosperm are essential for normal seed development. Gib-berellin A3 (GA3) increases the unsaturation of fatty acid(FA) in barley aleuronic layers, and this response exacer-bates the disruption of endoplasmic reticulum functionunder heat shock (Grindstaff, Fielding & Brodl 1996). Theapplication of exogenous GA3 to ga1–3 seedlings results indrastic changes in the transcription of WRINKLED1(WRI1), a central regulator of FA synthesis (Zentella et al.2007). Less attention has been focused on the role ofDELLAs in regulating the positive and negative down-stream factors that determine the final FA storage of a seed.

Correspondence: L. Jiang. e-mail: [email protected]; J. Peng.e-mail: [email protected]

Plant, Cell and Environment (2012) 35, 2155–2169 doi: 10.1111/j.1365-3040.2012.02546.x

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© 2012 Blackwell Publishing Ltd 2155

Based on the result of microarray analysis which indi-cated that DELLA proteins down-regulate several GDSL-type genes in imbibed seeds, as well as in young Arabidopsisflower buds (Cao et al. 2006), GA signals may modulateseed FA metabolism. This is because GDSL-type proteins,which have a serine-containing GDSL motif close to theN-terminus, five conserved blocks (I to V), four strictly con-served residues (Ser, Gly,Asn, and His in blocks I, II, III andV, respectively) and a Ser-Asp-His triad in the amino acidsequences, are active in the hydrolysis and synthesis oflipids and esters (Beisson et al. 1997). This class of enzymesis widely present in microbe and plant species. Importantmembers of this class include Aeromonas hydrophilialipases/acyltransferase, Vibrio parahaemolyticus hemolysin/phospholipase, Xenorhabdus luminescens lipase, Brassicanapus proline-rich protein, Vibrio mimicus arylesterase andStreptomyces rimosus lipase (Akoh et al. 2004). Studieshave shown that GDSL hydrolases have a flexible activesite that appears to change conformation with the presenceand binding of different substrates. Physiologically, plantGDSL-type genes are mainly involved in the regulation ofplant growth and development. Up to 108 GDSL-typegenes are present in Arabidopsis, which display remarkablestructural diversity, with intron numbers ranging from 0 to13. The genes, some of which are arranged in tandem, areasymmetrically distributed in chromosomes 1 and 5 (Ling2008). Presently, plant GDSL-motif enzymes have not beenproven to have any lipase activity. The Arabidopsis GDSLLIPASE-LIKE 1 (GLIP1) is involved in ethylene signallingand may mediate the production of systemic signalling mol-ecules (Kwon et al. 2009), whereas GLIP2 plays a role inresistance against Erwinia carotovora by negative regula-tion of auxin signalling (Lee et al. 2009). Overexpressionof Arabidopsis thaliana LTL1, a salt-induced gene thatencodes a GDSL motif, increases salt tolerance in yeast andtransgenic plants (Naranjo et al. 2006).At present, there is apaucity of information regarding the upstream regulators ofthe GDSL-type genes and their role in determining seed FAstorage and cellular signalling.

In Arabidopsis, the total seed oil content among differentaccessions varies widely from 33 to 43%. In contrast, the FAcomposition is much more conservative (O’Neill et al.2003). In plant, FA synthesis starts with the formation ofmalonyl-CoA from acetyl-CoA, which is catalyzed byacetyl-CoA carboxylase (ACCase). FA synthases catalyzethe transfer of the malonyl moiety to the acyl carrierprotein (ACP) by adding two carbons to the growing chain.This leads to the formation of C16:0-ACPs and C18:0-ACPs, which further elongate and unsaturate to formvarious FA derivatives from the acyl chains. These nascentFAs are then transported into the cytoplasm for lipid pro-duction (Harwood 1996; Mu et al. 2008; Baud & Lepiniec2009). ACCase is an important enzyme that controls theinitial step of FA synthesis. At least 24 enzymes and sub-units are involved in the pathway. The genes that encodemost of the enzymes have been characterized and clonedin Arabidopsis (Baud & Lepiniec 2009). FA biosynthesisis regulated by several key transcription factors (TFs),

including WRI1, LEAFY COTYLEDON1 (LEC1), LEC2,ABSCISIC ACID INSENSITIVE3 (ABI3) and FUSCA3(FUS3).The WRI1 gene encodes a transcriptional regulatorof the AP2/EREB family that targets enzymes involved inlate glycolysis and in the plastidial FA biosynthetic network(Focks & Benning 1998; Cernac & Benning 2004; Baud et al.2009). Both LEC1 and LEC2 function as positive regula-tors upstream of WRI1, ABI3 and FUS3, which jointly helpcontrol the expression of seed storage proteins and thegenes related to FA (Kroj et al. 2003; Kagaya et al. 2005; Muet al. 2008; Pan et al. 2010). All these TFs have been shownto participate in a regulatory cascade that controls theexpression of genes involved in seed FA biosynthesis duringseed maturation.

The present study investigates genes important for seeddevelopment and FA metabolism among Arabidopsis withreduced DELLA function to examine the activity of fiveGDSL-type SFAR genes and their effect on seed FAstorage.

MATERIALS AND METHODS

Plant materials and growth conditions

The Arabidopsis ecotype Columbia (Col-0) was used forthe GA3 application and transformation studies. Lands-berg erecta (Ler) was used for the generation of Q/ga1–3(ga1–3, gai-t6, rga-t2, rgl1-1, and rgl2-1) and Q/WT (gai-t6,rga-t2, rgl1-1 and rgl2-1) mutants as well as GA3 applica-tion studies. The T-DNA insertion mutants (SALKmutants) obtained from the Arabidopsis BiologicalResources Center were all on the Col-0 background. TheSALK mutants that mutated at loci SFAR1 (At1g54790),SFAR2 (At1g58430), SFAR3 (At2g42990), SFAR4(Ag3g48460) and SFAR5 (At4g18970) were ordered. Theywere sfar1-1 (SALK_022225C), sfar1-2 (SALK_111581C),sfar1-3 (SALK_063590), sfar2-1 (SALK_075941),sfar3-1 (SALK_083121), sfar3-2 (SALK_123457), sfar4-1(SALK_075665), sfar4-2 (SALK_008418C), sfar5-1,(SALK_029865) and sfar5-2 (SALK_089605). The struc-tures of the five SFAR genes and the locations of T-DNAinsertions of the SALK mutants are illustrated inSupporting Information Fig. S1a. All T-DNA insertionSALK mutants were backcrossed thrice with Col-0. Thehomozygous T-DNA insertion lines were selected byPCR genotyping (Supporting Information Fig. S1b) andRT-PCR confirmation for null transcript of the respectiveSFAR gene (Supporting Information Fig. S1c). All theprimers used for the PCR and RT-PCR analysis arelisted in Supporting Information Tables S1 and S2. Thepurified homozygous mutants were used to analyse theseed FA content and produced the nine double mutants:sfar2-1 sfar3-2, sfar2-1 sfar4-1, sfar2-1 sfar5-1, sfar1-3sfar3-1, sfar1-1 sfar4-1, sfar1-3 sfar5-1, sfar3-1 sfar4-2,sfar3-1 sfar5-1 and sfar4-2 sfar5-1 by crossing and subse-quent PCR screening. The growing conditions for Arabi-dopsis were as our previous descriptions (Cao et al. 2005,2006).

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© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 2155–2169

Plasmid construction and plant transformation

The primers used to clone the cDNA of the five SFARs arelisted in Supporting Information Table S3. With SFAR2 asan example, a 1.309 Kb SFAR2 cDNA fragment amplifiedthrough PCR using KAPAHiFi™ DNA Polymerase wascloned into modified pCAMBIA1300 driven by 35S. Theseconstructs were verified by extensive restriction endonu-clease digestion and DNA sequencing and were then trans-formed into Agrobacterium tumefaciens strain GV3101,which was then used for the transformation of ArabidopsisWT (Col-0) plants via floral dip (Clough & Bent 1998). Thetransgenic plants were selected on solid Murashige andSkoog (MS) medium with 30 mg mL-1 hygromycin and veri-fied by PCR genotyping. T4 transgenic plants were gener-ated and then RT-quantitative PCR was applied to verifythe transgenic plants further.

Application of exogenous GA3 to plants

Distilled water, used as the control, was set as Level-1(0 mm) and a 500 mm GA3 solution was set as Level-2. Thedifferent levels of GA3 solution were applied to eight indi-vidual plants (Col-0) in one of three randomly arrangedblocks. At the bolting stage, the whole plants were sprayedwith 0 and 500 mm every other day until the first silique washarvested. Traits, such as plant height, branch number,number and length of siliques, and seed appearance, werethen recorded.

Seed morphology and oil bodies indeveloping seeds

The seeds for observation were selected from the siliquesharvested from the basal part of a major inflorescence. Sixseeds of each genotype (or treatment) were randomlyselected for sectioning.Perpendicular transections were pro-duced and the sections with the largest oval-shaped surfacearea were selected for quantification.Twenty cells that locatein the middle of a section were selected to count the oilbodies within a cell. The seeds were photographed using anOLYMPUS SZ 61 stereomicroscope (Tokyo, Japan). Theobservation of the oil bodies under transmission electronmicroscopy (TEM) (JEM-1230, Tokyo Japan) was per-formed following the description by Eastmond (2006).

Analysis of FAs

FA extraction and analysis were carried out following Muet al. (2008). About 10 mg mature seeds were prepared byheating intact seeds at 80 °C in a methanol solution con-taining 1 m HCl for 2 h. The FA methyl esters wereextracted with 2 mL hexane and 2 mL 0.9% (w/v) NaCl,and the organic phase was used for analysis by gas chroma-tography (GC), using methyl heptadecanoate as an internalstandard. The machine (SHIMADZU, Kyoto, Japan,GC-2014) was equipped with a flame ionization detectorand a 30 m (length) ¥ 0.25 mm (inner diameter) ¥ 0.5 mm

(liquid membrane thickness) column (Supelco wax-10,Supelco, Cat. no. 24079, Schnelldorf, Germany). The initialcolumn temperature was maintained at 160 °C for 1 min,then increased by 4 °C min-1 to 240 °C, and held for 16 minat the final temperature.After the run, peaks correspondingto each FA species were identified by their characteristicretention times. Concentrations of each sample were nor-malized against the internal control.

Analysis of gene expression by RT-PCR andRT-quantitative PCR

Flowers were tagged with different coloured threads toindicate the days after pollination. Only the seeds from thesiliques on primary shoots were harvested for RNA extrac-tion. All the RNA samples utilized for verifying the trans-genic plants were isolated from the young leaves at therosette stage. The RNA samples were isolated using theInvisorb Spin Plant RNA Mini Kit (Invitek, Berlin,Germany) following the manufacturer’s instructions. Theprocedures of semiquantitative RT-PCR (sRT-PCR) andRT-quantitative PCR (RT-qPCR) were according to ourprevious descriptions (Pak et al. 2009).All primer pairs usedin the RT-PCR and RT-qPCR analysis are listed in Support-ing Information Table S2.

Analysis of seed germination rate andseedling establishment

Arabidopsis seeds placed on MS medium containing 6%(w/v) glucose were kept at 4 °C for 3 d to synchronize ger-mination. The seed germination frequency (defined asradicle emergence) was scored daily. Arabidopsis plants(3.5 weeks old) on the MS medium were carefully trans-ferred into MS solution containing 15% (w/v) polyethyleneglycol 6000 (PEG6000), dehydrated, and harvested 20 minand 1 h after the PEG6000 treatment for RNA analysis.

Statistical analysis

Completely randomized block design with at least threebiological replicates for each experiment was applied in thepresent study. The baseline and threshold cycles (CT value)were automatically determined using Bio-Rad iQ Software(version 3.0, Hercules, CA, USA). The relative amounts ofexpressed RNA were calculated using the method by Livak& Schmittgen (2001). Data were classified with Win-Exceland analysed via analysis of variance (ANOVA) using theSPSS (version 8.0, SPSS Inc., Chicago, IL, USA) statisticalpackage. Comparisons between the treatment means weremade using Tukey’s test at a probability level of P � 0.05.

RESULTS

Loss of DELLA function changes seedmorphology and FA storage

To determine whether the loss of DELLA function affectsseed morphology and FA storage, WT plants (Ler) were

GA regulates seed fatty acid storage via SFARs 2157


compared with a Quadruple/ga1–3 (Q/ga1–3) mutant thatcontains the loss-of-function gai-t6, rga-t2, rgl1-1 and rgl2-1alleles in the GA-deficient background (ga1–3). To excludeeffects of GA deficiency, the WT (Ler) was also compared

with a Q/WT mutant that contains the loss-of-function gai-t6, rga-t2, rgl1-1 and rgl2-1 alleles in the WT (Ler) back-ground. As shown in Fig. 1, the loss of DELLA functioncaused obvious changes in seed morphology. Both the

Figure 1. Comparison of seed morphology among the WT (Ler), the Q/ga1–3 mutants (ga1–3, gai-t6, rga-t2, rgl1-1 and rgl2-1) of the Lerbackground, the Q/WT mutants (gai-t6, rga-t2, rgl1-1 and rgl2-1) of the Ler background, the Ler plants treated with GA3 (500 mm), the WT(Col-0) and the Col-0 plants treated with GA3 (500 mm). Bar = 500 mm. GA3, gibberellin A3; Ler, Landsberg erecta; WT, wild type.

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Q/ga1–3 and the Q/WT plants had greener seeds on the16th day after pollination (DAP), shorter siliques and lessnumerous seeds of a silique upon maturation (data notshown). Both mutants had lower total FAs per milligramof seeds. Approximately 1 mg of seeds of Q/ga1–3 andQ/WT contained only 246.11 and 242.18 mg, respectively, oftotal FAs, which was significantly lower compared with303.38 mg of the WT seeds (Table 1). Notably, the loss ofDELLA function changed the FA composition. Comparedwith the WT (Ler), the DELLA mutants had FA compo-sitions with increased C18:1 and C20:1, and decreasedC18:2 and C18:3 (Table 2). Aside from these changes, theloss of DELLA function resulted in significant increases inplant height, silique number and a significant reduction inbranch number (Table 1).

GA triggers DELLA degradation to decrease DELLAfunction. We sprayed exogenous GA3 (500 mm) on the WTplants. Both the Ler and Col-0 plants that were treated withexogenous GA3 had shorter siliques and fewer seeds persilique (data not shown). The average weight per thousandseeds of the treated Col-0 plants was 33.10 mg, 62.2% morethan that of the untreated plants. The weight per thousandseeds of the treated Ler was 6.89% more than that of the

untreated control. The GA3-treated plants of both the Lerand Col-0 ecotypes had significantly decreased total seedFA (mg per mg) featured with enlarged proportions ofC18:1 and C20:1, a decreased proportion of C18:2 andC18:3 (Tables 1 & 2; Supporting Information Table S4-1,2).However, the changes were moderate, relative to that of theDELLA mutants (Table 2).

Overall, the loss of DELLA function through DELLAmutation or exogenous GA3 treatment reduces the totalseed FA storage, changes the seed FA composition andcauses a variety of morphologic changes.

Q/ga1–3 oil bodies are smaller than those ofLer upon maturation

The structure of seed oil bodies were examined to investi-gate the subcellular structural features that lower the FAcontent of DELLA mutant seeds. The oil body sizes of Lerand Q/ga1–3 mutants were compared. At 12 DAP, theQ/ga1–3 seeds had larger developing oil bodies than the Lerseeds (Fig. 2). In the mature seeds, however, the Q/ga1–3 oilbodies were 0.68 (�0.03) mm along the long axis and 0.48(�0.04) mm along the short axis on average, much smaller

Table 1. Comparison for plant morphological traits and seed FA storage among WT (Col-0 and Ler), the WT plants treated with 500 mmGA3, and the Q/ga1–3, Q/WT mutants

Traits Col-0 Col-0 + GA3 Ler Ler + GA3 Q/ga1–3 Q/WT

Plant height (cm) 24.75 � 1.23 37.60 � 4.63* 17.50 � 2.18 28.95 � 2.89* 29.76 � 3.05* 30.03 � 2.98*Silique number 23.38 � 1.51 31.25 � 4.46* 21.52 � 3.25 39.78 � 3.56* 43.28 � 4.62* 43.98 � 4.35*Branch number 4.63 � 0.52 4.63 � 0.92 4.68 � 0.57 4.78 � 0.48 1.51 � 0.83* 1.55 � 0.78*1000 seed weight (mg) 19.92 � 1.74 33.10 � 2.28* 18.58 � 1.63 19.86 � 1.58 19.36 � 1.86 19.52 � 1.79Total FAs (mg per mg) 297.36 � 4.62 253.22 � 8.09* 303.68 � 7.08 248.32 � 4.87* 246.11 � 4.56* 242.18 � 4.96*Total FAs (mg per seed) 5.92 � 0.38 8.38 � 0.49* 5.64 � 0.35 4.93 � 0.33* 4.76 � 0.32* 4.73 � 0.38*Oil content (%) 29.74 � 1.98 25.32 � 1.69* 30.37 � 1.98 24.83 � 1.86* 24.61 � 1.69* 24.22 � 1.59*

Asterisk (*) indicates the significant difference (P � 0.05) compared with the respective control.FA, fatty acid; GA3, gibberellin A3; Ler, Landsberg erecta; WT, wild type.

Table 2. Reduction between the values of individual FA species of the SFAR gain-of-function plants and those of their correspondingcontrols (mol %)

16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:0 22:1 24:0

Col-0 + GA3 -0.79 -0.18 +2.51 -2.99 -0.18 -0.11 +1.87 -0.03 +0.04 -0.03Ler + GA3 +0.28 +0.66 +6.84 -2.87 -7.44 -0.08 +2.42 +0.06 -0.20 -0.01Q/ga1–3 +0.52 +0.61 +7.14 -3.02 -7.17 -0.08 +2.33 +0.16 -0.22 0.01Q/WT +0.20 +0.62 +8.58 -3.49 -7.52 -0.11 +1.34 -0.09 -0.32 -0.0235S::SFAR1 +0.14 -0.12 +0.54 -1.41 -0.22 -0.10 +1.32 -0.12 -0.17 -0.0935S::SFAR2 -0.16 -0.17 +1.39 -1.38 -0.64 -0.14 +1.23 -0.11 -0.11 -0.0735S::SFAR3 -0.18 -0.09 +0.47 -1.18 -0.31 -0.09 +1.48 -0.10 -0.19 -0.0235S::SFAR4 -0.64 -0.11 +1.59 -0.68 -1.27 -0.18 +1.13 -0.11 -0.18 -0.0835S::SFAR5 +0.41 -0.17 +0.29 -2.31 -2.89 -0.11 +1.03 -0.10 -0.16 -0.01

A genotype/treatment with a solid star ( ) has Ler as the control, whereas a genotype/treatment with a hollow star ( ) has Col-0 as thecontrol.The deduction is based on the data in Supporting Information Tables S1-2 and S2-2, where each value is an average of three biologicalrepetitions. Data for each overexpressed SFAR are based on an average of five to six independent transgenic lines.The bold font indicates theSFAR feature of FA composition.FA, fatty acid; Ler, Landsberg erecta; SFAR, Seed Fatty Acid Reducer; WT, wild type.



than the 0.95 (�0.11) mm ¥ 0.62 (�0.06) mm Ler oil bodies(Fig. 2; Supporting Information Table S5-1).

Relief of DELLA-dependent repressionup-regulated the TFs that regulate seeddevelopment and genes that catalyze varioussteps of FA biosynthesis

The expression of the key TFs (WRI1, FUS3, LEC1, LEC2and ABI3), which are important for embryogenesis andseed development in Arabidopsis, was studied by perform-ing quantitative RT-PCR using the RNAs extracted fromseeds at 6, 10, 12, 14 and 16 DAP (upper panel of Fig. 3). InWT (Ler), the expression of LEC1 and LEC2 peaked earlyat 6 DAP stage. In contrast, the expression of FUS3 andABI3 peaked late at 14 and 16 DAP, respectively. Theexpression of WRI1 peaked at 10 DAP, later than that ofLEC1 and LEC2, but earlier than FUS3 and ABI3. Relativeto the WT, the Q/ga1–3 mutation caused more than twofoldhigher LEC1 and LEC2 transcripts at 10 DAP. It resulted in1.7-fold higher FUS3 transcripts and nearly threefoldhigher WRI1 transcripts at 14 and 16 DAP, and 2.5-foldhigher ABI3 transcripts at 16 DAP.

YUC4, YUC2, IAA17, ACS4, oleosin and 2S3 are down-stream targets of LEC2 (Stone et al. 2008). Resulting fromthe up-regulated LEC2 expression, these genes were also

up-regulated during seed development, in particular at 14DAP (Supporting Information Fig. S2).

The expression of genes that encode the critical FA bio-synthetic enzymes CAC2, CAC3, BCCP2 (subunits ofACCase), MOD1, KASII, CDS2, FAB2, FatA, FAD2,FAD3 and FAE1 were also examined (middle and bottompanels of Fig. 3). In the WT, the expression of all of thesegenes peaked relatively early, either at 6 DAP, such as theexpression of CAC2, CAC3, BCCP2, MOD1, KASII, CDS2and FatA, or at 10 DAP, such as the expression of FAB2,FAD2, FAD3 and FAE1. Relative to the WT, the Q/ga1–3mutation led to higher transcripts of most of these genes atrelatively later stages. It resulted in more than twofoldincrease in the expression of CAC2, BCCP2, MOD1, CDS2,FatA, FAD3 and FAE1 transcripts at 12 and 14 DAP.Among the genes that encode the critical FA synthesisenzymes, KASII was the exception; its expression wasslightly down-regulated in the Q/ga1–3 mutant at almost alltime points.

The expression patterns of these TFs and FA biosyntheticgenes following treatment of the Ler plants with exogenousGA3 (500 mm) mirrored the results obtained from theQ/ga1–3 mutant, although the increase in transcriptionlevels relative to the control was greater in the Q/ga1–3mutant plants than the GA3-treated Ler plants (SupportingInformation Fig. S3).

Figure 2. Subcellular differences in seed cells from the WT (Ler) and the Q/ga1–3 mutant at 12 DAP and mature seed stage. Arrowsindicate lipid droplets (L), aleurone grains (A) and globoids (G). Bar = 1 mm. DAP, days after pollination; Ler, Landsberg erecta; WT, wildtype.

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Overall, the loss of DELLA function up-regulated theexpression of TFs and their corresponding downstreamtargets, which implies an increase of seed FA synthesis.

Relief of DELLA repression up-regulated fiveGDSL-type SFAR genes that function inreducing seed FA storage

Based on microarray analysis, Cao et al. (2006) reportedthat DELLA proteins down-regulate GDSL-type genes inunopened flowers and/or imbibed seeds. We designatedthese genes as Seed Fatty Acid Reducer (SFARs), specifi-cally, SFAR1 for At1G54790, SFAR2 for At1G58430, SFAR3for At2G42990, SFAR4 for At3G48460 and SFAR5 forAt4G18970. The SFAR expression in developing Q/ga1–3seeds was examined at 6, 10, 12, 14 and 16 DAP.Those of theQ/WT were examined at 12, 14 and 16 DAP. Figure 4 clearlyshows that the SFARs in both the Q/ga1–3 and the Q/WTwere significantly up-regulated at 14 DAP by up to 60-foldmore than in the WT (Ler). Again, the GA3 treatment(500 mm) produced similar changes in SFAR expression.However, their peak increases were much lower (Support-ing Information Fig. S4).

Interruption or overexpression of the SFARsaltered total seed FA content and composition

To clarify further the role of five SFARs in seed FA storage,the total seed FA content and FA species composition of

WT (Col-0), sfar single and double mutants, the sfar doublemutants sfar2-1 sfar3-1, and sfar4-1 sfar5-1 plants treatedwith GA3 (500 mm) and the transgenic lines that overexpressindividual SFARs were compared (Fig. 5; Supporting Infor-mation Table S6). As shown in Fig. 5, the interruption of anindividual SFAR gene resulted in significant increases intotal seed FA from 9.1 to 16.9%, depending on the locusinterrupted. In contrast, the ectopic SFAR overexpressionresulted in significant reductions in total seed FA from 13.4to 25.1%, depending on the overexpressed gene. The over-expression of any of the five SFARs resulted in the changeof FA composition, featured with an increase of C18:1 andC20:1, and a decrease of C18:2 and C18:3 proportions(Table 2 & Supporting Information Table S6).

Double mutations of the SFARs were generated to deter-mine if the SFAR loci had additive effects on seed FAstorage. Except for the double mutant sfar1 sfar2 that wasdifficult to produce because of the low recombination rate,nine double mutants were generated and harvested for seedFA analysis. On average, the double mutants showed higherincreases in total seed FA (23.7%) compared with the singlemutants (13.4%). The double mutant sfar1-3 sfar3-1 exhib-ited a 31.8% increase in total seed FA compared with theWT (Fig. 5; Supporting Information Table S6-1).

The seed morphology and oil body structure of the WT(Col-0), the SFAR gain-of-function transgenic plants(35S::SFAR1), and the loss-of-function mutants (sfar2-1sfar3-2) were further compared. As shown in Fig. 6, theseed morphology of the 35::SFAR1 plants and sfar2-1

Figure 3. Comparison of the relative transcription levels of the TFs that regulate embryogenesis and seed development (top panel), andthe genes that encode enzymes critical to FA biosynthesis (middle and bottom panels) between the WT (Ler) and the Q/ga1–3 mutants atdifferent seed developmental stages. The transcription levels are relative to the WT (Ler) at 6 DAP, which is set to 1. FA, fatty acid; DAP,days after pollination; Ler, Landsberg erecta; TF, transcription factor; WT, wild type.



sfar3-2 plants were not visibly different from that ofthe WT (Col-0). However, significant differences wereobserved among the WT, 35S::SFAR1 and sfar2-1 sfar3-2plants in terms of average number and size of oil bodies inthe seed cells. A sfar2-1 sfar3-2 seed cell contained anaverage of 171.06 (�14.72) visible oil bodies, less thanthe 386.66 (�19.52) oil bodies in WT seed cells and the453.91 (�12.72) oil bodies in 35S::SFAR1 seed cells. On theother hand, the sfar2-1 sfar3-2 oil bodies measured 1.69(�0.23) mm along the long axis and 0.87 (�0.12) mm alongthe short axis, much larger than the 0.69 (�0.08) mm ¥ 0.61(�0.05) mm WT oil bodies and the 0.48 (�0.03) mm ¥ 0.31(�0.02) mm 35S::SFAR1 oil bodies (Supporting Informa-tion Table S5-2).

Therefore, gain of SFAR function reduces seed FAstorage and oil body size, whereas loss of SFAR functionincreases seed FA storage and oil body size. Furthermore,the SFAR loci have an additive effect on seed FA content.

SFAR gene overexpression enhanced thegermination rate and young seedlingestablishment in a stressed environment

Considering FA metabolism may be involved in stress-resistance mechanisms, and SFAR overexpression changesthe seed FA composition in the transgenic plants, we inves-tigated whether SFAR gene overexpression enhances thegermination rate and seedling establishment under stressed

environments. On medium containing 6% (w/v) glucose,60% of the seeds from the 35S::SFAR4 transgenic line #12and 50% of the seeds from the 35S::SFAR4 transgenic line#23 germinated on the third day after sowing, whereas only3% of the WT seeds germinated. Seven days after sowing,only 35% of the WT seed germinated, whereas the germi-nation rates of transgenic lines #12 and #23 were 98 and97%, respectively (Fig. 7a). In 6% (w/v) glucose, about 55 to65% of the transgenic plants reached the two-leaf stage 3.5weeks after sowing. By contrast, the WT plants failed todevelop normal cotyledons and green leaves under thesame stressful condition (Fig. 7b). The overexpression ofthe other four Arabidopsis SFAR genes, viz., SFAR1,SFAR2, SFAR3 and SFAR5, had similar effects on glucosetolerance during seed germination (Table 3). We also com-pared between the WT and sfar loss-of-function mutants fortolerance against osmotic stress; and we didn’t find anysignificant differences in germination rate and young seed-ling establishment (data not shown).

The transient expressional differences between genesrelated to osmotic stress in the WT (Col-0) and the35S::SFAR4 plants were compared using sRT-PCR(Fig. 7c).The osmotic stress caused by short-term PEG6000treatment induced the differential expression of genes suchas ABA1, ABA2, ABA3, ABI1, ADH, Rab18, CBL1, CBL9and NCED3 between the WT and the transgenic plants.After 60 min of PEG6000 treatment, the genes had higherexpression levels in the WT (Col-0) than in the transgenic

Figure 4. Comparison of the relative transcription levels of SFARs between the WT (Ler) and the Q/ga1–3 mutant at different seeddevelopmental stages. The transcription levels of the WT at 6 DAP are set to 1 for the upper panel. The transcription levels of the WT at12 DAP are set to 1 for bottom panel. DAP, days after pollination; Ler, Landsberg erecta; SFAR, Seed Fatty Acid Reducer; WT, wild type.

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plants, which indicates that the transgenic plants havereduced sensitivity to osmotic stress.

DISCUSSION

Previous studies reported that the GA signal regulatesplant growth and development, such as seed germination,stem elongation and leaf expansion, as well as trichome,flower and fruit development (Swain et al. 1997; King et al.2001; Sasaki et al. 2003; Cheng et al. 2004; Sun et al. 2004;Fleet & Sun 2005; Yamauchi et al. 2007). However, littleattention has been paid on the effects of GA signalling onthe regulation of FA storage in seeds and the downstreamfactors that positively or negatively determine seed FAstorage capacity. In the present study, the knockout ofDELLA proteins or the application of GA3 to WT plantssignificantly reduced seed FA storage in Arabidopsis. Thiseffect on seed FA requires the action of SFAR becausethe FA composition in the DELLA loss-of-function plantsclearly featured an ‘SFAR footprint,’ that is, increasedC18:1 and C20:1, and decreased C18:2 and C18:3, accom-panied by a reduction in the total seed FA content(Table 2). On the other hand, the GA3-treated sfar2-1sfar3-2 and/or sfar4-2 sfar5-1 double mutant plants had a

lower FA content than the untreated double mutantplants. They had the ‘SFAR footprint’ nevertheless, indi-cating that GA would have up-regulated unmutatedSFARs, greater in number than the genes mutated. Theup-regulation of these SFARs offset the increase of seedFA in the double mutants that would otherwise beobserved (Fig. 5). The best way to demonstrate the SFARrequirement of GA is to treat SFAR-silenced plants withGA3 and the resulting seed FA content. However, thistreatment is technically difficult to perform. Firstly, thegeneration of sfar1 sfar2 is difficult because of the closelinkage of the two genes on one chromosome. Secondly,the exact number of SFARs acting on the GA signallingpathway is still unknown. GA may affect the seed FAcontent via SFARs (with the ‘SFAR footprint’). However,GA may also affect the seed FA content independent ofSFARs. As shown in Table 1, the GA signal affects manyplant traits. Traits such as plant height, as well as siliqueand branch numbers affect the seed oil content, a typicalquantitative trait regulated by many factors. Moreover,GA functions via the DELLA-dependent pathway aswell as independently in Arabidopsis (Cao et al. 2005).Nonetheless, the GA-SFAR pathway is an importantGA-mediated regulator of seed FA content.

Figure 5. Comparison of the seed FA content between the WT (Col-0) and the SFAR single gene mutants, the transgenic plants thatoverexpress the respective single SFAR genes (a–e) and the double mutants (f). The dashed lines indicate the level of seed FA content inWT (Col-0). The asterisks (*) indicate significant difference (P � 0.05) compared with the WT. FA, fatty acid; SFAR, Seed Fatty AcidReducer; WT, wild type.



The loss of DELLA function up-regulated the TFs, suchas LEC1, LEC2, FUS3, ABI3 and WRI1, which regulateembryonic and seed development (upper panel of Fig. 3;Supporting Information Fig. S3). In Arabidopsis, LEC1 is akey regulator of FA biosynthesis, and LEC1 overexpressionincreased the expression of many FA biosynthetic genes

involved in the condensation, chain elongation and desatu-ration of FA biosynthesis (Mu et al. 2008). The LEC2protein induces the maturation of many traits, auxin activ-ity, and the expression of the other seed regulators likeFUS3 and ABI3, which redundantly induce the expressionof seed storage proteins, degrading chlorophyll and

Figure 6. Comparison of the seed morphology and oil body structure among the WT (Col-0), the gain-of-function line thatoverexpresses SFAR1 and the loss-of-function line sfar2-1 sfar3-2. Arrows indicate lipid droplets (L), aleurone grains (A) and globoids(G). Bar = 500 mm for the left panels; bar = 1 mm for the right panels. SFAR, Seed Fatty Acid Reducer; WT, wild type.

2164 M. Chen et al.


anthocyanins in dry seeds, and confers sensitivity to ABA(Luerssen et al. 1998; To et al. 2006). Moreover, LEC2 regu-lates seed FA metabolism by targeting WRI1, which con-verts sucrose into precursors of TAG biosynthesis (Cernac& Benning 2004; Baud et al. 2007) by targeting oleosin,which maintains oil bodies as small single units and pre-vents their coalescence during seed desiccation (Siloto et al.2006). The sizes of the oil bodies in the plants with reducedDELLA function and/or increased SFAR expression weresmaller than that in WT (Figs 2 & 6). This may arise from

two reasons. Firstly, LEC2 and its target gene oleosin areup-regulated in these plants. Secondly, the SFARs, whichmay involve FA hydrolyzation before free FAs are incor-porated into lipid droplets, are also up-regulated in theseplants. However, Siloto et al. (2006) also reported that theinterruption of oleosin with RNAi produces unusually largeoil bodies that correlate with lower seed lipid content. Incontrast to their results, the current observations indicatethat larger oil bodies correlate with higher final seed FAcontent. LEC2 is also involved in the auxin response (Stone

Figure. 7. Comparison of the germination rate (a) and establishment of young seedlings (b) between the WT (Col-0) and the transgeniclines (#12, #23) that overexpress SFAR4 on MS medium containing 6% (w/v) glucose. (c) RT-PCR results comparing the gene expressioninduced by osmotic stress between the WT (Col-0) and the transgenic line #12 that overexpresses SFAR4. The expression levels of thegenes were compared at 0, 20 and 60 min after PEG6000 treatment. The differentially expressed genes are highlighted in the white lineboxes. MS, Murashige and Skoog; PEG6000, polyethylene glycol 6000; WT, wild type.

Table 3. Comparison of germination rate(%) on MS medium containing 6% (w/v)glucose between the WT (Col-0) and thetransgenic lines that overexpress SFAR1,SFAR2, SFAR3 and SFAR5

1 DAS 2 DAS 3 DAS 4 DAS 5 DAS 6 DAS 7 DAS

WT(Col-0) 0 � 0 0 � 0 3 � 1 5 � 2 15 � 2 23 � 3 35 � 435S::SFAR1 #6 0 � 0 0 � 0 45 � 3 95 � 4 97 � 3 98 � 2 98 � 235S::SFAR1 #15 0 � 0 0 � 0 50 � 3 87 � 5 95 � 3 95 � 4 98 � 235S::SFAR2 #1 0 � 0 0 � 0 35 � 2 80 � 5 90 � 5 95 � 3 97 � 335S::SFAR2 #8 0 � 0 0 � 0 40 � 3 75 � 5 90 � 5 93 � 4 97 � 235S::SFAR3 #2 0 � 0 0 � 0 55 � 3 85 � 4 93 � 4 97 � 3 98 � 135S::SFAR3 #8 0 � 0 0 � 0 50 � 3 78 � 4 90 � 5 95 � 4 98 � 235S::SFAR5 #3 0 � 0 0 � 0 50 � 2 85 � 4 90 � 3 95 � 3 98 � 235S::SFAR5 #5 0 � 0 0 � 0 55 � 2 80 � 3 86 � 5 97 � 2 98 � 1

DAS is days after sowing.# is the individual transgenic line number.The germination rates of two 35S::SFAR4 transgenic lines are presented in Fig. 7a.SFAR, Seed Fatty Acid Reducer; WT, wild type.



et al. 2008). Therefore, fewer branches were observed in theDELLA mutant plants, possibly due to enhanced apicaldominance (Table 1). The GA3 treatment did not reducebranching in the WT plants because the branches andlateral buds should have already formed before the appli-cation of GA3 (Table 1).

The up-regulation of TFs during seed development by theloss of DELLA function resulted in increased transcriptionof genes that catalyze various steps in the formation ofdifferent FA species, which include CAC2, CAC3, BCCP2,MOD1, FAB2, CDS2, FAD2, FAD3 and FAE1 (middle andbottom panels of Fig. 3; Supporting Information Fig. S3). Ofthese enzymes, CAD2, CAC2 and BCCP2 are subunits ofACCase, which catalyze the initial synthesis step, the for-mation of malonyl-CoA from acetyl-CoA.The MOD1 func-tions as an enoyl-ACP reductase. FAB2 (SSI2) encodes astearoyl-ACP desaturase (Lightner et al. 1994). The CDS2enzyme determines the phosphatidate cytidylyl transferaseactivity and is involved in the phospholipid biosyntheticprocess. FAD2 is essential for polyunsaturated lipid synthe-sis (Okuley et al. 1994) and FAD3 is responsible for thesynthesis of 18:3 FAs from phospholipids (Shah, Xin &Browse 1997). FAE1 is required for the synthesis of verylong chain FAs in seeds and it is presumed to be a condens-ing enzyme that extends the chain length of FAs from C18to C20 and C22 (James et al. 1995). Both SFAR gain-of-function plants and DELLA loss-of-function plants had asignificantly increased proportion of C18:1 and C20:1. Theup-regulated expressions of the genes such as FAB2 andFAE1 could be the probable reason responsible for thefeature (Table 2; Fig. 3; Supporting Information Fig. S3).

The up-regulation of the TFs and the downstream FAbiosynthesis genes induced by the relief of DELLA repres-sion implies higher seed FA storage. However, the totalseed FA decreased significantly (Table 1; Supporting Infor-mation Table S4). This may be because the loss of DELLAfunction also accelerates the seed FA breakdown byup-regulating SFARs.The formation of seed FA should be adynamic balance between synthesis and breakdown. SFARswere up-regulated in the Q/ga1–3 plants from 12 DAP andpeaked at 14 DAP (Fig. 4). Previous studies have shownthat embryo morphogenesis is accomplished at 6 DAP, andthe accumulation of storage compound starts from 7 DAPand is completed at 20 DAP, which is essential for seed FAaccumulation from 12 DAP to 16 DAP (Goldberg et al.1994; Baud et al. 2002; Fait et al. 2006; Baud & Lepiniec2009). Most of the aforementioned FA synthesis genes wereup-regulated at 12 DAP or at earlier stages, whereas theSFAR genes were drastically up-regulated at 14 DAP. Inline with this observation, the Q/ga1–3 mutant had larger oilbodies than the WT at 12 DAP, but smaller oil bodies at themature stage after the drastic SFAR expressional peak at 14DAP (Figs 2 & 4). Taken together, FA synthesis and break-down, the opposing processes that determine the finalquantity of seed FA storage, are subjected to global regu-lation by TFs during seed development.

Our results demonstrate that SFARs, the GDSL-motifgenes, involve seed FA breakdown and lead to a less seed

FA storage (Fig. 5). SFARs might cause less free FAs to beincorporated into lipid droplets, where TAG is the mainconstituent. To the best of our knowledge, there has notbeen any evidence demonstrating that a plant GDSL-motifgene has any kind of lipase activity. Our experiment indi-rectly confirmed that SFARs are not lipases. If they were,higher free FA contents in seeds of those SFAR gain-of-function genotypes would have been detected. In fact,SFARs cause less seed FA storage. We show that somehowSFARs involve the seed FA breakdown. However, the exactrole of SFAR remains unclear and need further investiga-tion. So far, the b, a and w-oxidation pathways are under-stood for FA decomposition and b-oxidation pathway is thegeneral model of FA catabolism in planta (Harwood 1988;Graham & Eastmond 2002).

In addition to the change in seed FAs, the loss of DELLAfunction also produced enlarged seeds, with a fewer numberof seed sets and shorter siliques. More photosynthateswould have been transported to these larger seeds. Inter-estingly, the application of GA3 did not alter the shape ofthe Col-0 ecotype seeds, but altered those of the Lerecotype. Moreover, both the Q/ga1–3 and Q/WT mutationchanged the seed shape from oval to round (Fig. 1). Differ-ences in the seed formation were observed between the twoecotypes in response to the loss of DELLA function.

Interestingly, the transgenic line that overexpresses one ofthe SFAR esterase genes was much less sensitive to stressfulenvironments, as reflected by their much higher germinationrate and better young seedling establishment than the WT(Col-0) (Fig. 7a,b). On the other hand, the loss-of-functionsfar mutants were not significantly different from the WTplants in terms of stress tolerance. Higher stress tolerancemay result from the ‘SFAR footprint’, that is, a change in FAcomposition characterized by increased C18:1 and C20:1,and decreased C18:2 and C18:3. The ‘SFAR footprint’was only found in SFAR gain-of-function plants, whereasno significant differences in seed FA composition wereobserved between the sfar mutants and the WT plants (Sup-porting InformationTable S6-1).FAs are major componentsof the phospholipid bilayer and are involved in several cellmembrane functions. FA metabolism is involved in thestress-resistance mechanisms of some species (Horikawa &Sakamoto 2009).A decrease in polyunsaturated FAs report-edly reduces the osmotic stress tolerance of transgenicworms (Horikawa & Sakamoto 2009;Tazearslan et al. 2009).However, the role of FA unsaturation in stress toleranceand the interaction between FA metabolism and stressresponses in plants remain poorly understood.

In Arabidopsis, there are 108 GDSL-type genes (Ling2008). The five SFARs that were characterized in this paperare regulated by GA signalling.The regulation of the rest ofthe GDSL-type genes, and whether they modulate seed oilformation remain interesting questions.

ACKNOWLEDGMENTS

We thank Prof Dr Xiangdong Fu for providing the Q/WTmutant used in the study.The work of our lab was supported

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by the Natural Science Foundation of China (Grant nos.30971700 and 31171463) and Zhejiang Province (Grant no.Z3100130), the Fundamental Research Funds for theCentral Universities (2012FZA6011 and 2012XZZX012),and the Special Grand National Science and TechnologyProject (Grant no. 2009ZX08009-076B).

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Received 23 February 2012; received in revised form 14 May 2012;accepted for publication 18 May 2012

SUPPORTING INFORMATION

Additional supporting information may be found in theonline version of this article.

Figure S1. Identification of the SALK mutants at the SFARloci.Figure S2. Comparison of the relative transcription levels ofthe LEC2 targeting genes between the WT (Ler) and theQ/ga1-3 mutant. The transcription levels are relative to WT(Ler) at 6 DAP, which is set to 1.Figure S3. Comparison of the relative transcription levels ofthe TFs that regulate embryogenesis and seed developmentand the genes that encode enzymes needed for FA biosyn-thesis at different seed developmental stages between theWT (Ler) and GA3-treated WT plants. The transcriptionlevels are relative to the WT (Ler) at 10 DAP, which is setto 1.Figure S4. Comparison of the relative transcription levels ofthe five GDSL-type genes at different seed developmentalstages between the WT (Ler) and GA3-treated WT plants.The transcription levels are relative to the WT (Ler) at 10DAP, which was set to 1.Figure S5. Comparison of the germination rates of the WT(Col-0) and the SFAR transgenic lines.Table S1. Primers used in genotyping the SALK mutants.Table S2. Primers used in the RT-PCR and RT-qPCRanalyses.

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Table S3. Primers used for cloning the cDNA of the fiveGDSL-type genes.Table S4. Comparison of the seed FA composition of theQ/ga1–3, Q/WT, WT (Col-0 and Ler) and the WT plantstreated with 500 mm GA3 (S4-1 in mg per mg and S4-2 inmol %).

Table S5. Oil body statistics (S5-1 is the comparisonbetween Ler and the Q/ga1-3 mutant; S5-2 is thecomparison among Col-0, 35S::SFAR1 and sfar2-1sfar3-2).Table S6. FA composition of the SFAR mutants and thetransgenic lines (S6-1 in mg mg-1 and S6-2 in mol %).



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