plants-05-00016.pdf

plantsArticlePaired Hierarchical Organization of 13-Lipoxygenasesin ArabidopsisAdeline Chauvin1,2,Aurore Lenglet2,Jean-Luc Wolfender1and Edward E.Farmer2,*1School of Pharmaceutical Sciences,University of Lausanne,University of Geneva,quai Ernest-Ansermet 30,CH-1211 Geneva 4,Switzerland;(A.C.);jean-luc.wolfenderunige.ch(J.-L.W.)2Department of Plant Molecular Biology,University of Lausanne,CH-1015 Lausanne,Switzerland;aurore.lengletunil.ch*Correspondence:edward.farmerunil.ch;Tel.:[phone]These authors contributed equally to this work.Academic Editor:Eleftherios P.EleftheriouReceived:10 November 2015;Accepted:18 March 2016;Published:24 March 2016Abstract:Embryophyte genomes typically encode multiple 13-lipoxygenases(13-LOXs)that initiatethe synthesis of wound-inducible mediators called jasmonates.Little is known about how theactivities of these different LOX genes are coordinated.We found that the four 13-LOX genes inArabidopsis thaliana have different basal expression patterns.LOX2 expression was strong in softaerial tissues,but was excluded both within and proximal to maturing veins.LOX3 was expressedmost strongly in circumfasicular parenchyma.LOX4 was expressed in phloem-associated cells,incontrast to LOX6,which is expressed in xylem contact cells.To investigate how the activities of thesegenes are coordinated after wounding,we carried out gene expression analyses in 13-lox mutants.This revealed a two-tiered,paired hierarchy in which LOX6,and to a lesser extent LOX2,controlmost of the early-phase of jasmonate response gene expression.Jasmonates precursors producedby these two LOXs in wounded leaves are converted to active jasmonates that regulate LOX3 andLOX4 gene expression.Together with LOX2 and LOX6,and working downstream of them,LOX3 andLOX4 contribute to jasmonate synthesis that leads to the expression of the defense gene VEGETATIVESTORAGE PROTEIN2(VSP2).LOX3 and LOX4 were also found to contribute to defense againstthe generalist herbivore Spodoptera littoralis.Our results reveal that 13-LOX genes are organised in aregulatory network,and the data herein raise the possibility that other genomes may encode LOXsthat act as pairs.Keywords:jasmonic acid;jasmonate;oxylipin;eicosanoid;wounding;defense;herbivore1.IntroductionLipoxygenases(LOXs)function to produce lipid mediators that operate in a broad range ofprocesses,many of which are related to defense in animals 1 and in plants 2.The Arabidopsisthaliana genome encodes six LOXs of which four are 13-LOXs,where“13”refers to the carbon atomin polyunsaturated 18-carbon fatty acids that are preferentially oxygenated by the LOX.13-LOXsincorporate molecular oxygen into-linolenic acid to produce its 13(S)-hydroperoxide 3,a moleculethat is transformed into jasmonates which regulate wound-induced defense gene expression 4,5.Tocomplete jasmonate synthesis,fatty acid hydroperoxides formed through LOX action are dehydratedand cyclized to form the intermediate 12-oxo-phytodienoic acid(OPDA)in reactions catalysed byallene oxide synthase(AOS)and auxiliary proteins 6,7.Further transformations of OPDA then resultin the production of jasmonic acid(JA)and its biologically active derivatives,chief among which isjasmonyl-isoleucine(JA-Ile)8.Plants 2016,5,16;doi:10.3390/ 2016,5,162 of 13Jasmonates(and/or their immediate precursors)produced in response to wounding do notstay where they are formed and can be transported efficiently within tissues.Following wounding,jasmonates/jasmonate precursors produced via LOX6 action in the vasculature of Arabidopsis leavesare highly mobile and move radially outwards from veins into the mesophyll 9.In additionto jasmonate mobility,many 13-LOXs are themselves jasmonate-inducible.Therefore,in theory,jasmonates produced via the activity of any 13-LOX could be dispersed to different cell types capableof expressing other LOXs that also make jasmonate precursors.This raises an obvious question:howis the activity of the four 13-LOX genes in Arabidopsis coordinated?The roles of 13-LOXs in jasmonate-controlled defense responses have been studied in numerousplants,including,but not restricted to potato 10,wild tobacco 11,tomato 12,rice 13,andmaize 14,as well as in Arabidopsis,a plant in which systematic LOX gene mutagenesis has beenemployed 15.Intriguingly,while all four 13-LOXs encoded in the A.thaliana genome contribute to thesynthesis of jasmonic acid 15,they each appear to have somewhat different functions in physicallydamaged leavesthe subject of the present work.For example,in addition to the initiation of JAsynthesis in wounded leaves 16,17,LOX2 also plays a minor role in JA synthesis in undamagedleaves distal to wounds 18.Furthermore,close to the site of damage,LOX2-derived hydroperoxidesare also channelled into the synthesis of arabidopsides,galactolipids that carry one or more esterifiedOPDA or dinor-OPDA molecules 17,1921.Consistent with arabidopsides being defensive secondarymetabolites,plants lacking LOX2 were more susceptible to the lepidopteran herbivore Spodopteralittoralis than is the wild type 17.LOX6 also plays a role in leaf defense.The experiments that revealed this role involved thegenetic removal of each of the three other 13-LOXs through producing a lox2 lox3 lox4 triple mutant.In this plant,LOX6 functioning alone was capable of maintaining the defense of emerging leavesand shoot apical tissues in Arabidopsis rosettes 15.Interestingly,the relative impact of LOX6 in earlywound-stimulated jasmonate production in leaves increases with the distance from damage sites.Thatis,LOX6 was necessary for most of the rapid distal expression of the regulatory gene JASMONATEZIM-DOMAIN 10(JAZ10)when the rosette was wounded 15,making this LOX of particular relevancein studies of long distance wound signalling.Finally,the LOX3/LOX4 pair contributes approximately 20%of the total JA pool that accumulatesin leaves in the first three minutes after wounding 15,however,no roles for these two LOXs in leavesare known.Here,we investigated the relative contributions of LOX2,LOX3,LOX4,and LOX6 to eachothers expression,as well as to the expression of a defense gene.We then used herbivory assays toinvestigate LOX3 and LOX4 function in leaves.Our results revealed a lipoxygenase network thatoperates to coordinate jasmonate synthesis and defense responses in wounded leaves.2.Results and Discussion2.1.13-LOX Expression Patterns in Unwounded RosettesThe expression patterns of Arabidopsis LOXs have been examined at the seedling stage 22,butequivalent data for leaves were lacking.Is each 13-LOX expressed in a different leaf cell type?Tocharacterize basal 13-LOX gene activity in unwounded leaves,each promoter was fused to a secretable-glucuronidase(GUS)reporter gene.LOX6,principally expressed in xylem contact cells 9,15,servedas a comparison with other 13-LOXs,as shown in Figure 1.GUS staining in younger leaves wasstronger than in older leaves for all 13-LOX promoters,and sections of younger leaves were compared.LOX2 had the only promoter among the four that was widely active in most tissues except in andnear maturing veins.Because of this,transversal sections for visualizing LOX2 reporter expressionwere cut nearer the leaf tip than for the other reporters(red bars in Figure 1).LOX2 protein is readilydetectable in leaves 23 and LOX2 expression was strong in mesophyll cells,bundle sheaths,andleaf-tip vasculaturebut only at a distance from maturing veins.LOX3 activity was perivascular andstrongest in the xylem region,with weaker expression in the mesophyll.LOX4 promoter activity wasPlants 2016,5,163 of 13strongest in small cells in the phloem region.These might be companion cells,although their identitywas not verified.Phloem is a known region of JA synthesis enzyme localisation 24.We noted thatLOX2 expression was almost a“mirror image”of the LOX3,LOX4,and LOX6.The expression of thesethree LOXs(unlike for LOX2)was strong in the maturing vasculature of expanding leaves.Plants 2016,5,16 3 of 13 although their identity was not verified.Phloem is a known region of JA synthesis enzyme localisation 24.We noted that LOX2 expression was almost a“mirror image”of the LOX3,LOX4,and LOX6.The expression of these three LOXs(unlike for LOX2)was strong in the maturing vasculature of expanding leaves.Figure 1.13-lipoxygenases(13-LOX)promoter-driven-glucuronidase(GUS)activity in 3.5 week-old plants.Upper images:rosettes.Scale bar=1 cm.Red bars indicate approximate section locations shown in lower images.E=epidermal cell;X=xylem region;P=phloem region.Scale bars for sections=30 m.Note that the LOX2 section was cut nearer the leaf tip than the other LOX sections.Notably,all 13-LOXs are expressed in vascular tissues with only basal LOX2 expression excluded from maturing veins.LOX3 and LOX6 expression is strongest near the xylem.LOX4 is the only 13-LOX to show basal expression almost exclusively in the phloem region.In terms of cellular space covered by promoter activities,LOX4 and LOX6 in unwounded leaves display a relatively small basal promoter activity domain,whereas the other two 13-LOXs(LOX2 and LOX3)have more extensive basal activity domains(Figure 1).GUS staining in the rosettes of wounded plants is shown in the Appendix(Figure A1).2.2.LOX2 and LOX6 Regulate LOX3 and LOX4 Expression LOX6 transcripts are not wound-inducible in roots 25 or leaves 9,15.LOX2 expression was investigated in the lox6 single mutant and the lox3 lox4 double mutant.LOX2 remained wound-inducible in each of the genetic backgrounds,however,in the absence of the functional LOX6 gene,there was weakly reduced LOX2 expression following wounding(Figure 2).LOX6 activity therefore contributes to LOX2 gene expression in wounded leaves.The possibility that there are compensatory effects whereby above-WT activity of a particular 13-LOX gene is stimulated by mutation of one or more of its homologues was tested.Figure A2 shows that basal LOX3 or LOX4 expression was not affected in unwounded leaves in the lox2-and lox6-containing backgrounds.Figure 1.13-lipoxygenases(13-LOX)promoter-driven-glucuronidase(GUS)activity in 3.5 week-oldplants.Upper images:rosettes.Scale bar=1 cm.Red bars indicate approximate section locationsshown in lower images.E=epidermal cell;X=xylem region;P=phloem region.Scale bars forsections=30 m.Note that the LOX2 section was cut nearer the leaf tip than the other LOX sections.Notably,all 13-LOXs are expressed in vascular tissues with only basal LOX2 expression excludedfrom maturing veins.LOX3 and LOX6 expression is strongest near the xylem.LOX4 is the only13-LOX to show basal expression almost exclusively in the phloem region.In terms of cellular spacecovered by promoter activities,LOX4 and LOX6 in unwounded leaves display a relatively small basalpromoter activity domain,whereas the other two 13-LOXs(LOX2 and LOX3)have more extensivebasal activity domains(Figure 1).GUS staining in the rosettes of wounded plants is shown in theAppendix(Figure A1).2.2.LOX2 and LOX6 Regulate LOX3 and LOX4 ExpressionLOX6 transcripts are not wound-inducible in roots 25 or leaves 9,15.LOX2 expressionwas investigated in the lox6 single mutant and the lox3 lox4 double mutant.LOX2 remainedwound-inducible in each of the genetic backgrounds,however,in the absence of the functionalLOX6 gene,there was weakly reduced LOX2 expression following wounding(Figure 2).LOX6activity therefore contributes to LOX2 gene expression in wounded leaves.The possibility that thereare compensatory effects whereby above-WT activity of a particular 13-LOX gene is stimulated bymutation of one or more of its homologues was tested.Figure A2 shows that basal LOX3 or LOX4expression was not affected in unwounded leaves in the lox2-and lox6-containing backgrounds.Plants 2016,5,164 of 13Plants 2016,5,16 4 of 13 Figure 2.13-LOX gene expression in different 13-lox mutant backgrounds.LOX2 expression analysed in WT,in lox6A and in lox3B lox4A double mutants.LOX3 and LOX4 expression was analyzed in WT,lox2-1,and lox6A single mutants,and the lox2-1 lox6A double mutant.LOX6 expression was analyzed in lox2-1 and the lox3B lox4A double mutant.Leaves 8(wounded)and 13(distal)were snap-frozen before(unfilled bars)and 1 h after the wounding(filled bars).Data are from three(controls)and three to four(wounded)biological replicates(SD).Letters(a,b,and c)refer to significant differences(ANOVA and t-test;p 0.05).LOX3 and LOX4 transcript levels in the WT and both lox2 and lox6 single mutants were similar in the wounded leaf,while LOX6 was found to be required for full,wound-induced LOX3 and LOX4 transcript levels in the distal leaf(Figure 2).In the wounded leaf,the double mutant lox2 lox6 displayed 2-fold lower inductions of LOX3 and LOX4 transcripts compared to the WT.In the distal leaf,LOX3 and LOX4 gene expression was reduced by 97.2%and 96.3%,respectively,in the lox2 lox6 double mutant relative to the WT.In the distal leaf,the lox6 single mutant displayed an approximately 20-fold induction of LOX3 transcripts,but in lox2 these transcripts were induced to a far higher level(approximately 100-fold)that is to a level similar to that in the WT.Similarly,a strong effect on distal LOX4 transcript accumulation was observed in lox6 compared to lox2.LOX6 was not wound-inducible in the WT or in lox mutant backgrounds(Figure 2).The low transcript levels and associated large error bars for this LOX limit the interpretation of this result.Figure 2.13-LOX gene expression in different 13-lox mutant backgrounds.LOX2 expression analysedin WT,in lox6A and in lox3B lox4A double mutants.LOX3 and LOX4 expression was analyzed in WT,lox2-1,and lox6A single mutants,and the lox2-1 lox6A double mutant.LOX6 expression was analyzed inlox2-1 and the lox3B lox4A double mutant.Leaves 8(wounded)and 13(distal)were snap-frozen before(unfilled bars)and 1 h after the wounding(filled bars).Data are from three(controls)and three to four(wounded)biological replicates(SD).Letters(a,b,and c)refer to significant differences(ANOVA andt-test;p 0.05).LOX3 and LOX4 transcript levels in the WT and both lox2 and lox6 single mutants were similarin the wounded leaf,while LOX6 was found to be required for full,wound-induced LOX3 andLOX4 transcript levels in the distal leaf(Figure 2).In the wounded leaf,the double mutant lox2 lox6displayed 2-fold lower inductions of LOX3 and LOX4 transcripts compared to the WT.In the distalleaf,LOX3 and LOX4 gene expression was reduced by 97.2%and 96.3%,respectively,in the lox2 lox6double mutant relative to the WT.In the distal leaf,the lox6 single mutant displayed an approximately20-fold induction of LOX3 transcripts,but in lox2 these transcripts were induced to a far higher level(approximately 100-fold)that is to a level similar to that in the WT.Similarly,a strong effect on distalLOX4 transcript accumulation was observed in lox6 compared to lox2.LOX6 was not wound-induciblePlants 2016,5,165 of 13in the WT or in lox mutant backgrounds(Figure 2).The low transcript levels and associated large