Sławomir Borek Stanisława Pukacka Krzysztof Michalski. Introduction

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1 At Physiol Plnt () 34:99 6 DOI.7/s ORIGINAL PAPER Regultion y surose of storge ompounds rekdown in germinting seeds of yellow lupine (Lupinus luteus L.), white lupine (Lupinus lus L.) nd Anden lupine (Lupinus mutilis Sweet). II. Moiliztion of storge lipid Słwomir Borek Stnisłw Pukk Krzysztof Mihlski Reeived: 9 June / Revised: Otoer / Aepted: 6 Deemer / Pulished online: 8 Deemer Ó The Author(s). This rtile is pulished with open ess t Springerlink.om Astrt Reserh of the regultory funtion of surose in storge lipid rekdown ws onduted on isolted emryo, exised nd whole s of three lupine speies grown in vitro on medium with 6 mm surose or without the sugr. Lk of surose in the medium used signifint inrese in totl lipid ontent in yellow, white nd Anden lupine isolted ut in Anden lupine nd exised, lipid level ws lerly lower in rohydrtes defiient onditions. Surose used no signifint effet on ftty ids spetr. The min ftty id in yellow lupine seeds ws linolei id, in white lupine olei id nd in Anden lupine oth olei nd linolei ids. The min phospholipid in orgns of three lupine speies ws phosphtidylholine. In sugr-defiient onditions, ontent of phosphtidylholine nd some others phospholipids ws deresed. The peulir fetures of regultion y sugrs of storge lipid rekdown in germinting lupine seeds nd indution of utophgy in young rohydrte strved is disussed. Keywords Autophgy Ftty id Legume Phospholipid Sugr strvtion Communited y S. Weidner. S. Borek (&) Deprtment of Plnt Physiology, Adm Mikiewiz University, ul. Umultowsk 89, 6-64 Poznn, Polnd e-mil: orek@mu.edu.pl S. Pukk Institute of Dendrology, Polish Ademy of Sienes, ul. Prkow 5, 6-35 Kórnik, Polnd K. Mihlski Plnt Breeding nd Alimtiztion Institute, ul. Strzeszyńsk 36, Poznn, Polnd Introdution In mture lupine seeds, the min storge ompound is protein, minly gloulins (Rtjzk et l. 999; Durnti et l. 8). In yellow lupine seeds, protein ontent my reh out 45 of dry mtter (Cerletti 98), in white lupine seeds up to 38 (Mohmed nd Rys-Durte 995) nd in Anden lupine seeds 4 5 (Sntos et l. 997). Lupine seeds ontin lrge quntities of storge lipid s well. In yellow lupine, lipid ontent is out 6 of dry mtter, in white lupine seeds 7 4 nd in Anden lupine seeds up to (Borek et l. 9). In this se, Anden lupine seeds re similr to soyen seeds, whih my ontin 6 of storge lipid (Zhou et l. 6). Crohydrte ontent in lupine seeds is on verge out 36.5 of dry mtter, of whih 6 is fier (Hedley ). Lupine seeds ontin no strh (Borek et l. 6, ; Durnti et l. 8). Investigtions onerning storge lipid rekdown in germinting lupine seeds hve een onduted nerly exlusively on yellow lupine seeds, i.e. the speies tht umulte smll mount of lipid in seeds. It hs een shown tht in yellow lupine seeds, oil odies re loted minly in, ut quite numerous oil odied re present in s well. During germintion storge lipid is quikly moilized nd lmost ll odies in emryo dispper fter four dys of germintion (Borek et l. 6, ). In germinting yellow lupine seeds, strong onnetions etween pthwys of storge lipid rekdown nd pthwys of mino ids synthesis hve een deteted. Storge lipid is used s respirtory sustrte or is onverted into sugrs, ut some prt of lipid derived ron skeletons is used for mino ids synthesis (minly sprgine, glutmine nd glutmte) (Borek et l. 3; Borek nd Rtjzk ). Moreover, when sprgine synthesis is

2 At Physiol Plnt () 34:99 6 disrupted (y L-methionine sulfoximine n inhiitor of glutmine synthetse), hnges in lipid rekdown re oserved. In yellow nd white lupine nd, slight inrese in lipid utiliztion hs een deteted, ut in Anden lupine nd, L-methionine sulfoximine uses signifint enhnement in lipid utiliztion (Borek et l. ). In reserh of ron flow from storge lipid into mino ids (s well s into sugrs), the rdiolelled ette s the simplest ftty id ws used (Borek et l. 3; Borek nd Rtjzk ). In reserh of regultion of storge lipid rekdown, surose is tken under onsidertion. This sugr is very importnt euse of storge lipid is synthesized from surose in developing seeds (Weer et l. 5; Bud et l. 8) nd surose is one of the min end produts of storge lipid degrdtion in germinting seeds (Grhm 8; Quettier nd Estmond 9). Otherwise, surose nd gluose re importnt signling moleules in plnt metolism regultory network nd these sugrs might ontrol plnt ell metolism y modifition of expression of mny genes (Smeekens et l. ). Experiments regrding regultory funtion of surose in lipid metolism in lupine seeds re not numerous nd hve een onduted lmost only on yellow lupine seeds. Ultrstruture investigtions hve showed tht oil odies in nd in re smller, less numerous or disppered when sugrs level dereses in tissues (Borek et l. 6, ). Lipse nd tlse tivity inreses in sugr-depleted onditions (Borek et l. 6), ut enzymes involved in further steps of lipid rekdown (ytosoli onitse, isoitrte lyse, NADP? -dependent ytosoli isoitrte dehydrogense) re more tive in surose-fed tissues (Borek nd Nu ). Moreover, it hs een proved tht inrese or derese of enzyme tivity is used y modifition of gene expression. Lipse mrna level is higher nd mrna levels for ytosoli onitse nd NADP? -dependent isoitrte dehydrogense re lower in yellow lupine nd in whih sugrs ontent is deresed (Borek nd Nu ). Cytosoli onitse nd NADP? -dependent isoitrte dehydrogense re importnt enzymes in the ove-mentioned pthwys of ron flow from storge lipid into mino ids in germinting seeds of yellow lupine (Borek et l. 3, ; Borek nd Rtjzk ). Stimultory effet of surose on these two enzymes tivity is orrelted with the enhned ron flow from lipid to mino ids, euse mino ids synthesis from lipid derived ron skeletons is signifintly higher in surose-fed nd (Borek nd Rtjzk ). Sine lupine seeds ontin lrge protein quntities, they hve een used mostly in reserh onerning storge protein nd mino ids metolism. Experiments regrding storge lipid metolism in lupine seeds re not numerous, so dt presented in this pper enlrge our knowledge out storge lipid rekdown during germintion nd its regultion y surose. This work is the ontinution of desried ove reserh onduted on germinting yellow lupine seeds nd the next elements of storge lipid rekdown were investigted in this speies. However, the very importnt im of the reserh ws heking how surose regultes lipid rekdown in lupine seeds, whih umulte muh more this storge ompound thn yellow lupine seeds. Sine investigtions were onduted on seeds of three lupine speies, i.e. yellow lupine (lipid ontent out 6 of dry mtter), white lupine (7 4) nd Anden lupine (out ), to define the regultory funtion of surose, the isolted, exised nd s were grown in vitro on medium with 6 mm surose or without the sugr. Surose in this onentrtion dded to the medium used signifint inrese in solule rohydrte nd strh levels in growing in vitro lupine, nd s. Lk of surose in the medium used onsiderle derese in solule rohydrte nd strh ontent in tissues, espeilly in isolted (Borek et l. 6). In this pper, the totl lipid level, ftty ids spetrum, nd phospholipids ontent re presented. Mterils nd methods Plnt mteril Yellow, white nd Anden lupine seeds were surfesterilized in. HgCl for, 5 nd min, respetively, nd llowed in the drk to imie for 4 h t 5 C. Emryo nd isolted from imied seeds, s well s whole imied seeds deprived of their ots, were pled on sterilized filter pper (Whtmn no. 3) in sterile tues ove Heller s medium (Heller 954) in two trophi vrints: with 6 mm surose (?S) nd without surose (-S). Isolted, exised nd s were ultured in vitro for 96 h in the drk t 5 C. All experiments were onduted on isolted orgns s well s on orgns euse it enled to detet undesirle effet of injury, i.e. isoltion of orgns. Totl lipid determintion Totl lipids were extrted from nd of ir dry nd imied (4 h) yellow, white nd Anden lupine seeds s well s from orgns grown in vitro for 96 h using hloroform:methnol (:, v/v) ontining.5 utylted hydroxytoluene (BHT), ording to Allen et l. (966), s desried y Pukk (99). The mounts of lipid were determined grvimetrilly.

3 At Physiol Plnt () 34:99 6 Ftty ids determintion Spetrum of totl ftty ids were determined in emryo nd of ir dry nd imied (4 h) yellow, white nd Anden lupine seeds s well s in orgns grown in vitro for 96 h ording to Borek et l. (9). Ftty ids (s methyl esters, fter trnsmethyltion of the extrted lipids with 5 mm KOH in methnol for 5 min t 7 C nd extrted in hexne) were determined y using n Agilent 689 gs hromtogrph, olumn 3 m DB5, temperture C, nd flme ioniztion detetor (FID). nd exised, the derese in totl lipid level ws smller thn in orgns. The lerest exmples for this were yellow lupine exised or Anden lupine isolted (Fig. ). Surose dded to the medium (?S) used no signifint hnges in totl lipid level in yellow, white nd Anden lupine. Distint differenes in totl lipid ontent were visile in isolted. Lipid level ws signifintly higher in white nd Anden lupine surose strved (-S) isolted thn in fed with surose (?S) ones (Fig. ). The differenes were even more pronouned when the totl Phospholipids determintion Phospholipids were determined in liquots of the lipid extrts (desries ove) seprted y one-dimensionl TLC in hloroform:methnol:eti id:wter (85:5: :3.5, v/v; Nihols et l. 965), using originl phospholipid stndrds. Spots ontining phospholipids nd deteted with iodine vpor were srped off for nlysis of phosphorus. The phosphorus ontent ws estimted ording to Ames (966). mg lipid mg - FW,9,8,7,6,5,4,3,, yellow lupine,4,3,, isolted dry seeds imied seeds +S S Sttistil nlysis The results re the men ± SD of three independent experiments with two or three replitions eh. Signifine of differenes etween men vlues ws determined with Student s t test. The sttistil nlysis were onneted only to ssessing the signifine in the differenes etween orgns grown in vitro on medium ontining 6 mm surose (?S) nd orgns grown on medium without surose (-S). Results In mture lupine seeds, storge lipid ws umulted oth in nd in. In yellow lupine seeds, whih ontin only out 6 of lipid in dry mtter, the lipid level ws lmost the sme in nd in, ut in white lupine seeds (lipid ontent 7 4) nd Anden lupine seeds (lipid ontent out ), ontined more lipid thn (Fig. ; dry seeds). After 4 h of imiition, the totl lipid ontent in nd ws onsiderly lower thn in dry seeds nd (Fig. ), ut it is rther ovious result euse of wtering of tissues. Aprt from the in vitro ulture onditions, huge derese (ompring to nd of imied seeds) in totl lipid ontent ws oserved in 96-h of yellow, white nd Anden lupine. Similr derese ws noted in yellow nd Anden lupine. In in vitro grown isolted mg lipid mg - FW mg lipid mg - FW,8,6,4,,,8,6,4,,,8,6,4,,,8,6,4, emryo white lupine,6,4, emryo Anden lupine emryo isolted isolted isolted isolted dry seeds imied seeds +S S dry seeds imied seeds +S S exised exised exised Fig. Totl lipid ontent in yellow, white nd Anden lupine nd of dry nd imied (4 h) seeds nd in orgns grown in vitro for 96 h on medium with 6 mm surose (?S) or without the sugr (-S). Smll grphs re the mgnifition of totl lipid ontent in nd isolted. Sttistil signifine t P B.5 () or t P B. ()

4 At Physiol Plnt () 34:99 6 lipid ontent ws lulted on dry mtter. Then the lipid ontent ws onsiderly higher in surose strved ( S) yellow, white nd Anden lupine isolted (higher lipid level y 43, 44 nd 7, respetively). In grown in vitro yellow nd white lupine (oth nd exised ), lipid level ws similr in?s nd -S orgns nd differenes were not sttistilly signifint. Only in Anden lupine the lipid level ws lower in sugrs depletion onditions ( S) in tissues nd the differene in ws highly sttistilly signifint (Fig. ). In nd of yellow lupine dry nd imied seeds, the previling ftty id ws linolei id (Fig. ); in white lupine seeds, it ws olei id (Fig. 3), nd in Anden lupine seeds, it ws olei nd linolei ids (Fig. 4). During 4 h of imiition in nd of ll three lupine speies, no hnges in ftty id spetr were deteted (Figs., 3, 4). However, during 96 h of growth, ler hnges were oserved in nd isolted ompring to ftty ids spetr in of dry nd imied seeds. In yellow lupine, linolei id deresed nd the min ftty id ws linoleni id (Fig. ). In isolted emryo, the previling ftty id ws still linolei id, ut perentge of linoleni id inresed (Fig. ). In white lupine, olei id deresed, wheres linoleni id inresed (Fig. 3). In isolted, olei id deresed nd the previling ftty id ws linolei id (Fig. 3). In Anden lupine nd isolted, ler derese in olei id ws oserved nd the min ftty id ws linolei id (Fig. 4). Contrry to ove desried hnges ourring in, no suh ler hnges were oserved in grown in vitro yellow, white nd Anden lupine (Figs., 3, 4). Tking under ount the trophi onditions of in vitro ulture, it n e onluded tht surose used no mny signifint hnges in ftty ids spetr. These hnges were deteted minly in yellow nd white lupine nd nd were onneted minly with ftty ids whih ontent in tissues ws low, for exmple plmitoolei id, eiosenoi id or erui id (Figs.,, 3,, 4, ). Only one hnge ws onneted with the previling ftty id, nmely, the perentge of linolei id ws lower in of yellow lupine s grown on medium without surose ( S; Fig. ). In nd of dry nd imied seeds s well s in orgns grown in vitro of eh of the three lupine speies, the min phospholipid ws phosphtidylholine (Figs. 5, 6, 7 ). Similrly to totl lipid level (Fig. ), huge derese in the ontent of eh phospholipid ws deteted during 4-h imiition (Figs. 5, 6, 7). Additionl derese in phospholipids ontent ws oserved fter 96 h of growth (Figs. 5,, 6,, 7, ) yellow lupine plmiti id plmitoolei id steri id olei id linolei id linoleni id eiosenoi id eheni id erii id isolted exised Fig. Ftty id omposition (perentge of totl integrted peks on hromtogrphs) in yellow lupine nd of dry nd imied (4 h) seeds () nd in () nd () grown in vitro for 96 h on medium with 6 mm surose (?S) or without the sugr (-S). Sttistil signifine t P B.5 () or t P B. () Surose dded to the medium (?S) used signifint derese in severl phospholipids ontent, ut the most frequent hnges were noted in phosphtidylholine ontent. In rohydrte defiieny onditions ( S) phosphtidylholine ontent ws deresed in yellow, white nd Anden lupine nd in white nd Anden isolted (Figs. 5, 6, 7). In yellow nd white lupine nd exised, no hnges or slight derese in phosphtidylholine ontent ws noted nd the hnges were not sttistilly signifint (Figs. 5, 6). The most signifint hnges in phospholipids ontent were deteted in Anden lupine

5 At Physiol Plnt () 34: white lupine plmiti id plmitoolei id steri id olei id linolei id linoleni id eiosenoi id eheni id erii id Anden lupine plmiti id plmitoolei id steri id olei id linolei id linoleni id eiosenoi id eheni id erii id isolted isolted exised exised Fig. 3 Ftty id omposition (perentge of totl integrted peks on hromtogrphs) in white lupine nd of dry nd imied (4 h) seeds () nd in () nd () grown in vitro for 96 h on medium with 6 mm surose (?S) or without the sugr (-S). Sttistil signifine t P B.5 () or t P B. () Fig. 4 Ftty id omposition (perentge of totl integrted peks on hromtogrphs) in Anden lupine nd of dry nd imied (4 h) seeds () nd in () nd () grown in vitro for 96 h on medium with 6 mm surose (?S) or without the sugr (-S). Sttistil signifine t P B.5 () or t P B. () nd exised. In these, the ontent of eh phospholipid ws lower in sugrs depletion onditions in tissues (-S; Fig. 7). Disussion The regultory funtion of surose in storge lipid rekdown during lupine seeds germintion ws investigted. In literture, there re mny dt, whih hve proved tht in rohydrte depletion onditions the enhnement of storge ompounds moiliztion ours or other ell ompounds degrdtion tkes ple. An exmple for the regultion y sugrs of storge ompounds rekdown might e the enhnement of storge protein moiliztion in surose strved nd during yellow lupine seeds germintion (Borek nd Rtjzk ; Borek et l., ) nd intensified mino ids utiliztion s respirtory sustrtes (Borek et l. ; Morkuns et l. 3). It hs een desried in detils how sugrs regulte strh tolism in germinting erel seeds (Thoms nd Rodriquez 994). Finlly, storge lipid moiliztion ws retrded y exogenously pplied sugrs in Aridopsis (To et l. ).

6 4 At Physiol Plnt () 34: yellow lupine 8 7 white lupine phosphtidylinositol phosphtidylholine phosphtidylglyerol phosphtidylethnolmine phosphtidi id phosphtidylinositol phosphtidylholine phosphtidylglyerol phosphtidylethnolmine phosphtidi id,8,6,5,4,,,,8,6,5,,4,5, isolted isolted,5,6,4,5,,8,6,5,4, exised exised Fig. 5 Phospholipid ontent in yellow lupine nd of dry nd imied (4 h) seeds () nd in () nd () grown in vitro for 96 h on medium with 6 mm surose (?S) or without the sugr (-S). Sttistil signifine t P B.5 () Fig. 6 Phospholipid ontent in white lupine nd of dry nd imied (4 h) seeds () nd in () nd () grown in vitro for 96 h on medium with 6 mm surose (?S) or without the sugr (-S). Sttistil signifine t P B.5 () Our previous reserh on yellow lupine germinting seeds hs proved tht storge lipid is degrded fster upon sugr-defiient onditions in tissues. Ultrstruture oservtions hve showed tht in surose-strved (extly in root meristemti zone ells), oil odies dispper during 96-h germintion period while in they re smller nd less numerous (Borek et l. 6, ). Lipse nd tlse (enzymes onneted with the eginning steps of storge lipid rekdown) tivity is lso higher in orgns grown in vitro on medium without surose (Borek et l. 6). Ctlse is ssoited with the ftty ids -oxidtion nd rise in its tivity n e treted s n indiret proof for intensifition of the ftty ids -oxidtion in sugr-strved tissues (Dieuide et l. 99, 993). In this pper, the effet of surose nutrition on the totl lipid level in, nd s of three lupine speies is presented. In yellow nd white lupine, hnges used y surose were not signifint. Anden lupine ontined less lipid when they were grown on medium without surose ( S). However, in surose strved ( S) yellow, white nd Anden lupine isolted the lipid level ws remrkly higher thn in fed with surose (?S; Fig. ). Despite of signifint hnges used y surose in storge lipid ontent, there were no numerous hnges in ftty ids spetr (Figs.,, 3,, 4, ). Inrese in

7 At Physiol Plnt () 34: FW nmol Pi mg - FW nmol Pi mg 8 6 4,5,,5,,5,5,5,5 Anden lupine phosphtidylinositol phosphtidylholine phosphtidylglyerol phosphtidylethnolmine phosphtidi id isolted exised lipid level in S isolted is onsistent with our previously mde ultrstruture oservtions. In white nd Anden lupine isolted more oil odies were oserved when they were grown on medium without surose ( S; Borek et l. ). This result is very diffiult to interpret t present stge of the reserh euse it is opposite to other literture dt whih desrie enhned lipid nd ftty ids degrdtion (Brouquisse et l. 99; Grhm et l. 994; Yu999; To et l. ) or inresed expression of genes involved in lipid degrdtion (Grhm et l. 994; Yu999; Gonzli et l. 6; Li et l. 6) in rohydrte depletion onditions. Now it is impossile to unmiguously explin the inresed lipid ontent in Fig. 7 Phospholipid ontent in Anden lupine nd of dry nd imied (4 h) seeds () nd in () nd () grown in vitro for 96 h on medium with 6 mm surose (?S) or without the sugr (-S). Sttistil signifine t P B.5 () or t P B. () surose-strved lupine isolted (Fig. ). Only suppositions or hypothesis re possile. Mye suh extrordinry higher ontent of lipid in surose strved is result of utophgy? Ultrstruture oservtions hve showed tht in sugr-depleted onditions huge inrese in vuoliztion ours in the root meristemti zone ells (Borek et l. 6,, ). Enlrgement of vuoles size is one of the effets of utophgy ourring in sugrs strved ells (Yu 999). Mye peroxisomes (glyoxysomes) or some enzyme proteins involved in lipid rekdown re degrded in this proess nd sine it in surose strved ( S) isolted emryo remined more lipid. This sequene of events in lupine ws proposed nd desried in detils in our erlier pper (Borek t l. ). Supposition tht in plnts might ourred speilized forms of utophgy, whih re similr to mentioned ove pexophgy ws lredy proposed y Thompson nd Vierstr (5). Dt presented in this pper indiretly support the hypothesis. One of result used y utophgy triggered y rohydrte strvtion is derese in phospholipid ontent euse of mssive memrne lipids degrdtion (Auert et l. 996; Inoue nd Moriysu 6). In white nd Anden lupine S isolted, signifint derese in phosphtidylholine ontent ws oserved (Figs. 6, 7) even though the lipid level ws higher (Fig. ). The issue disussed ove definitely needs dditionl reserh, espeilly on ultrstruture level with the use of onnmyin A nd ysteine protese inhiitor E-64 whih would llow nlyzing the utophgy t remrkly detiled level (Bsshm 7). Summrizing the dt presented in this pper, it n e onluded tht rohydrte level in tissues is very importnt gent in regultion of storge lipid rekdown in yellow, white nd Anden lupine germinting seeds. Regultion y surose of storge lipid moiliztion in germinting lupine seeds hs some peulir fetures. The exmple for this might e the higher lipid ontent in surose strved isolted. This speil feture of the regultion of lipid rekdown y surose ourred in ll three studied lupine speies nd ws independent of storge lipid mount in seeds. A very interesting nd needing further reserh is utophgy indued y rohydrte depletion in young during lupine seeds germintion. Aknowledgments We thnk Dr. Stnisłw Stwiński, Hed of Plnt Breeding Sttion Smolie Division in Przeędowo (Murown Goślin, Polnd), for providing lupine seeds. This work ws prtilly supported y grnt no. P6A 4 9 from Polish siene funding in yers 5 8. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution Nonommeril Liense whih permits ny nonommeril use, distriution, nd reprodution in ny medium, provided the originl uthor(s) nd soure re redited.

8 6 At Physiol Plnt () 34:99 6 Referenes Allen CF, Good P, Dvis HF, Chisum P, Fowler SD (966) Methodology for the seprtion of plnt lipids nd pplition to spinh lef nd hloroplst lmelle. J Am Oil Chem So 43:3 3 Ames DN (966) Assy of inorgni phosphte, totl phosphte nd phosphtses. Methods Enzymol 8:5 8 Auert S, Gout E, Bligny R, Mrty-Mzrs D, Brrieu F, Alouvette J, Mrty F, Doue R (996) Ultrstruturl nd iohemil hrteriztion of utophgy in higher plnt ells sujeted to ron deprivtion: ontrol y the supply of mitohondri with respirtory sustrtes. J Cell Biol 33:5 63 Bsshm DC (7) Plnt utophgy more thn strvtion response. Curr Opinion Plnt Biol : Bud S, Dureuq B, Miquel M, Roht C, Lepinie L (8) Storge reserve umultion in Aridopsis: metoli nd developmentl ontrol of seed filling. The Aridopsis ook. Am So Plnt Biol. doi:.99/t.3 Borek S, Morkuns I, Rtjzk W, Rtjzk L () Metolism of mino ids in germinting yellow lupine seeds III. Brekdown of rginine in sugr-strved orgns ultivted in vitro. 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