Control of ascorbic acid synthesis and accumulation and glutathione by the incident light red/far red ratio in Phaseolus vulgaris leaves

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1 FEBS Letters 8 (9) 8 journl homepge: Control of scoric cid synthesis nd ccumultion nd glutthione y the incident light red/fr red rtio in Phseolus vulgris leves Crlos G. Brtoli,, Edurdo A. Tmussi, Fnello Diego, Christine H. Foyer Instituto de Fisiologí Vegetl, Fcultd Ciencis Agrris y Forestles, Universidd Ncionl de L Plt, CCT-CONICET, cc7 9 L Plt, Argentin School of Agriculture, Food nd Rurl Development, The University of Newcstle upon Tyne, Newcstle upon Tyne NE 7RU, UK rticle info strct Article history: Received 9 Octoer 8 Revised Novemer 8 Accepted 4 Novemer 8 Aville online 6 Decemer 8 Edited y Ulf-Ingo Flügge Keywords: Phytochrome Light cclimtion Pyridine nucleotide Ascoric cid synthesis Glutthione Respirtion L-glctone-,4-lctone dehydrogense The effects of red/fr red () rtios on lef scorte (AA) nd glutthione (GSH) ccumultion were exmined in common en (Phseolus vulgris L.). Growth under low rtios resulted in shde phenotype nd much lower lef AA nd GSH contents thn high () rtios. Photosynthesis rtes were unffected y chnges in the rtio ut lef respirtion rtes, pyridine nucleotide pools nd ntioxidnt enzyme ctivities were decresed under the low regime. The GSH pool chnged slowly in response to ltered rtios ut lef scorte cclimted over single photoperiod. We conclude tht light qulity signls, prticulrly rtios, re importnt regultors of ntioxidnt synthesis nd ccumultion. These cclimtory chnges re n erly response to chnging light environment. Ó 8 Federtion of Europen Biochemicl Societies. Pulished y Elsevier B.V. All rights reserved.. Introduction The red/fr red () rtio of incident light is key environmentl signl tht llows leves to perceive the presence of other leves in the vicinity, either from the prent plnt or from their ner neighours []. When exposed to low rtio leves orchestrte chnges tht enle them to void shding y competitors []. Plnts experiencing FR-enriched cnopy typiclly show petiole elongtion nd or lef re increment chnges []. The phytochrome fmily of photoreceptors tht sor R nd FR wvelengths in plnts re signl trnsducers in these responses []. Ascoric cid (AA) nd glutthione (GSH) re uiquitous nd undnt metolites prticipting of mny protective mechnisms in leves. AA functions not only s n ntioxidnt ut it is lso cofctor in numer of enzymtic rections [4]. In prticulr, AA is the co-fctor involved in the ctlysis of -oxociddependnt dioxygense (ODD) rections in plnts nd nimls []. The ODD fmily of enzymes re responsile for the synthesis of wide rnge of secondry metolites prticulrly hormones []. AA lso prticiptes in processes involved in the dissiption of excess excittion energy in the chloroplsts, such s xnthophyll cycle [6] nd wter wter cycle [7]. We hve previously shown Corresponding uthor. Fx: E-mil ddress: crlos.rtoli@gro.unlp.edu.r (C.G. Brtoli). tht growth irrdince nd respirtion re importnt fctors tht regulte the AA content of Aridopsis leves [8]. We showed tht incresing growth irrdince progressively elevted lef AA contents. Moreover, we provided evidence tht regultion of L-glctone-,4-lctone dehydrogense (L-GlLDH), which ctlyses the lst rection of the synthetic pthwy, y light nd respirtory controls ws n importnt determinnt of the extent of lef AA ccumultion [8]. In this study we hve further documented the effects of light, exploring the effects of light qulity rther thn quntity in determining AA ccumultion in leves. In prticulr, we hve nlysed the effects of the rtio of incident light, which is key shde signl, on the regultion of low moleculr ntioxidnt contents in leves.. Mterils nd methods.. Plnt mteril nd experimentl set-up Phseolus vulgris L. (cv. TUC ) plnts were cultivted under two light rtios: () =. nd () =. tht simulte sun nd shde light qulity, respectively, while mintining the sme photosynthetic photon flux density (PPFD, lmol photon m s ). Plnts were grown under h photoperiod t C for 7 or dys. At these times, some tches of plnts were trnsferred from sun ( =.) to shde ( =.) growth 4-79/$4. Ó 8 Federtion of Europen Biochemicl Societies. Pulished y Elsevier B.V. All rights reserved. doi:.6/j.feslet.8..4

2 C.G. Brtoli et l. / FEBS Letters 8 (9) 8 9 conditions for further or dys. The unifolite leves only were used in the following nlyses fter dys... Light tretments The light from tungsten hlogen lmps ws pssed through solutions of Cu SO 4 or green filter to generte sun nd shde, rtios, respectively. The wvelengths of sorption were chosen s previously descried y other collegues [9], with Cu SO 4 preferentilly soring FR (7 nm) light while the green filter preferentilly sors R (6 nm) rdition. Spectrl energy distriution ws continuously mesured with spectrordiometer (Instrumenttion Specilities Co., Lincoln, NE, USA) nd PPFD ws mesured with quntum sensor (LI-9 SA, Li-Cor, Lincoln, NE, USA). Blue light rdition ws similr under oth tretments. The distnce etween lmps nd the plnts ws djusted dily in order to ensure comprle irrdince (PPFD) levels under oth tretments... Chlorophyll determintions Chlorophyll ws determined fter extrction with dimethylformmide []..4. Antioxidnt determintions Reduced nd oxidised scoric cid (AA nd DHA, respectively) were determined s descried in [8] nd reduced nd oxidised glutthione (GSH nd GSSG, respectively) were determined s descried in []. L-GlLDH ws extrcted nd mesured ccording to []. Dehydroscorte reductse (DHAR) nd glutthione reductse (GR) were mesured essentilly s descried in [,4]... Nucleotide pyridines determintions NAD(P)/NAD(P)H contents were mesured spectrophotometriclly s descried in []..6. Photosynthesis nd respirtion mesurements The net CO ssimiltion rtes of unifolite leves were mesured using n infr red gs nlyser (Cirs-, PP Systems Ltd.) with stndrd lef cuvette (PLC6). Photosynthetic electron trnsport rtes (ETR) nd photochemicl quenching (qp, the percentge of open PSII centres) were mesured in the sme leves with portle modulted chlorophyll fluorometer (FMSII, Hnstech, UK). The ETR nd qp prmeters were clculted s descried in [6]. The net CO ssimiltion rtes nd chlorophyll fluorescence mesurements were performed under the growth conditions (temperture, PPFD nd rtio) for ech tretment. Respirtion ws mesured using Clrk-type electrode (Hnstech, UK) in lef disks hrvested from drk-dpted leves [8]..7. Inhiition of photosynthetic electron trnsport y -(,4- dichlorophenyl)-,-dimethylure (DCMU) Photosynthetic electron trnsport ws inhiited y supplying lm DCMU vi the roots for 48 h prior to the nlysis. The extent of inhiition of the photosynthetic electron trnsport chin ws determined using the ETR mesurement, s descried ove..8. Sttisticl nlysis The dt were nlysed using stndrd ANOVA method. The dt shown re the men vlues otined from t lest four independent experiments with P 6... Results nd discussion.. Lef responses to different rtios The common en plnts showed typicl responses to the different light environment tretments: leves produced under light enriched in FR hd lower chlorophyll contents nd lower specific lef weights. They lso hd longer petiole lengths thn leves produced under high rtio regime (Tle ). Plnts tht were trnsferred from growth in the sun (.) environment to the shde (.) environment for dys prior to mesurement displyed n intermedite plnt sun/shde phenotype (Tle )... Effects of the rtio on lef ntioxidnt contents Leves under shde (.) environment hd on verge 8% lower AA content thn leves under sun (.) environment (Fig. A). The mounts of DHA were similr under ll light tretments. When plnts were trnsferred from the sun to the shde rtio regime the lef AA content decresed (Fig. A). In ddition, lef GSH contents were lower when plnts were grown under the shde (.) environment (Fig. B) conditions, nd the level of GSSG ws similr under oth regimes. The content of AA ws reduced to out % t the end of the drk period (. nd. lmol g FW for sun nd shde light environment, respectively) nd the oxidised stte ws similr for leves under oth tretments showing % of the oxidised form. The concept tht light is key environmentl fctor controlling lef ntioxidnt contents is widely ccepted [7]. However, while the effects of light quntity re well documented, there is no literture informtion on the effects of light qulity on the undnce of lef ntioxidnts. The dt presented in Fig. demonstrte tht high rtios fvour ccumultion of AA nd GSH. The high rtios simulte the light qulity experienced y the plnt under conditions when plnts might e exposed to full sunlight, sitution where the potentil for excess irrdince nd photoinhiition is high. In this context high AA nd GSH contents would confer pre-emptive dvntge so tht the photosynthetic tissues could support light-induced increses in oxidtive lod. AA is essentil for the production of zexnthin nd therml excittion energy dissiption y non-photochemicl quenching [8]. AA nd GSH prticipte the wter wter cycle which is mechnism for the dissiption of excess reducing power in chloroplsts [7]. In order to confirm tht rtio of incident light ws n importnt environmentl trigger controlling the lef AA nd GSH contents s well s the reduced/oxidised rtios, we performed short-term trnsition experiments trnsferring plnts from the high to the pplied low rtio conditions, for short periods nd the cclimtion process ws then followed. Accordingly plnts grown under the sun (.) were trnsferred to the shde (.) on dy of the experiment in drkness t the end of Tle The effect of different light rtios on the chlorophyll content, specific lef weight nd petiole length of common en leves. Plnts were cultivted under either sun (.) or shde (.) rtios t the sme PPFD ( lmol photon m s ) for dys. At the dy 7 tch of plnts ws trnsferred from. to. light rtio (..) for further dys. rtio Chlorophyll content (lgmg FW) Specific lef weight (mg cm ) Petiole length (mm)..4 ±. 8.8 ± ±.... ±.4 6. ±. 6. ± ±.c. ±.c 64. ± 6. Dt with similr letter represent sttisticlly homogenous groups (ANOVA,

3 C.G. Brtoli et l. / FEBS Letters 8 (9) 8 A DHA (μmol g - FW) B GSH (nmol.g - FW) GSSG (nmol g - FW).. to.... to.. C 7 6. D 4 c. DHA (μmol g - FW) GSH (nmol g - FW) GSSG (nmol g - FW).. to.... to.. Fig.. The effect of the incident light rtio on the ntioxidnt contents of common en leves. In (A nd B) plnts were cultivted t n overll intensity of lmol photon m s for dys. Some tches of plnts were then trnsferred t dy 7 from the high to the low light rtio (..) condition for dys. Lef AA nd DHA contents (A) nd GSH nd GSSG contents (B) were then mesured. Results in (C) nd (D) show the effect on lef AA/DHA nd GSH/GSSG contents, respectively, fter trnsfer from high to low rtios for single photoperiod ( h). Plnts were cultivted t rtio of. or. for dys. One tch of plnts ws trnsferred t dy from the rtio of. to rtio of. (..). The results re the men vlues otined from four independent the photoperiod. Antioxidnt contents were then mesured t the end of the following photoperiod (i.e. h into the photoperiod). While the lef chlorophyll contents nd the lef petiole lengths were not modified significntly in this short period fter trnsfer (dt not shown), the mounts of AA nd DHA responded rpidly to the ltered sun nd shde environments, reching vlues tht were similr to those ttined in the previous studies on longer term trnsfer exposures to the different light qulity environments (Fig. C). In contrst, GSH nd GSSG contents were not modified in this short term experiment (Fig. D). The oserved chnges in lef AA contents were chieved over reltively short time period (i.e. h). This oservtion suggests tht light qulity, i.e. rtio is key determinnt of the extent of lef AA ccumultion nd tht the regultory effect of rtio on AA synthesis is triggered erly in the cclimtiztion response to the chnging light environment. Moreover, these short term trnsition experiments provide evidence tht the oserved rtio effects on AA occur in dvnce of (nd re therefore lrgely independent of) other cclimtory modifictions in for exmple lef chlorophyll contents, specific lef weight, etc... Effect of rtio on respirtion nd photosynthesis Lef ntioxidnt homeostsis is directly influenced y photosynthesis nd respirtion. AA synthesis is tightly linked to the mitochondril electron trnsport chin [] ecuse L-GlLDH, which ctlyses AA synthesis, is locted in the inner mitochondril memrne. The oxidtion of L-glctone-,4-lctone y L-GlLDH feeds electrons into the cytochrome c pool in the mitochondril electron trnsport chin. This reltionship is importnt in cclimtion to environmentl cues [9]. Leves otined from plnts grown under high irrdinces show incresed respirtion rtes nd they lso hve higher L-GlLDH ctivities nd AA concentrtions [8]. The lef respirtion rte is lower in leves grown under shde light conditions (Tle ), consistent with the low lef AA levels oserved in the sme conditions (i.e..). Lef respirtion mesured in plnts trnsferred from sun to shde for only one photoperiod show n intermedite rte (dt not shown). Lef Tle The effect of different light rtios on drk respirtion nd photosynthesis. Plnts were cultivted under either sun (.) or shde (.) rtios t the sme PPFD ( lmol photon m s ) for dys. rtio Respirtion rte (lmol O g FW h ) Net photosynthetic rte (lmol CO g FW h ) ETR (lmol electrons m s ) qp reltive units 8.8 ±. 9 ± 7 9 ±.9 ±.. 9. ±. 84 ± 8 98 ±.9 ±. Dt with similr letter represent sttisticlly homogenous group (ANOVA,

4 C.G. Brtoli et l. / FEBS Letters 8 (9) 8 Tle The effect of different light rtios on lef L-GlLDH, DHAR nd GR ctivities. Plnts were cultivted under either sun (.) or shde (.) rtios t the sme PPFD ( lmol photon m s ) for dys. On dys 7 nd fter sowing some of the tches of plnts grown under the sun conditions (.) were trnsferred to shde (.) conditions, so tht effects of the trnsition to shde rtios could e determined dys nd dy fter trnsfer, respectively. In ll cses smples were hrvested for nlysis on the th dy fter sowing. rtio L-GlLDH ctivity DHAR ctivity (lmol g FW min ) GR ctivity..44 ±..9 ±. 4.4 ±..4 ±.. ±.. ±.7.. d nd. ±. nd.7 ±. nd.6 ±.6.. d.96 ±. nd.8 ±. nd.94 ±. nd..7 ±..9 ±.. ±.. ±.4.8 ±..8 ±. nd mens not determined. Dt with similr letter represent sttisticlly homogenous group (ANOVA, P 6,). The results re the men vlues otined from four independent A 4. B... DHA (μmol g - FW) GSH (nmol g - FW) GSSG (nmol g - FW).... Fig.. The effect of the photosynthetic inhiitor DCMU on lef ntioxidnt contents. Plnts were grown for dys under the different light rtios nd then one tch of plnts ws treted with lm DCMU. Lef ntioxidnt contents were mesured dys lter. (A) AA/DHA contents nd (B) GSH/GSSG contents. Dshed rs represent the DCMU tretment. Asterisks denote the dt tht re sttisticlly different from the control (without DCMU) (ANOVA, P 6,). The results re the men vlues otined from four independent L-GlLDH, which is n integrl component of the respirtory electron trnsport chin, hd much lower ctivities in leves under the shde light environment (Tle ). These results suggest tht down-regultion of mitochondril metolism my e key process contriuting to the lower lef AA contents under the shde light regime. The ctivity of the photosynthetic electron trnsport chin lso influences AA ccumultion in Aridopsis leves []. However, the dt shown in Tle show tht photosynthesis is not involved in the cclimtion response to light rtio, s it ws similr under oth light qulity conditions. Net CO ssimiltion rtes, nd Tle 4 Effect of light rtio on the NAD(P)/NAD(P)H contents in common en leves. Plnts were cultivted under either sun (.) or shde (.) rtios t the sme PPFD ( lmol photon m s ) for dys. At the dy tch of plnts ws trnsferred from to. light rtio (.) nd nucleotides were mesured y the end of the photoperiod (i.e. h). rtio NAD NADH NADP NADPH (nmol g FW) 74. ± 7.6 ± 9 8. ± ± ± 4.7 ± 6.8 ± ± ± 4.7 ± 7. ± 8 6. ± Dt with similr letter represent sttisticlly homogenous groups (ANOVA, ETR nd qp vlues were similr in plnts grown under the sun nd shde conditions. The differences in lef AA content oserved under different light tretments re therefore not directly linked to photosynthesis. When plnts were treted with DCMU, n inhiitor of the electron trnsport chin for 48 h, photosynthesis ws inhiited (dt not shown) nd lef AA contents fell to similr vlues under oth light regimes (Fig. A). These results suggest tht process linked to photosynthesis is required to support high levels of lef AA ccumultion. Surprisingly, DHA content incresed under sun ut not under shde light environmentl conditions when photosynthesis ws inhiited. A cndidte process tht might e ffected y the previling rtio of the light environment is the regenertion of AA from DHA through the AA/GSH cycle s this is NADPH-dependent rection sequence. The dt in Fig. B show tht lef GSH contents re decresed when plnts were grown under shde rtio conditions. In ddition, the shde leves hve less DHAR nd GR ctivities suggesting tht the cpcity for AA regenertion from DHA is decresed when the rtio is low (Tle ). Furthermore, n smll GSH increse ws oserved in leves under shde rtio when ETR is locked (Fig. B). Under the sun rtio it is possile tht the flux of electrons to NADPH nd hence to AA regenertion is greter thn under the shde rtios. In support of this hypothesis, the totl lef pyridine pools re incresed nd the NADH/NAD nd NADPH/NADP rtios re higher under the sun conditions of high light rtios (Tle 4).

5 C.G. Brtoli et l. / FEBS Letters 8 (9) 8 4. Conclusions The dt presented here demonstrte tht the rtio of incident light modultes lef ntioxidnt contents. The lef AA pool (ut not lef GSH) ws chnged y the signlling system fter single photoperiod. We conclude tht shde cclimtion response medited y the signlling system hs mjor control over the extent of AA ccumultion in leves. The rpid cclimtion of the AA pool to the rtio of incident light nd the effects of the rtio on respirtion, L-GlLDH, DHAR nd GR ctivities suggest direct effects on AA synthesis s well the regenertion of AA from its oxidised forms. Tken together, these results suggest tht the incident rtio might ct s n lrm signl, conveying informtion relevnt to the possiility of n enhnced risk of photoinhiition nd oxidtive lod compred to tht experienced in shde environment. Finlly, tretments similr to those used here might e pplied not only to oost the content of lef ntioxidnts ut lso s posthrvest tretment to mintin or enhnce the ntioxidnt content of stored vegetles. Acknowledgements C.G.B. nd E.A.T. re reserchers of CONICET (Argentin). D.F. is fellowship student of CICBA (Argentin). This work ws supported y CONICET (Grnt PIP 97 to CGB). The uthors re grteful to Dr. Jun José Guimet from INFIVE (Universidd Ncionl de L Plt, Argentin) nd to Dr. Rodolfo Snchez from IFEVA (Universidd de Buenos Aires, Argentin) for the stimulting discussion of the results. References [] Bllré, C. (999) Keeping up with the neighours: phytochrome sensing nd other signlling mechnisms. Trends Plnt Sci. 4, 97. [] Vndenussche, F., Pierik, R., Millenr, F.F., Voesenek, L.A.C.J. nd Vn Der Streten, D. () Reching out of the shde. Curr. Opin. Plnt Biol. 8, [] Frnklin, K. nd Whitelm, G.C. () Phytochromes nd shde-voidnce. Responses Plnts. Ann. Bot. 96, [4] Foyer, C.H. nd Noctor, G. () Redox Homeostsis nd ntioxidnt signling: metolic interfce etween stress perception nd physiologicl responses. Plnt Cell 7, [] Arrigoni, O. nd De Tullio, M.C. () Ascoric cid: much more thn just n ntioxidnt. Biochim. Biophys. Act 69, 9. [6] Niyogi, K.K. () Sfety vlves for photosynthesis. Curr. Opin. Plnt Biol., [7] Asd, K. (999) The wter wter cycle in chloroplsts: scvenging of ctive oxygens nd dissiption of excess photons. Annu. Rev. Plnt Physiol. Plnt Mol. Biol., [8] Brtoli, C.G., Yu, J., Gómez, F., Fernández, L., McIntosh, L. nd Foyer, C.H. (6) Inter-reltionships etween light nd respirtion in the control of scoric cid synthesis nd ccumultion in Aridopsis thlin leves. J. Exp. Bot. 7, 6 6. [9] Brreiro, R., Guimet, J.J., Beltrno, J. nd Montldi, E.R. (99) Regultion of the photosynthetic cpcity of primry en leves y the red:fr-red rtio nd photosynthetic photon flux density of incident light. Physiol. Plnt. 8, 97. [] Inskeep, W. nd Bloom, P.R. (98) Extinction coefficient of chlorophylls nd in N,N-dimethylformmide nd 8% cetone. Plnt Physiol. 77, [] Griffith, O.W. (98) Determintion of glutthione nd glutthione disulfide using glutthione reductse nd -vinylpyridine. Anl. Biochem. 6, 7. [] Brtoli, C.G., Pstori, G. nd Foyer, C. () Ascorte iosynthesis in mitochondri is linked to the electron trnsport chin etween complexes III nd IV. Plnt Physiol., 4. [] Brtoli, C.G., Simontcchi, M., Tmussi, E.A., Beltrno, J., Montldi, E.R. nd Puntrulo, S. (999) Drought nd wtering-dependent oxidtive stress: effect on ntioxidnt content in Triticum estivum L leves. J. Exp. Bot., 7 8. [4] De Gr, L., Pcioll, C., De Tullio, M.C., Motto, M. nd Arrigoni, O. () Ascorte-dependent hydrogen peroxide detoxifiction nd scorte regenertion during germintion of highly productive mize hyrid: evidence of n improved detoxifiction mechnism ginst rective oxygen species. Physiol. Plnt. 9, 7. [] Quevl, G. nd Noctor, G. (7) A plte reder method for the mesurement of NAD, NADP, glutthione, nd scorte in tissue extrcts: Appliction to redox profiling during Aridopsis rosette development. Anl. Biochem. 6, [6] Rosenqvist, E. nd vn Kooten, O. () Chlorophyll fluorescence. generl description nd nomenclture in: Prcticl Applictions of Chlorophyll Fluorescence in Plnt Biology (DeEll, J.R. nd Toivonen, P.M.A., Eds.), Kluwer Acdemic Pulishers, Dordrecht, Netherlnds. [7] Smirnoff, N. () Ascorte iosynthesis nd function in photoprotection. Phil. Trns. Roy. Soc. Biol. Sci., [8] Goln, T., Müller-Moulé, P. nd Niyogi, K.K. (6) Photoprotection mutnts of Aridopsis thlin cclimte to high light y incresing photosynthesis nd specific ntioxidnts. Plnt Cell Environ. 9, [9] Millr, A.H., Mittov, V., Kiddle, G., Hezlewood, J.L., Brtoli, C.G., Theodoulou, F.L. nd Foyer, C.H. () Control of scorte synthesis y respirtion nd its implictions for stress responses. Plnt Physiol., [] Yut, Y., Mied, T., Rpolu, M., Nkmur, A., Motoki, T., Mrut, T., Yoshimur, K., Ishikw, T. nd Shigeok, S. (7) Light regultion of scorte iosynthesis is dependent on the photosynthetic electron trnsport chin ut independent of sugrs in Aridopsis. J. Exp. Bot. 8,