The Role of Cu in Respiration of Pea Plants and Heterotrophically Growing Scenedesmus Cells

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The Rle f Cu in Respiratin f Pea Plants and Hetertrphically Grwing Scenedesmus Cells Matilde Barón Ayala* and Gerhard Sandmann Lehrstuhl Physilgie und Bichemie der Pflanzen, Universität Knstanz,

1 The Rle f Cu in Respiratin f Pea Plants and Hetertrphically Grwing Scenedesmus Cells Matilde Barón Ayala* and Gerhard Sandmann Lehrstuhl Physilgie und Bichemie der Pflanzen, Universität Knstanz, D-7750 Knstanz, Bundesrepublik Deutschland Z. Naturfrsch. 43c, (1988); received December 7, 1987/February 10, 1988 C u Deficiency, Cytchrme Oxidase, Mitchndria, Pea Plants, Scenedesmus In Scenedesmus abut half f N A D H xidatin prceeds via a cyanide-sensitive and the ther half via a cyanide-insensitive respiratry pathway. In cntrast, respiratin is cmpletely cyanide sensitive in pea indicating that the alternative respiratry pathway is absent. C u deficiency in pea plants and in hetertrphically grwn Scenedesmus cells interferes with respiratry activity f mitchndria. In bth rganisms, the cyanide-sensitive N A D H xidatin was strngly decreased during cultivatin in lw C u media. C u sensitivity was als bserved fr the alternative respiratry pathway in Scenedesmus. These results suggest that a Cu-cntaining cmpnent is invlved in the alternative respiratry pathway. This is the main reasn why alternative respiratin cannt be regarded as a cmpensatin fr lw cytchrme-xidase activities during C u starvatin. The C u dependency f the cyanide-sensitive respiratin was lcated at the site f cytchrme xidase. A strng crdinatin f the bisynthesis f the Cu-cntaining cytchrme-xidase cmplex was evident. W h e n the endgenus C u pl was lw, frmatin f cytchrme aa3, anther cmpnent f cytchrme xidase, was als decreased. Intrductin Many redx reactins are catalyzed by enzymes which cntain redx-active metals as functinal grups [1]. Besides f Fe, Cu plays an essential rle in the electrn-transfer reactins f chlrplasts and mitchndria. In the latters, the prminent Cu-cntaining prtein is cytchrme xidase, the terminal xidase f the respiratry electrn-transprt chain [2]. It cntains the cytchrmes a, a3 and tw atms f cpper [3], Studies with yeast [4], sycamre cells [5], clver [6, 7] and a green alga [8] have shwn that Cu deficiency inhibits frmatin f cytchrme xidase. This decreased synthesis in general results in lw respiratin rates with a subsequent negative impact n the energy metablism. Similar Cu-deficiency effects n synthesis and functin f cytchrme xidase have als been bserved with animal mitchndria [9], In a recent publicatin, we have investigated the influence f Cu deficiency n phtsynthesis and respiratin in the green alga Dunaliella and shwn that bth prcesses are strngly affected [8]. In additin t direct effects f Cu deficiency n cytchrme * O n leave frm: stación xperimental del Zaidín, CSIC, Granada, Spain. Reprint requests t Dr. G. Sandmann. Verlag der Zeitschrift für Naturfrschung, D-7400 Tübingen /88/ S 01.30/0 xidatin, we fund an indirect ne due t inhibited phtsynthesis which supplies carbhydrates, a substrate fr the respiratry prcess. This dependency is characteristic fr auttrphic rganisms. Therefre, we extended ur studies n hetertrphic cultures f Scenedesmus and reprt here n the influence f Cu deficiency n respiratin f this green alga cultivated n exgenus sugar in the dark. Furthermre, Scenedesmus exhibits cyanide-insensitive respiratin [10]. It has been discussed that a Cu-cntaining redx cmpnent participates in this alternative pathway [11, 12]. If this is the case, Cu deficiency shuld als decrease cyanide-insensitive respiratin. The results n respiratry activity f Scenedesmus will be cmpared t data btained with strngly Cu-depleted pea plants. Materials and Methds Pea seeds (Pisum sativum, cv. Prgress) were germinated in mistened sand at 28 C fr ten days and then hydrpnically grwn in a Hewitt full nutrient slutin supplemented with different Cu 2 + cncentratins; 0.03 pm fr deficient plants and 1 pm fr the cntrl plants. The details have been described recently [13]. The plants dented as strngly Cu depleted riginated frm seeds prduced by Cu-deficient plants. Scenedesmus acutus (strain a, Algal Culture Cllectin, University f Göttingen) was grwn Dieses Werk wurde im Jahr 2013 vm Verlag Zeitschrift für Naturfrschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.v. digitalisiert und unter flgender Lizenz veröffentlicht: Creative Cmmns Namensnennung-Keine Bearbeitung 3.0 Deutschland Lizenz. This wrk has been digitalized and published in 2013 by Verlag Zeitschrift für Naturfrschung in cperatin with the Max Planck Sciety fr the Advancement f Science under a Creative Cmmns Attributin-NDerivs 3.0 Germany License. Zum ist eine Anpassung der Lizenzbedingungen (ntfall der Creative Cmmns Lizenzbedingung Keine Bearbeitung ) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsfrmen zu ermöglichen. On it is planned t change the License Cnditins (the remval f the Creative Cmmns License cnditin n derivative wrks ). This is t allw reuse in the area f future scientific usage.

M. Barn Ayala and G. Sandmann Rle f Cu in Respiratin 439 under hetertrphic cnditins in the dark. Grwth cnditins and cmpsitin f the sterile cultivatin medium were as described [14].

2 M. Barn Ayala and G. Sandmann Rle f Cu in Respiratin 439 under hetertrphic cnditins in the dark. Grwth cnditins and cmpsitin f the sterile cultivatin medium were as described [14]. The cmpsitin f the nutrient slutin was mdified by the additin f glucse (5 g/1), vitamin B I2 (30 nm), and ribflavin (42.5 pm). The cpper cntent f the standard medium was 10~ 6 M, and in the Cu-deficient cultures as indicated. The algae were harvested after a 8-day grwth perid. Cell mass f cultures was determined as packed cell vlume (pcv) in graduated micrcentrifuge tubes f 80 pi capacity. lectrn-transfer activities In vitr respiratry electrn-transprt activities were measured ut with washed mitchndria frm pea leaves and Scenedesmus cells. Pea mitchndria were islated as described by Duce et al. [15]. Scenedesmus mitchndria were btained accrding t Sharpless and Butw [16]. The cells were brken with glass beads (0.5 mm diameter) fr 30 sec at maximum speed with a Braun- Merckenschläger hmgenizer in an islatin medium cntaining 0.3 M srbitl, 0.5 MM DTA, and 25 mm HPS buffer, ph 7.0. Frm the resulting hmgenate, mitchndria were islated by differential centrifugatin. Fr determinatin f cytchrme aa 3, the mitchndria were further purified n a density gradient with three steps f 30%, 45%, and 55% (w/w) sucrse. Centrifugatin was fr 90 min at 100,000 x g in a swing-ut rtr. The mitchndrial fractins were cllected between the 45% and 55% sucrse steps. Respiratry electrn-transprt activities frm NADH t xygen were determined in an assay medium cntaining 1 mm NADH, 0.1 mm ADP and aliquts f the rganelle fractin in a reactin medium cntaining 0.3 M sucrse, 10 MM KCl, 5 MM MgCl 2, 10 mm KH 2 P0 4, and 10 mm HPS buffer, ph 7.2. Oxygen uptake was recrded with a Clarktype xygen electrde. Cytchrme-xidase activity was determined as KCN-sensitive xidatin f reduced hrse-heart cytchrme c-550 in a dublebeam spectrphtmeter at 550 nm, as previusly described [8]. The reactin mixture cntained 40 mm Tris-HCl buffer, ph 7.3, 1 mm DTA, 1% Tritn X-100, 20 pm reduced cytchrme c-550, and the rganelle preparatin. Determinatin f redx cmpnents The amunts f redx prteins were determined by ptical difference spectrscpy in a duble-beam spectrphtmeter. Reduced minus xidized difference spectra f cytchrme aa 3 were btained with mitchndria purified by sucrse-density centrifugatin. Fr recrding the spectra, the mitchndria were suspended in Tris-HCl buffer, ph 7.5 cntaining 1% Tritn X-100, and the sample reduced with dithinite leaving the reference xidized. Fr quantitatin f cytchrme aa 3, a differential extinctin cefficient ( nm) f 13 mm cm -1 was used. Assays fr ther parameters The Cu cntent f Scenedesmus cells and pea leaves were measured with a Varian atmic-absrptin spectrphtmeter (Md. A A 575) equipped with a carbn-rd atmizer. Befre use, the leaves were dried (12 h, 120 C), pwdered and suspended in 2 N HNO3. Intact Scenedesmus were suspended in 2 N HN0 3 and measured directly. Prtein cncentratin was determined by the methd f Bradfrd [17]. Results and Discussin The standard Cu cncentratin f the medium fr unrestricted frmatin f Cu-cntaining prteins in Scenedesmus is 1 pm [18]. A decrease f the Cu supply by '/t r Vw resulted in a strng depletin f the endgenus Cu accumulated by the cells (Fig. 1). In Scenedesmus the maximum Cu-sequestering capacity was almst 4-fld higher than in Dunaliella, anther green alga [8]. Hwever, when Cu is limiting the endgenus cncentratins tend t be similar. A direct effect f this Cu depletin n the respiratry capacity culd be bserved. In mitchndria islated frm Scenedesmus cells with an ptimum Cu supply the rate f respiratry NADH xidatin was highest and decreased with decreasing Cu cncentratins. The lwest respiratin values fr Scenedesmus grwn with n added Cu t the nutrient was abut 40% f the respiratin f mitchndria frm Cu-supplemented cntrl cells (Table I). Under these cnditins, grwth was abut 60%. In cntrast t higher plants [19], nly half f the respiratry activity was sensitive t cmparably high KCN cncentratins in Scenedesmus (Table I). This cntributin is due t the cytchrme-xidase reac-

3 M. Barn Ayala and G. Sandmann Rle f Cu in Respiratin tained after further purificatin f mitchndria n a sucrse gradient indicating that it is caused by an 1000 alternative branch f the respiratry pathway rather CL than by xidizing enzymes lcated n cntaminating \ micrsmal membranes. The cntributins f the 500 cyanide-sensitive cytchrme xidase and the c SHAM-sensitive alternative xidase n xygen-dependent NADH xidatin were equal in ScenedesRespi rati n.c mus cultures. This is a similar cntributin by the X alternative xidatin pathway as reprted fr heter t_ Q. trphic cultures f the alga uglena [16]. en Inhibitin f cytchrme xidatin by grwing O sycamre cells with cyanide [20] r by grwing yeast " i8 in lw Cu media [21] activated the alternative ressi i ï 3Í?ÍÍ5. ääis spiratry pathway in bth rganisms. Hwever, in :hrr ne C y l b e «Scenedesmus regardless whether the cells have been x Idaise grwn in high r lw Cu media bth respiratry path. e? 4000 ways are perating and bth pathways were inhibited \ _ by Cu deficiency t the same extent as the ttal Z 2000 respiratry activity. This result demnstrates that in Scenedesmus the alternative respiratin pathway is c unable t cmpensate fr inhibited cytchrme xi10"8 10"7 10"6 datin during Cu deprivatin. The Cu sensitivity f Cu c n c e n t r a t i n ^ ] cyanide-insensitive NADH xidatin indicates a parfig. 1. ndgenus C u cntent, respiratry N A D H xida- ticipatin f a Cu-cntaining cmpnent in this alternative pathway. A stepwise decrease f the Cu suptin and cytchrme-xidase activity in hetertrphically grwn Scenedesmus cells supplemented with different C u ply frm 10~6 M t 10-8 M by Vw resulted in ttal recncentratins. spiratin rates which were 20 t 30% lwer after each depletin step (Fig. 1). The same can be bserved fr the activity f cytchrme xidase which was parallely decreased t the same extent. Table I. N A D H - d e p e n d e n t respiratin f mitchndria Cu cntent islated frm Scenedesmus cells grwn under nrmal and Cu-deficient cnditins. Respiratin NADH 02 (nml/mg prt x h) untreated + K C N (0.5 mm) + S H A M (15 mm) N C u added 1 ÍM CU Cntributins f different respiratry pathways [%] CN~-sensitive SHAM-sensitive tin which can be ttally inhibited by cyanide. The ther part f NADH-dependent xygen uptake is sensitive t salicylhydrxamic acid (SHAM), an'inhibitr f an alternative terminal xidase [12]. This SHAM-sensitive respiratry xygen uptake is re- The degree f Cu depletin in pea plants is dependent n the Cu supplementatin f this medium and n the rigin f the seeds. Seeds frm Cu-deficient plants nly cntain V\ f the Cu which accumulates in seeds frm ptimal supplied plants [13]. Fr ur experiments we used a Cu supplementatin f 1 pm fr the cntrl plants and 0.03 pm fr Cu-depleted plants in rder t ensure the frmatin f a minimum amunt f plastcyanin which is necessary fr phtsynthesis and unrestricted plant grwth [13]. Accrding t the grwth cnditins, the leaf Cu-cntent varied between 10 and 35 nml/mg fresh weight (Table II). This resulted in a decrease f mitchndrial respiratry N A D H xidatin by 20% in the Cu-depleted pea plants with a leaf Cu-cntent f 19.9 nml/mg fresh weight and a decrease f 50% in case f strngly Cu-starved pea plants with a leaf Cu-cntent f Cytchrme-xidase activity measured with mitchndria frm the tw different

4 M. Barn Ayala and G. Sandmann Rle f Cu in Respiratin 441 Cu-deficient cultures was lwered almst t the same extent as the verall respiratin rate. In pea mitchndria nt nly the cytchrme-xidase reactin but als the respiratry electrn transfer frm NADH t xygen was cmpletely inhibited by KCN. In this respect, the mitchndria frm pea resemble the nes frm wheat in which n alternative respiratin was detected [19]. In pea mitchndria, the decrease f respiratry NADH xidatin during Cu deficiency culd be attributed t the inhibited cytchrme-xidase reactin in depleted and strngly depleted plants (Table II). Cmpared t the cntrl, the relative decrease f NADH xidatin and cytchrme xidatin was very similar. This Cu-dependency f the cytchrme-xidase reactin is typical fr many higher and lwer plants [4, 6 8]. Hwever, in Cu-depleted sycamre cells, xidative and phsphrylatin activities were similar t Cu-supplied cells [5]. Althugh cytchrme aa 3 -cntent was decreased, the higher turnver number f cytchrme xidase in Cu-deficient mitchndria cmpensated fr this lss. Cytchrme a cmpnent f the Cu-cntaining cytchrme-xidase cmplex, was determined in islated mitchndria frm Scenedesmus and pea plants (Table III). In bth rganisms the amunt f cytchrme aa 3 decreased cncurrently with the Cu supply. Apparently, the frmatin f the cytchrme-xidase cmplex is crdinated in pea and Scenedesmus. When single cmpnents such as Cu are missing, the synthesis f the ther cmpnents is als prevented. Therefre, we culd mnitr a de- Table III. Cntent f cytchrme aa 3 (nml/mg prt) in cntrl and Cu-deficient mitchndria f Scenedesmus and pea plants. Cu status f the rganism Scenedesmus Pisum sativum ptimal supplied depleted /0.13* * Frm pea cultures with strng Cu deficiency due t the use f Cu-depleted seeds. creased synthesis f the cytchrme-xidase cmplex by determinatin f the cytchrme aa 3 -cntent. ither fr Scenedesmus r pea we fund a very gd relatinship between the endgenus Cu in the cells and the cytchrme aa 3 -cntent. This resembles very much the significant crrelatin between endgenus Cu pls and the frmatin f plastcyanin, anther Cu-cntaining prtein in pea chlrplasts [13]. Fr bth rganisms, we bserved a strnger decrease f the cytchrme aa 3 -cntent than f respiratry NADH xidatin during Cu deprivatin. This was als reprted fr the green alga Dunaliella [8]. These results suggest that mitchndria f higher and lwer plants cntain a surplus f cytchrme aa 3 ver the ther respiratry cmpnents. This is in agreement with an accumulatin f cytchrme aa 3 ver b- r c-type cytchrmes in Candida [22] and the cnclusin drawn by Ducet and Rsenberg [23] that cytchrme aa 3 is in excess in plant mitchndria. Table II. Cu cntent and respiratin f mitchndria islated frm pea plants grwn with high and lw Cu supply. Cntrl Cu depleted Strngly Cu depleted* Cu cntent f leaves (nml/g fresh weight) Respiratin with NADH as substrate** (pml 0 2/mg prt x h) Cytchrme-xidase activity (pml cyt/mg prt x h) * These plants were grwn frm Cu-depleted seeds in media cntaining 0.03 pm Cu 2+. ** The reactin is cmpletely inhibited by 0.5 mm KCN.

5 442 Amng the Cu-dependent reactins, respiratin is ne f the mst imprtant targets fr Cu deficiency. Therefre, in higher r lwer plants Cu-limited grwth has a direct impact n the energy metablism. Mst likely, plant mitchndria pssess a certain excess f Cu-cntaining cytchrme xidase in rder t alleviate temprary Cu shrtages. Algae are able t perate an alternative respiratry pathway. Hwever, this pathway cannt take ver when cytchrme-xidase activity is decreased in lw Cu M. Barn Ayala and G. Sandmann Rle f Cu in Respiratin 442 mitchndria because the alternative respiratry pathway itself is sensitive t Cu deprivatin, t the same extent as cytchrme xidase. Acknwledgements This wrk was supprted by a grant frm the Spanish ducatin and Science Ministry t M. B. A. We thank Prf. P. Böger fr his interest in the curse f this wrk and Mrs. S. Kuhn fr excellent technical assistance. [1] G. Sandmann and P. Böger, in: ncyclpedia f Plant Physilgy, Vl. 15B, Inrganic Plant Nutritin (A. Läuchli and R. L. Bieleski, eds.), pp , Springer Verlag, Berlin [2] C. D. Walker and J. Webb, in: Cpper in Sils and Plants (J. F. Lneragan, A. D. Rbsn, and R. D. Graham, eds.), pp , Academic Press, Sydney [3] B. T. Strey, in: The Bichemistry f Plants (P. K. Stumpf and.. Cnn, eds.), Vl. 2, pp , Academic Press, New Yrk [4]. Keyhani and B. Chance, FBS Lett. 17, (1971). [5] B. Bligny and R. Duce, Plant Physil. 60, (1977). [6] C. D. Walker and J. F. Lneragan, Ann. Bt. 47, (1981). [7] J. M. Hill, J. xp. Bt. 24, (1973). [8] G. Sandmann, Physil. Plant. 65, (1985). [9] C. B. Lawrence, N. T. Davies, C. F. Mills, and F. Nicl, Bichim. Biphys. Acta 809, (1985). [10] S. Scherer,. Stürzl, and P. Böger, Physil. Plant. 60, (1984). [11] C. Lance, M. Chauveau, and P. Dizengremel, in: Higher Plant Cell Respiratin (R. Duce and D. A. Day, eds.), pp , Springer Verlag, Berlin [12] J. N. Siedw and D. A. Berthld, Physil. Plant. 66, (1986). [13] M. Barn Ayala and G. Sandmann, Physil. Plant., in press (1988). [14] G. Sandmann and P. Böger, Phtsynth. Res. 2, (1981). [15] R. Duce, A. L. Mre, and M. Neuburger, Plant Physil. 60, (1977). [16] T. K. Sharpless and R. A. Butw, J. Bil. Chem. 245, (1970). [17] M. M. Bradfrd, Anal. Bichem. 77, (1976). [18] G. Sandmann and P. Böger, Z. Pflanzenphysil. 98, (1980). [19] A. H. Gldstein, J. O. Andersn and R. G. McDaniel, Plant Physil. 67, (1981). [20] J. P. Blein, Plant Sei. Lett. 19, (1980). [21] J. A. Dwnie and P. B. Garland, Bichem. J. 134, (1973). [22]. Keyhani, Bichem. Biphys. Res. Cmmun. 93, (1980). [23] G. Ducet and A. J. Rsenberg, Annu. Rev. Plant Physil. 13, (1962).

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