ferredoxin ferredoxin-nadpreductase photosynthetic ... · 1.18.1.2); n-terminal, amino-terminal;...

7
Plant Physiol. (1991) 96, 1207-121 3 0032-0889/91/96/1 207/07/$01 .00/0 Received for publication November 13, 1990 Accepted March 29, 1991 Ferredoxin and Ferredoxin-NADP Reductase from Photosynthetic and Nonphotosynthetic Tissues of Tomato1 Laura S. Green2, Boihon C. Yee, Bob B. Buchanan*, Kaeko Kamide, Yukika Sanada, and Keishiro Wada Department of Plant Biology, University of California, Berkeley, California 94720 (L.S.G., B.C. Y., B.B.B.), and Department of Biology, Faculty of Science, Kanazawa University, Marunouchi, Kanazawa 920 Japan (K.K., Y.S., K.W.) ABSTRACT Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and 11 were the most prevalent forms in leaves and green pericarp, and ferredoxin IlIl was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins 11 and IlIl plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins 1, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatog- raphy and by sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activ- ity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR 11 being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes. Ferredoxin, a small, low-potential iron-sulfur protein of chloroplasts, participates in a wide variety of photosynthetic reactions, including the reduction of NADP+, cyclic photo- phosphorylation, and the light regulation, via thioredoxin, of key enzymes of carbon dioxide assimilation. During photo- synthesis, electrons flow from PSI to ferredoxin and then to one of several acceptors, including NADP+, which is reduced via FNR3. Alternatively, ferredoxin may contribute its elec- trons directly to a variety of ferredoxin-dependent enzymes ' This study was supported by grants from the National Science Foundation to L.S.G. (Postdoctoral Fellowship in Plant Biology) and to B.B.B. (DCB 8815980), and by a grant from the Japanese Ministry of Education, Science and Culture to K.W. 2Present address: CSIRO, Division of Plant Industry, GPO Box 1600, Canberra, Australian Capital Territory 2601 Australia. 3Abbreviations: FNR, ferredoxin NADP+ oxidoreductase (EC 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higher plants investigated so far have at least two forms of chloroplast ferredoxin ( 13). The relative abundance of the isoforms is regulated both temporally and spatially during leaf development and in response to changes in light intensity during growth (9, 23). Recently, nonphotosynthetic tissues of higher plants have also been found to contain ferredoxin and FNR. Following their detection in corn roots (9, 20), and in a variety of other nonphotosynthetic tissues (references in ref. 15), these pro- teins were purified and characterized from roots of both radish (14, 25) and spinach (15) and from etiolated bean sprouts (6, 7). In these tissues, ferredoxin and FNR probably support ferredoxin-dependent biosynthetic processes, such as nitrogen assimilation, that take place in amyloplasts and proplastids (17, 24). It is not known, however, whether ferredoxin and FNR are required for the functioning of other types of non- green plastids. In this study, we demonstrate the presence of ferredoxin and FNR in pericarp of ripe tomato fruit (Lyco- persicon esculentum), a chromoplast-containing tissue, and compare the proteins to their counterparts from leaf, green pericarp, and root. MATERIALS AND METHODS Plant Material Cherry-type tomatoes (Lycopersicon esculentum cv VFNT) were grown in pots in a greenhouse under sunlight. Fruit was harvested either at the mature green stage (mature green stages 1-3, unripe pericarp) or when fully red (ripe pericarp) (1 1). For roots, the plants were grown hydroponically in Hoagland solution in the greenhouse. Where indicated, full-size toma- toes (cv Momotaro) were grown in the field during the sum- mer of 1989. Ferredoxin Purification Plant material was washed thoroughly in cold distilled water and prepared as follows before homogenization: leaves were deveined; green and red fruit were cut open and the locule and seeds removed; and roots were cleaned of older, woody tissue. Each kg of tissue was combined with 1 L of buffer (see below) and homogenized in a Waring Blendor at medium speed for 1 min. The homogenization buffer was varied for different tissues to control the pH of the resulting extract. The buffer used for leaves was 100 mm Tris-HCl (pH 7.8) and for red and green pericarp, 75 mm Tris (pH unadjusted). 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Page 1: Ferredoxin Ferredoxin-NADPReductase Photosynthetic ... · 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higherplants investigated

Plant Physiol. (1991) 96, 1207-121 30032-0889/91/96/1 207/07/$01 .00/0

Received for publication November 13, 1990Accepted March 29, 1991

Ferredoxin and Ferredoxin-NADP Reductase fromPhotosynthetic and Nonphotosynthetic Tissues of Tomato1

Laura S. Green2, Boihon C. Yee, Bob B. Buchanan*, Kaeko Kamide, Yukika Sanada, and Keishiro WadaDepartment of Plant Biology, University of California, Berkeley, California 94720 (L.S.G., B.C.Y., B.B.B.),

and Department of Biology, Faculty of Science, Kanazawa University, Marunouchi,Kanazawa 920 Japan (K.K., Y.S., K.W.)

ABSTRACT

Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) werepurified from leaves, roots, and red and green pericarp of tomato(Lycopersicon esculentum, cv VFNT and cv Momotaro). Fourdifferent ferredoxins were identified on the basis of N-terminalamino acid sequence and charge. Ferredoxins I and 11 were themost prevalent forms in leaves and green pericarp, and ferredoxinIlIl was the most prevalent in roots. Red pericarp of the VFNT cvyielded variable amounts of ferredoxins 11 and IlIl plus a uniqueform, ferredoxin IV. Red pericarp of the Momotaro cv containedferredoxins 1, II, and IV. This represents the first demonstrationof ferredoxin in a chromoplast-containing tissue. There were nomajor differences among the tomato ferredoxins in absorptionspectrum or cytochrome c reduction activity. Two forms of FNRwere present in tomato as judged by anion exchange chromatog-raphy and by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. FNR II had a lower apparent relative molecular weight,a slightly altered absorption spectrum, and a lower specific activ-ity for cytochrome c reduction than FNR I. FNR II could be apartially degraded form of FNR I. The FNRs from the differenttissues of tomato plants all showed diaphorase activity, with FNR11 being more active than FNR I. The presence of ferredoxin andFNR in heterotrophic tissues of tomato is consistent with theexistence of a nonphotosynthetic ferredoxin/FNR redox pathwayto support the function of ferredoxin-dependent enzymes.

Ferredoxin, a small, low-potential iron-sulfur protein ofchloroplasts, participates in a wide variety of photosyntheticreactions, including the reduction of NADP+, cyclic photo-phosphorylation, and the light regulation, via thioredoxin, ofkey enzymes of carbon dioxide assimilation. During photo-synthesis, electrons flow from PSI to ferredoxin and then toone of several acceptors, including NADP+, which is reducedvia FNR3. Alternatively, ferredoxin may contribute its elec-trons directly to a variety of ferredoxin-dependent enzymes

' This study was supported by grants from the National ScienceFoundation to L.S.G. (Postdoctoral Fellowship in Plant Biology) andto B.B.B. (DCB 8815980), and by a grant from the Japanese Ministryof Education, Science and Culture to K.W.

2Present address: CSIRO, Division of Plant Industry, GPO Box1600, Canberra, Australian Capital Territory 2601 Australia.

3Abbreviations: FNR, ferredoxin NADP+ oxidoreductase (EC1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenolindophenol.

in chloroplasts (1). All higher plants investigated so far haveat least two forms of chloroplast ferredoxin ( 13). The relativeabundance of the isoforms is regulated both temporally andspatially during leaf development and in response to changesin light intensity during growth (9, 23).

Recently, nonphotosynthetic tissues of higher plants havealso been found to contain ferredoxin and FNR. Followingtheir detection in corn roots (9, 20), and in a variety of othernonphotosynthetic tissues (references in ref. 15), these pro-teins were purified and characterized from roots ofboth radish(14, 25) and spinach (15) and from etiolated bean sprouts (6,7). In these tissues, ferredoxin and FNR probably supportferredoxin-dependent biosynthetic processes, such as nitrogenassimilation, that take place in amyloplasts and proplastids(17, 24). It is not known, however, whether ferredoxin andFNR are required for the functioning of other types of non-green plastids. In this study, we demonstrate the presence offerredoxin and FNR in pericarp of ripe tomato fruit (Lyco-persicon esculentum), a chromoplast-containing tissue, andcompare the proteins to their counterparts from leaf, greenpericarp, and root.

MATERIALS AND METHODS

Plant Material

Cherry-type tomatoes (Lycopersicon esculentum cv VFNT)were grown in pots in a greenhouse under sunlight. Fruit washarvested either at the mature green stage (mature green stages1-3, unripe pericarp) or when fully red (ripe pericarp) (1 1).For roots, the plants were grown hydroponically in Hoaglandsolution in the greenhouse. Where indicated, full-size toma-toes (cv Momotaro) were grown in the field during the sum-mer of 1989.

Ferredoxin Purification

Plant material was washed thoroughly in cold distilled waterand prepared as follows before homogenization: leaves weredeveined; green and red fruit were cut open and the loculeand seeds removed; and roots were cleaned of older, woodytissue. Each kg of tissue was combined with 1 L of buffer (seebelow) and homogenized in a Waring Blendor at mediumspeed for 1 min. The homogenization buffer was varied fordifferent tissues to control the pH ofthe resulting extract. Thebuffer used for leaves was 100 mm Tris-HCl (pH 7.8) and forred and green pericarp, 75 mm Tris (pH unadjusted). To

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Page 2: Ferredoxin Ferredoxin-NADPReductase Photosynthetic ... · 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higherplants investigated

Plant Physiol. Vol. 96, 1991

control polyphenol formation, roots were homogenized in100 mM Tris-HCl/100 mm sodium borate (pH 8.0), 20 mMdiethyldithiocarbamate, 0.1% (w/v) fl-mercaptoethanol, and1.5% (w/v) PVP (15). All buffers also contained 2 mm benz-amidine-HCl and 2 mm e-amino-n-caproic acid. The crudeextract was filtered through four layers of nylon mesh andeither subjected to ammonium sulfate fractionation or tobatchwise adsorption on DEAE cellulose.

In preparations with an ammonium sulfate precipitationstep, the crude extract was first clarified by centrifugation at15,000g for 15 min. The 30 to 90% ammonium sulfatefraction was collected by centrifugation, resuspended in aminimum volume of 30 mm Tris-HCl (pH 7.8), and dialyzedextensively against the same buffer.

For batchwise adsorption to DEAE-cellulose, the crudeextract was diluted 1:1 with cold distilled water, combinedwith 10 mL of wet DEAE-cellulose (pre-equilibrated with 20mM Tris-HCl, pH 7.8) for each L of extract, and stirred for 1h. The DEAE was washed extensively with 20 mM Tris-HCl(pH 7.8) and poured into a column (5 x 10 cm), and proteinwas eluted with 0.55 M NaCl in 50 mM Tris-HCl (pH 7.8).The eluate was concentrated by dialysis against solid sucroseand then dialyzed for 24 h against 30 mM Tris-HCl, pH 7.8.

After either ammonium sulfate fractionation or batchwiseadsorption on DEAE-cellulose, samples were loaded onto aDE52 (Whatman) column (2.5 x 40 cm) that had beenequilibrated with 30 mm Tris-HCl (pH 7.8) and washed with200 mL of this buffer. The column was then eluted with alinear gradient (1000 mL total) of0 to 0.6 M NaCl in the samebuffer. Active fractions were pooled, concentrated, and sub-jected to gel filtration on a Sephadex G-50 column (2.5 x 95cm) equilibrated with 200 mm NaCl, 50 mm Tris-HCl, pH7.8. After concentration and dialysis, the ferredoxin samplewas brought to 60% saturation for ammonium sulfate, loadedonto a Phenyl-Sepharose CL-4B column (1.5 x 9 cm; Phar-macia), and eluted with a reverse linear gradient (140 mLtotal) of 60% to 0% saturation ammonium sulfate in 30 mmTris-HCl (pH 7.8). Final purification of ferredoxin wasachieved by fast protein liquid chromatography with a mono-Q HR 5/5 column (Pharmacia) equilibrated with 50 mm Tris-HCl (pH 8.0) and eluted with a 0.2 to 0.5 M NaCl gradient inthis buffer.

FNR Purification

FNR was obtained from the ferredoxin preparations andseparated from ferredoxin at the DE52 column step. Fractionscontaining FNR activity, which eluted at approximately 0.2M NaCl, were pooled, concentrated, and subjected to gelfiltration on a Sephadex G-100 column (Pharmacia) equili-brated with 200 mm NaCl in 50 mm Tris-HCl (pH 7.8). TheFNR was next loaded onto a Red Matrex column (Bio-Rad,1.5 x 6 cm) and eluted with a linear gradient (200 mL total)of 0 to 0.5 M NaCl in 30 mm Tris-HCl, pH 7.8. Finalpurification was achieved by affinity chromatography on a

column (1 x 8 cm) of Chlamydomonas reinhardtii ferredoxinbound to cyanogen bromide-activated Sepharose (Pharmacia)as described by Huppe et al. (8).

Ferredoxin/FNR Assays

Ferredoxin was assayed by its ability to reduce horse heartCyt c (type VI; Sigma) in the presence ofNADPH and excessspinach leafFNR (12). FNR was assayed by the same methodexcept that FNR was omitted from the reaction mixture andwas replaced with excess spinach leaf ferredoxin. The reactionmixture contained, in a final volume of 1 mL, the sample tobe tested, 39 nmol Cyt c, 250 nmol NADPH, 50 ,mol Tris-HCl (pH 7.8), and either 2 nmol spinach leafferredoxin (FNRassays) or 0.1 nmol spinach leafFNR (ferredoxin assays). Thereduction of Cyt c was monitored by the increase in A550. Anextinction coefficient of 19.1 mm-' cm-' was used to calculatereduction rates.The diaphorase activity of FNR was assayed by following

the reduction of DCPIP essentially as described by Masaki etal. (12). The reaction mixture contained, in a final volume of1 mL, the sample to be tested, 50 ,mol Tris-HCl (pH 7.5),125 nmol NADPH, and 60 nmol of DCPIP. The reductionof DCPIP was followed by the decrease in A645, using anextinction coefficient of 22 mm-' cm-'. One unit of activityrepresents 1 ,umol of either Cyt c or DCPIP reduced per min.

Analytical Techniques

SDS-PAGE was carried out using the buffer system ofLaemmli (10), and proteins were visualized by staining withCoomassie brilliant blue G-250. Nondenaturing gel electro-phoresis was carried out using the pH 8 buffer system ofWilliams and Reisfeld (26). In these gels, ferredoxin wasidentified by its A420. For immunological analysis, the proteinwas transferred electrophoretically (22) to 0.1 ,um nitrocellu-lose (Schleicher and Schull) and incubated with rabbit anti-bodies raised against purified spinach leaf ferredoxin or FNR(both a gift of Professor R. Malkin). Bound antibody wasvisualized with either horseradish peroxidase or alkaline phos-phatase conjugated to goat anti-rabbit IgG, according to themanufacturer's instructions (Bio-Rad).

Protein concentration was determined by the method ofBradford (3) using a commercial kit (Bio-Rad) and bovinegamma globulin as the protein standard. Spectra were meas-ured with a Cary 214 recording spectrophotometer (Varian).

Protein Sequencing

For N-terminal amino acid sequence determinations, pu-rified ferredoxins were dialyzed exhaustively against distilledwater. Approximately 100 pmol (1.2 ,ug) of ferredoxin wereused for each determination. Sequences were determinedusing an Applied Biosystems Sequenator (Foster City, CA) atthe Cancer Research Laboratory of University of Californiaat Berkeley or at the Cancer Research Institute of KanazawaUniversity.

RESULTS

Ferredoxin Identification and Purification

Ferredoxin was found in all tomato tissues examined andbehaved similarly during purification to its counterpart fromother sources. In each case, tomato ferredoxin fractionated as

1 208 GREEN ET AL.

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Page 3: Ferredoxin Ferredoxin-NADPReductase Photosynthetic ... · 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higherplants investigated

TOMATO FERREDOXIN AND FNR

a single species up to the mono-Q chromatography step, whenactivity typically separated into two or more peaks, eacheluting at a different salt concentration (Fig. 1). The ferredoxinactivity from tomato leaves separated into two peaks, themajor form (ferredoxin I) eluting at a slightly higher saltconcentration than the minor form (ferredoxin II). The fer-redoxin activity of green pericarp was also resolved into twopeaks, eluting at the same salt concentrations as the leafforms.On the basis of activity, the yield of ferredoxin I was abouttwice that offerredoxin II from either leaves or green pericarp.The two forms of ferredoxin could also be distinguished bydifferences in their mobility on SDS-PAGE (Fig. 2); ferre-doxin I migrated as a broad band at a position near that ofpure spinach leaf ferredoxin (apparent Mr 18,000), whileferredoxin II ran as a tight band with an apparent Mr of 9,000.The smearing of ferredoxin I on denaturing gels probablyresulted from an anomalous interaction of the protein withSDS, as has been observed before with leaf ferredoxin (28).The purified tomato ferredoxin I ran as a single tight band onnative gels (data not shown). The ferredoxin purified fromtomato roots eluted as single broad peak from the mono-Qcolumn (Fig. 1) and ran as a highly diffuse band in SDS-PAGE (Fig. 2). On the basis of these distinguishing character-istics, the root protein was designated ferredoxin III.The number of different forms of ferredoxin obtained from

ripe pericarp was more variable, especially for the VFNT cv.

8LEAVES j 0.7

6 0.5

4ofE ~~~~~~0.3

0.1Q- 0) GREEN PERICARPa)4 - 0.4O0E2 O.2C-47 ROOTS

MII 0.60

0~~~~

-b ~~~~~0.42 0.2

0 R5 10 15 20

Fraction NO.Figure 1. Profile of cherry tomato ferredoxins (cv VFNT) on mono-Qchromatography. Roman numerals indicate activity peaks corre-sponding to ferredoxins 1,1II, III, and IV. Data derived from preparationsinvolving ammonium sulfate fractionation (leaves, green pericarp,roots) or batchwise adsorption to DEAE cellulose (ripe pericarp).

TOMATOLEAF

k D Stds LEAF I H

GREENPERICARP ROOT

III I

RIPEPERICARP

II IV

68- __

43- loonow

17---12 -

6.5-

Figure 2. SDS-PAGE of cherry tomato ferredoxins (cv VFNT) stainedwith Coomassie brilliant blue G-250. Each lane contains 6 to 8 mg ofthe indicated protein. Molecular mass standards are as follows: BSA(68 kD), ovalbumin (43 kD), myoglobin (17 kD), Cyt c (12 kD), andbovine trypsin inhibitor (6.5 kD). Data derived from preparationsinvolving ammonium sulfate fractionation.

Here the activity generally eluted in two or three peaks fromthe mono-Q column, but the peak positions and the relativeabundance of the different forms changed from one prepara-tion to the next. Of four separate VFNT ripe pericarp prepa-rations, all contained ferredoxins that comigrated in SDS-PAGE with ferredoxin I (designated ferredoxin IV because ofdifferences in amino acid sequence-see below) and ferre-doxin II (Fig. 2). In addition to these two forms, somepreparations yielded a root-type ferredoxin and a very minorspecies that appeared to be unique (data not shown); neitherofthese forms was sequenced. The pattern with the Momotarocv was, by contrast, more reproducible, as forms I, II, and IVwere consistently observed in native gel electrophoresis ofpartially purified extracts from ripe pericarp (Fig. 3). Interest-ingly, ferredoxin IV, which was observed as a minor form ingreen fruit of the Momotaro variety, became the major formin fully ripened fruit. The root-type ferredoxin, form III, wasnot observed in ripe fruit of the Momotaro cv.

Despite the heterogeneity displayed during anion-exchangechromatography and SDS-PAGE, the different tomato ferre-doxins were indistinguishable by most other criteria. With theexception of some root and red fruit preparations whoseabsorption was masked by UV-absorbing, nonprotein mate-rial (probably polyphenols), all ferredoxins purified from to-mato tissues had absorption spectra and A420/A276 ratios sim-ilar to those of spinach leaf ferredoxin (Table I). Likewise, alltomato ferredoxins tested cross-reacted on Western blots withan antibody raised against spinach leaf ferredoxin (data notshown). In the Cyt c reduction assay carried out with spinachleaf FNR, the purified tomato ferredoxins typically had spe-cific activities comparable with that of spinach leaf ferredoxin(Table I). The exception was the root-type ferredoxin, formIII, which often displayed a higher specific activity than theother forms.The yield of ferredoxin from tomato tissues other than

1 209

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Page 4: Ferredoxin Ferredoxin-NADPReductase Photosynthetic ... · 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higherplants investigated

Plant Physiol. Vol. 96, 1991

adi qei l.-eirr hr: F 1FtI Ii

* - e -1 i t C ( t;X r|. ,tF

leaves was very low. From each kg fresh weight of startingmaterial, leaves yielded an average of 23 mg of ferredoxin(the sum of both ferredoxins I and II, as determined byBradford analysis), whereas the corresponding values for greenpericarp, red pericarp, and root were 0.14, 0.07, and 0.05 mg,respectively.

Protein Sequences

To determine whether the heterogeneity displayed by thetomato ferredoxins resulted from differences in primary struc-ture or from post-translational modification, the N-terminalsequence was determined for each of the major forms. Figure4 shows the sequences for cv Momotaro. Ferredoxin I yieldedthe same sequence whether purified from leaves or greenpericarp. Likewise, the tissue of origin did not affect thesequence of ferredoxin II; the protein yielded the same se-quence whether purified from leaves, green pericarp, or redpericarp. Ferredoxins I and II differed at positions 2, 14, 17,23, 27, and 30. Ferredoxin IV, a red fruit form that resembledferredoxin I in SDS-PAGE analysis (Fig. 2), differed fromferredoxin I in its N-terminal sequence (Fig. 4). FerredoxinIV shared with forms I and II the amino acids invariant inmost other ferredoxins (Fig. 4, boxed positions 3-5, 10, 12)and also showed sequence identity with the other two tomatoferredoxins at positions 1, 6 to 9, 15, 16, and 18 to 22(indicated by boldface type). However, ferredoxin IV hadsubstitutions at two positions that were conserved betweenferredoxins I and II (positions 11 and 13) and, in addition,differed from ferredoxin I at positions 2, 14, and 17 and fromferredoxin II at positions 14, 23, 27, and 30. On the basis of

Table I. Characteristics of Ferredoxins from Cherry Tomato (cvVFNT)The specific activities represent the average values from two

preparations except where indicated. The spectral ratios given aretypical for a given preparation. Data are pooled from preparationsinvolving ammonium sulfate fractionation and batchwise adsorptionto DEAE cellulose.

Ferredoxin Source Type A420/A276 Specific Activity

units mg'Spinach leaf 0.48 1.5Tomato

Leaf 0.56 3.611 0.57 2.7

Green pericarp 1 0.55 2.711 0.56 2.4

Ripe pericarp 11 0.38 1.5aIII 0.31 17.1aIV 0.40 3.7a

Root IlIl 0.097b 16.9a Values are for individual preparations. b Preparation was ho-

mogeneous in SDS-PAGE but contained UV-absorbing material, prob-ably polyphenols.

the sequence data obtained so far, ferredoxins I, II, and IVseem to be equally distinct from one another. It should benoted that although ferredoxin IV ran as a doublet in SDS-PAGE (Fig. 2), it yielded a single N-terminal sequence. VFNTferredoxins were also sequenced and were quite similar tothose obtained from cv Momotaro, differing only at position9 (which was P for VFNT) and possibly position 11 (whichcould not be determined) of ferredoxin IV (Fig. 4). No mean-ingful sequence data were obtained for the root-type ferre-doxin III.

FNR Purification

FNR was present in all tissues examined (cv VFNT). Inpreparations from both green and ripe pericarp, the FNR

L A S Y K V K

I A T Y K V K

IV: A T Y K V K

10

L I T E G P

L I T P E G P

L I TLP S G A

I E F E C P

3DD

F E F D C P D

V E F D C P D

30

D V Y I L D Q A E E

D V S I L D R A E T

D V Y I L D Q A E E

Figure 4. N-terminal amino acid sequences for tomato ferredoxinsfrom cv Momotaro. The sequences were identical in cv VFNT exceptfor ferredoxin IV, which differed at position 9 (P for cv VFNT) andpossibly position 11 (could not be determined). Boxes indicate aminoacids conserved in most plant ferredoxins sequenced so far. Boldletters indicate additional amino acids conserved in all three tomatoferredoxin sequences. Data pooled from preparations involving am-monium sulfate fractionation and batchwise adsorption to DEAEcellulose.

GREEN ET AL.1210

1! ji!

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Page 5: Ferredoxin Ferredoxin-NADPReductase Photosynthetic ... · 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higherplants investigated

TOMATO FERREDOXIN AND FNR

activity split into two peaks on the DE52 column (Fig. 5),whereas leaf and root preparations yielded only a single peakof activity. When two peaks were present, the peak eluting atthe lower salt concentration was designated FNR I and thesecond peak was designated FNR II. On the basis of activity,the ratio ofFNR I/FNR II on the DE52 column was 1.9 and3.7 for green and ripe pericarp, respectively. The peaks werepurified separately through subsequent fractionation steps.The specific activities of the purified tomato FNRs were

generally comparable with that of spinach leaf FNR (TableII). The yield of FNR from tomato leaf averaged 13.8 mgenzyme per kg (initial fresh weight). In contrast, the yieldsfrom green and ripe pericarp (form I and II combined) weremuch lower; 0.05 and 0.02 mg, respectively, per kg of startingmaterial. Tomato root yielded about 1.0 mg FNR per kg offresh weight, a large amount considering the low amount offerredoxin recovered from this tissue. The mg of ferredoxinrecovered per mg of FNR from leaves, green pericarp, redpericarp, and root was 1.7, 2.7, 4.2, and 0.047, respectively.When analyzed by SDS-PAGE, FNR I from both green

and ripe pericarp migrated with an apparent Mr of 34,700,whereas FNR II from these tissues had an apparent Mr of30,900 (Fig. 6). Leaf FNR comigrated with FNR II and rootFNR with FNR I. FNR I (from roots) showed an absorptionspectrum typical of higher plant FNRs with peaks at 275,385, and 455 nm and a shoulder at 480 nm (data not shown).The absorption spectrum ofFNR II (from leaves) was similarexcept that the A385 peak was shifted to A375, suggesting thatthe structure ofthe protein was slightly aberrant. The leafandroot FNRs had A455/A275 ratios of 0.11 and 0.14, respectively.The FNRs from green and ripe pericarp were contaminatedwith UV-absorbing, nonprotein material that partially ob-scured the absorbance pattern of the flavin. These prepara-

5

4

3~

EC-0

C\j

2

0

!I

LEAVES-

A280nm:.................

FNR '- NoCI- Activity

40 ,,-80---120..

*- -;-'---40 80 120

15

10

5

Table II. Catalytic Activities of Cherry Tomato (cv VFNT)Ferredoxin-NADP+ Reductases

Data derived from preparations involving ammonium sulfate frac-tionation.

Specific ActivityFNR Sample

Cyt c reduction" Diaphoraseb

units mg-'Spinach

Leaf 16.4 176Tomato

Leaf II 13.2 184Green pericarp 1 19.4 100

I1 10.5 435Ripe pericarp 1 47.0 160

11 21.8 475Root 25.0 130

a Values are the average of three determinations. b Values arethe average of two determinations except for the spinach leaf en-zyme, which is the average of four determinations.

tions nevertheless had specific activities comparable with theroot and leaf enzymes and migrated as single bands duringSDS-PAGE.

All of the tomato FNR preparations showed diaphoraseactivity (Table II). In general, FNR II showed higher diaphor-ase activity relative to Cyt c reduction than did FNR I.

DISCUSSION

The present results demonstrate that tomato plants containfour different forms of ferredoxin. The forms could be distin-

- 0.7

-0.5 2._.

-0.3 0

- 0.1 Figure 5. Profile of cherry tomato ferredoxin-NADP+ reductases (cv VFNT) on DE52 chro-matography. Roman numerals indicate activitypeaks corresponding to FNRs and II. Dataderived from preparations involving ammoniumsulfate fractionations.

1 0.7

1 0.52._.

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Fraction No.

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Page 6: Ferredoxin Ferredoxin-NADPReductase Photosynthetic ... · 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higherplants investigated

Plant Physiol. Vol. 96, 1991

guished by differences in primary structure, as evidenced byN-terminal sequences, and in charge, as evidenced by behav-ior during anion exchange chromatography and native gelelectrophoresis. The abundance of the different forms variedfrom tissue to tissue, with ferredoxins I and II being mostprevalent in leaves and green pericarp, and ferredoxin III inroots. Ripe pericarp of VFNT tomato yielded variableamounts of ferredoxins II and III and an additional form,ferredoxin IV, that was not detected in the other tissues. Theexistence of a novel ripe fruit ferredoxin was also observed inthe Momotaro cultivar, confirming that a specific ferredoxinoccurs in chromoplast-containing tissues. Other than one ortwo discrepancies (at position 9 and possibly position 11 ofform IV) that may reflect cultivar differences, the ferredoxinsof the two cultivars showed identical N-terminal amino acidsequences. The occurrence of proline at position 9 of VFNTferredoxin IV is unusual in that this position is generallyoccupied by threonine. The only other known exceptionsamong higher plants are two radish root ferredoxins withglycine at position 9 (25). The differences in the apparentabundance of ferredoxins I and III in ripe Momotaro fruit as

compared with VFNT may also result from cv differences orfrom variation in the ripening of small versus full-sized fruits.The yield of ferredoxin from the nonphotosynthetic tissues oftomato (roots and ripe pericarp) was less than 1% of thatobtained from tomato leaves.As found for other higher plants, the ferredoxin isoforms

of tomato were indistinguishable on the basis of absorptionspectra and activity. Other investigators have likewise beenunable to demonstrate a difference between ferredoxin iso-forms in their interaction with PSI (reduction of NADP+) or

glutamate synthase (25). It is unclear, then, why differentforms of ferredoxin are made or why their abundance variesfrom tissue to tissue. One possibility is that there are func-

tional differences between the ferredoxins that are not de-tected by the in vitro assays used to date. Alternatively, thedifferent proteins present may reflect the mechanism by whichplants control the amount of ferredoxin present in a giventissue. Although it is possible that the ferredoxin isoformscould be generated by differential splicing of a single gene,genomic ferredoxin sequences obtained so far imply that thedifferent proteins are encoded by separate genes (4) (T. Hase,personal communication). If separate ferredoxin genes areused to provide for tissue-specific levels of expression, theaccumulation of neutral mutations over evolutionary timewould result in tissue-specific isoforms that are not necessarilyfunctionally different (9, 25). The basis for the formation oftissue-specific forms of plant ferredoxin thus remains open.FNR was also purified, in the current study, from leaves,

roots, and green and red pericarp of tomato. Two forms werepresent as judged by anion exchange chromatography andSDS-PAGE. FNR II had a lower apparent Mr, a slightly alteredabsorption spectrum, and a lower specific activity for Cyt creduction than FNR I. Such differences suggest that FNR IIcould be a partially degraded form ofthe native enzyme, FNRI. FNR II, on the other hand, had a somewhat higher dia-phorase activity than FNR I. Limited proteolysis of FNR,resulting in physically altered but still fully functional forms,has been reported for spinach leaf preparations (5). Themolecular weight of FNR I is very close to that previouslyreported for unpurified tomato FNR (18). In contrast to thenonphotosynthetic FNR isolated from etiolated bean sprouts(6), all of the FNRs from green and non-green tissues oftomato plants showed significant diaphorase activity.The yield of FNR from ripe pericarp was especially low-

about 0.1% that from leaves. The low apparent abundance ofFNR in ripe pericarp may explain why it was not detected ina previous study of tomato ripening (18). The yield of FNRobtained from tomato roots, on the other hand, was highrelative to the amount offerredoxin recovered from this tissue.This phenomenon has also been observed for roots of radishand spinach (14, 15). Whether the high yield of FNR fromroots is of physiological significance is not known.The presence ofboth FNR and ferredoxin in nonphotosyn-

thetic tissues of tomato is consistent with the existence of aredox pathway (consisting ofNADPH, FNR, and ferredoxin)that supplies reduced ferredoxin to ferredoxin-dependent en-zymes. In cyanobacteria, such a pathway supplies the reducingequivalents needed for nitrogen fixation (21, 27) via a specificferredoxin (19). Ferredoxin-dependent nitrite reductase (EC1.7.7.1) and glutamate synthase (EC 1.4.7.1) have been de-scribed in tissues containing amyloplasts (2, 16), and there isrecent evidence for the occurrence of the latter enzyme inripe tomato fruit (F. Gallardo, F.R. Canton, A. Garcia-Gu-tierrez, F. Canovas, personal communication). It appears,therefore, that plant cells depend on ferredoxin and FNR forthe assimilation of inorganic nitrogen irrespective of the typeof plastid they contain.

ACKNOWLEDGMENTS

We thank Michael Moore for sequence determinations, FrankMurillo for preparing the figures, Bernie Tower for clerical assistance,and Prof. S. Migita for use of the sequenator at the Cancer ResearchInstitute of Kanazawa University.

1212 GREEN ET AL.

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Page 7: Ferredoxin Ferredoxin-NADPReductase Photosynthetic ... · 1.18.1.2); N-terminal, amino-terminal; DCPIP, 2,6-dichlorophenol indophenol. in chloroplasts (1). All higherplants investigated

TOMATO FERREDOXIN AND FNR

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