Properties of Synthesis of Chlorophyll a from Chlorophyll b in ...

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biolom, Inc

Vol. 269, No. 35, Issue of September 2, pp. 22034-22038, 1994 Printed in U.S.A.

Properties of Synthesis of Chlorophyll a from Chlorophyll b in Cucumber Etioplasts* (Received for publication, April 28, 1994, and in revised form, June 20, 1994)

Hisashi ItoS, Shinichi Takaichis, Hideo Tsujiq, and Ayumi Tanaka From the Department of Botany, Faculty of Science, Kyoto University, Kyoto 606-01,J a p a n a n dthe $Biological Laboratory, Nippon Medical School, Kawasaki 211, Japan

Chlorophyll a accumulatedwhenchlorophyllide b was incubated withisolated cucumber etioplasts in the dark. When ['4Clchlorophyllide b was used as the substrate for chlorophyll synthesis, ['4C]chlorophylla was formed,showingthatchlorophyll a wassynthesized from the exogenously added chlorophyllide b, not by light-independent reduction of endogenous protochlorophyllide. The reaction studied showed an optimal pH of 7.5 and required both the soluble and membrane fractions of etioplasts together with ATP. Time course experiments showed that chlorophyll a began to accumulate later thanchlorophyll b. Chlorophyllide b esterified with geranylgeraniol accumulatedfirst, but there was little accumulation of chlorophyll a with an unhydrogenated prenyl side chain. No chlorophyllidea was detected during incubation. These observations indicate that esterified chlorophyll b was convertedto chlorophyll a. This conversion would play an important rolein the use of chlorophyllb for thesynthesis of chlorophyll a in the reconstruction of photosystems. Higher plants and green algae have chlorophyll (Chl)' b as an accessory light-harvesting pigment. Chl a a n d b differ only i n the presence of a methyl group at pyrrole ring I1 in Chl a a n d that of an aldehyde group in Chl b. Chl b is thought to be synthesized bythe oxidation of this methyl group with 0, to an aldehyde group (1, 2). Despite the lack of direct experimental evidence, it has been generally accepted that Chl b or chlorophyllide (Chlide) b is biosynthetically derived from Chl(ide) a (3). The recent discovery of protochlorophyllide (Pchlide) b (4) suggests the conversion of Pchlide a to Pchlide b. In any case, of the methyl group of pyrrole Chl b is synthesized by oxidation ring I1 of Chl a or its precursors, although it is not clear at which step of the chlorophyll biosynthetic pathway the oxidation takes place.The conversion of Chl b to Chl a has not been thought to occur.

* This work was supported by Grant-in-aid 06259208 for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom correspondenceshould be addressed: Dept. of Botany, Faculty of Science, Kyoto University, Kyoto 606-01 Japan. Tel. 81-75753-4246; Fax: 81-75-753-4122. 1 Present address: Dept. of Botany, Kobe Women's University, Kobe 654, Japan. The abbreviations used are: Chl, chlorophyll;Chlide, chlorophyllide; Pchlide, protochlorophyllide; HPLC, high performance liquid chromatography; GG, geranylgeraniol;DHGG, dihydrogeranylgeraniol;THGG, tetrahydrogeranylgeraniol;Ph, phytol; GGPP, geranylgeranyl pyrophosphate; LHCII, Chl alb-protein complexof photosystem 11; CP1, P700-Chl a-protein complex; CPa, Chl a-protein complexes of photosystem 11.

Kupke and Huntington (5) reported that Chl a increased accompanied by a parallel decrease inChl b during dark incubation after a short period of illumination. The same results with rye ( 6 ) ,cucumber (71, and were reported by other workers tobacco (8). If Chl had been precisely determined and dark synthesis of Chl had not occurred, these results would suggest t h e conversion of Chl b to Chla. In spite of these observations, Chl b has not been considered tobe converted to Chla because the formyl groupof Chl b is known to be difficult to reducea to methyl group. However, in a previous study (9), we observed that Chl a accumulated when Chlide b was incubated with isolated cucumber etioplasts, indicating the conversion of Chl b to Chl a. In this study, we confirmedourpreviousresultsusing [l4C1Chl a and showed that both the membrane and soluble fraction are required forthe reaction. MATERIALSANDMETHODS Isolation of Etioplasts-Cucumber seeds (Cucumis satiuus L.cv. Aonagajibai) were soaked for about 3 h and germinated on moist vermiculite in the dark at 28 "C. About 400 etiolated cotyledons were cut from 6-day-oldseedlings and homogenized with a Waring blender in 80 ml of a buffer containing 50 mM Hepes-NaOH (pH 8.0), 0.5 M sorbitol, 5 mM cysteine, 2 mM EDTA, and 0.2% bovineserum albumin. The homogenate was filtered through a nylon mesh and centrifuged at 200 x g for 2 min. The supernatant was recentrifuged at 2,000 x g for 7 min. The resultant pellet (etioplast) was used for in organelle assay.All the procedures were done under a dim green safelight to prevent the reduction of Pchlide. Etioplast Lysis and Fractionation-The etioplast pellet was resuspended in an incubation buffer containing 50 mM Hepes-KOH (pH 7.51, 10 mM MgCl,, 10 mM ATP and was centrifuged at 10,000 x g for 10min. The supernatant (stroma) and pellet (membranes) were used. Preparation ofChl b-Chl b was prepared from spinach according to Omata and Murata (10) with slight modifications as described previously (9). Preparation of Chlide b-Thirty g of garland chrysanthemum leaves purchased at a local market werehomogenized in 300mlof 100% acetone with a Waring blender and centrifuged at 4,000 x g for 10 min. The pellet was homogenized completely with a Potter-Elvehjem-type glass homogenizer in 100%acetone and centrifuged at 10,000 x g for 10 min. The pellet was washed with 100% acetone and centrifuged until Chl had been completely removed.The resultant pellet was dried under reduced pressure to obtain acetone powder. A 0.5-g aliquot of the acetone powder was stirred in 10 ml of 20 mM phosphate buffer (pH 7.0) containing 0.2% TritonX-100 for 2 hat room temperature. The incubation mixture was centrifuged at 10,000 X g for 20 min. Although the supernatant had low chlorophyllase activity, it was almost free from Chl. The supernatant was mixed with 0.5 mg of Chl b dissolved in 300 plof diethyl ether with vigorous stirring, and the mixture was incubated at 28 "C for 3 hwith shaking. The reaction was stopped by adding 60 ml of acetone, and the mixture was centrifuged a t 10,000 x g for 10 min. The supernatant was washed twicewith an equal volume of hexane to remove unhydrolyzed Chl b from the acetone phase. The aqueous-acetone phase was diluted with NaC1-saturated water, and Chlide b was transferred to diethyl ether. The diethyl ether solution containing Chlide b was washed twice with NaC1-saturated water to remove Triton X-100 completelyand dried under N, (11).Chlide b was quantified by spectrophotometry with a molar absorption coefficient of

22034

Conversion of Chlorophyll b to Chlorophyll a

22035

Chl b (12). Theyield of Chlide b was about 10%. I n Organelle Conversion of Chlide b to Chl a-Unless otherwise noted, Chl synthesis was performed as follows. An etioplast pellet prepared from about 100 cotyledons was gently suspended in 0.5 ml of a reaction buffer(50 mM Hepes-KOH pH 7.5,0.5 M sorbitol, 10 mM MgCl,, 10 mMATP, 0.2% bovine serum albumin) containing 1pg of Chlide b and was incubated in the dark for 1.5 h at 28 "C. When etioplast subfractions were used instead of etioplasts, sorbitol and bovine serum albumin were removed from the reaction mixture. HighPerformanceLiquidChromatography (HPLCI Analysis of Chl(ide)-After incubation of Chlide b with etioplasts, 4 volumes of acetone were added, andthe mixture was centrifugeda t 5,000 x g for 5 n i n . Two ml of hexane was added to the supernatant. After removal of the esterified Chls with hexane. Chlides were transferred to diethyl ether. After evaporationof the solvents, the Chl(ide)s were dissolved in a small volume of acetone. The Chls were analyzed by HPLC on a n U octadecyl silica column (6 mm, inner diameter,x 150 mm) using 100% 0 5 10 15 methanol as the elution agenta t a flow rate of 1.5 ml/min at 30 "C (13). Retention Time (min) Chls were detectedby their absorption at 663 nm and quantifiedfrom thechromatographicpeakareasaftercalibration of the chromatoFIG.1. HPLC elution profiles of Chls. One pg of Chlide b was graphicresponsewithknownquantities of therelevantpigment. incubated with isolated cucumber etioplastsin the dark for 1.5 h. The Chlides were separated on a polyethylene column (4.6 mm, inner diam- pigments were extracted and eluted fromoctadecyl silica column with eter, x 150 mm) using50% acetone as the elution agent a t a flow rate of 100% methanol at a flow rate of 1.5 mumin at 30 "C and detected by 30 pl/min a t 20 "C (14). absorption at 663 nm. A, before incubation; B , after incubation. 1 , Chl Mass Spectroscopy-Molecular mass was analyzedby a Hitachi (To- bGc;2, Chl bDHGG; 3, Chl bmGc; 4 , Chl b,; 5 , Chl aGG;6, Chl a,,; 7, Chl kyo) model "2500 double-focusing gas chromatograpWmass spectrom-a;, 8, Chl aph. eter equipped with a field desorption apparatus (15). Preparation of ['4CIChl 6-In order to obtain Chl b with highspecific appearing after the incubation had the same retention time as radioactivity and efficient incorporation of exogenous labeled 5-aminolevulinicacidintoChl, cotyledons were fed withlabeled5-aminoChl a. As we showed in a previous study (91, the absorbance, levulinic acid in the presenceof gabaculine, a n inhibitor of glutamate- excitation, and fluorescence spectra of this peak wereidentical 1-semialdehyde aminotransferase (16, 171, to inhibit the synthesis of to those of Chl a. Chl b can undergo chemical reduction of the endogenous 5-aminolevulinic acid. A considerable amountof Chl a esformyl group to a hydroxymethylgroup, which producesa prodterifiedwithgeranylgeraniol (GG), dihydrogeranylgeraniol,tetrahyof drogeranylgeraniol, and phytol(Ph) wasfound during the early phase of uct thathas spectroscopic properties nearly identical to those greening, and Chl b, and Chl aGGwere not fully resolved by HPLC. Chl a. In order to make sure that the incubation product is Chls were first separated by thin layer chromatography, which enabled different from 7-hydroxymethyl-Chl, we synthesized 7separation of Chl b,, and Chlacc, and this wasfollowed by purification hydroxymethyl-Chl by reducing Chl b with sodium borohydride of Chl b,, by HPLC. and analyzed it by HPLC. The retention time of 7-hydroxyThree pairs of cotyledons with 1-cm hypocotyls were cut from etiomethyl Chl was 6.74 min whereas thatof Chl a was 13.42 min. lated cucumber seedlings, and the cotyledons were painted with500 p~ region of the incubation gabaculine using a brush. Next 25 pCi (925 kBq) of [4-'4C15-amino- The mass spectrum in the higher mass levulinic acid was dissolved in 1ml of distilled water, in which the pH product showed an ion at mlz 870 (data not shown). This was had been adjusted to 7.0 with KOH, and fed through the hypocotyls identical to themolecular mass of pheophytin a, indicating the under illumination (75 microeinsteins/m'/s) at 28 "C for 24 h. After reduction of a formyl group of Chl b to a methyl group. The ion illumination, Chls were extracted with acetone and transferred to hexcorresponding t o the molecular ion of Chl a was not detected, ane. After evaporation, Chls weredissolved in diethyl ether and sepaprobably due to difficult ionization of a small quantity of the rated by thin layer chromatography on a cellulose plate with hexane/ 2-propanol (20:l). Chl b was eluted from the plate with diethyl ether, product. From these observations, we considered this peak to dried, and then solubilized in acetone and subjected to HPLC as debe Chl a . When 1pg of Chlide b was incubatedfor 90 min with scribed above. Eluates corresponding to Chl b were collected and dried. etioplasts, 58 ng of Chl a and 65 ng of Chl b accumulated. Preparation of ['4CIChlide b-Garland chrysanthemum leaves were Twelve percent of the exogenous Chlide b had been esterified, homogenized in 20 mM Tris-HC1 (pH 8.01, 10 mM MgC1, and filtered and half had been converted to Chl a. The yield vaned with through a layer of nylon mesh. The homogenate was centrifuged at each experiment, probably due t o differences in the intactness 5,000 x g for 10 min. The pellet (chloroplast membranes) was suspended of the etioplasts. in 100% acetone with a glass homogenizer and centrifuged a t 10,000 x g for 10 min. The procedure was repeated five times, and the pellet was Although angiosperms are not thought to synthesize Chl in dried under reduced pressure. The resultant powder showed high chlo- thedark,thereare some reportsthatsuggestthe lightrophyllaseactivityalthoughtherewascontaminationwithasmall independent reduction of Pchlide in angiosperms (19, 20). To amount of Chl. We used this powder to prepare [14C]Chlideb because fromendogenous the contaminating Chl a does not interfere with the results of experi- ensure that Chl a wassynthesizednot ments when ['*C]Chlide b is used as the substrate for Chl a synthesis. Pchlide but from exogenous Chlide b, 4,000 cpm of [l4C1Chlide b was used as the substrate for Chl synthesis (Fig. 2). The Labeled Chl b was dissolved in a solution containing 20 mM citrateNaOH (pH 7.0) and 40% acetone and incubated with 0.25 g of the radioactivity in the fourth fraction before incubation (Fig. 2 A ) powder for 1.5 h (18).After incubation, acetone was added to bring the could be attributed to [l4C1Chlideb. Incorporation of radioaclevel up to 80%, and Chlide b was separatedas described in the prep- tivity of 579 cpm into Chl a (27th and 28th fractions) and 539 aration of Chlide b. Most of the Chl b was hydrolyzed to Chlide b. b (16th and 17th fractions) (Fig. 2 B ) appeared cpm into Chl Determination of Radioactivity of Chl-After incubation of 4,000 cpm after 90 min of incubation. This indicates clearly that exogeof [l4C1Chlide b with etioplasts, Chls were analyzed by HPLC, and nous Chlide b was converted t o Chl a. fractions were collected every 30 s. The radioactivity of each fraction was measured in a liquid scintillation counter (Aloca LSC 900). Optimum Conditions-Chlide b wasincubatedwith etioplasts a t various pH (data not shown). The ratioof Chl a to Chl RESULTS b was high at pH 7.5, indicating the optimum pHfor Chl b to

I*

Evidence for Conversion of Chlide b to Chl a-One pg of Chl a conversion is 7.5. Chlide b wasincubated with cucumber etioplastsprepared When ATP was not added, little Chl wasdetected (Table I). from about 100 cotyledons in thereaction buffer for 1.5 h, and This is consistent with theobservation that GG may be phosthe pigments were analyzed by HPLC (Fig. 1). One ofthe peaks phorylated to geranylgeranyl pyrophosphate (GGPP) by ATP


22036

z""lA

1

400

150-

"-c

h

K

300

Chla Chlb

Jz 200

Q

-

=

0 50

0

0 0.0

0.5

1 .O

2.0

1.5

2.5

Chlorophyilideb (pg)

500

- B 400

Chi a

Chl b

300

FIG.3.Accumulation of Chl on increasing the amount of exogenous Chlide b. Isolatedcucumber etioplasts wereincubated with variousconcentrations of Chlide b. Pigments weredeterminedand quantified by HPLC. 150

200 -

11.5

Fraction Number FIG.2. Accumulation of labeled Chl. ['4C1Chlideb was incubated with etioplasts. Pigments were analyzed by HPLC, and fractions were collected every 30 s. The radioactivity of each fraction was measured with a liquid scintillation counter. A, before incubation; B , after incubation. TABLE I Effects of exogenous Ph, ATP, and NADPH on accumulation of Chls Chlide b was incubated with etioplasts andaddendum. Pigments were determined and quantifiedby HPLC. Phwas dissolved in ethanol and added. ~

Addendum ng

Phytol" 0 nM 70 nM ATP 0 mM 10 r n M NADPH" 0lIU.l ll"

Chl

Chl a

Chl b

0

30

60

90

120

Incubation Time (min) FIG.4. Accumulation of Chl derivatives after incubation for various periods. Chlide b was incubated with etioplasts for various periods. Total Chl (01,Chl up,, (H), unhydrogenated Chl a ( 0 ,Chl b, (A), and unhydrogenatedChl b (A) were determined and quantified by HPLC. The rest of the added Chlideb (x) was quantifiedby its relative fluorescence in the acetone phase, after esterified Chls had beentransferred to hexane.

ng

concomitant lossof Chlide b. The accumulation of unhydrogenated Chl b reached maximum after 20 min of incubation and then decreased. Chl b increased during the first 60 min and 3.3 10.0 13.3 thengradually decreased.Theseobservations suggestthat 51.0 106.3 157.3 Chlide b was esterified with GG to Chl b , and thenreduced t o Chl b,. This isconsistent with thedetection of unhydrogenated 72.4 45.8 26.6 119.5 101.4 18.1 Chl b in greening cotyledons, which suggests that Chl bphis formed from Chlb , (13). Unhydrogenated Chla did not appear Reaction buffer contains 10 mM ATP. during the first 20 min and then began to accumulate although and then incorporated to Chl (21). The total Chl exhibited no the level was very low compared with that of unhydrogenated significant changes by the additionof Ph, Thisis probably due Chl b. Chl aphwas also notfound for 20 min and thenincreased to the largepool of endogenous GG. up t o 90 min. In order to examine whether NADPH is a reductant for conIn order to know whether Chlide b was converted to Chlide a version of Chl b to Chl a, itseffect on the accumulation of Chl before esterification, we tried to separate the Chlides after 30 was investigated. NADPH stimulated Chl a accumulation, in- min of incubation (Fig. 5).Alittle Chlide b remained, butChlide dicating its involvement in the reduction of Chl b to Chl a. a did not accumulate. However, Chl b to Chl a conversion occurred without exogenous The distributionof the enzymes for Chl b to Chl a conversion NADPH. Although the reduction of Chl b , t o Chl b , required was investigated by incubation with stromal and membrane NADPH (22), the reduction proceeded without the addition of fractions (Table 11).Chl b could accumulate when Chlide b was NADPH to our system, suggesting that endogenous NADPH incubated with the membranefraction, but Chl a was not obhad been used. Characterization-The accumulation of Chl a and b in the served. This indicates thatesterification of Chlide b occurs on presence of various concentrations of Chlide b was investigated the membrane. When Chlide b was incubated with the stromal (Fig. 3). Chl b accumulation was proportional to the amountof fraction, a very small amount of Chl b accumulated due to Chlide b in thereaction mixture while that of Chl a leveled off synthesis by the membrane contaminating the stromal fraction.WhenChlide b was incubated with both stromal and with a high level of Chlide b. Fig. 4 shows the time course of accumulation of Chls and membrane fractions, accumulation of Chl b was greatly acceltheir precursors. Chlide b decreased rapidly after the start of erated and that of Chl a was induced. Chl a and b also accufraction were incubation, and almost all the Chlide b disappeared after 30 mulated when the membrane and heated stromal b increased with a incubated together. min of incubation. Unhydrogenated Chl 145.5 158.3

41.5 39.3

104.0 119.0

Conversion of Chlorophyll Chlorophyll b to

a

22037

vinylPchlide inadditionto monovinyl Pchlide, andthese Pchlides arereduced to monovinyl and divinyl Chlide (14, 24). Although there are various Chl derivatives with chemically different structures, the amount of Chl in greening tissue is generally determined with a molar extinction coefficient of 3vinyl, 8-ethyl monovinyl Chl a,,, (251, which would cause some error in the determination. A large amount of Pchlide would also interfere with theexact determination of Chl. In order t o show the conversion of Chl b to Chl a, first we confirmed that theincubation product is differentfrom 7hydroxymethyl Chl by their retention times on HPLC.Next, we carried out a n experiment using etioplasts and [l4C1Chlideb. When [l4C]Ch1ide b was incubated with etioplasts, ['4ClChl a was formed. This observation showed that Chlide b is the substrate for Chl a. The observed increase in Chl a with a concomitant decrease in Chl b during dark incubation of greening tissues would be due to theconversion of Chl b to Chl a. Fig. 4 shows that unhydrogenated Chl b appeared first, and 0 10 20 30 40 then Chl b,, accumulated with a decrease of unhydrogenated Retention Time (min) Chl b. This indicates that Chlide b was esterified with GGPP FIG.5. HPLC elution profilesof Chlides. Chlides were eluted from a polyethylene column with 50% acetone at a flow rate of 30 pllmin at and then successively reduced to Ph. The amount of unhydro20 "C and detected by absorption at 666 nm. Chlide a and b were genated Chla was very small compared with thatof Chl b, and prepared from Chla and b by hydrolysis with chlorophyllase. A, Chlide Chl a was synthesized after a considerable amount of Chl b a ; B , Chlide b; C , Chlides after 30 min of incubation of Chlide b with accumulated. Chlide a was not detected when Chl a was being etioplasts. synthesized (Fig. 5). These data suggest that Chlide b was TABLEI1 esterified before conversion to Chlide a, and then Chl b was Accumulation of Chl after incubation of Chlide b with converted to Chl a. In order toknow whether exogenous Chl b subfractionated etioplasts can be converted to Chl a directly, Chl b solubilized in 0.1% Membrane and stromalfractions were prepared from etioplasts rupTriton X-100 or suspended in25% glycerol was incubated with tured by osmotic shock and centrifuged. Pigments were determined and quantified by HPLC. In Experiment A, Chlide b was incubated with etioplasts. After incubation, Chl a could not be detected. But stromal fraction, membrane fraction,or their mixture, and in Experi- this does not necessarily deny theconversion of esterified Chl b ment B, Chlide b was incubated with membrane suspendedin stromal to Chl a. Eichacker et al. (26) reported that when Chlide a and fraction heated at 100 "C for 1 min or unheated. phytyl pyrophosphate were added t o the in vitro translation Chl a Chl b system of Chl a apoprotein Chl a was synthesized and Chl a apoprotein accumulated, butexogenous Chl a could not induce ng ng ng Exp. A a similar response. Added insoluble Chl and esterified Chl in Membrane 22.3 0.0 22.3 plastid seem t o be of different character, perhaps in their loca2.5 Stromal fraction 2.5 0.0 tion in the plastid. 73.0 5.5 67.5 Membrane + stromal fraction Phosphorylation of GG t o GGPP, esterification of Chlide b Exp. B Membrane 36.0 1.5 34.5 with GGPP, reduction of the prenyl side chain of Chl b, to Chl Membrane + stromal fraction 64.0 9.3 73.3 b,,, and thereduction of the aldehyde group of Chl b to methyl Membrane + heated stromal fraction 41.0 6.8 34.2 group all occurred. When Chlide b is converted to Chl a in isolated etioplasts, ATP is used for the formation of GGPP. Without exogenous ATP, Chls do not accumulate, indicating DISCUSSION that the endogenous ATP pool is very small in etioplasts. We When etiolated tissues illuminated for a short period were could not showwhether ATP was necessaryfor the reduction of incubated in the dark, the content of Chl b decreased whilethat the aldehyde group, becauseesterification did not proceed of Chl a increased. Tanaka and Tsuji (7) reported that when 4-h without ATP. The requirement of ATP for the conversion reilluminated cucumber cotyledons were incubated in the dark mains to be studied. NADPH did not show any effects on esfor 24 h, Chl a increased by 9.2 pglg, fresh weight, while Chl b terification of Chlide a with GGPP when they were incubated decreased by 12.5 pg/g, fresh weight. These changes in Chl a with the membrane of barley etioplasts (22). However, exogand b during dark incubation do not necessarily indicate the enously added NADPH stimulated the accumulation of total conversion of Chl b to Chl a , because errors in Chl determina- Chls as well as conversion of Chl b to Chla (Table I). Thiswould tion and light-independent reductionof Pchlide cannot be ex- be due to theeffect of NADPH on preventing thebreakdown of cluded. Adamsonet al. (19)reported that when barley seedlings ChUide) or on stimulating the formation of Chl. NADPH ingrown for 5 days in daylight were transferred to the dark, bothcreased the accumulationof Chl a remarkably, which suggests Chl a and b increased. They suggested that the increase in Chl the requirementof the reductantfor the conversion of Chl b t o content was caused by light-independent reduction of Pchlide. Chl a. Further study is needed to clarify the role of NADPH. On the other hand,it is well known that chemically heterogeArgyroudi-Akoyunoglou et al. (27) suggested that Chl a on neous Chls accumulate during the early phase of greening. the light-harvesting Chl a lb-protein complex of photosystem I1 Chlide is esterified with GG, and the prenyl side chain is re- (LHCII) is reused in the formation of new photosystem I1 units duced to Ph successively. A considerable amount of Chl that in the dark. Previously, we also reported (28, 29) that when accumulates during greening has a prenyl side chain thatnotis greening cotyledons were transferred t o the dark, P700-Chl fully hydrogenated (13). Protoporphyrin is a 3,s-divinyl com- a-protein complex (CP1) and Chl a-protein complexes of photopound, and Chla is a 3-vinyl, 8-ethyl monovinyl compound (4, system I1 (CPa)increasedwith a concomitantdecrease in 23). Cucumber seedlings have been reported to have some di- LHCII. As Chl is not synthesized in the dark, these changes in Chlide a

22038


7. Tanaka, A., and Tsuji, H. (1981) Plant Physiol. (Rocku.)68, 567-570 Chl-protein content indicate that Chl a on LHCII was used for 8. Ikegami, I., Kamiya, A,, and Hase, E. (1984) Plant Cell Physiol. 25, 343-348 the formation of CP1 and CPa. However, it was not clear how 9. Ito, H., Tanaka, Y., Tsuji, H., and Tanaka, A. (1993)Arch. Biochem. Biophys. Chl b on LHCII was metabolized during Chl redistribution 306,14%151 from LHCII to CP1 and CPa,because CP1 and CPabind only 10. Omata, T., and Murata, M. (1980) Photochem. Photobiol. 31, 183-185 11. Klein, A. O., and Vishniac, W. (1961) J. Biol. Chem. 236,2544-2547 Chl a. If Chl b could not be converted to Chi a, Chl b would be 12. Holt, A. S., and Jacobs, E. E. (1954)Am. J. Bot. 41, 710-717 released from LHCII apoprotein andpooled in thylakoidmem- 13. Shioi, Y., and Sasa, T. (1983) Biochim. Biophys. Acta 756, 127-131 branes as free Chl. This free Chl is toxic for chloroplasts be- 14. Shioi, Y., and Beale, S . I. (1987) Anal. Biochem. 162, 493-499 15. Takaichi, S. (1993) Org. Mass Spectrom. 28, 785-788 cause it generates radicaloxygen, which damages chloroplasts. 16. Rando, R. R. (1977) Biochemistry 16,4604-4610 Chl b to Chl a conversion would also play an important role 17. Comveau, J. L., and Beale, S . I. (1986) Plant Sci. (Limerick)45, 9-17 in the adaptationof the photosynthetic apparatus to light con- 18. Holden, M. (1961) Biochem. J. 78, 359-364 19. Adamson, H., Griffiths, T., Packer, N., and Sutherland, M. (1985) Physiol. ditions. It is well known that the Chl a / b binding complex of Plant. 64, 345-352 LHCII decreases and the number of core complexes increases in 20. Walmsley, J., and Adamson, H. (1989) Physiol. Plant. 77,312-319 Riidiger, W., Benz, J., and Guthoff, C. (1980) Eur. J. Biochem. 109, 193-200 high light intensity(30,311. Chl b to Chla conversion would be 21. 22. Soll, S., Schultz, G., Riidiger, W., and Benz, J. (1983) Plant Physiol. (Rocku.) activated during adaptation from low light intensity t o high 71,849-854 23. Whyte, B. J., and Griffiths, W.T. (1993)Biochem. J. 291,939-944 light intensity and reduce theexcess free Chl b.

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