acyl lipids, pigments, gramine developing leaves barley ... · ammonia was quantified by the...

Plant Physiol. (1981) 67, 646-6540032-0889/81/67/0646/09/$00.50/0

Acyl Lipids, Pigments, and Gramine in Developing Leaves ofBarley and Its Virescens Mutant'

Received for publication May 16, 1980 and in revised form September 30, 1980

LAWRENCE W. THOMSON AND SAUL ZALIKDepartment ofPlant Science, University ofAlberta, Edmonton, Alberta, T6G 2E3, Canada

ABSTRACT

Changes in acyl lipids and pigments during leaf development in avirescens barley mutant (M) and the normal (N) were studied. Apical 3-cmleaf segments were extracted with chloroform-methanol, the extracts werepurified on Sephadex G-25 columns, and the polar Upids were separatedon two-dimensional-thin layer chromatography silica gel plates. The pig-ment reining on the Sephadex column was identified as flavonoids anda zone on the TLC plates which did not correspond to the usual standardswas identified as gramine. Quantification of acyl lipids by either polar headgroup analysis or fatty acid analysis using heptadecanoate as an internalstandard gave similar results. The per cent of the total lipid extractquanted for the M between 4 and 8 days ranged from 46 to 65% and thatfor the N ranged from 60 to 68%. Of these, acyl lipids represented 37 to48% in the M and 43 to 50% in the N. By 8 days, mono- and digalacto-syldiglyceride (MG and DG) accounted for 45 and 25% of the total acyllipid of both the M and N. For the period of study here, this representeda 4-fold increase in MG and a 2.5-fold increase in DG in the M but only a1.8-fold increase for MG and DG in the N. These increases were closelycorrelated with the increases in chlorophyll. Chlorophyli increased sharplybetween 4 and 6 days for the N, whereas, in the M, it rose from 7 to 50%relative to the normal by 8 days. The proportions of the various fatty acidswere unique for the lipld classes. The only major quantitative change for afatty acid was for hexadecanoate in phosphatidylglycerol which increasedfrom 5% at 4 days to 25 to 30% by 8 days. Relative to the N, the carotenoidcontent of the M increased from 14 to 50% between 4 and 8 days. In boththe M and N, the increase in a8-carotene and chlorophyll were closelycorrelated.

Chloroplast thylakoid membranes contain a unique lipid com-position which is ubiquitous to higher plants and differs consid-erably from that of other cellular membranes. The individual lipidclasses have been studied extensively and include the pigmentsand the major acyl lipids which represent approximately 25 and50%Yo, respectively, of the total chloroplast lipid (34). The acyl lipidfraction is comprised predominately of the galactolipids MG2 andDG, which account for 40 to 45% of the total lipid, and has arestricted phospholipid composition (34). The chloroplast acyllipids also contain very high levels of polyunsaturated fatty acids,which are concentrated primarily in the major galactolipids, and

' This work was supported by a grant to S. Z. from the Natural Sciencesand Engineering Research Council of Canada.

2 Abbreviations: MG, monogalactosyldiglyceride; 5-ALA, 5-&.amino-levulinic acid; DG, digalactosyldiglyceride; LHC a/b, light-harvestingchlorophyll a/b; PC, phosphatidylcholine; PE, phosphatidylethanolamine;PG, phosphatidylglycerol; PI, phosphatidylinositol; SL, sulfolipid; 2DTLC, two-dimensional thin-layer chromatography.

an unique fatty acid, trans-3-hexadecanoic acid, which is foundspecifically in photosynthetic tissue and is esterified primarily toPG (20). This specialized acyl lipid composition has led to thesuggestion that lipids play a vital role in photosynthesis.The chloroplast is very active in cellular lipid biosynthesis; the

complete biosynthetic pathway from 5-ALA to Chl (8) and thesynthesis of carotenoids occur within the plastid (18). Also, theplastid has been suggested to be the major, if not the sole, site ofcellular de novo fatty acid biosynthesis (36). The full assembly ofthe complex chloroplast acyl lipids, however, appears to requireenzyme systems associated with both the plastid and the cytoplasm(17,46). Studies using inhibitors specific for cytoplasmic ribosomesindicated the formation of 5-ALA (26) and the fatty acid desatu-rase enzyme activity (35) are dependent upon protein synthesis bythe cytoplasmic ribosomes. Nuclear mutations controlling thesteps between protophorphyrin IX and protochlorophyllide (51)and the desaturation and cyclization enzymes in carotenoid bio-synthesis (10) indicate nuclear control over major portions of thepigment biosynthetic pathways. Likewise, genetic studies under-taken to modify the fatty acid composition in seed oils indicatednuclear control of elongase and desaturase enzymes (12). A nu-clear mutant has been described in the control ofMG biosynthesisin greening barley seedlings (52) and for MG and DG levels inwheat endosperm (21).We present here a detailed leaf acyl lipid and pigment analysis

of Gateway barley and its virescens mutant during the initialstages of chloroplast development. The virescens mutation waspreviously shown to be due to a single recessive nuclear genewhich is partially self-correcting (45). Analysis of leaf ultrastruc-ture (24), lamellar proteins (24), and photoreductive activities (22)showed a general time-lag in plastid development of the mutant.This time-lag in its development makes it a useful alternate systemof study to those which have examined the greening of etioplastsand "static" mutations during the development of the chloroplaststructure and function. In addition, because the chloroplast has avery active role in cellular lipid metabolism, these studies are ofinterest in elucidating the respective roles of the chloroplast andcytoplasm in lipid metabolism as well as in the further character-ization of the virescens mutant.

MATERIALS AND METHODS

Plant Material. Barley (Hordeum vulgare cv. Gateway) and itsChl-deficient mutant which has previously been described wereused here. Seeds of Gateway and the mutant were surface-steri-lized with 5% NaOCl and grown in vermiculite. The two lineswere grown simultaneously under continuous light at 90 t&E/m2.s (fluorescent cool-white) and 20 C. The apical 3-cm leaf segmentswere used for lipid and pigment analysis except, when indicated,an additional 3-cm segment towards the basal region adjacent tothe apical segment was also studied.

Solvents. All solvents were of analytical reagent grade. Acetone,chloroform, hexanes, ligroine (petroleum ether; b.p., 63-75 C),

646 www.plantphysiol.orgon March 14, 2020 - Published by Downloaded from Copyright © 1981 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

LIPID COMPOSITION OF BARLEY LEAVES

methanol, and l-propanol were redistilled before use. Phenol usedfor sugar determinations was redistilled and allowed to crystallize.

Lipid Extraction. Seedlings were harvested at 4, 6, and 8 daysafter planting. The leaf segments, approximately 0.8 g for 4-dayand 0.5 g for 6- and 8-day analyses were steamed for 10 min todenature lipases. The samples were extracted with approximately15 ml chloroform:methanol (2:1, v/v) using a Ten Broeck tissuegrinder. Cellular material was pelleted by centrifugation at10,000g. The pellet was resuspended in chloroform:methanol (2:1,v/v) and the suspension was filtered through Whatman No. 1

filter paper with several washings. The filter paper containing thepigment-free residue was saved for nitrogen determinations. Thefiltrate plus initial supernatant were pooled and made to volume(25 ml). A 2-ml aliquot was withdrawn for Chl estimations and 20ml for lipid estimations. The nonlipid contaminants were removedusing the method of Williams and Merrilees (53), which involvedthe addition of 1 g Sephadex G-25 to each lipid extract. Tofacilitate swelling of the Sephadex, 0.2 ml water was added. Thelipids were washed from the Sephadex with 100 ml chloroform.Monitoring further washes containing methanol (chloroform:methanol, 2:1, v/v) by TLC revealed only faint traces of somelipid components but also showed contaminants about the origin.A yellow pigment remained in the column. It was not eluted withmethanol but was removed with water. The 100-ml chloroformwash was concentrated on a rotary vacuum evaporator and madeto volume (2 ml) with chloroform. For total lipid weight deter-mination, 600-id samples in duplicate were transferred to pre-weighed vials and concentrated to dryness with nitrogen. Thesamples were further dried over silica gel in a vacuum desiccatorfor approximately 3 days for constant weight determination.

Separation of Polar Lipids. The polar lipids were separated by

Frontt

:1i

C/M/H20

65:25:4

C/M/I P/NH40H

_ Front

NIl.

Pigment

Front

65: 35:0.5:5

FIG. 1. Outline of a representative two-dimensional thin layer chro-matogram of the polar lipids isolated from leaf segments of 4-, 6-, and 8-day mutant and normal barley seedlings. C, chloroform; M, methanol; IP,isopropylamine; NL, neutral lipids.

Table I. Gramine Content ofApical 3-cm Leaf Segments of 4- and 7-Day-old Mutant and Normal Barley Seedlings.

Gramine Content inDays

Mutant Normal

tgigfresh wt

48 756 ± 34 1,083 ± 177" 1,000 1,566

Values are means ± SE of four determinations.b Single determinations.

Table II. Estimation of Individual Lipid Constituents by either Phosphorusor Galactose Analysis and with Heptadecanoate Internal Standard

Seedling PI PC PE PG MG DG SL

nmol/gfresh wtMutant, 6-day'Head groupb 208.1 1209 571.6 244.2 1366 1082 304.8Acyl groupc 182.9 1181 539.0 238.2 1807 1081 330.0

Mutant, 8-day8Head groupb 228.7 1202 559.0 265.9 2059 1141Acyl groupc 165.0 1052 500.2 200.3 1913 1244 361.6

Normal, 6-day8Head groupb 233.8 1390 461.4 529.1 3418 3111Acyl groupc 249.0 1482 473.0 466.8 3848 2530 697.5

Normal, 8-day8Head groupb 272.2 1394 488.8 444.4 3874 2999Acyl groupc 249.5 1225 509.1 497.1 3750 2761 514.28 Aliquots are from the same total lipid extract of apical 3-cm leaf

segments.b Galactose and phosphorus estimates are averages of two determina-

tions for the mutant and single determinations for the normal.c Single determinations.

40_Protein

Lipid -~e

30-L~~~~~~ ~i-pid

20_C~~~~~~~~~~~~~

10-

0.0 2 4 6 6

Days continuous light

FIG. 2. Protein and lipid content of the apical 3-cm leaf segments of4-, 6-, and 8-day mutant (M) and normal (N) barley seedlings. Afterchloroform:methanol (2:1, v/v) extraction, protein in the residue wasestimated using a micro Kjeldahl digest. Total lipid content was estimatedgravimetrically.

2D TLC on glass plates (20 x 20 cm) coated with a 250-,um layerof Silica Gel HR (Merck) using the solvent system outlined byAllen and Good (1). A 200-p1 aliquot of the total lipid extract,containing 0.7 to 1 mg, was applied under a nitrogen atmosphere.

Identification of Phospholipids, Glycolipids, and Gramine. Alllipid areas were outlined by spraying the plate lightly with 50%oH2SO4 and charring in an oven at 180 C for 15 min. Authenticsamples, fatty acid analysis, IR spectral analysis, specific colortests, and comparisons to the migration patterns shown in pub-lished chromatograms were used to identify lipid classes andindividual lipid spots.

Lipid Quantification. Quantification of the lipid from the nor-mal and mutant seedlings was based on their fatty acid contentusing heptadecanoic acid as an internal fatty acid standard (1).The lipids were also quantified on the basis of their sugar (39)and phosphorus (6) content in some instances.

Lipid areas were detected by spraying the plates with Rhoda-mine 6G (Allied Chemical) and viewing immediately under short-wave UV. Esterification was achieved by adding 5 ml 5% H2SO4in methanol and the internal standard to 15-ml screw-capped(with Teflon liners) test tubes containing the sample plus absorb-ent. Following heating for 2 h at 70 C, the contents were allowedto cool and then diluted with 5 ml water, and the methyl esterswere extracted with three washings of hexane. The hexane was

o 80

c5PE

OPCOPI 1JGramine

x-origin

64--7Plant Physiol. Vol. 67, 1981

I

www.plantphysiol.orgon March 14, 2020 - Published by Downloaded from Copyright © 1981 American Society of Plant Biologists. All rights reserved.


THOMSON AND ZALIK


FIG. 3. Chl content of the apical 3-cm leaf segments of 4-, 6-, and 8-day mutant (M) and normal (N) barley seedlings. An aliquot of thechloroform:methanol extract (2:1, v/v) was concentrated to dryness andresolubilized in 80% acetone, and Chl was estimated according to Amnon(3).

evaporated under a stream of N2 and the methyl esters were takenup in methanol.The fatty acid methyl esters were analyzed on an Aerograph

model 200 gas chromatograph equipped with a hydrogen flame-ionizing detector. A coiled stainless steel column (1.7 m x 3 mm)packed with 10%o ethylene glycol adipate on Anakrom SD (90-100 mesh; Analabs) was used with nitrogen as carrier. The columntemperature was programmed at 8 C/min from 100 to 195 C withthe injector temperature held constant at 240 C.

Chi Determination. The 2-ml aliquot of the chloroform:metha-nol extract was concentrated to dryness under dim light andresolubilized in 80% acetone. The Chl was determined accordingto Amnon (3). The revised coefficients of Jeffery et al. (23) wereused to quantify the Chi separated by TLC.

Nitrogen Analysis. The nitrogen content of the pigment-freeresidue was determined using a micro Kjeldahl digest and theammonia was quantified by the phenol-hypochlorite procedure(32).Gramine Analysis. Standards and sample were located on the

TLC plates by spraying lightly with the xanthydrol solution (0.1%xanthydrol in 95% ethanol and 5% concentrated HCI). The SilicaGel HR was extracted with two successive 2-ml portions of 6 NHCI. To the pooled supernatants, 1 ml xanthydrol reagent wasadded and gramine was estimated as described by Moore et al.(33).

Samples for IR spectral analysis of gramine were isolated fromboth normal and mutant barley leaves using the method ofSchneider et al. (42). The gramine fraction was subjected to the2D TLC system, as outlined for lipid separations, except isopro-pylamine was not included in the second solvent system. Thegramine area was visualized by exposure to iodine vapor andeluted from silica gel with chloroform:methanol (1:1; v/v) concen-trated to dryness and resolubilized in a small volume of carbontetrachloride. The IR spectrum was determined with a PerkinElmer 421 grating spectrophotometer.

Analysis of Pigment Remaining on Sephadex Column. Pigmentsremaining on the G-25 column were completely eluted with 25 mlof distilled H20. The eluent was concentrated by freeze-dryingand redissolved in a small volume of water. Aliquots were appliedto Silica Gel HR TLC plates and screened according to theprocedures outlined by Egger (14). Separations were obtained

with the solvent system, ethyl acetate: butanone: formic acid: H20(50:30:10:10, v/v) developed for strongly polar plant phenolicderivatives, including flavone-glycosides and anthocyanidins.The extract was further studied by one (14)- and two-dimen-

sional paper chromatography (31), as well as by visible and UVspectral analysis.

Carotenoid Estimation. All procedures were carried out underdim light. The barley leaf segments were initially extracted with80o acetone; however, a reddish pigment remained in the residue.Further extraction of the residue with chloroform:methanol (2:1,v/v), followed by 2D TLC and visible spectral analysis, showedthe pigment consisted mainly of ,8-carotene. Traces of Chl werealso evident. Therefore, samples for quantitative carotenoid deter-minations were initially extracted with chloroform:methanol (2:1,v/v), concentrated to dryness under nitrogen and resuspended in80%1o acetone. The carotenoids were purified and quantified essen-tially by the procedures outlined by Jeffrey et al. (23). Goodseparations were obtained with up to 5 ,ug total pigment appliedto each TLC plate.

RESULTS

Lipid Extractions. The three frequently used procedures for theextraction and purification of lipids (7, 15, 53) were compared.Although similar total lipid estimates were obtained by all three,the Williams and Meerles method (53) was chosen because theSephadex method was preferable to the phase separation proce-dures.

Flavonoids. Analysis of the water-soluble pigments which re-mained on the Sephadex column, after lipid extraction, by one(14)- and two-dimensional paper chromatography (31) and spec-tral scans of the eluted zones (25) revealed that the pigments wereflavones and flavonols and were not lipid breakdown products.

Gramine. An outline of the total leaf lipids separated by 2DTLC is shown in Figure 1. The area identified as gramine has notbeen, to our knowledge, identified in leaf lipid studies. Charringwith 50Yo H2SO4 readily outlined all areas as shown in Figure 1except gramine, which developed a rose color upon standingovernight. The gramine area was readily outlined with iodinevapors and was visible as a dull reddish color on plates sprayedwith Rhodamine 6G and viewed under UV. The gramine bandwas not detected using identical extraction procedures with spin-ach leaves (data not shown) and with rye seedlings (47). Thissuggests its occurrence is specific to barley and not due to tech-nique. The unknown co-chromatographed with authentic gramine(K&K Laboratories) and also with the gramine fraction preparedfrom barley leaves according to the procedure of Schneider et al.(42). The gramine area also gave a positive reaction with Van Urkreagent spray for indoles. The IR spectrum of both authenticgramine and the unknown were similar. This evidence suggeststhe unknown is gramine or a closely related indole compound.

Recovery of authentic gramine from the Sephadex G-25 columnwas estimated at 95%, suggesting that the leaf gramine contentcould be quantified from the chloroform-methanol extract. Asshown in Table I, the gramine content of the mutant was approx-imately two-thirds that of the normal and increased in bothseedling types as the leaves matured.

Lipid Identification. The areas outlined in Figure I are repre-sentative of both mutant and normal seedlings at the growth stagesanalyzed. PI, PC, PE, and PG gave the characteristic blue colorspecific for phospholipids when sprayed with molybdenum bluereagent, PE developed the red-violet color characteristic of freeamino groups when sprayed with ninhydrin, and PC gave theorange color characteristic of choline when sprayed with Dragen-dorffs reagent (44). PI co-chromatographed with authentic PI(plant source, Applied Science). Fatty-acid analysis of PG fromgreen leaf material revealed the presence of trans-3-hexadecanoicacid, unique to PG. Early color development during charring with

648 Plant Physiol. Vol. 67, 1981




48

44.

40-

Mu t a n t

pMG

/

/ ~~~~~~~

//ooo P C~~P

>~~~~~~~~~_~~ S L~~~ ~~~ ~PGrIs I

o 36.0

x

¢ 32-

1,28-

._I

-; 24.a

0Ec

20.

16

12

8.

4-

v i4 6


2 4 6Days continuous light

FIG. 4. Acyl lipid content of the apical 3-cm leaf segments of 4-, 6-, and 8-day mutant and normal barley seedlings. The lipids were separated by 2D-

TLC as outlined in Figure and quantified using heptadecanoic acid internal standard as described. Data represent single determinations.

50%o H2SO4 gave a purple color characteristic of glycolipids andsterols for MG, DG, and SL. The two spots on the diagonal belowMG were also initially purple and are likely cerebrosides (1). Thefatty-acid composition of MG, DG, and SL was characteristic ofthese lipids from green tissue (29, 34) and the 2D TLC separationpatterns were similar to those reported for spinach by Allen andGood (1) except for the presence of gramine.

Lipid Quantification. Quantification of the acyl lipid constitu-ents by analysis of either the polar-head groups or fatty acidmoieties using heptadecanoic acid as the internal standard gaveresults that were generally in close agreement (Table II). Theseresults suggest minimal co-migration of phospholipid and galac-tolipid classes and show good agreement for the two methods.Recovery of lipids from the TLC plates estimated by phosphorusanalysis ranged from 77 to 86% for four determinations.Changes in Lipid Constituents. The protein content of the

residue insoluble in lipid solvents and the total lipid content ofleaf segments harvested at 4, 6, and 8 days is shown in Figure 2.The normal contained consistently more lipid and protein over

the growth stages. Increases were evident in the mutant throughoutthe growing period; however, for the normal, the increases were

most marked between 4 and 6 days. The lipid content of themutant averaged 67% of the normal.Chl accumulation showed a sharp increase in the normal be-

tween 4 and 6 days with a leveling off by 8 days. The increase inthe mutant was most pronounced between 6 and 8 days (Fig. 3).

The Chl content of the mutant increased from 7 to 50%o relative tothe normal.Marked increases in the major chloroplast acyl lipids, MG, and

DG (Fig. 4), also occurred at the growth stages where Chl accu-

mulation was most pronounced. These data represent a differentexperiment than those in Table II and show a larger increase inthe major galactolipids; however, similar trends were evident inthe two experiments. The MG:DG ratio was 1.6, 1.8, and 1.6 at 4,6, and 8 days for the normal and 1.2, 1.6, and 1.9, respectively, forthe mutant. The minor chloroplast lipids PG and SL showed a

slight increase or little change with increasing age. The majorlipids of the nonchloroplast membranes, PC and PE, (34) de-creased throughout the development period. The decrease was

most evident for PC (Fig. 4). The mutant contains proportionallymore of the major acyl lipids of the nonchloroplast membranes,PE and PC, at 4 days (Fig. 5). However, in both plant types, themajor galactolipids MG and DG accounted for approximately 45and 25%, respectively, of the total acyl lipids by 8 days (Fig. 5).The adjacent leaf segments between 3 and 6 cm from the apical

tip (Fig. 6) were lower in galactolipid content than the correspond-ing more mature apical segments. Large increases occurred as thesegments matured. The major nonchloroplast membrane lipids,PC and PE, contents, however, were similar on a fresh weightbasis to those of the apical 3 cm leaf segments for both the normaland mutant.

Variations in Fatty Acid Composition of Acyl Lipids. The fatty

369

32-

° 28.x

£ 24-

a

N 20-

- 16_-

a

12-

I

Normal _MG

PC

> ~~~~~~SL0 ~ *- PI

0Ec

8_

4-

0 1 I 8

649Plant Physiol. Vol. 67, 1981



THOMSON AND ZALIK

5

4

/O

3

2

51

41

/O

3

2

1'

84

Days cont inuous Ilight

FIG. 5. Acyl lipid content expressed as a proportion of total acyl lipidcontent for the apical 3-cm leaf segments of 4-, 6-, and 8-day mutant andnormal barley seedlings. Data calculated from Figure 4.

acid compositions of the acyl lipids, except for PG, were qualita-tively similar; however, the proportions of the representative fattyacids present were unique to the individual lipid classes (Figs. 7-13). MG in both plant types was highly unsaturated with linolenicacid (18:3) contributing approximately 90%o of the fatty-acid con-tent (Fig. 7). DG was also highly unsaturated, with 18:3 repre-senting close to 80%1o (Fig. 8). A distinguishing feature between thetwo galactolipids was the higher percentage of palmitic acid (16:0)in the DG, representing close to 18%, whereas, in MG, 16:0 waspresent in only trace amounts. Palmitic and linolenic were themajor acyl moieties of sulfolipid representing 35 and 60%o, respec-tively (Fig. 9). Trans-3-hexadecanoic acid was unique to PG,initially representing approximately 5% in both plant types at 4days and increasing to 25 to 30%o by 8 days (Fig. 10). A corre-sponding decrease from approximately 40 to 20%o occurred inpalmitic acid. Linolenic acid was also a major constituent of PGrepresenting 35 to 40%o. The major nonchloroplast lipids weremore saturated; linoleic acid (18:2) represented 40 to 50%o in bothPC and PE (Figs. 12 and 13), except for PC of the normal at 8days, in which case 18:3 became the major constituent at 38%(Fig. 12). Linolenic acid otherwise comprised 25 to 35% andpalmitic acid comprised 20 to 25% of the fatty acids for both theselipid classes.For all the lipid classes, only minor quantitative fatty-acid

differences were evident between the normal and the mutant andminor quantitative variations occurred as the seedlings matured,except as indicated for trans-3-hexadecanoic acid in PG. Thetrans-3-hexadecanoic acid content of PG from 6-day mutant

seedlings sampled from the second 3-cm leaf segment was presentat approximately half (6%) the level present in the normal (12%)(Fig. 11). The level in both plant types was approximately thesame by 8 days; however, the quantity was less than the levelobtained in the more mature leaf segments.

Carotenoids. Although there was variation in the intensity andsize of some pigment zones, they were present in all growth stagesof the mutant and the normal. Recoveries from the TLC plates,estimated on a Chl basis using the extinction coefficients ofJefferyet al. (23), were greater than 92% except for 4- and 5-day mutants,which averaged 75%. The low levels of the latter are perhaps dueto the very low levels of Chl in the mutants.

fl-Carotene was the dominant carotenoid in the normal, increas-ing from approximately 50 to 80 ,ug/g fresh weight between days4 to 6 (Fig. 14). This represents 40 and 50%, respectively, of thetotal carotenoids (Table III). At days 4 and 5, lutein and violax-anthin were the major carotenoids of the mutant comprisingapproximately 30%o; however, by 6 days, fl-carotene became dom-inant, increasing to 25 .tg/g fresh weight by 8 days and represent-ing 45% of the total carotenoid composition (Fig. 14; Table III).Lutein was the major xanthophyll in the normal throughout thedevelopment period, comprising 22 to 28% of the carotenoidcontent. This was followed by neoxanthin, representing 16 to 21%and violaxanthin declining from 16% at 5 days to 8% at 8 days(Fig. 14; Table III). Similar levels of neoxanthin, violaxanthin,and lutein accumulated in the mutant at 6 and 8 days with eachrepresenting 15 to 23% of the total carotenoid content (Fig. 14;Table III). The lutein and neoxanthin content of the mutant wasabout 25 and 50%o, respectively, ofthe levels reached in the normal.However, the ratio of carotenoids to total Chl, which was initiallyhigher in the mutant at 25%, was similar for both plant types by6 days, representing 10%o of the total Chl (Table III). The Chl alb ratio also declined in the mutant during the 4- to 6-day interval,approaching the value of 3 which is comparable to that of thenormal (Table III).

DISCUSSION

Since the Chi, lipid and protein content varied considerablyduring the developmental study (Figs. 2 and 3) and the leaves ofthe mutant and normal seedlings were similar in dimension andfresh weight per leaf, the comparison data were presented on afresh weight basis.The marked variation in the acyl lipids and pigments in corre-

lation with the changes in leaf ultrastructure, lamellar proteins(24), and photoreductive activities (22) indicate the major cellularprocess during the developmental study was the formation of themassive chloroplast internal membrane network. The changeswere most marked for the mutant, although a developmentalsequence was also evident in the normal between 4 and 6 days.Cellular fractionation studies have shown the unique chloroplastlipid composition (19) and plastid fractionation studies have re-vealed further differences between the envelope and the internallamellar membrane (11, 37). The increased galactolipid contentand corresponding decline in the major phospholipids, PC andPE (Figs. 4 and 5) likely reflects an increased chloroplast mem-brane biosynthesis relative to other cellular membranes. Othershave reported similar fmdings with greening etiolated leaves (5,40, 43, 50). The 4- and 2.5-fold increase in MG and DG, respec-tively, in the mutant (Figs. 4 and 5) exceeds the increases reportedfor greening etiolated tissue and is comparable to the increasesreported by Leech et al. (29) in sampling leaf segments of youngcorn leaves. The photoreductive activities of the normal expressedon a lamellar protein basis declined between 4 and 6 days (22),during which interval a considerable increase in Chl and a 1.8-fold increase in both MG and DG was evident (Fig. 4). This mayindicate that chloroplast membrane growth is still occurring inthenormal between 4 and 6 days. The decline in activities on a

Individual lipid class/Total acyl lipid

Mutant

,*O MGW0_ ____~~~~DG!0

=~~~~~~~~~~PC10

/ _ _ _ - _ 5~~~S P E0

P

4 6 8

Norma I

i0_

4- -------

t0_

0-

j~~~

n. I0 F--- I

650 Plant Physiol. Vol. 67, 1981

11



Plant Physiol. Vol. 67, 1981 651LIPID COMPOSITION OF BARLEY LEAVESAnl

36_

32._

24.

20.00

v-x

¢ 16.£to0

E 12.

.-8= 8-

0E 4.

0 28_

x

- 24_00

16..0Ec 12..

4.

v".

6 8Days continuous light Days continuous light

Acyl lipid content of the second 3-cm leaf segments of 6- and 8-day mutant and normal barley seedlings. Quantified as outlined in

Table III. Pigment Ratios Estimatedfrom Sucrose 2D TLC PlatesPigment extracts were prepared from the apical 3-cm leaf segments of normal (N) and mutant (M) barley

seedlings harvested at daily intervals from 4 to 8 days. The data were calculated from Figure 14.

Pigment Extracts

Days Chl a/b Carotenoid/Chl ,B-Carotene Lutein Violaxan- Neoxanthinthin

M N M N M N M N M N M N

ratio %

4 5.16 2.98 0.25 0.12 25 40 32 28 30 14 13 175 3.35 2.86 0.20 0.12 26 42 30 26 33 16 10 166 2.84 3.24 0.10 0.10 36 48 19 23 23 8 21 217 2.76 3.11 0.09 0.08 41 52 21 22 18 7 19 198 2.73 3.15 0.08 0.10 45 50 16 25 15 8 23 16

lamellar protein basis may reflect increased lamellar protein con-

tent due to an increase in the LHC a/b protein complex whichhas been hypothesized to add in discrete units to the photosystemreaction centers (4). Another possibility could be the accumulationof stroma lipids, presumably containing acyl lipids, which havealso been found to contribute to a significant proportion of theplastid lipid complement and are suggested to be lipoproteincomplexes in transit between the different chloroplast membranesystems (37). The increase in the MG:DG ratio during the greening

process has been reported by others and interpreted to reflect therelative amounts of chloroplast lamellae and envelope (34).The very low increase in the other characteristic chloroplast

acyl lipids, PG and SL, has also been shown by others analyzinggreening etiolated leaves (5, 40, 50) and developing leaf segments(29). Any increases in the PG and SL content are likely maskedby the massive changes in the other cellular constituents duringchloroplast development inasmuch as PG and SL each contributeonly about 5% of the total chloroplast membrane lipids (34).

M u t a n t

o MG

..DG

,..,P

I

WI~~~~

*- SLI=====PE

8I P

No r mal .MG

.'~~~~~~~~~.

,. D

PoG.PI

/1

/.

WI

PC

"PEPG

& ~~~PlI

FIG. 6.Figure 4.

d* .

gj 0I I

AI



100.18: 3

80.

60.

40.

20.

Normal MGMutant MGMutant MG

1823

18:1

II IIi 8 i

Days continuous tight

5.

4-

18:2 2-16:0

1-1:0

4

Days continuous tight

9Normal SL

60.18:3

50_

40_

1 6 : 0~~~~~1

30-

20.

10

5-

- * 8 : 2~~~~1:I-182

Mutant SL

1883

f16:0

1888.~~~~~8:

THOMSON AND ZALIK Plant Physiol. Vol. 67, 1981

10

o/4

x. 34

Normal PG Mutant PG

o 50-

o- 40-18:3

16:3

o0 30-16:1

!0- 20- I160

160

10 182 0 18:2

24 6 ~~ ~~ ~ ~~~~~24 6 8Days continuous tight

40.

30.

"I

20.

10.

Normal b PG

40.

18:3 30.16:0

,I* 1 6 1 20-

el 1 8:2101

18:1I1 8=== :0

4 6 8

Mutant b PG

118:3

1 6:016:0

5-u------a 1 82

I 8: I

V! 1 i I


a 8

12

Normal PC Mutant PC

50_ 50-

I0_ 18:3 40- -t8 2

10- 18:230

16:0 16:0

10_ 20_

0_ 10_

0- ' 18:0' - 8.1I I~~~~-6:2 4 6 8 2 4 6 8


13

50.

40.

/0 30_

20.

10.

Normal PE

50.

18:2 40.

30-

18:3

6:020.

10_

18.1I 8:0r _~~~~~t~

Mutant PE

18:3

18:1

16:0

!G.16.,04 4i 2 4 6 8 ; la

Days continuous light Days contInuous light

FIGS. 7-13. Changes in the fatty acid composition of acyl lipids (obtained as in Fig. I) from the apical 3-cm leaf segments of 4-, 6-, and 8-day mutantand normal barley seedlings. Figure II (mutant b and normal b) is from the second 3-cm leaf segment.

652

100-

80-

60.

40-

20-

5-

4-

3-

2-

1..

00

50..

40.

30-

20.

10_

5-

p 0 1 f. i p

Nor mal MG

5

41

7.34

21

1




80-

70_

60.

"50-

400,

0.

* 30.0-

0

20.

10

0I,'0',

0

C

10

4 5 6 7 8


Days Continuous lightFIG. 14. Chl and carotenoid content of the apical 3-cm leaf segments

of normal and mutant barley seedlings sampled at daily intervals from 4to 8 days. The pigments were separated by 2D-TLC on powdered sucrose

plates and quantified by their extinction coefficients (23).

Increases, however, were detected in studies on isolated plastids(30, 43).Although the individual acyl lipids all contained the same

qualitative fatty-acid composition, except for hexadecanoic acidin PG (Figs. 10 and I 1), the amounts of the individual fatty acidswere unique to the lipid classes (Figs. 7-13). The findings here arein close agreement with those obtained for wheat and barley (5,43) and show a higher level of unsaturation in PG and SL thanthat found in maize, bean, and pea (29, 40, 50). Very little changein fatty acid composition except for PG occurred over the periodof the study presented here and in other studies on greeningetiolated leaves. However, Leech et al. (29), on sampling successivesections of developing maize leaves, found a marked increase inunsaturation during plastid maturation. Trans-3-hexadecanoicacid increased markedly in both mutant and normal between 4and 8 days (Figs. 10 and 1 1) and was specifically associated withPG. This is in agreement with other reports (20). Although theexact role of this unique fatty acid is not known, it is foundspecifically in photosynthetic tissue, but it is uncertain whether itis essential for photosynthetic activity and grana stacking (9, 13,40).The same major carotenoids, fl-carotene, lutein, violaxanthin,

and neoxanthin, have been found to be ubiquitous to all greentissue of higher plants and are suggested to be located exclusivelyin the chloroplast (18). The deficiency of the mutant was againevident, and the total carotenoid content increased from 14 to50%, relative to the normal during the period of analysis. A closecorrelation between the 8-carotene concentration and that of Chla + b occurred in the mutant sampled at daily intervals from 4 to8 days, with a correlation coefficient of+ 0.99. A close correlationwas also evident in the normal during the period of pigmentaccumulation between 4 and 6 days. This correlation would beexpected during the formation of the chloroplast internal mem-brane network inasmuch as fl-carotene is a constituent of bothphotosystem reaction centers and the LHC a/b complex (49).Keck et al. (28) also obtained a high correlation for fl-caroteneand Chl in three soybean genotypes having different Chl concen-trations. The increase in f-carotene content relative to the totalcarotenoid concentration in the mutant (Table III) likely reflectsthe increased concentration of carotenoids in the chloroplastlamella relative to the envelope. Chloroplast envelopes have beenshown to be enriched in xanthophylls relative to fl-carotene (11,23). The increased xanthophyll content in the mutant between 5and 8 days (Fig. 14) probably reflects the formation of the LHCa/b complex because the complex has been shown to contain allthe carotenoids (49). During this growth period, there was also adecrease in the Chl a/b ratio (Table III) which is used as a markerfor the formation of the LHC a/b complex (4). Recent proposalson the organization of carotenoids within chloroplast membranessuggest that they occur either as pigment protein complexes (2,27) or within the lipid bilayer (38). In our study, the incompleteextraction of f-carotene with 80%o acetone and its complete re-moval with chloroform:methanol suggest that at least a fraction ofthe f-carotene is in close association with protein in vivo.

In summary, acyl lipid and pigment compositional studiescorroborate the previous findings that the virescens mutant dis-plays a general lag in chloroplast development. A previous studysuggested the initial pigment deficiency of the virescens barleywas not the result of a blockage in the biosynthetic pathway ofChl because the addition of 5-ALA increased the Chl content tothe same extent in both the normal and the mutant (41). Low Chlcontent which is not due to a blockage in Chl synthesis is commonto many pigment-deficient mutants. The deficiency is suggestedto be one of several pleiotropic responses resulting from the failureto make a specific gene product that is required for normalchloroplast formation (16, 24). Although one may speculate as tothe cause of the lower chloroplast acyl lipid and pigment content

Normal

p-carotene

chl a

lutein

neoxanthin

chl b

viola xanth in

vr.. . . . ~~~~~~~~-.-T

653Plant Physiol. Vol. 67, 1981



654 THOMSON

in the mutant, the results perhaps best reflect a close coordinationof the synthesis of macromolecules during plastid development.Moreover, the results provide further evidence that lipid compo-nents other than pigments, play an active role in the assembly ofthe photosynthetic mechanism.

In the succeeding paper (48), studies on acetyl-CoA carboxylaseactivity in developing leaves and isolated chloroplasts of the twoplant types are presented.

Acknowledgment-The assistance of Mr. Barry Zytaruk in the preparation of thefigures is gratefully acknowledged.

LITERATURE CITED

1. ALLEN CF, P GOOD 1971 Acyl lipids in photosynthetic systems. MethodsEnzymol 13: 523-547

2. ANDERSON JM, JC WALDRON, SW THORNE 1978 Chlorophyll-protein complexesof spinach and barley thylakoids. FEBS Lett 92: 227-233

3. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase inBeta vulgaris. Plant Physiol 24: 1-15

4. ARNTZEN CJ 1978 Dynamic structural features of chloroplast lamellae. In DRSanadi, LP Vernon, eds, Current Topics in Bioenergetics, Vol 8B. AcademicPress, London, pp 111-160

5. BAHL J, B FRANCKE, R MONEGER 1976 Lipid composition of envelopes, prola-mellar bodies, and other plastid membranes in etiolated, green and greeningwheat leaves. Planta 129: 193-201

6. BARTLErT GR 1959 Phosphorus assay in column chromatography. J Biol Chem234: 466-468

7. BLIGH EG, WS DYER 1959 A rapid method of total lipid extraction andpurification. Can J Biochem Physiol 37: 911-917

8. BoGoRAD L 1976 Chlorophyll biosynthesis. In TW Goodwin, ed, Chemistry andBiochemistry of Plant Pigments, Vol 1. Academic Press, London, pp 64-148

9. BOLTON P, J WHARuE, JL HARWOOD 1978 The lipid composition of a barleylacking chlorophyll b. Biochem J 174: 67-72

10. DAvIEs BH 1977 Carotenoids in higher plants. In M Tevini, HK Lichtenthaler,eds, Lipids and Lipid Polymers in Higher Plants. Springer-Verlag, New York,pp 199-217

11. DouCE R, RB HoLTz, AA BENSON 1973 Isolation and properties of the envelopeof spinach chloroplasts. J Biol Chem 248: 7215-7222

12. DOwNEY RK, DI MCGREGOR 1976 Breeding for modified fatty acid composition.In H Smith, ed, Commentaries in Plant Science. Pergamon Press, Oxford, pp153-169

13. DUVAL JC, A TREMOLIERES, JP DUBACQ 1979 The possible role of transhexa-decenoic and phosphatidylglycerol in the light reactions of photosynthesis.FEBS Lett 106:414-418

14. EGGER K 1969 Plant phenol derivatives. In E Stahl, ed, Thin-Layer Chromatog-raphy. Springer-Verlag, New York, pp 687-706

15. FOLCH J, M LEES, GHS STANLEY 1957 A simple method for the isolation andpurification of total lipids from animal tissues. J Biol Chem 226: 497-509

16. GILLHAM NW, JE BOYNTON, NH CHUAS 1978 Genetic control of chloroplastproteins. In DR Sanadi, LP Vernon, eds, Current Topics in Bioenergetics, Vol8B. Academic Press, London, pp 211-260

17. GIVAN CV, JL HARWOOD 1976 Biosynthesis of small molecules in chloroplastsof higher plants. Biol Rev 51: 365-406

18. GOODWIN TW 1976 Distribution ofcarotenoids. In TW Goodwin, ed, Chemistryand Biochemistry of Plant Pigments, Vol 1. Academic Press, London, pp 225-261

19. HAAS RE, E HEINZ, G Popovici, G WEISSENBOCK 1979 Protoplasts from oatprimary leaves as a tool for experiments on the compartmentation in lipid andflavonoid metabolism. Z Naturforsch 34: 854-864

20. HARWOOD JL, AT JAMES 1975 Metabolism oftrans-3-hexadecenoic acid in broadbean. Eur J Biochem 50: 325-334

21. HERNANDEz-Lucuz C, RF DECALEYA, P CARBONERO, F GARCIA-OLMEDO 1977Control of galactosyl diglycerides in wheat endosperm by group 5 chromo-somes. Genetics 85: 521-527

22. HORAK A, S ZALIK 1975 Development of photoreductive activity in plastids of avirescens mutant of barley. Can J Bot 53: 2399-2404

23. JEFFREY SW, R DoUCE, AA BENSON 1974 Carotenoid transformations in thechloroplast envelope. Proc Natl Acad Sci USA 71: 807-810

24. JHAMB S, S ZALIK 1973 Soluble and lamellar proteins in seedlings of barley andits virescens mutant in relation to chloroplast development. Can J Bot 51:

LND ZALIK Plant Physiol. Vol. 67, 1981

2147-215425. JuRD L 1962 Spectral properties of flavanoid compounds. In TA Geissman, ed,

The Chemistry of Flavonoid Compounds. Pergamon Press, New York, pp107-155

26. KANNANGARA CG, SP GOUGH 1979 Biosynthesis of 5-aminolevulinate in green-ing barley leaves. II. Induction of enzyme synthesis by light. Carisberg ResCommun 44: 11-20

27. KE B 1971 Carotenoproteins. Methods Enzymol 23A: 624-63628. KECK RW, RA DILLEY, CF ALLEN, S BIGGS 1970 Chloroplast composition and

structure differences in a soybean mutant. Plant Physiol 46: 692-69829. LEECH RM, MG RUMSBY, WW THOMSON 1973 Plastid differentiation, acyl lipid

and fatty acid changes in developing green maize leaves. Plant Physiol 52: 240-245

30. LEESE BM, RM LEECH 1976 Sequential changes in the lipids of developingproplastids isolated from green maize leaves. Plant Physiol 5: 789-794

31. MABRY TJ, KR MARKHAM, MB THOMAS 1970 The Systematic Identification ofFlavonoids. Springer-Verlag, New York, pp 3-15

32. MITCHELL HL 1971 Microdetermination of nitrogen in plant tissues. J Assoc OffAnal Chem 55: 1-3

33. MOORE RM, JD WILLIAMS, J CHIA 1967 Factors affecting concentrations ofdimethylated indole alkylamines in Phalaris tuberosa L. Aust J Biol Sci 20:1131-1140

34. MUDD JB, RE GARCIA 1975 Biosynthesis of glycolipids. In T Galliard, ElMercer, eds, Recent Advances in the Chemistry and Biochemistry of PlantLipids. Academic Press, London, pp 161-201

35. MURPHY DJ, PK STUMPF 1979 Light-dependent induction of polyunsaturatedfatty acid biosynthesis in greening cucumber cotyledons. Plant Physiol 63: 328-335

36. OHLROGGE JB, DN KUHN, PK STUMPF 1979 Subcellular localization of acylcarrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad SciUSA 76: 1194-1198

37. POINCELOT RP 1973 Isolation and lipid composition of spinach chloroplastenvelope membranes. Arch Biochem Biophys 159: 134-142

38. ROHMER M, P BOUVIER, G OURISSON 1979 Molecular evolution of biomem-branes: structural equivalents and phylogenetic precursors of sterols. Proc NatlAcad Sci USA 76: 847-851

39. ROUGHAN PG, RD BATT 1968 Quantitative analysis of sulfolipid (sulfoquino-vosyl diglyceride) and galactolipids (monogalactosyl and digalactosyl diglyc-eride) in plant tissues. Anal Biochem 22: 74-88

40. ROUGHAN PG, NK BOARDMAN 1972 Lipid composition of pea and bean leavesduring chloroplast development. Plant Physiol 50: 31-34

41. SANE PV, S ZALIK 1970 Metabolism of acetate-2-'4C, glycine 2-'4C and leucine-U-'4C, and effect of 5-aminolevulinic acid on chlorophyll synthesis in Gatewaybarley and its mutant. Can J Bot 48: 1171-1178

42. SCHNEIDER EA, RA GIBSON, F WIGHTMAN 1972 Biosynthesis and metabolism ofindol-3yl-acetic acid. The native indoles of barley and tomato shoots. J ExpBot 23: 152-170

43. SELLDEN G, E SELSTAM 1976 Changes in chloroplast lipids during the develop-ment of photosynthetic activity in barley etiochloroplasts. Physiol Plant 37: 35--41

44. SKIPSKI VP, M BARCLAY 1969 Thin-layer chromatography of lipids. MethodsEnzymol 14: 530-598

45. STEPHANSEN K, S ZALIK 1971 Inheritance and qualitative analysis of pigments ina barley mutant. Can J Bot 49: 49-51

46. STUMPF PK 1977 Lipid biosynthesis in higher plants. In TW Goodwin, ed,Internat Rev Biochem. Biochemistry of Lipids, II, Vol 14, University ParkPress, Baltimore pp 215-237

47. TiHOMSON LW, S ZALIK 1973 Lipids in rye seedlings in relation to vernalization.Plant Physiol 52: 268-273

48. THOMSON LW, S ZALIK 1981 Acetyl-CoA carboxylase activity in seedlings andchloroplasts of barley and its virescens mutant. Plant Physiol 67: 655-661

49. THORNBER JP 1975 Chlorophyll-proteins: light-harvesting and reaction centercomponents of plants. Annu Rev Plant Physiol 26: 127-158

50. TREMOLIERES A, M LEPAGE 1971 Changes in lipid composition during greeningof etiolated pea seedlings. Plant Physiol 47: 329-334

51. VON WETTSTEIN D, A KAHN, OF NIELSEN, S GOUGH 1974. Genetic regulation ofchlorophyll synthesis analyzed with mutants in barley. Science 184: 800-802

52. VON WETTSTEIN D, KW HENNINGSEN, JE BOYNTON, GC KANNANGARA, OFNIELSON 1971 The genetic control of chloroplast development in barley. InNK Boardman, AW Linnane, RM Smillie, eds, Autonomy and Biogenesis ofMitochondria and Cbloroplasts. North-Holland, Amsterdam, pp 205-223

53. WILLIAMS JP, PA MERRILEES 1980 The removal of water and nonlipid contami-nants from lipid extracts. Lipids 5: 367-370

A



acyl lipids, pigments, gramine developing leaves barley ... · ammonia was quantified by the...

Documents