Mutants of Sweetclover (Melilotus alba) LackingChlorophyll b",2STUDIES ON PIGMENT-PROTEIN COMPLEXES AND THYLAKOID PROTEIN PHOSPHORYLATION
Received for publication October 3, 1984 and in revised form December 10, 1984
JOHN P. MARKWELL*, ANDREW N. WEBBER,3 AND BRIDGET LAKEDepartment ofAgricultural Biochemistry and School ofBiological Sciences, University ofNebraska,Lincoln, Nebraska 68583-0718
ABSTRACT
Mutants of sweetclover (Melilotus alba) with defects in the nuclearch5 locus were examined. Using thin-layer chromatography and absorp-tion spectroscopy, three of these mutants were found to lack chlorophyll(Chl) b. One of these three mutants, U374, possessed thylakoid mem-branes lacking the three Chl b-containing pigment-protein complexes(AB-1, AB-2, and AB-3) while still containing A-1 and A-2, Chl acomplexes derived from photosystems I and II, respectively. Completesolubilization and denaturation of the thylakoid proteins from this mutantrevealed very little apoprotein from the Chl b-containing light-harvestingcomplexes, the major thylakoid proteins in normal plants. The normaland mutant sweetclover plants had active thylakoid protein kinase activ-ities and numerous polypeptides were labeled following incubation with1y-32PIATP. With the U374 mutant, however, there was very littledetectable label co-migrating with the light-harvesting complex apopro-teins on polyacrylamide gels. The Chl b-deficient chlorina-f2 mutant ofbarley (Hordeum vulgare) also had an active protein kinase activitycapable of phosphorylating numerous polypeptides, including ones mi-grating with the same mobility as the light-harvesting complex apopro-teins. These results indicate that the sweetclover mutants may be usefulsystems for studies on the function and organization of Chl b in thylakoidmembranes of higher plants.
not photochemically impaired (4), but the ultrastructure of itsthylakoid membrane network is different from that of normalplants (9, 22). The photosynthetic membranes of this mutantlack the Chl b-containing pigment-protein complexes (1, 24),although the Chl b-binding apoproteins are synthesized andpresent in diminished amounts (3).There are several reasons why the chlorina-f2 mutant may not
be the best mutant for biochemical and photochemical research.The chlorina-f2 barley was selected as a spontaneous mutantfound as a single plant in the F2 population following a cross oftwo different barley cultivars (13) and thus there is no singleparental type to use for control studies. We have found that this
Table I. Ratio ofChl a/b in Various ch5/chS Mutants ofSweetclover
Strain Genotype No of Average Chl a/b SDDeterminationsNormal +/+ 10 2.81 0.20U394 ch4/ch4 6 5.91 0.95U374 ch5/ch5 10 24.7 5.7U398 ch5/ch5 5 23.8 3.5T159 ch5/ch5 5 24.4 3.3
The major photosynthetically active pigments in most higherplants are Chl a and b. The latter molecule appears to functionsolely as an accessory pigment, harvesting light energy but notplaying a direct role in photochemical energy transduction. Moststudies on the location and role of Chl b in higher plants haveutilized mutants deficient in this pigment. The most useful andwidely studied mutant (e.g. 5, 10, 22, 24) has been the chlorina-f2 barley (Hordeum vulgare). This mutant was first reported in1950 (11) and the absence of Chl b confirmed in 1962 (13). It is
' Supported by United States Department of Agriculture, Science andEducation Competitive Research Grant No. 83-CRCR-1-1346, and bythe University of Nebraska-Lincoln Research Council. Paper No. 7604,Journal Series, Nebraska Agricultural Experiment Station.
2 Inquiries concerning seed of sweet clover plants used in these studiesshould be addressed to: Dr. H. J. Gorz, USDA-ARS, or Dr. F. A. Haskins,Department of Agronomy, University of Nebraska, Lincoln, NE 68583-0915.
3 Recipient of a Wain Fellowship from the U.K. Agricultural ResearchCouncil.
FIG. 2. Pigment protein complexes fraction-l Al _ ^1ated in duplicate from normal (A) and U374
Ai b- l mutant (B) plants of sweetclover. SolubilizationAglw |ofisolated thylakoid membranes, electrophoretic
-_ IAII fractionation on a polyacrylamide gel, and no-menclature of the complexes are as described in
l l l_reference 19. Each lane was loaded with 10 or7.5 ug Chl for normal or 'mutant samples, re-spectively.
Department ofAgronomy, University of Nebraska-Lincoln. Seedof normal and mutant barley (Hordeum vulgare) were the gift ofDr. Harry Highkin, California State University at Northridge.Plants were grown in a glasshouse in a soil-vermiculite mixturefor approximately 4 weeks before use. Leaves were harvested and
X1 / I\ / IA I thylakoid membranes isolated as previously described (18).J /\\Y \ I Room temperature absorption spectra were obtained with a Cary
219 spectrophotometer interfaced to an Apple II computer.\ Thylakoid membranes were analyzed for constituent pigment-
LO
Uj~~~~~~~~~~~~~~~L
400450500550 600650700WAVELENGTH (nm) <
FIG. 3. Room temperature absorption spectra of pigment-proteino|\complexes fractionated from isolated thylakoids of normal sweetclover lplants. Identification: A, A-l; B, AB-1; C, AB-2; D, AB-3. <
Limutant is susceptible to rust infection, making it difficult to grow >these plants to maturity in a healthy state. Also, the presence ofthe apoproteins of the Chl b-containing pigment-protein com--Iplexes is undesirable in the barley mutant and may give the |\thylakoid membrane different properties than would be observedin their total absence. Other mutants lacking Chl b have been Breported in higher plants such as Arabidopsis thaliana (14), pea(Pisum sativum) (12), maize (Zea mays) (15, 20), wheat (Triti-cum aestivum) (6, 7), and sweetclover (Melilotus alba) (8, 21,23). In general, these mutant strains have not been as well studiedor characterized as the chlorina-f2 barley. The data in this reportconstitute more detailed studies on sweetclover mutants lacking 400 450 500 550 600 650 700Chl b and suggest their usefulness in future research. WAVELENGTH (nm)
MATERIALS AND METHODS FIG. 4. Room temperature absorption spectra of pigment-proteinSeeds of normal and mutant sweetclover (Melilotus alba) were complexes fractionated from isolated thylakoids of U374 mutant of
the gift of Dr. H. J. Gorz, USDA-ARS, and Dr. F. A. Haskins, sweetclover. Identification: A, A-l; B, A-2.
FIG. 5. Polypeptides fractionated from isolated thylakoid membranesof normal (A) and chlorina-f2 mutant (B) barley and from normal (C)and U374 mutant (D) sweetclover. The lane to the immediate right ofeach sample contained a lesser amount of same sample (25 versus 150,Lg protein) labeled with (y-32P]ATP. The numbers to the left of the gelindicate the migration of proteins ofknown molecular mass (M, x 10-3).
protein complexes by solubilization with SDS and electrophoresison polyacrylamide gels (19). Pigment-protein complexes wereexcised and their absorption spectra determined in the gel slices.Chl concentrations and the ratio of Chl a/b were determinedusing the method ofArnon (2). Chl a and b were chromatograph-ically identified by extraction into 80% acetone and then diethylether. Samples were spotted onto silica gel 60 TLC plates (MCBManufacturing Chemists, Inc.) and developed with n-hex-ane:diethyl ether:acetone (60:30:20, v/v/v).
Thylakoid polypeptides were phosphorylated by incubationwith ['y-32P]ATP as described previously (17). Labeled and un-labeled membranes were solubilized by incubation at 60'C for 1
h in 10% glycerol, 2.5% SDS, 50 mM DTT, 62.5 mM Tris-HCl(pH 6.8). Solubilized proteins were fractionated on a gel contain-ing a linear gradient of 8% acrylamide and 3 M urea to 15%acrylamide and 6 M urea. The stacking gel contained 4% acryl-amide and 8 M urea. The buffer system used throughout was thatof Laemmli (16). Following electrophoresis, the gels were stainedwith Coomassie Brilliant Blue R250 and dried under vacuum.Autoradiography was performed using Kodak XAR-5 film.Most chemicals were purchased from Sigma Chemical Co.
Acrylamide was purchased from Bethesda Research Laboratoriesand [y-32P]ATP was purchased from New England Nuclear Corp.
RESULTS
Wild-type sweetclover and mutant strains were grown forapproximately 4 weeks. One mutant strain was defective in thech4 locus, whereas three were defective in the ch5 locus. The
FIG. 6. Autoradiogram of the gel in Figure 5 showing phosphopro-teins fractionated from isolated thylakoids of normal (A) and chlorina-f2 mutant (B) barley and from normal (C) and U374 mutant (D)sweetclover.
mutants are homozygous recessive; complementation crosseswere used to determine the locus of mutation (8, 21). The ratioof Chl a/b found in these plants using the assay and equationsof Arnon (2) are shown in Table I. The normal plants used inour study had a Chl a/b ratio of approximately 3, similar to thatusually found in C3 plants. All ofthe mutants had elevated ratios,indicating a lower than normal proportion ofChl b. The mutantswere grouped into two classes; deficient in Chl b or lacking Chlb. Mutant strain U394 (ch4/ch4) was deficient in Chl b andusually had Chl a/b ratios of approximately 6. Mutant strainsU374, U398, and T 159 (all ch5/ch5) routinely had Chl a/b ratiosgreater than 10, indicating that very little, if any, Chl b waspresent. The concentration of total Chl in the leaves of theseplants has previously been reported (23). Since the assay used inour study (2) is not able to give accurate Chl a/b ratios above 6,we used TLC as an alternate criterion for the presence or absenceof Chl b in these mutants. The normal plant was used as areference for authentic Chi a and Chl b. Strain U394, with a ChIa/b ratio of approximately 6, was found to contain a pigmentwhich migrated with the same mobility as Chl b; however, theother three mutants contained no such pigment.
Absorption spectra of thylakoid membranes isolated from thenormal and U374 strains substantiate the absence of Chl b fromthe mutant membranes (Fig. 1) since they are greatly reduced inthe absorption components at approximately 475 and 650 nmwhich are due to the presence ofChl b in the normal membranes.Analysis ofthe pigment-protein composition ofthese membranesis also consistent with a loss of Chl b in the U374 strain (Fig. 2).The three Chl b-containing pigment-protein complexes presentin the normal membranes (AB- 1, AB-2, and AB-3) are undetect-able in the U374 mutant which contains only the Chl a-contain-
ing A-I and A-2 complexes derived from PSI and PSII, respec-tively. The A-2 complex is obscured by the AB-3 complex in thefractionation pattern from normal plants (20). The results ob-tained with the U374 mutant are identical to the fractionationpatterns previously observed for the Chl b-lacking chlorina-f2mutant of barley (1, 20). Also, the absorption spectra of thefractionated pigment-protein complexes from the normal (Fig.3) and U374 (Fig. 4) sweetclover are similar to those publishedfor other higher plants ( 19, 20).The polypeptide composition and protein phosphorylation
patterns of thylakoid membranes from normal and U374 sweet-clover were compared with normal and chlorina-f2 barley. Thestained polypeptide pattern (Fig. 5) reveals that the U374 mutantcontains fewer polypeptides migrating with mobility similar tothe light-harvesting complex apoprotein (Mr approximately28,000) than the normal sweetclover thylakoid membranes. Thishas also previously been reported for the chlorina-f2 mutant ofbarley (5). Autoradiography of the fractiornted polypeptidesfrom membranes incubated with ['_-32P]ATP (Fig. 6) reveals thatboth sets of mutant and normal sweetclover plants contain activethylakoid protein kinase activities and that numerous thylakoidpolypeptides can act as substrates for this activity. As is apparent,the U374 sweetclover mutant appears to contain less phospho-protein migrating with the same mobility as the Mr = 28,000light-harvesting complex apoproteins than the chlorina-f2 mu-tant of barley.
DISCUSSION
We propose that the ch4 and ch5 mutants of sweetcloverwould be good alternate plant systems to the chlorina-f2 barleyfor studies on the organization and role ofChl b. The ch5 mutantsappear to be defective in a single nuclear gene as determined bycomplementation tests (8, 21) and appear to contain no Chl b.As in the chlorina-f2 mutant of barley (24), the absence of Chlb from the ch5/ch5 mutants results in an altered absorptionspectrum and the loss of the Chi b-containing pigment-proteincomplexes. The normally present A- 1 and A-2 pigment-proteincomplexes (20) are still present in the U374 mutant thylakoids.The sweetclover mutant also appears to have less protein mi-grating with the same mobility as the light-harvesting complexapoproteins relative to the normal plant than does the barleymutant.
It has been previously reported that the thylakoid proteinkinase activity of the chlorina-f2 mutant of barley is greatlyreduced and that the light-harvesting complex apoproteins arenot labeled (10). We previously reported finding protein kinaseactivity in chlorina-f2 thylakoids (25) and the present data cor-roborate the presence of protein kinase activity in the thylakoidsof both the barley and sweetclover mutants. It also appears thatthe phosphoproteins of the chlorina-f2 mutant barley may in-clude some of the light-harvesting complex apoproteins, knownto be present in amounts less than in the normal plants. Incontrast to the chlorina f-2 mutant of barley, the U374 mutantof sweetclover appears to have less phosphoprotein co-migratingwith the apoproteins of the light-harvesting complex.We feel that these data sufficiently characterize the Chl b-less
mutant of sweetclover to suggest its future usefulness as analternative to the chlorina f-2 barley in studies on photosynthesis
and thylakoid membrane function and organization.Acknowledgments-The authors are grateful to Ms. Kelli Stahlnecker and Dr.
Richard Dam for assistance with some of the experiments.
LITERATURE CITED
1. ANDERSON JM, JC WALDRON, SW THORNE 1978 Chlorophyll-protein com-plexes of spinach and barley thylakoids. Spectral characterization of sixcomplexes resolved by an improved electrophoretic procedure. FEBS Lett92: 227-233
2. ARNON DI 1949 Copper enzymes in isolated chloroplasts: polyphenoloxidasein Beta vulgaris. Plant Physiol 24: 1-15
3. BELLEMARE G, SG BARTLETT, NH CHUA 1982 Biosynthesis of chlorophyll a/b-binding polypeptides in wild type and the chlorina f2 mutant of barley. JBiol Chem 257: 7762-7767
4. BOARDMAN NK, HR HIGHKIN 1966 Studies on a barley mutant lackingchlorophyll b. I. Photochemical activity of isolated chloroplasts. BiochimBiophys Acta 126: 189-199
5. BURKE JJ, KE STEINBACK, CJ ARNTZEN 1979 Analysis of the light-harvestingpigment-protein complex of wild type and a chlorophyll b-less mutant ofbarley. Plant Physiol 63: 237-243
6. DuYSEN ME, TP FREEMAN, ND WILLIAMS, LL OLSON 1984 Regulation ofexcitation energy in a wheat mutant deficient in light-harvesting pigmentprotein complex. Plant Physiol 76: 561-566
7. FREEMAN TP, ME DUYSEN, NH OLSON, ND WILLIAMS 1982 Electron transportand chloroplast ultrastructure of a chlorophyll deficient mutant of wheat.Photosynth Res 3: 179-189
8. GENGENBACH BG, HJ GORZ, FA HASKINS 1970 Genetic studies of inducedmutants in Melilotus alba. II. Inheritance and complementation of chloro-phyll-deficient mutants. Crop Sci 10: 154-156
9. GOODCHILD DJ, HR HIGHKIN, NK BOARDMAN 1966 The fine structure ofchloroplasts in a barley mutant lacking chlorophyll b. Exp Cell Res 43: 684-688
10. HAWORTH P, DJ KYLE, CJ ARNTZEN 1982 Phosphorylation of the light-harvesting complex in wild-type, chlorophyll b-less, and intermittent light-grown barley. Arch Biochem Biophys 218: 199-206
12. HIGHKIN HR, NK BOARDMAN, DJ GOODCHILD 1969 Photosynthetic studieson a pea-mutant deficient in chlorophyll. Plant Physiol 44: 1310-1320
13. HIGHKIN HR, A FRENKEL 1962 Studies on growth and metabolism of a barleymutant lacking chlorophyll b. Plant Physiol 37: 814-820
14. HIRONO Y, GP REDEI 1963 Multiple allelic control of chlorophyll b level inArabidopsis thaliana. Nature 197: 1324-1325
15. HOPKINS WG, DB HAYDEN, MG NEUFFER 1980 A light-sensitive mutant inmaize (Zea mays L.) 1. Chlorophyll, chlorophyll-protein and ultrastructuralstudies. Z Pflanzenphysiol 99: 417-426
16. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685
17. MARKWELL JP, NR BAKER, M BRADBURY, JP THORNBER 1984 Use of zincions to study thylakoid protein phosphorylation and the state 1-state 2transition in vitro. Plant Physiol 74: 348-354
18. MARKWELL JP, NR BAKER, JP THORNBER 1982 Metabolic regulation of thethylakoid protein kinase. FEBS Lett 142: 171-174
19. MARKWELL JP, S REINMAN, JP THORNBER 1978 Chlorophyll-protein com-plexes from higher plants: a procedure for improved stability and fractiona-tion. Arch Biochem Biophys 190: 136-141
20. MILES CD, JP MARKWELL, JP THORNBER 1979 Effect of nuclear mutation inmaize on photosynthetic activity and content of chlorophyll-protein com-plexes. Plant Physiol 64: 690694
21. RONNENKAMP RR, HJ GORZ, FA HASKINS 1975 Genetic studies of inducedmutants in Melilotus alba. IV. Inheritance and complementation of sixadditional chlorophyll-deficient mutants. Crop Sci 15: 187-188
22. SIMPSON D 1980 Freeze-fracture studies on barley plastid membranes IV.Analysis of freeze-fracture particle size and shape. Carlsberg Res Commun45: 201-210
23. SPECHT JE, FA HASKINS, HJ GORZ 1975 Contents of chlorophylls a and b inchlorophyll-deficient mutants of sweetclover. Crop Sci 15: 851-853
24. THORNBER JP, HR HIGHKIN 1974 Composition of the photosynthetic appa-ratus of normal barley leaves and a mutant lacking chlorophyll b. Eur JBiochem 41: 109-116
25. THORNBER P, J MARKWELL, N BAKER, M BAKER 1982 Protein kinase in thebarley chlorina-f2 mutant. Plant Physiol 69: S-31
