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Characterization of Chloroplasts in a Tinged (tn) Mutant of Maize (Zea mays L.)
J.PlantPhysiol. Vol. 134.pp. 81-84 (1989)
Characterization of Chloroplasts in a Tinged (tn) Mutant of Maize (Zea mays L.) P. A.
TAYLOR
and W. G.
HOPKINS
Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7 Received August 19, 1988 . Accepted October 17, 1988
Summary Thylakoid composition and photochemical properties of a yellow-green tinged (tn) allele at the oil-yellow locus of maize (Zea mays L.) are described. Phenotypic alterations in the mutant seedling include lower chlorophyll content, elevated chlorophyll alb ratios and suppressed accumulation of chlorophyll alb light-harvesting pigment protein (LHCII). Loss of LHCII is accompanied by a decrease in the content of both of the two LHCII polypeptides. Rates of electron transport are increased by up to 200 percent and there is an almost complete loss of Mg2+ -mediated control over excitation energy distribution. These results are consistent with the argument that mutations at the oil-yellow locus represent an over-expression of the normal photoadaptation mechanisms.
Key words: Zea mays L., chloroplasts, electron transport, light·sensitive, LHCII, photoadaptation. Introduction In earlier publications, Hopkins et al. have described a chlorophyll b-deficient maize mutant designated Oil-yellow, yellow-green (Oy-ygl +) (1980 a, 1980 b). The principal effect of this mutation is to suppress the accumulation of the chlorophyll alb light harvesting complex, LHCII.Oy-ygl + chloroplasts exhibit a several fold increase in electron transport activity at saturating light intensities, a virtual absence of grana stacking and a loss of Mg2 +-mediated control of excitation energy distribution. Unlike other chlorophyll b-deficient mutants, the expression of the Oy·yg phenotype is light intensity-dependent. Hopkins et al. (1980 a, 1980 b) suggested that the wild-type allele at this locus may be responsible for normal adaptation to high light and that the Oy-yg mutation represents an extreme expression of that control. More recently, Greene et al. (1987) reported evidence that the Oy-yg mutation results in a selective reduction of the LHCI and of mobile LHcn complexes, both characteristic of the photoadaption response. In order to obtain further information about the oil-yellow locus and its possible involvement in the photoadaptation response, we have examined thylakoid composition and photochemical properties of another allele at that locus which is designated tinged (tn). © 1989 by Gustav Fischer Verlag, Stuttgart
Materials and Methods Plant material and growth conditions The material used in this study was a yellow-green mutant of Zea mays L. designated as the tinged (tn) allele at the oil-yellow locus of chromosome 10 (D.B. Walden, personal communication). Control seedlings were Ohio 43 x tn. Seedlings were grown in soil in controlled environment chambers under a 16 hour photoperiod. The day (night) temperature regime was 28°C (26°C). Light was provided by high output fluorescent lamps supplemented with incandescent (15 to 20 percent of total wattage). Intensity was eighter 95 or 480 /Lmol quata m -2 S-I. Intensity was varied by adjusting the lamp to plant distance. These two intensities were selected since they represent the extremes for expression of the Oy-yg mutant as reported by Hopkins et al. (1980 a}. Seedlings were usually in the 3rd or 4th leaf stage 10 days after sowing. The primary leaf was excluded from all analyses. Light intensity was measured as photosynthetically active radiation (PAR) with a Licor Model LI-185 quantum sensor (Lambda Instruments, Lincoln, Nebraska).
Chloroplast isolation Mesophyll chloroplasts were isolated by gringing in a cold mortar and pestle with a medium consisting of 50mM Natricine (pH 7.8), 0.6 M sucrose, 10 mM NaCI and 5 % (w/v) PVP-40 (Hopkins et
82
P. A. TAYLOR and W. G. HOPKINS
al., 1980 a). The homogenate was filtered through two layers of Miracloth and centrifuged for 10 min at 1500 x g and 4°C. The pellet was washed with unbuffered 10 mM NaCI, centrifuged 10 min at 3000 xg and 4°C and resuspended in minimal volume of 1 mM Natricine, pH 7.0, containing 10 mM NaCI. Chlorophyll a and b were determined in 80 percent acetone by the method of Arnon (1949). SDS-Acrylamide gel electrophoresis
Membrane chlorophyll-protein complexes were resolved by SDSpolyacrylamide gel electrophoresis according to Hayden and Hopkins (1976, 1977). Washed thylakoids were suspended in 0.1 M Tris-HCI (pH 8.0) containing 0.5 % SDS (SDS: ChI = 8: 1) for 1 hour at 25°C in the dark. Following electrophoresis, gels were scanned with a Joyce-Lobel densitometer. Relative proportions of the chlorophyll-protein complexes were estimated on the basis of the area under the curve in densitometer tracings. Lipid extracted thylakoids were solubilized according to the procedures of Henriques et al. (1975). The solubilized protein was electrophoresed on a 10-20% polyacrylamide gradient slab gel using the discontinuous buffer system of Laemmli (1970). Gels were fixed in acetic acid: methanol: water (7.5: 20: 72.5), stained one hour with 0.1 % Coomassie Brilliant Blue R250 in acetic acid: methanol: water (1 : 4: 5) and destained in the fixing solution. Photochemical activities
Electron transport was measured either polarigraphically with a Clark-type oxygen electrode or sprectrophotometrically as reduction of 2,6-dichlorophenol indophenol (DCPIP) as described by Hopkins et al. (1980 b). Fluorescence emission kinetics were measured as previously described (Hopkins et aI., 1980 b) Chloroplasts were diluted to a concentration of 10/Lg chlorophylllmL in 10 mM Na-tricine, pH 7.8 containing 10 mM NaCI. Excitation light provided by a Fiber-Lite (Model 170-D) high intensity illuminator was passed through a Corning CS4-96 blue filter. Chlorophyll fluorescence was maeasured at right angles by a Phototops UDT-500 photodiode (United Detector Technology) blocked with a corning CS2-S8 red filter. The output was recorded on a Houston 2000 fast response X - Y plotter.
Results
Light intensity and chlorophyll content The tn phenotype is a morphologically normal, yellowgreen seedling. In preliminary experiments, the chlorophyll contents of 10 day old tn seedlings grown at either 95 or 480 /Lmol quanta m- 2s- l were found to be significantly reduced compared to controls grown at the same light intensities (data not shown)_ The reduced chlorophyll content is reflected in the lower chlorophyll yield in the isolated mesophyll chloroplast fraction (Table 1). The yield of chlorophyll from Table 1: Chlorophyll yield and chlorophyll alb ratios of mesophyll chloroplast fractions isolated from 10 day old control and tn seedlings grown under two different light intensities. Values for chlorophyll yield are mgts.e. per g fresh weight of starting material. Light Intensityl
Chlorophyll Yield control tn
95 O.800tO.OS O.120±O.Ol 480 0.680±O.Ol 0.060±O.Ol 1 /Lmol quanta m - 2 S - 1 PAR.
green
ChI alb tn
2.9±0.03 3.2±O.06
6.S±0.28 10.6±O.53
the mutant seedlings grown a low light intensity was only 15 percent of the control while a t high light intensity the yield was further reduced to 9 percent of the control. Chorophyll alb ratios were significantly higher in chloroplasts from mutant seedlings grown at either light intensity, indicating a preferential reduction in the chlorophyll b content. Although chlorophyll accumulation is marginally lower in seedlings grown at the higher of the two light intensities, the suppression of chlorophyll accumulation in the tinged mutant appears to be relatively independent of light intensity within the range of 95 to 480 /tmole quanta.
Chlorophyll-protein complexes Chlorophyll-protein complexes of the SDS-solubilized thylakoid membranes from both mutant and control seedlings were resolved by acrylamide gel electrophoresis_ The relative proportions of the LHcn and CPI complexes as a function of light intensity are summarized in Table 2. The relative proportion of the chlorophyll b-containing LHCn is markedly lower in the mutant seelings compared with the control, regardless of light intensity_ At low light intensity, the relative proportion of CPI is nearly 50 percent higher in the mutant than in the control. Growth at high light intensity causes a reduction in the relative proportion of CPI as well as a further reduction in the proportion of LHcn in the mutant. There was a concomitant increase in the proportion of free pigment (data not shown). This result is consistent with the observed increase in the chlorophyll alb ratios under the same conditions (Table 1). The ratio of CPII LHCn remains relatively constant in both the mutant and the controls, regardless of light intensity_ A reduction in the relative proportion of LHcn is commonly accompanied by a loss of one or both of its major polypeptides (Burke et aI., 1978; Hopkins et aI., 1980 a). Thylakoid polypeptides from chloroplasts of seedlings grown at both 95 /tmol quanta and 480 /tmol quanta were resolved by SDS-polyacrylamide gel electrophoresis. The results are shown in Fig. 1. The indentity of the LHcn apoprotein was confirmed by co-electrophoresis with purified LHcn (Burke et aI., 1978). The polypeptide profiles of the yellow-green mutant seedlings reveal only small amounts of the 23 kD and 25 kD LHCn apoprotein when compared with the normal pattern of the controls. This is true of seedlings grown at either light intensity. In both cases, the ob-
Table 2: Relative proportions of chlorophyll-protein complexes resolved by SDS-acrylamide gel electrophoresis. Tinged (tn) and control seedlings were grown at the indicated light intensities for 10 days. Relative proportions are based on the area under the curve on densitometric tracings. Proportions are given as percent of the total area±s.e. Light Intensityl
LHCn2
Cpe
CPI LHcn tn control control tn control tn 9S 12.9t 1.7 33.0tO.3 21.9t 1.5 1S.0t 1.0 1.7 0.5 480 6.4tO.7 32.3± 1.9 13.0tO.2 12.8tO.S 1.9 0.4 1 /Lmol quanta m - 2 S - 1 PAR. 2 percentage of total.
tn
mutant of maize
83
Table 4: The effect of Mi+ on variable fluorescence yield in mesophyll chloroplasts isolated from control and tn seedlings grown at two different light intensities. Light intensity I 95 control tn 480 control
6F/Fo (+Mi+) 6F/Fo (_Mg2+) 6F/Fo (+Mg2+) 6F/Fo (-Mi+)
1.5 ±0.062 1.2±0.06 1.4 ± 0.04 tn 1.4±0.08 I /Lmol quanta m - 2 S- I PAR. 2 mean ± s.e.
25
•.
~
..
II.>
A
1.6 1.4 1.5 1.1
measured in chloroplasts isolated from seedlings grown at either of the light intensities. These results are shown in Table 3. Although we experienced quantitative variation among isolations, the trends were consistent. Rates of electron transport in chloroplasts from seedlings grown at 95 ",mol quanta were, on a unit chlorophyll basis, roughly 1.25 to 1.9 times greater in the mutant chloroplasts than in the controls. In chloroplasts from seedlings grown at 480 ",mol quanta, electron transport was, with the exception of water to DCPIP transport, approximately twice as fast in the mutants. Mutant chloroplasts also show a loss of Mg2+mediated control of variable fluorescence yield (Table 4).
_
2~·
2.4 ± 0.23 1.7±0.12 2.1±0.07 1.5±0.06
c
B
o
11-
E
Fig. 1: SDS-polyacrylamide slab gel electrophoresis of thylakoid polypeptides from tinged (tn) and control mesophyll chloroplasts grown at 95 and 480 /Lmol quanta m- 2s- 1 PAR. Lane A, purified LHCII; lane B, tn (95/Lmole quanta); lane C, control (95/Lmole quanta); lane D, tn (480 /Lmol quanta); lane E, control (480 /Lmol quanta). Table 3: Photosynthetic electron transport activities of mesophyll chloroplasts isolated from control and mutant (tn) seedlings grown at two light intensities. Each value is the mean of 6 measurements, using 3 separate chloroplast preparations. Bracketed values indicate the range between high and low values. Light intensity I 95 control
tn
DCPIPH2 - My2 883(203} 465(83} H 20 -DCPlp3 202 (24) 272 (68) H 20-My2 34 (8) 44 (11) I /Lmol quanta m -2 S-I PAR. 2 /Lmol O 2 consumed mg chl- I hr - I. 3 /Lmol DCPIP reduced mg chl- I hr - I.
480 tn 345(69} 628(188} 182(22} 192 (58) 73(11} 151 (30)
control
served decrease in the amount of LHCII in chloroplasts from the mutant seedlings (Table 2) is accompanied by a corresponding decrease in both associated polypeptides (Fig. 1).
Photochemical properties of isolated mesophyll chloroplasts The impact of the observed reduction in amount of LHCII was assessed in two ways. First, electron transport rates were
Discussion The tinged mutant is allelic to the Oil-yellow, yellowgreen mutant previously described by Hopkins et aI. (1980 a, 1980 b). In many respects, the impact of both mutations on chloroplast composition and function appears to be similar. At relatively high light intensities, the phenotypes of both mutations are characterized by lower chlorophyll content and higher chlorophyll alb ratios. There is a marked decrease in the relative content of light harvesting pigment protein (LHCII) and a decline in the amount of CPI (PSI and LHCI). Also in both mutations, the loss of LHCII is accompanied by a decrease in the content of the two polypeptides normally associated with LHCII. There is an approximately two-fold increase in the rate of electron transport at saturating light internsities and almost complete loss of M g2+ -mediated control over variable fluorescence yield. Both of these results are consistent with a loss of the chlorophyll b-containing LHCII complex, but without any significant loss of the photochemical reaction centers. Phenotypic expression of the Oy-yg mutant is light intensity dependent. At low light intensities, the Oy-yg mutant generally resembles the controls (Hopkins et aI., 1980 a). Chlorophyll yield in the mesophyll chloroplast fraction of Oy-yg seedlings grown under low light was more than 60 percent of control and alb ratios were 3.3 against 2.9 for the controls. The relative proportions of both LHCII and CPI were similar in mutants and controls. In the present study of the tn mutant, however, at 95 ",mol quanta m-Is- I , the mutant had only 15 percent the amount of chlorophyll in the mesophyll chloroplast fraction and the chlorophyll alb ratio was more than twice that of the controls. Similarly, our results for the chlorophyll protein complexes, thylakoid poly-
84
P. A. TAYLOR and W. G. HOPKINS
peptide analysis and photochemical measurements all indicate that the tn allele expresses more strongly at low light levels than the Oy-yg allele previously described (Hopkins et al., 1980a, 1980b). Although expression of the tn allele appears to be less sensitive to light intensity than the Oyyg allele, this is probably due only to the fact that it already expresses well under low light. The oil-yellow locus has been mapped to chromosome 10 in the nuclear genome of maize. The most pronounced impact of both the Oy-yg and tn alleles is to suppress the accumulation of LHcn apoprotein, which is known to be nuclear-encoded and synthesized on cytoplasmic ribosomes (Apel and Kloppstech, 1978; Schmidt et al., 1980). It is tempting to speculate that the oil-yellow locus might be the site of the gene for the LHcn apoprotein. The gene could also playa regulatory role in the post-translational uptake of the precursor protein into the chloroplast. Although the identity of the light-sensitive sequence remains unkown, the similarities in phenotypic expression between the tn allele describe here and the Oy-yg allele described earlier (Hopkins et al., 1980 a, 1980 b) suggest that sensitivity to light intensity is an inherent charateristic of the oil-yellow locus. It is well established that plants can adjust their photosynthetic apparatus in response to changing light intensities. The normal response to high light intensities is a reduction in the amount of chlorophyll b-containing antenna complexes in the thylakoid membrane (Boardman, 1977; Greene et al., 1988). The results of this study add further support to the suggestion that mutations at the oil-yellow locus represent an over-expression of the normal photoadaptation mechanism (Hopkins et al., 1980 b; Greene et al., 1987). Acknowledgements We thank Dr. D. B. Walden for providing the tn seed. This work was supported by an operating grant to W.G.H. from the Natural Sciences and Engineering Research Council of Canada.
References ApEL, K. and K. KLOPPSTECH: The plastid membranes of barley. Light-induced appearance of mRNA coding for the apoprotein
of the light-harvesting chlorophyll alb binding protein. Eur. Biochem. 85, 581-588 (1978).
J.
ARNON, D.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physio!. 24, 1-15 (1949). BOARDMAN, N. K.: Comparative photosynthesis of sun and shade plants. Ann. Tev. Plant Physio!. 28, 355-377 (1977).
BURKE, J. J., c. L. DITTO, and C. J. ARNTZEN: Involvement of the light-harvesting complex in cation regulation of excitation energy distribution in chloroplasts. Arch. Biochem. Biophys. 187, 252 - 263 (1978).
GREENE, B., D. R. ALLRED, D. MORISHIGE, and L. A. STAEHELIN: A light-sensitive photoregulatory mutant in maize deficient in LHCI and the "mobile" chlorophyll alb LHCIr. In: BIGGINS, J. (ed.): Progress in Photosynthesis Research. Vo!' II, 697-700. Martinus Nijhoff, Dordrecht (1987). - - - - Hierarchical response of light harvesting chlorophyllproteins in a light-sensitive chlorophyll b-deficient mutant of maize. Plant Physio!. 87, 357 -364 (1988). HAYDEN, D. B. and W. G. HOPKINS: Membrane polypeptides and chlorophyll protein complexes of maize mesophyll chloroplasts. Can. J. Bot. 54, 1684-1689 (1976). - - A second distinct chlorophyllprotein complex in maize mesophyll chloroplasts. Can. J. Bot. 55,2525-2529 (1977). HENRIQUES, F., W. VAUGHAN, and R. PARK: High resolution gel electrophoresis of chloroplast membrane polypeptides. Plant Physiol. 55,338 -339 (1975). HOPKINS, W. G., J. B. GERMAN, and D. B. HAYDEN: A light-sensitive mutant in maize (Zea mays L.) II. Photosynthetic properties. Z. Pflanzenphysio!. 100, 15-24 (1980 a). HOPKINS, W. G., D. B. HAYDEN, and M. G. NEUFFER: A light- sensitive mutant in maize (Zea mays L.) r. Chlorophyll, chlorophyllprotein and ultrastructural studies. Z. Pflanzenphysio!. 99, 417-426 (1980 b).
LAEMMLI, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680-685 (1970). SCHMIDT, G. W., S. BARTLETT, A. R. GROSSMAN, A. R. CASHMORE, and N. H. CHUA: In vitro synthesis, transport and assembly of the constituent polypeptides of the light-harvesting chlorophyll alb protein complex. In: LEAVER, C. J. (ed.): Genome organization and expression in plants. 337 - 351. Plenum, New Yorkl London (1980). VENABLE, J. H. and R. COGGESHALL: A simplified lead citrate stain for use in electron microscopy. J. Cell Bio!. 25, 407 -408 (1965).
Characterization of Chloroplasts in a Tinged (tn) Mutant of Maize (Zea mays L.) P. A.
TAYLOR
and W. G.
HOPKINS
Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7 Received August 19, 1988 . Accepted October 17, 1988
Summary Thylakoid composition and photochemical properties of a yellow-green tinged (tn) allele at the oil-yellow locus of maize (Zea mays L.) are described. Phenotypic alterations in the mutant seedling include lower chlorophyll content, elevated chlorophyll alb ratios and suppressed accumulation of chlorophyll alb light-harvesting pigment protein (LHCII). Loss of LHCII is accompanied by a decrease in the content of both of the two LHCII polypeptides. Rates of electron transport are increased by up to 200 percent and there is an almost complete loss of Mg2+ -mediated control over excitation energy distribution. These results are consistent with the argument that mutations at the oil-yellow locus represent an over-expression of the normal photoadaptation mechanisms.
Key words: Zea mays L., chloroplasts, electron transport, light·sensitive, LHCII, photoadaptation. Introduction In earlier publications, Hopkins et al. have described a chlorophyll b-deficient maize mutant designated Oil-yellow, yellow-green (Oy-ygl +) (1980 a, 1980 b). The principal effect of this mutation is to suppress the accumulation of the chlorophyll alb light harvesting complex, LHCII.Oy-ygl + chloroplasts exhibit a several fold increase in electron transport activity at saturating light intensities, a virtual absence of grana stacking and a loss of Mg2 +-mediated control of excitation energy distribution. Unlike other chlorophyll b-deficient mutants, the expression of the Oy·yg phenotype is light intensity-dependent. Hopkins et al. (1980 a, 1980 b) suggested that the wild-type allele at this locus may be responsible for normal adaptation to high light and that the Oy-yg mutation represents an extreme expression of that control. More recently, Greene et al. (1987) reported evidence that the Oy-yg mutation results in a selective reduction of the LHCI and of mobile LHcn complexes, both characteristic of the photoadaption response. In order to obtain further information about the oil-yellow locus and its possible involvement in the photoadaptation response, we have examined thylakoid composition and photochemical properties of another allele at that locus which is designated tinged (tn). © 1989 by Gustav Fischer Verlag, Stuttgart
Materials and Methods Plant material and growth conditions The material used in this study was a yellow-green mutant of Zea mays L. designated as the tinged (tn) allele at the oil-yellow locus of chromosome 10 (D.B. Walden, personal communication). Control seedlings were Ohio 43 x tn. Seedlings were grown in soil in controlled environment chambers under a 16 hour photoperiod. The day (night) temperature regime was 28°C (26°C). Light was provided by high output fluorescent lamps supplemented with incandescent (15 to 20 percent of total wattage). Intensity was eighter 95 or 480 /Lmol quata m -2 S-I. Intensity was varied by adjusting the lamp to plant distance. These two intensities were selected since they represent the extremes for expression of the Oy-yg mutant as reported by Hopkins et al. (1980 a}. Seedlings were usually in the 3rd or 4th leaf stage 10 days after sowing. The primary leaf was excluded from all analyses. Light intensity was measured as photosynthetically active radiation (PAR) with a Licor Model LI-185 quantum sensor (Lambda Instruments, Lincoln, Nebraska).
Chloroplast isolation Mesophyll chloroplasts were isolated by gringing in a cold mortar and pestle with a medium consisting of 50mM Natricine (pH 7.8), 0.6 M sucrose, 10 mM NaCI and 5 % (w/v) PVP-40 (Hopkins et
82
P. A. TAYLOR and W. G. HOPKINS
al., 1980 a). The homogenate was filtered through two layers of Miracloth and centrifuged for 10 min at 1500 x g and 4°C. The pellet was washed with unbuffered 10 mM NaCI, centrifuged 10 min at 3000 xg and 4°C and resuspended in minimal volume of 1 mM Natricine, pH 7.0, containing 10 mM NaCI. Chlorophyll a and b were determined in 80 percent acetone by the method of Arnon (1949). SDS-Acrylamide gel electrophoresis
Membrane chlorophyll-protein complexes were resolved by SDSpolyacrylamide gel electrophoresis according to Hayden and Hopkins (1976, 1977). Washed thylakoids were suspended in 0.1 M Tris-HCI (pH 8.0) containing 0.5 % SDS (SDS: ChI = 8: 1) for 1 hour at 25°C in the dark. Following electrophoresis, gels were scanned with a Joyce-Lobel densitometer. Relative proportions of the chlorophyll-protein complexes were estimated on the basis of the area under the curve in densitometer tracings. Lipid extracted thylakoids were solubilized according to the procedures of Henriques et al. (1975). The solubilized protein was electrophoresed on a 10-20% polyacrylamide gradient slab gel using the discontinuous buffer system of Laemmli (1970). Gels were fixed in acetic acid: methanol: water (7.5: 20: 72.5), stained one hour with 0.1 % Coomassie Brilliant Blue R250 in acetic acid: methanol: water (1 : 4: 5) and destained in the fixing solution. Photochemical activities
Electron transport was measured either polarigraphically with a Clark-type oxygen electrode or sprectrophotometrically as reduction of 2,6-dichlorophenol indophenol (DCPIP) as described by Hopkins et al. (1980 b). Fluorescence emission kinetics were measured as previously described (Hopkins et aI., 1980 b) Chloroplasts were diluted to a concentration of 10/Lg chlorophylllmL in 10 mM Na-tricine, pH 7.8 containing 10 mM NaCI. Excitation light provided by a Fiber-Lite (Model 170-D) high intensity illuminator was passed through a Corning CS4-96 blue filter. Chlorophyll fluorescence was maeasured at right angles by a Phototops UDT-500 photodiode (United Detector Technology) blocked with a corning CS2-S8 red filter. The output was recorded on a Houston 2000 fast response X - Y plotter.
Results
Light intensity and chlorophyll content The tn phenotype is a morphologically normal, yellowgreen seedling. In preliminary experiments, the chlorophyll contents of 10 day old tn seedlings grown at either 95 or 480 /Lmol quanta m- 2s- l were found to be significantly reduced compared to controls grown at the same light intensities (data not shown)_ The reduced chlorophyll content is reflected in the lower chlorophyll yield in the isolated mesophyll chloroplast fraction (Table 1). The yield of chlorophyll from Table 1: Chlorophyll yield and chlorophyll alb ratios of mesophyll chloroplast fractions isolated from 10 day old control and tn seedlings grown under two different light intensities. Values for chlorophyll yield are mgts.e. per g fresh weight of starting material. Light Intensityl
Chlorophyll Yield control tn
95 O.800tO.OS O.120±O.Ol 480 0.680±O.Ol 0.060±O.Ol 1 /Lmol quanta m - 2 S - 1 PAR.
green
ChI alb tn
2.9±0.03 3.2±O.06
6.S±0.28 10.6±O.53
the mutant seedlings grown a low light intensity was only 15 percent of the control while a t high light intensity the yield was further reduced to 9 percent of the control. Chorophyll alb ratios were significantly higher in chloroplasts from mutant seedlings grown at either light intensity, indicating a preferential reduction in the chlorophyll b content. Although chlorophyll accumulation is marginally lower in seedlings grown at the higher of the two light intensities, the suppression of chlorophyll accumulation in the tinged mutant appears to be relatively independent of light intensity within the range of 95 to 480 /tmole quanta.
Chlorophyll-protein complexes Chlorophyll-protein complexes of the SDS-solubilized thylakoid membranes from both mutant and control seedlings were resolved by acrylamide gel electrophoresis_ The relative proportions of the LHcn and CPI complexes as a function of light intensity are summarized in Table 2. The relative proportion of the chlorophyll b-containing LHCn is markedly lower in the mutant seelings compared with the control, regardless of light intensity_ At low light intensity, the relative proportion of CPI is nearly 50 percent higher in the mutant than in the control. Growth at high light intensity causes a reduction in the relative proportion of CPI as well as a further reduction in the proportion of LHcn in the mutant. There was a concomitant increase in the proportion of free pigment (data not shown). This result is consistent with the observed increase in the chlorophyll alb ratios under the same conditions (Table 1). The ratio of CPII LHCn remains relatively constant in both the mutant and the controls, regardless of light intensity_ A reduction in the relative proportion of LHcn is commonly accompanied by a loss of one or both of its major polypeptides (Burke et aI., 1978; Hopkins et aI., 1980 a). Thylakoid polypeptides from chloroplasts of seedlings grown at both 95 /tmol quanta and 480 /tmol quanta were resolved by SDS-polyacrylamide gel electrophoresis. The results are shown in Fig. 1. The indentity of the LHcn apoprotein was confirmed by co-electrophoresis with purified LHcn (Burke et aI., 1978). The polypeptide profiles of the yellow-green mutant seedlings reveal only small amounts of the 23 kD and 25 kD LHCn apoprotein when compared with the normal pattern of the controls. This is true of seedlings grown at either light intensity. In both cases, the ob-
Table 2: Relative proportions of chlorophyll-protein complexes resolved by SDS-acrylamide gel electrophoresis. Tinged (tn) and control seedlings were grown at the indicated light intensities for 10 days. Relative proportions are based on the area under the curve on densitometric tracings. Proportions are given as percent of the total area±s.e. Light Intensityl
LHCn2
Cpe
CPI LHcn tn control control tn control tn 9S 12.9t 1.7 33.0tO.3 21.9t 1.5 1S.0t 1.0 1.7 0.5 480 6.4tO.7 32.3± 1.9 13.0tO.2 12.8tO.S 1.9 0.4 1 /Lmol quanta m - 2 S - 1 PAR. 2 percentage of total.
tn
mutant of maize
83
Table 4: The effect of Mi+ on variable fluorescence yield in mesophyll chloroplasts isolated from control and tn seedlings grown at two different light intensities. Light intensity I 95 control tn 480 control
6F/Fo (+Mi+) 6F/Fo (_Mg2+) 6F/Fo (+Mg2+) 6F/Fo (-Mi+)
1.5 ±0.062 1.2±0.06 1.4 ± 0.04 tn 1.4±0.08 I /Lmol quanta m - 2 S- I PAR. 2 mean ± s.e.
25
•.
~
..
II.>
A
1.6 1.4 1.5 1.1
measured in chloroplasts isolated from seedlings grown at either of the light intensities. These results are shown in Table 3. Although we experienced quantitative variation among isolations, the trends were consistent. Rates of electron transport in chloroplasts from seedlings grown at 95 ",mol quanta were, on a unit chlorophyll basis, roughly 1.25 to 1.9 times greater in the mutant chloroplasts than in the controls. In chloroplasts from seedlings grown at 480 ",mol quanta, electron transport was, with the exception of water to DCPIP transport, approximately twice as fast in the mutants. Mutant chloroplasts also show a loss of Mg2+mediated control of variable fluorescence yield (Table 4).
_
2~·
2.4 ± 0.23 1.7±0.12 2.1±0.07 1.5±0.06
c
B
o
11-
E
Fig. 1: SDS-polyacrylamide slab gel electrophoresis of thylakoid polypeptides from tinged (tn) and control mesophyll chloroplasts grown at 95 and 480 /Lmol quanta m- 2s- 1 PAR. Lane A, purified LHCII; lane B, tn (95/Lmole quanta); lane C, control (95/Lmole quanta); lane D, tn (480 /Lmol quanta); lane E, control (480 /Lmol quanta). Table 3: Photosynthetic electron transport activities of mesophyll chloroplasts isolated from control and mutant (tn) seedlings grown at two light intensities. Each value is the mean of 6 measurements, using 3 separate chloroplast preparations. Bracketed values indicate the range between high and low values. Light intensity I 95 control
tn
DCPIPH2 - My2 883(203} 465(83} H 20 -DCPlp3 202 (24) 272 (68) H 20-My2 34 (8) 44 (11) I /Lmol quanta m -2 S-I PAR. 2 /Lmol O 2 consumed mg chl- I hr - I. 3 /Lmol DCPIP reduced mg chl- I hr - I.
480 tn 345(69} 628(188} 182(22} 192 (58) 73(11} 151 (30)
control
served decrease in the amount of LHCII in chloroplasts from the mutant seedlings (Table 2) is accompanied by a corresponding decrease in both associated polypeptides (Fig. 1).
Photochemical properties of isolated mesophyll chloroplasts The impact of the observed reduction in amount of LHCII was assessed in two ways. First, electron transport rates were
Discussion The tinged mutant is allelic to the Oil-yellow, yellowgreen mutant previously described by Hopkins et aI. (1980 a, 1980 b). In many respects, the impact of both mutations on chloroplast composition and function appears to be similar. At relatively high light intensities, the phenotypes of both mutations are characterized by lower chlorophyll content and higher chlorophyll alb ratios. There is a marked decrease in the relative content of light harvesting pigment protein (LHCII) and a decline in the amount of CPI (PSI and LHCI). Also in both mutations, the loss of LHCII is accompanied by a decrease in the content of the two polypeptides normally associated with LHCII. There is an approximately two-fold increase in the rate of electron transport at saturating light internsities and almost complete loss of M g2+ -mediated control over variable fluorescence yield. Both of these results are consistent with a loss of the chlorophyll b-containing LHCII complex, but without any significant loss of the photochemical reaction centers. Phenotypic expression of the Oy-yg mutant is light intensity dependent. At low light intensities, the Oy-yg mutant generally resembles the controls (Hopkins et aI., 1980 a). Chlorophyll yield in the mesophyll chloroplast fraction of Oy-yg seedlings grown under low light was more than 60 percent of control and alb ratios were 3.3 against 2.9 for the controls. The relative proportions of both LHCII and CPI were similar in mutants and controls. In the present study of the tn mutant, however, at 95 ",mol quanta m-Is- I , the mutant had only 15 percent the amount of chlorophyll in the mesophyll chloroplast fraction and the chlorophyll alb ratio was more than twice that of the controls. Similarly, our results for the chlorophyll protein complexes, thylakoid poly-
84
P. A. TAYLOR and W. G. HOPKINS
peptide analysis and photochemical measurements all indicate that the tn allele expresses more strongly at low light levels than the Oy-yg allele previously described (Hopkins et al., 1980a, 1980b). Although expression of the tn allele appears to be less sensitive to light intensity than the Oyyg allele, this is probably due only to the fact that it already expresses well under low light. The oil-yellow locus has been mapped to chromosome 10 in the nuclear genome of maize. The most pronounced impact of both the Oy-yg and tn alleles is to suppress the accumulation of LHcn apoprotein, which is known to be nuclear-encoded and synthesized on cytoplasmic ribosomes (Apel and Kloppstech, 1978; Schmidt et al., 1980). It is tempting to speculate that the oil-yellow locus might be the site of the gene for the LHcn apoprotein. The gene could also playa regulatory role in the post-translational uptake of the precursor protein into the chloroplast. Although the identity of the light-sensitive sequence remains unkown, the similarities in phenotypic expression between the tn allele describe here and the Oy-yg allele described earlier (Hopkins et al., 1980 a, 1980 b) suggest that sensitivity to light intensity is an inherent charateristic of the oil-yellow locus. It is well established that plants can adjust their photosynthetic apparatus in response to changing light intensities. The normal response to high light intensities is a reduction in the amount of chlorophyll b-containing antenna complexes in the thylakoid membrane (Boardman, 1977; Greene et al., 1988). The results of this study add further support to the suggestion that mutations at the oil-yellow locus represent an over-expression of the normal photoadaptation mechanism (Hopkins et al., 1980 b; Greene et al., 1987). Acknowledgements We thank Dr. D. B. Walden for providing the tn seed. This work was supported by an operating grant to W.G.H. from the Natural Sciences and Engineering Research Council of Canada.
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