Planta

Planta (1985)164:480-486

9 Springer-Verlag 1985

Comparison of photosynthetic parameters of an aurea mutant (Su/su) of tobacco and the wild-type by the photoacoustic method O. Canaani, Z. Motzan and S. Malkin Biochemistry Department, Weizmann Institute of Science, Rehovot 76100, Israel

Abstract. Oxygen evolution and energy storage yields in tobacco (Nicotiana tabacum L.) wild-type (cv. John Williams Broadleaf) and a mutant (Su/ su) deficient in chlorophyll were compared using the photoacoustic technique. Oxygen-evolution and energy-storage quantum yields in the mutant were higher when measured in red light (640-690 nm) than green or blue light (540 nm and 440 nm, respectively), indicating that carotenoids in this mutant do not transfer energy efficiently to the photochemical reaction centers. It is suggested that carotenoids may play a role in protecting the photosynthetic apparatus against damage by high energy fluxes. In the wild-type, the oxygenevolution yield did not change drastically throughout the visible spectrum. The mutant had a higher quantum yield of oxygen evolution than the wildtype. Similarly maximum rates obtained from saturation curves for the mutant were more than twice higher per leaf area and about five times higher per chlorophyll, as compared to the wild-type. Key words: Blue drop - Mutant (Nicotiana) - Nicotiana (aurea mutant) - Photoacoustics - Photosynthesis (oxygen evolution, energy-storage yield).

Introduction Burk and Menser (1964) discovered in John Williams Broadleaf tobacco a mutant, designated Su/ su and possessing unusual physiological and biochemical properties. It has a low level of chlorophyll, rendering its leaves a yellow-green (" aurea") color, and the ratio of total carotenoids/total chlorophyll is greater than in green (wild-type) plants (for a review, see Schmid 1971). Under high light, Abbreviation

." PS =

photosystem

the mutant exhibits higher photosynthetic rates, measured as CO 2 fixation on a chlorophyll basis, than does the wild-type (Schmid and Gaffron 1967a, b; Schmid 1967). Its chloroplasts contain very few grana and most of the thylakoids are unstacked, resembling in structure the photosynthetic apparatus in cells of blue-green algae (Schmid et al. 1966). Chloroplasts isolated from the mutant give a higher saturation rate in the Hill reaction than chloroplasts prepared from wild-type plants (Homann and Schmid 1967; Okabe et al. 1977). Finally, the photosynthetic unit size of the mutant is smaller than in the wild-type, allowing for a more efficient light-energy conversion at high light levels (Schmid and Gaffron 1968; Schmid 1971; Okabe et al. 1977). In previous studies in which net photosynthesis was measured, either manometrically or by 14C uptake, corrections had to be made accounting for respiration and dark CO2 fixation. By using the photoacoustic technique, as developed in our laboratory (Bults et al. 1982; Poulet et al. 1983; Canaani et al. 1984), we were able to measure the relative quantum yield of photosynthetic oxygen evolution directly in intact leaves by using a modulation technique, as well as to estimate the extent of energy storage in the intermediates of photosynthesis. Performing photoacoustic experiments at 22 Hz (Canaani et al. 1982a, b, 1983) allows us to separate completely the effects of respiration and dark fixation of CO2 on the rate of photosynthesis. These two processes have much slower rate-limiting constants, and would not contribute to the photoacoustic signal at this frequency range. Therefore, problems such as the Kok effect, variations in rates of respiration from experiment to experiment, variations in dark fixation between wild-type and mutant (Schmid and Gaffron 1967a, b) are eliminated. Another advantage is that the results on quantum yield are direct and

O. Canaani et al. : Comparison of photosynthetic parameters of an aurea mutant (Su/su)

do not require measurements of the absorbed light energy. In this report, a comparative photoacoustic study of the Su/su tobacco mutant and the wildtype is described. We monitored the quantum yield as well as the rate-saturation curve of photosynthesis. In the mutant, oxygen-evolution yield was much lower in either the green (520 nm) or the blue (440 nm) spectral region compared with the red region (640-690 nm). Similar differences were also observed in the quantum-yield spectra of photochemical energy storage. In contrast, the wildtype showed the behaviour typical of normal, green plants with only slight variations of the quantum yield in different spectral regions. Photosynthetic saturation curves again indicated higher maximal rates of oxygen evolution in the mutant leaf compared with wild-type tobacco both on the basis of leaf area and even more dramatically on the basis of chlorophyll content. Materials and methods Seeds of the John Williams Broadleaf cultivar of tobacco (Nicotiana tabacum L.) and of the aurea mutant Su/su were a gift from Dr. G.H. Schmid, University of Bielefeld, FRG. All plants were grown in a greenhouse in natural light at 25+ I ~ and an energy fluence rate of 200 W m - 2. Growing conditions were as described by Schmid (1967). The plants were grown in pots in soil. Yellow-green and green leaves in the middle part of the stem of two-month-old mutant and wild-type plants, respectively, were used and l-am-diameter discs were cut and used immediately for experiments. Chlorophyll was determined by extracting the leaf discs in 100% acetone and diluting the extract with water to 80% acetone. Total chlorophyll content was determined by measuring absorption at 663 and 645 nm by the method of Arnon (1949). Carotenoid content was determined in the same pigment extracts and calculated according to the method of Lianen-Jensen and Jensen (1971). Measurements of oxygen-evolution yield and energy-storage yield (" photochemical loss") were carried out in our photoacoustic apparatus at 23_+ 1~ C and ambient CO 2 concentrations, as described in Bults et al. (1982). The light source for the modulated beam was a directcurrent Xenon lamp (450 W; XBO, ozone free, Osram, WestBerlin, Germany) and a monochromator (Bausch and Lomb, Rochester, N.Y., U S A ; 10 nm band-pass). The light was modulated by passing it through a rotating wheel chopper (Laser Precision, Utica, N.Y., USA) resulting in equal light and dark periods. The source for the background light was a slide projector (direct current, 250 W; Victor, Stockholm, Sweden). The photoacoustic signal is measured with the modulated light. In order to get a reference signal for the photoacoustic experiment, a background light of constant and high energy flux is added to the modulated beam. The modulated light is absorbed by a leaf disc and causes the release of both modulated heat and modulated oxygen evolution (Bults et al. 1982). Diffusion of heat and oxygen from the chloroplats to the cell surface causes periodic expansion and contraction of an inner air phase near the cell surface, thus creating a pressure wave which is transduced into an acoustic signal. The acoustic wave propagates itself outside the leaf and is detected by a micro-

481

phone (Knowles, Franklin Park, Ill., USA) and preamplified and processed by a two-phase lock-in amplifier (Ortholock 9502; Brookdeal, Bracknell, UK). At low frequency of modulation (400 Hz), at which modulated oxygen evolution is negligible. Modulated oxygen evolution was monitored at 22 Hz. The variability from one experiment to another was in the order of 10-15%.

Results

Oxygen evolution. The relative quantum-yield spectrum of oxygen evolution in wild-type tobacco and in the Su/su mutant is shown in Fig. 1. At 640-680 nm, oxygen yield in the mutant was higher by a factor of 1.5 than in the wild type. Leaves of both types exhibited a drastic decrease in the yield at 2 > 690 nm - the well-known " r e d d r o p "

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Fig. 1. Relative oxygen-evolution quantum yield in arbitrary units (a.u.) calculated as (Aox/ApT divided by 2) for wild-type (John Wiliam Broadleaf) tobacco ( o - - o ) and the Su/su mutant ( o - - o ) leaves as function of the wavelength of modulated light. Aox= Photoacoustic signal caused by 0 2 evolution; Ap~ = photoacoustic signal caused by photothermal conversion. Saturating light, 400-600nm, 400W/m2; modulated light, 650 nm, 10 W/m 2 frequency of modulation, 22 Hz. At each wavelength of modulated light, the leaf disc is irradiated for about 1 min to obtain steady-state 0 2 evolution. Then, the non-modulated, saturating, background light is added to the modulated light for 15 s to obtain the photothermal signal

O. Canaani et al. : Comparison of photosynthetic parameters of an aurea mutant (Su/su)

482

Table 1. Ratio of quantum yields of oxygen evolution at various exciting wavelengths for wild-type tobacco and the Su/su mutant. Results are given for four determinations; values are means _+SD

4.0

-$

3.0 2.5

Wavelengths compared (nm)

Ratio of quantum yields of O z evolution

i

Mutant Su/su

Wild type

K

680/650 470/440 680/520 680/470 680/440

1.10 __+0.10 0.72 + 0.05 2.99__+ 0.41 2.10__+0.12 1.54 __+0.10

1.05 -t- 0.14 0.84__+ 0.04 1.41 + 0.26 1.40-t-0.10 1.22 • 0.11

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2.0 I.-

(reviewed by Myers 1971). In contrast to the wildtype leaf which had only a moderate decrease of activity below 580 nm, the mutant exhibited a rather sharp drop. Comparisons of the oxygen yield showed that in wild-type tobacco this yield in the red spectral region (640-680 nm) was about 1.2 times higher than in the Soret region (440 rim), and about 1.4 times higher than at 520 nm. However, in the mutant the difference was more striking. The oxygen yield at 680 nm was about 1.5 times higher than at 440 nm, and 3.0 times higher than at 520 nm. A comparison of the ratio of typical oxygen-evolution yield values at several different wavelengths in the wild-type and the mutant (Fig. 1, Table 1) indicates that chlorophyll a and chlorophyll b contribute with a very similar photochemical efficiency in both plant types, as shown by the similar ratio of activity at 650 nm and 680 nm (characteristic of chlorophyll b and chlorophyll a absorption, respectively). It may be concluded that in the mutant both chlorophyll a and chlorophyll b are very effective in trapping red light while the accessory pigments absorbing at 500-540 nm (most probably carotenoids) transfer energy at a lower efficiency to the photochemical reaction centers, as compared to the wild-type. Similar conclusions have been drawn by Schmid and Gaffron (1967a, b, 1968) and Okabe etal. (1977). The total chlorophyll content (per unit leaf area) was found to be 17.5 _+2 [.tg/cm 2 in the mutant and 44.2 + 4 I,tg/cm 2 in the wild-type tobacco; the ratio of chlorophyll a/chlorophyll b was 3.4-t-0.1 and 2.4-t-0.3, respectively. In addition, the ratio of total carotenoids to chlorophyll in the mutant was increased by a factor of 1.7 as compared with the green leaf although the absolute amount of carotenoids was greater in the latter. The ratio of the photothermal signal to the fluence of incident light at each wavelength was deter-

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WAVELENGTH (nm)

Fig. 2. Relative percent absorption (ApT/I) in wild-type tobacco (I t ) and the Su/su mutant (o o) as function of the modulated-light wavelength. I=incident energy fluence rate. Light conditions and modulation frequency as in Fig. 1

mined in the mutant as well as in the wild-type (Fig. 2). The different data obtained for the two plant types may be partly a result of the variation in relative absorption, and partly of possible differences in the localization and specific organization of the blue-absorbing pigments (i.e., most probably, carotenoids).

Energy storage. The amplitude of the oxygen-evolution component relative to that of the photothermal signal decreases as the modulation frequency increases, as a consequence of the diffusion of oxygen and of electron-transfer reactions. At high frequency of modulation, only the photothermal signal is therefore detected. The difference between the photothermal signals in the presence and absence of photosynthetically saturating, non-modulated background light is proportional to the amount of energy stored in the photosynthetic process. This difference divided by the maximum photothermal signal is defined as "photochemical loss" (Malkin and Cahen 1979), and is equal to cAE/hv, where (0 is the quantum yield, AE is the energy stored in unit reaction cycle, h the Planck constant, and v the light frequency. A comparison of the quantum-yield spectra of photochemical energy storage for wild-type tobacco and the Su/su mutant is shown in Fig. 3. The mutant had a somewhat higher energy-storage yield than the wildtype at 680 nm, but showed a drop in the yield below 640 nm and reached very low values in the range of 450-530 nm. The energy-storage yield in the mutant at the Soret peak (440 nm) was half of that at the red peak (680 nm) and more strikingly, the yield of energy storage at 510 nm was only a quarter of that at 680 nm. In contrast, the quantum yield of photochemical energy stored in

O. Canaani et al. : Comparison of photosynthetic parameters of an aurea m u t a n t

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Fig. 3. Relative quantum yields for energy storage ( " p h o t o chemical loss", P.L., divided by 2) for wild-type tobacco ( o - - o ) and the Su/su m u t a n t ( o - - o ) as function the wavelength of the modulated light. Illumination parameters as in Fig. 1. Modulation frequency, 640 Hz

the wild-type tobacco was only slightly higher in the 680-nm peak than the 440-nm peak. Thus, a similar picture arises from the energy-storage yield spectrum as from the oxygen-evolution yield spectrum. In the mutant, pigments absorbing in the green region are not effective in contributing to the photochemical processes which produce highenergy storage products. The drop in the quantum yield of energy storage in the 450- to 520-nm region in the mutant is even more pronounced than the drop in the oxygen-evolution quantum-yield spectrum.

Maximal rates of photosynthesis. The photoacoustic signals are usually obtained under light-limiting conditions. In this case, the ratio of the oxygenevolution signal and the energy of absorbed light is proportional to the quantum yield (Poulet et al. 1983). U p o n addition of different energy fluence rates of non-modulated background light, the oxygen contribution to the photoacoustic signal decreases because of a lowered quantum yield of oxygen production which finally, at saturating light, approaches zero. The dependence of the oxygen yields on different energy fluence rates of non-modulated background light for both wild-type and Su/su tobacco is shown in Fig. 4. It is observed that the quantum yield in the wild-type decreases with a steeper slope than in the mutant. These curves reflect the "differential" quantum yields (i.e., the derivative of the light-saturation curve of photosynthesis rate versus energy flux; see Poulet et al. 1983). The initial extrapolated value for I--+0 corresponds also to the ordinary maximum quantum yield. Therefore, by integration of the curves

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Energy fluence rate of background saturating [igM ( W / m 2) Fig. 4. Relative quantum yields of oxygen evolution for wildtype tobacco ( e - - e ) and the Su/su mutant ( o - - o ) as function of the fluence of background light. Modulated light (fluence and frequency) as in Fig. 1

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Energy fiuencerate of background saturating light ( W / m 2) Fig. 5, Relative rate of oxygen evolution per leaf area as function of the energy fluence rate of the background light in wildtype ( o - - e ) and Su/su ( o - - o ) tobacco. These rates were obtained by integration of the curves in Fig. 4

in Fig. 4, we obtained the relative rate-saturation curves, or more exactly the ratio of the rate to the maximal quantum yield, for the wild-type tobacco and the mutant (Fig. 5). From these curves it can be seen that the maximum rate in the mutant per unit leaf area is more than two times higher than in the wild-type. Comparison on a chlorophyll-content basis gives a rate of about five times higher in the mutant relative to the wild-type. These results are in excellent agreement with the observations of Schmid and Gaffron (1967a, b) who used 14C fixation for measuring net photosynthetic rate and showed that in red light the mutant had about twice as hlgh a maximal rate per unit area as the wild-type.

484

O. Canaani et al. : Comparison of photosyntheticparameters of an aurea mutant (Su/su)

Discussion

Quantum-yield spectra of oxygen evolution measured photoacoustically are shown here for intact leaves of the wild-type and the Su/su mutant of tobacco. It is demonstrated that in the mutant, photosynthetic activity is higher in the red spectral region (600-690 nm) compared with the blue one (450-550 nm). The photoacoustic technique also demonstrates that in the mutant the maximal rate of 0 2 evolution is fivefold higher (on a chlorophyll basis) than that of the wild-type. In a similar manner, Schmid and Gaffron (1967a, b) observed that the maximal rate of CO 2 fixation measured in red light was two or three times higher (on a leaf-area basis) in the chlorophyll-deficient plants than in the wild-type. Okabe et al. (1977) reported that the maximal rate of oxygen evolution in the Su/su mutant was five times higher (on a chlorophyll basis) than in the green control. Since, in both cases, results were obtained in red light it was concluded that the increased carotenoid/chlorophyll ratio was of no special advantage to the Su/su plant. The striking new features observed in the mutant in this work is the strong suppression of both oxygen evolution and especially of energy-storage yield in the blue spectral region (450-550 nm) compared with the red one (600-690 nm) although the Su/su mutant had a higher content of carotenoids on a chlorophyll basis, compared with the wildtype. This difference in carotenoid content was calculated from the absorption spectra of extracted leaf pigments and had been observed previously (Schmid 1971). Similarly, absorption spectra of chloroplasts obtained from the wild-type and the Su/su mutant indicated a suppressed Soret band in the wild-type which was explained by the lower content of carotenoids and by the shape of these chloroplasts (Melis and Thielen 1980). One possibility to explain the blue drop in oxygen evolution and energy storage is to assume that excess carotenoids in the mutant were closely associated with photosystem (PS) II rendering its absorption crosssection much larger than PS I. In this case, the distribution of excitation energy between the two photosystems would be unbalanced and the yield of electron transport would be limited by the fraction of light absorbed by the smaller PS I. Emerson enhancement (Myers 1971) of 0 2 evolution is a sensitive indicator of the imbalance of excitation energy distribution between the two photosystems (Canaani and Malkin 1984). Therefore, we measured the Emerson enhancement of modulated O2 evolution by background far-red light as function of modulated excitation wavelength, both in the

wild-type and the mutant. The same enhancement spectrum was observed in the two plants (not shown) and was very similar to that published previously in tobacco leaves (Canaani and Malkin 1984). The alternative possibility is that the fraction of blue light absorbed by P S I is larger than that absorbed by PS II. This was checked by determining Emerson enhancement of modulated 0 2 evolution measured at 650 nm upon addition of background blue light in the range of 450-550 nm. No enhancement was observed in this case in the two species (data not shown). Therefore it appears unlikely that a change in the distribution of excitation energy between the two photosystems is the cause for the blue drop in photochemical activity. An alternative explanation for the blue drop is that the excess carotenoids in the mutant are not closely associated with the photosystems, possibly residing outside the thylakoid membrane and serving as screening pigments. In order to examine this possibility, and try to locate the excess carotenoids in the mutant, we used electron-microscopy of thin sections of mutant and wild-type leaves, respectively. In preliminary work (data not shown) we have found that the carotenoids in the mutant seem to be organized in globules in the interthylakoid spaces similar to the green alga Dunaliella bardawil, a photosynthetic organism which has a unique property of being able to accumulate large amounts of p-carotene at high energy fluence rates (Ben-Amotz et al. 1982). The p-carotene in D. bardawil is accumulated in globules in the interthylakoid spaces of the chloroplast and may protect against injury by high irradiance when the chlorophyll content per cell is low (Ben-Amotz and Avron 1983). The carotenoid globules might serve a similar function in the Su/su mutant. However, in future research a biochemical analysis of carotenoid distribution in different chloroplast fractions (envelope, thylakoids, globules) should be conducted to verify this suggestion. The observation that the blue drop was more pronounced in the energy-storage quantum-yield spectrum than in that of the oxygen-evolution one, may reflect a large contribution of energy-storing processes associated primarily with P S I which is activated by longer-wavelength light (2 > 680 nm). The prominent charactertistic of the Su/su mutant is the smaller photosynthetic unit size in comparison with the wild-type, leading to a more efficient use of solar energy at high energy fluence rates (Schmid and Gaffron 1968; Schmid 1971; Okabe et al. 1977). The mutant, when grown at high fluences, also contains only small amounts of the light-harvesting chlorophyll a/b protein

O. Canaani et al. : Comparison of photosynthetic parameters of an aurea mutant (Su/su) c o m p l e x ( R e m y a n d Bebee 1974; V e r n o t t e et al. 1976) a n d u n d e r certain conditions, the light-harvesting chlorophyll p o l y p e p t i d e s o f 2 5 0 0 0 - 2 9 0 0 0 m o l e c u l a r weight are absent ( K o i v u n i e m i et al. 1981). It a p p e a r s t h a t the Su/su m u t a n t has undergone a change in the architecture o f its p h o t o s y n t hetic a p p a r a t u s so t h a t it is better a d a p t e d to its n a t u r a l e n v i r o n m e n t a l conditions, e.g. high inadiations. It got rid o f a large extent o f its light-harvesting p i g m e n t s which are superfluous for activity at such irradiations; it d e v e l o p e d a specialized system for p r o t e c t i o n against c h l o r o p h y l l p h o t o o x i d a t i o n at high irradiances (e.g., excess c a r o t e n o i d s in int e r t h y l a k o i d globules) a n d it has also a different internal c h l o r o p l a s t structure (mostly single thylakoids with no grana). T h e possible functional adv a n t a g e s o f these structural changes are still unk n o w n . A current m o d e l in p h o t o s y n t h e s i s (Andersson a n d A n d e r s o n 1980) is b a s e d on a spatial s e p a r a t i o n between the p h o t o s y s t e m s so t h a t PS I I is m a i n l y located in the g r a n a a n d P S I in the s t r o m a , a n d the light-harvesting c h l o r o p h y l l a/b c o m p l e x is m o b i l e between PS I I a n d P S I (Barber 1982). In the case o f the m u t a n t , this structural s e p a r a t i o n is n o t expected to occur. This could h a v e i m p o r t a n t implications on the m e c h a n i s m o f energy distribution between the two p h o t o s y s t e m s . Therefore, it a p p e a r s t h a t u n d e r s t a n d i n g the consequences o f the unique structure o f the p h o t o s y n - . thetic m e m b r a n e a n d regulation o f the electront r a n s p o r t system in the m u t a n t should be an objective for future research. In conclusion, the a u r e a t o b a c c o m u t a n t Su/su was s h o w n to h a v e very high o x y g e n - e v o l u t i o n rates because it has a small, highly effective p h o t o synthetic unit. C a r o t e n o i d s do n o t p l a y a m a j o r role as light-harvesting p i g m e n t s b u t m a y p e r h a p s serve to p r o t e c t the small p h o t o s y n t h e t i c unit against d a m a g e b y high light energies. This work was partly supported by the U.S.-Israel Agricultural Research and Development Fund - BARD (Project 1-388-81). Thanks are due to Prof. G.H. Schmid for the gift of seeds.

References Andersson, B., Anderson, J.M. (1980) Lateral heterogeneity in the distribution of chlorophyll - protein complexes in the thylakoid membranes of spinach chloroplasts. Biochim. Biophys. Acta 593, 427-440 Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts, poly-phenol oxidase in Beta vulgaris. Plant Physiol. 24, I 15 Barber, J. (1982) Influence of surface charges on thylakoid structure and function. Annu. Rev. Plant Physiol. 33, 261-295 Ben-Amotz, A., Avron, M. (1983) On the factors which deter-

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mine massive fl-carotene accumulation in the halotolerant alga Dunaliella bardawil. Plant Physiol. 72, 593-597 Ben-Amotz, A., Katz, A., Avron, M. (1982) Accumulation of fl-carotene in halotolerant algae: purification and characterization offl-carotene-rich globules from Dunaliella bardawil (Chlorophyceae). J. Phycol. 18, 529-537 Bults, G., Horwitz, B.A., Malkin, S., Cahen, D. (1982) Photoacoustic measurements of photosynthetic activities in whole leaves photochemistry and gas exchange. Biochim. Biophys. Acta 679, 452 465 Burk, L., Menser, H.A. (1964) A dominant aurea mutation in tobacco. Tobacco Sci. 8, 101-104 Canaani, O., Barber, J., Malkin, S. (1984) Evidence that phosphorylation and dephosphorylation regulate the distribution of excitation energy between the two photosystems of photosynthesis in vivo: photoacoustic and fluorimetric study of an intact leaf. Proc. Natl. Acad. Sci. USA 81, 1614-1618 Canaani, O., Cahen, D., Malkin, S. (1982a) Photosynthetic chromatic transitions and Emerson enhancement effects on intact leaves studied by photoacoustics. FEBS Lett. 150, 142-146 Canaani, O., Cahen, D., Malkin, S. (1982b) Use of photoacoustic methods in probing development of the photosynthetic apparatus in greening leaves. In: Cell function and differentiation, pt. B, pp. 299-308, Akoyunoglou, G., Evangelopoulos, A.E., Georgatsos, J., Palaiologas, G., Trakatellis, A., Tsiganos, C.P. eds. Alan R. Liss, New York Canaani, O., Cahen, D., Malkin, S. 0983) Photoacoustics as a probe for photosynthetic O z evolution and energy storage in an intact leaf - distribution of excitation energy between PS II and PS I. Proc. VI Int. Congr. on Photosynthesis, Brussels, Belgium, pp. III.4.331-334, Sybesma, C., ed. Martinus Nijhoff/Dr. Junk, The Hague Canaani, O., Malkin, S. 0984) Distribution of light excitation in an intact leaf between the two photosystems of photosynthesis; changes in absorption cross-sections following state 1-state 2 transitions. Biochim. Biophys. Acta 766, 513-524 Homann, P.H., Schmid, G.H. (1967) Photosynthetic reactions of chloroplasts with unusual structures. Plant Physiol. 42, 1619-1632 Koivuniemi, P.J., Tolbert, N.E., Carlson, P.S. (1981) Characterization of the thylakoid membranes of the tobacco aurea mutant Su/su and of three green revertant plants. Planta 151, 40-47 Lianen-Jensen, S., Jensen, A. (1971) Quantitative determination of carotenoids in photosynthetic tissues. Methods Enzymol. 23, 586~602 Malkin, S., Cahen, D. 0979) Photoacoustic spectroscopy and radiant energy conversion: theory of the effect with special emphasis on photosynthesis. Photochem. Photobiol. 29, 803-813 Malkin, S., Laser-Ross, N., Bults, G., Cahen, D. (1981) Photoacoustic spectroscopy in photosynthesis. Proc. V Int. Congr. on Photosynthesis, pp. 1031-1042, Akoyunoglou, G., ed. Balaban International Science Service, Philadelphia, Pa., USA Melis, A., Thielen, A.P.G.M. (1980) The relative absorption cross-sections ofphotosystems 1 and photosystem II in chloroplasts from three types of Nieotiana tabacum. Biochim. Biophys. Acta 589, 275-286 Myers, J. (1971) Enhancement. Annu. Rev. Plant Physiol. 22, 289-312 Okabe, K., Schmid, G.H., Straub, J. (1977) Genetic characterization and high efficiency photosynthesis of an aurea mutant of tobacco. Plant Physiol. 60, 150-156 Poulet, P., Cahen, D., Malkin, S. (1983) Photoacoustic detec-

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O. Canaani et al. : Comparison of photosynthetic parameters of an aurea mutant (Su/su)

tion of photosynthetic oxygen evolution from leaves - quantitative analysis by phase and amplitude measurements. Biochim. Biophys. Acta 724, 433-446 Remy, R., Bebee, G. (1974) Membrane proteins of higher plant chloroplasts related to photochemical systems and membrane stacking. Proc. II Int. Congr. on Photosynthesis, pp. 1675-1684, Avron, M., ed. Elsevier, Amsterdam Schmid, G.H. (1967) The influence of different light intensities on the growth of the tobacco aurea mutant Su/su. Planta 77, 76-94 Schmid, G.H. (1971) Origin and properties of mutant plants: yellow tobacco. Methods Enzymol. 23, 171-194 Schmid, G.H., Gaffron, H. (1967 a) Light metabolism and chloroplast structure in chlorophyll-deficient tobacco mutants. J. Gen. Physiol. 50, 563-583

Schmid, G.H., Gaffron, H. (1967b) Quantum requirement for photosynthesis in chlorophyll-deficient plants with unusual lamellar structures. J. Gen. Physiol. 50, 2131-2144 Schmid, G.H., Gaffron, H. (1968) Photosynthetic units. J. Gen. Physiol. 52, 212-239 Schmid, G., Price, J.M., Gaffron, H. (1966) Lamellar structure in chlorophyll deficient but normally active chloroplasts. J. Microsc. Paris 5, 205-212 Vernotte, C., Briantais, J,-M., Remy, R. (1976) Light harvesting pigment protein complex requirement for spill-over changes induced by cations. Plant Sci. Lett. 6, 135-141

Received 10 January 1984; accepted 23 January 1985