- Email: [email protected]
The fine structure of chloroplasts in a barley mutant lacking chlorophyll B
684
D. J. Goodchild,
H. R. Highkin
and N. K. Boardman
Thanks are expressed to Dr R. D. Cahn for supplying the extract fractions and to Rosemary Amlie and Shelley Noon for technical assistance. This research was supported by U.S. Public Health Service Grant GM-10060 and by the Harvey Bassett Clarke Foundation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
CAHN, R. D. and CAHN, hf. B., Proe. h’atl Acad. Sci. U. S. 55, 106 (1966). COON, H. G., Proc. Nat! Acad. Sci. U.S. 55, 66 (1966). GROBSTEIN, C. and COHEN, J. H., Unpublished observations. ~ Science 150, 626 (1965). HAUSCHKA, S. D. and KONIGSBERG, I. R., Proc. Natl Acad. Sci. U.S. 55, 119 (1966). KALLMAN, I;., Unpublished observations. KALLMAN, F., EVANS, J. and WESSELLS, N. K., In preparation. PARKER, R. C., Methods of Tissue Culture, 3rd ed. Harper and Row, New York, 1961. RUTTER, W. J., WESSELLS, N. K. and GROBSTEIN, C., J. Natf Cancer Inst. 13, 51 (1964). WESSELLS, N. K., Proc. Natl Acad. Sci. U.S. 52, 252 (1964). WESSELLS, N. K., .I. CeKBiol. 20, 415 (1964). WESSELLS, N. K. and WILT, F. H., J. Mol. Biol. 13, 767 (1965). WOOD, G. C. and KEECH, M. K., Biochem. J. 75, 588 (1960).
THE
FINE
STRUCTURE MUTANT
D. J. GOODCHILD, C.S.I.R.O.,
OF CHLOROPLASTS
LACKING
CHLOROPHYLL
H. R. HIGHKIN Division
of Plant Received
Industry,
IN A BARLEY B
and N. K. BOARDMAN Canberra,
Australia
May 9, 19662
VIABLE mutants lacking chlorophyll
b have been found in barley (Hordeum vulgare) [5] and Chlorella pyrenoidosa [l], but only with the barley mutant have good photosynthetic rates been reported [4]. Recently, it was shown that chloroplasts isolated from the barley mutant were active in the Hill reaction [2]. Microscopic examination of the isolated chloroplasts under phase contrast showed no significant differences in the appearance of chloroplasts from the normal and mutant plants [a]. The present investigation was undertaken to compare the organization of the lamellar structure of the chloroplasts of the mutant and normal strains. The present studies show that in the absence of chlorophyll b grana formation is impaired to some extent, and the chloroplasts in mutant plants appear to be more disorganized than those in normal plants. Maferials and Mefhods.-The two strains of barley (Hordeum vulgare) used in these studies were described previously [3, 41. One of these, No. 2 was shown to lack chlorophyll b [2, 41 or at the very least, the ratio of chlorophyll a/chlorophyll [3],
Arabidopsis
fhaliana
1 On leave from Calif., U.S.A. e Revised version Experimental
Department received
Cell Research
of Biology, July
43
12, 1966.
San
Fernando
Valley
State
College,
Northridge,
~Sfrucfure
Fig.
I.--Electron
micrograph
of chloroplasfs
of a chloroplast
lacking
of a mutant
chlorophyll
barley
leaf.
685
b
x 16,500.
Insert,
x 30,000.
b is greater than 1000 [2]. The other strain, Lyon, contained both chlorophyll a and chlorophyll b in normal amounts. Both strains were grown in a 16 hr photoperiod of natural light, supplemented with artificial light. The temperature was maintained at 21°C for 8 hr and 16°C for 16 hr. For electron microscopy, squares of tissue (approximately 1 mm x 1 mm) were cut from the central portion of the second leaf of a mutant or normal barley plant and fixed in 2.5 per cent (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7, for 6 hr at 4°C. The tissue was washed, fixed at 4°C for 2 hr in 2 per cent potassium permanganate in 0.1 M phosphate buffer pH 7, dehydrated in alcohol, and embedded in epon. Sections were cut with an LKB “ultrotome” using a diamond knife. Sections were examined without further treatment in a Siemens Elmiskop 1 electron microscope. Preliminary observations suggested that the grana of the mutant chloroplasts contained fewer lamellae than the grana of normal chloroplasts. In order to investigate differences in the number of lamellae and to avoid, as far as possible, selection by the microscope operator, chloroplasts were photographed at random, the one requirement being that an entire chloroplast could be photographed at a magnification of 10,000 x . Approximately 50 photographs of normal and 50 of mutant chloroplasts at an enlargement of 3 x were made from sectioned material from two separate embeddings of plant material, grown at different times. These photographs were examined for distinctness and resolution of lamellae and 18 photographs of normal barley chloroplasts, and 17 of the mutant were finally selected. The photographs were submitted Experimental
Cell Research
43
686
D. J. Goodchild,
Fig. 2.-Electron
micrograph
H. R. Highkin
of a chloroplast
and N. K. Boardman
of a normal barley leaf. x 16,500. Insert x 30,000.
to two independent observers who had no previous knowledge of chloroplast structure. The observers were asked to count the numbers of single lamellae and groups of lamellae per chloroplast as well as the number of lamellae per group. These results were analysed statistically. TABLE
I. Average numbers Total lamellae/ chloroplast
of lamellae
Grana/ chloroplast
and granaa
Lamellae/ granum
in normal Single lamellae/ chloroplast
and mutant
chloroplasts.
Grana/chloroplast with 8 or more lamellae
Normal
260.67 Mufanf 176.59
49.44
5.07
12.83
7.17
33.94
3.90
46.41
1.18
15.50***
1.17***
Difference
84.08***
- 33.5s***
5.99***
a For the purposes of this table, a granum is defined as a group of lamellae containing 2 or more single lamellae. ***Highly significant, P c 0.001. The significance levels from the analysis are attached to the differences of the absolute values. Experimental
Cell Research
43
Structure
of chloroplasts
lacking
chlorophyll
687
b
Results.-Representative electron micrographs of chloroplasts from normal and mutant barley leaves are shown in Fig. 1 and 2. The lamellar structure of the mutant chloroplast appears to be disorganized in comparison with the normal chloroplast. There appears to be fewer grana in the mutant chloroplast as well as fewer lamellae per granum. The results of the lamellar counts made by Observer A are shown by the histogram in Fig. 3. It is apparent that there are a larger number of single laMUTANT
NORMAL
LAMELLAE/GROUP
Fig. 3.-Histogram and the average
showing the average number number of lamellae per group.
of grana
(groups
of lamellae)
per chloroplast
mellae in the mutant chloroplasts and smaller numbers of grana with three or more lamellae. The results of the statistical analysis of the data of Observer A are given in Table I. All differences between the normal and mutant chloroplasts were highly significant. As well as showing that the normal chloroplast contained more grana per chloroplast and more lamellae per granum, Table I also indicates that the normal chloroplast had a higher average content of lamellae per chloroplast. The difference in the number of grana per chloroplast with eight or more lamellae is particularly striking. The statistical analysis of the data of Observer B also showed that all differences were highly significant. Discussion.-The lamellar system of barley chloroplasts, in common with most higher plant chloroplasts, is differentiated into grana and non-grana regions. In the grana-containing chloroplast, chlorophyll appears to be confined mainly, if not solely, to the grana regions [7, 81, and it seems reasonable to suppose that the lack of one of the chlorophyll pigments would preferentially influence the structure of the grana. The striking differences between the normal and mutant chloroplasts are in the number of lamellae per granum and the number of grana per chloroplast. It has been postulated that chlorophyll is localized in the double membranes, which are formed as a result of the pairing of the single membranes of neighbouring grana lamellae [6, 91. It seems, therefore, that the pairing of the grana lamellae is affected by the absence of chlorophyll b, and this results in impaired grana formation. The mutant chloroplast gives the impression of being more disorganized. The inter-grana or stroma lamellae extend over greater distances before becoming associated with a granum. It is apparent, however, that chlorophyll b is not an essential structural Experimental
Cell Research
43
A. Forer
688
component of the grana of the higher plant chloroplast. Studies at high resolution of the ultrastructure of the membranes of the mutant and normal chloroplasts are proposed. We wish to thank Mr G. A. McIntyre for the statistical Paulin for assistance with electron microscopy. This work a grant from the National Science Foundation (GB3341).
analysis and Miss M. A. was supported in part by
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
ALLEN, M. B., Brookhaven Symp. Biof. 11, 339 (1959). BOARDMAN, N. K. and HIGHKIN, H. R., Biochim. Biophys. Acta (1966). HIGHKIN, H. R., Plant Physiol. 25, 294 (1950). HIGHKIN, H. R. and FRENKEL, A. W., Pfanf Physiof. 37, 814 (1962). HIRONO, Y. and REDEI, G. P., Nafure 197, 1324 (1963). MENKE, W., Ann. Reo. Plant Physiof. 13, 38 (1962). STRUGGER, S., Naturwiss. 37, 166 (1950). THOMAS, J. B., POST, L. C. and VERTREGT, N., Biochim. Biophys. Acfa WEIER, T. E., ENGELBRECHT, A. H. P., BISALPUTRA, T. and BENSON, Suppl. 40, xxviii (1965).
A SIMPLE
CONVERSION
PHASE-CONTRAST
OF REFLECTING
CONDENSERS
LIGHT
13, 20 (1954). A. A., Plant
Physiol.
LENSES INTO
FOR ULTRAVIOLET
IRRADIATIONS A.
Biological Foundation, Received
THIS article
In press.
FORER
Institute of the Car&berg Copenhagen, Denmark May
17,
1966
describes a simple way to use reflecting lenses as phase-contrast condensers, primarily for use with ultraviolet-microbeam equipment. When the visible light image of the microbeam source is focused onto the specimen, phase-contrast microscopy is needed in order to observe the specimen. The existing microbeam equipment achieves phase-contrast by the use of additional components; these components cost money, they take up space, and they require adjustment (see [3,8,14] for reviews of microbeam equipment). For example, the irradiations are usually performed with a reflecting lens which is used as either the objective or the condenser. When the reflecting lens is used as the objective, a standard phase-contrast lens is used as the condenser; the phase-shift normally provided by the objective’s phase plate is then obtained by means of an auxiliary optical system between the reflecting lens and the observer [6, 7, 10, 11, 121. When the reflecting lens is used as the condenser, a standard phase-contrast lens is used as the objective; the annulus of illuminating Experimental
Cell Research
43
D. J. Goodchild,
H. R. Highkin
and N. K. Boardman
Thanks are expressed to Dr R. D. Cahn for supplying the extract fractions and to Rosemary Amlie and Shelley Noon for technical assistance. This research was supported by U.S. Public Health Service Grant GM-10060 and by the Harvey Bassett Clarke Foundation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
CAHN, R. D. and CAHN, hf. B., Proe. h’atl Acad. Sci. U. S. 55, 106 (1966). COON, H. G., Proc. Nat! Acad. Sci. U.S. 55, 66 (1966). GROBSTEIN, C. and COHEN, J. H., Unpublished observations. ~ Science 150, 626 (1965). HAUSCHKA, S. D. and KONIGSBERG, I. R., Proc. Natl Acad. Sci. U.S. 55, 119 (1966). KALLMAN, I;., Unpublished observations. KALLMAN, F., EVANS, J. and WESSELLS, N. K., In preparation. PARKER, R. C., Methods of Tissue Culture, 3rd ed. Harper and Row, New York, 1961. RUTTER, W. J., WESSELLS, N. K. and GROBSTEIN, C., J. Natf Cancer Inst. 13, 51 (1964). WESSELLS, N. K., Proc. Natl Acad. Sci. U.S. 52, 252 (1964). WESSELLS, N. K., .I. CeKBiol. 20, 415 (1964). WESSELLS, N. K. and WILT, F. H., J. Mol. Biol. 13, 767 (1965). WOOD, G. C. and KEECH, M. K., Biochem. J. 75, 588 (1960).
THE
FINE
STRUCTURE MUTANT
D. J. GOODCHILD, C.S.I.R.O.,
OF CHLOROPLASTS
LACKING
CHLOROPHYLL
H. R. HIGHKIN Division
of Plant Received
Industry,
IN A BARLEY B
and N. K. BOARDMAN Canberra,
Australia
May 9, 19662
VIABLE mutants lacking chlorophyll
b have been found in barley (Hordeum vulgare) [5] and Chlorella pyrenoidosa [l], but only with the barley mutant have good photosynthetic rates been reported [4]. Recently, it was shown that chloroplasts isolated from the barley mutant were active in the Hill reaction [2]. Microscopic examination of the isolated chloroplasts under phase contrast showed no significant differences in the appearance of chloroplasts from the normal and mutant plants [a]. The present investigation was undertaken to compare the organization of the lamellar structure of the chloroplasts of the mutant and normal strains. The present studies show that in the absence of chlorophyll b grana formation is impaired to some extent, and the chloroplasts in mutant plants appear to be more disorganized than those in normal plants. Maferials and Mefhods.-The two strains of barley (Hordeum vulgare) used in these studies were described previously [3, 41. One of these, No. 2 was shown to lack chlorophyll b [2, 41 or at the very least, the ratio of chlorophyll a/chlorophyll [3],
Arabidopsis
fhaliana
1 On leave from Calif., U.S.A. e Revised version Experimental
Department received
Cell Research
of Biology, July
43
12, 1966.
San
Fernando
Valley
State
College,
Northridge,
~Sfrucfure
Fig.
I.--Electron
micrograph
of chloroplasfs
of a chloroplast
lacking
of a mutant
chlorophyll
barley
leaf.
685
b
x 16,500.
Insert,
x 30,000.
b is greater than 1000 [2]. The other strain, Lyon, contained both chlorophyll a and chlorophyll b in normal amounts. Both strains were grown in a 16 hr photoperiod of natural light, supplemented with artificial light. The temperature was maintained at 21°C for 8 hr and 16°C for 16 hr. For electron microscopy, squares of tissue (approximately 1 mm x 1 mm) were cut from the central portion of the second leaf of a mutant or normal barley plant and fixed in 2.5 per cent (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7, for 6 hr at 4°C. The tissue was washed, fixed at 4°C for 2 hr in 2 per cent potassium permanganate in 0.1 M phosphate buffer pH 7, dehydrated in alcohol, and embedded in epon. Sections were cut with an LKB “ultrotome” using a diamond knife. Sections were examined without further treatment in a Siemens Elmiskop 1 electron microscope. Preliminary observations suggested that the grana of the mutant chloroplasts contained fewer lamellae than the grana of normal chloroplasts. In order to investigate differences in the number of lamellae and to avoid, as far as possible, selection by the microscope operator, chloroplasts were photographed at random, the one requirement being that an entire chloroplast could be photographed at a magnification of 10,000 x . Approximately 50 photographs of normal and 50 of mutant chloroplasts at an enlargement of 3 x were made from sectioned material from two separate embeddings of plant material, grown at different times. These photographs were examined for distinctness and resolution of lamellae and 18 photographs of normal barley chloroplasts, and 17 of the mutant were finally selected. The photographs were submitted Experimental
Cell Research
43
686
D. J. Goodchild,
Fig. 2.-Electron
micrograph
H. R. Highkin
of a chloroplast
and N. K. Boardman
of a normal barley leaf. x 16,500. Insert x 30,000.
to two independent observers who had no previous knowledge of chloroplast structure. The observers were asked to count the numbers of single lamellae and groups of lamellae per chloroplast as well as the number of lamellae per group. These results were analysed statistically. TABLE
I. Average numbers Total lamellae/ chloroplast
of lamellae
Grana/ chloroplast
and granaa
Lamellae/ granum
in normal Single lamellae/ chloroplast
and mutant
chloroplasts.
Grana/chloroplast with 8 or more lamellae
Normal
260.67 Mufanf 176.59
49.44
5.07
12.83
7.17
33.94
3.90
46.41
1.18
15.50***
1.17***
Difference
84.08***
- 33.5s***
5.99***
a For the purposes of this table, a granum is defined as a group of lamellae containing 2 or more single lamellae. ***Highly significant, P c 0.001. The significance levels from the analysis are attached to the differences of the absolute values. Experimental
Cell Research
43
Structure
of chloroplasts
lacking
chlorophyll
687
b
Results.-Representative electron micrographs of chloroplasts from normal and mutant barley leaves are shown in Fig. 1 and 2. The lamellar structure of the mutant chloroplast appears to be disorganized in comparison with the normal chloroplast. There appears to be fewer grana in the mutant chloroplast as well as fewer lamellae per granum. The results of the lamellar counts made by Observer A are shown by the histogram in Fig. 3. It is apparent that there are a larger number of single laMUTANT
NORMAL
LAMELLAE/GROUP
Fig. 3.-Histogram and the average
showing the average number number of lamellae per group.
of grana
(groups
of lamellae)
per chloroplast
mellae in the mutant chloroplasts and smaller numbers of grana with three or more lamellae. The results of the statistical analysis of the data of Observer A are given in Table I. All differences between the normal and mutant chloroplasts were highly significant. As well as showing that the normal chloroplast contained more grana per chloroplast and more lamellae per granum, Table I also indicates that the normal chloroplast had a higher average content of lamellae per chloroplast. The difference in the number of grana per chloroplast with eight or more lamellae is particularly striking. The statistical analysis of the data of Observer B also showed that all differences were highly significant. Discussion.-The lamellar system of barley chloroplasts, in common with most higher plant chloroplasts, is differentiated into grana and non-grana regions. In the grana-containing chloroplast, chlorophyll appears to be confined mainly, if not solely, to the grana regions [7, 81, and it seems reasonable to suppose that the lack of one of the chlorophyll pigments would preferentially influence the structure of the grana. The striking differences between the normal and mutant chloroplasts are in the number of lamellae per granum and the number of grana per chloroplast. It has been postulated that chlorophyll is localized in the double membranes, which are formed as a result of the pairing of the single membranes of neighbouring grana lamellae [6, 91. It seems, therefore, that the pairing of the grana lamellae is affected by the absence of chlorophyll b, and this results in impaired grana formation. The mutant chloroplast gives the impression of being more disorganized. The inter-grana or stroma lamellae extend over greater distances before becoming associated with a granum. It is apparent, however, that chlorophyll b is not an essential structural Experimental
Cell Research
43
A. Forer
688
component of the grana of the higher plant chloroplast. Studies at high resolution of the ultrastructure of the membranes of the mutant and normal chloroplasts are proposed. We wish to thank Mr G. A. McIntyre for the statistical Paulin for assistance with electron microscopy. This work a grant from the National Science Foundation (GB3341).
analysis and Miss M. A. was supported in part by
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
ALLEN, M. B., Brookhaven Symp. Biof. 11, 339 (1959). BOARDMAN, N. K. and HIGHKIN, H. R., Biochim. Biophys. Acta (1966). HIGHKIN, H. R., Plant Physiol. 25, 294 (1950). HIGHKIN, H. R. and FRENKEL, A. W., Pfanf Physiof. 37, 814 (1962). HIRONO, Y. and REDEI, G. P., Nafure 197, 1324 (1963). MENKE, W., Ann. Reo. Plant Physiof. 13, 38 (1962). STRUGGER, S., Naturwiss. 37, 166 (1950). THOMAS, J. B., POST, L. C. and VERTREGT, N., Biochim. Biophys. Acfa WEIER, T. E., ENGELBRECHT, A. H. P., BISALPUTRA, T. and BENSON, Suppl. 40, xxviii (1965).
A SIMPLE
CONVERSION
PHASE-CONTRAST
OF REFLECTING
CONDENSERS
LIGHT
13, 20 (1954). A. A., Plant
Physiol.
LENSES INTO
FOR ULTRAVIOLET
IRRADIATIONS A.
Biological Foundation, Received
THIS article
In press.
FORER
Institute of the Car&berg Copenhagen, Denmark May
17,
1966
describes a simple way to use reflecting lenses as phase-contrast condensers, primarily for use with ultraviolet-microbeam equipment. When the visible light image of the microbeam source is focused onto the specimen, phase-contrast microscopy is needed in order to observe the specimen. The existing microbeam equipment achieves phase-contrast by the use of additional components; these components cost money, they take up space, and they require adjustment (see [3,8,14] for reviews of microbeam equipment). For example, the irradiations are usually performed with a reflecting lens which is used as either the objective or the condenser. When the reflecting lens is used as the objective, a standard phase-contrast lens is used as the condenser; the phase-shift normally provided by the objective’s phase plate is then obtained by means of an auxiliary optical system between the reflecting lens and the observer [6, 7, 10, 11, 121. When the reflecting lens is used as the condenser, a standard phase-contrast lens is used as the objective; the annulus of illuminating Experimental
Cell Research
43