Abnormal chloroplast structures in a mutant of Chlamydomonas reinhardi

Experimental

ABNORMAL

Cell Research 93 (1975) 240-244

CHLOROPLAST

STRUCTURES

OF CHLAMYDOMONAS C. S. NICHOLSON-GUTHRIE.’

IN A MUTANT

REINHARDI

F. R. TURNER and G. A. HUDOCK

Department of Zoology, Indiana University,

Bloomington,

IN 47401, USA

SUMMARY Atypical, highly organized, chloroplast structures, which occurred as tubules or spirals and appeared to be composed of a double membrane, were found in a mutant of Chlamydomonas reinhardi. These structures were light-induced, but their appearance was not directly related to the presence of chlorophyll.

The details of the connection between chloroplast structure and function are not completely understood. For example, the relationship between the specific arrangement of the inner chloroplast membranes (lamellae) and chlorophyll synthesis or photosynthesis is unknown. One approach to investigate this relationship is to study mutants of algae or higher plants which have atypical chloroplast structure. The most common type of normal chloroplast structure is a lamellar system composed of a number of long, flattened sacs (thylakoids) which are arranged in parallel in closely packed stacks (grana). The number of thylakoids per granum is species specific

[IIIn the unicellular alga, Chlamydomonas reinhardi, which has only one large, cupshaped chloroplast, each granum is composed of two to ten thylakoids [24]. Mutants of this organism which show atypical r Present address: 700 Drexel Drive, Evansville, IN 47712, USA. Reprint requests to this address. Exptl Cell Res 93 (1975)

chloroplast structure generally have some recognizable form of lamellae, namely disorganized granal arrangement, a decrease in thylakoid material, or just remnants of thylakoids [3-g]. We report a mutant (Rev) of C. reinhardi, containing numerous, highly organized, inner chloroplast structures which do not resemble typical lamellar membranes. (An electron microscopic exhibit of the chloroplast structures in the Rev mutant was presented at the XIII international congress of genetics at Berkeley, Calif., in August, 1973.) MATERIALS

AND METHODS

The Rev mutant was selected in the following manner. Strain y-l, which has normal chloroplast structure and chlorophyll content when grown in the light [3,9], was irradiated with ultraviolet light [lo]. From the irradiated cultures, a mutant, Y-Y, was isolated which lacked lamellae and chlorophyll in the light and dark [ 111.Cells of y-y reverted infrequently (
Abnormal structures in a Chlamydomonas mutant

241

Table 1. Spectral characteristics of light-grown Chlamydomonas mutants Total chlorophyll (pg/108 cells)

Ratio Chl. a :B

Rev

1.58 0.31

3.2 2.0

Y-Y

06

0.9-l .O’

Strain Y-1

Peaks 360-500 (nm) 435 438 (major) 470 (minor) 447 (major) 474 (minor)

Absorbance” 435 nm

447 nm

0.182 0.065

0.138 0.061

0.034

0.043

Data are given for one of the Rev mutants. a Optical density units for 106cells/ml acetone extract. b Cultures of y-y usually contained no chlorophyll. Infrequently, some cultures had barely detectable amounts (0.03-0.05 pg chlorophyll/lOB cells); green revertant cells were not found in these cultures. c Ratio was determined from y-y cultures with barely detectable amounts of chlorophyll.

spectral curve in the carotenoid absorbing region (360-500 nm). Although cells of Rev and y-y had two peaks in that region in contrast to the one peak of y-l, the major peak of Rev was closer to that of y-l. Note that approximately equal concentrations of chlorophyll a and B were found in y-y, but twice as much chlorophyll d was found in Rev. The ultrastructure of light-grown strains of wild-type and y-l cells are similar and have been described in detail [3, 93. The intracellular structures (nuclei, nucleoli, mitochondria, Golgi bodies, and eyespots), flagella, and sizes or shapes of the cells of light-grown cultures of y-y and Rev were comparable to those of wild type and y-l [lo]. With regard to the chloroplasts of these four strains, there were no discernible differences in the pyrenoid bodies, starch plates, shapes of the chloroplast, and outer chloroplast membranes. The only structural difference in the strains was in the inner membranes of the chloroplasts. The lamellae of wild type and y-l cells RESULTS AND DISCUSSION were packed in stacks [3, 9, lo]. The muIn general the pigment pattern of the Rev tant, y-y, lacked lamellae but appeared to mutants was intermediate between those of have tiny remnants of lamellae throughout y-l and y-y (table 1). When compared with the chloroplast ([lo, 111, a manuscript on y-l, Rev had less chlorophyll and a different the ultrastructure of y-y is in preparation).

liquid, enriched medium (0.2% anhydrous sodium acetate, 0.2 % Difco bacto-tryptone, and 0.2 % Difco yeast extract). Under these conditions the division time of y-l was around 9 h, or two to three times faster than the division times ofRev ory-y [lo]. In contrast toy-1,Rev and y-y did not grow in an inorganic salt medium with 5 % CO2 unless acetate was added. For pigment analysis a total of at least lo* cells in mid-logarithmic growth phase (0.2-0.3 optical density units at 750 nm with a Bausch and Lomb 20 Colorimeter and 13 mm diameter tube) were extracted at least 3 times in an ice bath with a total of 10 ml 80 % acetone. The absorbances of the extracts between 360 and 800 nm were determined with a Cary Model 14 recording spectrophotometer and 10 mm path length. Total chlorophyll and ratio of chlorophyll (C&l.) a :E were determined by the methods of Arnon [12] and MacKinney [13], respectively. For electron microscopy, cells were grown in test tubes containing 10 ml enriched medium to mid- to late logarithmic growth phase. The cells were collected on a Millipore filter and exposed to the vapors of a 2% 0~0, solution for 10 min. After covering the cells with a thin layer of melted 2% agar, they were fixed with osmium zinc iodide reagent [14] overnight in the dark at room temperature, rinsed, and subsequently stained with 0.5% uranyl acetate for 6-12 h. Samples were embedded in Spurr low viscosity medium, and thin sections were poststained with 0.5% uranyl acetate and lead citrate before examination with a Hitachi HU-11-C electron microscope. Alternately, the cells were fixed in 2% aqueous KMnO, at 4°C for 1 h. Embedding and section staining were as described for the osmium zinc iodide fixed cells.

Exprl Cell Res 93 (1975)

242

Nicholson-Guthrie,

Turner

and Hudock

Figs !A.

Atypical chloroplast structures in the Rev mutant. In fig. la Rev cell containing numerous atypical structures in the cup-shaped chloroplast is shown. The structures are randomly oriented (figs I. 2) and resemble tubules in longitudinal sections (fig. 3). In cross sections (fig. 4) the structures appear as a double membrane shaped as spirals (S), several concentric circles (C), or double rings (R). Cells were fixed with osmium zinc iodide (figs 1,2,3) or KMnO, (fig. 4). Fig. 1, xl 1200; fig. 2, x 15000; fig. 3, x28000; fig. 4, x70000.

The Rev mutants lacked lamellae remnants, and, instead, had numerous chloroplast structures (figs 1, 2) which appeared as tubules in longitudinal sections (fig. 3) and as spirals or concentric circles in cross sections (fig. 4). Some structures extended across the chloroplast (fig. 1) and appeared to be composed of a double membrane (fig. 4). Rev cells with the structures generally had few lamellae (fig. l), but both atypical structures and typical lamellae, usually disExptlCellRes

93 (1975)

organized, were seen in the same chloroplast . The spirals indicate an ordered pattern for the structures, but the structures lack apparent organization with relation to each other. The random arrangement of the structures within the chloroplast is demonstrated in figs 1 and 2. The presence of the chloroplast structures depended upon growth conditions. Light-grown cultures contained the structures, whereas dark-grown cells lacked

Abnormal structures in a Chlamydomonas mutant them, even when the dark-grown cells synthesized chlorophyll. When the structures were absent, the chloroplast contained sparse, disorganized lamellae, frequently occurring as single thylakoids. The mutation responsible for the atypical structures is relatively stable. The Rev mutants have been transferred to fresh medium over 40 times, and the tubules and spirals are still present. Yet, reversion of cells containing the atypical structures to cells lacking them occurred. Light-grown Rev cultures, originally isolated from single colonies containing the chloroplast structures, were found to revert such that a culture had two types of cells-those having the atypical tubules and spirals and those lacking them and containing sparse, disorganized lamellae. Furthermore, cultures differed in the proportion of cells which contained the atypical structures. For example, the structures were found in lo-20% of the cells of one culture but in 90% or more of the cells of another culture. Both cultures were derived from the original clone of Rev cells. The fact that the spiral structures are found in some cells but not in other cells of the same culture is evidence that the atypical structures are not due to artifacts caused by fixation or staining techniques. If the structures were the result of a technique, all Rev cells should have them. Also, as stated previously, dark-grown Rev cells which synthesized chlorophyll had no atypical chloroplast structures but sparse lamellae as demonstrated by both fixation methods. The atypical spirals or tubules were not found in the control cells, wild type and y-l; the chloroplast and other cellular structures appeared as reported by others [3, 91. By using two methods of fixation and control cells, the atypical chloroplast structures do not appear to be artifacts.

243

Rev is a derivative of y-l, originally selected from wild-type strain, 137~. Another wild type C. reinhardi strain, 90, exists which is morphologically similar to and mates with strain 137~[IO]. The two strains differ in DNA replication during meiosis [15, 161. Ultraviolet irradiation of strain 90 produced a mutant with decreased chlorophyll content and atypical chloroplast structures comparable to those found in Rev [IO]. Similar atypical chloroplast structures were seen in other ultraviolet-induced, chlorophyll deficient mutants, originating from y-l cells. The fact that these atypical spirals and tubules were found in mutants originating from two different wild type strains suggests that induction of these structures in C. reinhardi is not an infrequent event. Tetrad analysis of crosses between strain 137~ wild type and y-y gametes resulted in aberrant genetics, namely reciprocal crosses produced different results [lo, 231. When the mating type of y-y was minus (paternal), the tetrads were mainly composed of 2 green: 2 yellow colonies. The mating type marker segregated 2 plus : 2 minus and independently of the color phenotype. In the reciprocal cross, when y-y was mating type plus (maternal), the majority of the meiotic products were green colonies and lethal progeny; the majority of the viable progeny were mating type minus. The yellow color phenotype, lack of chlorophyll, of y-y cells was directly related to lack of lamellae [lo]. Since Rev is a revertant of y-y and aberrant genetics were found for the color phenotype of y-y, unusual inheritance is suspected for the atypical chloroplast structures in Rev. Studies on the inheritance of these atypical tubules are in progress. The relationship of these light-induced structures to lamellae and chlorophyll synthesis is not clear. Since the structures Exptl Cell Res 93 (1975)

244

Nicholson-Guthrie,

Turner und Hudock

were found in cultures which contained chlorophyll, they apparently do not result from a block in chlorophyll synthesis. Von Wettstein [17] concluded from studies on corn and barley that the normal arrangement of lamellae requires chlorophyll. Studies on C. reinhardi mutants indicated that at least 75% of the chlorophyll could be lost without grossly affecting chloroplast structure [4]. Yet Rev, containing 21% chlorophyll content, had atypical lamellar arrangement. Concentric lamellae in Euglena [18, 193, spheroid grana in higher plants [17, 20,211, and coiled thylakoids in a brown, saprophytic orchid [22] were reported. The numerous spiral structures in Rev more closely resemble those in the orchid than in the other organisms. However, chloroplast structures existing as spirals or tubules, as found in Rev, have not been reported. Are the chloroplast structures in Rev atypical thylakoid membranes? The spiral, tubular elements might represent coiled, unstacked thylakoid sheets. If the structures are thylakoid in nature, it is not clear why both atypical (tubules and spirals) and typical lamellae were found in the same chloroplast . Understanding the nature of these chloroplast structures and their relationship to lamellae and chlorophyll may help elucidate membrane structure and function in chloroplasts.

Exprl Cd Res 93 (1975)

This work was supported in part by an NIH Training Grant fromUSA, the National Institutes of General Medical Sciences

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ReceivedJune11 1974 Revised version received December 27, 1974