Radiation Studies in Lens culinaris: Ultrastructure of Chloroplast in the Leaves of the Normal and Mutated Plants1

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1 Cytologia 44: 13-19, 1979 Radiation Studies in Lens culinaris: Ultrastructure of Chloroplast in the Leaves of the Normal and Mutated Plants1 S. S. N. Sinha Department of Botany, Ranchi University, Ranchi, India Received March 10, 1977 During the last few years, a number of papers dealing with the fine structure of the chloroplast have been published (Muhlethaler 1955, Von Wettstein 1957 a and b, Sager 1959, Weir 1961, Menke 1962 and Thomson 1965). They have de scribed grana and stroma lamellae in different plants. Von Wettstein (1960, a and b) in barely, Walles (1965) in Helianthus annus and Boynton (1966) in Tomato have dealt with the fine structure of chloroplast in the leaves of chlorophyll mutants. In the present work efforts have been made to study the fine structure of chlo roplast in the normal green leaf and in the leaf of a mutated plant (whitish yellow in colour) found in the M2 generation of a population grown from irradiated seeds of Lens culinaris with a dose of 12kr. gamma rays. Experimental work Plants were raised in greenhouse (Temp. 22 Ž }2ß, day-light supplemented by mercury vapour lamp 500 watts). When the plants were three weeks old, the mature leaves measuring about 5mm in length were collected from different parts of the plants. Selected leaf areas, which were without any distortion and fully flattened, were cut into small peices (1 ~2mm) They were fixed with osmic tetraoxide (ph 7.2) using phosphate buffer for 2 hours at 0 Ž. This was followed by dehydration in a graded series ( %) of alcohols at minimum interval of half an hour. Three changes of absolute alcohol at 45 minutes interval were made and the tissue was allowed to come to room temperature. The tissue was gradually in filtrated with Epon solution and allowed to stand overnight in 100% resin. Then it was transferred to freshly prepared Epon and was embedded in small capsules. These capsules were left for 48 hours in an incubator at 60 Ž. The sections were cut on an LKB Ultratome III with glass knives. The sections were examined and photographed on Siemens Elmiskop IA, operating at 80kv with double condenser and a 20ƒÊ objective aperture. The thickness of the section varied between 40mƒÊ and 50mƒÊ judged by their interference colour (grey, gold). Results Chloroplasts from green leaves of normal plants are lens shaped in most 1 Part of the Ph. D. thesis.

2 14 S. S. N. Sinha Cytologia 44 Fig. 1. Section of the leaf of control plant showing membrane (M), peristome (P), grana (G), electron dense globuli (GL) and stroma lamellae (SL). ~70,000.

3 1979 Radiation Studies in Lens culinaris: Ultrastructure of Chloroplast 15 Fig. 2. Section of the leaf of mutant plant showing grana (G) and osmophillic globules (OG). ~ 70,000.

16 S. S. N. Sinha cytologia 44 cases. (Fig. 1) The chloroplasts are surrounded by a two-fold membrane (M), beneath the membrane (M) is peristome (Pr) (Wettstein's term).

4 16 S. S. N. Sinha cytologia 44 cases. (Fig. 1) The chloroplasts are surrounded by a two-fold membrane (M), beneath the membrane (M) is peristome (Pr) (Wettstein's term). Grana (G) and Stroma lamellae (SI) are aggregated in layers, traversing the entire chloroplast in a more or less regular way, parallel to the longer axis of the chloroplast. In most cases, the grana and stroma lamellae occupy about 50% of the chloroplast, the rest being stroma. Occasionally a granum oblique or at right angle to the longer axis of the chloroplast is formed. The grana lamellae are nearly 70 to 90A thick and are stacked in groups of 15 thylakoids. The stroma lamellae are thinner than the grana lamellae (40 to 45A). Electron dense globuli (Gl) which vary from 400 to 1200A in diameter occur in stroma. They seem to be carotinoid in nature or they might be lipids (Fig. 1). Starch grains are frequently present and in most cases are more than one. Chooroplast from leaves of M2 mutants (Fig. 2) are irregular in shape, char acterised by projections and invaginations. The plastids contain swollen grana with expanded thylakoid cavities, between the grana the stroma lamellae seen degenerated into small vesicles still more or less linearly disposed. Plastids which are nearly normal in shape contain degenerated vesicular lamellar material arranged in primary layers. The vesicle become more dispersed towards the periphery of the plastids. The grana when present are composed of many aggregated grana lamellae which are distorted. Grana structure is variable, it may be the morphology of a stack of thylakoids with distorted shape or presumably a mass of barely resolvable lamellae. Intermediate stages can be seen. Partially or diffused grana exist adjacent or connected to grana of nearly normal structures. Grana are interconnected by stroma lamellae in some cases, but generally these are vesiculate and feebly developed and grana are to some extent isolated from each other. Incipient grana can be located along the primary lamellar layers. Abnormally large and irregularly constituted grana are common, whereas chlo roplast with a few primary lamellar layers, lacking grana are frequent. Osmiophilic globules are present, often in groups. Such a group of globules regularly arranged in three rows occupies a site A on the degenerated stroma lamellae. This site appears to be that of a degenerated granum. The same plate at B shows similar globules present at such a site. In mutant chloroplasts the osmiophilic globules are always regularly arranged in this way at or near the end of the granum in rows of two or more, whereas in the normal plastids they are scattered. In the leaves of the mutant plants (Fig. 2) the mature plastids with conditions ranging from the total absence of ultrastructures upto fully developed deformed grana and stroma lamellae, and situation intermediate between the two have been recorded. Such plastids with differential development of ultrastructures inside may lie side by side in the same cell of the leaf. Starch grains are totally absent. Discussion The existing difference between the chloroplasts of the normal leaves and leaves from the mutant plants may be attributed to the change in the nature of genes,

5 1979 Radiation Studies in Lens culinaris: Ultrastructure of Chloroplast 17 controlling the development of chloroplasts. But it appears that possibly such an abnormality is caused by genetic blocking of the production of some enzyme controlling the development of the lamellar system. If so it is remarkable that in the same mutant plant, or in the same cell plastids with degenerated or structureless grana are present side by side with plastids whose grana are fully developed. The total absence of ultrastructures inside some mature plastids seems to indicate that such a block may come into action at the earliest stage of development. The diffuse internal structure of the grana concerned cannot be attributed to poor fixation because its occurrence is very common and other organelles-nuclei, mitochondria etc. exhibit normal morphology in the same cell. The possibility of oblique sections cannot be ruled out, but as they are very numerous in mutant leaves as compared with the controls, the possibility of grana being disintegrated cannot be ignored completely. Vesicles present in the chloroplast may be the outcome of lamellar disintegration Shaw and Manocha (1965), Von Wettstein (1959 b) and Menke (1962) have pointed out the possibility of the localization of the chlorophyll molecule within the parti tion formed by the fused margins of adjacent thylakoids comprising the chloroplst grana. The presence of degenerating grana with no chlorophyll in the leaves of the mutant plants is in accordence with these findings. Robbelen (1959) with lutescens mutants of Arabidopsis has demonstrated a secondary destruction of previously formed chloroplast lamellae by separation of the thylakoids. Complete degeneration of the lamellar system follows. Such a type of degeneration can be compared with the vesiculate degeneration observed here. The presence of osmiophilic globules in definite rows near the end of the granum may also be associated with the development of the lamellar system, the linear array of globules possibly indicating the line of potential lamellar materials. Chlorosis is induced in leaves of normal genotype of several plants species by mineral deficiencies, Shaw and Manocha (1965) by induced senescence in detached leaf of wheat and Thomson et al. (1966) by treatment with ozone on bean leaves have shown a relationship between disruption of the lamellar system of chloroplast and chlorosis. The large grana, larger than the controls, in the leaves of the mutant plants appear to have developed by an abnormal function of the grana development system. The early stages of grana formation located along the "primary layers indicate the fact that the formation of the granum starts in the normal way, but then is ar rested perhaps due to enzyme starvation, at a particular stage, the consequence of gene mutation, either it is little developed or it is abnormal. The total absence of ultrastructure indicates that such a block can be effective at a very early stage of grana development. Von Wettstein (1957 a and b, 1959 a and b) has pointed out that in barley the different mutants albina, xantha and viridis show blocks at different stages of de velopment of ultrastructures of plastids. Leyfort (1957) and Boynton (1966) in tomato has reported such blocks at different points in the developmental chain of the plastid structure in the leaves of the mutant plants. From the above results it may be concluded that irradiation causes changes in

6 18 S. S. N. Sinha Cytologia 44 the nature of the gene or genes controlling chloroplast development. Such a change brings disturbances in the development of the grana and consequently the chlo rophyll synthesis is blocked at a particular stage, causing the leaves to suffer from chlorosis. Most of the plastids grow upto maturity in spite of lack of differentia tion in the linner structure. Thus the growth of the entire chloroplast appears to be independent of its ultrastructural differentiation. Such a result has been reported by Von Wettstein (1959 a and b) also. In lentils the very rare occurrence of xantha or albina mutants may be due to more than one gene responsible for controlling the development of the ultrastructure of plastids. The presence of mature plastids with differential development of ultra structure in the same cell of the leaf may be due to such a mutation being effective at individual level. Summary Leaves of a mutant plant (whitish yellow in colour) were studied with electron microscope. The ultrastructure of the plastids of the chlorophyll mutants showed deformed or degenerated grana with disintegration of stroma lamellae. This may be attributed to the changes in the nature of the gene, controlling the development of the chloroplast. Such an abnormality may be caused by genetic blocking of the production of some enzyme controlling the development of the lamellar system. Complete degeneration of the lamellar system follows. Vesicles present in the chloroplast may be the out come of lamellar disintegration. In lentil the very rare occurrence of xantha or albina mutants may be due to more than one gene respon sible for controlling the development of the ultrastructures of plastids. Acknowledgement I express my heartfelt thanks to Professor M. B. E. Godward for her kindness during the preparation of the thesis. Bibliography Boynton, J. E Chloroplast deficient mutant in tomato requiring vitamin B I. Hereditas 56: Leyfort, M Structure fine die chloroplast normal chez Lycopersicum esculentum: liaisons grana. C. R. Acad. Sc. 244: Menke, W Structure and chemistry of plastids. Ann. Rev. Plant Physiology. 13: Muhlethaler, H Structure of chloroplast. Int. Rev. Cytol. 4: Robbelen, C Untersuchungen uber die Entwicklung der submikroscopishen Chloroplasten -structur in Elattfarbmutanten von Arabidopsis thaliana. Z. Vererbungslehre. 90: Sager, R The architecture of the chloroplast in relation to its photosynthetic activities. Brookhaven Symposium in Biology 11: Shaw, M. and Manocha, M. S Fine structure in detached senescing wheat leaves. Cand. Journ. Bot. 43: Thomson, W. W The ultrastructure of Phaseolus vulgaris chloroplast. Journal of Expt. Botany 16:

7 1979 Radiation Studies in Lens culimaris: Ultrastructure of Chloroplast 19 Thomson et al Effect of ozone on the fine structure of the palisade parenchyma cells of bean leaves. Cand. Journ. Bot. 44: Von Wettstein 1957.a. Chlorophyll letale und der submikroskopische Formwechsel der Plastiden. Expt. Cell Research 12: b. Genetic and submicroscopic cytology of plastids. Hereditas 43: a. The effect of genetic factors on the submicroscopic structure of the chloroplast, J. Ultrastructure Research 3: b. The formation of plastids structures. Brookhaven Symposium in Biology. 2: a. Multiple allelism in induced chlorophyll mutants II. Error in the aggregation of the lamellar discs in the chloroplast, Hereditas 46: b. Multiple allelism in induced Chlorophyll mutants of barley. Hereditas 46: Walles, B Plastids structure of carotenoids deficient mutants of sunflower. Hereditas 53: Weir, T. E The ultramicrostructure of starch free Chloroplast of fully expanded leaves of Nicotiana rustica. American J. Bot. 48:

THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L.

New Phytol (1974) 73, 139-142. THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L. BY JEAN M. WHATLEY Botany School, University of Oxford (Received 2 July 1973) SUMMARY Cells in