Light Saturation of Growth and Photosynthesis of the Shade Plant Marchantia Polymorpha

0 R. MACHE AND S. LOISEAUX Laboratoire de Physiologie Vigitale, Universite I de Grenoble (38) , France SUMMARY The growth rate of the shade plant Marchantia was at its maximum for a low illumination, 2-3 x io8 lx, and was inhibited by an excess of light. Photosynthesis by intact thalli and by isolated chloroplasts of Marchantia was saturated by a light intensity of 2-3 x io3 lx. These isolated chloroplasts were able to carry on satisfactory rates of photosynthesis, up to 35 fiM CO2/h/mg chlorophyll. The Hill reaction and photosystem II were also saturated by the same light intensities, demonstrating that the factor limiting the light saturation of photosynthesis is located in the chloroplast. The structure of chloroplasts was strongly modified by an excess of light, small grana and fret membranes being replaced by continuous grana. - MATERIALS AND METHODS Female thalli of Marchantia polymorpha were grown on Petri dishes as previously indicated (Mache & Loiseaux, 1972) but were illuminated at different light intensities as described under Results. The material taken for photosyntheric studies was grown on vermiculite in a greenhouse using the same nutrient solution as for cultures on Petri dishes. Isolation of chloroplasts The method as previously described (Mache & Loiseaux, 1972) was used with the difference that 0-5 M cacodylate was employed instead of 0-2 M. The isolated chJoroplasts used for studying the CO, fixation were prepared according to the method previously used (Mache & Loiseaux, 1972). For the Hill Reaction and study of photosystem II, chloroplasts were extracted as follows: harvested thalli were washed, chilled and ground for 15 s in a Waring blender with twice their volume of a solution containing 50 mM phosphate buffer at pH 7-0, 50 mM KC1, 5 mM MgCl2, 0-4 M sucrose. The slurry was filtered through 8 layers of cheese cloth. The filtrate was centrifuged for 1 min at 2000 g. The pellet was resuspended in the grinding solution. All operations were conducted at about 2 C. Fixation of CO2 The fixation of CO8 by isolated chloroplasts and adult thalli was studied by the method previously described (Mache & Loiseaux, 1972), but thalli were immersed in a final volume of 2 ml. Chlorophyll determination The chlorophyll content was determined according to Arnon's procedure (Arnon, 1949). Hill reaction and photosystem We followed the methods employed by Hoober, Siekevitz & Palade (1969). Assays were conducted with chloroplast suspensions containing 20-30 fig chlorophyll. Light saturation curves of growth Light intensity had an effect on the growth of Marchantia and a saturation curve was drawn (Fig. 1) using the length of thallus as a measure. The length of thalli at low illumination (below 750 Ix) was not as reliable as other measures since etiolation occurred: thalli were longer than expected, narrower and thinner. Propagulae of Marchantia grown under different light intensities, with a photoperiod of 14 h light, 10 h darkness, showed a maximum of growth for 5-6 x io3 lx when 15 days old (Fig. 1). After 26 days of growth, saturation was obtained with 2-3 x io8 lx as seen in Fig. 2, where length increments have been reported between the 15th and the 26th days of growth. This demonstrates 2 phases of growth occurring in our experiments: phase I up to the 15th day and phase II afterwards. At the 26th day the saturation curve is an hyperbola as shown by reciprocal plot of Light saturation of Marchantta Fig. i. Dependence of the length of thalli on light intensity after 15 days of growth (phase I). Fig. 2. Effect of light intensity on the length increment of thalli between the 15th and the 26th day of growth (phase II). lengths and light intensities (Fig. 3, B). This is not the case during phase I (Fig. 3, A). From these observations it follows that light operates on Marchantia's. growth through at least 2 light-limited mechanisms at the beginning of growth whereas there is only one light-limited factor afterwards. Inhibition effect of excess of light Growth of Marchantia was inhibited by light intensities of 6x io3 lx and above, during phase II (Fig. 2), and its external morphology was modified. This inhibiting effect has often been observed with Marchantia grown on agar under continuous illumination, with lower light intensities in this case, 4 x io3 lx after 6 days of growth. Thalli became thicker, brittle, yellowish and curved down into the medium. o 125 x 3 JO 25 Fig. 3. Reciprocal plot of light intensities and lengths, A reported in Fig. i (phase I) and B obtained after 26 days of growth (phase II). Electron microscopy was carried out in order to see whether any ultrastructural change could be detected in plants grown for 3 3 daysat8'5 x io3lx compared with plants grown at 2 x io3 lx. Surprisingly, chloroplasts were effectively modified by an excess of light. Dividing cells at the edge of the thallus were the most interesting as they showed different aspects and stages of chloroplast transformation. Dividing nuclei are normal as are mitochondria (Fig. 7) and Golgi bodies but chloroplasts are very different from those observed in controls. The most striking change is the numerous invaginations of the inner membrane in the whole surface of the chloroplasts. Invaginations (see arrow Fig. 10) are tubular and irregular-looking like those of mitochondria when cut tangentially (Fig. n ) . Another, probably more important, change occurs in the organization of the lamellae which is completely modified. This is particularly evident in young chloroplasts (Fig. 8) where thylakoids extend from one side of the chloroplast to the other, grouped in parallel strands. The partitions seem to be maintained as in normal grana. In older chloroplasts, probably normal at first, starch grains seem to resorb and normal grana tend to disappear, being replaced by very long groups of thylakoids. Small globules or tubules of unknown nature are seen (double arrow Figs. 10, 11) scattered in the plastid stroma. Large lipid globules are found in the cytoplasm. Only chloroplasts seem to be structurally modified by light in excess. Light saturation of photosynthesis in vivo It was interesting to study the saturation of photosynthesis and to see whether saturation occurs at the same light intensity as for growth and whether any inhibition appears at high illuminations. If saturation of photosynthesis occurs in vivo, for the same light intensities as it Fig. 4. Dependence of the rate of C0a fixation by intact thalli of Marchantia on light intensity. Fig. 5. Dependence of the rate of COt fixation by isolated chloroplasts of Marchantia on light intensity. occurs in the second phase of growth it would mean that photosynthesis is the limiting factor. Experiments were conducted with adult thalli, grown under 2 x io3 lx and photosynthesis was measured as indicated in Material and Methods. To be sure that limitation of C 0 2 diffusion did not occur when thalli of Marchantia were imm (...truncated)