Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive γ-Irradiation of the Seed

Abstract

Spinach seeds (Spinacia oleracea L.) given massive doses of y-irradiation (500 krad) germinate and form a seedling with two green cotyledons and a radicle, but develop no further. Irradiated cotyledons show no increase in cell number or total DNA over a 7-day period in the light, while in control cotyledons there is a small increase in cell number and large increases in total DNA and chloroplast number. The chloroplasts of irradiated cotyledons are delayed in their division, become greatly enlarged and contain large amounts of starch. The whole population of chloroplasts subsequently undergoes a wave of division. The daughter chloroplasts show normal thylakoid development, but have some abnormal structural features caused by the radiation stress. Information on the effect of X-irradiation, ultraviolet irradiation, and 5-fluorodeoxyuridine on chloroplast replication and on chloroplast and nuclear DNA synthesis was obtained from cultured spinach leaf discs. It appears that chloroplast replication is more resistant to ionizing radiation than cell division and can proceed in the absence of nuclear DNA synthesis and greatly reduced chloroplast DNA synthesis.When immature spinach leaf discs are cultured in the light, there is normally a close relationship between chloroplast DNA synthesis and chloroplast replication during cell growth (21, 23. 24). The two processes are not irrevocably coupled. Rates of chloroplast DNA synthesis in dark-grown spinach discs are comparable to those in light-grown discs, though the division rate is greatly reduced (24). Boasson and Laetsch (1) found that lightstimulated chloroplast replication could occur in the presence of the DNA inhibitor, FdUrd,' in tobacco leaf discs. They suggested that chloroplasts contained multiple copies of chloroplast DNA and could divide a number of times without additional DNA synthesis. It is now established that there are many DNA copies in chloroplasts (16). More recently, it has been shown that in Euglena plastids can replicate using a reduced number of genomes (17).In higher plants, relationships between chloroplast replication and cell growth (2, 14, 22), and chloroplast replication and nuclear DNA have been found (6,24). Recently gibberellic acid has been shown to stimulate cell elongation, chloroplast numbers, and the rate of synthesis of both mainband DNA and organelle-rich satellite DNA in cucumber hypocotyls (15).It was the purpose of this investigation to examine the regulatory relationships between cell growth, chloroplast DNA synthesis, nuclear DNA synthesis and chloroplast replication and development. In the first instance, we used cultured spinach Abbreviation: FdUrd: 5-fluorodeoxyuridine. 41 discs, but this report is primarily concerned with the growth and development of chloroplasts in spinach cotyledons that have developed from heavily y-irradiated seed. These are -'y-plantlets" similar to those studied by Haber (I 1). MATERIALS AND METHODSGrowth of Leaf Discs and Cotyledons. Methods used in growing spinach plants (Spinacia oler...

Plastid Replication and Development in the Life Cycle of Higher Plants

“…In both dividing and expanding cells, the increases in chloroplast number are from the division of chlorophyll-con taining plastids (6, 57). No evidence of either proplastids or etioplasts can be found in light and electron microscope observations of young emerged leaves, but chloroplast dumbbells are present at high frequencies in these tissues (15,64). Table 1 also provides data on the approximate number of chloroplasts present in the cells of both young and mature leaves.…”

“…The results of experiments in which X and 'Y irradiation have been used to inhibit DNA synthesis in leaf discs and cotyledons of spinach indicate that chloroplast division is less sensitive to ionizing radiation than is cell division (64). A factor contributing to the greater resistance of plastid division to radiation is the possibility that this event can occur without concomitant replication of ptDNA (63,80).…”

“…This means that during leaf growth a doubling of chloroplast numbers occurs at the time of the last chloroplast division cycle. In most leaves there is a gradient in cell maturity between the tip and the base of leaves so that chloroplast formation continues to occur at the leaf base at a time when it has ceased at the leaf tip (58,64).…”

Significance of structural variation in thylakoid membranes in maintaining functional photosystems during reproductive growth

“…Such stress response reactions are apparently controlled by complex interactions between nuclear and chloroplastic signals and involve intrinsic factors that mediate growth phases. The interrelation between the mesophyll cell cycle and the development and degradation of chloroplasts was monitored in spinach cotyledons (Rose and Possingham ) and rice coleoptiles (Inada et al ). However, little information is available on the changes in chloroplasts during the phase change from vegetative to reproductive growth.…”