Phytochrome and greening in etioplasts

This thesis is concerned with the role played by phytochrome (P) in the development of etioplasts into chloroplasts.Previously dark-grown maize seedlings are not as sensitive as pea seedlings to very low fluences of red light (R) with regard to induction of rapid chlorophyll (Chl) accumulation in white light (WL), but a very low fluence response (VLFR) has been established in this plant species as well. Much higher fluences of a second R pre-irradiation are required to give an additional effect (low fluence response or LFR). When the effect of far-red light (FR) as such is accounted for, the effects of both a first and a second R pre- irradiation are 60-80% reversible by FR in maize seedlings. In high irradiance WL, the lag phase of Chl accumulation is of considerably longer duration. This indicates that photodestruction of Chl plays a role in the occurrence of a lag phase in Chl accumulation. R has a relatively large effect in high irradiance WL (Chapter 3).Phytochrome (P) was measured spectrophotometrically for the first time in purified etioplast preparations obtained in complete darkness from dark-grown seedlings (D etioplasts) (Chapter 4). The P content of etioplast preparations from R pre-irradiated seedlings marginally exceeded that of D etioplasts. While the total P content of maize leaves, as measured in homogenates, decreased after R irradiation as a result of Pfr dark destruction, the P content of etioplasts from similar seedlings remained constant.Attempts to demonstrate a physiological effect of etioplast- associated P were not successful. Preliminary studies on ultrastructural development of etioplasts (Chapter 5) showed that the invitro development during 1 h WL did not completely parallel development insitu . An effect of invivo R pre-irradiation on prolamellar body transformation, which was evident insitu , was not observed invitro . Insitu , formation of incipient grana in WL was stimulated by R pre-irradiation, however, isolated etioplasts proved incapable of forming incipient grana.In the dark, following a short irradiation, regeneration of phototransformable protochlorophyll(ide) (PChl(ide)) was observed in isolated etioplasts (Chapters 6 and 7). However, regeneration kinetics differed from those invivo and no effect of invivo R pre-irradiation could be demonstrated. Invivo , the rate of PChl(ide) regeneration was increased by Pfr (Chapter 6).Wavelength shifts of the 77K fluorescence emission maxima of newly formed chlorophyll(ide) (Chl(ide)) after a short irradiation were studied in leaves and isolated etioplasts. Derivative spectroscopy and curve fitting were applied to study kinetics of these shifts (Chapter 7). The first shift, a red shift, was slower in isolated etioplasts than in leaves. No effect of R pre-irradiation was observed on the rate of this shift. The subsequent blue shift, the so-called Shibata shift, was more rapid, but less complete in isolated etioplasts than in leaves. Whereas in leaves the rate of the Shibata shift was increased by Pfr, this was hardly, if at all, detectable in isolated etioplasts. The amount of phototransformable PChl(ide) decreased and the rate of the Shibata shift increased during storage of isolated etioplasts at 4 °C in darkness. Newly formed Chl(ide) proved unstable in isolated etioplasts.The above results point to a decisive influence of the cytoplasm on the development of etioplasts in WL. In this respect, polypeptides of Chl-protein complexes synthesized in the cytoplasm may play an important role. However, a direct influence of etioplast-associated P in the development of etioplasts into chloroplasts, e.g. on permeability of the etioplast envelope, can not be excluded. Evidence for such an effect is found in the observation that the potentiating effect of a R pre-irradiation with regard to rapid Chl accumulation in WL is still partially reversible by FR after a dark period of 24 h. While Pfr in bulk P had already disappeared due to dark destruction after 4 h of darkness, the amount of P associated with etioplasts appeared not to decrease (see above). It is attractive to attribute at least that part of R potentiation which shows a long-term reversibility by FR, to apparently relatively stable etioplast-associated Pfr.The results are discussed in relation to the phytochrome transport model of Raven and Spruit (Chapter 8). It is concluded that, though they do not provide a direct support for the model, they are not in disagreement with it. The transport model still appears to give an attractive explanation for a number of P responses, such as the VLFR and the Zea P paradox.