Chlorophyll formation and phytochrome

The rôle of phytochrome in the regeneration of protochlorophyll (Pchl) in darkness following short exposures to light, as well as in the accumulation of chlorophyll- a (Chl- a ) in continuous light in previously dark-grown seedlings of pea, bean, and maize has been the subject of the present investigation.The in vitro red absorption peak of Chl- a was situated at shorter wavelengths, if a dark period was inserted between the moment of Pchl phototransformation and that of pigment extraction (Chapter 5, fig. 8). There was a considerable pigment photobleaching during Pchl photoconversion in high quantum flux densities (2.2 x 10 4ergs/cm 2sec) of red light (fig. 10). The effectivity of red (651 nm) light in Pchl phototransformation surpassed that of blue (442 nm) light about two times (fig. 11.).The initial rates and ultimate level of Pchl regeneration were strongly depressed in older leaves (Chapter 6). In young seedlings Pchl regeneration in darkness after an illumination, generally, stopped after having reached its initial level (e.g. figs. 16, 18, and 19), which level (either expressed as pigment content per g fr. w. or per constant number of leaves) was not affected by simultaneous leaf growth over a prolonged period (fig. 54). Thus, we concluded that the number of Pchl regeneration sites per cell is constant during the first hours of greening. Red-far red control of the initial rates of Pchl regeneration could not be demonstrated (figs. 16, 17, 18, and table 1). In older maize leaves, an effect attributable to phytochrome was observed upon the final level of Pchl, reached in prolonged darkness (fig. 18). In intact bean seedlings, however, even five repetitive exposures to red and red followed by far red, at 2-hour intervals, did not significantly alter the final level of Pchl (fig. 19).The duration of the lag phase in Chl- a formation was increased, and the ultimate rate of Chl- a accumulation depressed in pea seedlings continuously exposed to white fluorescent light of high intensity (60,000 ergs/cm 2sec) (figs. 23 and 25) which we ascribe to photodestruction of freshly formed chlorophyllous pigments (Chapter 7). The duration of the lag phase in Chl- b formation was even more sensitive than that of Chl- a formation to the intensity of the light (fig. 24).In detached leaves and leaves of seedlings with the cotyledons removed, Chl- a formation was very poor as compared with leaves on intact plants (section 7.2.1.2.). Chl- a synthesis of detached leaves was only partly restored by sucrose supply. Application of δ-aminolevulinic acid had no stimulating effect on the rate of Chl- a formation in the light (fig. 26 and section 8.2. 1.) nor on the accumulation of Pchl in darkness (section 7.3.). This renders unlikely that synthesis of this compound is a bottleneck in Chl- a formation.Continuous red light (646-651 nm) was the most effective wavelength range for Chl- a formation and accumulation of carotenoids; they were much weaker in the blue (442 nm) (section 7.2.2.). The nature of the photoreceptor pigment(s) involved could not be established with certainty (sections 7.2.2. and 7.3). The increase in fresh weight of pea plumules during continuous illumination is most likely mediated by phytochrome (fig. 36). Electron micrographs demonstrated that internal structural development of etioplasts was especially rapid in continuous blue light (section 7.2.3). During the first hours of greening in white light, three photoreceptor systems may be simultaneously active.In intact seedlings the stimulatory effect of brief pre-exposures to red light on rapid Chl- a accumulation in continuous light was retained during a dark period of at least 48 hours (Chapter 8, figs. 38 and 40). This may be related to the irreversible concomitant light induced rise in fresh and dry weight of leaves (figs. 48 and 41, respectively), which may be paralleled by equally irreversible growth and development of etioplasts (section 9.3). Excised leaves were completely insensitive to irradiations of the type, inductive in intact plants (fig. 39).Considerable differences in sensitivity to red light (fig. 42) were not accompanied by similar differences in spectrophotometrically demonstrable phytochrome (fig. 43).Pea leaves were found extremely light sensitive (fig. 44): even relatively short exposures to weak green 'safelight' induced rapid Chl- a accumulation in subsequent continuous white light (fig. 45). The action spectrum pointed to phytochrome as the photoreceptor pigment (fig. 46).Induction by red light was hardly reversible by subsequent far red in various cultivars of pea and bean, and in young maize seedlings (table 4), owing to considerable inductive capacity of far red (Chapter 9). Far red reversibility of the effect induced by red increased considerably with increasing duration of dark incubation between pre-irradiation and continuous white light (fig. 48). Fairly complete red-far red reversal occurred in plants de-etiolated by pre-irradiation some hours prior to the inductive treatment (fig. 50). Even relatively short exposures to green safelight caused de-etiolation with concomitant increase in subsequent red- far red antagonism (fig. 52). We define as de-etiolated the state of a completely dark-grown seedling treated with a photobiologically inductive amount of light (see p. 1, and p. 77).In order to explain the difference between completely dark-grown and deetiolated seedlings in their sensitivity to far red induction and red-far red photoreversibility, a model is presented, involving transport of phytochrome during de-etiolation to receptor sites of restricted capacity which then become activated to initiate the physiological response (figs. 57 and 58, section 9.1).As for the rôle of phytochrome in the greening process, it is concluded that the biosynthetic pathway leading to Pchl and Chl- a is not directly under phytochrome control. However, P fr is postulated to increase the capacity of the biosynthetic system forming Pchl by stimulating synthesis of structural proteins, enabling rapid build-up of the photosynthetic apparatus as soon as Chl molecules are being continuously supplied by phototransformation of Pchl. During this process, Chl is supposed to be detached from the Pchl regeneration sites, and the availability of empty regeneration sites is supposed to activate Pchlbiosynthesis.Phytochrome-mediated enhancement of the rate of phytolization might well be another factor favouring Chl- a accumulation in continuous illumination by facilitating the protection of the freshly formed chlorophylls from photodestruction. A similar function may be ascribed to certain red light induced carotenoid pigments, at least for greening in light with an appreciable content of shorter wavelengths.