Helga Kasemir

A TWOFOLD ACTION OF PHYTOCHROME IN CONTROLLING CHLOROPHYLL a ACCUMULATION

Abstract— A pretreatment with light prior to continuous illumination with high intensity white light eliminates the lag phase in chlorophyll a accumulation and increases the steady‐state rate of chlorophyll a accumulation. In mustard seedlings (Sinapis alba L.) the effect of a pretreatment can be fully attributed to phytochrome. The effect of phytochrome on chlorophyll a accumulation is twofold. It is possible to separate the effect on the lag phase from the effect on the steady‐state rate of accumulation. While the effect on the lag phase is a relatively fast process (occurring within less than 3 h) the effect on the rate requires a considerable period of time (at least 12 h) to become manifest.

Control of chlorophyll synthesis by phytochrome : I. The effect of phytochrome on the formation of 5-aminolevulinate in mustard seedlings.

SummaryTreatment of mustard (Sinapis alba L.) seedlings with levulinate leads to the inhibition of chlorophyll synthesis and causes the accumulation of 5-aminolevulinate which is only formed in light. A stoichiometric relationship exists between the extent of inhibition of chlorophyll synthesis and 5-aminolevulinate accumulation. The formation of 5-aminolevulinate in continuous white light is increased by pre-irradiation. The effect of the preirradiation can be fully attributed to phytochrome. Under various light conditions the rate of 5-aminolevulinate formation in levulinate-treated seedlings is similar to the rate of chlorophyll accumulation in seedlings not treated with levulinate. This result supports the hypothesis that the phytochrome-controlled chlorophyll accumulation is regulated at the level of the formation of 5-aminolevulinate.

The capacity of chlorophyll-a biosynthesis in the mustard seedling cotyledons as modulated by phytochrome and circadian rhythmicity

Within the temporal pattern of “primary differentiation” the capacity of chlorophyll — a biosynthesis in the cotyledons ofSinapis alba L. seedlings is controlled by phytochrome (in continuous light) or by releasing the circadian rhythm either with lightdark cycles or by a light→dark transition. The sensor pigment for this process is phytochrome. It is very probable that in continuous light as well as under conditions under which the circadian rhythm plays the major part, the capacity of chlorophyll a biosynthesis is limited by the capacity of the biosynthetic step which produces 5-aminolaevulinate.

Phytochrome-mediated delay of plastid senescence in mustard cotyledons: changes in pigment contents and ultrastructure

Changes in pigment contents and ultrastructure have followed in cotyledons of mustard (Sinapis alba L.) seedlings during dark-mediated senescence. The seedlings were kept in white light for 7 d, treated with 5 min long wavelength far-red light and then kept in darkness up to 14 d after sowing. Under these conditions the chloroplasts remain stable for 2 d before a sequential plastidal disintegration commences. The data indicate a selective breakdown of the light-harvesting chlorophyll a/b protein. Phytochrome retards the differential loss of chlorophyll a, b and carotenoids and preserves the fine structure of chloroplasts.

Control of chlorophyll synthesis by phytochrome : III. Does phytochrome regulate the chlorophyllide esterification in mustard seedlings?

SummaryThe rate of chlorophyllide esterification in mustard cotyledons can be increased by a pretreatment with 5 min red light applied 24 h prior to the protochlorophyll(ide)→chlorophyll(ide) photoconversion at 60 h after sowing. Simultaneously the red light pulse pretreatment leads to a decrease of the total amount of chlorophyll(ide) a in darkness. It has been proven that phytochrome (Pfr) is the photoeffector for both. Since the amounts of esterified chlorophyllide are determined by the ratio [chlorophyll a]/[chlorophyllide a+chlorophyll a] it is assumed that Pfr increases the rate of esterification indirectly via stimulating the decrease of chlorophyll(ide) a. The regulation of chlorophyll synthesis by Pfr does not seem to involve a control of esterification. The duration of the chlorophyllide esterification differs from the duration of the Shibata shift although both are greatly shortened by the red light pulse pretreatment. The effect of 5 min red light on the duration of the esterification is fully reversible by 5 min far-red light while the reversibility with respect to the Shibata shift is lost within 2 min [Jabben, M. and H. Mohr, Photochem. Photobiol. 22, 55–58 (1975)]. We conclude that the control of the chlorophyllide esterification and the control of the Shibata shift cannot be traced back to the same initial action of Pfr.

PHYTOCHROME‐MEDIATED CONTROL OF PROLAMELLAR BODY REORGANIZATION AND PLASTID SIZE IN MUSTARD COTYLEDONS

Abstract— The development of plastids in the palisade parenchyma cells of the cotyledons of mustard seedlings (Sinapis alba L.) was studied by electron microscopy. In darkness the etioplasts undergo a sequence of morphogenic changes previously recognized in principle in bean and barley leaves, as summarized by Rosinski, J. and W. G. Rosen (1972) Quart. Rev. Biol.47, 160–190. From 12 to 36 h after sowing, an increase in the percentage of etioplast profiles with paracrystalline prolamellar bodies can be observed. Thereafter, the degree of organization and size of the prolamellar bodies decrease. 60 h after sowing, the etioplasts show only remnants of prolamellar bodies with irregularly spaced tubules. Continuous far‐red light, which is considered to operate via phytochrome, counteracts the decay of organization of the prolamellar body and strongly increases the size of the plastids. The effect of continuous far‐red light (onset of light 36 h after sowing) can be substituted by 12 h of far‐red light given between 36 and 48 h after sowing. It is shown with red and far‐red light pulses that the morphogenic effect of long‐term far‐red light on plastid size and appearance of the prolamellar body is exclusively due to phytochrome (Pfr). Changes by light in the amounts of protochlorophyll(ide) or chlorophyll(ide) do not affect these results. The action of Pfr on the structure of the prolamellar body is a relatively fast process, occurring within 3 h. Formation of thylakoids does not seem to be under phytochrome control. Rather, this response seems to be related to the protochlorophyll(ide)→ chlorophyll(ide) a transformation.

Coaction of three factors controlling chlorophyll and anthocyanin synthesis

In a three-factor analysis the rate of chlorophyll a (Chl) accumulation in excised mustard cotyledons was studied as a function of kinetin, light (operating through phytochrome, Pfr) and an excision factor. It was found that the three factors operate additively provided that the Pfr level is high enough. When the Pfr level is below approximately 1 per cent (ϕλ<0.01) the effectiveness of the excision factor decreases while the effect of kinetin remains additive. The observed additivity is explained by a model where the three factors operate independently through a common intermediate (presumably 5-aminolevulinate) in the biosynthetic chain leading to Chl. With regard to the coaction of the excision factor and phytochrome it is concluded that the production of the excision factor requires the operation of phytochrome (even though saturated at a low Pfr level) while the action of the excision factor is independent of phytochrome. This conclusion was confirmed by experiments in which the rate of light-mediated anthocyanin synthesis was measured in excised mustard cotyledons. The effect of excision in the case of anthocyanin formation differs kinetically from the effect of excision on Chl formation.

Control of chlorophyll synthesis by phytochrome : II. The effect of phytochrome on aminolevulinate dehydratase in mustard seedlings.

SummaryThe activity of aminolevulinate dehydratase in mustard (Sinapis alba L.) seedlings increases in continuous far-red light. The light effect can be attributed to phytochrome. The same was found for the accumulation of protochlorophyll(ide) if the seedlings were treated with 5-aminolevulinate. This result could indicate that a considerable portion of the aminolevulinate dehydratase is located in the plastids. No correlation exists between aminolevulinate dehydratase activity and the capacity of the mustard seedlings to form chlorophyll. In conclusion, the increase in enzyme activity is probably not involved in the phytochrome-mediated control of chlorophyll biosynthesis.

Coaction of light and cytokinin in photomorphogenesis.

Intact mustard seedlings were treated with zeatin and photomorphogenetically active light in different ways: (1) hormone treatment preceding light treatment, (2) light treatment preceding hormone treatment, (3) hormone and light applied simultaneously. Under all experimental conditions the effect of the hormone treatment is multiplicative to the light effect with regard to the increase of cotyledon area. However, the hormone effect is additive to the light effect with regard to increases of the level of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and carotenoid contents. Anthocyanin synthesis is inhibited by exogenous zeatin whereby the concentration response curves are similar, irrespective of the extent of anthocyanin formation mediated by light. However, an interaction was found in the sense that the responsiveness toward zeatin is decreased somewhat by the action of phytochrome. Our results show that the responsiveness to light (via the far-red-absorbing form of phytochrome; Pfr) is not changed by a preceding or simultaneous hormone treatment. Moreover, the responsiveness of the plant to exogenously applied zeatin is not affected — except in anthocyanin synthesis — by a preceding or simultaneous light treatment. We conclude from our results that the action of phytochrome on the developmental processes is not related to cytokinin levels.

Die Regulation von Chlorophyll- und Protein Gehalt in Farnvorkeimen Durch Sichtbare Strahlung

SummaryMorphogenesis and metabolism of the gametophytes (=sporelings) of the common male fern Dryopteris filix-mas are controlled by visible radiation. Short wavelengths visible radiation (=blue light) leads to an increase in protein synthesis and enables the formation of “normal” two dimensional prothallia. Under long wavelength visible radiation (=red light) the gametophytes will grow as cellular filaments whose protein contents are markedly lower even under conditions of equal rate of photosynthesis in red and blue (Ohlenroth and Hohr 1963). It is shown in the present paper that the chlorophyll contents of sporelings at the same age is always higher in blue light than in red light. However, the chloroplasts which originated under red light are not essentially different from those which originated under blue light as far as their photosynthetic efficiency is concerned (increase of dry weight per unit time and per unit chlorophyll under the same light conditions). Therefore the completely different morphogenesis of the sporelings in blue light and red light cannot be related to differences in structure and function of the chloroplasts.If we relate chlorophyll contents, total protein and dry weight of the sporelings (Figs. 5 and 6) we find relationships which are very similar to those which have been described by Böger (1964) and Pirson and Böger (1965) for unicellular green algae. These investigators related chlorophyll contents, chloroplast structure protein and dry weight (Table 2). We conclude from these data, that the blue light dependent increase of the relative protein contents of the sporelings might be due exclusively to an increase of chloroplast protein. We further conclude that the small chloroplasts which originate under red light are not qualitatively different from the larger chloroplasts which originate under blue light.It seems that blue light causes — as far as protein synthesis is concerned —two different processes in these sporelings: 1. The autonomous protein synthesis of the chloroplasts is increased. 2. A specific enzyme synthesis is started in the cytoplasm, possibly through an activation of potentially active genes (Ohlenroth and Mohr 1964). These enzymes which could not be detected directly hitherto, must be a prerequisite for the specific blue light dependent “normal” morphogenesis of the sporelings. Experiments with antimetabolites (Hotta and Osawa 1958, Bergfeld 1965) support this concept of a blue light dependent enzyme synthesis.SusammenfassungDurch kurzwellige sichtbare Strahlung (=Blaulicht) kann bei Farnvorkeimen die Proteinsynthese gesteigert werden; parallel dazu kommt es zur Ausbildung „normaler” zweidimensionaler Prothallien. Im längerwelligen Licht (=Hellrot) wachsen die Vorkeime highegen als Zellfäden. Ihr Proteingehalt ist bei gleicher Photosyntheseleistung wesentlich geringer (Zusammenfassung bei Ohlenroth und Mohr 1963).In der vorliegenden Arbeit wird gezeigt, daß auch der Chlorophyllgehalt gleichaltriger Vorkeime (bezogen auf Trockengewicht oder Gesamtprotein-N) im Blaulicht stets größer ist als im Hellrot. Die relativ kleinen Chloroplasten, die im Hellrot entstanden sind, unterscheiden sich aber hinsichtlich ihrer photosynthetischen „Leistungsfähigkeit” und hinsichtlich ihrer Protein-Chlorophyll-Relation nicht grundlegend von den im Blaulicht entstandenen, relativ großen Chloroplasten, so daß man keinen Grund hat, die völlig verschiedene Morphogenese der Vorkeime im Blaulicht und im Hellrot mit Unterschieden in Bau und Funktion der Chloroplasten in Beziehung zu bringen.Wenn man Chlorophyllgehalt, Gesamtprotein und Trockengewicht der Vorkeime in Beziehung brigt (Abb. 5 und 6), so erhält man ganz ähnliche Relationen, wie sie von Böger (1964) und Pirson und Böger (1965) bei einzelligen Grünalgen für den Zusammenhang von Chlorophyllgehalt, Chloroplastenstrukturprotein und Trockengewicht gefunden wurden (Tabelle 2). Wir ziehen daraus den Schluß, daß im Blaulicht die Bildung von Plastidenprotein besonders gefördert ist und daß die von uns registrierte Zunahme des relativen Proteingehalts der Vorkeime unter dem Einfluß von Blaulicht ausschließlich auf die durch Blaulicht gesteigerte plastidäre Proteinsynthese zurückzuführen ist.Es scheint, daß durch Blaulicht in den Vorkeimen im Zusammenhang mit der Proteinsynthese zwei verschiedene Vorgänge verursacht werden:1.Es wird die autonome Proteinsynthese der Chloroplasten gesteigert.2.Es wird eine spezifische Enzymsynthese im extraplastidären Cytoplasma ausgelöst („Aktivierung potentiell aktiver Gene”). Diese Enzyme müssen als die Voraussetzung für die blaulichtspezifische Morphogenese der Vorkeime angesehen werden. Bislang konnte diese Hypothese einer spezifischen extraplastidären Proteinsynthese lediglich durch Experimente mit Antimetaboliten gestützt werden.