H. Mohr

Expression of nuclear genes as affected by treatments acting on the plastids.

In a preceding paper (Oelmüller and Mohr 1986, Planta 167, 106–113) it was shown that in the cotyledons of the mustard (Sinapis alba L.) seedling the integrity of the plastid is a necessary prerequisite for phytochrome-controlled appearance of translatable mRNA for the nuclear-encoded small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCP). It was concluded that a signal from the plastid is essential for the expression of nuclear genes involved in plastidogenesis. The present study was undertaken to characterize this postulated signal. Chloramphenicol, an inhibitor of intraplastidic protein synthesis and Norflurazon, an inhibitor of carotenoid synthesis (to bring about photooxidative sensitivity of the plastids) were applied. We obtained the following major results. (i) After a brief period of photooxidative damage a rapid decrease of the above translatable mRNAs was observed. Conclusion: the signal is short-lived and thus required continually. (ii) Once the plastids became damaged by photooxidation, no recovery with regard to nuclear gene expression was observed after a transfer to non-damaging light conditions. Conclusion: even a brief period of damage suffices to prevent production of the signal. (iii) Chloramphenicol inhibited nuclear gene expression (SSU, LHCP) and plastidic development when applied during the early stages of plastidogenesis. Once a certain stage had been reached (between 36–48 h after sowing at 25° C) nuclear gene expression became remarkably insensitive toward inhibition of intraplastidic translation. Conclusion: a certain developmental stage of the plastid must be reached before the signal is released by the plastid. (iv) Under the growth conditions we adopted in our experiments the plastids in the mesophyll cells of mustard cotyledons developed essentially between 36 and 120 (-144) h after sowing. Only during this period could translatable mRNAs for SSU and LHCP be detected. Conclusion: the signal is released by the plastids only during this time span.

Photooxidative destruction of chloroplasts and its consequences for expression of nuclear genes

Expression of nuclear genes involved in plastidogenesis is known to be controlled by light via phytochrome. Examples are the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b binding protein of photosystem II (LHCP). In the present study we show that, beside phytochrome, the integrity of the plastid is essential for the expression of the pertinent nuclear genes as measured at the level of translatable mRNA. When the plastids are severely damaged by photooxidation in virtually carotenoid-free mustard (Sinapis alba L.) seedling cotyledons (made carotenoid-free by the application of Norflurazon, NF), almost no SSU, no SSU precursor, LHCP and LHCP precursor can be detected by immunological assays, and almost no translatable mRNA of SSU and LHCP can be found, although the levels and rates of phytochrome-mediated syntheses of representative cytoplasmic, mitochondrial and glyoxisomal enzymes are not adversely affected and morphogenesis of the mustard seedling proceeds normally (Reiß et al. 1983; Planta 159, 518–528). Norflurazon per se has no effect on the amount of translatable mRNA of SSU and LHCP as shown by irradiation of NF-treated seedlings with far-red light (FR) which strongly activates phytochrome but does not cause photooxidation in the plastids. It is concluded that a signal from the plastid is required to allow the phytochrome-mediated appearance of translatable mRNA for SSU and LHCP. Seedlings not treated with NF show a higher level of translatable mRNALHCP in red light (RL) compared to FR, whereas the mRNASSU levels are the same in RL and FR. These facts indicate that the level of translatable mRNALHCP is adversely affected if the apoprotein is not incorporated into the thylakoid membrane.

Die Beeinflussung der Farnsporen-Keimung [Osmunda cinnamomea (L.) undO. claytoniana (L.)] über das Phytochromsystem und die Photosynthese

SummaryThe control by light of the spore germination ofOsmunda cinnamomea L. andO. claytoniana L. has been investigated.This light control is complex, and in order to investigate this control quantitatively the process of germination had to be divided into at least two stages. The first stage of germination leads to the rupture of the exospore, the second step is characterized by the outgrowth of a rhizoid. In a very few percent of our spore population the first stage can occur in total darkness, the second step however does not ever occur in darkness.In the present paper it has been demonstrated, usingO. cinnamomea spores, that the first step of germination is phytochrome controlled, whereas the second step is under photosynthetic control. This was observed withO. claytoniana by a rough action spectrum and in experiments with a CO2-less atmosphere.The germinating spores of these two species ofOsmunda show a very similar behaviour as the spores of the mossFunaria hygrometrica (L.) Sibth. (Bauer andMohr 1959). They behave very differently compared with spores of other ferns, e.g.Dryopteris filix-mas (L.) Schott, where the whole process of germination is exclusively phytochrome controlled (Mohr 1956).

Photooxidative destruction of chloroplasts and its consequences for cytosolic enzyme levels and plant development

Mustard (Sinapis alba L.) seedlings were grown in the presence of herbicides (Difunon, Norflurazon) which inhibit carotenoid synthesis without affecting development, in darkness or in continuous far-red light. In strong white light (12,000 lx) the cotyledons of the herbicide-treated seedlings did not contain normal chloroplasts, but only small chlorophyll-free rudiments whose internal structure had almost disappeared. The plastid marker enzyme NADP-dependent glyceraldehyde-3-phosphate dehydrogenase was almost lacking. Plastid ribosomes and ribosomal RNAs were no longer detectable nor could synthesis of mature plastidal ribosomal RNAs be detected. Cytosolic ribosomes and rRNAs were not affected. Plastid DNA was apparently still intact as shown by restriction analysis. The appearance of marker enzymes of glyoxisomes, mitochondria and cytosol was not impaired while the level of marker enzymes of peroxisomes was drastically lowered. Accumulation of anthocyanin in mustard cotyledons was normal after a short, transient delay. Levels of representative enzymes of flavonoid biogenesis (phenylalanine ammonia-lyase, chalcone synthase) were somewhat increased rather than inhibited in the cotyledons of herbicide-treated, white-light-grown seedlings. The growth rate of hypocotyl and cotyledons was inhibited to the same extent in the herbicide-treated, white-light-grown seedling, although light inhibits growth of hypocotyls and promotes growth of cotyledons. Analysis of the data shows that photomorphogenesis of a herbicide-treated, white-light-grown seedling is normal, and is thus independent of plastid gene expression However, a ‘factor’ which coacts multiplicatively with phytochrome in determining the growth rate of the organs seems to originate from the plastids. Biogenesis of anthocyanin and synthesis of major enzymes of the flavonoid pathway are not affected adversely by a photooxidative elimination of plastid gene expression.

DIFFERENTIAL GENE ACTIVATION AS A MODE OF ACTION OF PHYTOCHROME 730

Abstract— Phytochrome‐induced photomorphogenesis in the mustard seedling (Sinapis alba L.) which can be regarded as being representative of the dicotyledonous seedlings has been analysed. In the present paper a number of arguments are presented, including data on RNA and protein synthesis and on the effects of actinomycin D and puromycin, which support the hypothesis that the ‘positive’ photoresponses of the seedling can be explained by a differential gene activation through P,30. ‘Positive’ photoresponses are those which are characterized by an initiation or an increase of biosynthetic or growth processes (e.g. biosynthesis of anthocyanin; growth of cotyledons). The lag‐phase of this type of photoresponse is rather long, ‘Negative’ photoresponses are those which are characterized by an inhibition of growth processes or other physiological processes like translocation. Here the lag‐phase is short. Inhibition of hypocotyl lengthening is a typical response of this sort. The concept of differential gene repression through P730 may serve as a working hypothesis to approach the causal analysis of phytochrome‐induced ‘negative’ photoresponses.

Inhibition of carotenoid biosynthesis by the herbicide SAN 9789 and its consequences for the action of phytochrome on plastogenesis

Treatment of the mustard (Sinapis alba L.) seedling with the herbicide SAN 9789 inhibits synthesis of colored carotenoids and interferes with the formation of plastid membrane lipids without affecting growth and morphogenesis significantly. In farred light, which is hardly absorbed by chlorophyll, development of plastid ultrastructure, synthesis of ribulosebisphosphate carboxylase and synthesis of chlorophyll are not affected by SAN 9789. It is concluded that normal phytochrome actions on plastid structural development, protein and chlorophyll syntheses are not affected by the absence of carotenoids provided that there is no significant light absorption in chlorophyll. The findings show that the inhibition of synthesis of one set of plastid membrane components (the carotenoids) does not stop synthesis of other components such as chlorophyll and does not halt membrane assembly. Supplementary experiments with the closely related compound SAN 9785, which affects the amount and composition of plastid lipids but not carotenoid and chlorophyll syntheses, suggest that the effect of the herbicide SAN 9789 is due exclusively to its inhibition of synthesis of colored carotenoids. In the presence of SAN 9789 white or red light at high fluence rate causes photodestruction of chlorophyll and ribulosebisphosphate carboxylase and photodecomposition of thylakoids. These effects are interpreted as resulting exclusively from the self-photooxidation and photosensitizing action of chlorophyll once the protection by carotenoids of chlorophyll against self- and sensitized photooxidation is lost.

THE MODE OF INTERACTION BETWEEN BLUE (UV) LIGHT PHOTORECEPTOR AND PHYTOCHROME IN ANTHOCYANIN FORMATION OF THE SORGHUM SEEDLING

Two non‐photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light‐mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (Pfr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without Pfr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.

KINETIC STUDIES TO INTERPRET ‘HIGH-ENERGY PHENOMENA’ OF PHOTOMORPHOGENESISL ON THE BASIS OF PHYTOCHROME

Abstract— Kinetic studies with the mustard seedling (Sinapis alba L.) support the hypothesis that the so‐called ‘high energy reaction’ of photomorphogenesis can be understood solely on the basis of phytochrome. Light‐induced anthocyanin synthesis (a typical ‘positive’ photoresponse(1) and light dependent inhibition of hypocotyl lengthening (a typical ‘negative’ photoresponse(1)) have been investigated. In order to explain the experimental data we have to assume that there are two different types of phytochrome 730 which differ greatly as far as their resistance to irreversible destruction is concerned. The existence of these two different types of phytochrome 730 has already been postulated on the basis of spectrophotometric measurements in vivo.(2)

THE CONTROL OF PLANT GROWTH AND DEVELOPMENT BY LIGHT

This article is devoted to photochemical reaction systems other than photosynthesis present in plants which enable normal growth and development to take place. In many higher plants (e.g. dicotyledonous seedlings) at least four different photochemical reaction systems are effective:

Action of light, nitrate and ammonium on the levels of NADH- and ferredoxin-dependent glutamate synthases in the cotyledons of mustard seedlings

In mustard (Sinapis alba L.) cotyledons, NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) is only detectable during early seedling development with a peak of enzyme activity occurring between 2 and 2.5 d after sowing. With the beginning of plastidogenesis at approximately 2 d after sowing, ferredoxindependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) appears while NADH-GOGAT drops to a very low level. The enzymes were separated by anion exchange chromatography. Both enzymes are stimulated by light operating through phytochrome. However, the extent of induction is much higher in the case of Fd-GOGAT than in the case of NADH-GOGAT. Moreover, NADH-GOGAT is inducible predominantly by red light pulses, while the light induction of Fd-GOGAT operates predominantly via the high irradiance response of phytochrome. The NADH-GOGAT level is strongly increased if mustard seedlings are grown in the presence of nitrate (15 mM KNO3,15 mM NH4NO3) while the Fd-GOGAT level is only slightly affected by these treatments. No effect on NADH-GOGAT level was observed by growing the seedlings in the presence of ammonium (15 mM NH4Cl) instead of water, whereas the level of Fd-GOGAT was considerably reduced when seedlings were grown in the presence of NH4Cl. Inducibility of NADH-GOGAT by treatment with red light pulses or by transferring water-grown seedlings to NO3--containing medium follows a temporal pattern of competence. The very low Fd-GOGAT level in mustard seedlings grown under red light in the presence of the herbicide Norflurazon, which leads to photooxidative destruction of the plastids, indicates that the enzyme is located in the plastids. The NADH-GOGAT level is, in contrast, completely independent of plastid integrity which indicates that its location is cytosolic. It is concluded that NADH-GOGAT in the early seedling development is mainly concerned with metabolizing stored glutamine whereas Fd-GOGAT is involved in ammonium assimilation.