R. Oelmüller

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.

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.

Physiological characterization of a plastidic signal required for nitrate-induced appearance of nitrate and nitrite reductases.

We compared the response of NO3--induced nitrate-reductase (NR) and nitrite-reductase (NIR) levels in virtually carotenoid-free far-red-light-grown mustard (Sinapis alba L.) cotyledons following a photooxidative treatment of the plastids. The cytosolic localization of NR and the plastidic localization of NIR were confirmed with this approach. Emphasis was on a plastidic factor previously postulated to be involved obligatorily in the transcriptional control of nuclear genes coding for proteins destined for the chloroplast. Photooxidative damage of the plastid would be to destroy the ability of the organelle to send off this signal. Dependency of NIR and NR induction by NO3-on the plastidic factor is described in detail, and it is concluded that requirement for the plastidic factor is relatively high in the case of NR while factor requirement to allow induction is low in the case of NIR. The data indicate that in the case of NIR the photooxidative damage done to the plastid also affects accumulation of the enzyme directly. Since this effect is absent in the case of cytosolic NR, induction of NR is a particularly suitable system for further molecular studies of the plastidic factor and its mode of action.

Regulatory factors involved in gene expression (subunits of ribulose-1,5-bisphosphate carboxylase) in mustard (Sinapis alba L.) cotyledons

Phytochrome-controlled appearance of ribulose-1,5-bisphosphate carboxylase (RuBP-Case) and its subunits (large subunit LSU, small subunit SSU) was studied in the cotyledons of the mustard (Sinapis alba L.) seedling. The main results were as follows: (i) Control of RuBPCase appearance by phytochrome is a modulation of a process which is turned on by an endogenous factor between 30 and 33 h after sowing (25° C). Only 12 h later the process begins to respond to phytochrome. (ii) The rise in the level of RuBP-Case is the consequence of a strictly coordinated synthesis de novo of the subunits. (iii) While the levels of translatable mRNA for SSU are compatible with the rate of SSU synthesis the relatively high LSU mRNA levels are not reflected in the rates of in-vivo LSU or RuBPCase syntheses. (iv) Gene expression is also abolished in the case of nuclear-encoded SSU if intraplastidic translation and concomitant plastidogenesis is inhibited by chloramphenicol, pointing to a “plastidic factor” as an indispensable prerequisite for expression of the SSU gene(s). (v) Regarding the control mechanism for SSU gene expression, three factors seem to be involved: an endogenous factor which turns on gene expression, phytochrome which modulates gene expression, and the plastidic factor which is an indispensable prerequisite for the appearance of translatable SSU mRNA.

Time course of competence in phytochrome-controlled appearance of nuclear-encoded plastidic proteins and messenger RNAs.

The phytochrome-controlled expression of genes coding for plastidic proteins was studied in mustard (Sinapis alba L.) seedling cotyledons in continuous red (R) and far-red (FR) light, i.e. under steady-state conditions with regard to phytochrome, and in darkness over a time span of 8 d after sowing (25° C). (i) The time courses of the levels of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBPCase) and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GPD) were found to be optimum curves. The time at which the optimum (peak) occurred was — independent of fluence rate — the same in R (strong phytochrome action, chlorophyll accumulation and photosynthesis) and FR (strong phytochrome action but no significant chlorophyll accumulation and no photosynthesis). The starting point (first detectable inccrease of enzyme level) was also endogenously fixed and not affected by light. However, the two enzymes differed insofar as the peak was at 4 d after sowing for RuBPCase activity and 4.5 d for GPD. Western blots of the small (SSU) and large (LSU) subunits of RuBPCase showed that enzyme activity and protein levels were correlated. It was concluded that a dramatic change of competence towards phytochrome had occurred and that this change was endogenous. This conclusion was confirmed by short-term induction experiments. In constant darkness (D) the low enzyme levels were saturation rather than optimum curves, presumably because enzyme turnover was lacking. (ii) The time course of accumulation of membrane components showed that chlorophyll and LHCP (light-harvesting chlorophyll a/b-binding protein of photosystem II) levels were closely correlated in R until 6 d after sowing. Thereafter the levels remained constant. The accumulation of membrane components was not related to the accumulation of Calvin-cycle enzymes. (iii) Time courses of the levels of translatable mRNAs, particularly SSU mRNA and LHCP mRNA were determined. In the case of SSU the maximum mRNA-level was found in R, FR and D around 3 d. This was compatible with the in-situ protein accumulation rate. Induction experiments with FR showed that accumulation of SSU mRNA followed the same rise and fall (peak at 3 d) as would be expected from the time course of mRNA levels and from enzyme-induction experiments. In the case of LHCP mRNA the peak was between 3 and 4 d in R, and was not well correlated with in-situ protein accumulation. Translatable LHCP mRNA was also formed in FR and in D-with a peak between 3 and 4 d-although LHCP protein was not detectable under these circumstances (because of the lack of chlorophyll). The data indicate that competence of gene expression towards phytochrome is determined endogenously. However, in the case of LHCP its appearance is not only limited by mRNA but also depends on the availability of chlorophyll.

Coaction of blue light and light absorbed by phytochrome in control of glutamine synthetase gene expression in Scots pine (Pinus sylvestris L.) seedlings

The level of plastidic glutamine synthetase (GS; EC 6.3.1.2) in the cotyledonary whorl of the Scots pine (Pinus sylvestris L.) seedling was previously reported to be regulated by light. In the present paper we report on the control by light of the GS transcript level. A full-length GS cDNA clone of Scots pine was isolated (pGS1), sequenced and employed to measure GS transcript levels. Using dichromatic light treatments it was found that the transcript level is regulated by phytochrome. The strong specific effect of blue light is to be attributed to an increase of the responsiveness to phytochrome. Since no direct correlation between the transcript level and the rate of GS protein synthesis was observed, it was concluded that GS gene expression is only coarsely regulated at the level of transcript accumulation. Synthesis of GS protein is by itself light-dependent (light-mediated fine tuning of gene expression). This control at the translational level is also exerted via phytochrome with blue light determining the reponsiveness of the process toward phytochrome. If the level of the far-red absorbing form of phytochrome (Pfr) is kept very low, blue light is not capable of bringing about synthesis of GS protein.

Signal storage in phytochrome action on nitrate-mediated induction of nitrate and nitrite reductases in mustard seedling cotyledons.

Application of nitrate leads to an induction of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of dark-grown mustard (Sinapis alba L.) seedlings, and this induction can strongly be promoted by a far-red-light pretreatment — operating through phytochrome — prior to nitrate application. This light treatment is almost ineffective — as far as enzyme appearance is concerned — if no nitrate is given. When nitrate is applied, the stored light signal potentiates the appearance of NR and NIR in darkness, even in the absence of active phytochrome, to the same extent as continuous far-red light. This action of previously stored light signal lasts for approx. 12 h.Storage of the light signal was measured for NR and NIR. The process shows enzyme-specific differences. Storage occurs in the absence as well as in the presence of nitrate, i.e. irrespective of whether or not enzyme synthesis takes place. The kinetics of signal transduction and signal storage indicate that the formation and action of the stored signal are a bypass to the process of direct signal transduction. Signal storage is possibly a means of enabling the plant to maintain the appropriate levels of NR and NIR during the dark period of the natural light/dark cycle.

Control by phytochrome of the appearance of ribulose-1,5-bisphosphate carboxylase and the mRNA for its small subunit.

We have measured levels of ribulose-1,5-bisphosphate carboxylase (RuBPCase) and levels of in-vitro-translatable mRNA for the small subunit (SSU) of RuBPCase up to 96 h after sowing in mustard (Sinapis alba L.) cotyledons, in order to investigate to what extent the rate of enzyme synthesis is related to the level of SSU-mRNA. Both enzyme and mRNA level are controlled strongly by phytochrome, but the rate of RuBPCase accumulation was found to be unrelated to the level of translatable SSU-mRNA. As an example, it was found that the amount of SSU-mRNA in far-red light (FR)-grown mustard seedlings doubles between 54 and 84 h after sowing while the rate of RuBPCase accumulation remains constant over this period. Since the holoenzyme shows zero turnover during this period it is concluded that the rate of enzyme synthesis remains constant although the level of SSU-mRNA increases strongly. Following an FR→dark transition, with different levels of physiologically active phytochrome (Pfr) established at the end of the light period, no correlation was found between the time course of mRNA levels in darkness and the rate of enzyme synthesis. Rather, the data indicate that there is at least one translational or post-translational regulatory step which is also phytochrome-dependent. It is concluded that coarse control of the appearance of translatable SSU-mRNA is essential for RuBPCase to appear at a high rate but that fine tuning by phytochrome of the actual appearance of RuBPCase is not transcriptional.

Carotenoid composition in milo (Sorghum vulgare) shoots as affected by phytochrome and chlorophyll.

The composition of coloured carotenoids in the milo shoot was investigated quantitatively (high performance liquid chromatography) during light-mediated plastidogenesis, including the time span of ‘photodelay’ as caused by medium and high light fluxes. It was found that as long as only the far-red-absorbing form of phytochrome operates, the carotenoid pattern remains virtually the same as in complete darkness (violaxanthin and lutein as major constituents, traces of β-carotene). On the other hand, the pattern changes dramatically in white or red light with increasing amounts of chlorophyll (lutein and β-carotene dominate, β-carotene showing the strongest relative increase). Photodelay during the early phase of plastidogenesis affects the carotenoid composition strongly. Increase of neoxanthin, violaxanthin and β-carotene contents are diminished while lutein accumulation proves resistant towards chlorophyll-mediated photoinhibition. The photodelay can be diminished by an appropriate light pretreatment. The data indicate that light-mediated control over carotenoid accumulation is exerted at three levels: i) a coarse control through phytochrome, ii) fine tuning in connection with chlorophyll accumulation, iii) stabilization of holocomplexes against photodecomposition.