Tris(methylthio)methane produced by Mortierella hyalina affects sulfur homeostasis in Arabidopsis.

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Incorporation of the sulfur from the fungal volatile into plant metabolism was shown by 34S labeling experiments. Under sulfur deficiency, TMTM down-regulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

PRH75, a new nucleus-localized member of the DEAD-box protein family from higher plants.

The putative RNA helicases of the DEAD-box protein family are involved in pre-mRNA splicing, rRNA maturation, ribosome assembly, and translation. Members of this protein family have been identified in organisms from Escherichia coli to humans, but except for the translation initiation factor 4A, there have been no reports on the characterization of other DEAD-box proteins from plants. Here we report on a novel member of the DEAD-box protein family, the plant RNA helicase 75 (PRH75). PRH75 is localized in the nucleus and contains two domains for RNA binding. One is located at the C terminus and is similar to RGG RNA-binding domains of nucleus-localized RNA-binding proteins. The other one is located between amino acids 308 and 622, a region containing the conserved motif VI characteristic of DEAD-box proteins and known as the RNA-binding site of eIF-4A. The N-terminal 81 amino acids are sufficient for nuclear targeting of the protein. Northern and Western blot analyses show that PRH75 is mainly expressed in young and rapidly developing tissues. The purified recombinant PRH75 has a weak ATPase activity which is barely stimulated by RNA ligands. The fractionation of spinach whole-cell extracts by glycerol gradient centrifugation and gel filtration on a Superdex 200 column shows that the protein exists in a complex of about 500 kDa. Possible biological functions of PRH75 as well as structure-function relationships in the context of its modular primary structure are discussed.

The Role of Plastids in the Expression of Nuclear Genes for Thylakoid Proteins Studied with Chimeric [beta]-Glucuronidase Gene Fusions.

We have analyzed plastid and nuclear gene expression in tobacco seedlings using the carotenoid biosynthesis inhibitor nor-flurazon. mRNA levels for three nuclear-encoded chlorophyll-binding proteins of photosystem I and photosystem II (CAB I and II and the CP 24 apoprotein) are no longer detectable in photobleached seedlings, whereas those for other components of the thylakoid membrane (the 33- and 23-kD polypeptides and Rieske Fe/S polypeptide) accumulate to some extent. Transgenic tobacco seedlings with promoter fusions from genes for thylakoid membrane proteins exhibit a similar expression behavior: a CAB-[beta]-glucuronidase (GUS) gene fusion is not expressed in herbicide-treated seedlings, whereas PC-, FNR-, PSAF-, and ATPC-promoter fusions are expressed, although at reduced levels. All identified segments in nuclear promoters analyzed that have been shown to respond to light also respond to photodamage to the plastids. Thus, the regulatory signal pathways either merge prior to gene regulation or interact with closely neighboring cis elements. These results indicate that plastids control nuclear gene expression via different and gene-specific cis-regulatory elements and that CAB gene expression is different from the expression of the other genes tested. Finally, a plastid-directing import sequence from the maize Waxy gene is capable of directing the GUS protein into the photodamaged organelle. Therefore, plastid import seems to be functional in photobleached organelles.

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis.

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.

Different blue-light requirement for the accumulation of transcripts from nuclear genes for thylakoid proteins in Nicotiana tabacum and Lycopersicon esculentum.

We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b-binding protein of photosystem II (CP24), the Rieske iron-sulphur protein of the cytochrome b6/f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum. The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue-UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

Natural inhibitors of germination and growth, VII synthesis of ribulosebisphosphate carboxylase in darkness and its inhibition by coumarin.

Cress (Lepidium sativum) seeds were germinated in darkness. Seedlings were investigated for soluble proteins by SDS-PAGE. Two proteins were identified by microsequencing: the small subunit of ribulosebisphosphate carboxylase (SSU) and the alpha subunit of the storage protein cruciferin. Net synthesis of small and large subunits of ribulosebisphosphate carboxylase (SSU and LSU) was investigated by Western blot. Net synthesis of both subunits was inhibited by coumarin. To the contrary, net synthesis of cruciferin was increased by coumarin. With specific cDNA probes, we determined steady state levels of the corresponding mRNAs (rbcS mRNA for SSU, rbcL mRNA for LSU). Both mRNAs can be detected in dry seeds; their amount increases during germination in the dark. Incubation with coumarin inhibits this increase. Inhibition of development by coumarin on the level of transcription is discussed.

Evidence that carotenoids are required for the accumulation of a functional photosystem II, but not photosystem I in the cotyledons of mustard seedlings.

If Norflurazon-treated mustard (Sinapis alba L.) seedlings are grown in low-fluence-rate white light, accumulation of carotenoids is completely inhibited, while levels of chlorophyll (Chl) a and b are comparable to those of control seedlings. Measurements of fluorescence yield and oxygen evolution indicate that carotenoid-free, green cotyledons are unable to perform leephotosynthesis in vivo. When thylakoid membranes were prepared and electron transport was measured in vitro, only PSI but not PSII activity was detected. Solubilization of the photosystems from thylakoid membranes and separation by sucrose-gradient centrifugation confirmed that PSII is absent in carotenoid-free seedlings, while PSI is present. Western blot analysis for representative proteins of the four photosynthetic complexes showed that subunits 1 and 2 of PSI, the Rieske-iron sulfur-protein, the α-subunit of the CF1 moiety of the ATP-synthase complex, cytochrome b 559 and the lumenal 33-kDa protein of the water-splitting apparatus of PSII are present in comparable amounts in Norflurazon-treated and control plants, while the amounts of Chl-binding proteins of PSII (the major light-harvesting Chl-a/b-binding protein of the antenna complex and the 51- and 44-kDa Chl-a-binding proteins) and two components of the PSII reaction center, (the D1 and D2 protein) are substantially reduced. The data indicate that accumulation of PSII polypeptides is either not inhibited or not completely inhibited in carotenoid-free mustard seedlings, but that assembly of a functional PSII complex does not occur. If Norflurazon-treated seedlings are transferred to water, lutein accumulates rapidly and reaches about 80% of the level detectable in control plants, while the level of other carotenoids is still less than 1%. The accumulation kinetics for lutein are similar to the kinetics for the appearance of PSII activity. This indicates that the availibility of lutein rather than that of other carotenoids might be rate-limiting for the appearance of PSII activity.

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 ß-carotene). On the other hand, the pattern changes dramatically in white or red light with increasing amounts of chlorophyll (lutein and ß-carotene dominate, ß-carotene showing the strongest relative increase). Photodelay during the early phase of plastidogenesis affects the carotenoid composition strongly. Increase of neoxanthin, violaxanthin and ß-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.

Mode of coaction between blue/UV light and light absorbed by phytochrome in light-mediated anthocyanin formation in the milo (Sorghum vulgare Pers.) seedling.

Anthocyanin formation in milo (Sorghum vulgare Pers.) seedlings (coleoptile, mesocotyl, taproot) occurs only in white light and blue/UV light (BL/UV), while red light (RL) and far-RL are totally ineffective. However, after a BL/UV pretreatment, the participation of phytochrome can be demonstrated. With a short-wavelength light source [peak emission in longwave UV (UV-A)], the mode of coaction between BL/UV and light absorbed by phytochrome (RL) was studied with the following principal results. (i) As soon as the seedling becomes competent to respond to UV-A (with regard to anthocyanin formation), the involvement of phytochrome can be detected. (ii) A 5-min pulse of UV-A has a strong effect on the anthocyanin synthesis in the milo mesocotyl. This effect is fully reversible if a long-wavelength far-RL pulse (RG9 light) is given immediately after the UV-A light pulse. (iii) When seedlings treated with 5 min of UV-A and 5 min of RG9 light are kept in darkness for 3 hr and then transferred to RL, anthocyanin appears. (iv) In continuous UV-A treatment, anthocyanin accumulation starts after a lag phase of 3.5 hr (25 degrees C). A RL pretreatment prior to the onset of UV-A treatment strongly increases anthocyanin accumulation in UV-A, though the lag phase is not affected. Moreover, a RL pretreatment does not affect the time course for escape from reversibility in UV-A. It is concluded from these data that BL/UV cannot mediate induction of anthocyanin synthesis in the absence of P(fr), the active form of phytochrome that absorbs maximally in the far-red. Rather, the action of BL/UV must be considered to establish responsiveness of the anthocyanin-producing mechanism to P(fr). P(fr) operates in this system via two different channels. As the effector of the terminal response, it sets in motion the signal-response chain that eventually leads to the appearance of anthocyanin. This is a slow process with a lag phase of the order of 3.5 hr. The second function of P(fr) is to determine the responsiveness to the effector P(fr) in mediating anthocyanin synthesis. This is a very fast and highly sensitive phytochrome action that can be detected readily within 1 min. However, as long as the plant has not received BL/UV, the strong effect of RL on the effectiveness of P(fr) remains cryptic. The effect of a RL pretreatment and the effect of a UV-A pretreatment on responsiveness towards P(fr) (or, effectiveness of P(fr)) were found to be totally independent of each other, even though it is the UV-A that permits operation of P(fr).

Inhibition and promotion by light of the accumulation of translatable mRNA of the light-harvesting chlorophyll a/b-binding protein of photosystem II.

The amount of in-vitro translatable mRNA of the light-harvesting chlorophyll a/b-binding protein (LHCP) of photosystem II strongly increases in darkness (D) after a 5-min red-light pulse while continuous illumination of mustard seedlings with far-red (FR), red or white light leads only to a slight increase in the amount of translatable LHCP-mRNA. No increase can be observed after a long-wavelength FR (RG9-light) pulse. However, a FR pretreatment prior to the RG9-light pulse strongly increase LHCP-mRNA accumulation in subsequent D. This is not observed in the case of the mRNA for the small subunit of ribulose-1.5-bisphosphate carboxylase. The increase of LHCP-mRNA in D after a FR pretreatment can be inhibited by a reillumination of the seedlings with FR. The inhibition of LHCP-mRNA accumulation during continuous illumination with FR and the strong increase in D following a FR illumination was found to be independent of chlorophyll biosynthesis since no correlation between chlorophyll biosynthesis and translatable LHCP-mRNA levels could be detected. Even strong changes in the amount of intermediates of chlorophyll biosynthesis caused by application of levulinic acid or 5-aminolevulinic acid did not affect LHCP-mRNA levels. Therefore, we conclude that the appearance of LHCP-mRNA is inhibited during continuous illumination, even though illumination leads to a storage of a light singal which promotes accumulation of translatable LHCP-mRNA in D.

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.

Heterotrimeric G-protein beta-subunit is localized in the plasma membrane and nuclei of tobacco leaves.

Heterotrimeric G-proteins are involved in a variety of cellular responses, but relatively little is known about their function and biochemistry in plants. Antibodies raised against the tobacco heterotrimeric G-protein beta-subunit (Gbeta) were used to analyse its distribution in tobacco leaves. In young tissue the protein level was relatively high, while it declined substantially during later stages of leaf development. Cell fractionation revealed that Gbeta is tightly associated with plasma membrane, but can also be detected in purified nuclei.

Induction versus modulation in phytochrome-regulated biochemical processes.

The time course of appearance of competence towards phytochrome (Pfr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated formation of anthocyanin (aglycone cyanidin) and NADP-dependent plastidal glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13). The experiments were performed to answer the following question: Does phytochrome act to turn responses on (induction), or - as an alternative - does phytochrome cause an amplification of processes already occurring in absolute darkness albeit at low rates once competence is reached (modulation)? The data show that in the case of GPD, phytochrome causes an amplification of the rate of synthesis once the competence point is reached at approximately 36 h after sowing at 25° C. In the case of anthocyanin, it was found that two distinct points of competence exist (26 h and 39 h after sowing, 25° C). In the case of 'early anthocyanin' (competence point at 26 h), synthesis does not occur in darkness without Pfr, while in the case of 'late anthocyanin' (competence point at 39 h), phytochrome causes an amplification of a process occurring in complete darkness albeit at a very low rate. It is concluded that in phytochrome-mediated photomorphogenesis, modulation as well as induction of biosynthetic processes plays a role.

Blue light is required for survival of the tomato phytochrome-deficient aurea mutant and the expression of four nuclear genes coding for plastidic proteins.

When dark-grown aurea mutant tomato seedlings which lack more than 95% of the phytochrome present in isogenic wild-type seedlings are kept in white or blue light, four nuclear-encoded transcripts coding for plastidic proteins (the light-harvesting chlorophyll a/b-binding protein of photosystem I and II [cab-PSII], plastocyanin and subunit 2 of photosystem I) are present in comparable amounts. These transcript levels in red light are strongly reduced in aurea seedlings when compared with those of wild type. Thus, blue light is required for normal expression of these genes in the mutant, while red light alone is not sufficient. Red light-grown aurea seedlings are very sensitive to blue light, even 10 minutes of blue light every day suffices to cause a measurable increase in cab-PSII transcript level. The action of blue light on the expression of cab-PSII in the mutant is under phytochrome control. After 8 days of blue light, phytochrome is almost as effective in inducing cab-PSII mRNA as in the isogenic wild type, whereas after 8 days of red light, only a small phytochrome response was observed in the mutant. It is concluded that blue light sensitizes the mutant to the residual phytochrome which allows normal gene expression and survival of the mutant under daylight conditions.

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

We compared the response of NO 3 (-) -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 NO 3 (-) 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.

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.

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.

Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons.

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.

Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gbeta). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 degrees C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100-400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored.

Photoreversibility of the Effect of Red and Green Light Pulses on the Accumulation in Darkness of mRNAs Coding for Phycocyanin and Phycoerythrin in Fremyella diplosiphon.

DNA fragments encoding a red light-inducible phycocyanin gene and a green light-inducible phycoerythrin gene have been used to investigate the effect of red and green pulses on the accumulation of phycocyanin and phycoerythrin mRNA in subsequent darkness. A red pulse promotes phycocyanin and suppresses phycoerythrin mRNA accumulation while a green pulse has an opposite effect on both transcript levels. The effect of a saturating light pulse is canceled by a subsequently given pulse of the other light quality. For a given mRNA, the positive and negative effects require the same fluence for saturation, whereas to saturate the phycoerythrin mRNA response requires at least twice as much light as to saturate the phycocyanin mRNA response. Calculations of the apparent extinction coefficients for the pigments mediating the light-regulated mRNA increase and decrease are of the order of 2 x 10(4) for phycocyanin mRNA and less than 10(4) for phycoerythrin mRNA. The data are consistent with the hypothesis that the light-induced increase and decrease of a particular phycobiliprotein mRNA is controlled by a single red/green photoreversible photosystem, but that phycoerythrin and phycocyanin mRNA levels are either controlled by two distinct photoreversible systems or that marked differences occur in the chain of events leading from photoperception to gene activation. These system(s) differ from most phytochrome systems in several ways: First, they remain fully on or off depending upon the light quality of the terminal irradiation. Second, they can be completely reversed by light of the appropriate wavelength after several hours of darkness without diminution of the effectiveness of the reversing light pulse. These two features argue against the existence of dark reversion or dark destruction of the biologically active moiety. Third, signal transduction is rapid-measurable mRNA changes occur even during a 10 minute irradiation.