Dark-grown Barley Seedlings Research Articles

Lil3 assembles as chlorophyll-binding protein complex during deetiolation

Dark-grown angiosperm seedlings are etiolated and devoid of chlorophyll. Deetiolation is triggered by light leading to chlorophyll dependent accumulation of the photosynthetic machinery. The transfer of chlorophyll to the chlorophyll-binding proteins is still unclear. We demonstrate here that upon illumination of dark-grown barley seedlings, two new pigment-binding protein complexes are de novo accumulated. Pigments bound to both complexes are identified as chlorophyll a and protochlorophyll a. By auto-fluorescence tracking and mass spectrometry, we show that exclusively Lil3 is the pigment-binding complex subunit in both complexes.

A mechanism for light-induced translation of the rbcL mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase in barley chloroplasts.

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.

Protein assembly and heat stability in developing thylakoid membranes during greening.

The development of the thylakoid membrane was studied during illumination of dark-grown barley seedlings by using biochemical methods, and Fourier transform infrared and spin label electron paramagnetic resonance spectroscopic techniques. Correlated, gross changes in the secondary structure of membrane proteins, conformation, composition, and dynamics of lipid acyl chains, SDS/PAGE pattern, and thermally induced structural alterations show that greening is accompanied with the reorganization of membrane protein assemblies and the protein-lipid interface. Changes in overall membrane fluidity and noncovalent protein-lipid interactions are not monotonic, despite the monotonic accumulation of chlorophyll, LHCII [light-harvesting chlorophyll a/b-binding (polypeptides) associated with photosystem II] apoproteins, and 18:3 fatty acids that follow a similar time course with highest rates between 12-24 h of greening. The 18:3 fatty acid content increases 2.8-fold during greening. This appears to both compensate for lipid immobilization by membrane proteins and facilitate packing of larger protein assemblies. The increase in the amount of protein-solvating immobile lipids, which reaches a maximum at 12 h, is caused by 40% decrease in the membranous mean diameter of protein assemblies at constant protein/lipid mass ratio. Alterations in the SDS/PAGE pattern are most significant between 6-24 h. The size of membrane protein assemblies increases approximately 4.5-fold over the 12-48-h period, likely caused by the 2-fold gain in LHCII apoproteins. The thermal stability of thylakoid membrane proteins increases monotonically, as detected by an increasing temperature of partial protein unfolding during greening. Our data suggest that a structural coupling between major protein and lipid components develops during greening. This protein-lipid interaction is required for the development and protection of thylakoid membrane protein assemblies.

Photodynamic action of uroporphyrin and protochlorophyllide in greening barley leaves treated with cesium chloride.

Incubation of greening barley leaves with cesium chloride (CsCl) results in photodynamic leaf lesions within 24 h due to an inactivation of uroporphyrinogen III decarboxylase, an enzyme of tetrapyrrole biosynthesis, and transient accumulation of uroporphyrin (ogen). To examine the mechanism of porphyrinogenesis, time kinetics of the accumulating tetrapyrrole intermediates uroporphyrin (ogen) and protochlorophyllide were performed with leaves which were cut from 7-day-old dark-grown barley seedlings and incubated in 15 mM CsCl or water under different light regimes. In the presence of CsCl chlorophyll and carotenoids accumulation was inhibited in the first 24 h of continuous light and the pigment content decreased dramatically during extended illumination. When CsCl=treated leaves were transferred to darkness, accumulated uroporphyrinogen was completely converted to protochlorophyllide. Low temperature fluorescence spectroscopy confirmed that uroporphyrinogen almost completely accumulated in the reduced form. The oxidised form, uroporphyrin, was detectable after 24 h of illumination. The photodynamic leaf lesions became visible at the same time. Protochlorophyllide synthesised from accumulated uroporphyrinogen III in dark incubated leaves had a fluorescence maximum at 635 nm which is indicative for its non-photoconvertible form. Re-illumination of the barley leaves resulted in a rapid degradation of proteins and pigments and an intense lipid peroxidation within less than two hours due to the photodestructive potential of non-metabolised protochlorophyllide.

Involvement of protein kinase and extraplastidic serine/threonine protein phosphatases in signaling pathways regulating plastid transcription and the psbD blue light-responsive promoter in barley.

We investigated the signaling pathways that control changes in plastid transcription in response to development and light. Plastid gene expression was analyzed in dark-grown barley (Hordeum vulgare L.) seedlings treated in vivo with an inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), or an inhibitor of protein kinases (K252a), followed by exposure of the seedlings to either red, blue, or white light. OA prevented blue light from activating the plastid pshD blue-light-responsive promoter (BLRP) and prevented red and blue light from activating the expression of the plastid-encoded rbcl and psbA and the nuclear-encoded RbcS and Lhcb genes. OA reduced total plastid transcription activity in dark- and light-grown seedlings by 77 to 80%, indicating that OA prevented light-responsive transcription by reducing total plastid transcription. In contrast, K252a activated the accumulation of mRNAs arising from the BLRP. Blue light in combination with K252a increased psbD mRNA levels in an additive manner. The results indicate that protein phosphatases 1 and/or 2A, which reside external to the organelle, are required for proper function of plastid transcription and chloroplast development, whereas a protein kinase represses the BLRP in plants grown in the dark.

Absolute Configuration of Protochlorophyllideaand Substrate Specificity of NADPH−Protochlorophyllide Oxidoreductase

Protochlorophyllide a was isolated from dark-grown barley (Hordeum vulgare L.) seedlings. The circular dichroism spectrum was identical with that of protochlorophyll a prepared from chlorophyll a b...

Proteolytic activity in intact barley etioplasts: endoproteolysis of NADPH-protochlorophyllide oxidoreductase protein

Crude and purified etioplastswere prepared from dark grown barley seedlings and compared for acid protease activity which liberates amino acids from added hemoglobin or endogenous etioplast proteins. Although crude etioplasts showed a high activity as measured by the ninhydrin method, no activity was detectable with intact etioplasts, indicating that etioplasts have no acid protease activity which releases amino acids and that the activity observed with the crude etioplasts is due to contamination with extra-plastidic proteases. Comparison of SDS-PAGE profiles of polypeptides from etioplast membranes before and after incubation at pH 7 revealed that NADPH-protochlorophyllide oxidoreductase (NPR) was preferentially degraded during incubation. Together, this suggests that etioplasts have an activity to hydrolyze polypeptides with no detectable amino acid liberation. At pH 5, however, some other polypeptides were hydrolyzed in addition to NPR. The hydrolysis of NPR was inhibited by N-ethylmaleimide (NEM), a thiol protease inhibitor.

Regulation of Light-Induced Chloroplast Transcription and Translation in Eight-Day-Old Dark-Grown Barley Seedlings

Plastid transcription and translation are light-activated in 8-day-old dark-grown barley (Hordeum vulgare L.) seedlings. Pretreatment of dark-grown seedlings with cycloheximide (inhibitor of cytoplasmic protein synthesis) abolished the activation of rbcL, psbA, and psaA-B transcription by light. In contrast, inhibition of plastid protein synthesis by chloramphenicol stimulated light-activated transcription of rbcL, psbA, and psaA-B. Light-induced transcription of the plastid genome occurred normally in the chlorophyll-deficient mutant xan-J(64). These results suggest that although the light-induced activation of plastid transcription is modulated by cytoplasmic and organellar protein synthesis, transcriptional activation is not dependent on the absorption of light by protochlorophyllide or the attainment of photosynthetic competence. In addition, plastid translation increased dramatically when 8-day-old dark-grown seedlings were illuminated and activation was dependent on cytoplasmic protein synthesis. Blockage of light-activated plastid transcription by Tagetin treatment (inhibitor of plastid RNA polymerase) did not attenuate the activation of plastid translation by light. These results suggest that while light simultaneously activates plastid transcription and translation, the rapid burst in plastid protein synthesis is due mainly to cytoplasmic-derived changes that regulate the rate of translation of pre-existing mRNAs.

Light-induced switch in barley psbD-psbC promoter utilization: a novel mechanism regulating chloroplast gene expression.

The synthesis of reaction center protein D2 and mRNAs which encode this protein are differentially maintained at high levels in mature barley chloroplasts. To understand the differential maintenance of psbD mRNA abundance, we have studied the transcription and the RNAs produced from the psbD-psbC operon in plastids of light and dark-grown barley seedlings. Ten psbD-psbC RNAs synthesized in dark-grown barley share four different 5'-ends, two of which arise by transcription initiation, and one of which is generated by 5'-processing of longer psbD-psbC transcripts. Illumination of dark-grown barley causes the decline of these ten transcripts, and the accumulation of two different psbD-psbC RNAs. Capping assays, in vitro transcription and RNA processing experiments and treatment of plants with tagetitoxin (a selective inhibitor of chloroplast transcription), indicate that the light-induced transcripts arise by transcription initiation. Run-on transcription and RNA quantitation experiments provide evidence that both light-induced transcription and RNA stability play roles in the accumulation of the light-induced RNAs. These data document a novel mechanism for regulating plastid gene expression involving a light-induced switch in psbD-psbC promoter utilization.

Light-induced transcription of chloroplast genes. psbA transcription is differentially enhanced in illuminated barley.

Transcription and Northern dot blot analyses were used to determine the relationship between light-induced changes in RNA levels and transcription in barley and maize plastids. Overall plastid transcription activity is near maximum and largely light independent in apical sections of 4.5-day-old dark-grown barley seedings. With continued development in the dark, transcription activity in barley plastids declined precipitously, but transcription could be reactivated if plants were illuminated. Illumination of 8-day-old dark-grown barley seedlings increased the transcription of psbA, rbcL, and 16 S rDNA; however, the stimulation of psbA transcription was 2-4-fold more than rbcL or 16 S rDNA. Differential enhancement of psbA transcription occurred when 4.5- or 8-day-old dark-grown seedlings were illuminated, and the influence persisted in plants illuminated for 72 h. Illumination of 8-day-old dark-grown barley caused an increase in psbA and rbcL mRNA levels, but the increase in psbA mRNA was greater than that of rbcL. Similarly, illumination of 9-day-old dark-grown maize increased plastid transcription activity and the levels of psbA and rbcL mRNA. These results indicate that light can modulate overall plastid transcription activity and differentially stimulate the transcription of genes including psbA.

Gabaculine inhibition of chlorophyll synthesis in light and darkness in intact barley ( Hordeum vulgare) seedlings

Dark chlorophyll accumulation in the first leaf of intact 5–6-day-old glasshouse-grown barley seedlings ( Hordeum vulgare) was inhibited by gabaculine (3-amino 2,3-dihydrobenzoic acid) (GAB) supplied via the roots. The inhibitory effect of gabaculine was eliminated by the addition of 5-aminolevulinic acid. Gabaculine also inhibited chlorophyll accumulation in intact dark-grown barley seedlings transferred to light. Inhibition of dark chlorophyll accumulation by gabaculine is evidence that the aminolevulinic acid precursor for light-independent chlorophyll synthesis is formed via the C-5 pathway.

Blue light regulates the accumulation of two psbD-psbC transcripts in barley chloroplasts.

Synthesis of D2, a Photosystem II reaction center protein encoded by psbD, is differentially maintained during light-induced chloroplast maturation. The continued synthesis of D2 is paralleled by selective light-induced accumulation of two psbD-psbC transcripts which share a common 5' terminus. In the present study, we examine the nature of the photoreceptor and the fluence requirement for psbD-psbC transcript induction. The light-induced change in psbD-psbC RNA population can be detected between 1 and 2 h after 4.5 day old dark-grown barley seedlings are transferred to the light. Light-induced transcript accumulation occurs normally in the chlorophyll-deficient barley mutant, xan-f10, indicating that light-activated chlorophyll formation and photosynthesis are not required for RNA induction. High fluence blue light fully induces psbD-psbC transcript accumulation; low or high fluence red or far-red light do not. However, psbD-psbC transcript accumulation elicited by blue light pulses can be partially attenuated if far-red light is given immediately following the blue light treatment. Thus, although blue light is needed to initiate transcript accumulation, phytochrome modulates the amplitude of the response. Pretreatment of dark-grown plants with cycloheximide blocks light-induced psbD-psbC transcript accumulation. This could implicate a blue-light responsive nuclear gene in the light-induced accumulation of the two psbD-psbC transcripts.

Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N',N'-tetracetic acid (EGTA). A free-Ca(2+) activity of less than 40 μM restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca(2+)-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca(2+) during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca(2+) addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.

Light-regulated translation of chloroplast proteins. I. Transcripts of psaA-psaB, psbA, and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings.

We have previously observed (Klein, R. R., and J. E. Mullet, 1986, J. Biol. Chem. 261:11138-11145) that translation of two 65-70-kD chlorophyll a-apoproteins of Photosystem I (gene products of psaA and psaB) and a 32-kD quinone-binding protein of Photosystem II (gene product of psbA) was not detected in plastids of dark-grown barley seedlings even though transcripts for these proteins were present. In the present study it was found that nearly all of the psaA-psaB transcripts in plastids of dark-grown plants were associated with membrane-bound polysomes. Membrane-associated polysomes from plastids of dark-grown plants synthesized the 65-70-kD chlorophyll a-apoproteins at low levels when added to a homologous in vitro translation extract capable of translation elongation. However, when etioplast membranes were disrupted with detergent, in vitro synthesis of the 65-70-kD chlorophyll a-apoproteins increased to levels observed with polysomes of plastids from illuminated plants. These results suggest that synthesis of the chlorophyll a-apoproteins of Photosystem I is arrested on membrane-bound polysomes at the level of polypeptide chain elongation. In addition to the selective activation of chlorophyll a-apoprotein translation, illumination also caused an increase in chloroplast polysomes (membrane-associated and stromal) and induced a recruitment of psbA and rbcL transcripts into chloroplast polysomes. These results indicate that in conjunction with the selective activation of chlorophyll a-apoprotein elongation, illumination also caused a general stimulation of chloroplast translation initiation.

Abscisic Acid Accumulation Is Not Required for Proline Accumulation in Wilted Leaves

Leaves from dark-grown barley (Hordeum vulgare L. var Larker) seedlings grown in the presence and absence of fluridone were used to determine whether or not abscisic acid (ABA) accumulation was necessary for proline to accumulate in wilted tissue. Wilted tissue (polyethylene glycol-treated) leaves from fluridone-grown seedlings did not accumulate ABA but did accumulate proline at a rate that was not different from the non-fluridone-treated leaves. Thus ABA accumulation is not required for wilting-induced proline accumulation in barley leaves. Proline accumulation in wilted leaves from the wilty tomato (Lycopersicon esculentum) mutant, flacca, was compared to that in the wild type, Rheinlands Ruhm. Proline accumulated in wilted leaves from flacca. The rate of accumulation was faster in flacca compared to the rate in the wild type because the wilty mutant wilted faster. ABA accumulated in wilted leaves from the wild type but not in the wilty mutant. This result is a further confirmation that ABA accumulation is not required for wilting-induced proline accumulation. These results are significant in that proline accumulation in barley leaves can be induced independently by any one of three treatments: wilting, ABA, or salt.

The light-dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center in barley. Evidence for translational control.

The light-dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center has been studied in greening barley (Hordeum vulgare L.) seedlings. Immunoblot analysis of total cellular protein fractions and immunogold labelling of the P700 chlorophyll a protein in ultrathin sections of Lowicryl-embedded leaf tissue revealed that the concentration of this chlorophyll-binding protein in plastids of dark-grown barley seedlings is below the limit of detection. Upon illumination with white light, a rapid accumulation of this protein is induced. This light effect seems not to be regulated at the level of transcription. The gene for the P700 chlorophyll a protein has been mapped within the large single-copy region of the plastid DNA of barley. High levels of transcripts of this gene are present already in dark-grown seedlings and remain fairly constant throughout an extended illumination period. Polysomes were isolated from etioplasts and chloroplasts. The same high relative concentration of mRNA encoding the P700 chlorophyll a protein was present in both polysome fractions. This result suggests that the light-dependent accumulation of the P700 chlorophyll a protein during chloroplast formation in barley seedlings is regulated at the translational, or posttranslational, level.

Regulation of chloroplast-encoded chlorophyll-binding protein translation during higher plant chloroplast biogenesis.

Etioplasts of 5-day-old dark-grown barley seedlings synthesize most of the soluble and membrane proteins found in chloroplasts of illuminated plants. Prominent among these proteins are the large subunit of ribulose bisphosphate carboxylase and the alpha- and beta-subunits of the chloroplast ATPase. However, etioplasts do not synthesize four chloroplast-encoded proteins which are major constituents of the chloroplast thylakoid membrane: two chlorophyll apoproteins of photosystem I (68 and 65 kDa) and two chlorophyll apoproteins of photosystem II (47 and 43 kDa). Pulse-labeling experiments show that the lack of radiolabel accumulation in the chlorophyll apoproteins in etioplasts is due to inhibition of synthesis rather than apoprotein instability. Illumination of 5-day-old dark-grown barley selectively induces synthesis of the plastid-encoded chlorophyll apoproteins and proteins of 32, 23, and 21 kDa. Synthesis of the chlorophyll apoproteins was significant in plants illuminated for 15 min and was near maximum by 1 h. The induction of photosystem I chlorophyll apoprotein synthesis was not accompanied by an increase in mRNA for these proteins. These results demonstrate that the synthesis of the plastid-encoded photosystem I chlorophyll apoproteins is blocked at the translational level in dark-grown barley. Translation of the chlorophyll apoproteins is induced rapidly by light with a time course which is similar to the light-dependent formation of chlorophyll from protochlorophyllide.

Photoreduction of protochlorophyllide and its relationship to delta-aminolaevulinic acid synthesis in the leaves of dark-grown barley (Hordeum vulgare) seedlings.

The photoreduction of protochlorophyllide (Pchl) in dark-grown leaves of barley (Hordeum vulgare) brings about the synthesis of delta-aminolaevulinic acid (AmLev). Manipulation of the Pchl level in the leaves by incubation in AmLev indicated that the production of AmLev was intimately related to the state of the Pchl reductase ternary complex. Free Pchl reductase that is unassociated with substrate/product appeared at first to be essential for the photoinduction of AmLev synthesis. Experiments on the photoreduction of Pchl in dark-grown leaves exposed to low-energy red-light, however, showed that photoreduction and AmLev synthesis would occur when the Pchl reductase, together with substrate, was maintained at relatively high endogenous concentration. Under such conditions the availability of free reductase protein would be negligible. An alternative scheme is presented, therefore, that can explain many, if not all, of the observations on AMLev synthesis and its close relationship to Pchl reduction, and which is based on a common supply of NADPH for the reduction of glutamate to AmLev and the synthesis of chorophyll(-ide).

Regulation of delta-aminolaevulinic acid synthesis and protochlorophyllide regeneration in the leaves of dark-grown barley (Hordeum vulgare) seedlings.

Laevulinic acid (Lev) was used to control the rate of protocholorophyllide (PChl) regeneration in the leaves of dark-grown seedlings of barley (Hordeum vulgare) after a brief light treatment. In the leaves given Lev, at concentrations that severely block the resynthesis of protochlorophyllide, there was a massive overproduction of delta-aminolaevulinic acid (AmLev) that was well in excess of that required for the regeneration of PChl observed in the control leaves. Lev, at low concentrations, slightly delayed regeneration and held up, rather than inhibited, the utilization of the AmLev, which accumulated in the tissues. The overproduction and uncontrolled formation of AmLev also occurred in dark-grown leaves treated with a high concentration of Lev and given a light treatment of just sufficient energy to photoreduce only small quantities of the endogenous PChl. Experiments in which a high level of free PChl was induced by incubating the leaves in AmLev indicated that the active species of PChl was that associated with, and bound to, the PChl reductase protein. The results strongly demonstrate a close relationship between the PChl-protein complex and the ability of the leaves to synthesize AmLev.

Regulation of synthesis of the photosystem I reaction center.

The in vivo biosynthesis of the P700 chlorophyll a-apoprotein was examined to determine whether this process is light regulated and to determine its relationship to chlorophyll accumulation during light-induced chloroplast development in barley (Hordeum vulgare L.). Rabbit antibodies to the 58,000-62,000-mol-wt apoprotein were used to measure relative synthesis rates by immunoprecipitation of in vivo labeled leaf proteins and to detect apoprotein accumulation on nitrocellulose protein blots. 5-d-old, dark-grown barley seedlings did not contain, or show net synthesis of, the 58,000-62,000-mol-wt polypeptide. When dark-grown barley seedlings were illuminated, net synthesis of the apoprotein was observed within the first 15 min of illumination and accumulated apoprotein was measurable after 1 h. After 4 h, P700 chlorophyll a-apoprotein biosynthesis accounted for up to 10% of the total cellular membrane protein synthesis. Changes in the rate of synthesis during chloroplast development suggest coordination between production of the 58,000-62,000-mol-wt polypeptide and the accumulation of chlorophyll. However, when plants were returned to darkness after a period of illumination (4 h) P700 chlorophyll a-apoprotein synthesis continued for a period of hours though at a reduced rate. Thus we found that neither illumination nor the rate of chlorophyll synthesis directly control the rate of apoprotein synthesis. The rapidity of the light-induced change in net synthesis of the apoprotein indicates that this response is tightly coupled to the primary events of light-induced chloroplast development. The data also demonstrate that de novo synthesis of the apoprotein is required for the onset of photosystem I activity in greening seedlings.