The occurrence and photoregulation of flavonoids in barley plastids

Whole-leaf extracts of etiolated or light-grown barley shoots contain the C-glycosylflavones saponarin, lutonarin and lutonarin 3′-methyl ether. Plastids isolated by aqueous techniques contain only saponarin. Contamination experiments using foreign flavonoids indicate that saponarin recovered from plastids is not a contaminant from other cellular fractions. In response to brief red light treatment 24 hr before harvest, saponarin levels are approximately doubled in whole-shoot extracts, but increased about 3.5 fold in plastids. This photocontrolled increase is far-red reversible. Thus saponarin is selectively accumulated in barley plastids and this accumulation is controlled by phytochrome.     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.     

Assembly of the barley light-harvesting chlorophyll a/b proteins in barley etiochloroplasts involves processing of the precursor on thylakoids

A barley gene encoding the major light-harvesting chlorophyll a/b-binding protein (LHCP) has been sequenced and then expressed in vitro to produce a labelled LHCP precursor (pLHCP). When barley etiochloroplasts are incubated with this pLHCP, both labelled pLHCP and LHCP are found as integral thylakoid membrane proteins, incorporated into the major pigment-protein complex of the thylakoids. The presence of pLHCP in thylakoids and its proportion with respect to labelled LHCP depends on the developmental stage of the plastids used to study the import of pLHCP. The reduced amounts of chlorophyll in a chlorophyll b-less mutant of barley does not affect the proportion of pLHCP to LHCP found in the thylakoids when import of pLHCP into plastids isolated from the mutant plants is examined. Therefore, insufficient chlorophyll during early stages of plastid development does not seem to be responsible for their relative inefficiency in assembling pLHCP. A chase of labelled pLHCP that has been incorporated into the thylakoids of intact plastids, by further incubation of the plastids with unlabelled pLHCP, reveals that the pLHCP incorporated into the thylakoids can be processed to its mature size. Our observations strongly support the hypothesis that after import into plastids, pLHCP is inserted into thylakoids and then processed to its mature size under in vivo conditions.     

Protochlorophyllide-independent import of two NADPH:Pchlide oxidoreductase proteins (PORA and PORB) from barley into isolated plastids

The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear-encoded protein that is post-translationally imported to the plastid. In barley and Arabidopsis thaliana , the reduction of Pchlide is controlled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORB (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in greenhouse conditions for 6 days. Alternatively, greenhouse-grown pea plants were incubated for 4 days in darkness before plastid isolation, or chloroplasts isolated from greenhouse-grown plants were incubated with Δ -aminolevulinic acid (ALA), an early precursor in the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the differentially treated plants, including those isolated from greenhouse-grown plants. The absence or presence of Pchlide did not significantly affect the import capacity of barley pPORA or pPORB. Assays performed on stroma-enriched fractions from chloroplasts and etioplasts of barley indicated that no post-import degradation of the proteins occurred in the stroma, irrespective of whether the incubation was performed in darkness or in light.     

NADPH:Protochlorophyllide oxidoreductase uses the general import route into chloroplasts

Chloroplast differentiation in angiosperm plants depends on the light-dependent conversion of protochlorophyllide to chlorophyllide by NADPH:protochlorophyllide oxidoreductase (PORA; EC 1.6.99.1), a nuclearly encoded protein. The protein import of the precursor form of PORA into plastids was shown previously to strictly depend on the presence of its substrate protochlorophyllide. PORA seemed to follow a novel, posttranslationally regulated import route. Here we demonstrate that the precursor of PORA from barley is imported into isolated barley plastids independently of protochlorophyllide. PORA as well as PORB import is competed for by the precursor of the small subunit of Rubisco. The data demonstrate that the PORA precursor uses the general import pathway into plastids. Furthermore, en route into chloroplasts the pea POR precursor can be cross-linked to the protein import channel in the outer envelope Toc75 from pea.     

Translation and stability of proteins encoded by the plastid psbA and psbB genes are regulated by a nuclear gene during light-induced chloroplast development in barley

We have characterized a nuclear mutant of barley, viridis-115, lacking photosystem II (PSII) activity and compared it to wild-type seedlings during light-induced chloroplast development. Chloroplasts isolated from wild-type and viridis-115 seedlings illuminated for 1 h synthesized similar polypeptides and had similar protein composition. After 16 h of illumination, however, mutant plastids exhibited reduced ability to radiolabel D1, CP47, and several low Mr membrane polypeptides, and by 72 h, synthesis of these proteins was undetectable. Immunoblot analysis showed that plastids of dark-grown wild-type barley lacked several PSII proteins (D1, D2, CP47, and CP43) and that 16 h of illumination resulted in the accumulation of these polypeptides. In contrast, these polypeptides did not accumulate in illuminated viridis-115 seedlings, although mutant plastids accumulated two PSII proteins that participate in oxygen evolution, oxygen-evolving enhancers 1 and 3. Northern analysis showed that the levels of psbA and psbB mRNA in mutant plastids were equal to or greater than levels in wild-type plastids throughout the developmental period examined here. These results indicate that the nuclear mutation present in viridis-115 affects the translation and stability of the chloroplast-encoded D1 and CP47 polypeptides and that its influence is expressed after the onset of light-induced chloroplast development.     

Division and DNA distribution in ribosome-deficient plastids of the barley mutant “albostrians”

The ribsome-deficient plastids of the albino leaves of the barley mutant albostrians divide at about the same rate as normal plastids and contain similar levels of plastids DNA to the normal plastids. Double-ring structures were observed around the neck of constricting dumbbell-shaped, ribosome-deficient plastids in the basal intercalary meristem of albino leaves. In the distal region of albino leaves the ribosome-deficient plastids contain a rudimentary thylakoid system often closely associated with DNA nucleoids. It is suggested that nuclear coded proteins synthesized within the cytoplasm are responsible for the formation of the double-ring structures and the rudimentary thylakoids of albino plastids.     

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15–24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16–72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution ofpsbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16–72 h. Toeprint analysis showed similar levels of translation initiation complexes onpsbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing onpsbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing onpsbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll will D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.     

Changes of Plastids During Xylem Differentiation in Barley Root

Plastids (eoplasts) are present in meristematic cells of prospectivecentral metaxylem in the barley root. Starch starts to be formedin plastids precisely after the cessation of mitotic activityand at the beginning of endomitotic growth. During secondarywall formation, the starch is gradually lost. Cavities are formedin plastids and signs of plastid degeneration are present fromthis stage of cell development. However, some intact globularplastids without starch are present until shortly before thefinal step of ontogeny, i.e. total destruction of protoplast. Hordeum distichum L., root, xylem, plastids, endomitotic growth, starch     

Isolation and characterisation of developing chloroplasts from light-grown barley leaves

Developing chloroplasts were isolated from the basal region of green barley ( Hordeum vulgare L. cv. Menuet) leaves and their ultrastructure and biochemical composition were compared to those of mature chloroplasts from the tip of the same leaves, using two methods of purification on sucrose and Percoll gradients.
When examined and compared to mature chloroplasts, the developing chloroplasts showed well-developed grana stacks, but these last organelles were 2-fold smaller and contained lower amounts of chlorohylls and polar lipids. Only traces of trans -3-hexadecenoic acid could be detected in phosphatidylglycerol of developing plastids. The protein content of these plastids was higher than in mature plastids and showed an increased proportion of polypeptides linked to P-700 chlorophyll α-protein. The photosynthetic activity of these plastids was about 2-fold lower and their photosystem 1/photosystem II ratio higher than in mature chloroplasts.     

Light-induced changes in the distribution of the 36000-Mr polypeptide of NADPH-protochlorophyllide oxidoreductase within different cellular compartments of barley (Hordeum vulgare L.)

Changes in the relative content of NADPH-protochlorophyllide oxidoreductase during the light-induced greening of barley plants were measured both in the total leaf extract as well as in intact and broken plastids. The enzyme protein was identified by its apparent molecular weight and its immunological crossreactivity with an antiserum directed against the NADPH-protochlorophyllide oxidoreductase. The monospecificity of the antiserum was tested by two different criteria: i. The antiserum was purified by affinity chromatography. ii. It was demonstrated that the antiserum crossreacts with only those polypeptides which appear to be enzymatically active. In the fraction of broken plastids isolated from leaves of briefly illuminated barley plants the concentration of the enzyme protein was reduced drastically. Our results indicate that this decrease in enzyme protein content is the consequence of an artificial proteolytic breakdown of the membrane-bound enzyme protein. In intact plastids and in the total leaf extract the concentration of the enzyme protein did not change dramatically during the first 4 to 6 h of illumination. However, when the exposure to continuous white light was extended further the concentration of the enzyme protein in intact plastids began to decline rapidly while in total leaf extracts the concentration remained almost constant for the next 10 h of light. These results indicate that part of the enzyme protein may be localized outside of the plastid compartment.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulfate     

Stoichiometric analysis of barley plastid ribosomal proteins

We analyzed the protein composition of plastid 70S ribosomes isolated from the stromal fractions of barley plastids by the radical-free and highly reducing method of two dimensional polyacrylamide gel electrophoresis (RFHR 2D-PAGE). Intactness of the ribosomes was confirmed by the poly(U)-directed phenylalanine polymerization activity and by the reassociation capacity of the subunits into 70S ribosomes. The small and large ribosomal subunits were composed of 23 and 36 proteins, respectively. In addition, one acidic protein associated with ribosomes in low salt buffer but released in high salt buffer was found. The plastid ribosomes contained relatively larger numbers of acidic proteins than prokaryotic ribosomes. Stoichiometric analysis revealed the presence of several ribosomal proteins in low copy numbers, indicating that the ribosomes of plastids were heterogeneous. We also investigated the protein composition of plastid ribosomes from greening barley leaves and found that it did not change during greening.     

Lipid biosynthesis by isolated barley chloroplasts in relation to plastid development

The effect of seedling age and of the time of greening on the incorporation of 1-¹⁴C-acetate into lipids by isolated barley (Hordeum vulgare cultivar Svalöf's Bonus) plastids was examined. The fatty acid synthesizing capacity of plastids isolated from 5-day-old seedlings did not increase markedly from zero to 36 hours of greening nor was a light stimulation of fatty acid synthesis observed. However, an increasing capacity for fatty acid synthesis and an increasing light stimulation of this process with greening were attained by the plastids isolated from 7-, 9-, and 11-day-old seedlings.     

Simultaneous Appearance of C-550 and Cytochrome b559HP (High Potential Form) in Barley Leaves Greened under Intermittent Light

Cytochrome b_559HP has been detected by spectrochemical assays in plastids of barley leaves greened under intermittent light (flashed leaves). The amount of cytochrome b_559HP in these plastids was nearly 10-fold lower than in normal chloroplasts when the results were expressed on plastid number. The amount of cytochrome b_559HP phototransformable at -170°C was similar, on a C-550 basis, in the plastids of flashed and normal green leaves. The appearance of the 2 components was simultaneous during the greening process under intermittent light and it is suggested that it was parallel to the increase of appressed regions in thylakoids. The illumination of flashed leaves under continuous light for 5 minutes allowed the appearance of a normal Photosystem-II activity, but had no effect either on the cytochrome b_559HP content of the plastids or on the photoreactivity of this component at low temperature.     

Protoporphyrinogen oxidation in chloroplasts and plant mitochondria,a step in heme and chlorophyll synthesis

The oxidation of protoporphyrinogen to protoporphyrin was demonstrated in greening plastids and mitochondria from greening barley shoots. The plastids, purified by sucrose gradient centrifugation, were essentially free of a mitochondrial marker enzyme. The plastid activity was destroyed by mild heating and was proportional to plastid concentration suggesting, an enzymatic reaction. Uroporphyrinogen I was not oxidized at an appreciable rate. Activity was also demonstrated in etioplasts and mitochondria from dark-grown barley, and in chloroplasts from commercial spinach leaves. The chelating agent 1,10-phenanthroline partially decreased activity in plant organelles, but cyanide did not. The plastid activity, like the activity in liver mitochondria, was readily demonstrable at pH 8.4 in the presence of glutathione as reducing agent. However, the plastid activity was markedly enhanced by assay at pH 7.0 and the absence of reducing agents. These properties distinguish the activity in plants from that previously described in mammalian mitochondria and photosynthetic bacteria.     

Plastid ultrastructure and DNA related to albinism in androgenetic embryos of various barley (Hordeum vulgare L.) cultivars

In order to understand the occurrence of albinism during androgenesis in barley, a number of plastid parameters were analyzed in microspore-derived embryos and androgenetic plantlets, and the results were compared in albino and non-albino producing cultivars. In the winter cv. Igri, plastids in microspore-derived embryos are characterized by numerous divisions, differentiated thylakoids, low amount of starch and a high DNA content examined by immunoelectron microscopy. After regeneration, the androgenetic plantlets were mostly chlorophyllous. In contrast, in the spring lines tested, the plastids of microspore-derived embryos were rarely dividing amyloplasts in which thylakoids and DNA were scarce and albino plantlets were mainly regenerated. After 2 weeks on the regeneration medium, plastids of Igri chlorophyllous androgenetic plantlets were typical chloroplasts, whereas in spring lines plastids of albino androgenetic plantlets were proplastids with the same characteristics as those in the corresponding microspore-derived embryos. These results strongly suggest that the origin of androgenetic albinism differs in winter and spring cvs.: in the winter cv. Igri plastid alteration may take place during the regeneration step of androgenesis whereas in the tested spring lines plastids are already affected in the microspore-derived embryos meaning that albinism is not initiated during regeneration but originates earlier during the androgenetic process likely as early as the sampling stage.     

The stromal protein large subunit of ribulose-1,5-bisphosphate carboxylase is translated by membrane-bound ribosomes.

Translation of the large subunit of ribulose-1,5-bisphosphate carboxylase (LSU) was investigated by labeling of isolated barley plastids with [35S]-methionine. In both chloroplasts and etioplasts, labeling of LSU was severely impaired if plastid membranes were removed from the reaction mixtures. Removal of membrane-bound polysomes with high salt or puromycin greatly decreased translation of LSU. Pulse-labeled chloroplast membranes were shown to release LSU if chased with unlabeled methionine in the presence of stroma. Immunoprecipitation detected higher amounts of labeled LSU translation intermediates associated with the membrane fraction than in the soluble fraction. We therefore conclude that, in plastids, membrane-bound polysomes are required not only for translation of membrane-intrinsic proteins but also for translation of a soluble protein.     

Light-dependent,but phytochrome-independent,translational control of the accumulation of the P700 chlorophyll-a protein of photosystem I in barley (Hordeum vulgare L.)

This work reports on the regulation of synthesis of the P700 chlorophyll-a apoprotein of photosystem I in barley. The mRNA for the P700 apoprotein is almost exclusively confined to the plastid membrane-bound polysomes. However, the mRNA for the 32-kDa herbicide-binding protein of photosystem II is found in both the soluble and membrane-bound polysomes.The mRNA for the P700 apoprotein is found in similar amounts in dark-grown and light-grown wild-type as well as mutant xantha-l⁸¹ barley. The latter mutant is deficient in chlorophyll biosynthesis. However, while wild-type leaves accumulate the P700 chlorophyll-a protein only in the light, mutant leaves never accumulate the P700 apoprotein.A more sensitive approach was taken using isolated plastids to study P700 apoprotein synthesis. Etioplasts did not synthesize detectable P700 apoprotein even when the etioplasts were exposed to light. However, only a 1-min exposure of leaves to light was necessary to induce P700 apoprotein synthesis by isolated plastids.Phytochrome involvement in controlling P700 apoprotein synthesis was tested by using red/farred light treatment of leaves. These treatments showed no far-red reversibility of red-induced P700-apoprotein synthesis in isolated plastids even after 3 h of darkness after the light treatments. From these data we conclude that the accumulation of P700 apopootein is not under the control of phytochrome and that the light induction of P700 apoprotein is most likely mediated through the protochlorophyllide/chlorophyllide system. This control, however, may also involve cytoplasmic signals as the synthesis of the P700 apoprotein is not turned on in illuminated etioplasts.