The Effect of Diuron and 5-Aminolevulinic Acid on the Maintenance of Stable Chlorophyll Content in Greening Barley Leaves

Chlorophyll (Chl) accumulation and delayed luminescence of PSII were compared in greening barley leaves pretreated and untreated with diuron (DCMU) in the etiolated state, and reactions of two photosystems were studied in the plastids isolated from the pretreated and untreated leaves. The effect of treatment in light of post-etiolated leaves after 40-h illumination with 5-aminolevulinic acid (ALA), on the content of Chl and its precursor, protochlorophyllide (PChld) was also studied. The pretreatment of etiolated leaves with DCMU did not affect the rate of greening and the stable level of Chl content in barley. ALA, when introduced to leaves after the termination of Chl accumulation, increased PChld, but not Chl level. We suppose that the primary cause of greening cessation in etiolated leaves is the inhibition and cessation of the synthesis of apoproteins of pigment–protein complexes. The exhaustion of binding sites for newly synthesized Chl molecules leads to their retention in the so-called retroinhibitory pool of Chl, thus resulting in the inhibition of ALA synthesis by a negative feedback mechanism.     

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.     

Tetrapyrrole biosynthesis in greening etiolated barley seedlings

Allyl isopropylacetamide (AIA) does not stimulate porphyrin biosynthesis in greening barley; AIA inhibits the synthesis of 5-aminolaevulinate (ALA) in plants and does not overcome the repression of ALA-synthetase. This indicates that the ALA synthesis system of green plants is regulated differently from ALA synthetase of mammalian systems. Laevulinic acid (LA) inhibited the biosynthesis of tetrapyrrole pigments in greening barley and diminished the insertion of ⁵⁵Fe into extractable protohaem, confirming that haem was synthesized at a time of little net increase in protohaem. ALA feeding increased iron incorporation into protohaem without increasing either extractable protohaem or cytochromes b and f. Since ALA feeding greatly increased the protochlorophyllide content of darkgrown plants and subsequent chlorophyll levels in the light, the regulation of haem pigment synthesis in plants occurs after protoporphyrin and protohaem synthesis and is likely to involve the turnover of protohaem produced in excess of haem protein requirements.     

Differential effects of gabaculin and laevulinic acid on protochlorophyllide regeneration

Abstract The effects of gabaculin (3-amino 2,3-dihydrobenzoic acid) and laevulinic acid on the regeneration of protochlorophyllide from exogenous δ-aminolaevulinic acid in leaves of dark-grown barley (Hordeum vulgare) after a brief light treatment were compared. Gabaculin, a potent inhibitor of chlorophyll biosynthesis, did not inhibit this process showing that it affects the formation of δ-aminolaevulinic acid rather than its further metabolism. Laevulinic acid, which is an inhibitor of δ-aminolaevulinic acid dehydratase, prevented regeneration of protochlorophyllide provided pools of intermediates in the biosynthetic sequence were depleted. Formation of relatively large amounts of protochlorophyllide in some experiments suggests a lack of control in the utilization of δ-aminolaevulinic acid for protochlorophyllide synthesis.     

The photochemical activities and electron carriers of developing barley leaves

The development of photochemical activities in isolated barley plastids during illumination of dark-grown plants has been studied and compared with the behaviour of plastocyanin, cytochromes f, b-559_LP, b-563 and b-559_HP and pigments P546 (C550) and P700. Electron-transport activity dependent on Photosystem 1 and cyclic photophosphorylation dependent on N-methylphenazonium methosulphate (phenazine methosulphate) were very active relative to the chlorophyll content after only a few minutes of illumination of etiolated leaves, and then rapidly declined during the first few hours of greening. By contrast, Photosystem 2 activity (measured with ferricyanide as electron acceptor) and non-cyclic photophosphorylation were not detectable during the first 2½h of greening, but then increased in total amount in parallel with chlorophyll. The behaviour of the electron carriers suggested their association with either Photosystem 1 or 2 respectively. In the first group were plastocyanin, cytochrome f and cytochrome b-563, whose concentrations in the leaf did not change during greening, and cytochrome b-559_LP whose concentration fell to one-half its original value, and in the second group were cytochrome b-559_HP and pigment P546, the concentrations of which closely followed the activities of Photosystem 2. Pigment P700 could not be detected during the first hour, during which time some other form of chlorophyll may take its place in the reaction centre of Photosystem 1. The plastids started to develop grana at about the time that Photosystem 2 activity became detectable.     

Changes in Chloroplast Lipids during the Development of Photosynthetic Activity in Barley Etio-Chloroplasts

The content of monogalactosyl diglyceride, digalactosyl diglyceride, sulfoquinovosyl diglyceride and phosphatidyl glycerol of gel-filtrated etio-chloroplasts isolated from greening barley seedlings was determined. The development of photosynthetic electron transport, measured as anthraquinone autooxidation, was simultaneously determined with an oxygen electrode. During the first hour of irradiation of the etiolated seedlings the lipid content of the plastids decreased rapidly. The decrease is interpreted as a chlorophyll sensitized photooxidation of the fatty acids of the diglycerides. With artificial electron donors an oxygen uptake was detected after 10 min of greening. With no donors added, a DCMU sensitive oxygen uptake was detected after 2 h. The level of DCMU inhibition increased as the plastid developed and total inhibition was obtained after 5 h. Between 2 and 6 h of greening the lipid content of the plastids stayed constant. During this greening period there was a correlation between the appearance of a DCMU sensitive electron transport and the accumulation of the trans-3-hexadecenoic acid of phosphatidyl glycerol. The trans-3-hexadecenoic acid was present already in the dark-grown seedlings but an increase in content did not occur until after 3 h. The lipid content increased after 6 h of greening. This increase coincided well in time with the formation of grana. The fatty acid composition of the individual lipids, with the exception of phosphatidyl glycerol, and the monogalactosyl diglyceride to digalactosyl diglyceride ratios did not change fundamentally during the greening.     

Nitrate reductase is not accumulated in chloroplast-ribosome-deficient mutants of higher plants

The activities of nitrite reductase (EC 1.7.7.1) are 60–70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants albostrians (Hordeum vulgare) and iojap (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of albostrians. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.Abbreviations CRM cross-reacting material - Mo-co molybdenum cofactor - NiR nitrite reductase - NR nitrate reductase     

Effect of chloramphenicol and cycloheximide on the induction of nitrate reductase and nitrite reductase in bean leaves

Summary In etiolated leaves of Phaseolus vulgaris L. cv. Prelude only low levels of NADH-nitrate oxidoreductase (E.C. 1.6.6.2; NAR) and reduced benzyl viologen-nitrite oxidoreductase (E.C. 1.6.6.4; NIR) could be detected, even in the presence of nitrate. When nitrate was available illumination of leaves of 10-day-old etiolated seedlings resulted in an induction of both NAR and NIR. In the absence of nitrate no induction of the enzymes took place, although greening of the leaves was normal. Chloramphenicol (CAP) and cycloheximide (CHI), applied at the beginning of the light period, inhibited the induction of both NAR and NIR. Administered after 24 h of illumination CHI still inhibited the induction of both enzymes whereas CAP was no longer inhibitory. The induction of NAR and NIR by nitrate in green leaves in light was inhibited by CHI but not by CAP. From these results it seems likely that both the enzymes NAR and NIR are synthesized on cytoplasmic ribosomes. Before the enzymes can be manufactured in the cytoplasm some chloroplast development is required.Abbreviations CAP chloramphenicol - CHI cycloheximide - G-6-P(-dh) glucose-6-phosphate (dehydrogenase) - NAR nitrate reductase - NIR nitrite reductase     

Plastid ultrastructure and photosynthesis in greening petaloid hypsophylls

The ultrastructural and biochemicalphysiological aspects of postfloral greening have been studied in hypsophylls of Heliconia aurantiaca Ghiesbr., Guzmania cf. x magnifica Richter and Spathiphyllum wallisii Regel. In all three species the greening of the hypsophylls is due to plastid transformation, chloroplast formation proceeding from the initially different types of plastids. The degradation process of the original plastid structures and the mode of thylakoid formation are distinct in each case. In none of the species do the transformed plastids look identical to the chloroplasts of the corresponding foliage leaves. On a chlorophyll basis, the rate of photosynthesis of the greened hypsophylls surpasses the rate of the leaves considerably in Spathiphyllum, but is much lower in Heliconia (no data for Guzmania). In all species, anatomy, plastid structure, pigments, 77° K-fluorescence emission, ribulose-1,5-bis-phosphate carboxylase activities and short-term photosynthesis ¹⁴CO₂-assimilation patterns prove the greened hypsophylls to be capable of providing additional carbon to the developing fruits, thus supplementing the import of organic matter from the foliage leaves.Abbreviations MDH malate dehydrogenase (EC 1.1.1.37) - PEPCase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

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.     

Ultrastructural demonstration of ferricyanide reductase (Diaphorase) activity in the envelopes of the plastids of etiolated barley (Hordeum vulgare L.) leaves

Electron-dense precipitate was found consistently in the plastid envelope compartment in etiolated barley (Hordeum vulgare L.) leaves, incubated prior to fixation with succinate or malate as substrates and ferricyanide as the electron acceptor. Sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide abolished this reaction, while KCN did not affect it. Prefixation with 0.1% glutaraldehyde followed by incubation in basic media did not change the fine structural localization of precipitate, whereas pretreatment with 1.25% glutaraldehyde resulted in aspecific precipitation. Omission of the subtrate from the medium brought about diminished or negative reaction. Our results indicate that a (possibly not yet assembled) nitrate reductase complex is present in the plastid envelope compartment, the diaphorase part of which is responsible for the observed precipitation.Abbreviations PCMB p-chloromercuribenzoate - NEM N-ethylmaleimide - NR nitrate reductase - SDH succinic dehydrogenase     

Light and Nitrate Requirements for Induction of Nitrate Reductase Activity in Hordeum vulgare

The importance of light to the induction of nitrate reductase activity in barley (Hordeum vulgare L.) was studied. Activity in etiolated leaves in darkness stayed at a low endogenous level even while large amounts of nitrate were actively accumulated. Light was required for any increase in activity, though the requirement may be satisfied to a limited extent before nitrate is available. Nitrate reductase activity was induced in the dark in green leaves which had not previously had nitrate but were supplied nitrate at the beginning of the dark period. If the nitrate then made available was sufficient, nitrate reductase activity increased until the effect of the previous light treatment was exhausted. Activity then decreased even though nitrate uptake continued. Upon returning the leaves to light, enzymatic activity increased again, as expected. Nitrate uptake was eliminated as an experimental variable by giving dark-grown plants nitrate, then detaching the leaves for induction studies. Under these conditions light saturation occurred between 3600 and 7700 lux at exemplary periods of illumination. At intensities of 3600 lux and above, activity increased sharply after a 6-hour lag period. As light intensity was decreased below 3600 lux the lag period became longer. Thus, when sufficient nitrate was available, the extent of induction of nitrate reductase activity was regulated by light.     

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.     

The effect of haem on chlorophyll synthesis in barley leaves

Exogenously supplied bovine haemin, fed to etiolated barley leaves, inhibited chlorophyll synthesis in leaves exposed to light. Haemin inhibited the regeneration of protochlorophyllide (P650) and the conversion of exogenously supplied δ-aminolaevulinate (ALA) to protochlorophyll (P630). The effect of haemin on chlorophyll production was overcome by incubating the leaves in water in the dark before light treatment, suggesting the operation of a rapid haem destruction mechanism in leaves. Protohaem turnover in dark-grown leaves was between 8 and 9 hr, based on the rate of degradation of erogenous haemin and the rate of protohaem breakdown in laevulinic acid (LA) treated leaves. The rate constant for haem destruction was 85 pmol/nmol/hr in the dark and 45 pmol/nmol/hr after 4 hr light. There was no evidence that light affects the synthesis of protohaem. It appears that the regulation of endogenous levels of protohaem is by breakdown and it is this mechanism which is under light control. Haem considerably decreased the incorporation of radioactivity from glycollate-[¹⁴C], glycine-[¹⁴C] and glutamate-[¹⁴C] into accumulated ALA in the presence of LA.     

Influence of Age and Light on the Distribution and Development of Nitrate Reductase in Greening Barley Leaves

The relation between leaf age and the induction of nitrate reductase activity by continuous and intermittent light was studied with barley seedlings (Hordeum vulgare L. cv. Club Mariout). In general, nitrate reductase activity declined as the period of growth in darkness was extended beyond 5 days. Maximum activity was found near the leaf tip while activity was lowest in the morphologically youngest tissue near the base of the lamina. Increased activity was observed after continuous illumination of dark-grown seedlings for 24 hours. The increase in activity in response to light was greatly reduced when the dark pretreatment period was extended beyond 8 days. The amount of nitrate reductase activity present in the different sections of the leaf was closely related to the amount of polyribosomes present. The pattern of chlorophyll accumulation closely parallelled that of increases in nitrate reductase activity. The initial lag in the induction of nitrate reductase activity was removed by a 10-minute light treatment 6 hours before placing dark-grown barley seedlings in light. The enzyme was also induced under flashing light with various dark intervals. These induction curves closely resembled those of chlorophyll accumulation under the same conditions. The development of photosynthetic CO₂ fixation follows the same induction pattern in this system. Our results suggest that photosynthetic products may be required for the induction of significant levels of nitrate reductase activity in leaves of dark-grown seedlings, although other light effects may not be discounted.     

Characterization and sequence of a novel nitrate reductase from barley

Summary Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.Scientific Paper No. 9101-14. College of Agriculture and Home Economics Research Center, Washington State University, Research Project Nos. 0233 and 0745     

Molecular biology, genetics and regulation of nitrite reduction in higher plants

Nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.6.6.1) carries out the six-electron reduction of nitrite to ammonium ions in the chloroplasts/plastids of higher plants. The complete or partial nucleotide sequences of a number of nitrite reductase apoprotein genes or cDNAs have been determined. Deduced amino acid sequence comparisons have identified conserved regions, one of which probably is involved in binding the sirohaem/4Fe4S centre and another in binding the electron donor, reduced ferredoxin. The nitrite reductase apoprotein is encoded by the nuclear DNA and is synthesised as a precursor carrying an N-terminal extension, the transit peptide, which acts to target the protein to, and within, the chloroplast/plastid. In those plants examined the number of nitrite reductase apoprotein genes per haploid genome ranges from one (barley, spinach) to four ( Nicotiana tabacum ). Mutants defective in the nitrite reductase apoprotein gene have been isolated in barley. During plastidogenesis in etiolated plants, synthesis of nitrite reductase is regulated by nitrate, light (phytochrome), and an uncharacterised 'plastidic factor' produced by functional chloroplasts. In leaves of green, white-light-grown plants up-regulation of nitrite reductase synthesis is achieved via nitrate and light and down-regulation by a nitrogenous end-product of nitrate assimilation, perhaps glutamine. A role for phytochrome has not been demonstrated in green, light-grown plants. Light regulation of nitrite reductase genes is related more closely to that of photosynthetic genes than to the nitrate reductase gene. In roots of green, white-light-grown plants nitrate alone is able to bring about synthesis of nitrite reductase, suggesting that the root may possess a mechanism that compensates for the light requirement seen in the leaf.     

Influence of nitrogen sources on chloroplast development in wheat seedlings

The effect of different nitrogen sources (ammonium, nitrate or both ions together) on plastid development in dark-grown and illuminated seedlings of wheat ( Triticum vulgare L. cv. Yecora) has been investigated. Plastids of plants grown in ammonium showed even in the dark a larger internal membrane length, higher ribulose bisphos-phate carboxylase activity and greater content of soluble proteins than plastids of plants grown in nitrate. After the first hour of illumination rudimentary thylakoids showing some joining points were observed in the ammonium plastids. After 10 h no prolamellar bodies were seen in the ammonium plastids, and the internal plastid membrane length was greater than in the other treatments. There was no light-induced increase in protein synthesis after illumination for 1 h. After 10 h the increase observed in protein synthesis was not followed by a response in the enzyme activity in any of the treatments. After 20 h the lag in the induction of ribulose bisphosphate carboxylase ceased, the enzyme activity and soluble proteins being higher in the leaves of ammonium seedlings than in those from nitrate. From the correlation obtained between the ultrastructural electron microscope observations and the enzymatic studies, it appears that ammonium nutrition has a positive influence on the formation of the plastid membrane system and on the onset of photosynthesis and, consequently, on the development of chloroplasts.     

Plastid development in primary leaves of Phaseolus vulgaris: The light-induced development of the chloroplast cytochromes

Summary Etioplasts obtained from the primary leaves of dark-grown bean plants contained cytochromes f, b-559_LP and b-563 in a molar ratio of approximately 1.0:2.0:1.5. On illumination of the plants there was a lag of between 10 and 15 h before these cytochromes increased in amount, but after 48 h they had increased from 6- to 10-fold on a per plastid basis. The presence of cytochrome b-559_HP in the plastids was first detected after 15 h of illumination, which coincided with the commencement of grana formation and the onset of a number of photosynthetic reactions in the greening leaves. After 48 h of illumination the molar ratio for cytochromes f, b-559_HP, b-559_LP and b-563 was 1.0:1.2:2.8:2.6.Agranal chloroplasts formed by the exposure of dark-grown plants to intense light flashes contained high amounts of cytochromes f, b-559_LP and b-563 but cytochrome b-559_HP could not be detected.As the light-induced formation of cytochromes f, b-559_LP and b-563 was substantially inhibited by D-threo chloramphenicol, but not by the L-threo isomer, it seems likely that their formation was dependent on 70S ribosomes. Both chloramphenicol isomers gave plastids which lacked cytochrome b-559_HP.     

Rapid separation of the plastid,mitochondrial, and cytoplasmic fractions from intact leaf protoplasts of Avena

Purified intact protoplasts were isolated from etiolated and greening leaves of Avena sativa. They were ruptured by forcing them through a 20-m aperture nylon net and immediately thereafter fractionated into a pure pellet of plastids (well above 70% of total plastids), a layer of mitochondria only slightly contaminated by other cellular constituents (about 50% of total mitochondria), and a cytoplasmic supernatant. This was achieved within 60 s by an integrated method of homogenation of protoplasts and centrifugal filtration of the homogenate on a gradient of silicone oils, contained together with the nylon net in 450 l microtubes, and verified by comparing the levels of activity of specific markers within the three fractions obtained. With appropriate modifications to immediately quench metabolic reactions within the fractions, this method allows the determination of metabolite levels within plastids, mitochondria, and the cytoplasmic compartment of intact protoplasts. The applicability of this technique is demonstrated by the determination of ATP in the plastids, mitochondria, and the cytoplasm of protoplasts obtained from etiolated and greening primary leaves of Avena. The levels of ATP, corrected for contamination of the fractions by each other, exhibit a pronounced transient increase during greening, especially within the cytoplasm.Abbreviations BSA bovine serum albumin - Cyt c cytochrome c - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - MES 2(N-morpholino) ethane sulphonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenol pyruvic acid - RuBP ribulose-1.5-bis-phosphate