Haem and chlorophyll formation in etiolated and greening leaves of barley

The amounts of protochlorophyllide (P650) and protohaem were measured in ageing dark-grown barley leaves. Maximum amounts of P650 and protohaem were found in 6- to 8-day-old material after which P650 declined rapidly and protohaem more slowly. In leaves exposed to light maximum chlorophyll was produced in 6-day-old material with progressively less the older the leaves. Haem concentrations increased in seedlings of all ages exposed to light. A lag phase was observed for both chlorophyll and haem formation in leaves given a light treatment. Haem, however, showed a slight yet sig nificant decline as chlorophyll production commenced. The results indicate that chlorophyll and haem synthesis share a common pool of δ-aminolae vulinic acid (ALA). At a certain stage of development, the magnesium porphyrin pathway diverts precursors away from haem synthesis. It is only when the ALA synthesising system is well developed that the production of ALA can satisfy pathways to both haem and chlorophyll. The observed changes in haem under certain conditions suggest that, as in animal systems, haem levels may regulate porphyrin formation (chlorophylls) by controlling the supply of ALA.     

Cytochromes of developing plastids of greening barley: Effects of inhibitors of haem synthesis

The properties of the cytochromes of etiolated barley plastids and 20 hr greened barley plastids were examined by potentiometric titration. In etiolate     

Influence of cesium on tetrapyrrole biosynthesis in etiolated and greening barley leaves

Cesium chloride (CsCl) treatment of greening primary leaves of barley for 8 h inhibited chlorophyl] accumulation in a concentration-dependent manner and led to the accumulation of excessive amounts of uroporphyrin(ogen) III (URO[gen]) and to a minor extent of heptacarboxylporphyrin(ogen). When dark-grown leaves were incubated with CsCl, accumulation of URO(gen) was observed only after feeding of the tetrapyrrole precursor 5-aminolevulinic acid. Western blot analysis showed no apparent difference in content of uroporphyrinogen decarboxylase (EC 4.1.1.37, UROD) or selected proteins involved in tetrapyrrole biosynthesis in extracts of CsCl-incubated (15 m M ) versus control leaves. UROD activity was drastically decreased upon CsCl treatment in leaves incubated in the dark or in the light (44 and 86%, respectively). Selected preceding enzymes of the tetrapyrrole biosynthetic pathway, 5-aminolevulinic acid dehydratase (EC 4.2.1.24, ALAD) and porphobilinogen deaminase (EC 4.3.1.8, PBGD), were influenced only to a minor extent under standard incubation conditions (15 m M CsCl). Furthermore, the ALA synthesizing capacity did not differ in leaves incubated with and without Cs⁻ cations. UROD activity of crude homogenates from control plants and after partial purification was reduced to 56 and 80%, respectively, upon addition of 10 m M CsCl. Equal concentrations of KCl were not inhibitory. Enzyme assays of the same barley extract in the presence of CsCl yielded no effect on ALAD and a minor loss of PBGD activity. The initial visible cytotoxic effect of CsCl appeared to be a selective inhibition of UROD resulting in accumulation of photosensitizing URO (gen). Consequences of the diminished UROD activity on early steps of the tetrapyrrole biosynthesis and its functional and regulatory significance for the porphyrin synthesis are discussed.     

The biosynthesis of coumarylagmatine in barley seedlings

An enzyme present in extracts of the shoots of barley seedlings has been shown to synthesize coumarylagmatine from p-coumaryl-coenzyme A and [U-¹⁴C]agmatine.     

Agmatine coumaroyltransferase from barley seedlings

4-Coumaroyl-CoA: agmatine N-4-coumaroyltransferase (agmatine coumaroyltransferase; ACT) from barley seedlings was purified with a 100-fold increase     

Relationships between glycollate and formate metabolism in greening barley leaves

The activities of enzymes catalysing glycollate oxidation, formate production and folate-dependent formate utilization were examined in the primary leaves of Hordeum vulgare cv Galt. Seedlings were grown for 6 days in darkness and then transferred to continuous light (500 μinsteins/m² per sec) for up to 5 days. Cell-free extracts of the primary leaves contained glycollate oxidase (EC 1.1.3.1), 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5, 10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) and ability to enzymically decarboxylate glyoxylate. These activities increased during greening and at the end of the light treatment were 70–450% higher than etiolated controls. Greened primary leaves also incorporated [¹⁴C]formate at rates that were three- to four-fold higher than shown by etiolated leaves. The specific activity of 10-formyltetrahydrofolate synthetase was decreased by 20–35% when the leaves were greened in the presence of 10 mM hydroxysulphonate. This inhibitor also reduced the incorporation of [¹⁴C]formate by up to 45%. A potential flow of carbon from glycollate to 10-formyltetrahydrofolate via glyoxylate and formate was suggested by the data.     

Proline biosynthesis in a proline-accumulating barley mutant

A barley (Hordeum vulgare L.) mutant, R5201, selected for resistance to 4? mM trans-4-hydroxyproline had a 3–6 fold increase in the soluble proline content of the leaf compared with the parent cultivar, Maris Mink. The mutant converted more [U-⁴C]glutamic acid to free proline in the leaves than Maris Mink but incorporation into protein proline was similar. Incorporation of radioactivity into proline was inhibited by exogenous proline more in Maris Mink than R5201, suggesting that feedback inhibition of proline biosynthesis is relaxed, but not absent in the mutant. When [1-¹⁴C]ornithine was the precursor, both R5201 and Maris Mink incorporated similar small amounts of label into soluble and protein proline. More protein proline was formed by both genotypes from labelled glutamic acid than from labelled ornithine. There may exist two routes of proline formation, where the glutamate pathway is synthetic and the ornithine pathway is catabolic.     

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.     

Carnitine content of greening Barley leaves

The carnitine content of etiolated barley leaves doubled on exposure to light. After 24 hr exposure the carnitine content was the same as that of barle     

Lysophosphatidylcholine biosynthesis in developing barley

Acetate-2-[¹⁴C] and choline-Me-[ ¹⁴C], absorbed through the stems of isolated barley heads, were used to label lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) of the endosperm tissue. Labelling of LPC occurred in barley heads at almost all stages of development but was at a maximum when the fr. wt of the seeds had attained ca 60–70% of their maximum wt. In time-course experiments labelling of PC from each substrate reached a maximum after 50 hr and then declined. Label in LPC, however, continued to accumulate throughout 72 hr. Stimulation of labelling of LPC from choline-Me-[¹⁴C] by sucrose was observed. A bound form of LPC (starch lipid) and a free form were distinguished by differential solvent extraction.     

Polyamines in barley seedlings

Polyamine levels in barley seedlings grown in the dark or in diurnal illumination have been determined, by direct dansylation, 3, 6 and 12 days after g     

Effect of carnitine on greening barley leaves

Carnitine increases chlorophyll production in greening barley leaves. [Methyl-¹⁴C]carnitine fed to greening leaves was not utilized as a carbon sou     

Glycine metabolism in etiolated barley leaves on exposure to light

Of a large number of amino acids examined, changes in glycine were the only ones which were correlated with the ability of dark-grown barley leaves to synthesise protochlorophyllide, δ-aminolaevulinic acid and chlorophyll on exposure to light. A rapid depletion was found in endogenous glycine in barley leaves after day 7. Illumination of the leaves increased the rate of glycine depletion. Glycine concentrations were high throughout the young leaf. The top and middle leaf sections however, which had maximal chlorophyll synthesising potential exhibited the most pronounced decrease in glycine as the leaf aged. Using glycine-[¹⁴C] pulse techniques the half life of glycine in 7 and 14-day-old dark-grown leaves was 3.5 and 4.4 min respectively. Light treatment lengthened the half life to 6.9 and 12.1 min in 7 day and 14-day-old-leaves. Sustained illumination continued to decrease glycine turnover.     

Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings

Intact etiolated bean (Phaseolus vulgaris L. cv. Limburgse vroege) seedlings were illuminated with red light (10.5 W·m^-2) for 10 min. After different time intervals ethylene production, and contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1-carboxylic acid were measured. The red-light-induced decrease of ethylene production in 8-d-old intact etiolated bean seedlings was fast, strong and long-lasting ad was mediated through the phytochrome system. This effect appeared to be strictly age-dependent, as it could not be detected in plants younger than 6 d or older than 11 d.The capacity for the conversion of ACC to ethylene was not affected by red light. The inhibitory effect of the light treatment on ethylene production could be related to a reduced free-ACC content. This reduction was a consequence of a temporary non-reversible increase of ACC malonylation and a long-lasting, for a certain time reversible, inhibition of ACC synthesis. The effect of a brief irradiation with red light on the decrease of ethylene production and free-ACC content was completed after about 2 h. Reversibility by far-red, however, persisted for at least 3 h, and was lost between 3 and 6 h.Abbrevation ACC 1-aminocyclopropane-1-carboxylic acid - M-ACC 1-(malonylamino)cyclopropane-1-carboxylic acid     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Oxygen incorporation in the biosynthesis of p-coumaric acid in seedlings of Zea mays

The results of experiments in which seedlings of Zea mays were grown in the light in an atmosphere enriched with oxygen-18 indicated that the hydroxyl oxygen atom in p-coumaric acid is derived from molecular oxygen.     

Metabolism of protein,peptides and aminoacids in ageing etiolated barley leaves

Both light- and dark-grown primary leaves of barley show a reduction in protein after about 7 days. With this reduction in protein there is a rise in t     

Succinyl-CoA synthetase in greening maize leaves

In extracts of greening maize leaves succinyl-CoA synthetase was present in both a particulate and a soluble fraction. Aqueous and non-aqueous fractionation together with determination of chlorophyll content and cytochrome oxidase activity indicated that the enzyme was neither located, nor originated in plastids. Pre-illumination of leaves caused only small increases in the activity of either the particulate or the soluble enzyme. The soluble enzyme was ATP specific and had a low affinity for succinate (Km = 63 mM).     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

Amitrole treatment of etiolated barley seedlings leads to deregulation of tetrapyrrole synthesis and to reduced expression of Lhc and RbcS genes

The effect of amitrole, known as an inhibitor of carotenoid biosynthesis, upon tetrapyrrole biosynthesis and its regulation has been studied. Etiolated barley (Hordeum vulgare L.) seedlings, grown in 125 μM amitrole, accumulated high levels of 5-aminolevulinate, Mg-protoporphyrin, Mg-protoporphyrin monomethyl ester, and protochlorophyllide. The amitrole-treated seedlings did not form paracrystalline prolamellar bodies, and the induction of Lhc and RbcS gene expression was reduced by non-photooxidative, low-intensity light. None of these events was observed upon treatment of the seedlings with 100 μM norflurazon, another inhibitor of carotenoid biosynthesis. The effect of amitrole cannot be explained solely by interaction with a presumed feedback inhibition of 5-aminolevulinate synthesis since incubation with amitrole and 5-aminolevulinate indicated that deregulation also occurs at later steps of tetrapyrrole biosynthesis. A possible relationship between this deregulation and ultrastructural changes is discussed. In connection with previously published data, we discuss Mg-protoporphyrin and its monomethyl ester as possible candidates for a “plastid signal” that operates as a negative factor, reducing the expression of Lhc and RbcS genes in this higher plant. Received: 27 June 2000 / Accepted: 12 October 2000