The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves

Application of levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [¹⁴C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of ¹⁴CO₂ evolution from labeled precursors, including amino and organic acids. Inhibition of ¹⁴CO₂ evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-¹⁴C]ALA or [1-¹⁴C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of ³²Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.     

The consequences of delayed greening during leaf development for light absorption and light use efficiency

Many species of rainforest plants have an unusual form of leaf development such that leaves delay greening until after full leaf expansion. Chlorophyll accumulation was measured during leaf development in five woody rainforest species, three with white young leaves, and two with ‘normal’ greening. In the three species with white leaves, the chlorophyll content of the expanding leaves was about 0.4mg dm^?2, whereas in the two species with green young leaves, chlorophyll content was about 2.1 mg dm^?2. Chlorophyll accumulation in greenhouse and field experiments was independent of light level. During leaf expansion, species with delayed chloroplast development only absorb 18–25% of the maximum possible light, compared with 80% for species with normal greening. Furthermore, species with delayed greening have low chlorophyll contents and reduced absorption for at least 30 d after full expansion. At a PPFD typical of the forest under story, the photosynthetic light use efficiency based upon incident radiation was 0.030–0.036 for species with delayed chloroplast development and 0.068–0.085 for the two species with normal greening. The lower light use efficiency of white species was primarily due to decreased light absorption. However, they also had a slightly lower light use efficiency based upon absorbed radiation, suggesting that development of other components of the photo-synthetic apparatus also may be delayed. Despite the fact that delayed greening decreases light absorption and light use efficiency during leaf development, it is extremely common in shade-tolerant species. We suggest that an advantage of delayed greening is that resources are not invested in the leaf until it is fully expanded and better defended from herbivores.     

Lipid Composition of Pea and Bean Leaves during Chloroplast Development

The changes in composition of the complex lipids were followed during the greening of dark-grown pea (Pisum sativum) and bean (Phaseolus vulgaris) seedlings. No significant changes in glycerolipid concentrations in the leaves were observed during the early stages of greening (0-8 hour for peas and 0-12 hour for beans). On further greening, there was an increase in the proportion of galactolipids and a decrease in the phospholipids. The fatty acid composition of the galactolipids remained constant during 24 hours of greening, but there was a slight increase in α-linolenic acid at 72 hours in the bean. The percentage of α-linolenic acid in the phospholipids and in sulfolipid showed a marked increase between 24 and 72 hours in the bean. Trans-δ³-hexadecenoic acid was the major fatty acid of phosphatidyl glycerol in bean leaves at 72 hours, but it was barely detectable at 24 hours. The lipid composition of greening leaves is discussed in relation to the fine structure and photochemical activity of the developing plastids.     

The biosynthesis of δ-aminolevulinic acid in greening maize leaves

In greening maize leaves δ-aminolevulinic acid (ALA) was not formed from succinyl-CoA and glycine as shown by the incorporation of [¹⁴C]-labeled     

Light-induced fluorescence decay during the greening of normal and lincomycin-treated maize leaves

Light-induced fluorescence decay was examined during the greening of control and lincomycintreated maize (Zea mays L.) leaves. Assuming that this decay to a first approximation is the result of two parallel first-order reactions, the fluorescence induction curves were linearized on the logarithm plot and the parameters were determined. The variable fluorescence increased, and the parameters of the two linear sections of the fluorescence decay—that is, the kinetics of the induction curves—changed during the greening of the control leaves. Lincomycin treatment caused some chlorophyll deficiency and the lowering of the chlorophyll a/b ratio, changed the fluorescence emission spectra and the effect of Mg²⁺ on the regulation of the excitation energy distribution. The structure of the thylakoids and the kinetics of the fluorescence decay were also changed in the treated leaves. The possible relationship between the change of the kinetics of the fluorescence decay and the change of spillover during greening and after lincomycin treatment is discussed.Abbreviations LHC light-harvesting complex - Chl chlorophyll - LM lincomycin - PS photosystem - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

The nature of formate metabolism in greening barley leaves

The metabolism of [³H]formate has been examined in etiolated and greening leaves of barley (Hordeum vulgare), dwarf bean (Phaseolus vulgarls), broad bean (Vicia faba) and corn (Zea mays). Tritium was extensively incorporated by primary leaves incubated for 20-min periods in light or dark. The organic acids and free amino acids were the principal products of formate metabolism but these and other products were more heavily labelled in green tissues. Time course experiments with barley leaves revealed a rapid labelling of serine, accompanied by increasing amounts of ³H in glycine and aspartate as the feeding period was extended. These amino acid products were formed throughout a 4-day greening period with an approximate doubling in total incorporation being due to large accumulations of tritiated glycine and aspartate. The involvement of tetrahydrofolate-dependent reactions in formate metabolism was indicated by inhibition of [¹⁴C] and [³H]formate incorporation by the folate antagonist, aminopterin. Labelling of glycine and serine was also strongly inhibited (up to 90%) when the leaves were incubated with increasing concentrations of isonicotinylhydrazide.     

Developmental changes in energy dissipation in etiolated wheat seedlings during the greening process

We studied the developmental changes in photosynthetic and respiration rates and thermal dissipation processes connected with chloroplasts and mitochondria activity in etiolated wheat (Triticum aestivum L., var. Irgina) seedlings during the greening process. Etioplasts gradually developed into mature chloroplasts under continuous light [190 μmol(photon) m^?2 s^?1] for 48 h in 5-day-dark-grown seedlings. The net photosynthetic rate of irradiated leaves became positive after 6 h of illumination and increased further. The first two hours of de-etiolation were characterized by low values of maximum (F_v/F_m) and actual photochemical efficiency of photosystem II (PSII) and by a coefficient of photochemical quenching in leaves. F_v/F_m reached 0.8 by the end of 24 h-light period. During greening, energy-dependent component of nonphotochemical quenching of chlorophyll fluorescence, violaxanthin cycle (VXC) operation, and lipoperoxidation activity changed in a similar way. Values of these parameters were the highest at the later phase of de-etiolation (4–12 h of illumination). The respiration rate increased significantly after 2 h of greening and it was the highest after 4–6 h of illumination. It was caused by an increase in alternative respiration (AP) capacity. The strong, positive linear correlation was revealed between AP capacity and heat production in greening tissues. These results indicated that VXC in chloroplasts and AP in mitochondria were intensified as energy-dissipating systems at the later stage of greening (after 4 h), when most of prolamellar bodies converted into thylakoids, and they showed the greatest activity until the photosynthetic machinery was almost completely developed.     

Light-induced de Novo Synthesis of Ribulose 1,5-Diphosphate Carboxylase in Greening Leaves of Barley

An antibody specific for ribulose 1,5-diphosphate carboxylase was used to isolate the enzyme from greening barley (Hordeum vulgare L.) leaves. The increase in enzymatic activity during greening was due to de novo synthesis of the enzyme. Increases in enzymatic activity were accompanied by corresponding increases in enzyme protein and by incorporation of radioactive leucine, all of which were inhibited by low concentrations of cycloheximide. ¹⁴C-Labeled amino acids were incorporated into the enzyme by covalent peptide bonding.     

Demographic consequences of chromatic leaf defence in tropical tree communities: do red young leaves increase growth and survival?

Background

Many tropical forest tree species delay greening their leaves until full expansion. This strategy is thought to provide newly flushing leaves with protection against damage by herbivores by keeping young leaves devoid of nutritive value. Because young leaves suffer the greatest predation from invertebrate herbivores, delayed greening could prevent costly tissue loss. Many species that delay greening also produce anthocyanin pigments in their new leaves, giving them a reddish tint. These anthocyanins may be fungicidal, protect leaves against UV damage or make leaves cryptic to herbivores blind to the red part of the spectrum.

Methods

A comprehensive survey was undertaken of seedlings, saplings and mature trees in two diverse tropical forests: a rain forest in western Amazonia (Yasuní National Park, Ecuador) and a deciduous forest in Central America (Barro Colorado Island, Panamá). A test was made of whether individuals and species with delayed greening or red-coloured young leaves showed lower mortality or higher relative growth rates than species that did not.

Key results

At both Yasuní and Barro Colorado Island, species with delayed greening or red young leaves comprised significant proportions of the seedling and tree communities. At both sites, significantly lower mortality was found in seedlings and trees with delayed greening and red-coloured young leaves. While there was little effect of leaf colour on the production of new leaves of seedlings, diameter relative growth rates of small trees were lower in species with delayed greening and red-coloured young leaves than in species with regular green leaves, and this effect remained when the trade-off between mortality and growth was accounted for.

Conclusions

Herbivores exert strong selection pressure on seedlings for the expression of defence traits. A delayed greening or red-coloured young leaf strategy in seedlings appears to be associated with higher survival for a given growth rate, and may thus influence the species composition of later life stages.     

Variations during leaf development of the relative amounts of two bean (Phaseolus vulgaris) chloroplast tRNAsPhe which differ in their minor nucleotide content

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNA^Phe species, namely tRNA^Phe1 and tRNA^Phe2. By sequence determination, we show that tRNA^Phe2 is identical to the previously sequenced tRNA^Phe1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAs^Phe vary during leaf development: in etiolated leaves the undermodified tRNA^Phe2 only represents 15% of total chloroplast tRNA^Phe, during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : II. Formation from Glutamate

Intact plastids from greening maize (Zea mays L.) leaves converted [¹⁴C]glutamate and [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg²⁺, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activity was obtained from plastids of leaves which had been illuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor.

The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation.

     

Plastid Development in Pisum sativum Leaves during Greening : I. A Comparison of Plastid Polypeptide Composition and in Organello Translation Characteristics

Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twentythree of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A⁺) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis.     

Mg-protoporphyrin-IX and delta-aminolevulinic acid synthesis from glutamate in isolated greening chloroplasts. Mg-protoporphyrin-IX synthesis

A crude plastid preparation from greening cucumber cotyledons was able to accumulate Mg-protoporphyrin-IX when incubated in the presence of glutamate. ¹⁴C from l-[U-¹⁴C]glutamate was incorporated into the porphyrin. The product was identified by its emission and excitation fluorescence spectra and by its chromatographic behavior on cellulose thin layers. The biosynthesis had a marked requirement for ATP and O₂. α,α′-Dipyridyl, a metal ion-chelating agent which had been shown to stimulate the synthesis of Mg porphyrins and phorbins in etiolated bean leaves and other whole tissues, stimulated likewise the in vitro synthesis of Mg-protoporphyrin-IX by isolated greening chloroplasts. Freezing and thawing, which destroy organelle integrity, abolished the ability of these isolated plastids to biosynthesize Mg-protoporphyrin-IX.     

Changes in the protein composition of cytoplasmic ribsomes during the greening of etiolated barley leaves

We examined changes in the protein composition of cytoplasmic ribosomes in etiolated barley leaves following illumination. Cytoplasmic ribosomes were isolated from greening barley leaves by sucrose density gradient centrifugation, and were analyzed by radical-free highly reducing polyacrylamide gel electrophoresis (RFHR-PAGE). Eighty-nine proteins were resolved from the ribosomal fraction; among them, 8 proteins changed their copy numbers depending on the stage of greening. We designated these as phase dependent ribosomal proteins (PD1–PD8). Two of the proteins (PD1 and 5) present in the ribosomes of etiolated leaves showed a decrease in level during greening. In contrast, the levels of 6 ribosomal proteins (PD2, 3, 4, 6, 7 and 8) increased as greening proceeded. N-terminal amino acid sequence of PD8 showed high homology to rat ribosomal protein L34. The ribosomal proteins that appeared after illumination were not found in any fraction of the etiolated leaves, suggesting that they were synthesized after the onset of illumination. Copy numbers of other ribosomal proteins did not change during greening.     

The development of photophosphorylation and photosynthesis in greening bean leaves

Photophosphorylation and oxygen evolution were measured in 8-day-old dark-grown bean leaves (Phaseolus vulgaris) after various times of greening in far red light and in white light. The sequence of development was the same for both greening regimes, but the processes were much more rapid in white light. The capacity for photophosphorylation, as assayed by the firefly luciferase assay, appeared after 12 hours in far red light. At this stage and for times up to 24 hours, photophosphorylation was not inhibited by 10⁻⁵m 3-(3,4-dichlorophenyl)-1,1-dimethylurea. At 24 hours, the capacity for oxygen evolution appeared and photophosphorylation became partially inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea at concentrations which inhibited oxygen evolution. In white light photophosphorylation appeared after 15 minutes, and oxygen evolution at one hour. Photophosphorylation became partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea when oxygen evolution appeared. Carbonylcyanide m-chlorophenyl-hydrazone inhibited photophosphorylation and photosynthesis at low concentrations, 10⁻⁵m, with immature leaves, but the leaves developed resistance to carbonylcyanide m-chlorophenyl-hydrazone as they greened.     

Control of chlorophyll production in rapidly greening bean leaves

The possible involvement of nucleic acid and protein synthesis in light-regulated chlorophyll formation by rapidly greening leaves has been studied.

Removing leaves from illumination during the phase of rapid greening results in a reduction in the rate of pigment synthesis; cessation occurs within 2 to 4 hours. Etiolated leaves which exhibit a lag in pigment synthesis when first placed in the light do not show another lag after a 4 hour interruption of illumination during the phase of rapid greening.

Actinomycin D, chloramphenicol, and puromycin inhibit chlorophyll synthesis when applied before or during the phase of rapid greening. Application of δ-amino-levulinic acid partially relieves the inhibition by chloramphenicol.

It is suggested that light regulates chlorophyll synthesis by controlling the availability of δ-aminolevulinic acid, possibly by mediating the formation of an enzyme of δ-aminolevulinate synthesis. This process may result from gene activation or derepression; the involvement of RNA synthesis of some sort is suggested by the inhibitory effect of actinomycin D on chlorophyll production by rapidly greening leaves.

     

Delayed greening,leaf expansion,and damage to sympatric<Emphasis Type="Italic"> Shorea</Emphasis> species in a lowland rain forest

The function of delayed greening in the seedlings of canopy tree species in a lowland tropical rain forest was examined in terms of its potential defensive value against herbivory. To explore the ecological and evolutionary backgrounds for delayed greening, we chose eight sympatric congeneric (Shorea) dipterocarp species that were either normal-greening or delayed-greening species. Expansion and toughening of leaves took approximately 30 days for all species, and did not differ between the normal- and delayed-greening species. The main factors that affected leaf damage during expansion were insect herbivory and fungal infection. Levels of leaf damage were significantly lower for delayed-greening species than for normal-greening species, but proportions of heavily damaged leaves and leaf abscission during expansion did not differ. In addition, no significant difference was found in damage levels on leaves (aged 1–2 months) of naturally occurring seedlings between normal- and delayed-greening species. Therefore, delayed greening may effectively reduce the level of leaf damage in young expanding leaves, but may not necessarily reduce leaf abscission and damage to mature leaves. The existence of delayed greening could not be simply explained by the phylogenetic and ecological backgrounds of the trees. Consequently, delayed greening may have a function in reducing damage during expansion, but more information (such as knowledge of the secondary metabolites involved in this phenomenon) is needed to explain fully why these species exhibit delayed greening.     

Relative Contribution of Ribulose 1,5-Diphosphate Carboxylase and Phosphoenolpyruvate Carboxylase to CO2 Fixation Activity in Roots of Dark-grown or Light-grown Lens culinaris Seedlings

The pathway of carbon assimilation in greening roots was compared to the pathway in leaves of Lens culinaris seedlings by means of labelling distribution analysis among the products of ¹⁴CO₂ fixation in vivo, and in vitro with ribulose 1,5-diphosphate as the substrate. In green leaves, CO₂ fixation via ribulose 1,5-diphosphate carboxylase predominated largely while, in green roots, this carboxylase activity and the phosphoenolpyruvate carboxylase contributed almost equally to the whole in vivo CO₂ fixation. A participation of the activities of both carboxylases according to the double carboxylation pathway in the synthesis of dicarboxylic acids (malate and aspartate) was demonstrated in vitro after 48 h of greening in roots but seemed to be absent in in vivo experiments.     

The Biosynthesis of delta-Aminolevulinic Acid in Higher Plants: II. Formation of C-delta-Aminolevulinic Acid from Labeled Precursors in Greening Plant Tissues

δ-Aminolevulinic acid was accumulated by greening cucumber (Cucumis sativus L. var. Alpha green) cotyledons, barley (Hordeum sativum var. Numar) leaves, and bean (Phaseolus vulgaris L. var. Red Kidney) leaves in the presence of various ¹⁴C-labeled precursors and levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase. The radioactivity in the accumulated δ-aminolevulinic acid was measured.     

Discrete character of the development of the photosynthetic apparatus in greening barley leaves

Biogenesis of the photosynthetic apparatus in greening etiolated leaves of barley (Hordeum vulgare L) was investigated by an approach permitting investigation of this process under conditions that minimize differences in plastid development. Distributions of barley leaves greening for 24 h as to chlorophyll content and of chloroplast grana as to number of thylakoids were shown to be of a multimodal character. The shape of time-course curves of chlorophyll accumulation in local sites of greening etiolated leaves was of a stepped or (at the end of greening) undulated character. The stepwise accumulation of chlorophyll was accompanied by wave-like changes in chlorophyll b/a ratio, intensity of low-temperature chlorophyll fluorescence and photosynthetic activity with minima at the time points of transition to accelerated chlorophyll accumulation. It is assumed that (1) development of the photosynthetic apparatus in local sites of greening etiolated leaves occurs stepwise, from one steady level to another, but not as gradually as is generally accepted, and (2) every separate step in development of the photosynthetic apparatus seems to begin with formation of photosystem cores and to end with the synthesis of light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.