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.     

Involvement of nitric oxide in light-mediated greening of barley seedlings

When seedlings are grown in the dark, proplastids of the developing leaf differentiate into etioplasts. Greening of etiolated plastids is stimulated by light, which is sensed by various types of photoreceptors. Nitric oxide (NO) has been shown to be a bioactive molecule that could take part in this light-mediated process in plants. In this paper, we show that emission of NO in barley seedlings increased concomitantly with increasing activities of nitric oxide synthase (NOS) during the greening. Treatment with sodium nitroprusside (SNP), a NO donor, increased the accumulation of chlorophyll contents, enhanced the accumulation of thylakoid membrane proteins, such as light harvesting complex of photosystem II (LHCII) and PSIA/B, and then improved the effective quantum yield of photosystem II (PSII) (Phi(PSII)) in the light. Instead, treatment with either NO scavenger 2-phenyl-4,4,5,5-tetramentylimidazoline-1-oxyl-3-xide (PTIO) or NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) retarded the greening of etiolated-seedlings. Moreover, sodium ferrocyanide, an analog of SNP, nitrite and nitrate, two NO-decomposition products did not have any effect on the greening process. These results indicated that NO, as an endogenous signaling molecule, participates in light-mediated greening of barley seedlings, and exogenous NO accelerates this process.     

Chlorophyll-protein complex composition and photochemical activity in developing chloroplasts from greening barley seedlings

The time course for the observation of intact chlorophyll-protein (CP) complexes during barley chloroplast development was measured by mild sodium dodecyl sulfate polyacrylamide gel electrophoresis. The procedure required extraction of thylakoid membranes with sodium bromide to remove extrinsic proteins. During the early stages of greening, the proteins extracted with sodium bromide included polypeptides from the cell nucleus that associate with developing thylakoid membranes during isolation and interfere with the separation of CP complexes by electrophoresis. Photosystem I CP complexes were observed before the photosystem II and light-harvesting CP complexes during the initial stages of barley chloroplast development. Photosystem I activity was observed before the photosystem I CP complex was detected whereas photosystem II activity coincided with the appearance of the CP complex associated with photosystem II. Throughout chloroplast development, the percentage of the total chlorophyll associated with photosystem I remained constant whereas the amount of chlorophyll associated with photosystem II and the light-harvesting complex increased. The CP composition of thylakoid membranes from the early stages of greening was difficult to quantitate because a large amount of chlorophyll was released from the CP complexes during detergent extraction. As chloroplast development proceeded, a decrease was observed in the amount of chlorophyll released from the CP complexes by detergent action. The decrease suggested that the CP complexes were stabilized during the later stages of development.Abbreviations Chl chlorophyll - CP chlorophyll-protein - CPI P700 chlorophyll-a protein complex of photosystem I - CPa electrophoretic band that contains the photosystem II reaction center complexes and a variable amount of the photosystem I light-harvesting complex - CP A/B the major light-harvesting complex associated with photosystem II - DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPC diphenyl carbazide - MV methyl viologen - PAR photosynthetically active radiation - PSI photosystem I - PSII photosystem II - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TEMED N,N,N,N-tetramethylethylenediamine - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 9949 of the Journal Series of the North Carolina Agricultural Research Service, Raleight, NC 27695-7601.     

The effect of copper on chlorophyll organization during greening of barley leaves

The effect of copper on chlorophyll organization and function during greening of barley was examined, using chlorophyll fluorescence and photoacoustic techniques. Copper was found to inhibit pigment accumulation and to retard chlorophyll integration into the photosystems, as evident from low temperature (77 K) fluorescence spectra. Resolution of the minimal fluorescence (F₀) into active and inactive parts, indicated a higher inactive fraction with copper treatment. This was attributed to chlorophyll molecules which failed to integrate normally, a conclusion supported by the longer fluorescence lifetime observed in copper treated plants. A lower ratio of chlorophyll a to b and fluorescence induction transients, showing accelerated Photosystem II closure, both indicate that copper treatment resulted in a larger light-harvesting antenna. Another effect of copper treatment was the suppression of oxygen evolution, indicating a decrease in photosynthetic capacity. We suggest that the non-integrated chlorophyll fraction sensitizes photodamage in the membrane, contributing to disruption of electron flow and pigment accumulation.     

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.     

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.     

Excitation kinetics of chlorophyll fluorescence during light-induced greening and establishment of photosynthetic activity of barley seedlings

Excitation kinetics based on feedback regulation of chlorophyll (Chl) fluorescence of leaves measured with the chlorophyll fluorometer, FluoroMeter Modul (FMM), are presented. These kinetics showed the variation of excitation light (laser power, LP) regulated by the feedback mechanism of the FMM, an intelligent Chl fluorometer with embedded computer, which maintains the fluorescence response constant during the 300-s transient between the dark- and lightadapted state of photosynthesis. The excitation kinetics exhibited a rise of LP with different time constants and fluctuations leading to a type of steady state. The variation of excitation kinetics were demonstrated using the example of primary leaves of etiolated barley seedlings (Hordeum vulgare L. cv. Barke) during 48 h of greening in the light with gradual accumulation of Chl and development of photosynthetic activity. The excitation kinetics showed a fast rise followed by a short plateau at ca. 30 s and finally a slow constant increase up to 300 s. Only in the case of 2 h of greening in the light, the curve reached a stable steady state after 75 s followed by a slight decline. The final LP value (at 300 s of illumination) increased up to 12 h of greening and decreased with longer greening times. The active feedback mechanism of the FMM adjusted the excitation light during the measurement to the actual photosynthetic capacity of the individual leaf sample. In this way, the illumination with excessive light was avoided. The novel excitation kinetics can be used to characterize health, stress, disease, and/or product quality of plant material.     

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     

Proteomic characterization of the greening process in rice seedlings using the MS spectral intensity-based label free method

Illumination-induced greening in dark-grown plants is one of the most dramatic developmental processes known in plants. In our current study, we characterized the greening process of rice seedlings using comparative proteome analysis. We identified 886 different proteins in both whole cell lysates of illuminated and nonilluminated rice shoots and performed comparative proteome analysis based on the MS spectral intensities obtained for unique peptides from respective proteins. Furthermore, the changes in the levels of individual proteins were then compared with those of the corresponding mRNAs. The results revealed well-coordinated increases in the enzymes involved in the Calvin cycle at both the protein and mRNA levels during greening, and that the changes at the mRNA level precede those at the protein level. Although a much lower effect of illumination was found on the enzymes associated with glycolysis and the TCA cycle, coordinated increases during greening were evident for the enzymes involved in photorespiration and nitrogen assimilation as well as the components of the chloroplastic translational machinery. These results thus define the differential regulation of distinct biological systems during greening in rice and demonstrate the usefulness of comprehensive and comparative proteome analysis for the characterization of biological processes in plant cells.     

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     

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.     

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.     

Gibberellic Acid effects on greening in pea seedlings

The effect of gibberellic acid (GA) on light-induced greening of etiolated pea plants (Pisum sativum [L.] cultivars Alaska and Progress) was characterized. Progress, a GA-deficient dwarf of Alaska, was found to accumulate chlorophyll and light harvesting chlorophyll protein associated with photosystem II (LHC-II) more rapidly than Alaska, Alaska treated with GA, or Progress treated with GA. A slightly lower chlorophyll content was noted after 24 hours of light induced greening for Alaska treated with GA relative to untreated Alaska. GA-treated Progress, Alaska, and GA-treated Alaska all gave essentially identical patterns for LHC-II accumulation. Similar patterns of LHC-II mRNA induction were found in all four treatments indicating that differences in mRNA induction did not cause differences in LHC-II accumulation. Chlorophyll and LHC-II accumulation in each treatment followed the same patterns of accumulation and a significant correlation (at the 0.01 level of significance) was found between chlorophyll and LHC-II content. Since Progress treated with GA accumulated LHC-II and chlorophyll in a manner similar to that of Alaska, it is clear that GA alters the process of greening either directly or indirectly.     

Protoheme turnover and chlorophyll synthesis in greening barley tissue

Studies in which ¹⁴C-labeled precursors were fed to etiolated barley leaves (Hordeum vulgare L. var. Proctor) yielded chlorophyll and protoheme having similar specific radioactivities. These findings indicate: (a) there appears to be a rapid turnover of protoheme in the absence of net synthesis; (b) both pigments probably originate from a single 5-aminolevulinic acid pool; (c) the efficient utilization of glutamate-1-¹⁴C and the relatively poor utilization of glycine-2-¹⁴C suggest that 5-aminolevulinic acid is probably synthesized by a pathway other than 5-aminolevulinic acid synthetase (succinyl CoA-glycine succinyltransferase) in agreement with previously published work; (d) protoheme turnover appears to be faster under conditions which allow for rapid chlorophyll accumulation; (e) difference spectra indicate that mitochondrial cytochromes make a relatively minor contribution to the total heme in barley leaves. These findings are discussed in the light of current knowledge about tetrapyrrole regulation in photosynthetic organisms.     

The greening process in plastids

Plastids in etiolatedAvena leaves were studied by electron microscopy of thin sectioned material fixed in glutaraldehyde and osmium tetroxide and embedded in Epon. Each plastid contains one—three prolamellar bodies. These are highly ordered systems, the membraneous component of which consists of interconnected tubules lying in the three major axes of a cubic lattice. Where three tubules (one in each axis of the lattice) meet and fuse at the corners of each unit cell, their unit membranes are smoothly confluent so that the principal curvatures of the membrane surface are of opposite sign at every point. A face view of a unit cell shows four tubules delimiting a circular opening of diameter 380 Å. The diameter of the tubules is 210 Å at their narrowest point, i. e. half way along the edges of the unit cells. The plastid stroma penetrates the prolamellar body via the 380 Å openings, and contributes ribosome—like particles to the system. These particles are centrally located, one in each unit cell. The literature on prolamellar bodies is reviewed, it is concluded that this type of organisation is widespread in plants. Structures with similar geometry are described, and it is suggested that the unit membranes of the lattice are laid down on contours of uniform “field” strength centred on the lattice of ribosome-like particles. The surface area of membrane in a prolamellar body is estimated.     

Accumulation of hydrogen peroxide and functioning of defense system in overwatered barley seedlings

The effects of overwatering (flooding) on the oxidative potential, the level of low-molecular-weight antioxidants, the content of stress proteins, and activities of antioxidant enzymes in green barley (Hordeum vulgare L.) seedlings were studied. Overwatering retarded barley seedling growth and induced hydrogen peroxide accumulation, a decrease in the total ascorbate content and an increase in the content of reduced glutathione (GSH), but it did not affect the content of oxidized glutathione (GSSG). After the cessation of stress factor action (post-stress period), the content of hydrogen peroxide declined to the initial level, the content of ascorbate reduced still stronger, whereas the content of GSH continued to rise. Under flooding conditions, activities of glutathione reductase (GR) and superoxide dismutase (SOD) increased. After the cessation of stress factor action, activities of these enzymes decreased but remained at rather high levels as compared with control. Activity of catalase (CAT) reduced during stress, whereas activity of ascorbate peroxidase (APX) was not essentially changed. In the post-stress period, CAT activity remained to be low; in contrast, APX activity increased. Barley seedling flooding induced the synthesis of stress proteins, HSP70 and dehydrins (DH). In the post-stress period, the content of stress proteins decreased; however, the content of DH in experimental leaves remained rather high. The results obtained indicate that barley defense system manifested a complex response to overwatering, which may be related to the oxygen shortage under stress conditions and sharp metabolism activation at re-aeration in the post-stress period.     

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.