Carotenoid Synthesis in Leaves of Etiolated Wheat Seedlings after Varying Light and Dark Treatments

A short impulse of red light has a varying effect on carotenoid synthesis in dark-grown wheat seedlings. Except for β-carotene, which remains unchanged in the dark (it increases in continuous light), the carotenoid synthesis shows the same tendency as during constant irradiation. Thus, lutein and neoxanthin slowly increases, while violaxanthin decreases. During a period of constant light following various periods of darkness after the short impulse of light, the pigment changes correspond to those occurring in the dark, but are much more pronounced. The changes are discussed on the basis of phytochrome action.     

Changes of Sucrose-Phosphate Synthase Activity in Barley Primary Leaves during Light/Dark Transitions

The activity of sucrose-phosphate synthase (SPS) in 9-day-old barley (Hordeum vulgare L.) primary leaves was measured over a 24-hour period. Extractable enzyme activity was constant in the light, decreased 50 to 60% during the first one-half hour of darkness, and then returned to full activity before the start of the normal light period. Decreases of SPS activity in the dark were fully reversed by less than 10 minutes of illumination. In contrast to results with barley, the measurable activity of SPS in soybean, spinach, and pea leaves was unchanged during the first hour of darkness. Changes of SPS activity in barley primary leaves were stable upon gel filtration. The exact biochemical mechanism responsible for the enzyme activity changes in barley leaf extracts is unknown. The above findings support the suggestion by de Fekete (1973 Eur J Biochem, 10: 73-80) that SPS is controlled by posttranslational protein modification. These results are discussed in relation to the regulation of photosynthetic sucrose metabolism.     

Characterization and Properties of a Protochlorophyllide Ester in Leaves of Dark Grown Barley with Geranylgeraniol as Esterifying Alcohol

The protochlorophyllide ester isolated from dark grown barley leaves was shown to contain geranylgeraniol as esterifying alcohol. No phytylester was found. The qualitative analyses were performed with combined gas chromatography-mass spec-trometry. Chromatographic separation and spectrofluorometric determination of the protochlorophyll and chlorophyll pigments before and after irradiation of the dark grown leaves with light flashes at 2°C showed that part of the protochlorophyllide ester was photoconverted to chlorophyll a.     

The Influence of Varying Light Intensities on the Photo-transformation of Protochlorophyllide636 in Dark Grown Wheat Leaves Treated with δ-Aminolevulinic Acid

Leaves treated with δ-aminoievulinic acid accumulate protochlorophyllide₆₃₆ in large amounts. Due to a continuous conversion of protochlorophyllide₆₃₆ (nonphototransformable) into protochlorophyllide₆₅₀ (phototransformable) in weak red light, the photoreduction of protochlorophyllide to chlorophyllide can proceed for at least 20 minutes and results in a chlorophyllide content of the leaves three times higher than that in untreated leaves. The half time for this chlorophyllide accumulation is 55 seconds. A photodestruction of the pigments takes place at high light intensities or if the content of protochlorophyllide₆₃₆ is high. The conversion of protochlorophyllide₆₃₆ to chlorophyllide is dependent on the light intensity used for phototransformation of protochlorophyllide₅₅₀ The conversion of PChlide₆₄₆ was not limiting for chlorophyllide formation within the range of the light intensity used. The extrapolation of a double reciprocal plot of chlorophyllide formation, rate versus light intensity gives a maximal value of 8.7 μg chlorophyllide per g fresh weight and min. The conversion of protochlorophyllide₃₆₃ to protochlorophyllide₆₅₀ is believed to depend on the available sites of an apophotoenzyme.     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The stability against high intensity irradiation (red light, 700 W m^?2) was investigated for the chlorophyll(ide) pigments formed after the primary photoreduction of the protochlorophyll(ide) in dark grown leaves of wheat. After photoreduction, most of the chlorophyll(ide) exists in a form with an absorption maximum at 684 nm. This form is gradually transformed into a form with an absorption maximum at 673 nm (the Shibata shift). It was possible to ascribe a specific photostability to each of the pigment forms. This photostability was higher for the 673-form than for the 684-form. A red-shift in the absorption maximum following upon the Shibata shift, reflects the successive transformation of the 673-form into other pigment forms, which were quite photostable at the intensity used.     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The effect of denaturing treatments on the stability against high intensity irradiation (red light, 700 W m^?2) was investigated in vivo for various chlorophyll forms in wheat. Three pigment forms were investigated: the 650-form (protochlorophyllide) present in dark grown leaves; the 684-form (chlorophyllide) formed within 5 s after photoreduction of the 650-form; and the 673-form (chlorophyll), into which the 684-form has been transformed 25 min after photoreduction of the 650-form. (The pigment forms are denoted by their absorption maxima in the red region before denaturation.) Two denaturing treatments were used: heat treatment (water of 55°C for 2 min) and freezing and thawing (freezing in liquid nitrogen followed by thawing in water of 25°C). Heat treatment as well as freezing and thawing caused a shift in the absorption peak of the two nonesterified pigment forms. The peak of of the chlorophyllide 684-form shifted to 673 nm and that of the protochlorophyllide 650-form to 636 nm. The absorption maximum of the chlorophyll 673-form was not affected by the above treatments. Heat treatment as well as freezing and thawing had profound effects on the structural organization of the plastid pigments, as shown by a decrease in the photostability. For the 684-form, heat treatment reduced the photostability by a factor of about 14 (half-life in strong light changed from 170 s to 12 s). Freezing and thawing also reduced the photostability, although the effect was less pronounced (c. 3–4 times decrease in half-life). Upon transformation of the chlorophyllide 684-form into the chlorophyll 673-form (the Shibata-shift) the pigments became less sensitive to light, and were no longer “aggregated” by heat treatment. The “aggregating” effect of freezing and thawing was still present after the Shibata shift. The results thus verify a clear difference in structural organization of the 684-form and the 673-form, since the two pigment forms were differently affected by heat treatment. The 650-form behaved similarly to the 684-form, although it appeared to be slightly less aggregated by heat treatment. — The decrease in photostability, caused by heat treatment of the 684-form, changed the kinetics for the photodecomposition from a first towards a second order reaction.     

Photosynthetic Characteristics of Spinach Leaves Grown with Different Nitrogen Treatments

Spinach plants (Spinacia oleracea L.) were grown hydroponicallywith different concentrations of nitrate nitrogen, ranging from0.5 to 12 mM, in a glasshouse under full sunlight. Using anopen gas exchange system, the rate of CO₂ assimilation, A, wasdetermined as a function of intercellular partial pressure ofCO₂, P_i, with a constant amount of absorbed light per unit Chl.When expressed on a leaf area basis, A measured at high irradianceand at p_i=500 µbar, was proportional to the in vitro rateof uncoupled whole-chain electron transport as well as to Chlcontent. There was a curvilinear relationship between the mesophyllconductance (the slope of the A : P_i curve near the CO₂ compensationpoint) and the in vitro RuBP carboxylase activity. The curvaturedid not appear to be due to enzyme inactivation in vivo in leaveswith high nitrogen contents. The curvature suggested the presenceof a CO₂ transfer resistance between the intercellular spacesand the site of carboxylation of 2.2 m² s bar mol^–1 CO₂,which is similar to that previously observed in wheat. Thisimplied that, while nitrogen deficiency increased the ratioof in vitro activity of electron transport to that of RuBP carboxylase,the two activities remained balanced in vivo. Irradiance response curves were determined by both net CO₂ andO₂ exchange. The two methods gave reasonable agreement at lightsaturation. The quantum yield measured by O₂ evolution was 0.090?0.003mol O₂ mol^–1 absorbed quanta, whereas after correctingfor p_i = 500µbar, the quantum yield for CO₂ assimilationwas only 82% of that measured by oxygen evolution. ²Present address: Plant Environmental Biology Group, ResearchSchool of Biological Sciences, The Australian National University,G.P.O. Box 475, Canberra, A.C.T. 2601, Australia. (Received July 29, 1987; Accepted November 2, 1987)     

Light-Dark Changes in Proline Content of Barley Leaves under Salt Stress

Proline accumulation in leaves of barley (Hordeum vulgare L. cv. Alfa) seedlings treated with 150 mM NaCl was promoted in the light and suppressed in the dark. The light/dark changes of proline content was enhanced with each 12 h light/12 h dark cycle and the proline content increased steadily. Root and shoot concentrations of Na⁺ and Cl^– in salt treated plants increased about 10 to 25 times as compared to the control. The content of these ions and the content of malondialdehyde were higher in the shoot of seedlings exposed to salt stress for 4 d in the light in comparison with the seedlings exposed to NaCl for 4 d in darkness. Light stimulated both ions and proline accumulation in the leaves and has no effect in the roots. Oxygen uptake was higher in the seedlings kept 4 d in the light which have higher endogenous free proline content. Chlorophyll fluorescence measurements showed that the photochemical activity of PS 2 slightly decreased as a result of salt stress and was not influenced by light regimes during plant growth.     

Expression of Genes in New Sprouts of Cunninghamia lanceolata Grown Under Dark and Light Conditions

Journal of Plant Growth Regulation - Chinese fir (Cunninghamia lanceolata) cotyledons are green in darkness due to the coexistence of functional dark-operative protochlorophyllide reductase (DPOR)...     

Developmental Changes in Amounts of Thylakoid Components in Plastids of Barley Leaves

Changes in the amounts of several components of the photosyntheticelectron-transport system during greening of etiolated barleyleaves were studied on a "per plastid" basis. P700 and Q_A, whichwere initially absent from etioplasts, appeared 2 h after thestart of illumination in complete complexes of PS I and PS II,respectively. From 6 h, they increased rapidly in amount witha constant stoichiometric ratio of 1:1. Amounts of Cyt f, Cytb₆, Cyt b-559 and FeS, initially present in etioplasts at levelsthat were one-third to half of those in mature chloroplasts,also increased rapidly after 6 h of illumination. The molarratio of Cyt f, Cyt b₆ and Cyt b-559 was the same in etioplastsand in mature chloroplasts, namely 1:2:2. After 4 h of illumination,levels of FeS increased at nearly the same rate as those ofthe PS I complex. The increase in levels of all components wasmarked after 6 h of illumination, probably due to the energysupplied by developing plastids that had just become photosyntheticallycompetent. The results are discussed in relation to the timeof appearance of chlorophyll-protein complexes and photochemicalactivities. ¹ Present address: Department of Botany, Faculty of Science,Kyoto University, Kyoto, 606-01 Japan.     

In Vivo Measurements of the Photo-Oxidation of Chlorophyll Pigments in Dark Grown Wheat Leaves after Treatment with δ-Aminolevulinic Acid

Dark-grown leaves of wheat fed with δ-aminolevulinic acid accumulate protochlorophyllide₆₃₆ in excess. After the leaves had been illuminated with high intensity red light (154 W × m^?2) for half a minute, a treatment which blocks the phototrans-formation protochlorophyllide chlorophyllide, the sensitivity of chlorophyllide and protochlorophyllide to light was examined. The decrease in pigment content, caused by photo-oxidation was found to be very close to a second order reaction. The second order “rate constant” for decrease in absorbance was found to be eight times greater for the formed chlorophyllide than for protochlorophyllide. The light intensity dependence of the decomposition was found to be linear within the intensity range used (E= 25 – 154 W × m^?2). In samples in which the pigments had been heat denatured, it was possible to photodecompose the chlorophyllide without affecting the protochlorophyllide. The results are discussed in connection with the theory of a photodynamic action involving oxygen in the singlet state (¹ΔO₂).     

Jasmonate-induced Changes in Flavonoid Metabolism in Barley (Hordeum vulgare) Leaves

The effects of jasmonic acid (JA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. A reversed-phase HPLC analysis revealed that the amount of a particular compound increased in excised barley leaf segments that had been treated with JA. This compound was purified and identified as 6´´´-feruloylsaponarin (1) by spectroscopic analyses and alkaline hydrolysis. A related compound, 6´´´-sinapoylsaponarin (2), was also found to accumulate in excised leaves independently of the JA treatment. The accumulation of these compounds was accompanied by a decrease in the saponarin (3) content. [8,9-¹³C]p-Coumaric acid and [2,3,4,5,6-²H]L-phenylalanine were effectively incorporated into the hydroxycinnamoyl moieties in 1 and 2, while the degree of incorporation of the labeled precursors into the saponarin part was small. These findings indicate that the hydroxycinnamoyl moieties of 1 and 2 are synthesized de novo from phenylalanine via the phenylpropanoid pathway, and that the saponarin part is mainly provided by the constitutive pool of 3.     

Developmental Effects of Sandoz 6706 on Activities of Enzymes of Phenolic and General Metabolism in Barley Shoots Grown in the Dark or under Low or High Intensity Light

The activities of three enzymes of phenolic biosynthesis and six of general metabolism were studied at 24-hour intervals between the 3rd and 8th day after planting in barley shoots treated with the chlorosis-inducing herbicide Sandoz 6706 and grown in the dark or under high or low intensity light. The herbicide had no effect on fresh weight or soluble protein (per shoot) in plants grown in the dark or under low intensity light, but slightly decreased these parameters in plants grown for more than 5 days under high intensity light. In dark-grown seedlings the herbicide had no detectable effects on plastid ultrastructure or on the activity of malate dehydrogenase, cytochrome c oxidase, NADP-cytochrome c reductase, triose phosphate isomerase, peroxidase, catalase, shikimate dehydrogenase, phenylalanine ammonia-lyase, or chalcone-flavanone isomerase. Under low intensity light, Sandoz 6706-treated plants developed plastids with single thylakoids extending across the organelle, and the activity of all enzymes examined was increased to varying degrees. When the herbicide-treated plants were grown under high intensity light, plastid lamellar organization was severely disrupted. Activities of shikimate dehydrogenase and chalcone-flavanone isomerase were markedly enhanced, phenylalanine ammonia-lyase activity slightly promoted, and catalase activity severely inhibited. The other enzymes were not appreciably affected by Sandoz 6706 under high intensity light. It is concluded that the changes in plastid ultrastructure and enzyme activities of the herbicide-treated plants are largely secondary photomorphogenetic or photooxidative responses in the carotenoid-free plants in which chlorophylls accumulate in reduced amounts (low intensity light) or are completely absent (high intensity light).     

The Activation State of Ribulose 1,5-bisphosphate Carboxylase in Maize Leaves in Dark and Light

A spectrophotometric procedure for assay of initial and totalactivity of ribulose 1,5-bisphosphate carboxylase in maize leaveswas established. The extraction of the crude enzyme from maizeleaf tissue, which was prefrozen in liquid nitrogen, desaltingof the extract, and assay of the enzyme was completed within3 min. From experiments adding deactivated ribulose 1,5-bisphosphatecarboxylase to the leaf tissue prior to extraction it was estimatedthat the maximum extent of activation during extraction, desaltingand assay was 8%. In predarkened leaves the enzyme showed 67to 84% of maximal activation while in preilluminated leavesthe enzyme showed 89 to 98% of maximal activation. These resultsindicate that deactivation of the enzyme in the dark is nota reason for the previous finding of a transient peak of ribulose1,5-bisphosphate in maize leaves during induction of photosynthesis[Usuda (1985) Plant Physiol. 78: 859–864]. This transientincrease in the substrate level upon illumination might be explainedby the presence of an unknown negative effector for ribulose1,5-bisphosphate carboxylase in vivo in leaf tissue in the dark,or limiting CO₂ supply to the enzyme during the induction period. (Received May 30, 1985; Accepted August 16, 1985)     

Two-Dimensional Electrophoretic Analysis of Salicylic Acid-Induced Changes in Polypeptide Pattern of Barley Leaves

The salicylic acid-induced changes in the polypeptide patterns of barley (Hordeum vulgare L.) leaves have been analysed using two-dimensional gel electrophoresis. An optimized 2-D PAGE protocol was used and gave reproducible 2-D gels from leaf crude protein extracts with a high number of detected polypeptides. When applied for 24 h SA affected the expression of a number of soluble proteins. Most of them appeared to be down-regulated. Although no abundant expression of specific proteins was observed, we detected three polypeptides that were present only in SA-treated leaves.     

Photochemical Properties of Photosystem 2 in Primary Leaves of Barley Seedlings Grown Under Various Blue or Red Irradiances

Photosystem 2 (PS2)-driven electron transfer was studied in primary leaves of barley (Hordeum vulgare L.) seedlings grown under various photon fluxes (0.3–170.0 mol m^–2 s^–1) of blue (BR) or red (RR) radiation using modulated chlorophyll fluorescence. The F_v/F_m ratio was 0.78–0.79 in leaves of all radiation variants, except in seedlings grown under BR or RR of 0.3 mol m^–2 s^–1. The extent of the photochemical phase of the polyphasic F_v rise induced by very strong white light was similar in leaves of all radiation treatments. Neither radiation quality nor photon flux under plant cultivation influenced the amount of non Q_B-transferring centres of PS2 except in leaves of seedlings grown under BR of 0.3 mol m^–2 s^–1, in which the amount of such centres increased threefold. Both BR and RR stimulated the development of photochemically competent PS2 at photon fluxes as low as 3 mol m^–2 s^–1. Three exponential components with highly different half times were distinguished in the kinetics of F_v dark decay. This indicates different pathways of electron transfer from Q_A ^–, the reduced primary acceptor of PS2, to other acceptors. Relative magnitudes of the individual decay components did not depend on the radiation quality or the photon flux during plant cultivation. Significant differences were found, however, between plants grown under BR or RR in the rate of the middle and fast components of F_v dark decay, which showed 1.5-times faster intersystem linear electron transport in BR-grown leaves.     

Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves

Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominant Mla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.