The After-Effect of Water Stress on Chlorophyll Formation during Greening and the Levels of Abscisic Acid and Proline in Dark Grown Wheat Seedlings

Cut seedlings of wheat plants (Triticum aestivum L. cv. Starke II Weibull) between 6 and 7 days old were water stressed in darkness by exposing them to air of 35% relative humidity 2.5 to 20 h. This treatment resulted in a water potential of -11 bars in the leaves after 20 h. The leaves were then rewatered and irradiated. The chlorophyll formation that took place in fully turgid leaves during the greening was markedly decreased in the case of the water-stress pretreatmet. and especially the lag phase was prolonged. The longer the stress pretreatment the more evident was the subsequent effect on chlorophyll formation. However, no linear relationship was found between the amount of stress and the chlorophyll content. Protochlorophyllide regeneration from endogenously formed δ-aminolevulinic acid was markedly decreased even after the shortest water-stress period. However, protochlorophyllide accumulation from exogenously supplied δ-aminolevulinic acid was only slightly decreased following the water-stress pretreatment. Further more, the ratio of protochlorophyllide₆₅₀ to protochlorophyllide₆₂₈ was slightly reduced by the same conditions. During the stress period both abscisic acid and proline were accumulated in the leaves. The content of abscisic acid increased up to six times the normal level during water stress lasting for 20 h. The increase of proline was about three-fold for similar treatment. After rewatering the leaves the levels of both abscisic acid and proline rapidly declined and reached. 10 h later, the levels found in unstressed seedlings. The increase in abscisic acid during water stress associated with impaired chlorophyll metabolism suggested that the after-effect of water stress might be linked to chlorophyll metabolism through abscisic acid or some of its metabolites. The changes in proline content open the possibility that this substance could function as a reserve substance for the formation of chlorophyll after the discon tinuation of the stress.     

Oxygen Consumption Stimulated by δ-Aminolevulinic Acid in Darkness and during Irradiation of Dark Grown Wheat Leaves

Dark grown wheat leaves (Triticum aestivum L. cv. Starke II Weibull), treated with δ-aminolevulinic acid in darkness, showed an increased oxygen uptake as measured by a Warburg method. The production of CO₂ was also increased in darkness, giving an RQ ? 1. The increased respiration was dependent on the treatment time as well as on the concentration of the δ-aminolevulinic acid. Potassium cyanide suppressed both the normal and the increased respiration. The treatment with δ-aminolevulinic acid caused accumulation of high amounts of protochlorophyllide. Levulinic acid suppressed the increased oxygen uptake as well as the protochlorophyllide accumulation in δ-aminolevulinic acid treated leaves. Irradiation rapidly decreased the protochlorophyllide content with a simultaneous increase in oxygen uptake over the dark value. The peak value of the increase in oxygen uptake was reached after about 5 min. The light induced oxygen uptake was dependent on the amount of PChlide present at the onset of irradiation. Also the CO₂ production was increased during the first minutes of irradiation but soon fell under the buffer control value. Neither potassium cyanide nor heat denaturation affected the oxygen uptake in light in contrast to the effect on the CO₂ production, which was blocked by heat denaturation. The increased oxygen uptake in light initially seems to be a purely photochemical process leading to a release of CO₂, which release is probably an enzymatic process induced by the photo-oxidative decomposition of pigment.     

Chloroplast culture VIII a new effect of kinetin in enhancing the synthesis and accumulation of protochlorophyllide invitro

By pretreating etiolated cucumber cotyledons with kinetin in the dark, it was observed that the plastids isolated from such tissues were 400% more active in the conversion of δ-aminolevulinic acid into protochlorophyllide, than plastids prepared from water-treated controls. The experimental evidence is consistent with the hypothesis that (a) the kinetin dark-pretreatment of the etiolated tissue, uncouples the joint biosynthesis of prothylakoids and protochlorophyll and results in the accumulation of excess prothylakoid membranes poorly supplied with protochlorophyllide (b) upon isolation of the plastids and incubation with δ-aminolevulinic acid, the latter is very rapidly converted into membrane-bound protochlorophyllide.     

The Influence of Protochlorophyllide, Formed from Exogenous δ-Aminolevulinic Acid, on Chlorophyll Synthesis in Leaves during Flash Illumination

In the presence of large accumulations of protochlorophyllide, derived from exogenous δ-aminolevulinic acid, chlorophyll synthesis in excised leaves of two varieties of barley was less than in untreated leaves. In oat leaves the accumulated protochlorophyllide, from exogenous δ-laminolevulinic acid, stimulated chlorophyll synthesis to above the control level. — These relationships could only be demonstrated when phtodestruction of pigments was minimised by the use of flash illumination (2 milliseconds every 3 minutes). — These was no evidence from in vivo absorption spectra that the pigments in the barley leaves were different to those in leaves studied by other workers. However, the presence of the accumulated protochlorophyllide appeared to prevent the shift of the chlorophyll absorption maximum from 673 nm to 677 nm. — Possible mechanisms of inhibition are discussed.     

Chloroplast culture IX chlorophyll(ide) a biosynthesis invitro at rates higher than inMvivo

Chlorophyll $\text{a}$ is the plant pigment which in nature catalyzes the conversion of solar energy into chemical energy. By pretreating etiolated cucumber cotyledons with kinetin and gibberellic acid in the dark, it was observed that the plastids which were isolated from such tissues, and incubated in a cofactor-fortified medium, under a repetitive light-dark regime, were capable of synthesizing chlorophyll(ide) $\text{a}$ from exogenous δ-aminolevulinic acid at a rate about twice as high as the highest rates observable in greening tissues $\text{in}\text{vivo}$.     

Influence of photodynamic processes induced by 2,2′-dipyridyl on the enzymatic system of chlorophyll biosynthesis

The influence of 2,2′-dipyridyl (2,2′-DP) on the activity of one of the enzymes at the initial stages of chlorophyll (Chl) biosynthesis, δ-aminolevulinic acid dehydratase (ALAD; δ-aminolevulinate hydro-lyase, EC 4.2.1.24), as well as on δ-aminolevulinic acid (ALA) accumulation was investigated in green barley (Hordeum vulgare L.) leaves. In seven-day-old green leaves treated with 3 mM 2,2′-DP for 17 h in darkness and subsequently irradiated with "white light" (15 W m-2) for 4, 8, and 24 h the ALAD activity was 51 % as compared to that in untreated leaves. At the same time, the ALA forming system was most sensitive to the photodynamic processes caused by 2,2′-DP. After 8 h of irradiation, ALA synthesis was entirely inhibited. After the treatment the leaves accumulated exceptionally high amounts of Chl precursors such as protoporphyrin IX (Proto), Mg-protoporphyrin IX (Mg-Proto), its monomethyl ester, and protochlorophyllide (Pchlide) that are photosensitizers of photodynamic processes in plants. A comparatively low Chl and carotenoid (Car) destruction was registered during the subsequent 4 and 8 h of irradiation. At the same time, the content of Chl precursors was negligible. The low photodestruction of Chl and Car included in pigment-protein complexes, against the background of fast porphyrin disappearance, and fast decrease of enzymatic activities at the initial stages of Chl production could mean that the photodynamic effect induced by porphyrins accumulated in the presence of 2,2′-DP affected first the Chl enzymatic system and did not change the pool of already synthesized photosynthetic pigments.     

CHLOROPHYLL AND CAROTENOID ACCUMULATION IN THREE CHLOROPHYLLOUS CALLUS PHENOTYPES OF GLYCINE MAX

Effects of exogenous carbohydrates and various medium supplements on chlorophyll and carotenoid accumulation in three chlorophyllous callus phenotypes of Glycine max (L.) Merrill were studied. Glucose (filtered), at 3%, supported the highest level of chlorophyll and carotenoids in the NG and Y phenotypes, while only moderate levels of chlorophyll accumulated in the LG phenotype. Sucrose (filtered and autoclaved), at 3%, supported phenotypical levels of chlorophyll and carotenoids for all phenotypes. Ascorbic acid, at 75 mg/l, stimulated chlorophyll-carotenoid accumulation for all phenotypes, while δ-aminolevulinic acid was slightly toxic for pigment biosynthesis in the NG and LG phenotypes. In contrast, δ-aminolevulinic acid supported chlorophyll-carotenoid accumulation in the Y phenotype. A negative correlation (r_XY) was evident between chlorophyll formation and callus growth for all phenotypes. Light intensity of 3,000 lux suppressed chlorophyll accumulation in the Y callus phenotype while an increase in pigment formation occurred in the NG and LG callus phenotypes. In comparison, light intensity of 700 lux supported chlorophyll accumulation in the Y callus phenotype. Carotenoid accumulation appeared to be coupled with chlorophyll formation in all callus phenotypes except for the Y callus phenotype when grown under the higher light intensity. All phenotypes accumulated chlorophylls a and b, α- and β-carotene, lutein plus zeaxanthin, violaxanthin and neoxanthin as indicated by their respective absorption maxima. Under cultural conditions, the genetic mutation (yll) in these soybean callus phenotypes appeared to regulate carotenoid formation which in turn influenced chlorophyll stability.     

The Role of Abscisic Acid in Senescence of Detached Tobacco Leaves

The role of abscisic acid in the regulation of senescence was investigated in detached tobacco leaves (Nicotiana rustica L.). Leaves senesced in darkness showed a sharp rise in abscisic acid level in the early stage of aging, followed by a rapid decline later. The same trend was found when leaves were aged in light, but the rise in abscisic acid occurred four days later than in darkness. Senescence was slower in light than in darkness, while salt stress accelerated the processes. Leaves treated with kinetin which senesced in light and darkness, did not show an increase in abscisic acid. Application of kinetin led to a transformation from free to bound ABA. These results may indicate that ABA and cytokinin are involved in a trigger mechanism which regulates senescence; the stage at which this trigger is activated determines the rate of senescence.     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

The Conversion of Protochlorophyllide636 to Protochlorophyllide630 in Leaves Treated with δ-Aminolevulinic Acid

Absorbancy changes in dark-grown, excised wheal leaves fed with δ-aminolevulinic acid are measured in vivo. The treatment with σ-aminolevulinic acid caused accumulation of protochlorophyllide, absorbing at 636 nm. After flashlight this form is found to convert in darkness to protochlorophyllide, absorbing at 650 nm. The conversion starts instantly after the leaves have been exposed to the flashlight, and the pre-existent pool of protocholorophyllidc absorbing at 650 nm will become emptied. The conversion is completed after 15–20 minutes, when a new pool of protochlorophyllide has been filled up. This new pool is transformed to chlorophyllide by a second flash and the sequence is repeated. The conversion may be composed of two reactions, a conclusion which can be drawn from the behaviour at different temperatures. One of these reactions is fairly temperature independent while the other is temperature dependent. The action of the protochlorophyllide holochrome is discussed.     

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 Influence of 8-Hydroxyquinoline on the Accumulation of Porphyrins in Dark-Grown Wheat Leaves Treated with δ-Aminolevulinic Acid

Dark-grown wheat leaves treated with δ-aminolevulinic acid and 8-hydroxyquinoline accumulated porphyrins, most of which were protochlorophyllide and magnesiumprotoporphyrin monomethylester. The ratio between these two components was dependent on the concentration of 8-hydroxyquinoline. Small amounts of other porphyrins could also be detected. The treatment with 8-hydroxyquinoline and the presence of large amounts of porphyrins other than protochlorophyllide did not influence the photoreduction of protochlorophyllide or the Shibata shift. 8-Hydroxyquinoline caused an inhibition of protochlorophyllide biosynthesis, which could be reversed by rinsing the leaves several times with phosphate buffer. Magnesiumprotoporphyrin monomethylester was then converted to protochlorophyllide. The reversal induced by washing was increased if the buffer contained iron. The possible function of iron in the chlorophyll metabolism and its role in the inhibition reactions with 8-hydroxyquinoline are discussed.     

Changes in Growth and Nitrogen Assimilation in Maize Plants Induced by NaCl and Growth Regulators

Experiments were conducted to determine the interactive effects of salinity and certain growth regulators on growth and nitrogen assimilation in maize (Zea mays L. cv. GS-2). 100 mM NaCl inhibited the biomass accumulation, chlorophyll and carotenoid contents in leaves, nitrate content and uptake and nitrate reductase activity. The application of kinetin, ascorbic acid and 10 and 50 μM abscisic acid in the first experiment and 50 and 100 μM abscisic acid in the second experiment induced a substantial increase in the above parameters, the effect was highest with abscisic acid in salinized as well as non-salinized plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

The hormonal regulation of bud outgrowth in Phaseolus vulgaris L.

Summary Aqueous solutions of indole acetic acid, kinetin, gibberellic acid and abscisic acid were applied singly and in combination to the decapitated stem stump of Phaseolus seedlings. Application of indole acetic acid will not completely replace the intact stem apex with regard to the inhibition of lateral bud extension. The greatest inhibition of bud growth is obtained when indole acetic acid is applied in combination with both kinetin and abscisic acid. Treatment with gibberellic acid causes massive bud growth even in the presence of indole acetic acid, kinetin and abscisic acid. Although both abscisic acid and kinetin have only a slight promoting effect on bud outgrowth when applied singly, these hormones will modify the effects of indole acetic acid and gibberellic acid.     

Regulation of Magnesium Chelatase Activity during Excessive Accumulation of Porphyrins in Green Barley Leaves

Activity of magnesium chelatase was studied in green barley leaves treated with 5-aminolevulinic acid (ALA). After this treatment, leaves accumulated excessive amounts of porphyrinic precursors of chlorophyll : protoporphyrin IX (PP), magnesium-protoporphyrin IX (MgPP), its monomethyl ester (MgPPE), and protochlorophyllide. The enzyme activity was found to be inversely dependent on the amount of MgPP formed from exogenous ALA. A conclusion was drawn about the existence of a mechanism for the regulation of the enzyme activity in vivo via its inhibition by the reaction product.     

Effects of Kinetin on Transpiration Rate and Abscisic Acid Content of Water Stressed Young Wheat Plants

Effects of kinetin on transpiration rate and abscisic acid content were determined. Leaves from 9-day-old wheat plants (Triticum aestivum L. cv. Weibull's Starke II) were used. —Transpiration rate decreased in excised leaves put in water, but it was maintained at a higher rate when kinetin was supplied. When excised leaves were water stressed by air-drying for 1 h, addition of kinetin resulted in a considerable stimulation of transpiration rate. The effect reached its maximum after 15 h and this level remained relatively unchanged for at least 10 h. Intact seedlings which were stressed before leaf excision, showed only a slight stimulation of kinetin on transpiration rate. — Abscisic acid content slowly increased up to three-fold in 2 days in excised leaves put in water. In excised and water-stressed leaves the abscisic acid content was reduced during the first 24 h and then increased. As the leaves were fully turgid, the increase could not have been caused by water stress. However, both in stressed and unstressed leaves kinetin addition reduced the increase in abscisic acid content. — It is suggested that the stimulation by kinetin on transpiration rate in excised and water stressed leaves was mainly due to the combined effect of (1) a reduction in the activity of endogenous cytokinins, (2) kinetin acting as a ‘substitute’ for the inactivated cytokinins but exerting a stronger effect on transpiration than the endogenous cytokinins, and (3) the ‘extra’ reduction in abscisic acid content caused by the kinetin treatment. Furthermore, the results indicate that changes in cytokinins might be partly responsible for the aftereffect on transpiration.     

Induction of delta-Aminolevulinic Acid Formation in Etiolated Maize Leaves Controlled by Two Light Systems

A short illumination of etiolated maize (Zea mays) leaves with red light causes a protochlorophyll(ide)-chlorophyll(ide) conversion and induces the synthesis of δ-aminolevulinic acid (ALA) during a subsequent dark period. In leaves treated with levulinic acid, more ALA is formed in the dark than in control leaves. Far red light does not cause a conversion of protochlorophyll(ide) into chlorophyll(ide) and does not induce accumulation of ALA in the dark. Both red and far red preilluminations cause a significant potentiation of ALA synthesis during a period of white light subsequent to the dark period. The results indicate a dual light control of ALA formation. The possible role of phytochrome and protochlorophyllide as photoreceptors in this control system is discussed.     

Hormonal Regulation, and Intracellular Localization of a 33-kD Cationic Peroxidase in Excised Cucumber Cotyledons

Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase (33-CPO) in excised cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. Compared to other hormones, such as kinetin, indoleacetic acid, gibberellic acid, and abscisic acid, ethylene was the only effective promoter of 33-CPO synthesis. The hypothesis that peroxidase plays a role in chlorophyll degradation was tested by comparing levels of 33-CPO in cotyledons treated with compounds thought to either retard (kinetin, indoleacetic acid and gibberellic acid), or promote (abscisic acid and methyl jasmonate [MJ]) senescence. It was concluded that 33-CPO did not play a role in senescence since no direct correlation between chlorophyll content and 33-CPO was observed. MJ was as effective as ethylene in inducing senescence. However, ethylene did not appear to be involved in the action of MJ. Using immunocytochemistry, 33-CPO was found to be located primarily around starch grains and near the plasmalemma. High levels of 33-CPO were also found in cells destined to be vascular tissue.     

The Relationship between Chlorophyllide Accumulation, the Amount of Protochlorophyllide636 and Protochlorophyllide650 in Dark Grown Wheat Leaves Treated with δ-Aminolevulinic Acid

The influence of different amounts of protochlorophyl-lide₆₃₆ and protochlorophyllide₃₅₀ on light Induced chloro-phyllide formation was. studied in wheat leaves treated with δ-aminolevulinic acid. The phototransformation of proto-chlorophyllide was performed with weak red light. This transformation is unaffected by the δ-aminolevulinic acid treatment, whilst the accumulation of chlorophyllide, both the rate and the amount, is greatly stimulated by moderate amounts of protochlorophyllide₆₃₆. The presence of large amounts of protochlorophyllide₆₃₆ decreases the rate of chlorophyllide formation, but increases the final amount of chloro-phyllide formed. A decreased level of protochlorophyllide₆₅₀, obtained by treatment with N_aN₃, results in a decreased transformation rate. Inhibitors; of protein synthesis do not seem to influence the transformation of protochlorophyllide₆₃₆ to chlorophyllide, suggesting that no new synthesis of protein is required. The experimental results indicate that the final steps in chlorophyll biosynthesis are protochlorpnyllide₆₃₆→ protochlorophyllide₆₅₀→ chlorophyllide → chlorophyll.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.