Conversion Of Chlorophyllide Research Articles

Stereoselective C3-substituent modification and substrate channeling by oxidoreductase BchC in bacteriochlorophyll a biosynthesis.

We report the invitro activity of recombinant BchC oxidoreductase involved inbacteriochlorophyll a biosynthesis. BchC of Rhodobacter capsulatus preferentially oxidizes 31 R-3-(1-hydroxyethyl)-chlorophyllide a and 31 R-3-(1-hydroxyethyl)-bacteriochlorophyllide a in the presence of NAD+ to 3-acetyl-chlorophyllide a and bacteriochlorophyllide a, respectively, leaving the unreacted 31 S-epimers. In the reverse reaction, BchC with NADH predominately produces 31 R-epimeric alcohols from the 3-acetyl-(bacterio)chlorins. BchC of Chlorobaculum tepidum demonstrates the same 31 R-selectivity, suggesting that utilization of 31 R-epimers in BchC-catalyzed reductions may be conserved across different phyla of photosynthetic bacteria. Additionally, the presence of BchC accelerates the 3-vinyl hydration by BchF hydratase of Chlorobaculum tepidum during conversion of chlorophyllide a to 3-acetyl-chlorophyllide a through 3-(1-hydroxyethyl)-chlorophyllide a, indicating that these enzymes work cooperatively to promote efficient bacteriochlorophyll a biosynthesis.

Arabidopsis noncoding RNA modulates seedling greening during deetiolation.

Seedling greening is essential for the survival of plants emerging from the soil. The abundance of chlorophyll precursors, including protochlorophyllide (Pchlide), is precisely controlled during the dark-to-light transition, as over-accumulation of Pchlide can lead to cellular photooxidative damage. Previous studies have identified and characterized multiple regulators controlling this important process. HID1 (hidden treasure 1) is the first noncoding RNA (ncRNA) found in photomorphogenesis. Under continuous red light, HID1 has been shown to inhibit hypocotyl elongation by repressing the transcription of PIF3 (phytochrome interacting factor 3). Here, we report that HID1 acts as a negative regulator of cotyledon greening. Knockdown of HID1 resulted in an increased greening rate of etiolated seedlings relative to wild type when exposed to white light. Genetically, HID1 acts downstream of PIF3 during the dark-to-light transition. The expression of HID1 is not regulated by PIF3 in the dark. Molecularly, the Pchlide content was reduced in dark-grown hid1 mutants than WT. Meanwhile, transcript levels of the protochlorophyllide oxidoreductases known to catalyze Pchlide to chlorophyllide conversion were significantly increased in hid1 seedlings. Thus, our study reveals an additional role of HID1 in the dark-to-light transition in Arabidopsis. Moreover, these results suggest HID1 could regulate distinct targets in different light-mediated developmental processes, and thus is essential to the control of these mechanisms.

Differential Molecular Responses of Rapeseed Cotyledons to Light and Dark Reveal Metabolic Adaptations toward Autotrophy Establishment.

Photosynthesis competent autotrophy is established during the postgerminative stage of plant growth. Among the multiple factors, light plays a decisive role in the switch from heterotrophic to autotrophic growth. Under dark conditions, the rapeseed hypocotyl extends quickly with an apical hook, and the cotyledon is yellow and folded, and maintains high levels of the isocitrate lyase (ICL). By contrast, in the light, the hypocotyl extends slowly, the cotyledon unfolds and turns green, the ICL content changes in parallel with cotyledon greening. To reveal metabolic adaptations during the establishment of postgerminative autotrophy in rapeseed, we conducted comparative proteomic and metabolomic analyses of the cotyledons of seedlings grown under light versus dark conditions. Under both conditions, the increase in proteases, fatty acid β-oxidation and glyoxylate-cycle related proteins was accompanied by rapid degradation of the stored proteins and lipids with an accumulation of the amino acids. While light condition partially retarded these conversions. Light significantly induced the expression of chlorophyll-binding and photorespiration related proteins, resulting in an increase in reducing-sugars. However, the levels of some chlorophyllide conversion, Calvin-cycle and photorespiration related proteins also accumulated in dark grown cotyledons, implying that the transition from heterotrophy to autotrophy is programmed in the seed rather than induced by light. Various anti-stress systems, e.g., redox related proteins, salicylic acid, proline and chaperones, were employed to decrease oxidative stress, which was mainly derived from lipid oxidation or photorespiration, under both conditions. This study provides a comprehensive understanding of the differential molecular responses of rapeseed cotyledons to light and dark conditions, which will facilitate further study on the complex mechanism underlying the transition from heterotrophy to autotrophy.

LTS3 gene controls light-independent chlorophyll biosynthesis in green algae Chlamydomonas reinhardtii

The genetic control of light-independent chlorophyll biosynthesis in plant cells has been investigated using Chlamydomonas reinhardtii Lts3-mutants defective in dark chlorophyll biosynthesis on the stage before protochlorophyllide to chlorophyllide conversion. In heterotrophic conditions the mutants are unable to synthesize chlorophyll and accumulate protoporphyrins, after illumination they are greening. The mutants were tested for pigment contents, activity of enzymes and expression of the genes, encoding these enzymes. The LTS3 gene has been identified by positional cloning, and the predicted LTS3 protein appeared to be a GATA transcription factor, which activate the expression of genes encoded chlorophyll biosynthesis enzymes: Mg-chelatase and glutamate 1-semialdehyde aminotransferase in the dark, and possibly, important for adaptation of plant cells for autotrophic conditions.

EFFECTS OF CA TREATMENT AND TEMPERATURE ON BROCCOLI COLOUR DEVELOPMENT

Broccoli combines high contents of vitamins, fibres and glucosinolates with a low calorie count and is sometimes referred to as the 'crown jewel of nutrition'. Colour is one of the most important quality attributes of broccoli, and yellowing due to senescence of broccoli florets is the main external quality problem in the broccoli supply chain. Controlled Atmosphere (CA) is a very effective method to maintain broccoli quality but the effects of CA on colour retention have not been studied extensively. The aim of this paper is to characterise the colour behaviour (measured by RGB colour image analysis) of broccoli as affected by CA and temperature. Data on colour behaviour and gas exchange were gathered for broccoli heads that were stored in containers at three temperatures and subjected to four levels of O 2 and three levels of CO 2 . Gas conditions and temperature have a clear effect on the colour change of broccoli especially at low O 2 in combination with high CO 2 . An integrated colour model is proposed that combines a colour model with a standard gas exchange model. The colour model is based on three differential equations describing the formation of (blue/green) chlorophyllide from the colourless precursor, the bidirectional conversion of chlorophyllide into (blue/green) chlorophyll, and the decay of chlorophyllide. During the first step of building the integrated model, gas exchange data were analysed simultaneously using multi response regression analysis. No fermentation was encountered for this batch of broccoli. During the second step it was found that only one of the reactions of the colour model, the decay of chlorophyllide, is affected by the gas conditions. In the final step, a multi-response approach was applied where gas exchange parameters were estimated using the gas exchange model, the colour parameters were estimated using the colour model with both models linked via the reaction rate constant affected by the gas conditions. Such a calibrated, integrated, model could be used as a tool for predicting colour change in the postharvest chain.

Development of colour of broccoli heads as affected by controlled atmosphere storage and temperature

Colour is one of the most important quality attributes of broccoli. Yellowing due to senescence of broccoli florets is the main external quality problem. Controlled atmosphere (CA) storage is a very effective method to maintain broccoli quality. The aim of this paper is to characterise the colour behaviour (measured by RGB colour image analysis) of broccoli as affected by CA and temperature. Data on colour behaviour and gas exchange were gathered for broccoli heads stored in containers at three temperatures and subjected to four levels of O 2 and three levels of CO 2 concentrations. An integrated colour model is proposed that combines a colour model with a standard gas exchange model. The colour model is based on an existing colour model that describes the formation of (blue/green) chlorophyllide from the colourless precursor, the bidirectional conversion of chlorophyllide into (blue/green) chlorophyll and the decay of chlorophyllide. A multi-response approach was applied, accounting for 92% of the variance. Gas exchange parameters were estimated using the gas exchange model, the colour parameters were estimated using the colour model. Both models are linked via the reaction rate constant that describes the decay of chlorophyllide, as this reaction rate constant was found to be affected by the gas conditions. The integrated model might be applied to predict colour changes of MAP packaged broccoli as a low level of O 2 and a high level of CO 2 will only affect colour retention at higher temperatures.

ブロッコリー（Ｂｒａｓｓｉｃａ ｏｌｅｒａｃｅａ Ｌ．）花らいにおけるクロロフィル代謝 Ｍｇ‐デキレターゼの関与

ブロッコリー花らいの脱緑機構を明らかにするため, 高速液体クロマトグラフィーを使用し, ブロッコリー花らいのクロロフィル代謝産物の分離を行った.エチレン処理, 無処理48時間の花らいから, クロロフィルa代謝産物であるクロロフィルドa, フェオフォルビドa, ピロフェオフォルビドa, C132-ヒドロキシクロロフィルa, フェオフィチンaが検出された.そこで, ブロッコリー花らいから調製した可溶性酵素を用いてクロロフィルドaからフェオフォルビドaへの変換過程を調査した.クロロフィルaから調製したクロロフィルン(Mg-クロリン)は, マグネシウムが外れることにより, 波長が658nmから, 687nmにシフトする性質を持っており, 脱離反応を687nmの変化によって調査した.粗酵素液を煮沸処理することで反応が進まないことからMg-デキレターゼによる酵素反応であることが明らかとなり, Cu-クロロフィリンを基質として用いても反応は認められなかったことからクロロフィルン(Mg-クロリン)を特異的に認識する事が明らかとなった.さらに, 全クロロフィル量とMg-デキレターゼ活性との間には, 高い負の相関関係が得られた : Y=-0.73X+97.37, r2=0.94(ただしXは相対的Mg-デキレターゼ活性, Yは相対的全クロロフィル量).この結果から, ブロッコリー花らいにおいてMg-デキレターゼ活性が増大することにより, 脱緑が促進されるものと考えられた.

A Novel Pathway of Chlorophyll Catabolism in Citrus unshiu Fruit: Enzymatic Conversion of Chlorophyllide a to Mg-C132-carboxyl-pyropheophorbide.

A Novel Pathway of Chlorophyll Catabolism in Citrus unshiu Fruit: Enzymatic Conversion of Chlorophyllide a to Mg-C132-carboxyl-pyropheophorbide.

Substrate Specificity of Chlorophyll(ide) b Reductase in Etioplasts of Barley (Hordeurn Vulgare L.)

Enzyme activity of chlorophyll(ide) b reductase is present in etioplasts. Recently the conversion of chlorophyllide b to chlorophyll a via 7(1)-hydroxychlorophyll a was demonstrated in barley etioplasts. We used zinc pheophorbide b for a detailed investigation of the reduction of the 7-formyl group to the 7(1)-hydroxy compound in intact barley etioplasts. The reaction proceeded likewise before esterification and after esterification with phytyl diphosphate. The metal-free pheophorbide b, that is not accepted by chlorophyll synthase for esterification, is reduced to 7(1)-hydroxypheophorbide a to a small extent. The zinc (13(2)S)-pheophorbide b is at least equally well accepted for reduction as the epimer with the 13(2)R configuration of natural chlorophyll b. The reaction requires NADPH or NADH, although the latter is less effective. ATP is not required for the first step to the 7(1)-hydroxy compound. The significance of chlorophyll b reduction for acclimation from shade to sun leaves and for chlorophyll degradation is discussed.

Chlorophyll breakdown in senescent leaves identification of the biochemical lesion in a stay-green genotype of Festuca pratensis Huds.

Chlorophyll breakdown in senescent leaves proceeds in essentially three steps: dephytylation by the action of chlorophyllase; conversion of chlorophyllide to phaeophorbide by Mg-dechelatase; and oxygenolytic cleavage of the chlorine-macrocycle by a newly discovered dioxygenase. The metabolic lesion responsible for high retention of chlorophyll during foliar senescence in a mutant genotype of meadow fescue (Festuca pratensis Huds.) was located in the third step of the breakdown pathway. Senescent leaves of both the normally yellowing reference genotype, c.v Rossa, and the non-yellowing mutant Bf993 were shown to be competent with regard to chlorophyllase and Mg-dechelatase. On the other hand, thylakoids isolated from senescent leaves of cv. Rossa were able to carry out oxygenolysis of phaeophorbide into a colourless fluorescent catabolite in vitro, whereas Bf993 thylakoids were deficient in this activity. It is concluded that the Sid locus, a mutant allele of which is responsible for the stay-green character, encodes or regulates the gene for, phaeophorbide a dioxygenase.

Clomazone Causes Accumulation of Sesquiterpenoids in Cotton (Gossypium hirsutumL.)

The herbicide clomazone caused ultrastructural damage to etioplasts of cotton cotyledons. Etioplast envelopes were irregular, prothylakoids were absent or irregular, stroma density was low, and there were abnormal stromal vesicles. Further damage occurred upon exposure to light. Clomazone greatly slowed the conversion of chlorophyllide to chlorophyll in cotton, suggesting that phytol synthesis was affected. Neither synthesis of protochlorophyllide nor phototransformation of protochlorophyllide to chlorophyllide was affected by clomazone. Clomazone completely inhibited carotenoid synthesis without an accumulation of phytoene. However, the sesquiterpenoids hemigossypol and the dimeric sesquiterpenoid gossypol accumulated in greater amounts in clomazone-treated than in control tissues. These results indicate that this herbicide inhibits synthesis of terpenoids after the formation of farnesyl pyrophosphate.

Breakdown of Chlorophyll in Chloroplasts of Senescent Barley Leaves Depends on ATP

Chloroplasts isolated from senescent mesophyll of barley primary leaves were tested for the production of one of the blue-fluorescent putative catabolites of chlorophyll, FC 2, of which a small pool is present in senescent organelles. When illuminated during incubation, intact chloroplasts produced this compound for up to 1h. The reaction was abolished by DCMU, an inhibitor of photosynthetic electron transport. In darkness, the reaction continued for at least 4h if the chloroplasts were supplemented with ATP. In presenescent chloroplasts the ATP-dependent accumulation of FC 2 occurred at a very low rate. The catalist(s) responsible for the reaction appears to be induced during the main period of chlorophyll breakdown: its activation, in leaves induced to senesce, was prevented by cycloheximide, an inhibitor of cytosolic protein synthesis. The activity described is likely to represent the key reaction in chlorophyll breakdown, namely the conversion of chlorophyllide into a product, FC 2, having an oxidatively opened tetrapyrrol ring system.

Bioengineering of photosynthetic membranes. Requirement of magnesium for the conversion of chlorophyllide a to chlorophyll a during the greening of etiochloroplasts in vitro

The massive conversion of delta-aminolevulinic acid (ALA) to protochlorophyllide (Pchlide) and the massive conversion of chlorophyllide a (Chlide a) to chlorophyll a (Chl a) are two essential conditions for the ALA-dependent assembly of photosynthetic membranes in vitro. In this work, we describe the development of a cell-free system capable of the forementioned biosynthetic activities at rates higher than in vivo, for the first 2 h of dark-incubation. The cell-free system consisted of (1) etiochloroplasts prepared from kinetin and gibberellic-acid-pretreated cucumber cotyledons, and (2) cofactors and additives described elsewhere and which are needed for the massive conversion of ALA to Pchlide, (3) high concentrations of ATP, MgCl(2), and an isoprenol alcohol such as phytol, were required for the massive conversion of Chlide a to Chl a. An absolute and novel requirement of Mg(2+) for the conversion of Chlide a to Chl a was also demonstrated. In addition to the role of phytol as a substrate for the conversion of Chlide a to Chl a, the data suggested that this alcohol may also be involved in the regulation of the reactions between ALA and Pchlide. It is proposed that during greening, the conversion of Chlide a to Chl a may follow different biosynthetic rates, having different substrate and cofactor requirements, depending on the stage of plastid development.

Inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl during greening of groundnut leaves

The site of inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl was found to be at the level of conversion of chlorophyllide (672 nm) to chlorophyll (678 nm) during greening of groundnut leaves. This inhibition was partially reversed by certain divalent cations.

Properties of chloroplasts and chloroplast fragments, as deduced from internal chlorophyll → chlorophyllide conversion

Summary 1. Fractions containing either small green particles with PS I or PS I + II properties, or «whole chloroplasts and large fragments» were isolated from spinach and endive leaf homogenates. 2. With spinach, internal chlorophyll → chlorophyllide conversion is shown to occur in small green particles dispersed in an aqueous medium and also, be it to a much lower degree, in «whole chloroplasts and large fragments». The effect can be increased by addition of Triton X-100 or acetone. 3. Reconversion of chlorophyllide into chlorophyll is shown to occur mainly in PS I particles, probably from stroma lamellae. 4. An aqueous extract from spinach acetone powder, containing dissolved chlorophyllase, more or less inhibits chlorophyllase activity in small spinach particles, while with «whole chloroplasts and large fragments» an increase occurs. Dissolved chlorophyllase is adsorbed by endive small green particles; a much lower adsorption occurs with «whole chloroplasts and large fragments». 5. Phytol inhibits chlorophyll → chlorophyllide conversion in green particles and in «whole chloroplasts and large fragments». 6. Oleic acid, at certain concentrations, increases chlorophyll → chlorophyllide conversion in small particles, while, at the same concentrations, an inhibition occurs with «whole chloroplasts and large fragments». 7. «Whole chloroplasts and large fragments» are «solubilized» completely, or nearly so, by 0.2 % Triton X-100; solubilization of small green particles is much less. 8. The results are discussed in terms of differences in lipid composition of the various membranes. The experiments provide new evidence for the hypothesis, put forward in an earlier paper (Terpstra, 1972), that «fraction 4 particles», with both PS I and PS II properties, are distinct particles, probably derived from stroma lamellae.

The Conversion of Photoinactive Protochlorophyllide633 to Phototransformable Protochlorophyllide650 in Etiolated Bean Leaves Treated with δ-Aminolevulinic Acid

The relationship of phototransformable protochlorophyllide to photoinactive protochlorophyllide has been studied in primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Various levels of photoinactive protochlorophyllide, absorbing at 633 nm in vivo, were induced by administering delta-aminolevulinic acid to the leaves in darkness. Phototransformable protochlorophyllide, absorbing at 650 nm in vivo, was subsequently transformed to chlorophyllide by a light flash, and the regeneration of the photoactive pigment was followed by monitoring the absorbance increase at 650 nm in vivo. A small increase in the level of protochlorophyllide(633) causes a marked increase in the extent of regeneration of protochlorphyllide(650) following a flash. High levels of the inactive pigment species, however, retard the capacity to reform photoactive protochlorophyllide. A nonstoichiometric and kinetically complex decrease in absorbance at 633 nm in vivo accompanied the absorbance increase at 650 nm. The half-time for protochlorophyllide(650) regeneration in control leaves was found to be three times longer than the half-time for conversion of chlorophyllide(678) to chlorophyllide(683) at 22 C. The results are consistent with the hypothesis that protochlorophyllide(633) is a direct precursor of protochlorophyllide(650) and that the protein moiety of the protochlorophyllide holochrome acts as a "photoenzyme" in the conversion of protochlorophylide to chlorophyllide.