Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

High-pressure liquid chromatography was used to separate chlorophyll derivatives in acetone extracts from senescing Citrus fruit peel, autumnal Melia azedarach L. leaves, and dark-held detached parsley (Petroselinum sativum L.) leaves. Chlorophyllide a and another polar, dephytylated derivative accumulated in large amounts in senescing Citrus peel, particularly in fruit treated with ethylene. Ethylene also induced a 4-fold increase in the specific activity of Citrus chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14). Detailed kinetics based on a hexane/acetone solvent partition system showed that the in vivo increase in dephytylated derivatives coincided with the decrease in total chlorophyll. Polar, dephytylated derivatives accumulated also in senescing Melia leaves. Senescing parsley leaves revealed a very different picture. The gradual disappearance of chlorophyll a was accompanied by an increase in pheophytin a and by the transient appearance of several phytylated derivatives. Only pheophytin a and an adjacent peak were left when all the chlorophyll a had disappeared. The pathways for breakdown of chlorophyll in the Citrus and parsley senescence systems are discussed.

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“…Meeting: Deutsche Botanische Gesellschaft, Sept. [12][13][14][15][16][17][18]1982, Freiburg, F.R.G., Abstr. 473.…”

Section: Methodsunclassified

“…(i) The chlorophyllase enzyme has been thoroughly purified and characterized (13,14), and its localization in chloroplasts seems well established (15,16). Correlative evidence in senescing leaves (17) and the upsurge of chlorophyllase in ethylene-treated Citrus fruit (16,(18)(19)(20) further support its role in senescent chlorophyll catabolism.…”

mentioning

confidence: 92%

Chlorophyll catabolism in senescing plant tissues: In vivo breakdown intermediates suggest different degradative pathways for Citrus fruit and parsley leaves

Amir-Shapira

Goldschmidt

Altman

1987

Proc. Natl. Acad. Sci. U.S.A.

182

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“…Chlase purified from a diatom, Phaeodactylum tricornutum, showed two bands, a major band estimated to be 43 kDa and a minor band of 46 kDa (10). The molecular mass of Chlases from Citrus was reported to be 35 and 27 kDa, depending on the species (8,11). It is unknown, however, whether the difference in their molecular masses is due to the difference of plant species or to the existence of different types of isozymes, or both.…”

mentioning

confidence: 99%

Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate

Tsuchiya

Ohta²,

Okawa³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

295

245

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Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase from Chenopodium album, screened the C. album cDNA library, and cloned a cDNA (CaCLH, C. album chlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP͞GTP-binding motif (P-loop). CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) in Arabidopsis thaliana (AtCLH1). We expressed the gene products of AtCLH1 and of CaCLH in Escherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on an Arabidopsis genomic fragment and expressed it in E. coli, demonstrating the presence of the second Arabidopsis CLH gene (AtCLH2). No typical feature of signal sequence was identified in AtCLH1, whereas AtCLH2 had a typical signal sequence for chloroplast. AtCLH1 mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h. AtCLH2, however, did not respond to MeJA.

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“…3.1.1.14) is involved in the metabolism of chlorophylls (Purvis andBarmore, 1981, andShimokawa, 1982) and some authors have found that this enzymatic activity increases notably with iron deficiency (Almela et al, 1983).…”

Section: Introductionmentioning

confidence: 96%

Iron deficiency in citrus limon: effects on photo‐chlorophyllase synthetic pigments and chlorophyllase activity

Fernández-López

Almela

López-Roca

et al. 1991

Journal of Plant Nutrition

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The photosynthetic pigment composition of chlorotic leaves of Citrus limon L. cv. Verna, grown in the field under iron deficiency conditions was determined. A Fe-polyflavonoid was used as fertilizer to control iron chloro-sis. The photosynthetic pigment content and the chlorophyllase activity were determined at 20 day intervals during the deficiency recovery period and compared to untreated similar material. The corresponding differences among treated and untreated control material were analyzed. Iron application increased the levels of all pigments, but the extent of the increase depended on the pigment affected. The chlorophylls/carotenoids and ß-carotene/xantophylls ratios were increased as chlorosis diminished. A multivariance analysis was performed with the data obtained which revealed that chlorophyll a and ß-carotene had the highest correlation coefficient. The chlorophyllase activity did not show significant changes, but it was lower in the treated leaves than in the untreated control leaves during all the sampling cycle.

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Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

Cited by 39 publications

References 13 publications

Chlorophyll catabolism in senescing plant tissues: In vivo breakdown intermediates suggest different degradative pathways for Citrus fruit and parsley leaves

Chlorophyll catabolism in senescing plant tissues: In vivo breakdown intermediates suggest different degradative pathways for Citrus fruit and parsley leaves

Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate

Iron deficiency in citrus limon: effects on photo‐chlorophyllase synthetic pigments and chlorophyllase activity

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