Citrus Chlorophyllase Dynamics at Ethylene-Induced Fruit Color-Break: A Study of Chlorophyllase Expression, Posttranslational Processing Kinetics, and in Situ Intracellular Localization

Abstract

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.

Biochemical characterisation of chlorophyllase from leaves of selected Prunus species--a comparative study.

“…In recent years, intensive efforts have been focused on deciphering the complex biological functions of Chlase and other crucial enzymes involved in chlorophyll turnover and maintaining its homeostasis at the physiological state of the cell, as well as during the adaptation of plants to a wide variety of biotic and abiotic stressors (Fernandez-Lopez et al, 1992;Karboune et al, 2005;Arkus & Jez, 2006;Azoulay-Shemer et al, 2008;Barry, 2009;Cowan, 2009;Beisel et al, 2010;Azoulay-Shemer et al, 2011;Banaś et al, 2011;Sytykiewicz et al, 2013). Surprisingly, numerous results regarding the participation of Chlase in senescence-induced chlorophyll diminution within different plant systems were often divergent or inconclusive (Wang et al, 2005;Ben-Yaakov et al, 2006;Criado et al, 2006;Hörtensteiner, 2006;Barry, 2009;Distefano et al, 2009;Gómez-Lobato et al, 2012).…”

“…Furthermore, it was established that molecular masses of Chlase F2 from the leaves of tested plant systems reached lower levels (35 kDa -European plum, 40 kDa -bird cherry, 60 kDa -sour cherry) when compared with fraction 1. Several studies demonstrated that molecular masses of chlorophyllases extracted from different plant species varied between 23.5 and 158 kDa (Fernandez-Lopez et al, 1992;Tang et al, 2004;Arkus et al, 2005;Lee et al, 2010;Azoulay-Shemer et al, 2008). Importantly, Arkus et al (2005) claim that Chlase isolated from plant tissues may undergo aggregation to high molecular weight structures.…”

Physiological and transcriptomic analysis of yellow leaf coloration inPopulus deltoidesMarsh

“…Chlase catalyzes the hydrolysis ester bond of Chl to yield Chlide and phytol, is thought to be the first enzyme in the Chl degradation [42]. Chlase activity is negatively correlated with Chl levels during citrus fruit color break and Chlase participate in Chl breakdown of citrus [15]. However, some evidence does not support that Chlase play a critical role in Chl degradation during leaf senescence [43-45].…”

“…In Arabidopsis mutant deficient in PPH (pheophytinase), Chl degradation is inhibited, and the plants exhibit a typeC stay-green phenotype during senescence [12]. Previous studies revealed that chlorophyllase (Chlase) is involved in Chl degradation in ethylene-treated citrus fruit and could regulate the balance between different plant defense pathways, enhance plant resistance to bacteria [13-15]. Recently, Mutants deficient in Chl biosynthesis and degradation have been identified in many yellow leaf plants, such as rice [16-19], Arabidopsis thaliana [20] and pak-choi [21].…”

Der Chlorophyllabbau in höheren Pflanzen – Phyllobiline als weitverbreitete, aber kaum sichtbare Zeichen von Reifung, Seneszenz und Zelltod

“…Überraschenderweise gibt es zwei unterschiedliche Wege in höheren Pflanzen für den Abbau von Chl a zu Pheo a (Abbildung ) . Einer dieser Wege ist in reifenden Zitrusfrüchten relevant, wo die Phytolesterfunktion von Chl a durch Chlorophyllase hydrolysiert wird, wobei Chlorophyllid a (Chlide a ) entsteht. Anschließend wird das Mg‐Ion entfernt, wobei Pheo a als Produkt gebildet wird .…”