Chl Degradation Research Articles

The Stay-Green Rice like (SGRL) gene regulates chlorophyll degradation in rice

The Stay-Green Rice (SGR) protein is encoded by the SGR gene and has been shown to affect chlorophyll (Chl) degradation during natural and dark-induced leaf senescence. An SGR homologue, SGR-like (SGRL), has been detected in many plant species. We show that SGRL is primarily expressed in green tissues, and is significantly downregulated in rice leaves undergoing natural and dark-induced senescence. As the light intensity increases during the natural photoperiod, the intensity of SGRL expression declines while that of SGR expression increases. Overexpression of SGRL reduces the levels of Chl and Chl-binding proteins in leaves, and accelerates their degradation in dark-induced senescence leaves in rice. Our results suggest that the SGRL protein is also involved in Chl degradation. The relationship between SGRL and SGR and their effects on the degradation of the light-harvesting Chl a/b-binding protein are also discussed.

Nitric oxide deficiency accelerates chlorophyll breakdown and stability loss of thylakoid membranes during dark-induced leaf senescence in Arabidopsis.

Nitric oxide (NO) has been known to preserve the level of chlorophyll (Chl) during leaf senescence. However, the mechanism by which NO regulates Chl breakdown remains unknown. Here we report that NO negatively regulates the activities of Chl catabolic enzymes during dark-induced leaf senescence. The transcriptional levels of the major enzyme genes involving Chl breakdown pathway except for RED CHL CATABOLITE REDUCTASE (RCCR) were dramatically up-regulated during dark-induced Chl degradation in the leaves of Arabidopsis NO-deficient mutant nos1/noa1 that exhibited an early-senescence phenotype. The activity of pheide a oxygenase (PAO) was higher in the dark-induced senescent leaves of nos1/noa1 compared with wild type. Furthermore, the knockout of PAO in nos1/noa1 background led to pheide a accumulation in the double mutant pao1 nos1/noa1, which retained the level of Chl during dark-induced leaf senescence. The accumulated pheide a in darkened leaves of pao1 nos1/noa1 was likely to inhibit the senescence-activated transcriptional levels of Chl catabolic genes as a feed-back inhibitory effect. We also found that NO deficiency led to decrease in the stability of photosynthetic complexes in thylakoid membranes. Importantly, the accumulation of pheide a caused by PAO mutations in combination with NO deficiency had a synergistic effect on the stability loss of thylakoid membrane complexes in the double mutant pao1 nos1/noa1 during dark-induced leaf senescence. Taken together, our findings have demonstrated that NO is a novel negative regulator of Chl catabolic pathway and positively functions in maintaining the stability of thylakoid membranes during leaf senescence.

Systematic Analysis of the Demetalation Kinetics of Zinc Chlorophyll Derivatives Possessing Different Substituents at the 3-Position: Effects of the Electron-Withdrawing and Electron-Donating Strength of Peripheral Substituents

Removal of the central metal from chlorophyll (Chl) molecules is biologically important in terms of production of the primary electron acceptors in photosystem-II photosynthetic reaction centers and the early stage in Chl degradation. The physicochemical properties on demetalation of chlorophyllous pigments are useful in the understanding of such reaction mechanisms in photosynthetic organisms. Here we analyzed the demetalation kinetics of a series of Zn-Chl derivatives with a systematic variation in the electron-withdrawing and -donating substituents at the 3-position of the chlorin macrocycle under acidic conditions to elucidate thoroughly the substitution effects on the demetalation properties of chlorophyllous pigments. Dehydrogenation of the aliphatic group (CH(2)CH(3) → CH═CH(2) → C≡CH) at the 3-position slowed the removal of the central zinc from the chlorin macrocycle. The gradual decrease in the demetalation rate constants of the three zinc chlorins originates from differences in the electron-withdrawing strength of the ethyl, vinyl, and ethynyl groups directly linked to the chlorin π macrocycle. Reduction of the 3(1)-carbonyl groups significantly increased the demetalation rate constants, and the relative ratios of the demetalation rate constants of the zinc chlorins possessing a carbonyl group to those possessing the corresponding hydroxy group were analogous in the cases of 3-formyl- and 3-acetyl-zinc chlorins. The demetalation rate constants of the seven Zn-Chl derivatives possessing various electron-withdrawing and -donating groups exhibited good correlation with the Hammett σ parameters of the 3-position substituents.

PHEOPHYTIN FORMATION WITH SENESCENCE IN STORED JAPANESE BUNCHING ONION

18 The shelf life of Japanese bunching onion (Allium fistulosum L.; JBO genome 19 FF) leaves diminished with yellowing as a consequence of chlorophyll (Chl) degradation, 20 which seems to be the main factor in yellowing of stored JBO leaves. The aim of this 21 study was to investigate pheophytin (Phy) formation as a Chl derivative during Chl 22 degradation in stored JBO leaves. A series of alien monosomic addition lines (AMALs, 23 FF+1A to FF+8A) of JBO, with extra chromosomes from shallot (A. cepa L., 24 Aggregatum group; genome AA) was used as supportive materials to study the 25 formation of Chl a derivatives and the activity of Chl-degrading enzymes during 26 storage. Chl contents in FF+3A and FF+5A decreased greatly during storage at 25°C, 27 whereas in FF+4A the reduction in Chl content was the lowest compared to the control, 28 FF. In JBO leaves, during Chl degradation, the presence of Phy a was prominent as Chl 29 derivatives, which was not recorded in other horticultural crops. Moreover, the 30 activities of Chl-degrading enzymes, especially Mg-dechelation, also progressively 31 increased during storage at 25 o C. The formation of Phy a in stored JBO leaves was 32 further investigated. By incubating the reaction mixture of crude enzyme extract and 33 Chl a, Phy a was formed by Mg-dechelating action. Especial significant was the greater 34 formation of Phy a in FF+3A than FF and FF+4A. Electron microscopic observation 35 elucidated the formation of plastoglobules (Pgs) in chloroplasts and its movement from 36 chloroplast to vacuole in JBO. A large number of Pgs in vacuole of FF+3A might relate 37 to the high Phy a formation with Chl degradation. These findings suggest that Chl a 38 could be degraded in part, through Phy a, and the vacuole along with the chloroplast 39 might be important sites for Chl degradation in JBO during storage. 40 41

Presence of Pheophytin and its Formation as a Chlorophyll Derivative in Selected Crop Species

Most of the green leaf vegetable and fruits lose their postharvest quality mainly due to yellowing as a consequence of chlorophyll (Chl) degradation. The study of Chl degradation of many plants have been revealed that the Chl degradation mainly progress through formation of chlorophyllide (Chlide) a. Recent studies have revealed that the Chl degradation could be progressing through formation of pheophytin (Phy) a as a Chl derivative. Present study was conducted to investigate the presence and formation of Phy a as Chl a derivative in selected stored plant species. Green tea leaves, bell pepper, parsley and Japanese bunching onion were used in the study. Presence of Chl a derivatives of 80% acetone extract of green tea leaves and Japanese bunching onion were detected using HPLC at the wave length of 663nm. Formation of Chl a derivatives were studied by incubating crude enzyme extract of bellpepper, parsley and bunching onion with Chl a and buffer solution. After one hour of incubation reaction was stopped by adding 80% acetone. Chl a derivatives were studied using HPLC at 663nm. Phy a level of processed green tea leaves was very high when compared to other plants. In parsley, bellpepper and Japanese bunching onion formation of Phy a was prominent. All these indicated that early studies of Chl degradation need further study to elucidate the mechanism of Chl degradation in green plants. The Journal of Agricultural Sciences, 2012, vol.7, no3, page 127-134 DOI: http://dx.doi.org/10.4038/jas.v7i3.4876

ChemInform Abstract: Demetalation of Chlorophyll Pigments

AbstractReview: 159 refs.

Demetalation of Chlorophyll Pigments

Natural chlorophylls (Chls) and bacteriochlorophyll (BChls), which are major pigments in photosynthesis, possess a central Mg (or Zn) in their cyclic tetrapyrrole macrocycles. Removal of the central metal atom from chlorophyllous pigments, called pheophytinization, is a biologically important reaction in that it allows production of the primary electron acceptors in photosystem II(-type) reaction centers and is one of the crucial steps in the Chl degradation pathway. Pheophytinization in processed and postharvest foods derived from vegetables and fruits has attracted considerable attention in agricultural and food chemistry from the viewpoints of maintenance of their color level and evaluation by consumers. In this review, we focus on in vivo demetalation reactions and demetalated products of chlorophyllous pigments. In addition, we summarize kinetic studies on in vitro demetalation of natural (B)Chl molecules and their synthetic analogs under acidic conditions.

Structure‐Dependent Demetalation Kinetics of Chlorophyll a Analogs under Acidic Conditions

Demetalation of chlorophyll (Chl) a and its analogs is an important reaction in oxygenic photosynthetic organisms, which produces the primary electron acceptors in photosystem II reaction centers and is crucial in the Chl degradation. From these viewpoints, demetalation reactions of four Chl a analogs, 3,8-divinyl-Chl a (DV-Chl a), 3-devinyl-3-ethyl-Chl a (mesoChl a), 13(2)-demethoxycarbonyl-Chl a (pyroChl a) and protochlorophyll a (PChl a), were kinetically analyzed under weakly acidic conditions, and were compared with that of Chl a. DV-Chl a exhibited slower demetalation kinetics than did Chl a, whereas demetalation of mesoChl a was faster than that of Chl a. The difference in demetalation kinetics of the three chlorophyllous pigments originates from the electron-withdrawing ability of the vinyl group as the peripheral substituent compared with the ethyl group. Removal of the electron-withdrawing and homoconjugating 13(2) -methoxycarbonyl group in Chl a (Chl a → pyroChl a) accelerated demetalation kinetics by two-fold. PChl a possessing the porphyrin-type skeleton exhibited slower demetalation kinetics than Chl a. The structure-dependent demetalation properties of Chl a analogs will be useful for understanding in vivo Chl demetalation reactions in oxygenic photosynthetic organisms.

Characterization of biogenic volatile organic compounds and membrane lipid peroxidation during leaf ageing in maize (Zea mays)

A study was conducted to access BVOCs emission pattern during leaf senescence in parallel with monitoring leaf Chl degradation and MDA accumulation. BVOCs emission rate and Chl content of leaves in the lower canopy position were significantly lower than leaves in the mid and up positions. While MDA and H2O2 content of the lower position leaves were significantly higher than the up and mid position leaves. Positive correlation between leaf volatile emission rate and chlorophyll content and negative correlation between leaf volatile emission rate and MDA and H2O2 content were observed. VOCs emission pattern during the cereal crop aging could provide more evidence for age-related resistance though the results here we show are in vitro.

Characterization of chlorophyll breakdown in green prickleyashes (Zanthoxylum schinifolium Zucc.) during slow drying

To investigate the possible mechanism of color degradation of green prickleyashes (Zanthoxylum schinifolium Zucc.) in slow drying, changes in their peel colors, enzyme activities, content of chlorophylls (Chls) and derivatives were evaluated. The results showed the peel color changed from brilliant green to black-brown and Chls underwent a rapid degradation. Enzyme activities changed as follows: chlorophyllase activity decreased; chlorophyll-degrading peroxidase (Chl-POD) activity as well as pheophorbidase (Pheidase) exhibited a biphase trend displaying an inverted “V” phase, and the increase in Chl-POD resulted in the accumulation of C132-hydroxy-chlorophyll a. Based on the study of enzyme activities and Chl degradation, conclusions were drawn that Chl-POD and Pheidase were considered as the key enzymes to promote chlorophyll breakdown. Compared with slow drying, fast drying could inhibit the two key enzyme activities and blockade the chlorophyll-degrading pathway, which was proposed to process green prickleyashes.

Overproduction of Chl b Retards Senescence Through Transcriptional Reprogramming in Arabidopsis

Leaf senescence is a developmentally and environmentally regulated process which includes global changes in gene expression. Using Arabidopsis as a model, we modified Chl arrangement in photosystems by overexpressing the catalytic domain (the C domain) of chlorophyllide a oxygenase (CAO) fused with the linker domain (the B domain) of CAO and green fluorescent protein (GFP). In these plants (referred to as the BCG plants for the B and C domains of CAO and GFP), the Chl a/b ratio was drastically decreased and Chl b was incorporated into core antenna complexes. The BCG plants exhibited a significant delay of both developmental and dark-induced leaf senescence. The photosynthetic apparatus, CO(2) fixation enzymes and the chloroplast structure were lost in wild-type plants during senescence, while BCG plants retained them longer than the wild type. Large-scale quantitative real-time PCR analyses of 1,880 transcription factor (TF) genes showed that 241 TFs are differentially expressed between BCG plants and wild-type plants at senescence, ∼40% of which are known senescence-associated genes (SAGs). Expression profiling also revealed the down-regulation of a large number of additional non-TF SAGs. In contrast, genes involved in photosynthesis were up-regulated, while those encoding Chl degradation enzymes were down-regulated in BCG plants. These results demonstrate that alteration of pigment composition in the photosynthetic apparatus retards senescence through transcriptional reprogramming.

Effect of Naringin Radical Formed by Peroxidase on Chlorophyll Degradation during Storage of Citrus nagato-yuzukichi Fruit

Free radical formation from flavonoids by peroxidase action and changes in flavonoid levels and peroxidase activity during storage were determined to clarify the involvement of naringin radical in chlorophyll (Chl) degradation in stored green Nagato-yuzukichi (Citrus nagato-yuzukichi hort. ex Y. Tanaka) fruit. Chl a was degraded by peroxidase-hydrogen peroxide system not in the presence of hesperidin but with naringin. However, radical formation of both naringin and hesperidin, which were the main flavonoids in the flavedo of Nagato-yuzukichi fruit, with peroxidase-mediated oxidation was found by electron spin resonance analysis. The naringin radical, which was formed by a radical forming reagent, potassium ferricyanide, was related to the formation of 132-hydroxychlorophyll (OHChl) with Chl a degradation, whereas the hesperidin radical hardly affected OHChl a formation. In addition, superoxide dismutase did not inhibit Chl a degradation by naringin radical formed by potassium ferricyanide. In Nagato-yuzukichi fruit, Chl-degrading peroxidase activity increased considerably during storage at 20°C, with a peak value on day 6 of storage in accordance with decrease in Chl content, after which it decreased. The contents of naringin and hesperidin, especially the former, decreased during storage at 20°C, but afterwards increased slightly. These findings suggest that the naringin radical formed by peroxidase could be partly involved in Chl degradation during storage in Nagato-yuzukichi fruit.

Functions of the water soluble chlorophyll-binding protein in plants

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700–900 μmol photons m −2 s −1. These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.

INVOLVEMENT OF CHLOROPHYLLASE ON CHLOROPHYLL DEGRADATION IN STORED BROCCOLI FLORETS AND ITS CONTROL BY UV TREATMENT

Yellowing is the most visible deterioration in broccoli (Brassica oleracea L. Italica Group) that usually occurs with the progress of chlorophyll (Chl) degradation. This study deals with the control of floret yellowing in stored broccoli by using UV-B irradiation at 0 (control) and 8.8 kJ.m -2 . The application of UV-B at 8.8 kJ.m -2 effectively delayed yellowing of broccoli florets. Chl a derivatives including chlorophyllide (Chlide) a, C13 2 -hydroxychlorophyll (C13 2 -OHChl) a and pheophytin (Phy) a levels in broccoli florets were decreased concomitantly with the enhancement of pheophorbide (Pheide) a and pyropheophorbide (Pyropheide) a levels especially in the control treatment. On the other hand, Chlide a, C13 2 -OHChl a and Phy a levels were increased in 8.8 kJ.m -2 UV-B treated broccoli during storage at 15°C. Two types of chlorophyllase (Chlase) were identified by molecular exclusion chromatography. Type I was suppressed in UV-B treated broccoli on day 4, and the Km value of Type I was lower than that of Type II. Thus, the Chlase activity, especially Type I, was effectively suppressed by UV-B treatment, whereas Type II Chlase may take in part of Chl degradation in stored broccoli florets.

PARTIALLY PURIFICATION OF MG-DECHELATASE IN RELATION TO CHLOROPHYLL DEGRADATION IN BROCCOLI (BRASSICA OLERACEA L. ITALICA GROUP) FLORETS

ISHS Southeast Asia Symposium on Quality and Safety of Fresh and Fresh-Cut Produce PARTIALLY PURIFICATION OF MG-DECHELATASE IN RELATION TO CHLOROPHYLL DEGRADATION IN BROCCOLI (BRASSICA OLERACEA L. ITALICA GROUP) FLORETS

Reverse Genetic Identification of CRN1 and its Distinctive Role in Chlorophyll Degradation in Arabidopsis

Recent identification of NYE1/SGR1 brought up a new era for the exploration of the regulatory mechanism of Chlorophyll (Chl) degradation. Cluster analysis of senescence associated genes with putative chloroplast targeting sequences revealed several genes sharing a similar expression pattern with NYE1. Further characterization of available T-DNA insertion lines led to the discovery of a novel stay-green gene CRN1 (Co-regulated with NYE1). Chl breakdown was significantly restrained in crn1-1 under diversified senescence scenarios, which is comparable with that in acd1-20, but much more severe than that in nye1-1. Notably, various Chl binding proteins, especially trimeric LHCP II, were markedly retained in crn1-1 four days after dark-treatment, possibly due to a lesion in disassociation of protein-pigment complex. Nevertheless, the photochemical efficiency of PSII in crn1-1 declined, even more rapidly, two days after dark-treatment, compared to those in Col-0 and nye1-1. Our results suggest that CRN1 plays a crucial role in Chl degradation, and that loss of its function produces various side-effects, including those on the breakdown of Ch-protein complex and the maintenance of the residual photosynthetic capability during leaf senescence.

Gibberellin retards chlorophyll degradation during senescence of Paris polyphylla

Chlorophyll (Chl) degradation was found to be related to the endogenous gibberellin (GA) content in shoots during senescence in the perennial plant Paris polyphylla var. yunnanensis (Franch.). Treatment with gibberellic acid (GA3) significantly increased the content of endogenous GAs (GA4 + GA7), retarded the senescence of shoots, and the degradation of proteins and Chl. Chlorophyllase, Mg-dechelation and peroxidase activities increased more in control plants than in those treated with GA3. GA3 treatment also protected lipoxygenase activity, which decreased significantly in control plants.

Continuous turnover of carotenes and chlorophyll a in mature leaves of Arabidopsis revealed by 14CO2 pulse-chase labeling.

Carotenoid turnover was investigated in mature leaves of Arabidopsis (Arabidopsis thaliana) by 14CO2 pulse-chase labeling under control-light (CL; 130 micromol photons m(-2) s(-1)) and high-light (HL; 1,000 micromol photons m(-2) s(-1)) conditions. Following a 30-min 14CO2 administration, photosynthetically fixed 14C was quickly incorporated in beta-carotene (beta-C) and chlorophyll a (Chl a) in all samples during a chase of up to 10 h. In contrast, 14C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the 14C fractions incorporated in beta-C and Chl a, whereas long-term HL acclimation significantly enhanced 14C labeling of Chl a but not beta-C. Consequently, the maximal 14C signal ratio between beta-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing alpha-carotene (alpha-C) together with reduced amounts of beta-C, remarkably high 14C labeling was found for alpha-C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum 14C ratios between carotenes and Chl a were 1:2 for alpha-C:Chl a and 1:5 for beta-C:Chl a in CL-grown lut5 plants, suggesting high turnover of alpha-C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.

Evaluation of photo-induced cellular damage using photoautotrophic cultures of puk-bung hairy roots as a sensing tool

In photoautotrophic cultures of pak-bung hairy roots, strong light irradiation (22 W/m(2)) increased the content of reactive oxygen species (ROS) in the cells, resulting in chlorophyll (Chl) degradation and DNA injury. The Chl degradation rate, R(D), can be used as a parameter to measure the cellular damage caused by photo-induced stress. The presence of the antioxidants ascorbic acid, chlorogenic acid or catechin reduced the R(D), while lowering the content of ROS and moderating the DNA injury.

Effects of postharvest ethanol vapor treatment on activities and gene expression of chlorophyll catabolic enzymes in broccoli florets

Postharvest yellowing of broccoli ( Brassica oleracea L. Italica Group) is an important indicator of quality deterioration and occurs with chlorophyll (Chl) degradation. Postharvest ethanol vapor treatment could delay the yellowing of broccoli florets, through the suppression of Chl degradation. As the first step in identifying the mechanism of this delay, the effects of postharvest ethanol vapor treatment on activities and gene expression of the Chl catabolic enzymes in broccoli were determined. Broccoli branchlets were placed in a perforated polyethylene bag with or without (control) an ethanol pad and stored at 20 °C in darkness. The Chl contents of the control broccoli florets decreased remarkably after three days in storage, whereas the contents of the ethanol-treated broccoli showed no significant changes except at day one. Changes in chlorophyllase activity in the ethanol-treated broccoli were similar to those of the control until three days in storage, but then the activity tended to decease. Mg-dechelatase and Chl-degrading peroxidase activities, which increased greatly with senescence in the controls, remained unchanged in the ethanol-treated broccoli. BoCLH1 expression showed changes almost similar to those of chlorophyllase activity. BoPAO expression in the control broccoli increased greatly at day one in storage and was maintained at a high level until three days. In contrast, this expression in ethanol-treated broccoli was suppressed until two days. BoRCCR expression in the control broccoli increased until two days in storage, while the expression in the ethanol-treated broccoli showed no change during storage. These results show that postharvest ethanol vapor treatment suppressed the activities and gene expression of Chl catabolic enzymes, resulting in delayed yellowing of broccoli florets.