Involvement of ethylene in chlorophyll degradation in peel of citrus fruits

The effect of ethylene on chlorophyll degradation in the peel of Robinson tangerine (X Citrus reticulata Blanco) and calamondin (X Citrofortunellamitis [Blanco] Ingram and Moore) fruits was studied. The chlorophyll degrading system in the peel of these two citrus species was not self-sustaining but required ethylene to function. Chlorophyll degradation ceased immediately when fruit were removed from ethylene and held in ethylene-free air at 0.2 atmospheric pressure. However, at atmospheric pressure, chlorophyll degradation continued for 24 hours in the absence of exogenous ethylene. Although chlorophyllase levels were negatively correlated with chlorophyll content in the peel (r = −0.981; P < 0.01), the level of chlorophyllase activity did not change when fruit were removed from ethylene, even though chlorophyll degradation had stopped. From these observations, it was concluded that ethylene is necessary for chlorophyll degradation in the two species of citrus studied, but its primary role is not solely for the induction of chlorophyllase activity.     

Effect of ethylene and 1-methylcyclopropene on chlorophyll catabolism of broccoli florets

Branchlets of broccoli (Brassica oleracea L.) were used to examine ethylene-stimulated chlorophyll catabolism. Branchlets treated with: 1) air (CK); 2) 1 µL·L^–1 1-methylcyclopropene (1-MCP) for 14 hr at 20 °C; 3) 1000 µL·L^–1 ethylene (C₂H₄) for 5 hr at 20 °C; or 4) 1-MCP then C₂H₄, were stored in the dark at 20 °C for up to 3 d. Chlorophyll (Chl) content and branchlet hue angle decreased during the storage period and 1-MCP treatment delayed this change. Chl degradation in broccoli was accelerated by exposure to C₂H₄, especially for Chl a. Prior treatment with 1-MCP prevented degreening stimulated by C₂H₄. Lipoxygenase activity was not altered by any of the treatments, however, 1-MCP with or without ethylene resulted in reduced activity of chlorophyllase (Chlase) and peroxidase (POD). Exposure to C₂H₄ stimulated Chlase activity and extended the duration of high POD activity. Treatment with 1-MCP followed by C₂H₄ resulted in reduced POD activity and delayed the increase in Chlase activity. The results suggest chlorophyll in broccoli can be degraded via the POD – hydrogen peroxide system. Exposure to C₂H₄ enhances activity of Chlase and extends the duration of high POD activity, and these responses may accelerate degreening. Treatment with 1-MCP delays yellowing of broccoli, an effect that may be due to the 1-MCP-induced reduction in POD and Chlase activities.     

Sugar regulation of plastid interconversions in epicarp of citrus fruit

Seasonal transformations between chloroplasts and chromoplasts, as measured by changes in chlorophyll content, in the epicarp of degreening and regreening Citrus sinensis (L.) Osbeck cv Valencia fruit closely parallelled the accumulation and later loss of soluble sugars. At any stage of development, reversing the relative soluble sugar content in the epicarp by culturing pericarp segments on agar media with low (15 millimolar) or high (150 millimolar) sucrose concentrations reversed the direction of change in chlorophyll content. Fruit of C. madurensis Lour., which mature year around and do not regreen, also accumulated soluble sugars in the pericarp as degreening was initiated.

The epicarp of C. sinensis fruit accumulated nitrogen, but total nitrogen concentrations and amino acid concentrations changed little, during degreening and regreening of C. sinensis fruit. Cessation of nitrogen fertilization reduced the tendency of pericarp segments to regreen in vitro during subsequent years, but regreening tendency was restored by inclusion of KNO₃ in the media.

It is concluded that chloroplasts become chromoplasts and citrus fruit degreen partially in response to the accumulation of sugars in the epicarp and that the reverse transformation accompanying regreening of certain citrus species occurs when accumulated sugars disappear. Change in nitrogen flux to the fruit is probably not a factor in regulating seasonal transformations, but an abundance of nitrogen in the epicarp diminishes the effects of high sugar concentrations in inducing transformation of chloroplasts to chromoplasts, thereby retarding degreening and promoting regreening.

     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

Inhibition of degreening in the peel of bananas ripened at tropical temperatures.

Changes in the plastid ultrastructure as revealed by thin-section electron-microscopy, chlorophyll a/b ratio, and the polypeptides of the thylakoid chlorophyll-protein complexes have been examined during the degreening of bananas (Musa AAA Group, Cavendish Subgroup) and plantains (Musa AAB Group, Plantain Subgroup) ripened at 20°C and 35°C. In bananas, where degreening is inhibited at temperatures above 24°C, ripening at the higher temperature results in a retention of thylakoid membranes, a relatively delayed breakdown in chlorophyll b, and a reduced dismantling of pigment-protein complexes. By contrast, in plantains, where degreening is complete within 4 days at both 20°C and 35°C, thylakoid membranes and their associated pigment-protein complexes are lost, and there is a rapid increase in chlorophyll a/b ratios at both ripening temperatures. It is suggested that the retention of thylakoid membranes is an important factor in the failure of Cavendish bananas to degreen when ripened at tropical temperatures, and that the degreening problem may be related to the comparatively high chlorophyll b content of the preclimacteric fruit.     

Effect of Cytokinins on Photosynthetic Pigments and Chlorophyllase Activity in in Vitro Cultures of Axillary Buds of Dianthus caryophyllus L.

The effect of benzyladenine (BA) and two phenylurea cytokinins, N-phenyl-N′-(2-chloro-4-pyridyl)urea (4-PU-30) and thidiazuron (TDZ), on the growth, photosynthetic pigment content, and activity of chlorophyllase (chlorophyll-chlorophylliodhydrolase, EC 3.1.1.14) of in vitro cultures of carnations was studied. All cytokinins caused a rise in the fresh weigth and a drop in the dry weight of leaf mass produced by the explanted buds. Both 4-PU-30 and TDZ increased the chlorophyll content and this correlated with changes in chlorophyllase activity. The effect of 4-PU-30 and TDZ was similar to that caused by BA but at 10-fold or 100-fold lower concentrations. The application of higher concentrations of the phenylurea cytokinins caused an increase in the chlorophyll a/chlorophyll b ratio. However, at equimolar concentrations, the purine and both phenylurea cytokinins had opposite effects, probably indirect and related to some malformations caused by phenylureas. 4-PU-30 increased, but TDZ decreased, photosynthetic membrane stability, which argues for a different molecular organization of the chloroplast membranes. Received February 26, 1996; accepted May 30, 1997     

The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis

A mutant of Arabidopsis thaliana with reduced content of C_18:3 and C_16:3 fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO₂-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.     

Probing the role of endogenous ethylene in the degreening of citrus fruit with ethylene antagonists

The ethylene antagonists, 2,5-norbornadiene (NBD) and silver nitrate, were used to probe the involvement of endogenous ethylene in the natural degreening of citrus fruit. Mature-green, detached Shamouti orange (Citrus sinensis L. Osbeck) fruit were treated with NBD vapor or dipped in solutions of silver nitrate. More than 80% of the chlorophyll was lost from control fruit after 8 days. NBD (0.11 mmole/liter) inhibited the loss of chlorophyll by 60%. NBD also antagonized the degreening induced by exogenous ethylene by 50%. Silver nitrate (0.1 mM) inhibited the loss of chlorophyll by 55%. Ethylene evolution of mature, green detached fruit was <2 nl.fruit^-1.h^-1 (ca. 13.5 nl.Kg^-1FW.h^-1) and did not change significantly for 7 days after harvest. NBD concentrations up to 0.22 mmole/liter did not enhance ethylene evolution. Not with-standing the extremely low amounts of ethylene evolved, the inhibition of degreening by NBD and silver nitrate suggests that endogenous ethylene is involved in the control of this process in mature citrus fruit.     

Studies on well coupled Photosystem I-enriched subchloroplast vesicles. Content and redox properties of electron-transfer components

Stable and well coupled Photosystem (PS) I-enriched vesicles, mainly derived from the chloroplast stroma lamellae, have been obtained by mild digitonin treatment of spinach chloroplasts. Optimal conditions for chloroplast solubilization are established at a digitonin/chlorophyll ratio of 1 ($\text{w}\text{w}$) and a chlorophyll concentration of 0.2 mM, resulting in little loss of native components. In particular, plastocyanin is easily released at higher digitonin/chlorophyll ratios. On the basis of chlorophyll content, the vesicles show a 2-fold enrichment in ATPase, chlorophyll-protein Complex I, P-700, plastocyanin and ribulose-1,5-bisphosphate carboxylase as compared to chloroplasts, in line with the increased activities of cyclic photophosphorylation and PS I-associated electron transfer as shown previously (Peters, A.L.J., Dokter, P., Kooij, T. and Kraayenhof, R. (1981) in Photosynthesis I (Akoyunoglou, G., ed.), pp. 691–700, Balaban International Science Services, Philadelphia). The vesicles have a low content of the light-harvesting chlorophyll-protein complex and show no PS II-associated electron transfer. Characterization of cytochromes in PS I-enriched vesicles and chloroplasts at 25°C and 77 K is performed using an analytical method combining potentiometric analysis and spectrum deconvolution. In PS I-enriched vesicles three cytochromes are distinguished: c-554 (E′₀ = 335 mV), b-559_LP (E′₀ = 32 mV) and b-563 (E′₀ = ? 123 mV); no b-559_HP is present (LP, low-potential; HP, high-potential). Comparative data from PS I vesicles and chloroplasts are consistent with an even distribution of the cytochrome b-563- cytochrome c-554 redox complex in the lateral plane of exposed and appressed thylakoid membranes, an exclusive location of plastocyanin in the exposed membranes and a dominant location of plastoquinone in the appressed membranes. The results are discussed in view of the lateral heterogeneity of redox components in chloroplast membranes.     

Ethylene Production and Leaflet Abscission in Mèlia azédarach L

Ethylene production or content was compared to leaflet abscission in detached, compound leaves of Mèlia azédarach L. In late autumn, when abscission was progressing from basal leaves upward, the oldest leaves both produced ethylene at the highest rates and abscised their leaflets first. When C₂H₄ levels were measured in intercellular air removed immediately after leaves were harvested, C₂H₄ levels were also highest in basal leaves and declined progressively in more apical leaves. Levels as high as 1.8 microliters C₂H₄ liter⁻¹ air were observed. Earlier in the season groups of leaves demonstrated a pattern of sequential initiation of abscission from base to apex, but the peak rates of C₂H₄ production followed an opposite trend, being highest in the youngest leaves. Peak rates of C₂H₄ production occurred after the initiation of leaflet abscission and presumably are related to either the auxin content or a climacteric-like, autocatalytic phase of C₂H₄ production not directly involved in the initiation of abscission. In these experiments, the early abscission of the older leaflets reflects their greater sensitivity to C₂H₄, presumably due to lower auxin content. C₂H₄ production rates in all experiments, with rare exceptions, exceeded 3 microliters per kilogram fresh weight per hour at least 24 hours before leaflet abscission reached 10%. This achieving of a threshold internal C₂H₄ level is viewed as an initiating event in leaflet abscission. Hypobaric conditions, to facilitate the escape of endogenous C₂H₄, delayed abscission compared to controls, and termination of hypobaric exposure allowed a normal progression of abscission as well as normal C₂H₄ synthesis rates. All of the data indicate that C₂H₄ initiates leaflet abscission in intact but detached leaves of Mèlia azédarach L. The seasonal patterns observed suggest that C₂H₄, in concert with those hormones which govern sensitivity to C₂H₄, regulate autumn leaf fall in this species.     

Control of Chlorophyll Degradation in Detached Leaves of Barley and Oat through Effect of Kinetin on Chlorophyllase Levels

Chlorophyllase seems to be responsible for the degradation of chlorophyll during senescence of detached leaves of barley (Hordeum vulgare) and oat (Avena sativa). Treatment at temperatures higher than 40°C — which protects against chlorophyll loss — lowers the level of chlorophyllase. Kinetin treatment lowers the level of chlorophyllase in barley leaves and prevents its rise in oat leaves after their detachment. Synthesis of proteins in both cytoplasm and chloroplast seems to be required in order to maintain a high level of Chlorophyllase in barley leaves after detachment.     

GUN4-porphyrin complexes bind the ChlH/GUN5 subunit of Mg-Chelatase and promote chlorophyll biosynthesis in Arabidopsis

The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranes—the site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O₂. To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding–deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mg-chelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex.     

Effect of gamma radiation on the ripening of bartlett pears

Gamma radiation at doses of 300 Krad or more inhibits the ripening of Bartlett pears (Pyrus communis L.). Immediately after irradiation there is a transitory burst of C₂H₄, which subsequently declines in fruits subjected to inhibitory doses. Ethylene production associated with ripening begins at the same time in unirradiated fruits and those subjected to noninhibitory doses, but the latter produces much more C₂H₄ at the climacteric peak. Fruits subjected to inhibitory doses produce low levels of C₂H₄ unless subjected to exogenously applied C₂H₄, whereupon they produce enough of the gas to induce ripening in unirradiated fruits.

Pears subjected to 300 and 400 Krad of gamma rays did not ripen even when held in a flowing atmosphere containing 1000 ppm of C₂H₄ for 8 days at 20°. It is concluded that the action of gamma rays on Bartlett pears involves both an inhibition of C₂H₄ production and a decreased sensitivity of the fruit to the ripening action of the gas. Ripening of Bartlett pears is inhibited by gamma radiation only when applied to preclimacteric fruit.

     

Fruit photosynthesis

Abstract. In addition to photosynthesis as in the leaf, fruit possess a system which refixes CO₂ from the mitochondrial respiration of predominantly imported carbon. This pathway produces malate by the action of phosphoenolpyruvate carboxylase, PEPC, (E.C. 4.1.1.31) and appears to be regulated primarily by the cytosolic concentration of HCO₃/CO₂ and malate. Malate is stored in the vacuole as malic acid, constituting a major carbon pool and a potential substrate for respiration. The PEPC in apple fruit proves to be an efficient form of the enzyme with low Michaelis constants, i.e. Km = 0.09 mol m^-3 PEP and 0.2 mol m^–3 HCO₃, and large Ki= 110 mol m^-3 HCO³. In fleshy fruit, chlorophyll and chloroplasts are unevenly distributed; they resemble the C₃ sun-type and arc concentrated in the perivascular tissue, with smaller chloroplasts, fewer grana per chloroplast and a larger degree of vacuolation than commonly found in a leaf of the same species. Fruit photosynthesis often compensates for respiratory CO₂ loss in the light. However, due to respiration in the dark, CO₂ loss is in excess of photosynthetic gain in the light, such that a continual loss of CO₂ was observed in the diurnal cycle and which is maintained throughout fruit development. The rate of CO₂ exchange decreases on a fresh weight or surface basis, but increases with fruit ontogeny on a per fruit basis, causing accumulation of several percent CO₂ in the internal cavity. Stomata are present in the outer epidermis of those fruits examined, but with a 10-to 100-fold lesser frequency than in the abaxial epidermis of leaf of the same species. The number of Stomata is set at anthesis and remained constant, while the stomatal frequency decreases as the fruit surface expands. Stomata are as sensitive as in leaves in the early stages of fruit development, but often are transformed into lenticels during fruit ontogeny, thereby decreasing the permeability of the outer epidermis. The discrepancy between the CO₂-concentrating mechanism provided by PEPC analogous to C₄/CAM Photosynthesis and the kinetics of fruit PEPC, characteristic of C₃/non-autotrophic tissue, suggests the definition of a new type of ‘fruit photosynthesis’ rather than its categorization within an existing type.     

Localization of chlorophyllase in the chloroplast envelope

Chlorophyllase catalyzes the first step in the catabolic pathway of chlorophyll. It is a constitutive enzyme located in chloroplast membranes. In isolated plastids the hydrolysis of the endogenous chlorophyll does not take place unless the membranes are solubilized in the presence of detergent. The structural latency of chlorophyllase activity appears to be due to the differential locations of substrate and enzyme within the plastids. Envelope membranes prepared from both chloroplasts and gerontoplasts contain chlorophyllase activity. The isolation of envelopes is associated with a marked increase in chlorophyllase activity per unit of protein. Yields of chlorophyllase and of specific envelope markers in the final preparations are similar, suggesting that the enzyme may be located in the envelope. It is hypothesized that the breakdown of chlorophyll during leaf senescence requires a mechanism that mediates the transfer of chlorophyll from the thylakoidal pigment-protein complexes to the sites of catabolic reactions in the envelope.Abbreviations ACT acyl CoA thioesterase - Chl chlorophyll - Chlide chlorophyllide - PC phosphatidylcholine     

Ethylene and carbon dioxide production by developing strawberries show a correlative pattern that is indicative of ripening climacteric fruit

Laser photoacoustic spectroscopy continuously quantified the ethylene (C₂H₄) produced by strawberry flowers and fruits developing in planta. C₂H₄ was first detected as flower buds opened and exhibited diurnal oscillations (to approximately 200 pl flower^?1 h^?1) before petal abscission. Exogenous application of silver thiosulphate (STS) to detached flowers inhibited petal abscission and flower senescence. In fruit, C₂H₄ production was maintained at a ‘low level’ (10–60 pl fruit^?1 h^?1) until fruit expanded when levels increased in a diurnal pattern (to 200 pl fruit^?1 h^?1). After expansion, C₂H₄ production declined to a low level until fruit attained the red‐ripe stage for at least 24 h. After this time, C₂H₄ levels increased linearly (no diurnal fluctuation) to approximately 1 nL fruit^?1 h^?1. Twenty‐four hours after the re‐initiation of C₂H₄ production by red fruit, CO₂ levels increased approximately three‐fold, indicative of a respiratory climacteric. STS applied to fruits developing in planta and dissected fruit parts ex situ established that C₂H₄ production is regulated by negative feedback until fruits had expanded. The C₂H₄ produced by red‐ripe fruit was regulated by positive feedback. Anti‐1‐amino‐cyclopropane‐1‐carboxylic acid oxidase IgG localization identified immunoreactive antigens of 40 and 30 kDa (M_r) within the fruit achenes of expanding and red‐ripe fruit. Analysis of dissected fruit showed that seed C₂H₄ accounts for 50% the C₂H₄ that is detectable from ripe fruit.     

Lipid and Fatty Acid composition of chloroplast envelope membranes from species with differing net photosynthesis

Lipid and fatty acid compositions were determined for chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L.) leaves. The lipid composition was similar in sunflower, spinach, and undifferentiated maize chloroplast envelope membranes and different in maize mesophyll chloroplast envelope membranes. The predominant lipid constituents in all envelope membranes were monogalactosyldiglyceride (27 to 46%), digalactosyldiglyceride (18 to 33%), and phosphatidylcholine (7 to 30%). The fatty acid composition was also similar in sunflower and spinach chloroplast envelope membranes in comparison to those from maize. The major acyl fatty acids of the chloroplast envelope membrane were palmitic (C_16:0, 41 and 36%) and linolenic (C_18:3, 29 and 40%) acids for spinach and sunflower; palmitic (77%) and stearic (C_18:0, 12%) acids for young maize; and palmitic (61%), stearic (14%), and linolenic (13%) acids for mature maize. The differences in lipid and acyl fatty acid compositions among these plants which vary in their rates of net photosynthesis were largely quantitative rather than qualitative.