Role of chlorophyllase in chlorophyll homeostasis and post-harvest breakdown in Piper betle L. leaf

Piper betle L., a dioecious shade-loving perennial climber is one of the important Pan-Asiatic plants. More than hundred landraces having marked variation in leaf chlorophyll (Chl) content are in cultivation in India. In this study, role of chlorophyllase (Chlase) in Chl homeostasis and post-harvest breakdown was investigated in two contrasting P. betle landraces Kapoori Vellaikodi (KV) with light green and Khasi Shillong (KS) with dark green leaves. The two landraces showed negative correlation between Chl content and Chlase activity in fresh as well as stored leaves. Accumulation of chlorophyllide a (Chlid a) was correlated with the level of Chlase activity, which was higher in KV than KS. The overall response of abscisic acid (ABA) and benzylaminopurine (BAP) was similar in KV and KS, however, the time-course was different. ABA-induced Chl loss was accompanied by rise in Chlase activity in KV and KS and the delay in Chl loss by BAP was accompanied by reduction in Chlase activity. While there were significant differences in Chlase activity in KV and KS, only minor differences were observed in the enzyme properties like pH and temperature optima, Km and Vmax. No landrace-related differences were observed on the effect of metal ions and functional group reagents/amino acid effectors on Chlase activity. These results showed that despite significant differences in Chl content and Chlase activity between landraces KV and KS, the properties of Chlase were similar. The findings show that in P. betle Chlase is involved in Chl homeostasis and also in Chl degradation during post-harvest storage and responds to hormonal regulations. These findings might be useful in predicting the stability of Chl during post-harvest storage and also the shelf-life in other P. betle landraces.     

The Pool of Chlorophyllous Pigments in Barley Seedlings of Different Ages under Heat Shock and Water Deficit

The effects of a high temperature (3 h, 40°C) and water deficit (45 h on 3% PEG 6000) on the pool of chlorophyllous pigments in the leaves of 4-, 7-, and 11-day-old barley (Hordeum vulgare L.) seedlings were studied. Heating resulted in a decrease in the total content of chlorophylls (Chl) (a + b) in 4-day-old plants but not in the older leaves. Water deficit induced an increase in the pigment content in young seedlings but reduced it in the leaves of 11-day-old plants. In young seedlings, hyperthermia and dehydration affected similarly Chl (a + b) degradation, leading to a marked inhibition of the chlorophyllase (Chlase) activity hydrolyzing Chl to chlorophyllides and phytol. In old leaves, an activation of this enzyme was observed. The stress factors under study affected different stages of pigment biosynthesis. High temperature inhibited the activity of dark and light stages of Chl(a + b) biosynthesis. Dehydration did not change markedly the resynthesis of protochlorophyllide, while the enzymes of the light stage of Chl biosynthesis were activated in young but inhibited in old barley leaves. The results thus obtained allowed us to conclude that heat treatment and dehydration specifically affected the Chl biosynthesis. At the same time, the Chlase response was nonspecific.     

Characterization and subcellular localization of chlorophyllase from Ginkgo biloba

Chlorophyllase (Chlase) catalyzes the initial step of chlorophyll (Chl)-degradation, but the physiological significance of this reaction is still ambiguous. Common understanding of its role is that Chlase is involved in de-greening processes such as fruit ripening, leaf senescence, and flowering. But there is a possibility that Chlase is also involved in turnover and homeostasis of Chls. Among the de-greening processes, autumnal coloration is one of the most striking natural phenomena, but the involvement of Chlase during autumnal coloration is not clear. Previously, it was shown that Chlase activity and expression level of the Chlase gene were not increased during autumnal coloration in Ginkgo biloba, indicating that Chlase does not work specially in the de-greening processes in G. biloba. In this study, we characterized the recombinant Chlase and analyzed its subcellular localization to understand the role of the cloned Chlase of G. biloba (GbCLH). GbCLH exhibited its highest activity at pH 7.5, 40 degrees C. Kinetic analysis revealed that GbCLH hydrolyzes pheophytin (Pheo) a and Chl a more rapidly than Pheo b and Chl b. Transient expression analysis of 40 N-terminus amino acids of GbCLH fused with GFP (green fluorescent protein) and subcellular fractionation showed that GbCLH localizes within chloroplasts. Together with our previous results, property of GbCLH and its location within the chloroplasts suggest that GbCLH plays a role in the turnover and homeostasis of Chls in green leaves of G. biloba.     

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.     

Effects of aluminium on photosynthetic performance in Al-sensitive and Al-tolerant maize inbred lines

Maize plant inbred lines, one Al-sensitive (B-73) and two Al-tolerant (F-2 and L-2039), were grown hydroponically in the presence of 200 μM Al. After 13 d of growth, root and shoot lengths, photosystem 2 (PS2) activity, chlorophyll (Chl) content, 5-aminolevulinic acid (5-ALA) synthesis rate, chlorophyllase (Chlase) activity, and N, Mg, Fe, and Mn contents in leaves were determined. PS2 activity and Chl content were most severely affected by Al in B-73, but F-2 was almost unaffected. This was in accordance with Al-accumulation in the plants. The observed changes in B-73 coincided with 5-ALA synthesis inhibition, Chlase activation, and leaf deprivation of Fe and Mg. In Al-treated L-2039 plants, the leaf Mg and Mn contents were decreased. Also, an excessive Chlase activation was found in Al-treated L-2039, without a substantial Chl loss. This may indicate the activation of different enzyme pools in tolerant and sensitive genotypes under low-stress conditions.     

Gender-specific responses of Piper betle L. to low temperature stress: changes in chlorophyllase activity

Gender based differences in response to low temperature stress in leaf chlorophyll (Chl), and carotenoids (Car) contents and chlorophyllase (Chlase) activity were monitored in male (Kapoori Vellaikodi and Madras Pan Kapoori) and female (Bangla Mahoba, Desi Bangla and Kaker) betel vine landraces. Although female plants contained nearly two fold more Chl than male counterparts, the low temperature induced Chl loss was comparable, however, male plants showed higher Chl a/b ratio than females. Chlase activity increased due to cold stress in all the landraces. Male plants always showed higher activities of Chlase, which may be one of the reasons for the rather low Chl contents in male plants.     

Ferredoxin limits cyclic electron flow around PSI (CEF-PSI) in higher plants--stimulation of CEF-PSI enhances non-photochemical quenching of Chl fluorescence in transplastomic tobacco

We tested the hypothesis that ferredoxin (Fd) limits the activity of cyclic electron flow around PSI (CEF-PSI) in vivo and that the relief of this limitation promotes the non-photochemical quenching (NPQ) of Chl fluorescence. In transplastomic tobacco (Nicotiana tabacum cv Xanthi) expressing Fd from Arabidopsis (Arabidopsis thaliana) in its chloroplasts, the minimum yield (F(o)) of Chl fluorescence was higher than in the wild type. F(o) was suppressed to the wild-type level upon illumination with far-red light, implying that the transfer of electrons by Fd-quinone oxidoreductase (FQR) from the chloroplast stroma to plastoquinone was enhanced in transplastomic plants. The activity of CEF-PSI became higher in transplastomic than in wild-type plants under conditions limiting photosynthetic linear electron flow. Similarly, the NPQ of Chl fluorescence was enhanced in transplastomic plants. On the other hand, pool sizes of the pigments of the xanthophyll cycle and the amounts of PsbS protein were the same in all plants. All these results supported the hypothesis strongly. We conclude that breeding plants with an NPQ of Chl fluorescence increased by an enhancement of CEF-PSI activity might lead to improved tolerance for abiotic stresses, particularly under conditions of low light use efficiency.     

Photodamage of the photosynthetic apparatus and its dependence on the leaf developmental stage in the npq1 Arabidopsis mutant deficient in the xanthophyll cycle enzyme violaxanthin de-epoxidase

The npq1 Arabidopsis mutant is deficient in the violaxanthin de-epoxidase enzyme that converts violaxanthin to zeaxanthin in excess light (xanthophyll cycle). We have compared the behavior of mature leaves (ML) and developing leaves of the mutant and the wild type in various light environments. Thermoluminescence measurements indicated that high photon flux densities (>500 micromol m(-2) s(-1)) promoted oxidative stress in the chloroplasts of npq1 ML, which was associated with a loss of chlorophyll and an inhibition of the photochemical activity. Illuminating leaf discs in the presence of eosin, a generator of singlet oxygen, brought about pronounced lipid peroxidation in npq1 ML but not in wild-type leaves. No such effects were seen in young leaves (YL) of npq1, which were quite tolerant to strong light and eosin-induced singlet oxygen. Non-photochemical energy quenching was strongly inhibited in npq1 YL and ML and was not improved with high-light acclimation. Our results confirm that the xanthophyll cycle protects chloroplasts from photooxidation by a mechanism distinct from non-photochemical energy quenching and they reveal that the absence of xanthophyll cycle can be compensated by other protective mechanisms. npq1 YL were observed to accumulate considerable amounts of vitamin E during photoacclimation, suggesting that this lipophilic antioxidant could be involved in the high phototolerance of those leaves.     

Effects of NaCl on photosynthetic pigments, saccharides, and chloroplast ultrastructure in leaves of tomato cultivars

In leaves of four tomato (Lycopersicon esculentum Mill.) cultivars (Red Cloud, Floradade, Peto 95, and Scorpio) the contents of chlorophyll (Chl) (a+b), Chl a, and -carotene decreased due to 100 mM NaCl treatment as compared with those of controls. The contents of soluble sugars and total saccharides were significantly increased in leaves of NaCl-treated plants, but the starch content was not significantly affected. Transmission electron microscopy indicated that in leaves of NaCl-treated plants, the chloroplasts were aggregated, the cell membranes were distorted and wrinkled, and there was no sign of grana and thylakoid structures in chloroplasts.     

Chlorophyllase Activity and Chlorophyll Content in Wild Type and eti 5 Mutant of Arabidopsis thaliana Subjected to Low and High Temperatures

Chlorophyll a (Chl a) content and chlorophyllase (Chlase) activity from leaves of wild type (WT) and the ethylene-insensitive mutant (eti 5) of Arabidopsis thaliana (L.) Heynh during temperature stress and plant recovery have been studied. The plants were subjected to temperatures of 4 °C (LT) and 38 °C (HT) for 24 h. Chl a gradually decreased somewhat during stress and in the first day of recovery, especially in HT-treated plants. At the end of the experimental period (1 d stress and 10 d recovery) Chl a content was lower in eti 5 plants than in WT ones. The Chlase in WT was more affected than in eti 5 plants during the temperature treatment and the recovery period. This revised version was published online in July 2006 with corrections to the Cover Date.     

The photoprotective role of epidermal anthocyanins and surface pubescence in young leaves of grapevine (Vitis vinifera)

BACKGROUND AND AIMS: Depending on cultivar, surfaces of young leaves of Vitis vinifera may be glabrous-green ('Soultanina') or transiently have anthocyanins ('Siriki') or pubescence ('Athiri'). A test is made of the hypothesis that anthocyanins and pubescence act as light screens affording a photoprotective advantage to the corresponding leaves, and an assessment is made of the magnitude of their effect. METHODS: Measurements were made on young leaves of the three cultivars in spring under field conditions. Photosynthetic gas-exchange and in vivo chlorophyll fluorescence were measured. Photosynthetic and photoprotective pigments were analysed by HPLC. KEY RESULTS: Compared with glabrous-green leaves, both anthocyanic and pubescent leaves had greater dark-adapted PSII photochemical efficiency and net photosynthesis. In leaves possessing either anthocyanins or pubescence, the ratio of xanthophyll cycle components to total chlorophyll, and mid-day de-epoxidation state of the xanthophyll cycle were considerably smaller, than in glabrous-green leaves. These differences were more evident in pubescent leaves, probably indicating that trichomes were more effective in decreasing light stress than anthocyanins in the epidermis. CONCLUSIONS: Light screens, especially in the form of pubescence, decrease the risk of photoinhibition whilst allowing leaves to maintain a smaller content of xanthophyll cycle components and depend less on xanthophyll cycle energy dissipation. This combination of photoprotective features, i.e. decreased photon flux to the photosynthetic apparatus and lower xanthophyll cycle utilization rates may be particularly advantageous under stressful conditions.     

Toxicity of Fusarium mycotoxins on maize plants

Synergistic effects of Fusarium — toxins mixture (moniliformin, fumonisin B₁, fusaproliferin, zearalenone, zearalenol and deoxynivalenol, each at concentration 3.5 μg mL-1) on maize plants of resistant and susceptible cultivars were studied. After 72-hour treatment the biomass production with both cultivars was approximately 6% lower than in the respective controls. In the resistant cultivar chlorophyll content was increased comparing to control, with higher increase in chlorophyll b. In susceptible cultivar chlorophyll content was slightly decreased, particularly with chlorophyll b.No significant differences between treated and non-treated plants were found in the cell ultrastructure. In susceptible plants the young root cells were more vacuolated and plasmolysis occurred in the cells of outer cortex. In leaves of this cultivar also disorganization of thylakoids in some chloroplasts was observed.     

Biosynthesis,accumulation and emission of carotenoids, α-tocopherol,plastoquinone, and isoprene in leaves under high photosynthetic irradiance

The localization of isoprenoid lipids in chloroplasts, the accumulation of particular isoprenoids under high irradiance conditions, and channelling of photosynthetically fixed carbon into plastidic thylakoid isoprenoids, volatile isoprenoids, and cytosolic sterols are reviewed. During leaf and chloroplast development in spring plastidic isoprenoid biosynthesis provides primarily thylakoid carotenoids, the phytyl side-chain of chlorophylls and the electron carriers phylloquinone K1, alpha-tocoquinone and alpha-tocopherol, as well as the nona-prenyl side-chain of plastoquinone-9. Under high irradiance, plants develop sun leaves and high light (HL) leaves with sun-type chloroplasts that possess, besides higher photosynthetic CO2 assimilation rates, different quantitative levels of pigments and prenylquinones as compared to shade leaves and low light (LL) leaves. After completion of chloroplast thylakoid synthesis plastidic isoprenoid biosynthesis continues at high irradiance conditions, constantly accumulating alpha-tocopherol (alpha-T) and the reduced form of plastoquinone-9 (PQ-9H2) deposited in the steadily enlarging osmiophilic plastoglobuli, the lipid reservoir of the chloroplast stroma. In sun leaves of beech (Fagus) and in 3-year-old sunlit Ficus leaves the level of alpha-T and PQ-9 can exceed that of chlorophyll b. Most plants respond to HL conditions (sun leaves, leaves suddenly lit by the sun) with a 1.4-2-fold increase of xanthophyll cycle carotenoids (violaxanthin, zeaxanthin, neoxanthin), an enhanced operation of the xanthophyll cycle and an increase of beta-carotene levels. This is documented by significantly lower values for the weight ratio chlorophylls to carotenoids (range: 3.6-4.6) as compared to shade and LL leaves (range: 4.8-7.0). Many plant leaves emit under HL and high temperature conditions at high rates the volatile compounds isoprene (broadleaf trees) or methylbutenol (American ponderosa pines), both of which are formed via the plastidic 1-deoxy-D: -xylulose-phosphate/2-C-methylerythritol 5-phosphate (DOXP/MEP) pathway. Other plants by contrast, accumulate particular mono- and diterpenes. Under adequate photosynthetic conditions the chloroplastidic DOXP/MEP isoprenoid pathway essentially contributes, with its C5 isoprenoid precusors, to cytosolic sterol biosynthesis. The possible cross-talk between the two cellular isoprenoid pathways, the acetate/MVA and the DOXP/MEP pathways, that preferentially proceeds in a plastid-to-cytosol direction, is shortly discussed.     

Plant aging increases oxidative stress in chloroplasts

Aging has received considerable attention in biomedicine, but little is known about the regulatory mechanisms responsible for the aging not associated with senescence in plants. This study provides new insights into the relationship between oxidative stress and plant aging, and points out chloroplasts as one of the target organelles of age-associated oxidative stress in plants. We simultaneously analyzed lipid oxidation, photosynthesis, chlorophyll content, de-epoxidation state of the xanthophyll cycle, and levels of chloroplastic antioxidant defenses such as beta-carotene and alpha-tocopherol in leaves of the same age in 1-, 3- and 7-year-old Cistus clusii Dunal plants growing under Mediterranean field conditions. Enhanced formation of malondialdehyde in leaves (2.7-fold) and chloroplasts (2.8-fold), decreased photosynthetic activity (25%), and lower chlorophyll (ca. 20%) and chloroplastic antioxidant defense levels (ca. 25%-85%) were observed in 7-year-old plants, when compared with 1- and 3-year-old plants. The differences observed, which were associated with plant aging, were only noticeable in mature nonsenescing plants (7-year-old plants). No differences were observed between pre-reproductive (1-year-old plants) and young plants (3-year-old plants). This study shows that from a certain age, oxidative stress increases progressively in chloroplasts as plants age, whereas photosynthesis is reduced. The results indicate that the oxidative stress associated with the aging in plants accumulates progressively in chloroplasts, and that the contribution of oxidative stress to aging increases as plants age.     

Enhanced Employment of the Xanthophyll Cycle and Thermal Energy Dissipation in Spinach Exposed to High Light and N Stress

The involvement of the xanthophyll cycle in photoprotection of N-deficient spinach (Spinacia oleracea L. cv Nobel) was investigated. Spinach plants were fertilized with 14 mM nitrate (control, high N) versus 0.5 mM (low N) fertilizer, and grown under both high- and low-light conditions. Plants were characterized from measurements of photosynthetic oxygen exchange and chlorophyll fluorescence, as well as carotenoid and cholorophyll analysis. Compared with the high-N plants, the low-N plants showed a lower capacity for photosynthesis and a lower chlorophyll content, as well as a lower rate of photosystem II photosynthetic electron transport and a corresponding increase in thermal energy dissipation activity measured as nonphotochemical fluorescence quenching. The low-N plants displayed a greater fraction of the total xanthophyll cycle pool as zeaxanthin and antheraxanthin at midday, and an increase in the ratio of xanthophyll cycle pigments to total chlorophyll. These results indicate that under N limitation both the light-collecting system and the photosynthetic rate decrease. However, the increased dissipation of excess energy shows that there is excess light absorbed at midday. We conclude that spinach responds to N limitation by a combination of decreased light collection and increased thermal dissipation involving the xanthophyll cycle.     

Effects of changes in growth temperature on photosynthesis and carotenoid composition in Zea mays leaves

The effects of changes in growth temperature on photosynthesis and carotenoid composition were examined in Zea mays L. leaves of different age and different developmental history. The plants were first grown at sub-optimal temperature (14°C) until the full development of the third leaf. At that time, the mature third leaf and the immature fourth leaf had a low chlorophyll (Chl) content, a low Chl a/b ratio, a high carotenoid/Chl a+b ratio, a high xanthophyll/β-carotene ratio, and about 80% of the xanthophyll cycle pool (violaxanthin [V] + antheraxanthin [A] + zeaxanthin [Z]) was in the form of zeaxanthin and antheraxanthin. When the temperature was increased from 14°C to 24°C for three days, increased Chl synthesis, accompanied by an increase in the Chl a/b ratio, took place. The ratios of lutein, neoxanthin, and V+A+Z to Chl a+b decreased markedly, whereas no significant changes appeared in the β-carotene/Chl a+b ratio. Furthermore, there was a sharp decrease in the xanthophyll/β-carotene ratio and most of zeaxanthin was converted to violaxanthin in the xanthophyll cycle. The third leaf and the tip segment of the fourth leaf, both expanded at 14°C, showed little difference in their pigment contents. However, the rate of CO₂ assimilation of the tip segment of the fourth leaf was nearly twice that of the third leaf on the third day at 24°C, while the photosynthetic activity was similar in both leaves before the transfer to 24°C. During the warm period at 24°C, new leaf tissue (basal segment of the fourth leaf and part of a fifth leaf) was formed. On the third day at 24°C, the pigment content of 24°C-grown leaf tissue did not differ much from that of 14°C-grown leaf tissue with the exception that the total carotenoid content was lower in the former as compared to the latter, mainly because of a lower V+A+Z content. The rate of CO₂ assimilation of 24°C-grown leaf tissue was comparable to that of the tip segment of the fourth leaf. Regardless of which leaf tissue is considered, reducing the temperature from 24°C to 14°C for 5 days slightly affected the pigment content, but violaxanthin was largely converted to zeaxanthin and antheraxanthin in the xanthophyll cycle. The results indicate that compared to old leaf tissue of mature leaves, physiologically younger leaf tissue of immature leaves is much more able to recover from depressions in the photosynthetic activity induced by growth at sub-optimal temperature when the plants experience optimal growth temperatures, but that factors other than the pigment content must determine this capability.     

The rapid yellowing of spruce at a mountain site in the Central Black Forest (Germany). Combined effects of Mg deficiency and ozone on biochemical, physiological and structural properties of the chloroplasts

Biochemical, physiological and ultrastructural changes of the chloroplasts were examined in the course of the rapid yellowing process of spruce (Picea abies (L.) Karst.) at a Mg-deficient and ozone polluted mountain site (Sch?llkopf mountain, Central Black Forest, Germany, 840 m a.s.l.). While at an early stage of yellowing the chlorophyll (Chl) content of the needles decreased slowly, significant changes occurred in the chloroplasts: The lability of the light-harvesting Chl a/b protein complex LHC II increased; the thylakoid cross-sectional area of chloroplasts in the outer mesophyll of the needles decreased, and their Chl fluorescence showed typical changes like the decrease of Fv/Fm and the increase of the photoinhibitory Fv quenching. Later on, the Chl content decreased rapidly, the changes in the chloroplasts continued and the needles turned yellow. Lutein and the pigments of the xanthophyll cycle were enhanced in relation to Chl a. Light and dark reactions of the xanthophyll cycle were highly active indicating efficient proton pumping and NADPH formation. The ratio of nonappressed to appressed thylakoid membranes increased with decreasing Fv/Fm suggesting that structural and fluorescence properties of the chloroplasts were related. The response of the needles to defined shading and improved Mg supply was also examined. The combined effects of strong sun light, low levels of non-Chl-bound Mg (Mg(free)) and ozone concentrations exceeding 80 microg m(-3) are shown to be necessary to induce the rapid yellowing process. For needles with Mg(free) < 0.12 mg g(-1) needle dry matter, the lability of the LHC II was correlated with the ozone concentration suggesting that the destabilization of the LHC II plays a central role in the rapid yellowing process.