Triton X-100 reacts with chlorophyll in the presence of chlorophyllase

Chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) catalyses the transesterification of chlorophylls with the surfactant Triton X-100, which is widely used in the preparation and study of this enzyme. The preparation and some properties of water-soluble tritonyl chlorophyllide esters are described. A mechanism for the role of Triton X-100 as an inhibitor in chlorophyllase-catalyzed hydrolysis and transesterification of chlorophylls is proposed. Bacteriochlorophyl a also has been employed as a substrate for green plant chlorophyllase.     

Involvement of Arabidopsis glutaredoxin S14 in the maintenance of chlorophyll content

Plant class‐II glutaredoxins (GRXs) are oxidoreductases carrying a CGFS active site signature and are able to bind iron–sulfur clusters in vitro. In order to explore the physiological functions of the 2 plastidial class‐II isoforms, GRXS14 and GRXS16, we generated knockdown and overexpression Arabidopsis thaliana lines and characterized their phenotypes using physiological and biochemical approaches. Plants deficient in one GRX did not display any growth defect, whereas the growth of plants lacking both was slowed. Plants overexpressing GRXS14 exhibited reduced chlorophyll content in control, high‐light, and high‐salt conditions. However, when exposed to prolonged darkness, plants lacking GRXS14 showed accelerated chlorophyll loss compared to wild‐type and overexpression lines. We observed that the GRXS14 abundance and the proportion of reduced form were modified in wild type upon darkness and high salt. The dark treatment also resulted in decreased abundance of proteins involved in the maturation of iron–sulfur proteins. We propose that the phenotype of GRXS14‐modified lines results from its participation in the control of chlorophyll content in relation with light and osmotic conditions, possibly through a dual action in regulating the redox status of biosynthetic enzymes and contributing to the biogenesis of iron–sulfur clusters, which are essential cofactors in chlorophyll metabolism.     

Functional organization of thylakoid membranes in viable pea mutants with low chlorophyll content

The functional organizations of thylakoid membranes from wild type pea ( Pisum sativum L. cv. Kapital) and two viable mutants with low chlorophyll (Chl) contents were compared. Nuclear mutations in mutants 7 and 42 led to two- and three-fold decrease in total chlorophyll content, respectively. In spite of low Chl content mutants showed 80% photosynthetic activity, biological productivity, and seed production. It has been shown that mutant membranes differed from that of wild type by Chl distribution between the pigment-protein complexes and by stoichiometry of the main electrontransport complexes. The ratio photosystem I (PSI): photosystem II (PSII): cytochrome (Cyt) bjf complex: Chl was 1:1.1:1.2:650 in wild type chloroplasts, 1:1.8:1.7:600 in mutant 7 , and 1:1.5:1.9:350 in mutant 42 . PSI- and PSII-dependent electron-transport activities were enhanced in the mutants per mg Chl in proportion to number of reaction centers. The activity of the non-cyclic electron-transport chain increased in proportion to PSII and Cyt bjf complexes. The amount of ATP synthetase per unit of Chl as estimated by H⁻ATPase activity was much greater in mutant thylakoids, which is favorable for photosynthetic energy transduction. The low content of the light-harvesting complexes (LHC) in mutants is compensated by an increase of the number of PSII and Cyt bjf complexes, which eliminates the bottleneck at the site of plastoquinone oxidation.     

Nondestructive determination of nitrogen and chlorophyll content in olive tree leaves and the relation with photosynthesis and fluorescence parameters

For Tunisian olive tree orchards, nitrogen deficiency is an important nutritional problem, in addition to the availability of water. Establishment of relationships between nutrients such as nitrogen and ecophysiological parameters is a promising method to manage fertilisation at orchard level. Therefore, a nitrogen stress experiment with one-year-old olive trees (Olea europaea L. ‘Koroneiki’ and ‘Meski’) was conducted with trees respectively subjected to four nitrogen supply regimes (23.96 meq l⁻¹, 9.58 meq l⁻¹, 4.79 meq l⁻¹ and 0 meq l⁻¹ NO₃ ⁻¹).     

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.     

Effect of certain dicarboxylic acid monoesters on growth,chlorophyll content,chlorophyllase and peroxidase activities,and gas-exchange of young maize plants

The effect of some dicarboxylic acid monoesters on growth, chlorophyll content, chlorophyllase (EC 3.1.1.14), and total peroxidase (EC 1.11.1.7.) activities was examined in detached and intact leaves of maize (Zea mays) plants grown in a greenhouse. The -monomethyl ester of itaconic acid (MEIA) at 1250 ppm had no effect on growth. However, application of the monoethyl ester of succinic (MESA) and monoethyl ester of adipic (MEAdA) acids (1250 ppm) resulted in an increased leaf area, fresh and dry weight of leaves and stems. These compounds retarded chlorophyll degradation in both detached and intact leaves. Chlorophyllase activity of the control and treated leaves was measured and related to chlorophyll content. Delaying of senescence by treatment with monoesters resulted in greater chlorophyll and protein content, compared with the control. However, the chlorophyllase activity/chlorophylla ratio in the treated plants decreased. Total peroxidase activity was higher in senescent leaves, but all treatments inhibited the increase of this enzyme activity. Prolonged carbon assimilative activity and enhanced leaf water use efficiency in treated plants was noted.     

Protein accumulation,RNA and soluble amino nitrogen content in developing endosperm of two varieties of Triticum aestivum with high and low protein seed

Summary The kinetics of protein accumulation, the variation in RNA, the soluble amino nitrogen content of developing endosperm of two varieties of Triticum aestivum, with high and low protein content in the mature seed, suggest a possible relation between maintenance of the RNA content and the ability to synthesize protein. A sudden halt in protein accumulation is observed as the RNA starts to decrease. The hypothesis is also advanced that maintenance of the RNA content might, in turn, be dependent on the presence, in the endosperm of developing wheat seed, of a certain level of soluble amino nitrogen which could then play the role of limiting factor for protein synthesis.Publication No. 491 from the Divisione Applicazione delle Radiazioni del C.N.E.N., SCN Casaccia, S.M. di Galeria, Rome, Italy.     

Effects of agronomic application of olive mill wastewater in a field of olive trees on carbohydrate profiles, chlorophyll a fluorescence and mineral nutrient content

Olive mill wastewater (OMW) management is a serious environmental issue for the Mediterranean area where there is the most production of olive oil. OMW contains a high organic load, substantial amounts of plant nutrients but also several compounds with recognized toxicity towards living organisms. Moreover, OMW may represent a low cost source of water. We studied the influence of irrigation with OMW (amounts applied: 30, 60, 100 and 150 m³ h⁻¹) in a field of olive trees on root colonization, photosynthesis, chlorophyll fluorescence, leaf nutrient concentration and soluble carbohydrate. The soil fatty acid methyl ester (FAME) 16:1ω5 was used to quantify biomass of arbuscular mycorrhizal (AM) fungi and the root FAME 16:1ω5 analysis was used as index for the development of colonization in the roots. Agronomic application of OMW decreased significantly the abundance of the soil FAME 16:1ω5 and the root FAME 16:1ω5 in the soil amended with 60, 100 and 150 m³ ha⁻¹ OMW. Decreased root FAME 16:1ω5 due to OMW amendment was associated with a significant reduction of tissue nutrient concentrations in the olive trees. The highest application of OMW to the soil reduced significantly the olive trees uptake of N, P, K, Ca, Mg, Fe, Cu, Mn and Zn. Land spreading of OMW increased concentration of soluble carbohydrate in the olive leaves, mostly due to decreased sink demand for carbon by the root. In the olive trees amended with 150 m³ ha⁻¹ OMW, net CO₂ uptake rate (A), quantum yield of photosystem II electron transport (Φ_PSII), maximal photochemical efficiency of photosystem II (Fv/Fm), photochemical quenching (q_p) and the electron transport rate (ETR) were significantly depressed, whereas non-photochemical quenching (NPQ) was found to increase. Taken with data from experiments in field conditions, our results suggest that agronomic application of OMW alters the functioning of arbuscular mycorrhizas and can even disrupt the relationship between AM fungi and olive trees.     

Rapid,nondestructive measurement of chlorophyll content in leaves with nonuniform chlorophyll distribution

A practical microcomputerized video image analysis method is described for quantifying leaf chlorophyll content without extraction. Chlorophyll concentration is estimated from densimetric measurements of whole, intact leaves. Direct comparison with conventional extraction measurements on Epipremnum aureum, a variegated species, verified the image analysis technique's accuracy. The inherent advantages with regard to the nondestructive and convenient nature of the measurement, and suitability for leaves with irregular chlorophyll distribution, are discussed.     

The stereospecific interaction between chlorophylls and chlorophyllase. Possible implication for chlorophyll biosynthesis and degradation.

Chlorophyllase-catalyzed hydrolysis and esterification of chlorophylls, bacteriochlorophylls, and their free acids, respectively, depend on the configuration around the C-13(2) atom of the corresponding substrate. The data suggest that the enzyme interacts preferentially with compounds having the isocyclic carbomethoxy and the C-17 propionic residues facing opposite sides of the porphyrin macrocycle. The relevance of this observation to chlorophyll biosynthesis and degradation in vivo is briefly discussed.     

Red cell metabolism in high and low glutathione goats.

The following biochemical features were measured in the red blood cells of high and low glutathione goats: reduced glutathione (GSH) stability; GSH regeneration; glucose consumption; lactate production; levels of adenosine triphosphate and 2,3-diphosphoglycerate, and the activities of nine different enzymes of Embden-Meyerhof and pentose phosphate pathways of glucose metabolism. Apart from significant differences in the GSH stability the results of all the tests were found to be similar in the two groups.     

L-Tryptophan synthesis from14C-Anthranilic acid in plants with high and low tryptophan content

The biosynthesis of L-tryptophan (L-trp) from anthranilic acid-¹⁴C (AA-¹⁴C) in. undamaged organs of the seedlings of kohlrabi and pea, with high L-trp content and ma ze plants, with low L-trp content was compared. As for maize the experiments were carried oiut with normal and opaque-2 phenotypes, both with the seedlings and with the ripening kernels. AA-¹⁴C is metabolized in the plants to L-trp pool (i.e. free and bound L-trp, and secondary metabolites) and to glycosyl esters of AA (i.e. to simple glucosyl ester in pea and kohlrabi and more complex glycosides in maize). In maize seedlings L-trp-¹⁴C is synthesized relatively less. (40% in the 1st and 2nd leaf and 33% in the 3rd leaf of the total radioactivity of the incorporated AA-¹⁴C is transferred into the L-trp-¹⁴C pool after 24 h) than in kohlrabi (52% in the hypocotyl and 85% in the cotyledons) and in pea (58% in the 1st and the 2nd internode and 85% in the 3rd and the 4th internode). Thede novo formation of L-trp-¹⁴C is stoped earlier in maize (after 5 h) than in kohlrabi (after 15 h). The level of free L-trp-¹⁴C is relatively low ill maize (15% and 13% of the total radioactivity of the incorporated AA-¹⁴C is converted to free L-trp-¹⁴C and remains in this form after 24 h) in comparison with kohlrabi (31% and 60%) and pea (30% and 49%). In spite of this the formation of L-trp-¹⁴C from AA-¹⁴C is sufficient in maize to incorporate L-trp both into the proteins and into a secondary metabolite that is not yet defined. At the period of seedlings the incorporation in maize of L-trp into the proteins (11% and 10% of the activity of the incorporated AA-¹⁴C) is comparable with that in kohlrabi (11% and 17%), and it is maximum in pea (29% and 36%). Maize, at the stage of germination, thus forms proteins rich in L-trp. The formation of free L-trp is approximately ten times lower in ripening kernels and in the leaves adjacent to the ear and it further decreases in the course of the ripening of the kernels. Although the activity of the biosynthesis of the AA-¹⁴C → L-trp-¹⁴C pathway is relatively lower in maize than in kohlrabi and pea, this pathway is most responsible for the differences in the content of L-trp in these plants. Neither amitrol nor histidine affected the biosynthesis of L-trp in kohlrabi; the interaction of the biosynthetic pathways of L-trp and histidine known in microorganisms is thus not important in a higher plant.     

Regulation and evolution of chlorophyll metabolism

Chlorophylls are the most abundant tetrapyrrole molecules essential for photosynthesis in photosynthetic organisms. After many years of intensive research, most of the genes encoding the enzymes for the pathway have been identified, and recently the underlying molecular mechanisms have been elucidated. These studies revealed that the regulation of chlorophyll metabolism includes all levels of control to allow a balanced metabolic flow in response to external and endogenous factors and to ensure adaptation to varying needs of chlorophyll during plant development. Furthermore, identification of biosynthetic genes from various organisms and genetic analysis of functions of identified genes enables us to predict the evolutionary scenario of chlorophyll metabolism. In this review, based on recent findings, we discuss the regulation and evolution of chlorophyll metabolism.     

Establishment and growth characteristics of a cell suspension culture of Marchantia polymorpha L. with high chlorophyll content

A green-pigmented cell suspension culture of Marchantia polymorpha was established using the medium of Murashige and Skoog with addition of organic acids of the tricarboxylic-acid cycle, vitamins and sugars plus sugar alcohols, exclusion of kinetin, and replacement of sucrose with glucose. In continuous light, the cells grew exponentially for ca. 10 days; in the dark, they grew only to a slight extent. The light-grown cells contained well-developed chloroplasts, and chlorophyll content reached almost twice that of the intact gametophyte.     

Water content of chlorophyll hydrate

Thermogravimetry shows that polycrystalline chlorophyll a is a chlorophyll dihydrate. Neither thermogravimetry nor differential thermal analysis indicates the existence of a stable chl a monohydrate. Moreover it is found that larger amounts of water-free chlorophyll a (>50 mg) cannot be prepared by application of vacuum and heat in a reasonable time, if chemical decomposition is to be avoided. Thin layers of polycrystalline chlorophyll a undergo spectroscopic changes depending on temperature and vacuum.     

Inheritance of cholesterol metabolism of probands with high or low cholesterol absorption

Heredity of cholesterol absorption and synthesis was studied in siblings of hypercholesterolemic probands with low and high serum cholestanol to cholesterol ratio (assumed to indicate low and high absorption of cholesterol, respectively). Cholesterol synthesis was assayed with sterol balance technique and measuring serum cholesterol precursor to cholesterol ratios (synthesis markers of cholesterol), and cholesterol absorption with measuring dietary cholesterol absorption percentage and serum plant sterol and cholestanol to cholesterol ratios (absorption markers of cholesterol). In the siblings of the low absorption families, cholesterol absorption percentage and ratios of absorption markers were significantly lower, and cholesterol and bile acid synthesis, cholesterol turnover, fecal steroids and ratios of synthesis markers significantly higher than in the siblings of the high absorption families. The ratios of absorption and synthesis markers were inversely interrelated, and they were correlated with cholesterol absorption and synthesis in the siblings. In addition, low absorption was associated with high body mass index, low HDL cholesterol, and serum sex hormone binding globulin levels, suggesting that low absorption was associated with metabolic syndrome. Intrafamily correlations were significant for serum synthesis markers, cholestanol, triglycerides, and blood glucose level. In conclusion, cholesterol absorption efficiency and synthesis are partly inherited phenomena, and they can be predicted by the ratios of non-cholesterol sterols to cholesterol in serum.