Photosynthetic CO(2) Fixation Products and Activities of Enzymes Related to Photosynthesis in Bermudagrass and Other Plants

After a 5-second exposure of illuminated bermudagrass (Cynodon dactylon L. var. `Coastal') leaves to ¹⁴CO₂, 84% of the incorporated ¹⁴C was recovered as aspartate and malate. After transfer from ¹⁴CO₂-air to ¹²CO₂-air under continuous illumination, total radioactivity decreased in aspartate, increased in 3-phosphoglyceric acid and alanine, and remained relatively constant in malate. Carbon atom 1 of alanine was labeled predominantly, which was interpreted to indicate that alanine was derived from 3-phosphoglyceric acid. The activity of phosphoenolpyruvate carboxylase, alkaline pyrophosphatase, adenylate kinase, pyruvate-phosphate dikinase, and malic enzyme in bermudagrass leaf extracts was distinctly higher than those in fescue (Festuca arundinacea Schreb.), a reductive pentose phosphate cycle plant. Assays of malic enzyme activity indicated that the decarboxylation of malate was favored. Both malic enzyme and NADP⁺-specific malic dehydrogenase activity were low in bermudagrass compared to sugarcane (Saccharum officinarum L.). The activities of NAD⁺-specific malic dehydrogenase and acidic pyrophosphatase in leaf extracts were similar among the plant species examined, irrespective of the predominant cycle of photosynthesis. Ribulose-1, 5-diphosphate carboxylase in C₄-dicarboxylic acid cycle plant leaf extracts was about 60%, on a chlorophyll basis, of that in reductive pentose phosphate cycle plants.     

Euploidy in Ricinus: EUPLOIDY EFFECTS ON PHOTOSYNTHETIC ACTIVITY AND CONTENT OF CHLOROPHYLL-PROTEINS

The effects of nuclear genome duplication on the chlorophyll-protein content and photochemical activity of chloroplasts, and photosynthetic rates in leaf tissue, have been evaluated in haploid, diploid, and tetraploid individuals of the castor bean, Ricinus communis L. Analysis of this euploid series revealed that both photosystem II (2,6-dichlorophenolindophenol reduction) and photosystem I oxygen uptake (N,N,N′,N′-tetramethyl-p-phenylenediamine to methyl viologen) decrease in plastids isolated from cells with increasingly larger nuclear complement sizes. Photosynthetic O₂-evolution and ¹⁴CO₂-fixation rates in leaf tissue from haploid, diploid, and tetraploid individuals were also found to decrease with the increase in size of the nuclear genome. Six chlorophyll-protein complexes, in addition to a zone of detergent complexed free pigment, were resolved from sodium dodecyl sulfate-solubilized thylakoid membranes from cells of all three ploidy levels. In addition to the P700-chlorophyll a-protein complex and the light-harvesting chlorophyll a/b-protein complex, four minor complexes were revealed, two containing only chlorophyll a and two containing both chlorophyll a and b. The relative distribution of chlorophyll among the resolved chlorophyll-protein complexes and free pigment was found to be similar for all three ploidy levels.     

Carbon dioxide metabolism in leaf epidermal tissue

A number of plant species were surveyed to obtain pure leaf epidermal tissue in quantity. Commelina communis L. and Tulipa gesnariana L. (tulip) were chosen for further work. Chlorophyll a/b ratios of epidermal tissues were 2.41 and 2.45 for C. communis and tulip, respectively. Phosphoenolpyruvate carboxylase, ribulose-1,5-diphosphate carboxylase, malic enzyme, and NAD⁺ and NADP⁺ malate dehydrogenases were assayed with epidermal tissue and leaf tissue minus epidermal tissue. In both species, there was less ribulose 1,5-diphosphate than phosphoenolpyruvate carboxylase activity in epidermal tissue whether expressed on a protein or chlorophyll basis whereas the reverse was true for leaf tissue minus epidermal tissue. In both species, malic enzyme activities were higher in epidermal tissue than in the remaining leaf tissue when expressed on a protein or chlorophyll basis. In both species, NAD⁺ and NADP⁺ malate dehydrogenase activities were higher in the epidermal tissue when expressed on a chlorophyll basis; however, on a protein basis, the converse was true. Microautoradiography of C. communis epidermis and histochemical tests for keto acids suggested that CO₂ fixation occurred predominantly in the guard cells. The significance and possible location of the enzymes are discussed in relation to guard cell metabolism.     

CHLOROPLAST STRUCTURE AND FUNCTION IN CULTURED TOBACCO TISSUE

A comparative study was made of the ability of cultured pith tissue, leaves of buds induced from callus, and mature leaf tissue of Nicotiana tabacum L. ‘Maryland Mammoth’ to fix carbon, as determined by light-induced C¹⁴O₂ incorporation. Photosynthetic ability was then correlated with the fine structure of chloroplasts from these tissues. The light to dark incorporation ratio for C¹⁴O₂ was at least 3 times as great in the leaf tissue as in growing cultured tissue. The chlorophyll content of the leaf tissue was 10 times as great. The carbon fixation pattern of all the tissues, as determined by radioautographs of chromatogramed extracts, was qualitatively the same. The rate of sucrose synthesis differed greatly, since 20% of the total radioactivity of the extracts from mature leaf tissue appeared in sucrose, while only 1.0% was found in sucrose from callus extracts. The incorporation of C¹⁴O₂ into sugars was inhibited in all the tissue by DCMU (3,4-dichlorophenyl,1, 1-dimethylurea). Cultured tissue past the log phase of growth was intermediate between the younger cultured tissue and the leaf tissue in its chlorophyll content and ability to incorporate C¹⁴O₂ in the light. Proplastids from dark-grown callus possessed stroma lamellae, but prolamellar bodies were not observed. The chloroplasts from growing callus were partially differentiated in comparison with chloroplasts from mature leaf tissue, since each granum had only 4-7 lamellae. Chloroplasts from callus past the log phase of growth were indistinguishable from those in mature leaves. This study establishes that the differentiation of chloroplasts in cultured tissue is a function of the growth rate of the tissue. The growth rate and degree of differentiation of the tissue can be regulated, so a well-defined system is available for the experimental study of chloroplast differentiation.     

Ribulose bisphosphate carboxylase/oxygenase content determined with [C]carboxypentitol bisphosphate in plants and algae

As is the case with spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), [¹⁴C]carboxyarabinitol bisphosphate (CABP) bound to purified Chlorella Rubisco with a molar ratio of unity to large subunit of the enzyme. The concentration of binding sites in extracts of photosynthetic organisms was determined by reacting the extracts with [¹⁴C]-carboxypentitol bisphosphate (CPBP) and precipitating the resultant Rubisco-[¹⁴C]CABP complex with a combination of polyethylene glycol-4000 and MgCl₂. Plots of the relationship between concentrations of [¹⁴C] CPBP in the reaction mixture and the precipitated [¹⁴C]CPBP gave a straight line and the concentration of binding sites were estimated by extrapolation to zero [¹⁴C]CPBP since the dissociation constant of CABP with Rubisco is 10⁻¹¹ molar. Spinach, pea, and soybean leaves contained 6.4 to 6.8 milligrams Rubisco per milligram chlorophyll, corresponding to 92 to 97 ribulose bisphosphate-binding sites per milligram chlorophyll. The Rubisco content of sunflower and wheat leaves was 5.3 to 5.5 milligrams per milligram chlorophyll. The concentrations in C₄ plants were not uniform and corn and Panicum miliaceum leaves contained 3 and 7 milligrams Rubisco per milligram chlorophyll. The Rubisco content of green algae was one-fifth to one-sixth that of C₃ plant leaves and was affected by the CO₂ concentration during growth. The content of Euglena and blue-green algae is also reported.     

Nitrogen deposition limits photosynthetic response to elevated CO2 differentially in a dioecious species

Sexual dimorphisms of dioecious plants are important in controlling and maintaining sex ratios under changing climate environments. Yet, little is known about sex-specific responses to elevated CO₂ with soil nitrogen (N) deposition. To investigate sex-related physiological and biochemical responses to elevated CO₂ with N deposition, Populus cathayana Rehd. was employed as a model species. The cuttings were subjected to two CO₂ regimes (350 and 700???mol?mol^?1) with two N levels (0 and 5?g?N?m^?2?year^?1). Our results showed that elevated CO₂ and N deposition separately increased the total number of leaves, leaf area (LA), leaf mass, net photosynthetic rate (P _n), light saturated photosynthetic rate (P _max), chlorophyll a (Chl a), and chlorophyll a to chlorophyll b ratio (Chl a/b) in both males and females of P. cathayana. However, the effects on LA, leaf mass, P _n, P _max, Chl a and Chl a/b were weakened under the combined treatment of elevated CO₂ and N deposition. Males had higher leaf mass, P _n, P _max, apparent quantum yield (??), carboxylation efficiency (CE), Chl a, Chl a/b, leaf N, and root carbon to N ratio (C/N) than did females under elevated CO₂ with N deposition. In contrast to males, females had significantly higher levels of soluble sugars in leaves and greater starch accumulation in roots and stems under the same condition. The results of the present work imply that P. cathayana females are more responsive and suffer from greater negative effects on growth and photosynthetic capacity than do males when grown under elevated CO₂ with soil N deposition.     

Arbuscular Mycorrhizal Fungi Alter Fractal Dimension Characteristics of Robinia pseudoacacia L. Seedlings Through Regulating Plant Growth,Leaf Water Status,Photosynthesis, and Nutrient Concentration Under Drought Stress

The influence of arbuscular mycorrhizal fungi (AMF), Funneliformis mosseae and Rhizophagus intraradices, on plant growth, leaf water status, chlorophyll concentration, photosynthesis, nutrient concentration, and fractal dimension (FD) characteristics of black locust (Robinia pseudoacacia L.) seedlings was studied in pot culture under well-watered, moderate drought stress, and severe drought stress treatments. Mycorrhizal seedlings had higher dry biomass, leaf relative water content (RWC), and water use efficiency (WUE) compared with non-mycorrhizal seedlings. Under all treatments, AMF colonization notably enhanced net photosynthetic rate, stomatal conductance, and transpiration rate, but decreased intercellular CO₂ concentration. Leaf chlorophyll a and total chlorophyll concentrations were higher in AM seedlings than those in non-AM seedlings although there was no significant difference between AMF species. AMF colonization improved leaf C, N, and P concentrations, but decreased C:N, C:P, and N:P ratios. Mycorrhizal seedlings had a larger FD value than non-mycorrhizal seedlings. The FD value was positively and significantly correlated to the plant growth parameters, photosynthesis, RWC, WUE, and nutrient concentration but negatively correlated to leaf/stem ratio, C:N and C:P ratios, and intercellular CO₂ concentration. We conclude that AMF lead to an improvement of growth performance of black locust seedlings under all growth conditions, including drought stress via improving leaf water status, chlorophyll concentration, photosynthesis, and nutrient uptake. Moreover, FD technology proved to be a powerful non-destructive method to characterize the effect of AMF on the physiology of host plants during drought stress.     

δ-Aminolaevulinate biosynthesis in the cyanobacterium Synechococcus 6301

The cyanobacterium (blue-green alga) Synechococcus 6301 incorporated a large amount of isotope from [1-¹⁴C] and [2-¹⁴C]acetate into phaeophorbide a obtained from chlorophyll a and into glutamatein cell protein; very little radioactivity was present in aspartate in cell protein. This distribution of isotope indicates that aspartate and the tetrapyrrole of chlorophyll a are not derived from a common C₄, precursor. The ratios of the specific radioactivities of phaeophorbide a to glutamate for organisms grown in the presence of 1-¹⁴C] and [2-⁴C ] acetate were 2.5:1 and 10:1 respectively. These are close to the theoretical values for the C₅, route to δ-aminolaevulinate which indicates that this is the only pathway to the tetrapyrrole precursor in Synechococcus 6301.     

Light- and MgCl2-dependent characteristics of four chlorophyll-protein complexes isolated from the marine dinoflagellate,Glenodinium sp.

Chlorophyll-protein complexes previously isolated from low-light (80 μE·m⁻²·s⁻¹) log cultures of the marine dinoflagellate, Glenodinium sp., were further characterized. SDS solubilization in combination with polyacrylamide gel electrophoresis in the presence of Deriphat 160-C resolved four discrete chlorophyll-protein bands. In order to elucidate the functional role of Glenodinium sp., room-temperature absorption and fluorescence spectra, protein composition, and pigment molar ratios were obtained for each complex. Results indicated that complex I was analogous to the green plant Photosystem I complex and was also associated with light-harvesting chlorophyll c₂. Complex II was highly enriched in chlorophyll c₂, devoid of peridinin, and demonstrated energy transfer from chlorophyll c to chlorophyll a within the complex, indicating the presence of a light-harvesting component. Based on peridinin: chlorophyll a ratios and fluorescence excitation spectra analyses for complexes III and IV, it was concluded that these complexes contained functional peridinin-chlorophyll a-protein complexes. Changing the ionic environment during isolation of the complexes, or altering the growth irradiance of Glenodinium sp. cultures, resulted in a significant alteration of distribution of chlorophyll a among the chlorophyll-protein complexes.     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

The effect of red and far red light on the desaturation of Fatty acids in barley leaves

Barley (Hordeum vulgare) leaf tissue was either (a) exposed to continuous red light or (b) exposed to red, far red, or red followed by far red light. The fatty acid composition and incorporation of acetate-2-¹⁴C into linolenate were determined. Changes occurred in the fatty acid composition of dark-grown barley leaves regardless of whether the plants were subsequently exposed to red light or whether the tissue remained in the dark. Measurements were also made of the fatty acids of the coleoptile. Red light treatment did not reduce the lag period for the synthesis of linolenate when chlorophyll synthesis was inhibited. It appears that the desaturation process per se in the synthesis of linolenate is not phytochrome-mediated but may appear to be phytochrome mediated if, possibly, galactolipid and chlorophyll syntheses occur concomitantly.     

P(700) activity and chlorophyll content of plants with different photosynthetic carbon dioxide fixation cycles

Representative plants containing either the reductive pentose phosphate cycle or the C₄ dicarboxylic acid cycle of photosynthetic carbon dioxide fixation have distinctly different contents of P₇₀₀ and chlorophylls a and b. With leaf extracts and isolated chloroplasts from C₄ cycle plants, the mean value of the relative ratio of P₇₀₀ to total chlorophyll was 1.83 and the mean value of the ratio of chlorophyll a to b was 3.89. The respective values in similar extracts and chloroplasts from pentose cycle plants are 1.2 and 2.78.     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.     

Chlorophyll synthesis and intracellular fluctuations of 5-aminolaevulinate formation during the regreening of nitrogen-deficient Chlorella fusca

Orange, chlorophyll-deficient cells of Chlorella fusca were obtained by prolonged exposure (6 wk) to light and CO₂ (1.5% in air) in a nitrogen-sparse medium: growth ceased after 6 days, chlorophyll formation after 3 days, and then chlorophyll degradation followed with a drop in chlorophyll a:b ratio. When the 6-wk-old cells were exposed to light in a nitrogen-rich medium and sparged with CO₂ (1.5% in air) rapid chlorophyll synthesis ensued with preferential synthesis of chlorophyll a. Regreening under these conditions was complete in approximately 24–30 hr and during this period no cell division occurred. We were unable to demonstrate 5-aminolaevulinate synthase (EC 2.3.1.37) in cell-free extracts of regreening Chlorella but demonstrated aminolaevulinate formation by whole regreening cells incubated in the presence of laevulinate, an inhibitor of aminolaevulinate dehydratase (EC 4.2.1.24). Chlorophyll synthesis was almost completely inhibited by 100 mm laevulinate, and a stoichiometric relationship exists between aminolaevulinate formation and the chlorophyll deficit caused by the presence of laevulinate: thus, the use of the inhibitor provides a true indication of the ability of the cells to form aminolaevulinate.Using this technique we found that chlorophyll synthesis during regreening appears to be regulated by the availability of aminolaevulinate since there was a correlation between the rate of aminolaevulinate and chlorophyll synthesis: both reached a maximum about halfway through the regreening period. It was not possible to decide whether the availability of aminolaevulinate was limited by the level or activity of aminolaevulinate synthase or by the supply of succinyl CoA. Regreening of orange Chlorella was inhibited by cycloheximide. Regreening of Chlorella can occur in the dark if vigorously sparged with oxygen so differing from greening of higher plants which is light dependent.Both [1,4-¹⁴C]succinate and [2-¹⁴C]glycine were incorporated into aminolaevulinate by partly regreened Chlorella fusca cells incubated in the presence of laevulinate.     

THE CHLOROPHYLL-PROTEIN COMPOUND OF THE GREEN LEAF

1. Aqueous extracts of spinach and Aspidistra leaves yield highly opalescent preparations which are not in true solution. Such extracts differ markedly from colloidal chlorophyll in their spectrum and fluorescence. The differences between the green leaf pigment and chlorophyll in organic solvents are shown to be due to combination of chlorophyll with protein in the leaf. 2. The effect of some agents on extracts of the chlorophyll-protein compound has been investigated. Both strong acid and alkali modify the absorption spectrum, acid converting the compound to the phaeophytin derivative and alkali saponifying the esterified groups of chlorophyll. Even weakly acid solutions (pH 4.5) denature the protein. Heating denatures the protein and modifies the absorption spectrum and fluorescence as earlier described for the intact leaf. The protein is denatured by drying. Low concentrations of alcohol or acetone precipitate and denature the protein; higher concentrations cause dissociation liberating the pigments. 3. Detergents such as digitonin, bile salts, and sodium desoxycholate clarify the leaf extracts but denature the protein changing the spectrum and other properties. 4. Inhibiting agents of photosynthesis are without effect on the absorption spectrum of the chlorophyll-protein compound. 5. The red absorption band of chlorophyll possesses the same extinction value in organic solvents such as ether or petroleum ether, and in aqueous leaf extracts clarified by digitonin although the band positions are different. Using previously determined values of the extinction coefficients of purified chlorophylls a and b, the chlorophyll content of the leaf extracts may be estimated spectrophotometrically. 6. It was found that the average chlorophyll content of the purified chloroplasts was 7.86 per cent. The protein content was 46.5 per cent yielding an average value of 16.1 parts per 100 parts of protein. This corresponds to a chlorophyll content of three molecules of chlorophyll a and one of chlorophyll bfor the Svedberg unit of 17,500. It is suggested that this may represent a definite combining ratio of a and b in the protein molecule.     

Mevalonate-activating enzymes in callus culture cells from Nepeta cataria

The 30000 g supernatants from cell-free extracts of Nepeta cataria leaf tissue and leaf callus tissue have mevalonic acid kinase, mevalonic acid phosphate kinase and mevalonic acid pyrophosphate decarboxylase activities. The callus tissue cell-free extract produced mevalonic acid pyrophosphate and isopentenyl pyrophosphate; however, very little mevalonic acid phosphate was observed. The leaf cell-free extracts incubated with [¹⁴C]-mevalonic acid produced higher amounts of mevalonic acid phosphate. When both the leaf cell-free extract and the callus cell-free extract were incubated with [¹⁴C]-mevalonic acid in the presence of iodoacetamide, the ion exchange column elution profile was cleaner, which was confirmed by PC. Apparently the callus tissue 30000 g supernatant contains mevalonic acid phosphorylating enzymes even though there is no production of the methyl cyclopentane monoterpenes.     

The P700-chlorophyll a-protein. Isolation and some characteristics of the complex in higher plants

A P700-chlorophyll a-protein complex has been purified from several higher plants by hydroxylapatite chromatography of Triton X-100-dissociated chloroplast membranes. The isolated material exhibits a red wavelength maximum at 677 nm, major spectral forms of chlorophyll a at 662, 669, 677, and 686 nm, a chlorophyll/P700 ratio of $\text{40–45}\text{1}$, and contains only chlorophyll a and β-carotene of the photosynthetic pigments present in the chloroplast. The spectral characteristics and composition of the higher plant material are homologous to those of the P700-chlorophyll a-protein previously isolated from blue-green algae; however, unlike the blue-green algal component, cytochromes f and b₆ are associated with the higher plant material. Evidence is presented that a chlorophyll a-protein termed “Complex I” which can be isolated from sodium dodecyl sulfate extracts of chloroplast membranes is a spectrally altered form of the eucaryotic P700-chlorophyll a-protein. The isolation procedure described in this paper is a more rapid technique for obtaining the heart of photosystem I than presently exists; furthermore, the P700 photooxidation and reduction kinetics in the fraction are improved over those in other isolated components showing the same enrichment of P700. It appears very probable that the heart of photosystem I is organized in the same manner in all chlorophyll a-containing organisms.     

Effects of No. 2 Fuel Oil, Nigerian Crude Oil, and Used Crankcase Oil on Attached Algal Communities: Acute and Chronic Toxicity of Water-Soluble Constituents

Water extracts of a no. 2 fuel oil, a Nigerian crude oil, and used crankcase oil were examined for their effects on algal communities in experiments lasting several weeks conducted under near-natural conditions. No. 2 fuel oil extracts depressed algal biomass (chlorophyll a) and resulted in blue-green algal (cyanobacterial) dominance and decreased diatom occurrence. Changes in concentrations of chlorophyll c, which was specific for diatoms in this work, and phycocyanin, which was specific for blue-green algae, confirmed the observations. Used crankcase oil extracts also depressed biomass, but Nigerian crude extracts did not, and both these extracts had less effect on community composition than did no. 2 fuel oil extracts. Photosynthetic ¹⁴C incorporation was both stimulated and depressed by exposure to extracts with hydrocarbon concentrations 0.038 to 0.124 mg/liter. Short-term exposure to higher concentrations (1.17 to 15.30 mg of hydrocarbons per liter) of no. 2 fuel oil extracts depressed photosynthetic ¹⁴C incorporation by Vaucheria-dominated communities in all tests but one. Toxicity was greater from extracts prepared in the light than from extracts prepared in the dark.     

Extinction coefficients of chlorophyll a and B in n,n-dimethylformamide and 80% acetone

We found inconsistencies in the commonly used data for chlorophyll analysis in 80% acetone. Recently developed extinction coefficients for chlorophyll b in N,N-dimethylformamide (DMF) based on values from 80% acetone are low as a result of these inconsistencies. We determined extinction coefficients of chlorophyll a (Chl a) and chlorophyll b (Chl b) in DMF for wavelengths of 618 to 665 nanometers. The simultaneous equations necessary for quantifying Chl a, Chl b, or total Chl in DMF in the absence of other chlorophyllous pigments are: Chl a = 12.70A_664.5 - 2.79A₆₄₇; Chl b = 20.70 A₆₄₇ - 4.62A_664.5; total Chl = 17.90A₆₄₇ + 8.08A_664.5, where A = absorbance in 1.00 centimeter cuvettes and Chl = milligrams per liter.

N,N-Dimethylformamide is a very convenient solvent for Chl extraction since it is effective on intact plant parts and Chl is quite stable in DMF. There was no difference in the amount of Chl extracted when plant tissue was stored for 1 or 3 days at three temperatures, with or without solvent added.

     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.