Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy

The Spectral Determination of Chlorophylls a and b, as well as Total Carotenoids, Using Various Solvents with Spectrophotometers of Different Resolution*

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) - 2.79A(647); Chl b = 20.70 A(647) - 4.62A(664.5); total Chl = 17.90A(647) + 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.

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Extinction Coefficients of Chlorophyll a and b in N,N-Dimethylformamide and 80% Acetone

The extraction of chlorophylls in higher plant tissue using N,N-dimethylformamide expedites the process and enables the determination of small samples with low pigment level.Absorption spectra of Chl a, Chl b, and Pchl and of their acidified derivatives, the phaeophytins, were recorded. Conversion of Chl b to its corresponding acidified product occurs much more slowly than that of Chl a and Pchl. When acidified, Pchl differs from Chl a and Chl b by the disappearance of the red band in the absorption spectrum. Specific extinction coefficients were determined and formulae for quantitative determination of pigments concentrations were developed. When concentrations of pigments are low, as in etiolated plant material, the absorption spectra of the chlorophylls can be distorted due to the presence of other substances simultaneously extracted; formulae for pigment determination under such circumstances were also derived.

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Formulae for Determination of Chlorophyllous Pigments Extracted with N,N-Dimethylformamide

Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins.

Photosynthetic pigments from etiolated cucumber (Cucumis sativus var. Beit Alpha improved, Hazera Co., Gedera) cotyledons were extracted by direct immersion of the intact cotyledons into the solvent N,N-dimethylformamide (DMF). The solvent is especially efficient when pigment concentration is low; time and tools are saved and the loss of pigment that usually occurs in more complicated extraction procedures is prevented. The specific absorption coefficient of chlorophyll a in DMF was also determined.

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Chlorophyll determination in intact tissues using n,n-dimethylformamide.

Cotyledons excised without the hypocotyl hook from 6-day-old etiolated cucumber (Cucumis sativus L. var. Elem) seedlings accumulated a significantly higher amount of chlorophyll than cotyledons excised with hooks or intact cotyledons. It was found that maximum ehancement of greening was achieved after 2 h of dark incubation following excision. Pretreatments with red light effected an additive rise in chlorophyll level in subsequent white light after a dark incubation, suggesting that the effects of excision and phytochrome on greening act independently. Etiolated seedlings were variously dissected before greening and it was found that enhancement occurred only when cotyledons were excised at the level of the hypocotyl hook or above it. Similar results were obtained when the dissected plants were pre-treated with red light.

A double enhancement of greening in the cotyledons of cucumber seedlings by excision and red light

The changes in morphology during dark germination and subsequent growth of cucumber (Cucumis sativus) seedlings in the light go through three different phases described as latent, active, and steady-state. This pattern is consistently observed for several related developmental processes. The latent period lasts about 2 days following water imbibition after which the following capabilities appear in concert: (a) root and stem elongation, (b) pigment synthesis including protochlorophyll, chlorophyll, carotenoid, and phytochrome, (c) synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase, and (d) enhancement of greening by excision. Following the active phase, which lasts for another 2 to 3 days, these processes slow to a steady-state. Inhibition of chlorphyll accumulation by SO2 was only observed for seedlings in the steady-state phase.

Rami Moran

Papers

Formulae for Determination of Chlorophyllous Pigments Extracted with N,N-Dimethylformamide

Chlorophyll determination in intact tissues using n,n-dimethylformamide.

A double enhancement of greening in the cotyledons of cucumber seedlings by excision and red light

Kinetics of Chlorophyll Accumulation and Other Growth Parameters

Developmental Stages of Cucumber Seedlings Kinetics of Chlorophyll Accumulation and Other Growth Parameters