Dynamics of the biosynthesis of native chlorophyll forms from initial stages to the completion of the greening process in etiolated leaves]

The succession of the formation of the native chlorophill forms and the development of the energy migration between them was studied by means of the comparison of three spectrum types: absorption, fluorescence and fluorescence excitation, and of their second derivatives. Quantitatively the chlorophyll accumulation in 9 native forms was followed by means of mathematical disintegration of the spectra using computer.     

Haem and chlorophyll formation in etiolated and greening leaves of barley

The amounts of protochlorophyllide (P650) and protohaem were measured in ageing dark-grown barley leaves. Maximum amounts of P650 and protohaem were found in 6- to 8-day-old material after which P650 declined rapidly and protohaem more slowly. In leaves exposed to light maximum chlorophyll was produced in 6-day-old material with progressively less the older the leaves. Haem concentrations increased in seedlings of all ages exposed to light. A lag phase was observed for both chlorophyll and haem formation in leaves given a light treatment. Haem, however, showed a slight yet sig nificant decline as chlorophyll production commenced. The results indicate that chlorophyll and haem synthesis share a common pool of δ-aminolae vulinic acid (ALA). At a certain stage of development, the magnesium porphyrin pathway diverts precursors away from haem synthesis. It is only when the ALA synthesising system is well developed that the production of ALA can satisfy pathways to both haem and chlorophyll. The observed changes in haem under certain conditions suggest that, as in animal systems, haem levels may regulate porphyrin formation (chlorophylls) by controlling the supply of ALA.     

Energy migration in photoactive complexes of chlorophyll precursor in etiolated leaves and spectroscopic characteristics of pigment forms]

Migration energy between three forms of the precursor of chlorophyll and chlorophyllide are measured and calculated. Absorption coefficients, fluorescence yields of pigment forms and dimensions of native complexes (20-22 molecules) are calculated.     

Photoconvertible and non-photoconvertible forms of protochlorophyll(ide) in etiolated bean leaves

Precursors of chlorophylls in etiolated bean leaves were studiedby a sensitive technique of dual wavelength scanning of thinlayer chromatograms of pigments. The photoconvertible pigmentswith absorption maxima at 650 and 638 nm, respectively, wereidentified as protochlorophyllide. A minor non-photoconvertiblepigment with a maximum at 628 nm was found to be protochlorophyll. (Received July 3, 1974; )     

Characterization of two phases of chlorophyll formation during greening of etiolated barley leaves

The esterification kinetics of chlorophyllide, obtained by a single flash of light, were investigated in etiolated barley ( Hordeum vulgare L.) and oat ( Avena sativa L.) leaves. A rapid phase, leading to esterification of 15% of total chlorophyllide within 15-30 s, was followed by a lag-phase of nearly 2 min and a subsequent main phase, leading to esterification of 85% of total chlorophyllide within 30-60 min. The presence of additional protochlorophyllide, produced in the leaves by incubation with 5-aminolevulinate, did not change the esterification kinetics. The rapid phase was identical after partial (11-15%) and full (>80%) photoconversion of protochlorophyllide; the ability for a second rapid esterification phase was restored in a dark period of at least 10 min. Cooling the leaves to 0 degrees C abolished the esterification of the main phase while the rapid phase remained unchanged. The prolamellar bodies were already in part transformed into prothylakoid-like structures within 2-5 min after a full flash but not after a weak flash (11% photoconversion); in the latter case, the corresponding transformation required a dark period of about 45 min. The existence of subcomplexes of prolamellar bodies containing NADPH:protochlorophyllide oxidoreductase and chlorophyll synthase in the ratio 7:1 is discussed.     

The effect of kinetin on chlorophyll synthesis in ageing etiolated barley leaves exposed to light

Etiolated barley seedlings lose the ability to produce chlorophyll and soluble protein on exposure to light with increasing age. Similarly, the production of δ-aminolaevulinic acid-dehydratase and succinyl-CoA synthetase is decreased in older etiolated leaves exposed to light. The rate of protochlorophyllide₆₅₂ regeneration decreased well before the rates of exogenous δ-aminolaevulinic acid conversion to protochlorophyllide₆₃₂ was affected by ageing. Application of kinetin retarded these ageing symptoms in the etiolated leaves.     

Chlorophyllides in green and etiolated leaves

Chlorophyllides, extracted from lyophylized fresh leaves of different species by absolute methanol, were separated from the chlorophylls by thin-layer chromatography. They amounted only to 0·04 to 0·1 per cent of the total amount of chlorophyll a on a molar basis. On account of their low concentration it was concluded—in contrast to an assumption made recently—that the 683 nm absorption band of mature leaves in vivo cannot be due to a chlorophyllide-holochrome.     

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2–20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990–1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955–960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.Abbreviations Pchl protochlorophyll and protochlorophyllide - Chld chlorophyllide - Chl chlorophyll     

Relationships between leaf chlorophyll content and spectral reflectance and algorithms for non-destructive chlorophyll assessment in higher plant leaves

Leaf chlorophyll content provides valuable information about physiological status of plants. Reflectance measurement makes it possible to quickly and non-destructively assess, in situ, the chlorophyll content in leaves. Our objective was to investigate the spectral behavior of the relationship between reflectance and chlorophyll content and to develop a technique for non-destructive chlorophyll estimation in leaves with a wide range of pigment content and composition using reflectance in a few broad spectral bands. Spectral reflectance of maple, chestnut, wild vine and beech leaves in a wide range of pigment content and composition was investigated. It was shown that reciprocal reflectance (R lambda)-1 in the spectral range lambda from 520 to 550 nm and 695 to 705 nm related closely to the total chlorophyll content in leaves of all species. Subtraction of near infra-red reciprocal reflectance, (RNIR)-1, from (R lambda)-1 made index [(R lambda)(-1)-(RNIR)-1] linearly proportional to the total chlorophyll content in spectral ranges lambda from 525 to 555 nm and from 695 to 725 nm with coefficient of determination r2 > 0.94. To adjust for differences in leaf structure, the product of the latter index and NIR reflectance [(R lambda)(-1)-(RNIR)-1]*(RNIR) was used; this further increased the accuracy of the chlorophyll estimation in the range lambda from 520 to 585 nm and from 695 to 740 nm. Two independent data sets were used to validate the developed algorithms. The root mean square error of the chlorophyll prediction did not exceed 50 mumol/m2 in leaves with total chlorophyll ranged from 1 to 830 mumol/m2.     

The importance of PS I chlorophyll red forms in light-harvesting by leaves

We have investigated the importance of the long wavelength absorbing spectral forms (red forms) of Photosystem I in photosynthetic light harvesting by leaves. To this end leaf spectra were simulated by using a linear combination of absorption (OD) spectra of purified Photosystem I, Photosystem II and LHC II, multiplied by an empirical multiple scattering chloroplast/leaf conversion function. In this way it is demonstrated that while the PS I red forms account for only about 4–5% of light absorption in a normal daylight environment, in different shadelight environments these long wavelength pigments may be responsible for up to 40% of total photon capture. In the context of maximising the photosynthetic quantum efficiency under the low light conditions of shadelight, this relative increase in the absorption cross section of PS I can be understood by considering the increased synthesis of the major PS II antenna complex, LHC II, known to occur in plants growing under these light conditions. It is demonstrated that for plants in a moderate to deep shadelight regime the PS II cross section needs to increase by 50% to 100% via LHC II synthesis to balance the increased PS I absorption by the red forms. The possibility that under shade light conditions the increased PS I cross section may serve in cyclic phosphorylation is also discussed.     

The photostability of different chlorophyll forms in dark grown leaves of wheat

Formulae were developed for calculation of the relative amount of different pigment forms of dark grown leaves of wheat, present before and after photoreduction of the protochlorophyllide. Three pigment forms were calculated from in vivo absorption spectra: the photoreducible protochlorophyllide with absorption maximum at 650 nm and the two chlorophyll(ide) forms with absorption maximum at 684 nm and 673 nm, respectively. The formulae were used to study the changes of the pigment forms at repeated photoreduction of the protochlorophyllide, and at a repeated treatment involving photoreduction of the protochlorophyllide followed by partial photo-decomposition of the chlorophyllide formed. Five consecutive photoreductions and reaccumulations of protochlorophyllide were carried out by high intensity irradiations of one second (red light, 700 W m^-2) given at intervals of 3 h. The results show that the pool size of reaccumulated protochlorophyllide decreased sharply with the number of photoreductions performed. The absorption spectrum of the chlorophyllide formed at each photoreduction proceeded through the Shibata shift (transformation of the 684-form to the 673-form) and the late red-shift (transformation of the 673-form to other pigment form(s) in the dark). High intensity irradiation for ten minutes (red light, 700 W m^-2) immediately after each phototransformation caused a photodecomposition of about three quarters of the newly formed chlorophyllide (which was in the 684-form) while the earlier formed chlorophyll(ide) (in the 673-form) appeared not to be decomposed. This partial photodecomposition of the chlorophyllide had no effect on further accumulation of protochlorophyllide in the dark, and the absorption spectrum of the remaining chlorophyllide proceeded through the Shibata shift. The partial photodecomposition caused an inhibition of the late red-shift, and the accumulated chlorophyll(ide) remained in the 673-form.     

Comparative investigation of the appearance of primary chlorophyllide forms in etiolated leaves,prolamellar bodies and prothylakoids

A comparative investigation of the first steps of chlorophyllide formation from protochlorophyllide in the etiolated leaves, prolamellar bodies and prothylakoids was performed by measuring fluorescence emission spectra. It was shown that the formation of the first fluorescent chlorophyllide forms from non-fluorescent intermediates is a complex process including several dark reactions with different temperature dependencies. When the temperature of samples which had been illuminated at 77 K was increased to 190 K, four primary chlorophyllide forms were found by Gaussian deconvolution of the 77 K emission spectra. They had fluorescence emission maxima at 690, 696, 684 and 706 nm, respectively. Two new forms of chlorophyllide - Chlide690 and Chlide706 - were found in addition to the major known forms. A prolonged exposure to 190 K as well as rise of the temperature to 253 K led to a disappearance of Chlide690. The fate of this form is not clear. Chlide696 and Chlide706 were transformed into Chld673 and Chld684, respectively, during the prolonged dark exposure at 253 K. The existence of two pathways of native short wavelength chlorophyllide forms formation was proposed with different temperature dependencies.     

Biosynthesis of chlorophyll from protochlorophyll(ide) in green plant leaves

Using spectral methods, the biosynthesis of protochlorophyll(ide) and chlorophyll(ide) in green plant leaves was studied. The main chlorophyll precursors in the green leaves (as in etiolated leaves) were photoactive photocholorophyll(ide) forms Pchl(ide)655/650(448) and Pchl(ide)653/648(440). The contributions into Chl biosynthesis of the shorter-wavelength precursor forms ,which were accumulated in darkened green leaves as well, were completely absent (of Pchl(ide) 633/628(440)) or insignificant (of Pchl(ide)642/635(444)).     

Native state,energetic interaction of chlorophyll precursors and intraplastid location of S-adenosyl-L-methionine: Mg-protoporphyrin IX methyltransferase in etiolated leaves

Low temperature fluorescence spectra (FS) and fluorescence excitation spectra (FES) of protoporphyrin IX (Proto), Mg-protoporphyrin IX and its monomethyl ester (MgProto-ME) and protochlorophyllide (Pchlide) in etiolated barley leaves treated with 5-aminolevulinic acid and/or 2,2'-dipyridyl were studied. The spectra of Proto and MgProto-ME showed a little dependence on temperature of registration and exhibited similarity to low temperature spectra in diluted organic and buffer solutions. However, a red wavelength shift for Soret bands of Proto and MgProto-ME was observed due to porphyrin interaction with bovine serum albumin in 0.05 M, Na2HPO4 solution at room temperature. Disaggregating treatments had no effect on Proto and MgProto-ME spectra in plants. These results suggested that in etiolated leaves Proto and MgProto-ME molecules were in a monomer state. The spectral properties of these molecules were determined by interaction of porphyrins with proteins and other plastid membrane components. The spectral analyses indicated an efficient energy migration from Proto and MgProto-ME molecules to active form of Pchlide which emitted at 656nm, and no energy transfer from carotenoids to porphyrins in vivo. These findings suggested that Proto and MgProto-ME from carotenoids, and close location of these porphyrins and photoactive Pchlide in etioplast membranes. The latter conclusion was strongly supported by an observation that in etiolated leaves, S-adenosyl-L-methionin:Mg-protoporphyrin IX methyltransferase, which converts MgProto into MgProtoME, were located not only in prothylakoids but also in prolamellar bodies containing photoactive Pchlide.     

Acclimation of chlorophyll biosynthetic reactions to temperature stress in cucumber (Cucumis sativus L.)

The adaptive responses of the greening process of plants to temperature stress were studied in cucumber (Cucumis sativus L. cv. Poinsette) seedlings grown at ambient (25 °C), low (7 °C) and high (42 °C) temperatures. Plastids isolated from these seedlings were incubated at different temperatures and the net syntheses of various tetrapyrroles were monitored. In plastids isolated from control seedlings grown at 25 °C, the optimum temperature for synthesis of Mg-protoporphyrin IX monoester or protochlorophyllide was 35 °C. Temperature maxima for Mg-protoporphyrin IX monoester and protochlorophyllide syntheses were shifted to 30 °C in chill-stressed seedlings. The net synthesis of total tetrapyrroles was severely reduced in heat-stressed seedlings and the optimum temperature for Mg-protoporphyrin IX monoester or protochlorophyllide synthesis shifted slightly towards higher temperatures, i.e. a broader peak was observed. To further study the temperature acclimation of seedlings with respect to the greening process, tetrapyrrole biosynthesis was monitored at 25 °C after pre-heating the plastids (28–70 °C) isolated from control, chill- and heat-stressed seedlings. In comparison to 28 °C-pre-heated plastids the percent inhibition of protochlorophyllide synthesis in 40 °C-pre-heated plastids was higher than for the control (25 °C-grown) in chill-stressed seedlings and lower than for the control in heat-stressed seedlings. Maximum synthesis of total tetrapyrroles and protoporphyrin IX was observed when chloroplasts were heated at 50 °C, which was probably due to heat-induced activation of the enzymes involved in protoporphyrin IX synthesis. Prominent shoulders towards lower or higher temperatures were seen in chill-stressed or heat-stressed seedlings, respectively. The shift in optimum temperature for tetrapyrrole biosynthesis in chill- and heat-stressed seedlings was probably due to acclimation of membranes possibly undergoing desaturation or saturation of membrane lipids. Proteins synthesized in response to temperature-stress may also play an important role in conferring stress-tolerance in plants. Received: 8 October 1998 / Accepted: 19 November 1998     

Global remote sensing of water–chlorophyll ratio in terrestrial plant leaves

I evaluated the use of global remote sensing techniques for estimating plant leaf chlorophyll a + b (C_ab; μg cm⁻²) and water (C_w; mg cm⁻²) concentrations as well as the ratio of C_w/C_ab with the PROSAIL model under possible distributions for leaf and soil spectra, leaf area index (LAI), canopy geometric structure, and leaf size. First, I estimated LAI from the normalized difference vegetation index. I found that, at LAI values <2, C_ab, C_w, and C_w/C_ab could not be reliably estimated. At LAI values >2, C_ab and C_w could be estimated for only restricted ranges of the canopy structure; however, the ratio of C_w/C_ab could be reliably estimated for a variety of possible canopy structures with coefficients of determination (R²) ranging from 0.56 to 0.90. The remote estimation of the C_w/C_ab ratio from satellites offers information on plant condition at a global scale.     

Analysis of the chlorophyll biosynthetic system in a chlorophyll b-less barley mutant

The chlorophyll b-less barley (Hordeum vulgare L.) mutant chlorina 2807 allelic to the well-known barley mutant chlorina f2 was studied. 5-Aminolevulinic acid at saturating concentration (40 mM) was introduced into postetiolated leaves of the mutant and its wild type, and the protochlorophyllide accumulation in the dark was measured. It was found that the activity of the enzyme system transforming 5-aminolevulinic acid into protochlorophyllide was the same in both types of plants. The activity of esterifying enzymes that catalyze attachment of phytol to chlorophyllide was analyzed by infiltration of exogenous chlorophyllides a and b into etiolated leaves. The reaction was shown to have close rates in the mutant and wild-type plants. In very early stages of greening of etiolated leaves, when the apoproteins of the light-harvesting complexes are not yet formed, appearance of chlorophyll b was clearly recorded in the wild-type plants, while in the mutant chlorina 2807 no indications of chlorophyll b were detected in any stage of greening. On the other hand, in the mutant as well as in the wild type an active reverse conversion of chlorophyll b into chlorophyll a was possible. It is concluded that (a) in the mutant chlorina 2807 the ability of the biosynthetic system to transform 5-aminolevulinic acid to chlorophyll a is fully preserved, (b) in the mutant the enzymes converting chlorophyll a into chlorophyll b are most likely absent or damaged, (c) the conversion of chlorophyll a into chlorophyll b and the reverse conversion of chlorophyll b into chlorophyll a are performed by different enzymes.