Spectral signatures of five hydroxymethyl chlorophyll a derivatives chemically derived from chlorophyll b or chlorophyll f

Photosynthesis Research - Chlorophylls (Chls) are pigments involved in light capture and light reactions in photosynthesis. Chl a, Chl b, Chl d, and Chl f are characterized by unique absorbance...     

Picosecond kinetics of chlorophyll and chlorophyll/quinone solutions in ethanol

The mechanism of quenching by quinones of the lowest excited singlet state of chlorophyll has been investigated using picosecond laser spectroscopy. With chlorophyll alone, laser excitation resulted in immediate (< 10 ps) bleaching of the 665 nm band and production of new absorption bands in the regions 460–550 and 800–830 nm. The lifetimes of these changes were greater than 500 ps. Addition of 2,6-dimethyl-benzoquinone caused quenching of these absorbance changes. No indication of chlorophyll cation radical formation was obtained. Thus, the interaction between quinone and the chlorophyll excited singlet state results in energy dissipation without measurable formation of radical species having lifetimes longer than 10 ps. This is in marked contrast to the quenching of the chlorophyll lowest triplet state by quinones, during which easily detectable stable radical formation has been observed.     

Spontaneous chlorophyll mutants of Pennisetum americanum: Genetics and chlorophyll quantities

Summary Thirteen spontaneously occurring chlorophyll deficient phenotypes have been described and their genetic basis was established. Ten of these — white, white tipped green, patchy white, white virescent, white striping 1, white striping 2, white striping 4, fine striping, chlorina and yellow virescent showed monogenic recessive inheritance and the remaining three — yellow striping, yellow green and light green seedling phenotypes showed digenic recessive inheritance. The genes for (i) white tipped green (wr) and yellow virescent (yv) and (ii) patchy white (pw) and white striping 1 (wst 1) showed independent assortment. Further, the genes for white (w), white tipped green (wr) and yellow virescent (yv) were inherited independently of the gene for hairy leaf margin (Hm).In the mutants — white tipped green, patchy white, white striping 1, white striping 2, fine striping, chlorina, yellow virescent, yellow striping, yellow green and light green phenotypes total quantity of chlorophyll was significantly less than that in the corresponding controls, while in white virescent there was no reduction in the mature stage. For nine of the mutants the quantity of chlorophyll was also estimated in F₁'s (mutant x control green). In F₁'s of six of the mutants — white tip, patchy white, chlorina, yellow virescent, fine striping and yellow striping the quantity of chlorophyll was almost equal to the wild type. In the F₁'s of three of the mutants — white striping 1, white striping 2 and light green an intermediate value between the mutant and wild types was observed. In yellow virescent retarded synthesis of chlorophyll, particularly chlorophyll a was observed in the juvenile stage. Reduced quantity of chlorophyll was associated with defective chloroplasts. In the mutants — white tipped green, white virescent, fine striping, chlorina, yellow striping, yellow green and light green defective plastids were also observed. In patchy white secondary destruction of chlorophylls and the presence of defective plastids were found to be associated with reduced chlorophyll quantity at maturity.Paper chromatographic studies of leaf flavonoids revealed some variation between the inbreds, but there were three common spots, 7, 8 and 9, except for PDP in which the spot 8 was absent. Chlorophyll deficient mutants differed from their respective controls in the absence of one or more of the spots present in the controls and in the presence of new spots in some of the mutants.Most of the chlorophyll mutants showed higher survival rate in the Kharif season than in Rabi season which was attributed to the higher mean day temperature and longer day light period in the Kharif season than in Rabi season.     

In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown

A central reaction of chlorophyll breakdown, porphyrin ring opening of pheophorbide a to the primary fluorescent chlorophyll catabolite (pFCC), requires pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR), with red chlorophyll catabolite (RCC) as a presumably PAO-bound intermediate. In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). Here, we show that RCCR-deficient Arabidopsis thaliana accumulates RCC and three RCC-like pigments during senescence, as well as FCCs and NCCs. We also show that the stereospecificity of Arabidopsis RCCR is defined by a small protein domain and can be reversed by a single Phe-to-Val exchange. Exploiting this feature, we prove the in vivo participation of RCCR in chlorophyll breakdown. After complementation of RCCR mutants with RCCRs exhibiting alternative specificities, patterns of chlorophyll catabolites followed the specificity of complementing RCCRs. Light-dependent leaf cell death observed in different RCCR-deficient lines strictly correlated with the accumulation of RCCs and the release of singlet oxygen, and PAO induction preceded lesion formation. These findings suggest that RCCR absence causes leaf cell death as a result of the accumulation of photodynamic RCC. We conclude that RCCR (together with PAO) is required for the detoxification of chlorophyll catabolites and discuss the biochemical role(s) for this enzyme.     

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.     

Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence

Chlorophyll fluorescence was used to estimate profiles of absorbed light within chlorophyll solutions and leaves. For chlorophyll solutions, the intensity of the emitted fluorescence declined in a log–linear manner with the distance from the irradiated surface as predicted by Beer's law. The amount of fluorescence was proportional to chlorophyll concentration for chlorophyll solutions given epi‐illumination on a microscope slide. These relationships appeared to hold for more optically complex spinach leaves. The profile of chlorophyll fluorescence emitted by leaf cross sections given epi‐illumination corresponded to chlorophyll content measured in extracts of leaf paradermal sections. Thus epifluorescence was used to estimate relative chlorophyll content through leaf tissues. Fluorescence profiles across leaves depended on wavelength and orientation, reaching a peak at 50–70 µm depth. By infiltrating leaves with water, the pathlengthening due to scattering at the airspace : cell wall interfaces was calculated. Surprisingly, the palisade and spongy mesophyll had similar values for pathlengthening with the value being greatest for green light (550 > 650 > 450 nm). By combining fluorescence profiles with chlorophyll distribution across the leaf, the profile of the apparent extinction coefficient was calculated. The light profiles within spinach leaves could be well approximated by an apparent extinction coefficient and the Beer–Lambert/Bouguer laws. Light was absorbed at greater depths than predicted from fibre optic measurements, with 50% of blue and green light reaching 125 and 240 µm deep, respectively.     

Enzymes of chlorophyll biosynthesis

The enzymes responsible for chlorophyll biosynthesis in plants, algae and cyanobacteria are identified and described, with emphasis on their protein composition and structure, required cofactors, physical and catalytic properties, protein-protein interactions and allosteric modulation of activity. Properties and features of the pathway that enable it to operate in a coordinated way while using unstable and light-sensitive intermediates in potentially hostile biochemical environments are discussed. The evolutionary relationships and possible origins of the chlorophyll biosynthetic enzymes are also discussed.     

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.     

Photoelectrolysis using chlorophyll electrodes

By the conversion of sunlight to chemical energy, photoelectrolysis was carried out using two different chlorophyll-redox compound lining electrodes. These electrodes were prepared by covering platinum plates with chlorophyll and naphthoquinone or anthrahydroquinone and a conducting adhesive. These electrodes exhibit a photoexciting property. The potential was found to shift to a less noble state when the system of the chlorophyll-naphthoquinone electrode was inserted into NAD solution with illumination. On the other hand, the photoexcitation of the system of the chlorophyll-anthrahydroquinone electrode inserted into ferrocyanide solution made the potential more noble. (If the potential in the dark is in the positive region of the scheme

and the potential moves in the positive direction when the light is turned on, it can be said to be more positive or more noble. But if the potential moves in the negative direction in the light, but remains in the positive region, it can be said to become less noble, but it is not suitable to say that it becomes more negative.) To make the potential difference between two electrodes as big as possible, various factors such as intensity of illumination, molar ratio of chlorophyll to naphthoquinone or anthrahydroquinone and concentration of redox compound in electrolyte were examined.A cell was set up by combining the system of the chlorophyll-naphthoquinone electrode in NAD solution with that of the chlorophyll-anthrahydroquinone electrode in ferrocyanide solution, and photoelectrolysis was carried out by closing the external circuit with illumination. The photovoltage between the two electrodes was 0.25 V and the current density was 8 μA/cm². It was found that NAD was reduced to form NADH at the chlorophyll-naphthoquinone electrode and ferrocyanide was oxidized to form ferricyanide at the chlorophyll-anthrahydroquinone electrode.     

Demetalation of chlorophyll pigments

Natural chlorophylls (Chls) and bacteriochlorophyll (BChls), which are major pigments in photosynthesis, possess a central Mg (or Zn) in their cyclic tetrapyrrole macrocycles. Removal of the central metal atom from chlorophyllous pigments, called pheophytinization, is a biologically important reaction in that it allows production of the primary electron acceptors in photosystem II(-type) reaction centers and is one of the crucial steps in the Chl degradation pathway. Pheophytinization in processed and postharvest foods derived from vegetables and fruits has attracted considerable attention in agricultural and food chemistry from the viewpoints of maintenance of their color level and evaluation by consumers. In this review, we focus on in vivo demetalation reactions and demetalated products of chlorophyllous pigments. In addition, we summarize kinetic studies on in vitro demetalation of natural (B)Chl molecules and their synthetic analogs under acidic conditions.     

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.     

Mapping QTLs for chlorophyll content and chlorophyll fluorescence in wheat under heat stress

Heat stress, one of the major abiotic stresses in wheat, affects chlorophyll fluorescence and chlorophyll content and thereby photosynthesis. To identify quantitative trait loci (QTLs) associated with these traits under terminal heat stress, 251 recombinant inbred lines (RILs) derived from a cross HD 2808/HUW510 were phenotyped. Using composite interval mapping, 40 QTLs were identified; 17 were related to conditions after timely sowing and 23 to heat stress after late sowing. The various parameters of chlorophyll fluorescence were associated with 23 QTLs, which were located on chromosomes 1A, 2A, 3A, and 2D and explained 3.67 to 18.04 % of phenotypic variation, whereas chlorophyll content was associated with 17 QTLs on chromosomes 2A, 2B, 2D, 5B, and 7A explaining 3.49 to 31.36 % of phenotypic variation. Most of the identified QTLs were clustered on chromosome 2D followed by 2A and 1A. The QTL Qchc.iiwbr-2A for chlorophyll content linked with marker gwm372 was stable over conditions and explained 3.81 to 18.05 % of phenotypic variation. In addition, 7 epistatic QTL pairs were also detected which explained 1.67 to 11.0 % of phenotypic variance. These identified genomic regions can be used in marker assisted breeding after validation for heat tolerance in wheat.     

Evaluating the relationship between leaf chlorophyll concentration and SPAD-502 chlorophyll meter readings

Relationships between chlorophyll concentration ([chl]) and SPAD values were determined for birch, wheat, and potato. For all three species, the relationships were non-linear with an increasing slope with increasing SPAD. The relationships for birch and wheat were strong (r ² ∼ 0.9), while the potato relationship was comparatively weak (r ² ∼ 0.5). Birch and wheat had very similar relationships when the chlorophyll concentration was expressed per unit leaf area, but diverged when it was expressed per unit fresh weight. Furthermore, wheat showed similar SPAD–[chl] relationships for two different cultivars and during two different growing seasons. The curvilinear shape of the SPAD–[chl] relationships agreed well with the simulated effects of non-uniform chlorophyll distribution across the leaf surface and multiple scattering, causing deviations from linearity in the high and low SPAD range, respectively. The effect of non-uniformly distributed chlorophyll is likely to be more important in explaining the non-linearity in the empirical relationships, since the effect of scattering was predicted to be comparatively weak. The simulations were based on the algorithm for the calculation of SPAD-502 output values. We suggest that SPAD calibration curves should generally be parameterised as non-linear equations, and we hope that the relationships between [chl] and SPAD and the simulations of the present study can facilitate the interpretation of chlorophyll meter calibrations in relation to optical properties of leaves in future studies.     

Evidence for a light-harvesting chlorophyll a-protein complex in a chlorophyll b-less barley mutant

Chloroplasts of a chlorophyll (Chl) b-less barley mutant were solubilized with digitonin and fractionated by polyacrylamide gel electrophoresis with sodium deoxycholate in the running buffer. By this procedure, in contrast to using sodium dodecylsulfate (SDS) for solubilization, a Chl a-protein analogous to the major light-harvesting Chl a-b protein complex from wildtype chloroplasts was recovered. This mutant Chl a-protein comprises about fifty percent of the total Chl a, and is very similar in carotenoid, amino acid, protein and polypeptide composition to the major wildtype antenna Chl a-b protein. The only major differences we have found is its instability in the presence of SDS and sensitivity to protease action. Even with deoxycholate, the mutant Chl a complex often dissociates during electrophoresis into two green bands. The lack of Chl b appears to affect the normal organization of Chl a and protein in such a way as to render the complex more unstable.CIW-DPB No. 917.