Studies on the Localization and Spectral Characteristics of the Fluorescence Emission of Differently Pigmented Wheat Leaves

Intensity, spectral characteristics and localization of the UV-laser (337 nm) induced blue-green and red fluorescence emission of green, etiolated and white primary leaves of wheat seedlings were studied in a combined fluorospectral and fluoromicroscopic investigation. The blue-green fluorescence of the green leaf was characterized by a maximum near 450 nm (blue region) and a shoulder near 530 nm (green region), whereas the red chlorophyll fluorescence exhibited maxima in the near-red (F690) and far-red (F735). The etiolated leaf with some carotenoids and traces of chlorophyll a, in turn, showed a higher intensity of the blue-green fluorescence with a shoulder in the green region and a strong red fluorescence peak near 684 to 690 nm, the far-red chlorophyll fluorescence maximum (F735) was, however, absent. The norfluorazone-treated white leaf, free of chlorophylls and carotenoids, only exhibited blue-green fluorescence of a very high intensity. In green and etiolated leaves the blue-green fluorescence primarily derived from the cell walls of the epidermis and the red fluorescence from the chlorophyll a of the mesophyll cells. In white leaves the blue-green fluorescence emanated from all cell walls of epidermis, mesophyll and leaf vein bundles. The shape and intensity of the blue-green and red fluorescence emission is determined by the reabsorption properties of chlorophylls and carotenoids in the mesophyll, thus giving rise to quite different values of the various fluorescence ratios F450/F690, F450/F530, F450/F735 and F690/F735 in green and etiolated leaves.     

UV-B response of green and etiolated barley seedlings

7-d-old etiolated and green barley seedlings (Hordeum vulgare L. cv. Alfa) were irradiated with UV-B for 30 min and then kept for 24 h in light or darkness. Chlorophyll (Chl) synthesis was inhibited by about 30 % as a result of UV-B irradiation, but there were no significant changes in photochemical activity measured by variable to maximum fluorescence ratio (F_v/F_m), quantum yield (Φ_PS2) and oxygen evolution rate. Electron transport of etiolated seedlings was similar to that of green ones, nevertheless, the Chl content was more then 2-fold lower. Ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunits were diminished as a result of UV-B irradiation in etiolated and green plants, especially in those kept in the darkness. Catalase activity decreased and total superoxide dismutase activity increased in green and etiolated plants following UV-B treatment. When benzidine was used as a substrate, an isoform located between guaiacol peroxidases 2 and 3 (guaiacol peroxidase X) appeared, which was specific for UV-B treatment. As a result of irradiation, the contents of UV-B absorbing and UV-B induced compounds increased in green seedlings but not in etiolated seedlings.     

Blue,Green and Red Fluorescence Signatures and Images of Tobacco Leaves*

Laser-induced fluorescence images of the leaf of an aurea mutant of Nicotiana tabacum were recorded for the blue and green fluorescence at 440 and 520 nm and the red chlorophyll fluorescence at 690 and 735 nm. The results obtained were compared with direct measurements of the fluorescence emission spectra of leaves using a conventional spectrofluorometer. The highest emission of blue (F440) and green fluorescence (F520) within the leaf was found in the leaf veins, particularly the main leaf vein. In contrast, the intercostal fields of leaves, which exhibited the highest chlorophyll content, showed only a very low blue and green fluorescence emission, which was much lower than the red and far-red chlorophyll fluorescence emission bands (F690 and F735). Correspondingly, the ratio of blue to red leaf fluorescence F440/F690 of upper and lower leaf side was much higher in the leaf veins (values 1.2 to 1.5) than in intercostal fields (values of 0.6 to 0.7). The results also demonstrated that in the intercostal fields the major part of the blue-green fluorescence was reabsorbed by chlorophylls and carotenoids. A partial reabsorption of the red fluorescence band near 690 nm by leaf chlorophyll took place, but did not affect the far-red fluorescence band near F735. As a consequence the chlorophyll fluorescence ratio F690/F735 exhibited significantly higher values in the chlorophyll-poor leaf vein regions (1.7 to 1.8) than in the chlorophyll-rich intercostal fields (0.8 to 1.3). Imaging spectroscopy of leaves was shown to be much more precise than the screening of fluorescence signatures by conventional fluorometers. It clearly demonstrated that the blue-green fluorescence and the red chlorophyll fluorescence of leaves exhibit an inverse contrast to each other. The advantage of the fluorescence imaging spectroscopy, which allows the simultaneous screening of the whole leaf surface and distinct parts of it, and its possible application in the detection of stress effects or local damage by insects and pathogens, is discussed.     

Soret absorption of intermediate(s) in protochlorophyllide to chlorophyllide photoreduction trapped at low temperature

Absorption spectra at ca 100 K from 400 to 750 nm and fluorescence emission spectra at 77 K from 600 to 750 nm were obtained from: 1) etiolated leaves of the H-ordeum vulgare L. (barley) mutant albozonata 2 and SAN 9789-treated Avena sativa L. (oat) with low levels of carotenoids, and 2) preparations of protochlorophyllide holo-chrome from Phaseolus vulgaris L. cv. Commodore (bean).
This allowed clear resolution for the first time of the Soret bands of the green pigments before and after light-induced accumulation of intermediate(s) in protochlorophyllide to chlorophyllide photoreduction and after conversion of the intermediate(s) to chlorophyllide by warming the samples to 233 K in darkness. Although the intermediate(s) differ(s) in absorption and fluorescence in the red wavelength region from both protochlorophyllide and chlorophyllide, the extinction in the Soret band is not distinguishable from that of chlorophyllide. These observations indicate that the C7-C8 double bond in ring IV of protochlorophyllide has been altered in intermediate(s) accumulated at low temperature in intense light, such that the transition state exhibits the character of a π complex.     

Inhibition by mevinolin of plant growth, sterol formation and pigment accumulation

To obtain information on the importance of a functional mevalonate synthesis for plant growth and development, we investigated the effect of mevinolin, a highly specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A reductase (the mevalonate-producing enzyme) on growth, sterol accumulation and pigment formation of radish seedlings (Raphanus sativus L. cv. Saxa Treib) and in part also wheat seedlings (Triticum aestivum L. cv. Kolibri). Mevinolin applied during germination inhibits root elongation and development of lateral roots in etiolated and light-grown radish seedlings. This effect cannot be overcome by exogenous GA₃, but by addition of mevalonic acid, the product of the internally inhibited reaction. This emphazises the specifity of the mevinolin effect and indicates that the biosynthesis of mevalonic acid is a mandatory requirement for root growth. In light-grown radish seedlings mevinolin also affects hypocotyl length-growth and inhibits sterol accumulation, but has little effect on the chlorophyll and carotenoid accumulation in the chloroplasts of the cotyledons. This indicates the possible presence of an independent mevalonate synthesizing pathway within the plastids and suggests a low transport rate of mevinolin from the radish roots to the cotyledons. When mevinolin is directly applied to the leaves at higher concentrations, it also reduces the light-induced chlorophyll and carotenoid accumulation as has been shown with etiolated primary leaves of wheat. This inhibition is age-dependent and proceeds to a higher extent in older than in younger etiolated leaf tissue. From our results we conclude that plastids possess an independent HMG-CoA reductase. In the cotyledons of radish, mevinolin seems to induce a senescence retardation and sun-type growth response, as has been evaluated by measuring the fast and slow chlorophyll fluorescence induction kinetics (Kautsky effect). These responses may be due to inhibitor-induced changes in the intracellular phytohormone balance.     

PHYTOCHROME DISTRIBUTION IN ETIOLATED GRASS SEEDLINGS AS ASSAYED BY AN INDIRECT ANTIBODY-LABELLING METHOD

The distribution of phytochrome in several etiolated grass seedlings (Avena saliva L., cvs. Garry and Newton; Secale cereale L., cv. Balbo; Hordeum vulgare L., cv. Harrison; Oryza sativa L; Zea mays L., cv. Golden Cross) was determined, by an indirect antibody-labelling method employing peroxidase as the ultimate label. Although the pattern of phytochrome distribution in etiolated shoots varies widely, it is nevertheless clear that, with the exception of corn, in which phytochrome is relatively uniformly distributed, the distribution of phytochrome is highly specific with respect both to organs and to cell types within an organ for a given species. Oat, rye, barley, and rice shoots all have high concentrations of phytochrome near the tips of their coleoptiles, as well as near the shoot apex itself. Rice, barley, and rye also have high concentrations of phytochrome in their leaf bases, but oat leaves are almost totally devoid of measurable phytochrome. An association of phytochrome with vascular tissue often occurs and is most pronounced in the rice shoot. Dark-grown roots were found to have high levels of phytochrome only in the root caps, with lesser amounts, if any, observed in other parts of the root.     

Discrete character of the development of the photosynthetic apparatus in greening barley leaves

Biogenesis of the photosynthetic apparatus in greening etiolated leaves of barley (Hordeum vulgare L) was investigated by an approach permitting investigation of this process under conditions that minimize differences in plastid development. Distributions of barley leaves greening for 24 h as to chlorophyll content and of chloroplast grana as to number of thylakoids were shown to be of a multimodal character. The shape of time-course curves of chlorophyll accumulation in local sites of greening etiolated leaves was of a stepped or (at the end of greening) undulated character. The stepwise accumulation of chlorophyll was accompanied by wave-like changes in chlorophyll b/a ratio, intensity of low-temperature chlorophyll fluorescence and photosynthetic activity with minima at the time points of transition to accelerated chlorophyll accumulation. It is assumed that (1) development of the photosynthetic apparatus in local sites of greening etiolated leaves occurs stepwise, from one steady level to another, but not as gradually as is generally accepted, and (2) every separate step in development of the photosynthetic apparatus seems to begin with formation of photosystem cores and to end with the synthesis of light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nonpigment components of the photochlorophyllide photoactive complex: studies of low-temperature blue-green fluorescence spectra

Fluorescence spectra in the blue-green region and excitation fluorescence spectra of green wheat leaves, etiolated wheat leaves and isolated inner etioplast membranes (prolamellar bodies and prothylakoids) were compared to specify the structure of the active protochlorophyllide pigment-protein complex of inner etioplast membranes. Three bands in the blue region at 420, 443 and 470 nm and a broader green band at 525 nm were found. Comparison of the emission and excitation spectra suggests that the main components responsible for the blue fluorescence of etioplast inner membranes are pyridine nucleotides and pterins. The green fluorescence (525 nm) excitation spectra of etiolated samples were identical to the excitation spectrum of flavin fluorescence. The fact confirms the suggestion that flavins are the constituents of the active protochlorophyllide-protein complex.     

INTRACELLULAR PHYTOCHROME DISTRIBUTION AS A FUNCTION OF ITS MOLECULAR FORM AND OF ITS DESTRUCTION

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red-absorbing Pr form to the far-red-absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments.     

Glutamate synthase isoforms in rice: immunological studies of enzymes in green leaf, etiolated leaf, and root tissues

Rabbit antiserum was raised against ferredoxin-dependent glutamate synthase (EC 1.4.7.1) purified from green leaves of Oryza sativa L. cv Delta. Ferredoxin-dependent glutamate synthase, detected in green leaf, etiolated leaf, and root tissues cross-reacted completely with the antiferredoxin glutamate synthase immunoglobulin G. In contrast, the immunoglobulin G did not cross-react with NADH-dependent (EC 1.4.1.14) and NADPH-dependent (EC 1.4.1.13) glutamate synthases found in nonphotosynthetic etiolated leaf and root tissues. In addition, ferredoxin-dependent glutamate synthase was separated and distinguished by its affinity to ferredoxin from NAD(P)H-dependent glutamate synthase on ferredoxin-Sepharose affinity chromatography. Based on the immunological studies, it is suggested that ferredoxin-dependent glutamate synthases in green leaf and etiolated leaf tissues are closely related proteins; in contrast, ferredoxin-dependent glutamate synthase in root tissue is a distinct protein from the leaf enzymes.     

Glutamine synthetases of green and etiolated leaves ofSinapis alba

Studies on the glutamine synthetases (GS, EC 6.3.1.2) of green (GS₂) and etiolated leaves (GS_et) ofSinapis alba L. (cv. Steinacher) revealed striking similarities between the respective enzyme proteins. The enzymes showed corresponding chromatographic properties, both on dimethylaminoethyl-Sephacel and on hydroxylapatite columns. The purified GS proteins were also identical with regard to the molecular weight of their subunits. Isoelectrofocusing of pure GS_et yielded two distinct polypeptide bands in the pH 5.6 region of the gels. This pattern corresponded to the two strong bands of GS₂. Two charge variants of GS polypeptides could be detected by Western-blot analysis of the soluble protein of green leaves using antibodies against mustard GS₂. In immunoprecipitation experiments, the holoenzymes of GS₂ and GS_et were recognized with identical affinities by this antiserum. We conclude that strong similarities exist between the proteins of the GS enzymes in green and etiolated leaves of mustard. Most probably only one GS form, namely the plastidic enzyme, can be found in the epigeal organs ofSinapis. The polypeptides of the GS₂ subunits showed no differences in the hydrophobicity of the polypeptide chains. Neither glucosyl nor mannosyl residues could be detected. Dedicated to Professor Dr. H. Mohr on the occasion of his 60th birthday     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

Monoclonal antibodies directed to phytochrome from green leaves of Avena sativa L. cross-react weakly or not at all with the phytochrome that is most abundant in etiolated shoots of the same species

Seven monoclonal antibodies (MAbs) have been prepared to phytochrome from green oat (Avena sativa L. cv. Garry) leaves. One of these MAbs (GO-1) cross-reacts with apoprotein of the phytochrome that is most abundant in etiolated oat shoots as assessed by immunoblot assay of fusion proteins expressed in Escherichia coli. The epitope for this MAb is located between amino acids 618 and 686 in the primary sequence of type 3 phytochrome (Hershey et al. 1985, Nucleic Acids Res. 13, 8543–8559), which is one of the predominant phytochromes in etiolated oats. Three other MAbs (GO-4, GO-5, GO-6) immunoprecipitate phytochrome isolated from green oat leaves, as evaluated by photoreversibility assay. GO-1, GO-4, GO-5 and GO-6 are therefore directed to phytochrome. While evidence obtained with the other three MAbs (GO-2, GO-7, GO-8) strongly indicates that they are also directed to phytochrome, this evidence is not as rigorous. Recognition of antigen by any of these seven MAbs is not significantly reduced by periodate oxidation, indicating that their epitopes probably do not include carbohydrate. All but GO-1 bind either very poorly or not at all the phytochrome that is abundant in etiolated oat shoots. These data reinforce earlier observations made with antibodies directed to phytochrome from etiolated oats, indicating (1) that the phytochromes that predominate in etiolated and green oats differ immunochemically and (2) that phytochrome preparations from green oat leaves contain very little of the phytochrome that is abundant in etiolated shoots. An hypothesis that these two immunochemically distinct phytochromes form heterodimers in vitroAbbreviations Da Dalton - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - HA hydroxyapatite - Ig immunoglobulin - MAb monoclonal antibody - SDS sodium dodecyl sulfate is supported by comparison of immunoblot data obtained with conventionally purified phytochrome from etiolated oats to that expressed as fusion protein in E. coli. This research was supported by the U.S. Department of Energy (contract DE-AC-09-81SR10925 to L.H.P.). We thank Dr. Lyle Crossland and Ms. Sue Kadwell for their assistance in the construction of the cDNA clones, and Dr. Gyorgy Bisztray for providing us with clone pCBP3712. Dr. Phillip Evans and Dr. Russell Malmberg kindly provided MAbs 4F3, 6F12 and 8C10, as well as a corresponding antigen preparation. The excellent technical assistance of Mrs. Donna Tucker and Mrs. Danielle Neal is gratefully acknowledged.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

Photosynthetic characteristics of detached barley leaves during greening in the presence of SAN 9785

The effects of the pyridazinone compound SAN 9785 on the photosynthetic competence of leaves, on the photochemical activity of isolated thylakoids and on the formation and spectral properties of chlorophyll-protein complexes were studied during a 72-h greening period of detached etiolated leaves of barley (Hordeum vulgare L. cv. Horpácsi kétsoros). It was established that i) the photosynthetic capacity of the leaves decreased considerably (by 80 and 90%, as determined by¹⁴CO₂ fixation and fast fluorescence induction measurements, respectively); ii) the photochemical activity of isolated thylakoids from water to potassium ferricyanide and from dichlorophenol indophenol/ascorbate to methylviologen exhibited only slight reductions when expressed on a chlorophyll basis compared with the control; iii) the slow fluorescence induction curves of the treated leaves demonstrated the presence of a peculiar fluorescence component interrupting the quenching of fluorescence at around 1 min illumination; iv) a shortage of the chlorophyll-protein complex of photosystem I (CPI) occurred with a higher content of the monomer of the light harvesting complex in the thylakoids of treated leaves; and v) the fluorescence spectrum of the CPI band present in treated leaves indicates the destruction of the structural integrity of this complex during isolation from the membrane.Abbreviations Chl chlorophyll - CPI, CPII chlorophyll-protein complexes of the reaction centres of PSI and PSII - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPIP 2,6-dichlorophenol indophenol - DPIPH₂ chemically reduced form of DPIP - F _o fluorescence of constant yield - F _v fluorescence of variable yield - F _i ,F _m mitial and maximum yield of fluorescence - LHCP³ monomer of the light-harvesting complex - LHCP² and LHCP¹ oligomers of the light-harvesting complex LHCP³ - PSI, PSII photosystems I, II - SAN 9785 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone, also known as BASF 13-338 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.     

Isolation and initial characterization of virescent mutants of Arabidopsis thaliana

In higher plants, development of the chloroplasts must be coordinated with development of the leaf. In order to study the signals that synchronize these two developmental processes, we have isolated virescent (delayed in greening) mutants of Arabidopsis thaliana. Two such mutants that have pale-green young leaves which gradually green more fully during leaf maturation have been partially characterized. The two, vir1 and vir2, are due to separate nuclear recessive mutations. The pale leaves of vir1 and vir2 both had reduced 77°K fluorescence emission at 730–734 nm relative to that at 686–687 nm, indicating a reduction in the relative amount of LHC I compared to WT. As leaves greened, the amount of LHC I increased to near wildtype levels. The shift in the fluorescence emission peak from 730 nm to 734 nm, characteristic of maturing LHC I, was seen for vir1, but not vir2, suggesting that vir1 is a regulatory mutant while vir2 may be defective in a specific aspect(s) of LHC I function.Abbreviations D dark - EMS ethyl methanesulfonate - er erecta - gl1 glabrous1 - L light - LHC I light harvesting complex of Photosystem I - LHC II light harvesting complex of Photosystem II - M2 second generation of mutagenized seed - M3 third generation of mutagenized seed - vir virescent - WT wildtype     

Characterization of protochlorophyllide and protochlorophyllide esters in roots of dark-grown plants

The protochlorophyll pools of roots of dark-grown wheat ( Triticum aestivum L. cv. Walde), maize ( Zea mays L. cv. Goldcrest) and wrinkledseeded pea ( Pisum sativum L. ssp. sativurh cv. Kelvedon Wonder) were investigated by high performance liquid chromatography (HPLC) and low temperature fluorescence spectroscopy. All roots contained protochlorophyllide and esterified protochlorophyllides (protochlorophylls) but with considerably larger relative amounts of the latter compared with etiolated leaves. The alcohol moieties of the 4 detected protochlorophylls were geranylgeraniol (GG), dihydrogeranylgeraniol (DHGG), tetrahydrogeranylgeraniol (THGG) and phytol. The relative amounts of the different protochlorophylls varied between the species. Protochlorophyllide and the 4 protochlorophylls all contained monovinyl forms. The divinyl forms could not be detected by our instruments. Wrinkledseeded pea contained in addition chlorophyll a , some unidentified chlorophylls and negligible amounts of chlorophyllide. Small amounts of carotenoids were found in roots of all investigated species. The carotenoids were the same as those found in green or etiolated leaves, but present in different relative amounts.     

Content of carotenoids during ageing and senescence of tobacco leaves with genetically modulated life-span

We exploited leaves of tobacco (Nicotiana tabacum L., cv. Wisconsin 38) with introduced chimeric construct consisting of SAG12 promoter fused with ipt gene for cytokinin synthesis and therefore prolonged life-span. As a control we used its wild type. In 12-week-old plants, the first leaves of control plants showed senescence symptoms at the time of sampling. Carotenoid content decreased with increasing leaf age both in control and in transgenic plants. On the other hand, the first leaves of transgenic plants demonstrated better antioxidant capacity represented by carotenoids compared to the leaves of control plants of the same age. They stayed still green at this age.