Dietary cyanidin 3-glucoside from purple corn ameliorates doxorubicin-induced cardiotoxicity in mice.

BACKGROUND AND AIMS: Anthracyclines are effective anticancer drugs that have improved prognosis of hundred thousand cancer patients worldwide and are currently the most common chemotherapeutic agents used for the treatment of blood, breast, ovarian and lung cancers. However, their use is limited because of a cumulative dose-dependent and irreversible cardiotoxicity that can cause progressive cardiomyopathy and congestive heart failure. Aim of the present study was to determine the cardioprotective activity of a dietary source of cyanidin 3-glucoside (C3G), such as purple corn, against doxorubicin (DOX)-induced cardiotoxicity in mice. METHODS AND RESULTS: In vitro studies on murine HL-1 cardiomyocytes showed that pretreatment with both pure C3G and purple corn extract improved survival upon DOX treatment. However, C3G and purple corn extract did not affect the cytotoxic effect of DOX on human cancer cell lines. We then validated in vivo the protective role of a C3G-enriched diet against DOX-induced cardiotoxicity by comparing the effect of dietary consumption of corn isogenic lines with high levels of anthocyanins (purple corn - Red diet - RD) or without anthocyanins (yellow corn - Yellow diet - YD) incorporated in standard rodent diets. Results showed that mice fed RD survived longer than mice fed YD upon injection of a toxic amount of DOX. In addition, ultrastructural analysis of hearts from mice fed RD showed reduced histopathological alterations. CONCLUSION: Dietary intake of C3G from purple corn protects mice against DOX-induced cardiotoxicity.

Comparative analyses of albumin/globulin grain proteome fraction in differentially salt-tolerant Tunisian barley landraces reveals genotype-specific and defined abundant proteins.

Salinity is one of the major abiotic stresses threatening crop production and yield worldwide. Breeding programmes are therefore needed to improve yield under cultivation in soil. Traits from locally adopted landraces provide a resource to assist breeding of novel elite genotypes. Here, we examine differentially expressed proteins by performing comparative proteomic profiling of the albumin/globulin grain fraction of Tunisian barley genotype landraces with contrasting salinity tolerance. Tunisian barley Boulifa (B, tolerant) and Testour (T, sensitive) mature grains were assessed in 2-DE profiles. Differentially expressed spots, with an abundance enhanced 1.5-fold in the grain, were subjected to MALDI TOF/TOF MS for identification. Distinctiveness between tolerant and sensitive genotypes was proved in the albumin/globulin fraction using PCA; 64 spots showed significant differential abundance. Increased accumulation of 40 spots was confirmed in Boulifa with, interestingly, four genotype-specific spots. Two of these four spots were sHSP. Proteins with highest abundance were serpin Z7, 16.9 KDa Class I HSP and phosphogluconolactonase 2. Proteins such as expansin, kiwellin, kinesin and succinyl-CoA ligase were identified for the first time in barley grain. Moreover, ß-amylase, LEA family and others were identified as abundant in Boulifa. On the other hand, proteins more accumulated in Testour are implicated mainly in ROS scavenging and protease inhibition. Our results clearly indicate proteomic contrast between the two selected genotypes. With identification of specific HSP, high abundant stress-protective and other defined proteins, we provide biochemical traits that will support breeding programmes to address the threat of salinity in agricultural production.

Reduction of Uroporphyrinogen Decarboxylase by Antisense RNA Expression Affects Activities of Other Enzymes Involved in Tetrapyrrole Biosynthesis and Leads to Light-Dependent Necrosis.

We introduced a full-length cDNA sequence encoding tobacco (Nicotiana tabacum) uroporphyrinogen III decarboxylase (UROD; EC 4.1.1.37) in reverse orientation under the control of a cauliflower mosaic virus 35S promoter derivative into the tobacco genome to study the effects of deregulated UROD expression on tetrapyrrole biosynthesis. Transformants with reduced UROD activity were characterized by stunted plant growth and necrotic leaf lesions. Antisense RNA expression caused reduced UROD protein levels and reduced activity to 45% of wild type, which was correlated with the accumulation of uroporphyrin(ogen) and with the intensity of necrotic damage. Chlorophyll levels were only slightly reduced (up to 15%), indicating that the plants sustained cellular damage from accumulating photosensitive porphyrins rather than from chlorophyll deficiency. A 16-h light/8-h dark regime at high-light intensity stimulates the formation of leaf necrosis compared with a low-light or a 6-h high-light treatment. Transgenic plants grown at high light also showed inactivation of 5-aminolevulinate dehydratase and porphobilinogen deaminase, whereas the activity of coproporphyrinogen oxidase and the 5-aminolevulinate synthesizing capacity were not altered. We conclude that photooxidation of accumulating uroporphyrin(ogen) leads to the generation of oxygen species, which destabilizes other enzymes in the porphyrin metabolic pathway. This porphyrin-induced necrosis resembles the induction of cell death observed during pathogenesis and air pollution.

Comparative analysis of the low molecular weight and enzymatic antioxidants in response to the phototoxicity of accumulating uroporphyrin and protochlorophyllide in barley leaves treated with cesium chloride.

Cesium chloride treatment of illuminated barley leaves leads to accumulation of uroporphyrinogen which is subsequently either oxidised to uroporphyrin in continuous light or converted to protochlorophyllide in darkness [Shalygo et al. (1998) J Photochem Photobiol 42: 151-158]. We were interested to elucidate the differences in the phototoxicity of uroporphyrin and protochlorophyllide in the CsCI-treated leaves. Photosensitization and the induction of oxidative stress responses in the barley leaves occurred much faster upon protochlorophyllide than upon uroporphyrin accumulation. We compared the time resolved changes in the pool sizes of low molecular weight antioxidants, such as ascorbate, glutathione and tocopherol, as well as of the enzymatic activities of catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase in illuminated barley leaves which accumulated uroporphyrin or protochlorophyllide. A rapid loss of the antioxidant levels correlated with the accumulation of reactive oxygen species. The contents of low molecular weight antioxidants and the activities of most of the antioxidative enzymes declined more rapidly in the presence of protochlorophyllide than of uroporphyrin. Due to its high lipophilicity, free protochlorophyllide is associated with biomembranes. Therefore, it is assumed that it exerts its phototoxic effects to membranes more rapidly than uroporphyrin.

Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase.

The patterns of secondary metabolites in leaves of yeast invertase-transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were analyzed. Plants expressing cytosolic yeast-derived invertase (cytInv) or apoplastic (cell wall associated) yeast invertase (cwInv) showed a characteristic phytochemical phenotype compared to untransformed controls (wild-type plants). The level of phenylpropanoids decreased in the cytInv plants but increased in the cwInv plants, which showed an induced de novo synthesis of a caffeic acid amide, i.e. N-caffeoylputrescine. In addition, the level of the coumarin glucoside scopolin was markedly enhanced. Increased accumulation of scopolin in the cwInv plants is possibly correlated with the induction of defense reactions and the appearance of necrotic lesions similar to the hypersensitive response caused by avirulent pathogens. This is consistent with results from potato virus Y-infected plants. Whereas there was no additional increase in the coumarins in leaves following infection in cwInv plants, wild-type plants showed a slight increase and cytInc a marked increase.

Immunomodulation of ABA function affects early events in somatic embryo development.

Immunomodulation of abscisic acid (ABA) function during somatic embryogenesis of Nicotiana plumbaginifolia has been used to demonstrate for the first time the effect of this phytohormone on early embryonic events. A homozygous transgenic line constitutively expressing an anti-abscisic acid (ABA) single chain fragment variable antibody in the endoplasmic reticulum was established. Development of somatic embryos from the transgenic line and the wild type was compared. The ABA biosynthesis mutants aba1 and aba2 and wild type cultures treated with the ABA biosynthesis inhibitor fluridone were also used for the comparative investigations. The development of embryonic structures was disturbed in the early stages of all cultures in which ABA function was blocked or which were ABA-deficient. After ABA complementation of the in vitro cell cultures normal somatic embryo development was restored.

Photodynamic action of uroporphyrin and protochlorophyllide in greening barley leaves treated with cesium chloride.

Incubation of greening barley leaves with cesium chloride (CsCl) results in photodynamic leaf lesions within 24 h due to an inactivation of uroporphyrinogen III decarboxylase, an enzyme of tetrapyrrole biosynthesis, and transient accumulation of uroporphyrin (ogen). To examine the mechanism of porphyrinogenesis, time kinetics of the accumulating tetrapyrrole intermediates uroporphyrin (ogen) and protochlorophyllide were performed with leaves which were cut from 7-day-old dark-grown barley seedlings and incubated in 15 mM CsCl or water under different light regimes. In the presence of CsCl chlorophyll and carotenoids accumulation was inhibited in the first 24 h of continuous light and the pigment content decreased dramatically during extended illumination. When CsCl=treated leaves were transferred to darkness, accumulated uroporphyrinogen was completely converted to protochlorophyllide. Low temperature fluorescence spectroscopy confirmed that uroporphyrinogen almost completely accumulated in the reduced form. The oxidised form, uroporphyrin, was detectable after 24 h of illumination. The photodynamic leaf lesions became visible at the same time. Protochlorophyllide synthesised from accumulated uroporphyrinogen III in dark incubated leaves had a fluorescence maximum at 635 nm which is indicative for its non-photoconvertible form. Re-illumination of the barley leaves resulted in a rapid degradation of proteins and pigments and an intense lipid peroxidation within less than two hours due to the photodestructive potential of non-metabolised protochlorophyllide.

Root exudation and root development of lettuce (Lactuca sativa L. cv. Tizian) as affected by different soils.

Development and activity of plant roots exhibit high adaptive variability. Although it is well-documented, that physicochemical soil properties can strongly influence root morphology and root exudation, particularly under field conditions, a comparative assessment is complicated by the impact of additional factors, such as climate and cropping history. To overcome these limitations, in this study, field soils originating from an unique experimental plot system with three different soil types, which were stored at the same field site for 10 years and exposed to the same agricultural management practice, were used for an investigation on effects of soil type on root development and root exudation. Lettuce (Lactuca sativa L. cv. Tizian) was grown as a model plant under controlled environmental conditions in a minirhizotrone system equipped with root observation windows (rhizoboxes). Root exudates were collected by placing sorption filters onto the root surface followed by subsequent extraction and GC-MS profiling of the trapped compounds. Surprisingly, even in absence of external stress factors with known impact on root exudation, such as pH extremes, water and nutrient limitations/toxicities or soil structure effects (use of sieved soils), root growth characteristics (root length, fine root development) as well as profiles of root exudates were strongly influenced by the soil type used for plant cultivation. The results coincided well with differences in rhizosphere bacterial communities, detected in field-grown lettuce plants cultivated on the same soils (Schreiter et al., this issue). The findings suggest that the observed differences may be the result of plant interactions with the soil-specific microbiomes.

Recent progress in liquid chromatography-based separation and label-free quantitative plant proteomics.

Recent innovations in liquid chromatography-mass spectrometry (LC-MS)-based methods have facilitated quantitative and functional proteomic analyses of large numbers of proteins derived from complex samples without any need for protein or peptide labelling. Regardless of its great potential, the application of these proteomics techniques to plant science started only recently. Here we present an overview of label-free quantitative proteomics features and their employment for analysing plants. Recent methods used for quantitative protein analyses by MS techniques are summarized and major challenges associated with label-free LC-MS-based approaches, including sample preparation, peptide separation, quantification and kinetic studies, are discussed. Database search algorithms and specific aspects regarding protein identification of non-sequenced organisms are also addressed. So far, label-free LC-MS in plant science has been used to establish cellular or subcellular proteome maps, characterize plant-pathogen interactions or stress defence reactions, and for profiling protein patterns during developmental processes. Improvements in both, analytical platforms (separation technology and bioinformatics/statistical analysis) and high throughput nucleotide sequencing technologies will enhance the power of this method.

Iron assimilation and transcription factor controlled synthesis of riboflavin in plants.

Iron homeostasis is vital for many cellular processes and requires a precise regulation. Several iron efficient plants respond to iron starvation with the excretion of riboflavin and other flavins. Basic helix-loop-helix transcription factors (TF) are involved in the regulation of many developmental processes, including iron assimilation. Here we describe the isolation and characterisation of two Arabidopsis bHLH TF genes, which are strongly induced under iron starvation. Their heterologous ectopic expression causes constitutive, iron starvation independent excretion of riboflavin. The results show that both bHLH TFs represent an essential component of the regulatory pathway connecting iron deficiency perception and riboflavin excretion and might act as integrators of various stress reactions.

Ectopic expression of EFFECTOR OF TRANSCRIPTION perturbs gibberellin-mediated plant developmental processes.

The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific alpha-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J. Biol. Chem. 273: 23313-23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.

Isolation, sequencing and expression of cDNA sequences encoding uroporphyrinogen decarboxylase from tobacco and barley.

We have cloned and sequenced a full-length cDNA for uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) and a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tobacco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and processed to ca. 39 kDa. A truncated cDNA, from which the putative transit peptide had been deleted, was used to over-express the mature UROD in Escherichia coli. Purified protein showed UROD activity, thus providing an adequate source for subsequent enzymatic characterization and inhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination. Maximum UROD-RNA levels were detected in the basal segments relative to the top of the leaf.

Coproporphyrinogen III oxidase from barley and tobacco--sequence analysis and initial expression studies.

Coproporphyrinogen III oxidase (coprogen oxidase; EC 1.3.3.3) is part of the pathway from 5-amino-levulinate to protoporphyrin IX which is common in all organisms and catalyses oxidative decarboxylation at two tetrapyrrole side chains. We cloned and sequenced full-length cDNAs encoding coprogen oxidase from barley (Hordeum vulgare L.) and tobacco (Nicotiana tabacum L.). They code for precursor peptides of 43.6 kDa and 44.9 kDa, respectively. Import into pea plastids resulted in a processed tobacco protein of approx. 39 kDa, which accumulated in the stroma fraction. Induction of synthesis of recombinant putative tobacco mature coprogen oxidase consisting of 338 amino-acid residues in Escherichia coli at 20 degrees C result in a catalytically active protein of approx. 39 kDa, while induction of its formation at 37 degrees C immediately terminated bacterial growth, possibly due to toxic effects on the metabolic balance of tetrapyrrole biosynthesis. The plant coprogen oxidase gene was expressed to different extents in all tissues investigated. This is most likely due to the differing requirements for tetrapyrroles in different organs. The steady-state level of mRNA did not significantly differ in etiolated and greening barley leaves. The content of coprogen oxidase RNA reached its maximum in developing cells and decreased drastically when cells were completely differentiated. Functioning of the two photosystems apparatus requires the synthesis of all pigment and protein components during plant development. It is speculated that the enzymes involved in tetrapyrrole synthesis are developmentally rather than light-dependently regulated. Regulation of these enzymes also guarantees a constant flux of metabolic intermediates and avoids photodynamic damage by accumulating porphyrins.

Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative defense system.

A full-length cDNA sequence encoding coproporphyrinogen oxidase was inserted in inverse orientation behind a CaMV promoter and transferred to tobacco (Nicotiana tabacum) by standard transformation techniques. Transformants showed reduced coproporphyrinogen oxidase activity and accumulation of photosensitive coproporphyrin(ogen), indicating antisense RNA expression. An inverse correlation was observed between the level of coproporphyrinogen oxidase and transformant phenotype. The latter is characterized by a broad range of growth retardation and necrosis, indicating oxidative leaf damage. Coproporphyrinogen is an apparent chromophore and its excitation finally leads to the production of reactive oxygen. Evidence is presented that indicates a direct correlation between the accumulation of non-metabolized coproporphyrinogen and oxidative damage to cellular structural components. Enzymatic and non-enzymatic antioxidants were investigated. Whereas superoxide dismutase activity increased in transgenic plants, catalase and ascorbate peroxidase activity remained constant. Tocopherol, rather than carotene or zeaxanthin, seemed to be involved in detoxification, indicating the putative localization and allocation of coproporphyrinogen. Expression of coproporphyrinogen oxidase antisense RNA did not significantly influence the level of other enzymes in the chlorophyll metabolic pathway, but deregulated gene expression of nuclear encoded plastid proteins. Accumulation of coproporphyrinogen and/or the resulting effects, such as oxidative stress, impairs a plastid/nuclear signal which may adapt gene expression to the plastid state.

Purification and characterization of sinapoylglucose:malate sinapoyltransferase from Raphanus sativus L.

1-O-Sinapoyl-ß-glucose:l-malate O-sinapoyltransferase (SMT; EC 2.3.1.) from cotyledons of red radish (Raphanus sativus L. var. sativus) was purified to apparent homogeneity with a 2100-fold enrichment and a 4% recovery. Apparent Mrs of 52 and 51, respectively, were determined by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On isoelectric focusing, the SMT resolved into two isoforms which, on SDS-PAGE, showed, slightly different Mrs (SMT I: Mr/isoelectric point = 51/5.75; SMT II: Mr/isoelectric point = 51.5/5.9). The highest activity of SMT was found at pH 6.0 (50% at pH 5.5 and pH 6.5). The temperature maxima in the presence of 10, 50, 100 and 250 mM malate were 22, 30, 35 and 37° C, respectively, with energies of activation of 55, 81, 96 and 121 kJ · mol(-1). The enzyme accepted all the hydroxycinnamic acid-glucose esters tested with relative ratios of initial velocity values of 100∶85∶45∶26∶2.6 of 1-O-sinapoyl-, 1-O-feruloyl-, 1-O-caffeoyl-, 1,2-di-O-sinapoyl-, and 1-O-(4-coumaroyl)-ß-glucose. It showed an absolute acceptor specificity for l-malate. d-Malate as second acceptor molecule in standard assays with l-malate inhibited the reaction velocity noncompetitively (K i = 215 mM). The substrate saturation curves were not hyperbolic. The data for sinapoylglucose indicated substrate activation; those for l-malate, substrate inhibition. Kinetic analysis suggests a random bi bi mechanism within two ranges of substrate concentrations, with a kinetically preferred pathway via the enzyme-sinapoylglucose complex indicating a slow-transition mechanism. This may be interpreted as hysteretic cooperativity with sinapoylglucose.

Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase.

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants.

The chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modified Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg-chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.

Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination.

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination.

Isolation and characterisation of tobacco (Nicotiana tabacum) cDNA clones encoding proteins involved in magnesium chelation into protoporphyrin IX.

We have identified cDNA clones encoding the two Mg chelatase subunits CHL I and CHL H from tobacco (Nicotiana tabacum) by screening a cDNA library with homologous cDNA fragments from Arabidopsis thaliana. A full-length Chl I cDNA clone encodes a peptide with 426 amino acids. The entire cDNA sequence encoding 1382 amino acid long CHL H was obtained by extension of a truncated cDNA fragment using the 'rapid amplification of cDNA ends' (RACE) method. Both genes Chl I and Chl H were strongly expressed in young leaves and to a lesser extent in mature leaves. Only traces of both transcripts were found in flowering organs. Southern blot analysis suggests that CHL I is encoded by a single gene and CHL H most likely by several genes.