Effect of different processing methods on antioxidant activity of underutilized legumes, Entada scandens seed kernel and Canavalia gladiata seeds.

The present study is proposed to determine the antioxidant activity of raw and processed samples of underutilized legumes, Entada scandens seed kernel and Canavalia gladiata seeds. The indigenous processing methods like dry heating, autoclaving and soaking followed by autoclaving in different solutions (plain water, ash, sugar and sodium bicarbonate) were adopted to seed samples. All other processing methods than dry heat showed significant reduction in phenolics (2.9-63%), tannins (26-100%) and flavonoids (14-67%). However, in processed samples of E. scandens, the hydroxyl radical scavenging activity and ß-carotene bleaching inhibition activity were increased, whereas, 2,2-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt (ABTS·(+)), ferric reducing antioxidant power (FRAP), metal chelating and superoxide anion scavenging activity were similar to unprocessed ones. In contrary, except dry heating in C. gladiata, all other processing methods significantly (P<0.05) reduced the 2,2'-diphenyl-1-picryl-hydrazyl (DPPH·) (20-35%), ABTS·(+) (22-75%), FRAP (34-74%), metal chelating (30-41%), superoxide anion radical scavenging (8-80%), hydroxyl radical scavenging (20-40%) and ß-carotene bleaching inhibition activity (15-69%). In addition, the sample extracts of raw and dry heated samples protected DNA damage at 10 µg. All processing methods in E. scandens and dry heating in C. gladiata would be a suitable method for adopting in domestic or industrial processing.

Genotypic differences in pod wall and seed growth relate to invertase activities and assimilate transport pathways in asparagus bean.

BACKGROUND AND AIMS: Coordination of sugar transport and metabolism between developing seeds and their enclosing fruit tissues is little understood. In this study the physiological mechanism is examined using two genotypes of asparagus bean (Vigna unguiculata ssp. sesquipedialis) differing in pod wall and seed growth rates. Pod growth dominates over seed growth in genotype 'Zhijiang 121' but not in 'Zhijiang 282' in which a 'bulging pod' phenotype is apparent from 8 d post-anthesis (dpa) onward. METHODS: Seed and pod wall growth rates and degree of pod-bulging were measured in the two genotypes together with assays of activities of sucrose-degrading enzymes and sugar content in pod wall and seed and evaluation of cellular pathways of phloem unloading in seed coat using a symplasmic fluorescent dye, 5(6)-carboxyfluorescein (CF). KEY RESULTS: Activities of cell wall, cytoplasmic and vacuolar invertases (CWIN, CIN and VIN) were significantly smaller in pod walls of '282' than in '121' at 10 dpa onwards. Low INV activities were associated with weak pod wall growth of '282'. In seed coats, CF was confined within the vasculature in '282' but moved beyond the vasculature in '121', indicating apoplasmic and symplasmic phloem unloading, respectively. Higher CWIN activity in '282' seed coats at 6-8 dpa correlated with high hexose concentration in embryos and enhanced early seed growth. However, CWIN activity in '282' decreased significantly compared with '121' from 10 dpa onwards, coinciding with earlier commencement of nuclei endoreduplication in their embryos. CONCLUSIONS: The study shows genotypic differences between 'bulging pod' and 'non-bulging' phenotypes of asparagus bean in sucrose metabolism in relation to the pathway of phloem unloading in developing seed coats, and to pod and seed growth. Low INV activity in pod wall corresponds to its shortened and weak growth period; by contrast, the apoplasmic path in the seed coat is associated with high CWIN activity and strong early seed growth.

Radiation induced alterations in Vigna radiata during in vitro somatic embryogenesis.

PURPOSE: Tissue culture has been exploited to understand molecular aspects of regeneration potential of the plants in normal and in stressed conditions. The present study describes ionizing radiation from (60)Co source as the stress stimulator to assess in vitro development of somatic embryo of Vigna radiata, a protein-rich pulse. MATERIALS AND METHODS: Callus culture was established, using leaves of V. radiata. Somatic embryogenesis was induced by manipulating plant hormones. Calli were exposed to gamma rays. Genomic DNA isolated from gamma-irradiated callus samples were subjected to random amplified polymorphic DNA analysis. A band of molecular weight 1440 bp was used as a probe and Southern hybridization was carried out. To determine alterations in DNA following irradiation, RAPD bands were cloned and sequenced from control and irradiated samples. Embryogenic calli were exposed to gamma irradiation and the effects were assessed immediately and after seven days of exposure. Phenotypic alterations were observed using scanning electron microscopy. RESULTS: Exposed calli revealed altered frequency of somatic embryo formation. Results showed that the 1440 bp molecular weight probe hybridized with bands of low molecular weight. DNA sequences from irradiated samples showed recombination when compared to control. Scanning electron micrography illustrated presence of transient pores on the exposed embryos. BLAST search of the DNA sequences showed partial homology with some sequences from Arabidopsis thaliana. CONCLUSION: The present report might help in designing a breeding program, where both radiation coupled with somatic embryogenesis could be employed to build up the desired variants.

Characterization and potential uses of Copaifera langsdorfii seeds and seed oil.

Copaifera langsdorfii (Desf.) Kuntze (copaiba) seeds are abundantly produced and have not yet been characterized. The seed oil presents a characteristic odor of coumarin (250.1+/-6.57 mg/g determined through LC). The fatty acid composition of the oil was determined through CG/FID, being 45.3% linoleic acid, 32.3% monounsaturated, and 22.4% saturated fat. For the lipid-free seeds, the total carbohydrate, protein and moisture were 75.4%, 6.8% and 14.8%, respectively. The C. langsdorfii xyloglucan had an intrinsic viscosity of 804 mL/g, and the average molar mass (Mw) was 7.82 x 10(5)g/mol and Rg of 65 nm. The degree of polydispersion was 1.7, indicating the polydisperse family of polysaccharides. Its homogeneity, low degree of polymer contaminants and high intrinsic viscosity and molecular mass, represent good potential as a thickening agent. The presence of coumarin and xyloglucan as major components of C. langsdorfii seeds denotes its potential for use in the cosmetic or pharmaceutical industries.

Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies.

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.

Developing embryos of Sesbania sesban have unique potential to photosynthesize under high osmotic environment.

This study has been carried out to investigate the photosynthetic activities in developing embryos of Sesbania sesban under a highly osmotic environment. In S. sesban, the embryo turns green/chlorophyllous at the early heart shape stage. Interestingly, despite being deeply embedded within the supporting tissues (several layers of pod wall, seed coat and endosperm) and developing in a highly osmotic environment, the growing embryo of the developing seed showed the presence of various components of photosynthetic machinery besides being chlorophyllous. The shade-adaptive nature of the photosynthetic machinery of the embryo is evident from (a) low chlorophyll a/b ratio, (b) photosystem (PS) II attaining maximal activity at low photon flux density and (c) lesser plastoquinone pool. The photosynthetic potential of the growing embryo seems to contribute towards seed filling as it has the potential not only to harvest light energy but also to fix CO2 as efficiently as other photosynthetic parts of S. sesban. In fact, ribulose-1,5 bisphosphate carboxylase purified from embryos manifested subunit composition similar to that of leaves. The PS II activity in leaves, cotyledonary leaves and pod wall declined sharply with increase in the level of NaCl and sucrose above 150 and 300 mmol, respectively. Amazingly, PS II activity in developing embryos was maximal in the presence of 250 mmol NaCl or 500 mmol sucrose and remained high even when NaCl and sucrose levels were increased to 500 and 1000 mmol, respectively. We hypothesize that the developing embryos have some factor(s) which protect(s) the photosynthetic machinery in an environment of high osmotic strength.

Molecular evidence for the inclusion of the Korean endemic genus " Echinosophora" in Sophora (Fabaceae), and embryological features of the genus.

Although Echinosophora Nakai has been known as a monotypic and endemic genus of Papillionoideae of Fabaceae in Korea, it has been controversial whether it is distinct from or merged with Sophora. To resolve this matter, we conducted molecular phylogenetic analyses using nucleotide sequence data from the plastid rbcL gene and trnL (UAA) intron. Parsimony analysis, using a total of 53 taxa of the Papillionoideae (including E. koreensis [Nakai] Nakai and several species of Sophora and related genera) and using 20 taxa of Caesalpinioideae and Mimosoideae as outgroups, showed that, although the examined species of Sophora are split into two clades, E. koreensis formed a common clade with S. tomentosa (the type species of the genus) and S. flavescens. E. koreensis therefore should be treated as S. koreensis Nakai, and the generic name Echinosophora be eliminated. We also investigated the embryology of S. koreensis (= E. koreensis) and S. flavescens and found that no differences existed between them. Our molecular study, like other studies, strongly suggested that Sophora is polyphyletic. In this study we presented a summary of embryological features of the core Sophora for future critical comparison with related and unrelated taxa.

Spermicidal activity of seed oil of Pongamia glabra.

The medicinal properties of seed oil of Pongamia glabra are well known in traditional Indian medicine. It has antimicrobial activity against several organisms. It is used in the treatment of herpes and scabies and, systemically, it is also used in the treatment of dyspepsia with sluggish liver. The present study demonstrates that in vitro, Pongamia oil has strong spermicidal activity.

Primary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds.

A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. The inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K(i )values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M(r) 22 h Da. The primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors.

Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legume seeds.

The temporal and spatial distribution of cysteine proteinases (CPRs) was analyzed immunologically and by in situ hybridization to identify the CPRs involved in the initiation of storage-globulin degradation in embryonic axes and cotyledons of germinating vetch (Vicia sativa L.). At the start of germination several CPRs were found in protein bodies in which they might have been stored in the mature seeds. Cysteine proteinase 1 was predominantly found in organs like the radicle, which first start to grow during germination. Cysteine proteinase 2 was also present at the start of germination but displayed a less-specific histological pattern. Proteinase B was involved in the globulin degradation of vetch cotyledons as well. The histological pattern of CPRs followed the distribution of their corresponding mRNAs. The latter were usually detected earlier than the CPRs but the in situ hybridization signals were histologically not as restricted as the immunosignals. Proteolytic activity started in the radicle of the embryonic axis early during germination. Within 24 h after imbibition it had also spread throughout the whole shoot. At the end of germination, newly synthesized CPRs might have supplemented the early detectable CPRs in the axis. In the cotyledons, only the abaxial epidermis and the procambial strands showed proteinase localization during germination. Both CPR1 and CPR2, as well as the less common proteinase B, might have been present as stored proteinases. Three days after imbibition, proteolytic activity had proceeded from the cotyledonary epidermis towards the vascular strands deeper inside the cotyledons. The histochemical detection of the CPRs was in accordance with the previously described histological pattern of globulin mobilization in germinating vetch [Tiedemann J, et al. (2000)]. A similar link between the distribution of CPRs and globulin degradation was found in germinating seeds of Phaseolus vulgaris L. The coincidence of the histological patterns of globulin breakdown with that of the CPRs indicates that at least CPR1, CPR2 and proteinase B are responsible for bulk globulin mobilization in the seeds of the two legumes.

Genetic selection for enhanced bioavailable levels of iron in bean (Phaseolus vulgaris L.) seeds.

The bioavailability of Fe from 24 select genotypes of bean (Phaseolus vulgaris L.) seeds containing a range of concentrations of Fe, myo-inositol pentaphosphate plus phytic acid (IP5+IP6), and tannins was studied using a rat model. Bean accessions, selected from field trials for their variations in Fe, phytate, and tannin seed concentrations, were grown in a greenhouse in nutrient solutions radiolabeled with (59)Fe. Mature seeds were autoclaved and lyophilized. Test meals (containing 1 g of dried bean, 0.5 g of sucrose, and 1 g of basal Fe-deficient diet) were fed to marginally Fe-depleted weanling rats over a 3-h period; rats were radioassayed in a gamma-spectrometer immediately after feeding and daily thereafter for the next 10 d. Radioiron retention data were used to calculate percent Fe absorption (i.e., Fe bioavailability) from the meals. Seed Fe concentrations ranged from 52 to 157 microg g(-)(1) dry weight. There was a tendency to also select for higher Zn concentrations in the beans when selecting for high Fe concentrations. The Fe bioavailability to rats from test meals depended on the genotype and varied from 53% to 76% of the total Fe. Bean genotypes with higher seed Fe concentrations resulted in increased amounts of bioavailable Fe to rats. There was no significant correlation between the Fe concentration in different bean genotypes and Fe bioavailability to rats attributable to variations in IP5+IP6 or tannins, even though these antinutrients varied widely (i.e., from 19.6 to 29.2 micromol of IP5+IP6 g(-)(1) and from 0.35 to 2.65 mg of tannins g(-)(1)) in the test meals. Other unknown seed factors (i.e., antinutrients or promoter substances) may be contributing factors affecting Fe bioavailability from bean seeds.

Mitogenic effect of Parkia speciosa seed lectin on human lymphocytes.

Mitogenic activity of a lectin, purified from Parkia speciosa seeds, on the isolated peripheral blood lymphocytes taken from normal blood donors and patients with esophageal carcinoma was examined using [3H]thymidine incorporation. The lectin increases the incorporation of [3H]thymidine into DNA of human lymphocytes. The activity of the lectin increased as its concentration was increased and then declined once the concentration passed an optimum point. The stimulant effect was also expressed using a proliferation index (PI): the ratio of [3H]thymidine incorporated into lymphocytes in the presence and absence of the lectin. The mitogenic activity of the lectin is comparable to those of the known T-cell mitogens, such as concanavalin A, phytohaemagglutinin, and pokeweed mitogen. Only slightly less responsiveness was observed in the case of lymphocytes from esophageal cancer compared to lymphocytes from normal donors.

Endogenous protein phosphorylation and protein kinase activity in winged bean.

In winged bean (Psophocarpus tetragonolobus) protein kinases (E.C. 2.7.1.37) were found in all tissues studied. There was a significant increase in kinase activity during seed development, with a concomitant enhancement in the phosphorylation of a number of polypeptides; this was reversed in germinating seed cotyledons. Protein phosphorylation was apparently correlated with the increase in the protein content of the developing seed and the growing axis. At least three distinct autophosphorylating proteins could be distinguished in the developing seeds after SDS-PAGE, indicating the presence of different types of protein kinases in winged bean.

Characterization of Phaseolus vulgaris cDNA clones responsive to water deficit: identification of a novel late embryogenesis abundant-like protein.

Six cDNA clones from Phaseolus vulgaris, whose expression is induced by water deficit and ABA treatment (rsP cDNAs) were identified and characterized. The sequence analyses of the isolated clones suggest that they encode two types of late-embryogenesis abundant (LEA) proteins, a class-1 cytoplasmic low-molecular-weight heat shock protein (lmw-HSP), a lipid transfer protein (LTP), and two different proline-rich proteins (PRP). One of the putative LEA proteins identified corresponds to a novel 9.3 kDa LEA-like protein. During the plant response to a mild water deficit (psi w = -0.35 MPa) all genes identified present a maximal expression at around 16 or 24 h of treatment, followed by a decline in expression levels. Rehydration experiments revealed that those genes encoding PRPs and LTP transiently re-induce or maintain their expression when water is added to the soil after a dehydration period. This is not the case for the lea genes whose transcripts rapidly decrease, reaching basal levels a few hours after rehydration (4 h). Under water deficit and ABA treatments, the highest levels of expression for most of the genes occur in the root, excluding the ltp gene whose maximum expression levels are found in the aerial regions of the plant. This indicates that for these genes, both water deficit and ABA-dependent expression are under organ-specific control. The data presented here support the importance of these proteins during the plant response to water deficit.

Cell-type specific, coordinate expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.

Several cDNA clones encoding two different ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) polypeptides denoted VfAGPC and VfAGPP were isolated from a cotyledonary library of Vicia faba L. Both sequences are closely related to AGPase small-subunit sequences from other plants. Whereas mRNA levels of VfAGPP were equally high in developing cotyledons and leaves, the mRNA of VfAGPC was present in considerable amounts only in cotyledons. During development of cotyledons, both mRNAs accumulated until the beginning of the desiccation phase and disappeared afterwards. The increase of AGPase activity in cotyledons during the phase of storage-product synthesis was closely followed by the accumulation of starch. The AGPase activity in crude extracts of cotyledons was insensitive to 3-phosphoglycerate whereas the activity from leaves could be activated more than five-fold. Inorganic phosphate inhibited the enzyme from both tissues but was slightly more effective on the leaf enzyme. There was a correlation at the cellular level between the distribution of VfAGPP and VfAGPC mRNAs and the accumulation of starch, as studied by in-situ hybridisation and by histochemical staining in parallel tissue sections of developing seeds, respectively. During the early phase of seed development (12-15 days after fertilization) VfAGPase mRNA and accumulation of starch were detected transiently in the hypodermal, chlorenchymal and outer parenchymal cell layers of the seed coat but not in the embryo. At 25 days after fertilization both synthesis of VfAGPase mR-NA and biosynthesis of starch had started in parenchyma cells of the inner adaxial zone of the cotyledons. During later stages, the expression of VfAGPase and synthesis of starch extended over most of the cotyledons but were absent from peripheral cells of the abaxial zone, provascular and procalyptral cells.

Ethylene regulates the expression of a cysteine proteinase gene during germination of chickpea (Cicer arietinum L.).

Synthetic oligonucleotides corresponding to conserved regions of cysteine proteinases were used as primers in the RT-PCR amplification of a fragment of cDNA corresponding to a region of a cysteine proteinase gene expressed during germination of chickpea (cac for Cicer arietinum cysteine proteinase). The identity of the PCR-amplified fragment was confirmed by sequencing and the fragment used as a probe to investigate the pattern of cac gene expression during germination and its hormonal regulation. The corresponding transcript is undetected in the seed during embryogenesis and before imbibition, being detected 24 h after imbibition. Ablation of the embryonic axis before imbibition results in a dramatic decrease in the amount of transcript detected. Expression of the cac transcript in excised cotyledons is restored in the presence of aqueous extracts from embryonic axes and also by incubating the excised cotyledons in 1 mM ethephon. Experiments with various known inhibitors of ethylene action indicate that ethylene activates the expression of cac gene in the cotyledons of chickpea during normal germination.

Endo-1,4-beta-glucanase in the cell wall of stems of auxin-treated pea seedlings.

Endo-1,4-beta-glucanase induced by treatment of pea seedlings with 2,4-D was extracted from a preparation of the walls of epicotyl cells. The beta-glucanase was purified by chromatography on DEAE-cellulose, affinity chromatography on Con A-Sepharose and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The activity of beta-glucanase was retained after removal of SDS and extraction from polyacrylamide gels. The band of a protein (46 kDa), that corresponded to the activity of endo-1,4-beta-glucanase, was injected directly into mice for preparation of antiserum and the protein was also subjected to amino acid sequencing after blotting onto a membrane. Western blot analysis showed that the antiserum obtained bound to a 46-kDa polypeptide and recognized endo-1,4-beta-glucanase. The N-terminal sequence of the 46-kDa polypeptide revealed some homology to abscission endo-1,4-beta-glucanases of bean and avocado fruit.

Developmental patterns of free and protein-bound biotin during maturation and germination of seeds of Pisum sativum: characterization of a novel seed-specific biotinylated protein.

Mature dry pea seeds contain three major biotinylated proteins. Two of these of subunit molecular mass about 75 kDa and 200 kDa are associated with 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) and acetyl-CoA carboxylase activities (EC 6.4.1.2) respectively. The third does not exhibit any of the biotin-dependent carboxylase activities found in higher organisms and represents the major part of the total protein-bound biotin in the seeds. This novel protein has been purified from a whole pea seed extract. Because in SDS/polyacrylamide gels the protein migrates with an apparent molecular mass of about 65 kDa, it is referred to as SBP65, for 65 kDa seed biotinylated protein. The molecular mass of native SBP65 is greater than 400 kDa, suggesting that the native protein assumes a polymeric structure, resulting from the association of six to eight identical subunits. The results of CNBr cleavage experiments suggest that biotin is covalently bound to the protein. The stoichiometry is 1 mol of biotin per 1 mol of 65 kDa polypeptide. The temporal and spatial pattern of expression of SBP65 is described. SBP65 is specifically expressed in the seeds, being absent from leaf, root, stem, pod and flower tissues of pea plants. The level of SBP65 increases dramatically during seed development. The protein is not detectable in very young seeds. Its accumulation pattern parallels that for storage proteins, being maximally expressed in the mature dry seeds. SBP65 disappears at a very high rate during seed germination. The level of free biotin has also been evaluated for various organs of pea plants. In all proliferating tissues examined (young developing seeds, leaf, root, stem, pod and flower tissues), free biotin is in excess of protein-bound biotin. Only in the mature dry seeds is protein-bound biotin (i.e. that bound to SBP65) in excess of free biotin. These temporal expression patterns, and the strict organ specificity for expression of SBP65, are discussed with regard to the possibility that in plants, as in mammals, biotin plays a specialized role in cell growth and differentiation.

Suppression of phaseolin and lectin in seeds of common bean, Phaseolus vulgaris L.: increased accumulation of 54 kDa polypeptides is not associated with higher seed methionine concentrations.

Suppression of phaseolin and lectin accumulation in common bean resulted in higher concentrations of bean seed polypeptides with apparent molecular weights of 54 kDa and from 70 to 84 kDa on SDS-polyacrylamide gel electrophoresis. Polypeptides of 54 and 56 kDa segregated as products of different alleles. Genes for the 54/56 kDa bands and phaseolin were estimated to be 26.2 +/- 3.7 map units apart. The 54 kDa band phenotype manifested by SDS-PAGE consisted of from one to three polypeptides of 54 kDa MW on 2D gels, and the 56 kDa phenotype consisted of one polypeptide of 56 kDa plus two minor polypeptides of 54-54.5 kDa molecular weight. The pKI of these polypeptides was approximately 5.25. The methionine content of the 54 kDa polypeptides of the cultivar Great Northern Star was 1.6 +/- 0.1 g/100 g protein, which was not statistically different from the value (1.5 +/- 0.1%) obtained for phaseolin isolated by the same procedure. F2 seeds deficient for phaseolin and lectin contained as much total N per g as wild-type seeds and were not shrunken, but contained 50% more free amino acids. F2 seeds from two of the three populations contained from 8 to 13% less methionine per mg total N.

Both internal and external regulatory elements control expression of the pea Fed-1 gene in transgenic tobacco seedlings.

In previous studies using leaves of light-grown transgenic tobacco plants, we have shown that sequences located within the transcribed region of the pea Fed-1 gene (encoding ferredoxin I) are major cis-acting determinants of light-regulated mRNA accumulation. However, we show here that these internal sequences are less important for the Fed-1 light response in etiolated tobacco seedlings than they are in green leaves and that upstream elements confer organ specificity and contribute significantly to Fed-1 light responses in etiolated material. Light effects mediated by upstream response elements are thus most pronounced during the initial induction of gene activity, whereas internal elements play a more prominent role in modulating Fed-1 expression once the gene is already active.