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Characterization of the 12S globulin complex of Brassica napus. Evolutionary relationship to other 11-12S storage globulins.

Cruciferin (12S globulin) is the major seed protein in Brassica napus (oil seed rape). It is synthesized during seed development and consists of six subunit pairs. Each of these pairs is synthesized as a precursor containing one alpha and one beta chain. At least three different precursors exist (P1-3), giving rise to four different mature subunits (cru1-4). Several cruciferin clones were isolated from a seed mRNA cDNA library. Comparison of the deduced amino acid sequences of these clones to amino acid sequences of purified cruciferin chains and peptides identified them as coding for cru2/3 and cru4 subunits. From the amino acid sequences deduced from two overlapping cDNA clones, the precursor of the cru4 subunit was shown to consist of 465 amino acid residues. Comparison of cruciferin and cruciferin-related sequences from B. napus and Arabidopsis thaliana, respectively, suggested that early during evolution the Brassicaceae family only possessed two types of 11-12S globulin genes, like the present-day Fabaceae.

Allergens↗

Recombinant lipoxygenases as biocatalysts for natural flavour production.

We identified a lipoxygenase expressed early during almond seed development. Biochemical and molecular characterisation showed that the enzyme produces almost exclusively 9-hydroperoxides which have been demonstrated to be important factors for the production of characteristic aromas in several fruits. An almond LOX cDNA was identified by RT-PCR using RNA extracted from immature almond seeds. Sequence analysis revealed that the identified gene is closely related to tomato fruit and potato tuber lipoxygenases. The isolated cDNA was cloned into pET24a and the expression of recombinant protein was induced in E. coli. The presence of an active LOX was confirmed in cells containing the recombinant vector. HPLC analysis of the reaction products of recombinant almond LOX confirmed that the isolated cDNA encodes a 9-LOX.

Catalysis↗

[Experiments with growing plants on orbital stations Saliut-5, Saliut-6 and Saliut-7].

The experiments with air-dry Crepis capillaris seeds flown on the spacecraft Soyuz-16 and orbital stations Salyut-5, Salyut-6 and Salyut-7 showed that the number of aberrant cells in the seedlings grown during flight (experimental) and after flight (flight control) was higher than that in the ground-based control. This number was greater in the experimental seedlings than in the flight controls. The plants Arabidopsis thaliana grew from cotyledons to flowers during flight. The seeds developed postflight exhibited a lower fertility and a higher frequency of recessive mutants (Experiment Svetoblock-1). The greater number of mutants persisted in the progeny of plants that completed their developmental cycle (Experiment Phyton-3). Inhibited viability of germs manifested as a reduced germination rate of flown seeds and a premature death of seedlings. In the first postflight generation the lesions produced by large chromosome aberrations were eliminated and the lesions caused by gene mutations and micro-aberrations were retained.

Chromosome Aberrations↗

A debranching enzyme deficiency in endosperms of the sugary-1 mutants of maize.

Many of the sugary-1 mutants of maize (Zea mays L.) have the highly branched water-soluble polysaccharide, phytoglycogen, in quantities equal to or greater than starch as an endosperm storage product in mature seeds. We find that all sugary mutants investigated are deficient in debranching enzyme [alpha-(1, 6)-glucosidase] activity in endosperm tissue 23 days postpollination and suggest that this deficiency is the primary biochemical lesion leading to phytoglycogen accumulation in sugary endosperms. This would indicate that the amylopectin component of starch depends on an equilibrium between the activities of branching enzymes introducing alpha-1,6 branch points into the linear alpha-1,4 glucans and debranching enzymes. The debranching enzyme activities from nonsugary endosperms can be separated into three peaks on a hydroxyapatite column. The sugary endosperm extracts lack one of these peaks of activity while the other two fractions have much reduced activity. The embryos of developing seeds (23 days after pollination) from both sugary and nonsugary genotypes have equivalent debranching activity. The debranching enzyme activity of developing endosperms is proportional to the number of copies (0 to 3) of the nonmutant (Su) allele present suggesting that the Su allele may be the structural gene for this debranching enzyme, although this is not definitive. This identification of debranching enzyme activity as being the biochemical lesion in sugary endosperms is consistent with several previous observations on the mutant.

Journal Article↗

Latest on enzymology of serotonin biosynthesis in walnut seeds.

Serotonin (5-HT) accumulation in walnut cotyledons is seen as a detoxification mechanism protecting the sensitive plant tissues of seeds from highly toxic ammonia concentrations following seed desiccation. Different metabolic pathways and cell compartments are involved in biosynthesis and storage of 5-HT. Ammonia fixation and incorporation into the indole moiety of tryptophan is followed by 5-HT biosynthesis via tryptamine in a two-step pathway with the adaptive tryptophan decarboxylase and the constitutive tryptamine 5-hydroxylase. Evidence is provided that tryptamine 5-hydroxylase is a member of the cytochrome P450 family which is involved in lipid hydroxylation processes in the very early period of seed development.

Ammonia↗

The molecular and genetic basis of ovule and megagametophyte development.

The formation of ovules is a key step in the plant life cycle which alternates between a diploid and haploid generation, the sporophyte and the gametophyte. The transitions between the two generations in the female occur in the ovule, the site of meiosis, female gametogenesis and double fertilization. The intimate association of sporophytic and gametophytic tissues in the ovule allows an investigation of their cellular interactions during ovule and seed development. In Arabidopsis, several sporophytically acting loci which primarily affect the morphogenesis of the integuments have been identified. Early acting genes may respond to positional information leading to a region-specific initiation of morphogenesis, whereas later acting genes affect cellular aspects of integument growth. Much less is known about the mechanisms controlling the development of the megagametophyte. Comparative morphology and genetic analyses suggests that regulatory mechanisms ensure a tight coordination of independently controlled cellular processes with cell specification and differentiation.

Plant Development↗

Acid phosphatases and seed shriveling in triticale.

Seed shriveling in the man-made intergeneric hybrid, triticale (x Triticosecale Wittmack) appears to be related to increased activity of endosperm acid phosphatases including para-nitrophenyl phosphatase, ATPase, ADPase, phosphatidic phosphatase, and glucose-1-phosphatase that occur specifically at later stages of seed development. These hydrolases may reduce endogenous substrates for starch synthesis, deplete energy supply for maintenance and biosynthesis of tissue growth, and deassemble membrane structures resulting in a partially filled endosperm and localized necrosis. Electrophoretic isozyme patterns of endosperm acid phosphatase exhibited distinctive differences between lines producing plump and shriveled seeds indicating a divergent role of the isozymes in these two different seed conformations.

Journal Article↗

The FUS3 transcription factor functions through the epidermal regulator TTG1 during embryogenesis in Arabidopsis.

Loss-of-function mutations in the FUSCA3 (FUS3) gene of Arabidopsis result in alterations in cotyledon identity, inability to complete late seed maturation processes, and the premature activation of apical and root embryonic meristems, which indicates that this transcription factor is an essential regulator of embryogenesis. Although FUS3 shows a complex pattern of expression in the embryo, this gene is only required in the protoderm to carry out its functions. Moreover, the epidermal morphogenesis regulator TRANSPARENT TESTA GLABRA1 (TTG1) is negatively regulated by FUS3 in the embryo. When a loss-of-function ttg1 mutation is introduced into a fus3 mutant, a number of fus3-related phenotypes are rescued, indicating a functional TTG1 gene is required to manifest the fus3 mutant phenotype. It therefore appears that one of the functions of FUS3 is to restrict the domain of expression of TTG1 during embryogenesis. The FUS3-TTG1 interaction is both maternal and zygotic, suggesting a complex relationship is required between these gene products to allow correct seed development.

Anthocyanins↗

The synthesis of phosphoenolpyruvate carboxylase in imbibing sorghum seeds.

When sorghum seeds were imbibed either in the light or in the dark, the presence of newly synthesized phosphoenolpyruvate carboxylase (PEPC) could be detected immunologically after approximately 6 h. In addition, both PEPC mRNA and enzyme activity were detected in extracts of dry seeds prior to inhibition. By contrast, ribulose-1,5-bisphosphate carboxylase mRNA, protein, and activity, as well as chlorophyll, were not detected even after 24 h of inhibition. These observations suggest that the nonphotosynthetic form of PEPC is synthesized during seed development and may play an important role in the germinative process.

Blotting, Northern↗

Iris melanocytoma: clinical features and natural course in 47 cases.

PURPOSE: To describe the clinical features, natural course, management and histopathologic features of iris melanocytoma. DESIGN: Single-center retrospective case series. METHODS PATIENT POPULATION: Forty-seven consecutive patients (47 eyes) with iris melanocytoma. INTERVENTION PROCEDURE: Data regarding patient and tumor features were analyzed for their impact on the main outcome measures using univariate and multivariate regression models. Kaplan-Meier estimates were used to analyze the main outcomes as a function of time. MAIN OUTCOME MEASURES: Increased intraocular pressure (IOP), tumor seeding, and tumor growth. RESULTS: Associated findings at initial presentation included iris stromal seeds in 20 patients (43%), and anterior chamber angle seeds in 12 (26%). Intrinsic vascularization and sector cataract were not seen in any eyes. The management at presentation included observation in 39 patients (83%), tumor removal by sector iridectomy/iridocyclectomy in 7 (15%), and enucleation for blind painful eye with secondary increased IOP in 1 (2%). The diagnosis was confirmed by histopathologic examination in 11 patients (23%). The mean follow-up was 58 months. Using Kaplan-Meier estimates, clinical evidence of growth was observed in 23% at 5 years, 48% at 10 years, and 74% at 15 years. New tumor seeds developed in 34% at 5 years, 63% at 10 years, and 75% at 15 years. Increased IOP was observed in 11% at 5 years, 11% at 10 years, and 55% at 15 years. CONCLUSIONS: Iris melanocytoma represented only 3% of all iris nevi. Related iris stromal and anterior chamber angle seeds were common, and secondary glaucoma occurred in 11% at 5 years. Growth was observed in 23% at 5 years but no malignant transformation was found.

Adolescent↗

Indole-3-acetic acid protein conjugates: novel players in auxin homeostasis.

Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates.

Arabidopsis↗

Patterns of protein oxidation in Arabidopsis seeds and during germination.

Increased cellular levels of reactive oxygen species are known to occur during seed development and germination, but the consequences in terms of protein degradation are poorly characterized. In this work, protein carbonylation, which is an irreversible oxidation process leading to a loss of function of the modified proteins, has been analyzed by a proteomic approach during the first stages of Arabidopsis (Arabidopsis thaliana) seed germination. In the dry mature seeds, the legumin-type globulins (12S cruciferins) were the major targets. However, the acidic alpha-cruciferin subunits were carbonylated to a much higher extent than the basic (beta) ones, consistent with a model in which the beta-subunits are buried within the cruciferin molecules and the alpha-subunits are more exposed to the outside. During imbibition, various carbonylated proteins accumulated. This oxidation damage was not evenly distributed among seed proteins and targeted specific proteins as glycolytic enzymes, mitochondrial ATP synthase, chloroplastic ribulose bisphosphate carboxylase large chain, aldose reductase, methionine synthase, translation factors, and several molecular chaperones. Although accumulation of carbonylated proteins is usually considered in the context of aging in a variety of model systems, this was clearly not the case for the Arabidopsis seeds since they germinated at a high rate and yielded vigorous plantlets. The results indicate that the observed specific changes in protein carbonylation patterns are probably required for counteracting and/or utilizing the production of reactive oxygen species caused by recovery of metabolic activity in the germinating seeds.

Arabidopsis↗

Allantoin and Allantoic Acid in Tissues and Stem Exudate from Field-grown Soybean Plants.

Samples of stem exudate and plant tissue collected from field-grown soybean (Glycine max [L.] Merr.) plants were analyzed for allantoin and allantoic acid. Nitrogen in nitrate plus amino acids exceeded ureide N concentration in stem exudate prior to flowering. During all of reproductive development (from about 40 days after planting until maturity), ureide N concentration was two to six times greater than amino acid plus nitrate N concentration. Allantoin and allantoic acid, not asparagine, are the principal forms of nitrogen transported from nodulated roots to shoots of the soybean plant. During pod and seed development ureide N comprised as high as 2.3, 37.7, and 15.8% of total N in leaf blades, stems + petioles, and fruits, respectively. The concentration of ureide in stems and fruits declined to nearly zero at maturity.Maximum ureide concentration of exudate collected from soybean nodules was 5.3 milligrams N per milliliter (94 micromoles allantoin per milliliter). This result supports evidence published by others that the site of allantoin synthesis is the nodule.

Journal Article↗

The development of juvenile plants of the hybrid orchid Bratonia after seed cryopreservation.

The development of juvenile plants of hybrid Bratonia orchid in vitro after seed storage in liquid nitrogen and the effect of nutrient medium composition on protocorm multiplication and plant regeneration were investigated. Cryopreservation did not inhibit the germination rate of seeds. Protocorms derived from cryopreserved seeds developed faster than protocorms from control (unfrozen) seeds during the first 45 days. But during further culturing, this tendency was not retained and finally protocorms from cryopreserved seeds had the same size as control ones. There were no significant differences in leaf number and shoot length between juvenile plants derived from unfrozen and cryopreserved seeds. We found that among four tested media liquid Morel medium was the most preferable for protocorm multiplication, and liquid ?S medium with half-strength macronutrients was the best one for the development of juvenile plants.

Adaptation, Physiological↗

Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis.

BACKGROUND: Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. RESULTS: High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources. CONCLUSION: AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds.

Arabidopsis↗

Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis.

The Arabidopsis thaliana LEC1 gene regulates embryo morphology and seed maturation. For a better understanding of its function, we isolated a carrot (Daucus carota L. cv. US-Harumakigosun) counterpart of this gene, C-LEC1, from a cDNA library of carrot somatic embryos, since carrot is a better model plant for preparing large quantities of somatic embryos at the same developmental stage. The predicted amino acid sequence of C-LEC1 is similar to that of LEC1 and contains regions that are conserved in the heme-activated protein 3 (HAP3) subunit of plants, animals and microorganisms. C-LEC1 expression was detected in embryogenic cells, somatic embryos, and developing seeds. In situ hybridization analysis revealed C-LEC1 expression in the peripheral region of the embryos but not in the endosperm. Expression of C-LEC1 driven by Arabidopsis LEC1 promoter was able to complement the defects of the Arabidopsis lec1-1 mutant. These results suggest that C-LEC1 is a functional homolog of Arabidopsis LEC1, an important regulator of zygotic and somatic embryo development.

Amino Acid Sequence↗

Molecular cloning and sequence analysis of a seed-expressed acyl carrier protein (ACP) gene from Brassica campestris (Agrani).

We describe here the nucleotide sequence of the Brassica campestris ACPSF1 gene which encodes a seed-expressed acyl carrier protein (ACP). The 3600 bp sequence consists of 1740 bp upstream of the translation start codon, 828 bp spanning the coding region which is interrupted by three introns and 1032 bp downstream of the stop codon. Using a ACPSF1 gene-specific probe, transcripts could be detected in developing seeds, but not in leaves. The gene is now the only known member that represents group I seed-expressed ACP multigene family of Brassica species. The 5' flanking sequence of the ACPSF1 gene was examined for putative transcriptional regulatory elements. A sequence alignment of the 5' flanking regions of the available seed-expressed ACP genes of Brassica species showed some conserved regions which might have some common regulatory significance.

Acyl Carrier Protein↗

Over-expression of a seed specific hevein-like antimicrobial peptide from Pharbitis nil enhances resistance to a fungal pathogen in transgenic tobacco plants.

Two hevein-like peptides from the seed of Pharbitis nil, designated Pharbitis nil antimicrobial peptide 1 (Pn-AMP1) and Pn-AMP2, had been purified previously. Both exhibit potent in vitro antifungal activity against a broad spectrum of phytopathogenic fungi. We now report the isolation of two cDNA clones, designated pnAMP-h1 and pnAMP-h2, and the corresponding genomic clones encoding these proteins from mature seeds of P. nil. Comparison of the deduced amino acid sequence to that of the mature protein suggests that the peptides are produced as a prepropeptide consisting of an N-terminal signal peptide, the mature protein and C-terminal domains. The transcripts of the two genes are accumulated seed--specifically, and the maximum transcripts are observed in the mid-to-late stage of seed development. Constitutive over-expression of the pnAMP-h2 cDNA in transgenic tobacco under the control of the cauliflower mosaic virus 35S promoter conferred enhanced resistance against the oomycete Phytophthora parasitica, the causal agent of black shank disease. Thus the Pn-AMPs may play a role in the protection of seeds and may be useful as a novel gene source to engineer plants resistant to fungal pathogens.

Amino Acid Sequence↗