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Expression patterns of duplicate tryptophan synthase beta genes in Arabidopsis thaliana.

Expression of the two Arabidopsis thaliana genes encoding tryptophan synthase beta (TSB1 and TSB2) was investigated by gene-specific RNA blot hybridization and reporter gene analysis. TSB1 mRNA abundance varies in an organ-specific manner, whereas TSB2 mRNA does not. Quantitative analysis of transgenic plants expressing TSB1 and TSB2 translational fusions to the beta-glucuronidase (GUS) gene (gusA) indicates that TSB1-GUS activity is 15-fold higher than TSB2-GUS. Histochemical analysis of these transgenic A. thaliana plants indicates that GUS expression occurs in a developmentally regulated manner. GUS activity driven from the TSB1 promoter is predominantly associated with the stem, root tips, foliar vasculature, mesophyll cells, base of developing seed pods, and tips of anther filaments in plants 15 d and older. Sections through the vegetative stem reveal GUS staining in all cell types including the shoot apical meristem. Although TSB2-GUS expression is consistently detected in root tips and at the base of developing seed pods, it is observed later in plant development than is TSB1-GUS expression.

Arabidopsis↗

Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds.

Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.

Acyl Coenzyme A↗

The pea gene NA encodes ent-kaurenoic acid oxidase.

The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

Blotting, Northern↗

Gene structure, protein structure, and regulation of the synthesis of a sulfur-rich protein in pea seeds.

Two low molecular weight pea seed albumins (Mr approximately 6000 and approximately 4000) have been characterized by protein, cDNA, and gene sequencing. Both proteins are encoded by separate regions of the same mRNA species. The initial translation product is a preproprotein from which a signal sequence is removed co-translationally. The resultant proprotein (PA1) is then cleaved post-translationally to yield the mature form of the two albumins (PA1a and PA1b). Comparison of cDNA and protein sequences suggests that at least four different PA1 genes are expressed in the pea genome. Both PA1a and PA1b have an unusually high cysteine content (7.5 and 16.2%, respectively). Pea seeds developing under suboptimal levels of sulfur nutrient supply contain reduced levels of PA1 mRNA and accumulate greatly reduced levels of PA1a and PA1b in the mature seed. In vitro transcription studies showed that this reduced level of PA1 mRNA resulted from reduced post-transcriptional stability rather than an altered rate of transcription of the PA1 gene. In contrast, during normal seed development, the level of PA1 mRNA seems to be under transcriptional control. Sequence comparisons reveal some homology between PA1 and a number of low molecular weight proteins from seeds of a wide range of mono- and dicotyledonous plants.

Albumins↗

Expression of a modified ADP-glucose pyrophosphorylase large subunit in wheat seeds stimulates photosynthesis and carbon metabolism.

ADP-glucose pyrophosphorylase (AGP) is the rate-limiting step in seed starch biosynthesis. Expression of an altered maize AGP large subunit (Sh2r6hs) in wheat (Triticum aestivum L.) results in increased AGP activity in developing seed endosperm and seed yield. The yield phenotype involves increases in both seed number and total plant biomass. Here we describe stimulation of photosynthesis by the seed-specific Sh2r6hs transgene. Photosynthetic rates were increased in Sh2r6hs-expressing plants under high light but not low light growth conditions, peaking at roughly 7 days after flowering (DAF). In addition, there were significant increases in levels of fructose, glucose, and sucrose in flag leaves at both 7 and 14 DAF. In seeds, levels of carbon metabolites at 7 and 14 DAF were relatively unchanged but increases in glucose, ADP-glucose, and UDP-glucose were observed in seeds from Sh2r6hs positive plants at maturity. Increased photosynthetic rates relatively early in seed development appear to be key to the Sh2r6hs enhanced yield phenotype as no yield increase or photosynthetic rate changes were found when plants were grown in a suboptimal light environment. These findings demonstrate that stimulation of biochemical events in both source and sink tissues is associated with Sh2r6hs expression.

Biomass↗

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean.

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C3 plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other 'C3-type' PEPC proteins more closely than those implicated in C4 or crassulacean acid metabolism.

Amino Acid Sequence↗

G-protein complex mutants are hypersensitive to abscisic acid regulation of germination and postgermination development.

Abscisic acid (ABA) plays regulatory roles in a host of physiological processes throughout plant growth and development. Seed germination, early seedling development, stomatal guard cell functions, and acclimation to adverse environmental conditions are key processes regulated by ABA. Recent evidence suggests that signaling processes in both seeds and guard cells involve heterotrimeric G proteins. To assess new roles for the Arabidopsis (Arabidopsis thaliana) Galpha subunit (GPA1), the Gbeta subunit (AGB1), and the candidate G-protein-coupled receptor (GCR1) in ABA signaling during germination and early seedling development, we utilized knockout mutants lacking one or more of these components. Our data show that GPA1, AGB1, and GCR1 each negatively regulates ABA signaling in seed germination and early seedling development. Plants lacking AGB1 have greater ABA hypersensitivity than plants lacking GPA1, suggesting that AGB1 is the predominant regulator of ABA signaling and that GPA1 affects the efficacy of AGB1 execution. GCR1 acts upstream of GPA1 and AGB1 for ABA signaling pathways during germination and early seedling development: gcr1 gpa1 double mutants exhibit a gpa1 phenotype and agb1 gcr1 and agb1 gcr1 gpa1 mutants exhibit an agb1 phenotype. Contrary to the scenario in guard cells, where GCR1 and GPA1 have opposite effects on ABA signaling during stomatal opening, GCR1 acts in concert with GPA1 and AGB1 in ABA signaling during germination and early seedling development. Thus, cell- and tissue-specific functional interaction in response to a given signal such as ABA may determine the distinct pathways regulated by the individual members of the G-protein complex.

Abscisic Acid↗

Contents and bioactivities of lunasin, bowman-birk inhibitor, and isoflavones in soybean seed.

It has been previously demonstrated that lunasin is a novel and promising cancer preventive peptide from soybean. The Bowman-Birk protease inhibitor (BBI) and isoflavones are well-studied substances from soy. This study evaluated the levels and bioactivities of these three compounds as affected by stages of seed development and sprouting under light and dark conditions. BBI and lunasin appear at 7 and 6 weeks, respectively, after flowering and increase as the seed matures. Daidzein and genistein both decrease during seed maturation. During sprouting under light, BBI increases up to the 6th day and decreases thereafter, disappearing at the 9th day after soaking. Under dark conditions, BBI increases up to the 7th day after soaking and decreases thereafter, disappearing at the 10th day. Lunasin starts to decrease at 2 days after soaking and disappears completely at 7 days under light and dark conditions. Daidzein and genistein increase continuously during the 10 days of soaking, and both increase more in the dark than under light conditions. Protein extracts from early seed development (2-5 weeks after flowering) suppress cell viability to a greater degree than those from later stages (6-9 weeks). Inhibition of foci formation by protein extracts from later stages is greater than those from earlier stages. Lunasin and BBI suppress foci formation more than the isoflavones. Sprouting decreases lunasin and BBI contents but increases isoflavones. Protein extracts from early soaking times inhibit foci formation more and suppress cell viability less than those from later soaking times. Light and dark conditions have no influence on the bioactivities of protein extracts. These data are useful in the preparation of soy fractions enriched in lunasin, BBI, and isoflavones and in making dietary recommendations.

Animals↗

Storage lipid accumulation and acyltransferase action in developing flaxseed.

Investigations of storage lipid synthesis in developing flaxseed (Linum usitatissimum) provide useful information for designing strategies to enhance the oil content and nutritional value of this crop. Lipid content and changes in the FA composition during seed development were examined in two cultivars of flax (AC Emerson and Vimy). The oil content on a dry weight basis increased steadily until about 20 d after flowering (DAF). The proportion of alpha-linolenic acid (alpha-18:3, 18:3cisDelta9,12,15) in TAG increased during seed development in both cultivars while the proportions of linoleic acid (18:2cisDelta9,12) and saturated FA decreased. The developmental and substrate specificity characteristics of microsomal DAG acyltransferase (DGAT, EC 2.3.1.20) and lysophosphatidic acid acyltransferase (LPAAT, EC 2.3.1.51) were examined using cultivar AC Emerson. The maximal acyltransferase specific activities occurred in the range of 8-14 DAF, during rapid lipid accumulation on a per seed basis. Acyl-CoA of EPA (20:5cisDelta5,8,11,14,17) or DHA (22:6 cis4,7,10,13,16,19) were included in the specificity studies. DGAT displayed enhanced specificity for alpha-18:3-CoA, whereas the preferred substrate of [PAAT was 18:2-CoA. Both enzymes could use EPA- or DHA-CoA to varying extents. Developing flax embryos were able to take up and incorporate these nutritional FA into TAG and other intermediates in the TAG-formation pathway. This study suggests that if the appropriate acyl-CoA-dependent desaturation/elongation pathways are introduced and efficiently expressed in flax, this may lead to the conversion of alpha-18:3-CoA into EPA-CoA, thereby providing an activated substrate for TAG formation.

Acetyl Coenzyme A↗

Development and Distribution of a Lectin from the Stems and Leaves of Dolichos biflorus.

The stems and leaves of the Dolichos biflorus plant contain a lectin that cross-reacts with antiserum against the seed lectin. This cross-reactive material (CRM) was followed during early seedling growth, stem elongation, and seed development using a specific radioimmunoassay.No CRM was detected in developing seeds, but very low levels were found in dormant and imbibed seeds. As germination proceeds, the CRM accumulates at the apex of both etiolated and green seedlings in the epicotyl and leaves. Lower amounts of CRM are found in the cotyledons and hypocotyl, but no CRM was detected in the roots.The amount of CRM in the first and second stem internodes increases during elongation and gradually declines after the completion of elongation. Approximately 80% of the CRM in the stems of 19-day-old Dolichos biflorus plants is associated with the elongating tissues. These results are discussed with respect to the possible roles of lectins in plants.

Journal Article↗

The lysine-dependent stimulation of lysine catabolism in tobacco seed requires calcium and protein phosphorylation.

The accumulation of free lysine in tobacco seed triggers the stimulation of lysine-ketoglutarate reductase, an enzyme that acts in lysine catabolism. The mechanism of lysine-ketoglutarate reductase stimulation was studied in two different systems: (1) developing seeds of wild-type plants in which the low basal lysine-ketoglutarate reductase activity can be stimulated by the exogenous addition of lysine; and (2) developing seeds of transgenic tobacco plants expressing a bacterial dihydrodipicolinate synthase in which lysine-ketoglutarate reductase activity is stimulated by endogenous lysine overproduction. In both systems, the stimulation of lysine-ketoglutarate reductase activity was significantly reduced when treated with the Ca2+ chelator EGTA. Moreover, the inhibitory effect of EGTA was overcome by the addition of Ca2+ but not Mg2+, suggesting that the lysine-dependent activation of lysine-ketoglutarate reductase requires Ca2+. This was further confirmed by a significant stimulation of lysine-ketoglutarate reductase activity following the treatment of wild-type seeds with ionomycin (an ionophore that increases Ca2+ flow into the cytoplasm). In addition, treatment of wild-type seeds with the protein phosphatase inhibitor okadaic acid triggered a significant induction in lysine-ketoglutarate reductase activity, whereas treatment of the transgenic seeds with the protein kinase inhibitor K-252a caused a significant reduction in its activity. Thus, we conclude that the stimulation of lysine-ketoglutarate reductase activity by lysine in tobacco seed operates through an intracellular signaling cascade mediated by Ca2+ and protein phosphorylation.

Calcium↗

Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation.

The synthesis of the major protein and lipid storage reserves during embryogenesis in oilseed rape (Brassica napus L., cv. Mikado) has been examined by biochemical, immunological and immunocytochemical techniques. The mature seeds contained about 45% (w/w) storage oil and 25% (w/w) protein. There were three major seed protein components, i.e. about 40-50% total protein was cruciferin, 20% was napin and 20% was a 18 kDa hydrophobic polypeptide associated with the proteinaceous membrane surrounding the storage oil bodies. Embryogenesis was divided into four overlapping stages with regard to the synthesis of these storage components: (1) for the first 3 weeks after flowering, little, if any, synthesis of storage components was observed; (2) storage-oil synthesis began at about week 3, and maximal rates were from weeks 4 to 7; (3) synthesis of the soluble storage proteins cruciferin and napin started at week 6 and rates were maximal between weeks 8 and 11; (4) the final stage was the synthesis of the 19 kDa oil-body polypeptide, which started at weeks 8-10 and was at a maximal rate between weeks 10 and 12. The synthesis of the 19 kDa oil-body protein therefore occurred independently of the synthesis of the soluble seed storage proteins. This former synthesis did not occur until shortly before the insertion of the 19 kDa polypeptide into the oil-body membrane. No evidence was found, either from sucrose-density-gradient-centrifugation experiments or from immunogold-labelling studies, for its prior accumulation in the endoplasmic reticulum. Conventional and immunogold-electron-microscopic studies showed that oil bodies were synthesized in the early to middle stages of seed development without a strongly electron-dense membrane. Such a membrane was only found at later stages of seed development, concomitantly with the synthesis of the 19 kDa protein. It is proposed that, in rapeseed embryos, oil bodies are initially formed with no proteinaceous membrane. Such a membrane is formed later in development after insertion by ribosomes of the hydrophobic 19 kDa polypeptide directly into the oil bodies.

Allergens↗

Ovule and embryo development, apomixis and fertilization.

Genetic analyses, particularly in Arabidopsis, have led to the identification of mutants that define different steps of ovule ontogeny, pollen stigma interaction, pollen tube growth, and fertilization. Isolation of the genes defined by these mutations promises to lead to a molecular understanding of these processes. Mutants have also been obtained in which processes that are normally triggered by fertilization, such as endosperm formation and initiation of seed development, occur without fertilization. These mutants may illuminate apomixis, a process of seed development without fertilization extant in many plants.

Arabidopsis↗

Abscisic acid and gibberellin differentially regulate expression of genes of the SNF1-related kinase complex in tomato seeds.

The SNF1/AMP-activated protein kinase subfamily plays central roles in metabolic and transcriptional responses to nutritional or environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, activating and anchoring subunits associate with and regulate the activity, substrate specificity, and cellular localization of the kinase subunit in response to changing nutrient sources or energy demands, and homologous SNF1-related kinase (SnRK1) proteins are present in plants. We isolated cDNAs corresponding to the kinase (LeSNF1), regulatory (LeSNF4), and localization (LeSIP1 and LeGAL83) subunits of the SnRK1 complex from tomato (Lycopersicon esculentum Mill.). LeSNF1 and LeSNF4 complemented yeast snf1 and snf4 mutants and physically interacted with each other and with LeSIP1 in a glucose-dependent manner in yeast two-hybrid assays. LeSNF4 mRNA became abundant at maximum dry weight accumulation during seed development and remained high when radicle protrusion was blocked by abscisic acid (ABA), water stress, far-red light, or dormancy, but was low or undetected in seeds that had completed germination or in gibberellin (GA)-deficient seeds stimulated to germinate by GA. In leaves, LeSNF4 was induced in response to ABA or dehydration. In contrast, LeSNF1 and LeGAL83 genes were essentially constitutively expressed in both seeds and leaves regardless of the developmental, hormonal, or environmental conditions. Regulation of LeSNF4 expression by ABA and GA provides a potential link between hormonal and sugar-sensing pathways controlling seed development, dormancy, and germination.

Abscisic Acid↗

Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil.

Lysophosphatidic acid acyltransferase acylates the sn-2 hydroxyl group of lysophosphatidic acid to form phosphatidic acid, a precursor to triacylglycerol. A cDNA encoding lysophosphatidic acid acyltransferase was isolated from developing seeds of meadowfoam (Limnanthes alba alba). The cDNA encodes a 281-amino acid protein with a molecular mass of 32 kD. The cDNA was expressed in developing seeds of transgenic high-erucic-acid rapeseed (Brassica napus) using a napin expression cassette. Erucic acid was present at the sn-2 position of triacylglycerols from transgenic plants but was absent from that position of seed oil extracted from control plants. Trierucin was present in the transgenic oil. Alteration of the sn-2 erucic acid composition did not affect the total erucic acid content. These experiments demonstrate the feasibility of using acyltransferases to alter the stereochemical composition of transgenic seed oils and also represent a necessary step toward increasing the erucic acid content of rapeseed oil.

Acyltransferases↗

Molecular composition and surface properties of storage lipid particles in wax bean (Phaseolus vulgaris).

Lipid particles have been isolated from seeds of wax bean (Phaseolus vulgaris), a species in which starch and protein rather than lipid are the major seed storage reserves. These lipid particles resemble oil bodies present in oil-rich seeds in that > 90% of their lipid is triacylglycerol. Moreover, this triacylglycerol is rapidly metabolized during seed germination indicating that it is a storage reserve. The phospholipid surfaces of oil bodies are known to be completely coated with oleosin which prevents their coalescence, particularly during desiccation of the developing seed. This would appear to be necessary since lipid is the major storage reserve in oil seeds, and there are very few alternate types of storage particles in the cytoplasm of oil seed endosperm to provide a buffer against coalescence of oil bodies by isolating them from one another. The present study indicates that the surfaces of lipid particles from wax bean are not completely coated with oleosin and feature regions of naked phospholipid. This finding has been interpreted as reflecting the fact that lipid particles in wax been seeds are less prone to coalescence than oil bodies of oil-rich seeds. This arises because the individual lipid particles are interspersed in situ among highly abundant protein bodies and starch grains and hence less likely to come in contact with one another, even during desiccation of the developing seed.

Amino Acid Sequence↗

Electrodiffusional uptake of organic cations by pea seed coats. Further evidence for poorly selective pores in the plasma membrane of seed coat parenchyma cells.

In developing seeds, the permeability of the plasma membrane of seed coat parenchyma cells is crucial for the supply of nutrients to the embryo. Here, we report characteristics of the transport of the organic cation choline and the basic amino acid L- histidine (His; cation at pH 5, electroneutral at pH 7) into isolated seed coats of pea (Pisum sativum). Supplied at sub-micromolar concentrations, choline(+) accumulated in the seed coat tissue 5.1 +/- 0.8-fold, His(+) 2.4 +/- 0.3-fold, and His(0) 1.3 +/- 0.2-fold. Taking into consideration that at pH 5 His influxes as a cation but effluxes as a neutral molecule, these accumulations are in reasonable agreement with (electro) diffusional uptake at the prevailing membrane potential of -55 +/- 3 mV. At a concentration of 100 mM, choline(+) and His(+), but not His(0), depolarized the membrane of the parenchyma cells and neither of the substrates was accumulated. At this concentration, the relative influx (the ratio of influx and external concentration, a measure for membrane permeability) of choline and His was approximately 10 micromol g(-1) fresh weight min(-1) M(-1), similar to that found for neutral amino acids, sucrose, glucose, and mannitol. At lower concentrations, the relative influx of choline(+) and His(+) increased because of increasingly more negative membrane potentials, giving rise to apparent saturation kinetics. It is suggested that transport of organic cations can proceed by a general, poorly selective pore in the plasma membrane of seed coat parenchyma cells. This pore is thought to be responsible for the unloading of a range of solutes that serve as nutrients for the embryo.

Algorithms↗

Multiple nuclear factors interact with upstream sequences of differentially regulated beta-conglycinin genes.

The expression of the alpha' and beta subunit genes of beta-conglycinin is differentially regulated during soybean embryo development. Although both are expressed solely in developing seeds during mid to late stages of embryo development, the alpha' subunit is expressed more highly on a per gene basis, and alpha' subunit mRNA begins to accumulate three to five days earlier than beta subunit mRNA. In cultured cotyledons, beta subunit gene(s) respond to changes in methionine or abscisic acid levels, whereas expression of the alpha' subunit gene(s) is unaffected by these changes. To investigate the mechanisms by which these genes are transcriptionally regulated, we examined the interactions of nuclear proteins with upstream sequences from the alpha' and beta subunit genes. Four distinct DNA binding factors were identified in nuclear extracts from developing soybean seeds. These factors are termed Soybean Embryo Factors (SEF) 1 through 4. SEF binding sites are distributed non-uniformly between the alpha' and beta subunit genes, and the amount of protein binding is modulated over the course of embryo development. DNA footprinting revealed the sequences recognized by three of these factors. Factors which behave in a manner similar to that of SEF3 were also identified in nuclear extracts from developing tobacco and sunflower seeds.

Antigens, Plant↗