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PubMed1994

Organ-specific and auxin-inducible expression of two tobacco par A-related genes in transgenic plants.

We have isolated four genomic DNA clones that contain the transcription initiation site of the parA gene(s) from a tobacco genomic library by hybridization with the 5' segment of the parA cDNA previously isolated. They were classified into two types on the basis of their nucleotide sequences. Southern blot analysis indicated that two types of clones were respectively derived from the two parental species of tobacco, Nicotiana tomentosiformis and Nicotiana sylvestris. The genes corresponding to these clones were designated as parAt and parAs, respectively, and the parA cDNA clone was shown to code for mRNA from parAt on the basis of its nucleotide sequence. The 5' regions about 400 nucleotides upstream from the transcription initiation sites of the parAt and parAs genomic clones were highly homologous to one another, but regions further upstream showed no significant similarity. The coding sequence of the GUS (beta-glucuronidase) reporter gene was linked to the 5'-upstream regions of parAt and parAs, and the sites of expression of these fusion genes were examined in transgenic tobacco plants. In the absence of auxins, both fusion genes were expressed in capsules at a late stage of seed development, mature seeds, a root apex and a root-hair zone whereas no significant expression was seen in other organs. Their expression was enhanced by 2,4-dichlorophenoxyacetic acid in most of the organs of tobacco. The results show that expression of these genes is regulated by both organ-specific and auxin-inducible mechanisms.

Amino Acid Sequence↗
PubMed1984

Effects of exogenous methionine on storage protein composition of soybean cotyledons cultured in vitro.

Supplemental methionine in a complete culture medium increased the methionine content of the protein fraction of cultured soybean (Glycine max L. Merrill) cotyledons (Thompson, Madison, Muenster 1981 Phytochemistry 20: 941-945). To explain the observed increase in protein methionine, we have measured the amounts and subunit compositions of 7S and 11S storage proteins and determined the amino acid compositions of the three major protein fractions (2-5S, 7S, 11S) of seeds developed on plants and of cultured cotyledons grown in the presence or absence of supplemental l-methionine. Development of cultured cotyledons was representative of development of seeds on plants. The ratios of 11S to 7S proteins, the subunit contents, and amino acid compositions of their storage protein fractions were similar, but not identical. Supplemental methionine increased the mole percent methionine in each of the three protein fractions of cultured cotyledons and changed the amounts of several other amino acids. Supplemental methionine inhibited expression of the 7S beta-subunit gene. Concomitant with the absence of the beta-subunit, which contains no methionine, was an increase in the ratio of 11S to 7S proteins, and an increase in the methionine content of the subunits composing these fractions. Inhibition of beta-subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression.

Journal Article↗
PubMed1990

Analysis of seed storage protein genes of oats.

We have isolated genomic clones encoding the two major classes of seed storage proteins in oats, the 12 S globulins and the avenins. The globulin genes encode glutamine-rich, sulfur-poor storage proteins that are highly conserved in sequence and structure. The globulin genes contain three short introns whose positions in the coding sequence are the same as in storage globulin genes in legumes and other dicots. The avenin genomic clone contains four tightly linked genes that belong to both of the two avenin gene subfamilies. The avenin genes encode glutamine-rich, lysine-poor proteins that vary in length due to differences in the number of peptide repeats. Although globulin and avenin genes are expressed coordinately during oat seed development, their promoter regions do not contain any conserved sequence elements that might determine developmental timing. Previous studies showed that there are roughly equal amounts of globulin and avenin mRNAs in developing oat seed, despite there being much more globulin than avenin in mature seed. Storage protein synthesis in oats must therefore be controlled partially by post-transcriptional mechanisms. Sequence analysis of globulin and avenin genes has provided several clues as to why globulin mRNAs may be translated more efficiently than avenin mRNAs.

Allergens↗
PubMed2004

Vicilin-like storage globulin from buckwheat (Fagopyrum esculentum Moench) seeds.

An 8S storage globulin from buckwheat seed, which resembles the structure and features common to the vicilin-like family of seed storage proteins, was analyzed for this paper. It was found that expression of the 8S globulin gene precedes that of the 13S globulin (the main buckwheat storage protein) and starts from an early stage of buckwheat seed development (9-11 days after flowering), continuing to accumulate throughout seed development to contribute approximately 7% of total seed proteins. This protein fraction might be more interesting for biotechnological application than the 13S buckwheat legumin consisting of 23-25 kDa subunits reported to be the major buckwheat allergen. A partial cDNA was also isolated, showing high homology with cDNAs coding for vicilin-like storage proteins from various plant species, and its expression profile throughout seed development as well as in different buckwheat tissues was analyzed.

Amino Acid Sequence↗
PubMed1998

Expression of lauroyl-acyl carrier protein thioesterase in brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation

Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids.

Journal Article↗
PubMed1982

Temperature and oxygen effects on C-photosynthate unloading and accumulation in developing soybean seeds.

The environmental sensitivity of the processes associated with the import of photosynthate by developing soybean seeds was investigated within intact fruit and with excised, immature embryos. Intact pods of field-grown (Glycine max [L.] Merr.) Amsoy 71 soybeans were subjected to localized regimes of 0, 21, or 100% O(2) and 15, 25, or 35 degrees C during pulsechase translocation experiments and, 2.5 hours later, the uptake and distribution of (14)C-photosynthate among dissected fruit tissues determined. In other experiments, excised embryos were incubated in [(14)C]sucrose solutions under various experimental conditions to separate the effects of these treatments on accumulation by the embryos from those which may operate on phloem unloading in the maternal seedcoat.Import of (14)C-photosynthate by intact soybean fruit was both temperature- and O(2)-dependent. This dependency was shown to occur only within the seeds; import by the pod walls was essentially insensitive to fruit temperature or O(2) treatments. The embryos of anaerobic fruit were completely unlabeled, regardless of fruit temperature. But under anaerobic in vitro incubation conditions, uptake of [(14)C]sucrose in excised embryos was only 30% less than that in aerobic in vitro conditions. The data suggest that, within intact fruit, anoxia prevented sucrose efflux from the seed coat phloem and any subsequent uptake by the embryo. The demonstrated energy dependence of phloem unloading may reflect requirements for membrane integrity or energy metabolism in the companion cell-sieve element complex, consistent with a facilitated unloading process.Collectively, these data characterize the environmental sensitivity of photosynthate import in developing soybean fruit. They imply that environmental regulation of import may occur at both the embryo level and at the phloem terminals within the seed coat.

Journal Article↗
PubMed2005

Seed maturation: developing an intrusive phase to accomplish a quiescent state.

Plants, as sessile life forms, have evolved diverse mechanisms to circumvent unfavourable growth conditions, among them interruption of the life cycle is one of the most successful strategies. During seed formation, embryo development can proceed through a maturation phase that allows the entry into a quiescent state and represents an evolutionary advantage, since it facilitates dispersal and resuming of growth under optimal environmental conditions. In the maturation phase different gene expression programmes devoted to the accumulation of storage compounds, acquisition of desiccation tolerance and entry into quiescence occur, which are highly coordinated and under fine regulatory control. This paper is focused on recent findings related to central transcription factors which regulate gene expression during maturation. The structure of established regulatory networks is presented based on extensive gene promoter analyses and the characterisation of mutants affecting seed development and maturation. Different aspects of gene regulation both in the embryo and endosperm are covered and comparisons between monocot and dicot species on the basis of current knowledge of the system are also presented.

Gene Expression Regulation, Developmental↗
PubMed1984

Concentrations of sucrose and nitrogenous compounds in the apoplast of developing soybean seed coats and embryos.

The apoplast of developing soybean (Glycine max cv Hodgson) embryos and seed coats was analyzed for sucrose, amino acids, ureides, nitrate, and ammonia. The apoplast concentration of amino acids and nitrate peaked during the most rapid stage of seed filling and declined sharply as the seed attained its maximum dry weight. Amino acids and nitrate accounted for 80 to 95% of the total nitrogen, with allantoin and allantoic acid either absent or present in only very small amounts. Aspartate, asparagine, glutamate, glutamine, serine, alanine, and gamma-aminobutyric acid were the major amino acids, accounting for over 70% of the total amino acids present. There was a nearly quantitative conversion of glutamine to glutamate between the seed coat and embryo, most likely resulting from the activity of glutamate synthase found to be present in the seed coat tissue. This processing of glutamine suggests a partly symplastic route for solutes moving from the site of phloem unloading in the seed coat to the embryo.

Journal Article↗
PubMed2005

A microarray-based comparative analysis of gene expression profiles during grain development in transgenic and wild type wheat.

Global, comparative gene expression analysis is potentially a very powerful tool in the safety assessment of transgenic plants since it allows for the detection of differences in gene expression patterns between a transgenic line and the mother variety. In the present study, we compared the gene expression profile in developing seeds of wild type wheat and wheat transformed for endosperm-specific expression of an Aspergillus fumigatus phytase. High-level expression of the phytase gene was ensured by codon modification towards the prevalent codon usage of wheat genes and by using the wheat 1DX5HMW glutenin promoter for driving transgene expression. A 9K wheat unigene cDNA microarray was produced from cDNA libraries prepared mainly from developing wheat seed. The arrays were hybridised to flourescently labelled cDNA prepared from developing seeds of the transgenic wheat line and the mother variety, Bobwhite, at three developmental stages. Comparisons and statistical analyses of the gene expression profiles of the transgenic line vs. that of the mother line revealed only slight differences at the three developmental stages. In the few cases where differential expression was indicated by the statistical analysis it was primarily genes that were strongly expressed over a shorter interval of seed development such as genes encoding storage proteins. Accordingly, we interpret these differences in gene expression levels to result from minor asynchrony in seed development between the transgenic line and the mother line. In support of this, real time PCR validation of results from selected genes at the late developmental stage could not confirm differential expression of these genes. We conclude that the expression of the codon-modified A. fumigatus phytase gene in the wheat seed had no significant effects on the overall gene expression patterns in the developing seed.

6-Phytase↗
PubMedDate not supplied

[Effect of high intensity magnetic field on the processes of early growth in plant seeds and development of honeybees].

The influence of magnetic field on the early growth processes in plant seeds and the postembryonic development of honeybees was studied. Some general trends in the effects of magnetic field and differences in the tolerance of plant seeds and developing honeybees to its action were revealed. Some factors that may be responsible for a low reproducibility of magneto-biological effects are discussed.

Abnormalities, Radiation-Induced↗
PubMed2005

Gene expression of ADP-glucose pyrophosphorylase and starch contents in rice cultured cells are cooperatively regulated by sucrose and ABA.

Six cDNA clones encoding two small subunits and four large subunits of ADP-glucose pyrophosphorylase (AGPase) were mined from the database of rice full-length cDNAs, cloned and subsequently named: OsAPS1, OsAPS2, OsAPL1, OsAPL2, OsAPL3 and OsAPL4. Expression patterns of the six genes were examined by Northern blot analysis with gene-specific probes. OsAPL3 was predominantly expressed in the middle phases of seed development, and OsAPS1, OsAPL1 and OsAPL2 were expressed later in seed development. OsAPS2 and OsAPL4 were constitutively expressed and these isoforms were coordinated with starch accumulation in the developing rice seed. In order to clarify the effect of sugars and plant hormones on AGPase gene expression more precisely, a rice cell culture system was used. OsAPL3 transcript significantly accumulated in response to increased levels of sucrose and abscisic acid (ABA) concentration in the medium; however, the transcripts of other AGPase genes did not show significant accumulation. Under identical conditions, starch contents in the cultured cells also increased. Interestingly, ABA alone did not affect the gene expression of OsAPL3 and starch content. Collectively, these results indicated that the expression level of OsAPL3 and starch content in the cultured cells were cooperatively controlled by alterations in the concentration of both sucrose and ABA.

Abscisic Acid↗
PubMed1984

Effect of phosphorus and zinc nutrition on soybean seed phytic Acid and zinc.

The relationships between nutrient P and Zn levels and the phytic acid, P, and Zn concentrations in soybean (Glycine max L. Merr. cv ;Williams 79') seed were studied. Phytic acid increased linearly from 4.2 to 19.2 milligrams per gram as nutrient P treatment was varied from 2.0 to 50 milligrams per liter and Zn was held constant at 0.05 milligrams per liter. Leaf P concentration during seed development was found to be closely related to the concentrations of seed P and phytic acid. Leaf and seed Zn concentrations both responded positively to increasing nutrient Zn treatment. The effects of P treatment on plant and seed P and phytic acid were largely independent of the effects of Zn treatment on leaf and seed Zn. Phytic acid to Zn molar ratios ranging from 3.6 to 33.8 were observed.The effects of nutrient P treatments on the concentrations of phytic acid, seed P, and leaf P were also studied in the P-sensitive (gene np) cultivars ;Harosoy' and ;Clark' and their respective P-tolerant (gene Np) near-isogenic lines L66-704 and L63-1677. In general, the positive relationships observed among nutrient P, leaf P, seed P, and phytic acid concentrations were similar to those observed in the studies with Williams 79. When fertilized with low or moderate nutrient P (2.5 and 25.0 milligrams P per liter, respectively) no significant differences in any parameter were observed between Harosoy or Clark and their respective P-tolerant isolines. When fertilized with high nutrient P (100 milligrams P per liter), Harosoy seed had a significantly higher concentration of phytic acid (30 milligrams per gram) than did seed of its P-tolerant near-isogenic line L66-704 (24.2 milligrams per gram phytic acid), whereas no significant difference was observed between Clark and its P-tolerant near-isogenic line L63-1677 (22.8 and 21.6 milligrams per gram, respectively). Variation in the phytic acid concentrations in the mature seed of the cultivars and isolines more closely paralleled leaf P concentrations observed during seed development (49 days after flowering), than those observed at the onset of seed development (14 days after flowering). Electrophoresis and ion-exchange chromatography revealed that partially phosphorylated intermediates do not appear when phytic acid accumulation is greatly reduced by limiting the nutrient P or when accumulation is greatly accelerated by excess P.

Journal Article↗
PubMed2004

Catalase activity and expression in developing sunflower seeds as related to drying.

Changes in catalase (CAT) activity and in CAT isoform pattern and expression were investigated in developing sunflower (Helianthus annuus L.) seeds during desiccation on the mother plant and after artificial drying on the flowerheads. Seeds regularly desiccated during their development on the mother plant and reached mass maturity at c. 42 d after flowering (DAF). Freshly harvested seeds did not germinate at any stage of development because they were dormant, but their dormancy was broken after 5-6 months of dry storage. Immature seeds were desiccation-tolerant at 24 DAF since they were able to germinate fully after artificial drying on the flowerheads followed by dry storage. CAT activity increased in non-dehydrated seeds during their development, reaching a maximum a little after seed mass maturity and after artificial drying in immature seeds. This stimulation of CAT activity by natural and artificial drying was related to changes in CAT isoform pattern. Of the four constitutive CAT subunits, that of 59 kDa was always present, but dehydration induced the synthesis of a 55 kDa subunit. This synthesis of the CAT 55 kDa subunit resulted from an activation of the CATA1 gene, suggesting that the regulation of catalase activity and synthesis by drying occurred at the transcriptional level. The increase in CAT activity induced by seed drying was associated with a decrease in hydrogen peroxide level and in lipid peroxidation. These results suggest that CAT plays a role during seed desiccation by preventing dehydration-related oxidative damage and that H(2)O(2) may play a role in the regulation of CAT gene expression and the transduction pathway of the dehydration signal.

Catalase↗
PubMed2002

Proteome analysis of grain filling and seed maturation in barley.

In monocotyledonous plants, the process of seed development involves the deposition of reserves in the starchy endosperm and development of the embryo and aleurone layer. The final stages of seed development are accompanied by an increase in desiccation tolerance and drying out of the mature seed. We have used two-dimensional gel electrophoresis for a time-resolved study of the changes in proteins that occur during seed development in barley (Hordeum vulgare). About 1,000 low-salt extractable protein spots could be resolved on the two-dimensional gels. Protein spots were divided into six categories according to the timing of appearance or disappearance during the 5-week period of comparison. Nineteen different proteins or protein fragments in 36 selected spots were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry (MS) or nano-electrospray tandem MS/MS. Some proteins were present throughout development (for example, cytosolic malate dehydrogenase), whereas others were associated with the early grain filling (ascorbate peroxidase) or desiccation (Cor14b) stages. Most noticeably, the development process is characterized by an accumulation of low-M(r) alpha-amylase/trypsin inhibitors, serine protease inhibitors, and enzymes involved in protection against oxidative stress. We present examples of proteins not previously experimentally observed, differential extractability of thiol-bound proteins, and possible allele-specific spot variation. Our results both confirm and expand on knowledge gained from previous analyses of individual proteins involved in grain filling and maturation.

Alleles↗
PubMed1988

Regulation of Acyl Carrier Protein Messenger RNA Levels during Seed and Leaf Development.

We have examined the expression of acyl carrier protein (ACP) mRNA levels and ACP activity in leaves, where fatty acids function primarily in membrane synthesis, and in developing soybean seeds, where fatty acids are primarily used for oil storage. An RNA probe transcribed from a synthetic spinach ACP-I gene hybridized on Northern blots to ACP mRNA from both seed and leaf tissue from soybean, spinach, and rapeseed. In each species, the ACP transcript from leaf was slightly larger than that from seed. Both the amounts of ACP protein and the levels of ACP mRNA were substantially higher in young leaf tissue of spinach and soybean when compared to mature leaf tissue. Light-grown spinach leaves also contained higher ACP activity and accumulated more ACP mRNA than dark-grown leaves. ACP mRNA levels measured in developing soybean seeds peaked at 20 days after flowering then decreased 10-fold by 70 days after flowering. In each tissue, the developmental changes in ACP protein levels can be accounted for by changes in ACP mRNA abundance. Comparison of the relative prevalence of mRNA and protein for ACP and lectin in soybean seeds suggests a major difference in mRNA translational efficiency and/or protein stability for these two proteins.

Journal Article↗
PubMed2006

Soybean ATP sulfurylase, a homodimeric enzyme involved in sulfur assimilation, is abundantly expressed in roots and induced by cold treatment.

Soybeans are a rich source of protein and a key feed ingredient in livestock production, but lack sufficient levels of cysteine and methionine to meet the nutritional demands of swine or poultry as feed components. Although engineering the sulfur assimilatory pathway could lead to increased sulfur-containing amino acid content, little is known about this pathway in legumes. Here, we describe the cloning and characterization of soybean ATP sulfurylase (ATPS), which acts as the metabolic entry point into the sulfur assimilation pathway. Analysis of the ATPS clone isolated from a soybean seedling cDNA library revealed an open-reading frame, encoding a 52 kDa polypeptide with an N-terminal chloroplast/plastid transit peptide, which was related to the enzymes from Arabidopsis, potato, human, and yeast. Soybean ATP sulfurylase was expressed in Escherichia coli and purified to apparent homogeneity. Based on gel-filtration chromatography, the enzyme functions as a 100 kDa homodimer. Analysis of genomic DNA by Southern blotting revealed that multiple genes encode ATP sulfurylase in soybean. Analysis of the transcript profiles retrieved from a soybean EST database indicated that ATP sulfurylase mRNA was most abundant in root tissue. Cold treatment induced mRNA accumulation and enhanced the specific activity of ATP sulfurylase in root tissue. Northern blot analysis indicated a decline in the ATP sulfurylase transcript levels during seed development. Likewise, ATP sulfurylase specific activity also declined in the later stages of seed development. Increasing the expression levels of this key enzyme during soybean seed development could lead to an increase in the availability of sulfur amino acids, thereby enhancing the nutritional value of the crop.

Chloroplasts↗
PubMed2004

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds.

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.

Amino Acid Sequence↗
PubMed2002

A. thaliana TRANSPARENT TESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins.

Seeds of the Arabidopsis thaliana transparent testa 1 mutant (tt1) appear yellow, due to the lack of condensed tannin pigments in the seed coat. The TT1 gene was isolated by reverse genetics using an En-1 transposon mutagenized A. thaliana population. TT1 gene expression was detected in developing ovules and young seeds only, and the gene was shown to encode a nuclear protein. Mutant seeds displayed altered morphology of the seed endothelium in which brown tannin pigments accumulate in wild-type plants, indicating that TT1 is involved in the differentiation of this cell layer. When overexpressed in transgenic A. thaliana plants, TT1 caused aberrant development and organ morphology. The protein contains a novel combination of two TFIIIA-type zinc finger motifs. Closely related motifs were detected in a number of putative proteins deduced from plant genomic and EST sequences. The new protein domain containing this type of zinc finger motifs was designated WIP, according to three strictly conserved amino acid residues. Our data indicate the existence of a small gene family in A. thaliana which is defined by the occurrence of the WIP domain. WIP genes may play important roles in regulating developmental processes, including the control of endothelium differentiation.

Amino Acid Sequence↗