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PubMed2005

Storage oil breakdown during embryo development of Brassica napus (L.).

In this study it is shown that at least 10% of the major storage product of developing embryos of Brassica napus (L.), triacylglycerol, is lost during the desiccation phase of seed development. The metabolism of this lipid was studied by measurements of the fate of label from [1-(14)C]decanoate supplied to isolated embryos, and by measurements of the activities of enzymes of fatty acid catabolism. Measurements on desiccating embryos have been compared with those made on embryos during lipid accumulation and on germinating seedlings. Enzymes of beta-oxidation and the glyoxylate cycle, and phosphoenolpyruvate carboxykinase were present in embryos during oil accumulation, and increased in activity and abundance as the seeds matured and became desiccated. Although the activities were less than those measured during germination, they were at least comparable to the in vivo rate of fatty acid synthesis in the embryo during development. The pattern of labelling, following metabolism of decanoate by isolated embryos, indicated a much greater involvement of the glyoxylate cycle during desiccation than earlier in oil accumulation, and showed that much of the (14)C-label from decanoate was released as CO(2) at both stages. Sucrose was not a product of decanoate metabolism during embryo development, and therefore lipid degradation was not associated with net gluconeogenic activity. These observations are discussed in the context of seed development, oil yield, and the synthesis of novel fatty acids in plants.

Brassica napus↗

PubMed1986

Rapeseed embryo development in culture on high osmoticum is similar to that in seeds.

The development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without high osmoticum (0.48 and 0.69 molar sorbitol) was compared with normal development in situ to investigate the role of a drying environment in embryo maturation. Sensitivity to osmoticum was assayed in terms of its ability to mimic normal development, i.e. to both suppress germination and maintain 12 S storage protein (cruciferin) synthesis at levels comparable to those seen in the developing seed. The osmotic conditions used block germination of predesiccation stage embryos but were not sufficient to prevent desiccation stage embryos from taking up water and germinating. At all stages tested, the osmotically treated embryos had approximately normal levels of cruciferin mRNA. Measurements of endogenous abscisic acid (ABA) levels by radioimmunoassay indicated that the osmotic effects on germination and gene expression were not mediated by elevated embryonic ABA. Comparison of the kinetics of osmotic and ABA effects on gene expression showed that the osmotic effect is more rapid. These results are consistent with the hypothesis that ABA acts by inhibiting water uptake, which mechanically prevents germination and affects gene expression in some unknown manner.

Journal Article↗

PubMed1987

Synthesis and protein body deposition of maize 15-kd zein in transgenic tobacco seeds.

The maize 15-Kd zein structural gene was placed under the regulation of French bean beta-phaseolin gene flanking regions. Agrobacterium tumefaciens-mediated transformation was used to insert the chimeric phaseolin-zein gene into the tobacco genome. Transgenic plants synthesized zein in a tissue-specific manner during the latter half of seed development. Transcription of the chimeric gene was initiated in phaseolin-derived sequences, and was terminated within the phaseolin gene 3' flanking region. Both zein- and phaseolin-derived polyadenylation signals were used in the processing of zein RNA in transgenic plant seeds. Zein accumulation, though subject to an 80-fold variation among 19 plants tested, could reach as much as 1.6% of the total seed protein in several plants. In developing tobacco seeds, zein was correctly processed by the removal of a 20-amino-acid signal peptide. Electron microscope immunogold localization of the zein expressed in embryo and endosperm tissue indicates that the monocot protein accumulates in the crystalloid component of vacuolar protein bodies. The density of gold label over the protein bodies is several fold greater in the embryo than the endosperm. Zein is found in roots, hypocotyls and cotyledons of germinating transgenic tobacco seeds.

Journal Article↗

PubMed1992

A Role for the Surrounding Fruit Tissues in Preventing the Germination of Tomato (Lycopersicon esculentum) Seeds : A Consideration of the Osmotic Environment and Abscisic Acid.

During tomato seed development the endogenous abscisic acid (ABA) concentration peaks at about 50 d after pollination (DAP) and then declines at later stages (60-70 DAP) of maturation. The ABA concentration in the sheath tissue immediately surrounding the seed increases with time of development, whereas that of the locule declines. The water contents of the seed and fruit tissues are similar during early development (20-30 DAP), but decline in the seed tissues between 30 and 40 DAP. The water potential and the osmotic potential of the embryo are lower than that of the locular tissue after 35 DAP also. Seeds removed from the fruit at 30, 35, and 60 DAP and placed ex situ on 35 and 60 DAP sheath and locular tissue are prevented from germinating. Development of 30 DAP seeds is maintained or promoted by the ex situ fruit tissue with which they are in contact. Their germination is inhibited until subsequent transfer to water, and germination is normal, i.e. by radicle protrusion, and viable seedlings are produced, compared with 30 DAP seeds transferred directly to water; more of these seeds germinate, but by hypocotyl extension, and seedling viability is very poor. Isolated seeds at 35 and 60 DAP re-placed in contact with fruit tissues only germinate when transferred to water after 7 d. At 30 DAP, isolated seeds are insensitive to ABA at physiological concentrations in that they germinate as if on water, albeit by hypocotyl extension. At higher concentrations germination occurs by radicle protrusion. Osmoticum prevents germination, but there is some recovery upon subsequent transfer to water. Seeds at 35 DAP are very sensitive to ABA and exhibit little or no germination, even upon transfer to water. The response of the isolated seeds to osmoticum more closely approximates that to incubation on the ex situ fruit tissues than does their response to ABA. This is also the case for isolated 60 DAP seeds, whose germination is not prevented by ABA, but only by the osmoticum; these seeds are inhibited when in contact with ex situ fruit tissues also. It is proposed that the osmotic environment within the tissues of the tomato fruit plays a greater role than endogenous ABA in preventing precocious germination of the developing seeds.

Journal Article↗

PubMed2006

Posttranslational regulation of pyruvate, orthophosphate dikinase in developing rice (Oryza sativa) seeds.

Pyruvate, orthophosphate dikinase (PPDK; E.C.2.7.9.1) is most well known as a photosynthetic enzyme in C4 plants. The enzyme is also ubiquitous in C3 plant tissues, although a precise non-photosynthetic C3 function(s) is yet to be validated, owing largely to its low abundance in most C3 organs. The single C3 organ type where PPDK is in high abundance, and, therefore, where its function is most amenable to elucidation, are the developing seeds of graminaceous cereals. In this report, we suggest a non-photosynthetic function for C3 PPDK by characterizing its abundance and posttranslational regulation in developing Oryza sativa (rice) seeds. Using primarily an immunoblot-based approach, we show that PPDK is a massively expressed protein during the early syncitial-endosperm/-cellularization stage of seed development. As seed development progresses from this early stage, the enzyme undergoes a rapid, posttranslational down-regulation in activity and amount via regulatory threonyl-phosphorylation (PPDK inactivation) and protein degradation. Immunoblot analysis of separated seed tissue fractions (pericarp, embryo + aleurone, seed embryo) revealed that regulatory phosphorylation of PPDK occurs in the non-green seed embryo and green outer pericarp layer, but not in the endosperm + aleurone layer. The modestly abundant pool of inactive PPDK (phosphorylated + dephosphorylated) that was found to persist in mature rice seeds was shown to remain largely unchanged (inactive) upon seed germination, suggesting that PPDK in rice seeds function in developmental rather than in post-developmental processes. These and related observations lead us to postulate a putative function for the enzyme that aligns its PEP to pyruvate-forming reaction with biosynthetic processes that are specific to early cereal seed development.

Gene Expression Regulation, Enzymologic↗

PubMed1995

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags, and expression levels of mRNAs.

Using a cDNA library prepared from poly(A)+ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, alpha-1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycine-rich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.

Blotting, Northern↗

PubMed2001

Immunohistochemical study of DNA methylation dynamics during plant development.

DNA methylation represents one of the key processes that play an important role in the transcriptional control of gene expression. The role of cytosine methylation in plant development has been demonstrated by at least three different kinds of evidence: parent-specific expression of some genes in developing seeds, control of flowering time and floral morphogenesis, and correlation with silencing of intrusive DNA sequences (mobile genetic elements and transgenes). In this work global changes in DNA methylation during seed germination and shoot apical meristem development in Silene latifolia have been studied using an indirect immunohistochemical approach. The data presented show that a rapid decrease in global DNA methylation during seed germination occurs first in endosperm tissue and subsequently in the hypocotyl. Using 5-bromo-2'-deoxyuridine pulses, it has been demonstrated that these demethylation events occurred before cell division had begun. In the early post-germination period, a decrease in DNA methylation was detected in cotyledons, also before cell division was observed. Taken together, these results indicate that DNA demethylation takes place in a non-replicative way, probably by an active mechanism. The central zone of the shoot apical meristem remains highly methylated during the whole period of vegetative growth and in this region, only a low cell division activity was found. However, upon the transition of the shoot apical meristem to the floral bud, the meristem both decreased its high methylation status and its cells started to divide. These data indicate that the central zone of the shoot apical meristem can represent a relatively quiescent 'germ-line' which is activated upon flowering to form spores and gametes.

Cell Differentiation↗

PubMed1988

Response of Two Wheat Cultivars to CO(2) Enrichment under Subambient Oxygen Conditions.

Two cultivars of wheat (Triticum aestivum L. cvs Sonoita and Yecora Rojo) were grown to maturity in a growth chamber within four sub-chambers under two CO(2) levels (350 or 1000 microliters per liter) at either ambient (21%) or low O(2) (5%). Growth analysis was used to characterize changes in plant carbon budgets imposed by the gas regimes. Large increases in leaf areas were seen in the low O(2) treatments, due primarily to a stimulation of tillering. Roots developed normally at 5% O(2). Seed development was inhibited by the subambient O(2) treatment, but this effect was overcome by CO(2) enrichment at 1000 microliters per liter. Dry matter accumulation and seed number responded differently to the gas treatments. The greatest dry matter production occurred in the low O(2), high CO(2) treatment, while the greatest seed production occurred in the ambient O(2), high CO(2) treatment. Growth and assimilation were stimulated more by either CO(2) enrichment or low O(2) in cv Yecora Rojo than in Sonoita. These experiments are the first to explore the effect of whole plant low O(2) treatments on growth and reproduction. The finding that CO(2) enrichment overcomes low O(2)-induced sterility may help elucidate the nature of this effect.

Journal Article↗

PubMed1989

Nuclear factors interact with a soybean beta-conglycinin enhancer.

Upstream sequences of the gene encoding the alpha' subunit of beta-conglycinin were analyzed for interactions with nuclear proteins from immature soybean seeds. Two factors were identified that interact with specific sequence elements within 257 base pairs 5' of the transcription start site. One factor, SEF 3, binds exclusively to a region composed of two elements located at -183 to -169 base pairs and -153 to -134 base pairs relative to the start of transcription. Each of these sites includes the hexanucleotide sequence AACCCA, which may serve as a primary recognition sequence. During seed development, SEF 3 binding activity was found to increase in soybean embryos during the time of beta-conglycinin synthesis and to decrease as seeds neared maturity. The position of the SEF 3 binding sequence corresponds with a previously reported seed-specific enhancer region, and it seems likely that this factor may act as a positive regulator of transcription of the beta-conglycinin, alpha' subunit gene in developing soybean seeds. The second factor, SEF 4, also binds within the -257 to -77 region but also interacts with sites located further upstream.

Antigens, Plant↗

PubMed2001

A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.

Adaptation, Physiological↗

PubMed2006

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli.

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.

Amino Acid Sequence↗

PubMed1983

Arginine catabolism in the cotyledons of developing and germinating pea seeds.

Arginine is the predominant free amino acid in the cotyledons of developing seeds of Pisum sativum L. cv Marzia. Breakdown of arginine was measured by injecting l-[guanido-(14)C]arginine into detached cotyledons. Cotyledons of developing seeds showed a low rate of (14)CO(2) evolution whereas a much higher rate of (14)CO(2) evolution was measured from cotyledons of seeds 4 days after the onset of germination. The activities of the catabolic enzymes arginase, urease, and ornithine aminotransferase were measured throughout development and germination. Arginase and ornithine aminotransferase were present at an early stage of development. Urease activity appeared later as the seeds started to desiccate. During germination, all three enzymes were present. The different course of activity of these enzymes indicates that they are controlled separately.To explain the simultaneous presence of arginine and arginase without arginine degradation in the cotyledons of developing seeds, we propose a different intracellular localization of substrate and enzyme. In cotyledons of germinating pea seeds, urease has an enzymic function in arginine degradation.

Journal Article↗

PubMedDate not supplied

Effect of high-temperature stress on the growth and seed characteristics of barley and cotton.

The existence of a sensitivity gradient to a uniform high-temperature stress applied at stages during seed maturation can be demonstrated in barley. The germination of freshly harvested seed is depressed following heat stress at 7--10 days after awn emergence, but is enhanced by the same stress applied 3 weeks after awn emergence. The depression is attributed to reduced viability associated with thermal injury. The stimulation following stress at more mature stages of seed development is related to a thinner seed coat, increased permeability as evidenced by faster imbibition rate, and decreased content of water-soluble inhibitors in the seed. These effects of environmental stress during seed maturation aid in explaining differences noted in the germinability at harvest of seed produced in successive years or produced in the same year at different locations. Seed of American Cotton, var. AC 134, was stressed at 50 degrees, 60 degrees, 70 degrees, 80 degrees, and 90 degrees C for 24 or 48 h before seeding. Stresses at 50 degrees to 70 degrees C increased seedling emergence and subsequent performance of the cotton plants, but higher temperatures caused thermal injury or killed the seed.

Edible Grain↗

PubMed1999

Isolation and partial characterisation of galactose-specific lectins from African yam beans, Sphenostyles stenocarpa Harms.

A new galactose-specific lectin was isolated from African yam bean (Sphenostyles stenocarpa Harms) by affinity chromatography on galactose-Sepharose 4B. SDS-PAGE analysis resulted in four polypeptide bands of approximately 27, 29, 32 and 34 kDa, respectively. Based on the analysis of carbohydrate content and native PAGE, it is likely that the Sphenostyles lectin is a tetrameric glycoprotein with M(r) of approximately 122 kDa. N-terminal protein sequencing of purified lectins from four different Sphenostyles accessions shows that the four polypeptides have largely identical amino acid sequences. The sequences contain the conserved consensus sequence F-F-LILG characteristic of legume lectins, as well as Phaseolus vulgaris proteins in the arcelin-alpha-amylase inhibitor gene family. The lectin agglutinates both rabbit and human erythrocytes, but with a preference for blood types A and O. Using Western blotting, the lectin was shown to accumulate rapidly during seed development, but levels dropped slightly as seeds attained maturity. This is the first time a lectin has been purified from the genus Sphenostyles. The new lectin was assigned the abbreviation LECp.SphSte.se.Hga1.

Amino Acid Sequence↗

PubMed2002

Sex ratio and reproductive effort in the dioecious Juniperus communis subsp. alpina (Suter) Celak. (Cupressaceae) along an altitudinal gradient.

The hypothesis that reproductive cost differs between sexes was tested in Juniperus communis subsp. alpina along an altitudinal gradient. Sex ratio (male : female) increased significantly with elevation, and above 2,600 m it was significantly male-biased. The reproductive effort was markedly greater for females than for males at all elevations. However, over 3 years of study, the growth of the females, measured as elongation of the main axes, was similar to that of the males. In both sexes, growth decreased with increasing elevation. Neither size of the ripe seed cones, nor the number of developed seeds per cone varied with elevation. The percentage of filled seeds was significantly greater at higher elevations indicating more favourable conditions for wind pollination in these stands. However, cone production decreased with elevation and so, reproductive success of J. communis subsp. alpina in Sierra Nevada decreases towards both upper and lower altitudinal distribution limits. The results do not support the hypothesis of differential reproductive cost between sexes; thus, alternative arguments to explain the altitudinal variation of sex ratio are discussed.

Acclimatization↗

PubMed2004

Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying.

Late-embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to a high level in desiccation-tolerant tissues and are thus prominent in seeds. They are expected to play a protective role during dehydration; however, functional evidence is scarce. We identified a LEA protein of group 3 (PsLEAm) that was localized within the matrix space of pea (Pisum sativum) seed mitochondria. PsLEAm revealed typical LEA features such as high hydrophilicity and repeated motifs, except for the N-terminal transit peptide. Most of the highly charged protein was predicted to fold into amphiphilic alpha-helixes. PsLEAm was expressed during late seed development and remained in the dry seed and throughout germination. Application of the stress hormone abscisic acid was found to reinduce the expression of PsLEAm transcripts during germination. PsLEAm could not be detected in vegetative tissues; however, its expression could be reinduced in leaves by severe water stress. The recombinant PsLEAm was shown to protect two mitochondrial matrix enzymes, fumarase and rhodanese, during drying in an in vitro assay. The overall results constitute, to our knowledge, the first characterization of a LEA protein in mitochondria and experimental evidence for a beneficial role of a LEA protein with respect to proteins during desiccation.

Amino Acid Sequence↗

PubMed2006

Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding beta-amyrin synthase and a triterpene carboxylic acid glucosyltransferase.

Saponaria vaccaria (Caryophyllaceae), a soapwort, known in western Canada as cowcockle, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). To improve our understanding of the biosynthesis of these saponins, a combined polymerase chain reaction and expressed sequence tag approach was taken to identify the genes involved. A cDNA encoding a beta-amyrin synthase (SvBS) was isolated by reverse transcription-polymerase chain reaction and characterized by expression in yeast (Saccharomyces cerevisiae). The SvBS gene is predominantly expressed in leaves. A S. vaccaria developing seed expressed sequence tag collection was developed and used for the isolation of a full-length cDNA bearing sequence similarity to ester-forming glycosyltransferases. The gene product of the cDNA, classified as UGT74M1, was expressed in Escherichia coli, purified, and identified as a triterpene carboxylic acid glucosyltransferase. UGT74M1 is expressed in roots and leaves and appears to be involved in monodesmoside biosynthesis in S. vaccaria.

Amino Acid Sequence↗

PubMed1984

Sink to source translocation in soybean.

The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [(14)C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [(14)C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.

Journal Article↗