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Biomedical subjects

J D Bewley

Publications and source records attributed to J D Bewley.

At least 19 recordsLinked to original sources
PubMed2001

Endo-beta-mannanase is present in an inactive form in ripening tomato fruits of the cultivar Walter.

Fruits of the tomato cultivar Walter undergo normal development to the red-ripe stage but, unlike those of the cultivar Trust, they do not produce any active endo-beta-mannanase. Reasons for this failure to produce the enzyme were sought. The cv. Walter contains genes for endo-beta-mannanase, as shown by Southern blot analysis, and transcripts for the enzyme are present in ripening fruits, as revealed using Northern hybridization. Moreover, the enzyme protein is detectable by Western blots using an endo-beta-mannanase-specific antibody from tomato. In addition, the inactive enzyme is localized appropriately in the wall regions of the outer layers of the fruit (skin and outer pericarp). Mixing inactive fruit extracts of cv. Walter, in excess, with extracts from cv. Trust fruits, which contain active enzyme, leads to an increase rather than a reduction in enzyme activity, showing that there are no inhibitors of endo-beta-mannanase in cv. Walter fruits. Similar results were obtained with fruits of the tomato cv. Heinz 1439. In contrast to the situation in fruits, the seeds of both cvs Walter and Heinz 1439 produce active enzyme, especially following germination.

Blotting, Southern↗
PubMed2001

The cloning and characterization of alpha-galactosidase present during and following germination of tomato (Lycopersicon esculentum Mill.) seed.

alpha-Galactosidase (EC 3.2.1.22) is present in the embryo, micropylar and lateral endosperm of seeds of tomato during and following germination. Its activity is unchanged even when germination of the seeds is prevented by an osmoticum. It is also present in the developing and mature dry seed. A cDNA clone for tomato seed alpha-galactosidase (LeaGal) has been isolated and the characteristics of the protein deduced; the predicted molecular mass of the mature enzyme is 39.8 kDa, with a pI of 4.91. The tomato alpha-galactosidase has a high homology (>62%) at the amino acid level with that of other plant alpha-galactosidases. A hydrophobic signal peptide region is identified which is indicative that the enzyme enters the lumen of the endoplasmic reticulum during its translation, prior to its export to the protein body or cell wall, the presumed sites of its substrates. Using amino acid alignment and phylogenetic analysis, key amino acids have been identified, and relationships to other alpha-galactosidases inferred. Southern hybridization analyses show that the enzyme is derived from a single gene (for which a partial sequence has been obtained) and yet there are at least three different isoforms within the seed; post-translational modifications are thus presumed to occur. From Northern hybridization studies it is evident that alpha-galactosidase transcripts are present in the lateral and micropylar endosperm during and following germination, and also to a lesser extent in the embryo.

Amino Acid Sequence↗
PubMed2000

Endo-beta-mannanase activity increases in the skin and outer pericarp of tomato fruits during ripening.

Activity of endo-beta-mannanase increases during ripening of tomato (Lycopersicon esculentum Mill.) fruit of the cultivar Trust. beta-Mannoside mannohydrolase is also present during ripening, but its pattern of activity is different from that of endo-beta-mannanase. The increase in endo-beta-mannanase activity is greatest in the skin, and less in the outer and inner pericarp regions. This enzyme is probably bound to the walls of the outermost cell layers of the fruit during ripening, and it requires a high-salt buffer for effective extraction. The enzyme protein, as detected immunologically on Western blots, is present during the early stages of ripening, before any enzyme activity is detectable. The mRNA for the enzyme is also present at these stages; endo-beta-mannanase may be produced and sequestered in a mature-sized inactive form during early ripening. Most non-ripening mutants of tomato exhibit reduced softening and lower endo-beta-mannanase activity, but a cause-and-effect relationship between the enzyme and ripening is unlikely because some cultivars which ripen normally do not exhibit any endo-beta-mannanase activity in the fruit.

Blotting, Northern↗
PubMed1997

Changes in late-embryogenesis-abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L. seeds.

In Ricinus communis L. (castor bean) endosperms, two classes of Late Embryogenesis Abundant (Lea) transcripts were first detected during mid-development (at 30-35 days after pollination, DAP) and peaked at 50 DAP, just prior to the onset of desiccation. Most of the Class I mRNAs declined substantially during desiccation itself; Class II mRNAs remained abundant in the mature dry (60 DAP) seed. Following imbibition, all Lea mRNAs abundant in the mature dry seed declined rapidly (within 5-24 h). Premature drying of developing 35-DAP seeds resulted in the loss of storage-protein mRNAs (Leg B Mat I); following rehydration, mRNAs encoding post-germinative proteins (Germ D91, D30 and D38) increased in the endosperm. The Lea mRNAs present in the developing fresh seed at 35 DAP were preserved, but did not increase in response to premature desiccation; upon rehydration these Lea mRNAs declined within 5 h. During seed development, substantial changes occurred in the synthesis of a subset of LEA proteins referred to as "dehydrins'; in particular, new dehydrin polypeptides were induced between 40 and 60 DAP. Such proteins were not as evident in prematurely dried endosperms. In contrast to the rapid loss of Lea mRNAs during germination, many of the dehydrin proteins abundant in the dried seed persisted following imbibition or rehydration.

Ricinus communis↗
PubMed1997

Molecular cloning of a cDNA encoding a (1-->4)-beta-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum).

Mannose-containing polysaccharides are widely distributed in cell walls of higher plants. During endosperm mobilization in germinated tomato seeds (1-->4)-beta-mannan endohydrolases (EC 3.2.1.78) participate in the enzymic depolymerization of these cell wall polysaccharides. A cDNA encoding a (1-->4)-beta-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and characterized. The amino acid sequence deduced from the 5'-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids determined directly from the purified enzyme. The mature enzyme consists of 346 amino acid residues, it has a calculated M(r) of 38,950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28-30% sequence identity with fungal (1-->4)-beta-mannanases, but more highly conserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1-->4)-beta-mannanase as a member of the family 5 group of glycosyl hydrolases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization analyses indicate that the enzyme is derived from a family of about four genes. Expression of the genes, as determined by the presence of mRNA transcripts in Northern hybridization analyses, occurs in the endosperm of germinated seeds; no transcripts are detected in hypocotyls, cotyledons, roots or leaves.

Amino Acid Sequence↗
PubMed1995

Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development.

cDNA clones for two isoforms of starch branching enzyme (SBEI and SBEII) have been isolated from pea embryos and sequenced. The deduced amino acid sequences of pea SBEI and SBEII are closely related to starch branching enzymes of maize, rice, potato and cassava and a number of glycogen branching enzymes from yeast, mammals and several prokaryotic species. In comparison with SBEI, the deduced amino acid sequence of SBEII lacks a flexible domain at the N-terminus of the mature protein. This domain is also present in maize SBEII and rice SBEIII and resembles one previously reported for pea granule-bound starch synthase II (GBSSII). However, in each case it is missing from the other isoform of SBE from the same species. On the basis of this structural feature (which exists in some isoforms from both monocots and dicots) and other differences in sequence, SBEs from plants may be divided into two distinct enzyme families. There is strong evidence from our own and other work that the amylopectin products of the enzymes from these two families are qualitatively different. Pea SBEI and SBEII are differentially expressed during embryo development. SBEI is relatively highly expressed in young embryos whilst maximum expression of SBEII occurs in older embryos. The differential expression of isoforms which have distinct catalytic properties means that the contribution of each SBE isoform to starch biosynthesis changes during embryo development. Qualitative measurement of amylopectin from developing and maturing embryos confirms that the nature of amylopectin changes during pea embryo development and that this correlates with the differential expression of SBE isoforms.

1,4-alpha-Glucan Branching Enzyme↗
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↗
PubMed1992

Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.).

During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media, yet that protein is not evident on stained gels until day 10. Thus, both transcriptional and posttranscriptional events appear to be important in determining the protein complement of these seed tissues. On the basis of storage protein and mRNA accumulation, mature (14 days) somatic embryos most closely resemble stage VI zygotic embryos. The results of the developmental comparison also suggest that the patterns of synthesis of the individual storage proteins (7S, 11S, or 2S) are regulated independently of each other during embryogenesis in alfalfa.

Journal Article↗
PubMed1992

Distribution of Cytosolic mRNAs Between Polysomal and Ribonucleoprotein Complex Fractions in Alfalfa Embryos : Stage-Specific Translational Repression of Storage Protein Synthesis during Early Somatic Embryo Development.

Cell-free translational and northern blot analyses were used to examine the distribution of storage protein messages in the cytoplasmic polysomal and mRNA-protein complex (mRNP) fractions during development of somatic and zygotic embryos of alfalfa (Medicago sativa cv Rangelander RL-34). No special array of messages was identified in the mRNP fraction; however, some messages were selectively enriched in either the polysome or mRNP fractions, and their distribution pattern varied quantitatively during development of the embryos. During the earliest stages of somatic embryo development, storage protein messages already were present, but there was no detectable accumulation of the proteins. Selective enrichment of messages for the 11S, 7S, and 2S storage proteins occurred in the mRNP fraction during the globular, heart, and torpedo stages of somatic embryogenesis, but the distribution pattern was shifted toward the polysomal fraction at the beginning of cotyledon development. Thus, there was translational repression of storage protein synthesis at the early stage of somatic embryo development that was relieved later. During the cotyledonary development stages in the somatic and zygotic embryos, storage protein synthesis and distribution of the messages were similar in that these specific messages were predominantly in the polysomal fraction.

Journal Article↗
PubMed1991

Enzymes of Nitrogen Assimilation Undergo Seasonal Fluctuations in the Roots of the Persistent Weedy Perennial Cichorium intybus.

Chicory (Cichorium intybus), a deep rooted weed, grows in regions with temperate climates. Seasonal partitioning of compounds between the root and shoot results in fluctuations in the soluble carbohydrate, nitrate, amino acid, and protein pools within the roots. The activities of nitrate reductase (NR) (EC 1.6.6.1), glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxin glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (GDH) (EC 1.4.1.2-4) vary throughout the year and coincide with seasonal alterations in nitrate, fructose, and sucrose. During the winter, NR, glutamine synthetase and ferrodoxin glutamate synthase activities increase in the root, while GDH displays the opposite trend with elevated activity in the summer months. All of these enzymes exhibit seasonal alterations in abundance as detected by Western blot analysis, increasing during the winter and, therefore, contributing to the seasonally dynamic protein pool. Extensive fluctuations in abundance and activity of these enzymes in the root occur during the spring and fall and coincide with shoot growth and senescence, respectively. Several observations indicate that posttranslational modifications of NR and GDH are taking place throughout the year; for example, NR is particularly unstable during the spring and fall, and seasonal GDH activity does not correlate with protein abundance.

Journal Article↗
PubMed1990

Distinction between the Responses of Developing Maize Kernels to Fluridone and Desiccation in Relation to Germinability, alpha-Amylase Activity, and Abscisic Acid Content.

Developing kernels of the maize (Zea mays) hybrid W64A x W182E germinated precociously following fluridone treatment. Likewise, following premature drying, the kernels germinated upon subsequent rehydration. Tolerance of the aleurone layer to premature desiccation considerably preceded that of the embryo. The increase in alpha-amylase activity following premature drying was substantial and was equal to, or exceeded, the increase which occurred following normal maturation drying. In contrast, there was only a small increase in enzyme activity, regardless of the concentration of the supplied gibberellic acid, following fluridone treatment. Both fluridone and drying cause a decrease in abscisic acid content within the developing kernels. While this decline in growth regulator may permit kernels to germinate, alone this is not sufficient to permit an increase in alpha-amylase activity. Thus drying is necessary to sensitize the aleurone layer to gibberellin, and thereby elicit enzyme synthesis. For this tissue to achieve its full potential to produce alpha-amylase, it must not only be free of the inhibitory effects of abscisic acid, but it must also be competent to respond to gibberellin.

Journal Article↗
PubMed1989

Developing Seeds of Ricinus communis L., When Detached and Maintained in an Atmosphere of High Relative Humidity, Switch to a Germinative Mode without the Requirement for Complete Desiccation.

Immature seeds of castor bean (Ricinus communis) removed from the capsule at 25 to 40 days after pollination (25-40 DAP) and placed in an atmosphere of high relative humidity undergo limited water loss, and germinate upon subsequent return to full hydration. This switch from a developmental to a germinative/growth mode at 40 DAP is reflected in a change in the types of proteins being synthesized in the endosperm; after partial drying, developmental protein synthesis ceases and germinative/growth-related proteins are produced. The nature and timing of these protein synthetic changes elicited upon imbibition are identical to those following premature desiccation/rehydration of 30 and 40 DAP seeds and upon imbibition of the mature dry seed. Enzymes involved in postgerminative reserve mobilization (l-leucyl-beta-naphthylamidase and isocitrate lyase) are induced upon imbibition, following partial drying at 40 DAP, to levels attained in the endosperms of germinated mature, and prematurely dried/rehydrated, seeds. The changes in protein synthesis resulting from partial drying are effected at the transcriptional and post-transcriptional level. Upon return to full hydration some new (i.e. germination and growth-related) mRNAs are synthesized, while others (associated with development) present in the partially dried endosperm decline. Thus developing seeds of castor bean do not have to experience substantial (whole seed) water loss to acquire the ability to germinate and grow upon subsequent imbibition. Seed detachment from the mother plant alone is not sufficient to elicit a switch to germination and growth processes. However, the length of time of detachment from the mother plant, in combination with some water loss may interact to elicit the "switch" from development to germination.

Journal Article↗
PubMed1988

Use of electrophoretic techniques in determining the composition of seed storage proteins in alfalfa.

Holoprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two-dimensional electrophoretic techniques. The variety of two-dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non-seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin-like globulin), alfin (a vicilin-like globulin) and a family of lower molecular weight albumins (LMW1-3). These comprise 30%, 10%, and 20%, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr approximately 150,000) composed of polypeptides ranging in size from Mr 14,000 to 50,000 (alpha 1-alpha 6; 50,000, 38,000, 32,000, 20,000, 16,000 and 14,000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1-A9; Mr 49,000 to 39,000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24,000, 23,000 and 20,000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1/B2). Subunits (Mr approximately 50,000-65,000) are assembled as trimers (8S) or larger oligomers (12S-15S) in mature seeds. The lower molecular weight albumins (LMW1-3) are acidic (pI less than 6), and consist of sets of disulphide-bonded polypeptides (Mr 15,000 and 11,000).

Amino Acids↗
PubMed1988

Abscisic Acid Is an Endogenous Inhibitor in the Regulation of Mannanase Production by Isolated Lettuce (Lactuca sativa cv Grand Rapids) Endosperms.

The production of mannanase, a cell-wall-degrading carbohydrase, can be manipulated in isolated lettuce (Lactuca sativa cv Grand Rapids) endosperms by changes in the volume of buffer in which they are incubated. The enzyme is produced when endosperms are incubated in a large volume, but not when incubated in a small volume, which is suggestive that an endogenous, diffusible inhibitor of mannanase production is being lost from the endosperms in a large volume (JD Bewley, P Halmer 1980/1981 Israel J Bot 29: 118-132). We have investigated the possibility that the phytohormone abscisic acid (ABA) is involved in this regulation of mannanase production in isolated lettuce endosperms. We find several correlations between the presence of the endogenous inhibitor and of ABA, i.e. (a) a ;leachate' prepared from isolated lettuce endosperms induces synthesis of ABA-specific proteins in barley aleurone layers, indicating that incubation of endosperms in a large volume results in the diffusion of ABA therefrom into the surrounding medium; (b) fractionation of the components of a leachate by either polyvinylpyrrolidone-chromatography of C(18) reversed-phase high performance liquid chromatography fails to separate the endogenous inhibitor from authentic ABA; and (c) changes in the incubation volume of endosperms result in changes in the amount of extractable ABA in the endosperms, as detected by ELISA. These results are consistent with a role for endogenous ABA in the regulation of mannanase production in isolated lettuce endosperms.

Journal Article↗
PubMed1987

Establishment of thermotolerance in maize by exposure to stresses other than a heat shock does not require heat shock protein synthesis.

Maize (Zea mays) seedlings were pretreated prior to heat shock with either a progressive water stress of -0.25 megapascal PEG/hour from 0 to -1.25 megapascal over a 6-hour time period, or various concentrations of copper, cadmium, or zinc for 4 days. When the subsequent heat shock of 40 or 45 degrees C was administered for 3 hours, the seedlings showed an induced thermotolerance to these temperatures, which were otherwise lethal to control (water grown) seedlings. Thermotolerance was exhibited by both the root and the shoot of pretreated seedlings, even though the water and heavy metal stresses were applied only to the roots. Neither of these pretreatments had induced the synthesis of detectable levels of heat shock proteins (Hsps) at the time of heat shock. Pretreatment of seedlings with a progressive heat shock of 2 degrees C/hour from 26 to 36 degrees C, which did induce Hsps 18, 70, and 84, resulted in tolerance of a severe water stress of -1.5, -1.75, or -2.0 megapascal for 24 hours. But these seedlings producing Hsps were no better protected against water stress than those pretreated with a progressive water stress which did not produce Hsps. Hsps appear not to act as general stress proteins and their presence is not always required for the establishment of thermotolerance.

Journal Article↗