Finite indentation of highly curved elastic shells.

Experimentally measuring the elastic properties of thin biological surfaces is non-trivial, particularly when they are curved. One technique that may be used is the indentation of a thin sheet of material by a rigid indenter, while measuring the applied force and displacement. This gives immediate information on the fracture strength of the material (from the force required to puncture), but it is also theoretically possible to determine the elastic properties by comparing the resulting force-displacement curves with a mathematical model. Existing mathematical studies generally assume that the elastic surface is initially flat, which is often not the case for biological membranes. We previously outlined a theory for the indentation of curved isotropic, incompressible, hyperelastic membranes (with no bending stiffness) which breaks down for highly curved surfaces, as the entire membrane becomes wrinkled. Here, we introduce the effect of bending stiffness, ensuring that energy is required to change the shell shape without stretching, and find that commonly neglected terms in the shell equilibrium equation must be included. The theory presented here allows for the estimation of shape- and size-independent elastic properties of highly curved surfaces via indentation experiments, and is particularly relevant for biological surfaces.

Brassinosteroids and gibberellins promote tobacco seed germination by distinct pathways.

Seed germination of Nicotiana tabacum L. cv. Havana 425 is determined by the balance of forces between the growth potential of the embryo and the mechanical restraint of the micropylar endosperm. In contrast to the gibberellin GA4, the brassinosteroid (BR) brassinolide (BL) did not release photodormancy of dark-imbibed photodormant seeds. Brassinolide promoted seedling elongation and germination of non-photodormant seeds, but did not appreciably affect the induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm. Brassinolide, but not GA4, accelerated endosperm rupture of tobacco seeds imbibed in the light. Brassinolide and GA4 promoted endosperm rupture of dark-imbibed non-photodormant seeds, but only GA4 enhanced betaGLU I induction. Promotion of endosperm rupture by BL was dose-dependent and 0.01 microM BL was most effective. Brassinolide and GA4 promoted abscisic acid (ABA)-inhibited dark-germination of non-photodormant seeds, but only GA4 replaced light in inducing betaGLU I. These results indicate that BRs and GAs promote tobacco seed germination by distinct signal transduction pathways and distinct mechanisms. Gibberellins and light seem to act in a common pathway to release photodormancy, whereas BRs do not release photodormancy. Induction of betaGLU I in the micropylar endosperm and promotion of release of 'coat-enhanced' dormancy seem to be associated with the GA-dependent pathway, but not with BR signalling. It is proposed that BRs promote seed germination by directly enhancing the growth potential of the emerging embryo in a GA- and betaGLU I-independent manner.

Antisense-transformation reveals novel roles for class I beta-1,3-glucanase in tobacco seed after-ripening and photodormancy.

Little is known about the molecular basis for seed dormancy, after-ripening, and radicle emergence through the covering layers during germination. In tobacco, endosperm rupture occurs after testa rupture and is the limiting step in seed germination. Class I beta-1,3-glucanase (betaGLU I), which is induced in the micropylar endosperm just prior to its penetration by the radicle, is believed to help weaken the endosperm wall. Evidence is presented here for a second site of betaGLU I action during after-ripening. Tobacco plants were transformed with antisense betaGLU I constructs with promoters thought to direct endosperm-specific expression. Unexpectedly, these transformants were unaffected in endosperm rupture and did not exhibit reduced betaGLU I expression during germination. Nevertheless, antisense betaGLU I transformation delayed the onset of testa rupture in light-imbibed, after-ripened seeds and inhibited the after-ripening-mediated release of photodormancy. It is proposed that betaGLU I expression in the dry seed contributes to the after-ripening-mediated release of seed dormancy.

Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence.

beta-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both beta-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.

Beta-1, 3-glucanase and chitinase transgenes in hybrids show distinctive and independent patterns of posttranscriptional gene silencing.

Nicotiana sylvestris Speg. & Comes transformed with a tobacco class-I beta-1,3-glucanase (GLU I ) cDNA driven by CaMV 35S RNA expression signals exhibits posttranscriptional gene silencing (PTGS) which is triggered between the cotyledon and two-leaf stages of seedling development and is postmeiotically reset to the high-expressing state during seed development. The incidence of GLU I PTGS in sibling plants differed for the two different transformants tested and increased with the number of T-DNA loci. Comparison of host class-I and class-II beta-1,3-glucanase gene expression suggests that a similarity of 60-70% in the coding-region is required for PTGS of the homologous host genes. The GLU I transformants exhibited a spatial gradient in PTGS, in which expression of the silent phenotype gradually increased in successive leaves toward the bottom of the plant. In contrast, transformants carrying an unrelated tobacco class I chitinase (CHN I) cDNA in the same expression vector exhibited discontinuous patterns of PTGS with adjacent high-expressing and silent leaves. The GLU I- and CHN I-specific patterns were maintained in hybrids homozygous for both T-DNA's indicating that two different transgenes present in the same genome can exhibit independent and distinctive patterns of PTGS. This implies that the nature of the transgene rather than a general pre-pattern of competence for PTGS or propagation of the silent state are important for pattern determination.

Sense transformation reveals a novel role for class I beta-1, 3-glucanase in tobacco seed germination.

'Coat-enhanced' seed dormancy of many dicotyledonous species, including tobacco, is released during after-ripening. Rupture of the endosperm, which is the limiting step in tobacco seed germination, is preceded by induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm where the radicle will penetrate. Treating after-ripened tobacco seeds with abscisic acid (ABA) delays endosperm rupture and inhibits betaGLU I induction. Sense transformation with a chimeric ABA-inducible betaGLU I transgene resulted in over-expression of betaGLU I in seeds and promoted endosperm rupture of mature seeds and of ABA-treated after-ripened seeds. Taken together, these results provide direct evidence that betaGLU I contributes to endosperm rupture. Over-expression of betaGLU I during germination also replaced the effects of after-ripening on endosperm rupture. This suggests that regulation of betaGLU I by ABA signalling pathways might have a key role in after-ripening.

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase.

Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination.

Distinct ethylene- and tissue-specific regulation of beta-1,3-glucanases and chitinases during pea seed germination.

The expression of beta-1,3-glucanase (betaGlu) and chitinase (Chn) was investigated in the testa, cotyledons, and embryonic axis of germinating Pisum sativum L. cv. 'Espresso generoso' seeds. High concentrations of betaGlu and Chn activity were found in the embryonic axis. Treatment with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that an early, 4-fold induction of betaGlu activity in the embryonic axis during the first 20 h after the start of imbibition is ethylene-independent. This initial increase was followed by a later 4-fold ethylene-dependent induction in the embryonic axis starting at 50 h, which is after the onset of ethylene evolution and after completion of radicle emergence. The betaGlu activity in cotyledons increased gradually throughout germination and was ethylene-independent. In contrast, the ethylene-independent Chn activity increased slightly after the onset of radical emergence in the embryonic axis and remained at a constant low level in cotyledons. Immunoinactivation assays and immunoblot analyses suggest that early betaGlu activity in the embryonic axis is due to a 54-kDa antigen, whereas late induction is due to a 34.5-kDa antigen, which is likely to be the ethylene-inducible class I betaGlu G2 described for immature pea pods. Increases in Chn in the embryonic axis were correlated with a 26-kDa antigen, whereas amounts of the additional 32- and 20-kDa antigens remained roughly constant. Thus, ethylene-dependent and ethylene-independent pathways regulate betaGlu and Chn during pea seed germination. The pattern of regulation differs from that of leaves and immature pods, and from that described for germinating tobacco seeds. The functional significance of this regulation and its underlying mechanisms are discussed.

Ethylene-responsive element binding protein (EREBP) expression and the transcriptional regulation of class I beta-1,3-glucanase during tobacco seed germination.

Class I beta-1,3-glucanase (betaGLU I) is transcriptionally induced in the micropylar endosperm just before its rupture prior to the germination (i.e. radicle emergence) of Nicotiana tabacum L. cv. 'Havana 425' seeds. Ethylene is involved in endosperm rupture and high-level betaGLU I expression; but, it does not affect the spatial and temporal pattern of betaGLU I expression. A promoter deletion analysis of the tobacco betaGLU I B gene suggests that (1) the distal - 1452 to - 1193 region, which contains the positively acting ethylene-responsive element (ERE), is required for high-level, ethylene-sensitive expression, (2) the regions - 1452 to - 1193 and -402 to 0 contribute to downregulation by abscisic acid (ABA), and (3) the region -402 to -211 is necessary and sufficient for low-level micropylar-endosperm-specific expression. Transcripts of the ERE-binding proteins (EREBPs) showed a novel pattern of expression during seed germination: light or gibberellin was required for EREBP-3 and EREBP-4 expression; EREBP-4 expression was constitutive and unaffected by ABA or ethylene; EREBP-3 showed transient induction just before endosperm rupture, which was earlier in ethylene-treated seeds and inhibited by ABA. No expression of EREBP- and EREBP-2 was detected. In contrast to betaGLU I, EREBP-3 and EREBP-4 were not expressed specifically in the micropylar endosperm. The results suggest that transcriptional regulation of betaGLU I could depend on: activation of ethylene signalling pathways acting via EREBP-3 with the ERE as the target, and ethylene-independent signalling pathways with targets in the proximal promoter region that are likely to determine spatial and temporal patterns of expression.

Latex allergen database.

Two-dimensional (2-D) electrophoresis followed by immunoblotting and N-terminal protein microsequencing were used to characterize and identify the IgE-reactive proteins of Hevea latex that are the main cause of the latex type I allergy affecting especially health care workers and spina bifida children. This approach generated a comprehensive latex allergen database, which facilitated the integration of most of the latex allergen data presented in the literature. The major latex allergens Hev b 1, Hev b 3, Hev b 6 and Hev b 7 have been localized on our 2-D maps. Moreover, we were able to identify six previously undescribed IgE-binding latex proteins, namely enolase, superoxide dismutase, proteasome subunit C5, malate dehydrogenase, triosephosphate isomerase and endochitinase. The generated latex 2-D maps will provide valuable information to develop strategies for the isolation of the novel IgE binding proteins in order to study the frequency of sensitization among both risk groups. Detailed knowledge of all proteins involved in latex allergy will allow better diagnosis of latex allergy and to monitor the success of prevention strategies that are needed to reduce the high prevalence of latex allergy among both risk groups.

Transcripts at the mating type locus of Cochliobolus heterostrophus.

The single mating type locus (MAT) of the heterothallic ascomycete Cochliobolus heterostrophus is composed of a pair of unlike sequences called idiomorphs, each of which encodes one MAT-specific gene (MAT-1 and MAT-2). MAT transcripts were observed in blots of poly(A)+ RNA isolated from cultures grown in minimal medium, but were not detectable after growth of the fungus in complete medium, suggesting that transcription of MAT is tightly regulated. The idiomorphs (MAT-1 = 1297-bp, MAT-2 = 1171-bp) encode transcripts of 2.2 kb (MAT-1) and 2.1 kb (MAT-2), which start 5' and end 3' of the idiomorph within sequences common to both mating types. Analyses of MAT-1 and MAT-2 cDNAs revealed obligatory splicing of one intron (55-bp in MAT-1, 52-bp in MAT-2) within each MAT-specific ORF and optional splicing of two introns (63 and 79-bp) in the long (approximately 0.55 kb) 5' untranslated leader sequences; the 3' untranslated region is 0.46 kb long. Transcription start sites were found 5' of, and within, the 79-bp intron. Optional splicing of the upstream introns and at least two transcription start sites result in three types of transcript: Type I with both 5' introns spliced, Type II with only the 63-bp intron spliced, and Type III with neither 5' intron spliced. The three transcript types are distinguished by various combinations of four short ORFs encoded by the corresponding genomic DNA, in the leader sequences of the MAT mRNAs. The transcript structure suggests several mechanisms by which expression of the MAT genes might be regulated at the level of translation during sexual development.

Class I [beta]-1,3-Glucanases in the Endosperm of Tobacco during Germination.

Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum L. cv Havana 425 seeds. Treatment with 10-5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of [beta]-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I [beta]-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of [beta]-1,3-glucanase accumulation but did not delay the onset of induction. Independent of the ABA concentration used, onset of endosperm rupture was correlated with the same [beta]-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I [beta]-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination.