Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25°C, but not at 15°C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15°C and 25°C.

The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25°C than at 15°C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15°C and 25°C after the puncture force was reduced to between 0.3 and 0.4 newtons.

Application of gibberellic acid₄₊₇ (100 microliters per liter) resulted in earlier germination at 15°C and 25°C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O₂ concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid₄₊₇, but only at 25°C. At 15°C, high O₂ concentrations slowed germination and endosperm strength decline.

     

Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method

Background and aims Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals (^•OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to ‘fingerprint’ ^•OH-attacked polysaccharides.Methods We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during ^•OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography.Key Results Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA–pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently.Conclusions GalA–pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents (^•OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by ^•OH. The evidence shows that ^•OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). ^•OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.     

A water relations analysis of seed germination rates

Seed germination culminates in the initiation of embryo growth and the resumption of water uptake after imbibition. Previous applications of cell growth models to describe seed germination have focused on the inhibition of radicle growth rates at reduced water potential (Ψ). An alternative approach is presented, based upon the timing of radicle emergence, to characterize the relationship of seed germination rates to Ψ. Using only three parameters, a `hydrotime constant' and the mean and standard deviation in minimum or base Ψ among seeds in the population, germination time courses can be predicted at any Ψ, or normalized to a common time scale equal to that of seeds germinating in water. The rate of germination of lettuce (Lactuca sativa L. cv Empire) seeds, either intact or with the endosperm envelope cut, increased linearly with embryo turgor. The endosperm presented little physical resistance to radicle growth at the time of radicle emergence, but its presence markedly delayed germination. The length of the lag period after imbibition before radicle emergence is related to the time required for weakening of the endosperm, and not to the generation of additional turgor in the embryo. The rate of endosperm weakening is sensitive to Ψ or turgor.     

Changes in the Endosperm Cell Walls of Two Datura Species before Radicle Protrusion

The possibility of an association between changes in cell walls of the micropylar portion of the endosperm and the induction of germination was explored in seeds of Datura ferox and Datura stramonium. The structure of the inner surface of the endosperm was studied by scanning electron microscopy and the composition of cell wall polysaccharides analyzed by gas chromatography and gas chromatography-mass spectrometry. Both scanning electron microscope images and chemical analysis showed changes in the micropylar portion of the endosperm in induced seeds before radicle protrusion. The inner surface of the endosperm appeared eroded, and in some areas, wall material seemed to be missing. The content of the main component of the cell wall polysaccharides, containing predominantly 4-linked mannose, decreased well before the emergence of the radicle through the endosperm. We propose that the degradation of a mannan type polysaccharide is an important factor in the reduction in mechanical strength of the endosperm, thus facilitating germination.     

beta-d-Glucan Antibodies Inhibit Auxin-Induced Cell Elongation and Changes in the Cell Wall of Zea Coleoptile Segments

Antiserum was raised against the Avena sativa L. caryopsis β-d-glucan fraction with an average molecular weight of 1.5 × 10⁴. Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1→3), (1→4)-β-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ β-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress-relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis β-d-glucans. These results support the idea that the degradation of (1→3), (1→4)-β-d-glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation.     

Xyloglucan oligosaccharides promote growth and activate cellulase: evidence for a role of cellulase in cell expansion

Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose₄· xylose₃ (XG7) core. The substituted oligosaccharides XG8 (glucose₄· xylose₃· galactose) and XG9n (glucose₄· xylose₃· galactose₂) were more effective than XG7 itself and XG9 (glucose₄· xylose₃· galactose· fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10⁻⁴ molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of [³H]xyloglucan to ethanol-soluble fragments. This suggest that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products). We suggest that the promotion of midchain xyloglucan cleavage, by loosening the primary cell wall matrix, explains the promotion of growth by the oligosaccharides.     

Role of Ethylene in Lactuca sativa cv ;Grand Rapids' Seed Germination

Promotion of thermoinhibited (30°C) lettuce (Lactuca sativa cv `Grand Rapids') seed germination by ethylene is similar to the action of the gas in other hormonal systems. Ethylene was more active than propylene and ethane was inactive. An inhibitor of ethylene production, aminoethoxy-vinylglycine, reduced ethylene evolution and germination. Inhibitors of ethylene action such as, 5-methyl-7-chloro-4-ethoxycarbonylmethoxy-2,1,3-benzothiadiazole, 2,5-norbornadiene, and silver thiosulfate inhibited germination and the effect was reversed by the addition of ethylene to the gas phase. The action of ethylene appears to be due to the promotion of radial cell expansion in the embryonic hypocotyl. The action of N6-benzyladenine and fusiccocin, which also overcome thermoinhibition, appears to be due to a promotion of hypocotyl elongation. None of the germination promoters studied appeared to function by lowering the mechanical resistance of the endosperm to embryonic growth. Data presented here are consistent with the view that ethylene plays a role in lettuce seed germination under thermoinhibited and normal conditions.     

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K_a) values of 5.2 × 10⁶ to 44 × 10⁶ M⁻¹ among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.     

ABA inhibits embryo cell expansion and early cell division events during coffee (Coffea arabica 'Rubi') seed germination

Background and Aims

Coffee seed germination represents an interplay between the embryo and the surrounding endosperm. A sequence of events in both parts of the seed determines whether germination will be successful or not. Following previous studies, the aim here was to further characterize the morphology of endosperm degradation and embryo growth with respect to morphology and cell cycle, and the influence of abscisic acid on these processes.

Methods

Growth of cells in a fixed region of the axis was quantified from light micrographs. Cell cycle events were measured by flow cytometry and by immunocytochemistry, using antibodies against β-tubulin. Aspects of the endosperm were visualized by light and scanning electron microscopy.

Key Results

The embryonic axis cells grew initially by isodiametric expansion. This event coincided with reorientation and increase in abundance of microtubules and with accumulation of β-tubulin. Radicle protrusion was characterized by a shift from isodiametric expansion to elongation of radicle cells and further accumulation of β-tubulin. Early cell division events started prior to radicle protrusion. Abscisic acid decreased the abundance of microtubules and inhibited the growth of the embryo cells, the reorganization of the microtubules, DNA replication in the embryonic axis, the formation of a protuberance and the completion of germination. The endosperm cap cells had smaller and thinner cell walls than the rest of the endosperm. Cells in the endosperm cap displayed compression followed by loss of cell integrity and the appearance of a protuberance prior to radicle protrusion.

Conclusions

Coffee seed germination is the result of isodiametric growth of the embryo followed by elongation, at the expense of integrity of endosperm cap cells. The cell cycle, including cell division, is initiated prior to radicle protrusion. ABA inhibits expansion of the embryo, and hence subsequent events, including germination.Key words: Abscisic acid, β-tubulin, Coffea arabica, coffee seed, cell morphology, germination, microtubules     

Production of Reactive Oxygen Intermediates (O2˙−, H2O2, and ˙OH) by Maize Roots and Their Role in Wall Loosening and Elongation Growth

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O₂^˙−) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals (^˙OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O₂^˙−, H₂O₂, and ^˙OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O₂^˙− production. (3) Experimental generation of ^˙OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by ^˙OH produced by endogenous cell wall peroxidase in the presence of NADH and H₂O₂. (4) Inhibition of endogenous ^˙OH formation by O₂^˙− or ^˙OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate ^˙OH, and to respond to ^˙OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that ^˙OH formation is essential for normal root growth.     

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA₄₊₇. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V₀ membrane sector of vacuolar H⁺-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V₁ sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.     

Gibberellins regulate seed germination in tomato by endosperm weakening: a study with gibberellin-deficient mutants

The germination of seeds of tomato [Lycopersicon esculentum (L.) Mill.] cv. Moneymaker has been compared with that of seeds of the gibberellin-deficient dwarf-mutant line ga-1, induced in the same genetic background. Germination of tomato seeds was absolutely dependent on the presence of either endogenous or exogenous gibberellins (GAs). Gibberellin A₄₊₇ was 1000-fold more active than commercial gibberellic acid in inducing germination of the ga-1 seeds. Red light, a preincubation at 2°C, and ethylene did not stimulate germination of ga-1 seeds in the absence of GA₄₊₇; however, fusicoccin did stimulate germination independently. Removal of the endosperm and testa layers opposite the radicle tip caused germination of ga-1 seeds in water. The seedlings and plants that develop from the detipped ga-1 seeds exhibited the extreme dwarfy phenotype that is normal to this genotype. Measurements of the mechanical resistance of the surrounding layers showed that the major action of GAs was directed to the weakening of the endosperm cells around the radicle tip. In wild-type seeds this weakening occurred in water before radicle protrusion. In ga-1 seeds a similar event was dependent on GA₄₊₇, while fusicoccin also had some activity. Simultaneous incubation of de-embryonated endosperms and isolated axes showed that wild-type embryos contain and endosperm-weakening factor that is absent in ga-1 axes and is probably a GA. Thus, an endogenous GA facilitates germination in tomato seeds by weakening the mechanical restraint of the endosperm cells to permit radicle protrusion.Abbreviations GA(s) gibberellin(s) - GA₃ gibberellic acid     

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : V. Water Relations of Imbibition and Germination

The initiation of radicle growth during seed germination may be driven by solute accumulation and increased turgor pressure, by cell wall relaxation, or by weakening of tissues surrounding the embryo. To investigate these possibilities, imbibition kinetics, water contents, and water (Ψ) and solute (ψ_s) potentials of intact muskmelon (Cucumis melo L.) seeds, decoated seeds (testa removed, but a thin perisperm/endosperm envelope remains around the embryo), and isolated cotyledons and embryonic axes were measured. Cotyledons and embryonic axes excised and imbibed as isolated tissues attained water contents 25 and 50% greater, respectively, than the same tissues hydrated within intact seeds. The effect of the testa and perisperm on embryo water content was due to mechanical restriction of embryo swelling and not to impermeability to water. The Ψ and ψ_s of embryo tissues were measured by psychrometry after excision from imbibed intact seeds. For intact or decoated seeds and excised cotyledons, Ψ values were >−0.2 MPa just prior to radicle emergence. The Ψ of excised embryonic axes, however, averaged only −0.6 MPa over the same period. The embryonic axis apparently is mechanically constrained within the testa/perisperm, increasing its total pressure potential until axis Ψ is in equilibrium with cotyledon Ψ, but reducing its water content and resulting in a low Ψ when the constraint is removed. There was no evidence of decreasing ψ_s or increasing turgor pressure (Ψ-ψ_s) prior to radicle growth for either intact seeds or excised tissues. Given the low relative water content of the axes within intact seeds, cell wall relaxation would be ineffective in creating a Ψ gradient for water uptake. Rather, axis growth may be initiated by weakening of the perisperm, thus releasing the external pressure and creating a Ψ gradient for water uptake into the axis. The perisperm envelope contains a cap of small, thin-walled endosperm cells adjacent to the radicle tip. We hypothesize that weakening or separation of cells in this region could initiate radicle expansion.     

Mechanism of seed priming in circumventing thermodormancy in lettuce

Lettuce (Lactuca sativa L. cv Minetto) seeds were primed in aerated solutions of 1% K₃PO₄ or water at 15°C in the dark for various periods of time to determine the manner by which seed priming bypasses thermodormancy. Seeds which were not primed did not germinate at 35°C, whereas those which were primed for 20 h in 1% K₃PO₄ or distilled H₂O had up to 86% germination. The rate of water uptake and respiration during priming were similar regardless of soak solution. Cell elongation occurred in both water and 1% K₃PO₄, 4 to 6 h prior to cell division. Both processes commenced sooner in water than K₃PO₄. Radicle protrusion (germination) occurred in the priming solution at 21 h in water and 27 h in 1% K₃PO₄.

Respiration, radicle protrusion and cell division consistently occurred sooner in primed (redried) seeds compared to nonprimed seeds when they were imbibed at 25°C. Cell division and elongation commenced after 10 h imbibition in primed (redried) seeds imbibed at 35°C. Neither process occurred in nonprimed seeds. Respiratory rates were higher in both primed and nonprimed seeds imbibed at 35°C compared to those imbibed at 25°C, although radicle protrusion did not occur in nonprimed seeds which were imbibed at 35°C. It is apparent that cell elongation and division are inhibited during high temperature imbibition in nonprimed lettuce seeds. Seed priming appears to lead to the irreversible initiation of cell elongation, thus overcoming thermodormancy.

     

Bgs3p,a putative 1,3-beta-glucan synthase subunit,is required for cell wall assembly in Schizosaccharomyces pombe

β-Glucans are the main components of the fungal cell wall. Fission yeast possesses a family of β-glucan synthase-related genes. We describe here the cloning and characterization of bgs3⁺, a new member of this family. bgs3⁺ was cloned as a suppressor of a mutant hypersensitive to Echinocandin and Calcofluor White, drugs that interfere with cell wall biosynthesis. Disruption of the gene is lethal, and a decrease in Bgs3p levels leads to rounded cells with thicker walls, slightly reduces the amount of the β-glucan, and raises the amount of α-glucan polymer. These cells finally died. bgs3⁺ is expressed in vegetative cells grown in different conditions and during mating and germination and is not enhanced by stress situations. Consistent with the observed expression pattern, Bgs3-green fluorescence protein (GFP-Bgs3p) was found at the growing tips during interphase and at the septum prior to cytokinesis, always localized to growth areas. We also found GFP-Bgs3p in mating projections, during the early stages of zygote formation, and at the growing pole during ascospore germination. We conclude that Bgs3p localization is restricted to growth areas and that Bgs3p is a glucan synthase homologue required for cell wall biosynthesis and cell elongation in the fission yeast life cycle.