Peroxidase activity develops in the micropylar endosperm of tomato seeds prior to radicle protrusion

Peroxidase activity developed specifically in the micropylar region of the endosperm of imbibed tomato seeds prior to radicle emergence. The activity was first detected approximately 24 h after the start of imibibition (6 h before radicle emergence) and increased markedly thereafter. In the lateral portion of the endosperm, peroxidase activity was undetectable for the first 2 d after the start of imbibition. Although the activity in the lateral endosperm became detectable 3 d after imbibition, the extent of the development of the activity was slight. The localization of peroxidase activity in the micropylar endosperm 2 d after the start of imbibition was confirmed by tissue printing analyses. When the endosperm tissues were wounded, there was an enhancement of the enzyme activity at the wounded region. H2O2 was formed at the expense of NADH only in the presence of Mn2+ and dinitrophenol by the extract from the micropylar endosperm in which peroxidase activity was present. The presence of H2O2 in the micropylar portion of the endosperm was shown histochemically. The possible functions of the peroxidases that develop in the endosperm of tomato seeds are discussed.     

Development of beta-1,3-glucanase activity in germinated tomato seeds

Laminarin-hydrolysing activity developed in the endosperm of tomato (Lycopersicon esculentum) seeds following germination. The enzyme was basic (pI>10) and the apparent molecular mass was estimated to be 35 kDa by SDS-PAGE. It was specific for linear beta-1,3-glucan substrates. Laminarin was hydrolysed by the enzyme to yield a mixture of oligoglucosides, indicating that the enzyme had an endo-action pattern. Thus, the enzyme was identified as beta-1,3- endoglucanase (EC 3.2.1.39). The activity of the enzyme developed in the endosperm after radicle protrusion (germination) had occurred and the enzyme activity was localized exclusively in the micropylar region of the endosperm where the radicle had penetrated. When the lateral endosperm region, where no induction of the enzyme occurred, was wounded (cut or punctured), there was a marked enhancement of beta-1,3-glucanase activity. Thus the post-germinative beta-1, 3-glucanase activity in the micropylar endosperm portion might be brought about by wounding resulting from endosperm rupture by radicle penetration.     

Temporal and spatial pattern of the biochemical activation of the endosperm during and following imbibition of tomato seeds

Electron microscopic observations of the endosperm of tomato ( Lycopersicon esculentum Mill.) seeds revealed that changes in the cell wall structures along with the vacuolation of protein bodies occurred in the micropylar portion of the endosperm prior to germination. No changes were detected at that time in the rest of the endosperm. Endo‐β‐mannanase activity was restricted to the micropylar region of the endosperm prior to germination. Cell wall digestion by this pregerminative mannanase seemed to be associated with the changes in cell wall structures occurring in the micropylar region prior to germination. The protein content in the micropylar part of the endosperm began to decrease shortly after imbibition and attained about 40% of the initial level by the time of radicle protrusion (38 h after imbibition). On the other hand, only slight changes in the content were detected in the lateral endosperm during the same time; the protein content in the lateral endosperm decreased only after germination started. In conformity with the results on protein contents, proteolytic activity began to develop first in the micropylar portion prior to germination, and then in the lateral portion after germination. Thus, the timing of the biochemical activation of the endosperm after imbibition differed between the micropylar and the lateral region. Some qualitative differences in patterns of polypeptides synthesized in vivo were detected, as analyzed by pulse‐labeling and fluorography, between the micropylar and the lateral portions of the endosperm of seeds imbibed for 25 h. This suggests that processes of the biochemical activation of the endosperm may be qualitatively, as well as quantitatively, different depending on the regions of the endosperm.     

The relationship between beta-mannosidase and endo-beta-mannanase activities in tomato seeds during and following germination: a comparison of seed populations and individual seeds

beta-Mannosidase and endo-beta-mannanase are involved in the mobilization of the mannan-containing cell walls of the tomato seed endosperm. The activities of both enzymes increase in a similar temporal manner in the micropylar and lateral endosperm during and following germination. This increase in enzyme activities in the micropylar endosperm is not markedly reduced in seeds imbibed in abscisic acid although, in the lateral endosperm, endo-beta-mannanase activity is more suppressed by this inhibitor than is the activity of beta-mannosidase. Gibberellin-deficient (gib-1) mutants of tomato do not germinate unless imbibed in gibberellin; low beta-mannosidase activity, and no endo-beta-mannanase activity is present in seeds imbibed in water, but both enzymes increase strongly in activity in the seeds imbibed in the growth regulator. For production of full activity of both beta-mannosidase and endo-beta-mannanase in the endosperm, this tissue must be in contact with the embryo for at least the first 6 h of imbibition, which is indicative of a stimulus diffusing from the embryo to the endosperm during this time. These results suggest some correlation between the activities of beta-mannosidase and endo-beta-mannanase, particularly in the micropylar endosperm, in populations of tomato seeds imbibed in water, abscisic acid and gibberellin. However, when individual micropylar endosperm parts are used to examine the effect of the growth regulators and of imbibition in water on the production of the two enzymes, it is apparent that within these individual seed parts there may be large differences in the amount of enzyme activity present. Micropylar endosperms with high endo-beta-mannanase activity do not necessarily have high beta-mannosidase activity, and vice versa, which is indicative of a lack of co-ordination of the activities of these two enzymes within individuals of a population.     

An Endo-[beta]-Mannanase Develops Exclusively in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence

A galactomannan-hydrolyzing enzyme that develops pregerminatively in the micropylar region of the endosperm of the tomato (Lycopersicon esculentum [L.] Mill.) seed was characterized. The enzyme was endo-[beta]-mannanase (EC 3.2.1.78), since it hydrolyzed galactomannan into oligosaccharides with no release of galactose and mannose. The mobility of this pregerminative enzyme in sodium dodecyl sulfate and native polyacrylamide gel electrophoresis was not identical to that of any of the three endo-[beta]-mannanases that develop in the same tissue (endosperm) after germination (H. Nonogaki, M. Nomaguchi, Y. Morohashi [1995] Physiol Plant 94: 328-334). There were also some differences in the products of galactomannan hydrolysis between the pregerminative and the postgerminative enzymes, indicating that the action pattern is different between the two types of enzymes. The pregerminative enzyme began to develop in the micropylar region of the endosperm at about 18 h postimbibition and increased up to the time immediately before radicle protrusion (24 h postimbibition). This enzyme was not present in the lateral part of the endosperm at any stage before or after germination. It is proposed that the enzyme develops prior to germination specifically at the micropylar region of the endosperm.     

Mechanism and control of Genipa americana seed germination

Genipa americana (Rubiaceae) is important for restoration of riparian forest in the Brazilian Cerrado. The objective was to characterize the mechanism and control of germination of G. americana to support uniform seedling production. Morphology and morphometrics of seeds, embryo and endosperm were assessed by light and scanning electron microscopy during germination. Imbibition and germination curves were generated and over the same time interval endosperm digestion and resistance were measured by puncture force analysis and activity assay of endo-β-mannanase (EBM) in water and in abscisic acid (ABA). The gene encoding for EBM was partially cloned and its expression monitored by quantitative real-time-polymerase chain reaction. Embryos displayed growth prior to radicle protrusion. A two-phase increase in EBM activity coincided with the two stages of weakening of the micropylar endosperm. The second stage also coincided with growth of the embryo prior to radicle protrusion. Enzyme activity was initiated in the micropylar endosperm but spread to the lateral endosperm. ABA completely inhibited germination by inhibiting embryo growth, the second stage of weakening and expression of the EBM gene, but EBM activity was not significantly inhibited. This suggests that a specific isoform of the enzyme is involved in endosperm weakening. EBM may cause a general 'softening' of micropylar endosperm cell walls, allowing the embryo to puncture the endosperm as the driving force of the decrease in puncture force.     

The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo-beta-mannanase activity

BACKGROUND AND AIMS: Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-beta-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate. METHODS: Activity of endo-beta-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy. KEY RESULTS: The activity of endo-beta-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination. CONCLUSIONS: The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion.     

Beta-mannosidase (EC 3.2.1.25) activity during and following germination of tomato (Lycopersicon esculentum Mill.) seeds. Purification,cloning and characterization

Beta-mannosidase, a high-salt-soluble enzyme, increases in activity in seeds of tomato prior to the completion of germination. This increase occurs in both the lateral and micropylar endosperm and becomes more evident during post-germinative seedling growth. The beta-mannosidase activity profile is similar to that of endo beta-mannanase although it is the first to increase in the lateral endosperm. Tomato seed beta-mannosidase was purified to homogeneity and its cDNA (LeMside1) obtained by 3'-RACE PCR using oligonucleotide sequences based on four peptide sequences obtained from the purified enzyme. The derived amino acid sequence of the tomato beta-mannosidase shows the enzyme is a member of the Glycosyl Hydrolases Family 1 (GHF1) but has a very low sequence identity with that of beta-mannosidases from non-plant sources; no other plant sequence for the enzyme is known. There appears to be only one gene encoding beta-mannosidase in tomato, the sequence of which has been determined (LeMSide2). Its expression occurs first in the micropylar endosperm, and then declines after germination. This is followed by an increase in its expression in the lateral endosperm, which precedes that of the gene for endo beta-mannanase. Expression of the beta-mannosidase gene increases appreciably in the growing seedling embryo. With this report, the cloning of all three of the enzymes involved in galactomannan mobilization (endo beta-mannanase, alpha-galactosidase and beta-mannosidase) in tomato seeds has now been achieved.     

A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (K(m)=9.86mM, V(max)=24.66 EU [DeltaAmin(-1)]) and catechol (K(m)=3.44mM, V(max)=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.     

Galactomannan hydrolyzing activity develops during priming in the micropylar endosperm tip of tomato seeds

Development of galactomannan hydrolyzing activity was followed in seeds of tomato [ Lycopersicon esculentum (L.) Mill. cv. Toyonishiki] during priming and germination. The activity developed in seeds that were being primed in polyethylene glycol (-0.8 MPa). The activity was detected exclusively in the endosperm portion just adjacent to the radicle tip. Part of the activity remained active after desiccation of the primed seeds. After transfer to water, the activity in the primed seeds immediately began to increase, while in unprimed seeds the beginning of the increase in activity was delayed by about 1 day. In scanning electron microscopy, the inner surface of the cell walls of the micropylar endosperm portion appeared eroded in primed seeds that had been imbibed in water for 16 h (before germination), but not in unprimed seeds imbibed for the same period. These results support the hypothesis that galactomannan hydrolyzing enzyme, which is believed to be responsible for breakdown of tomato endosperm cell walls and hence for the weakening of mechanical restraint against radicle growth, may be involved in the improved germination of primed tomato seeds.     

Cell-Wall Autohydrolysis in Isolated Endosperms of Lettuce (Lactuca sativa L.)

Cell walls prepared from the endosperm tissue of hydrated lettuce (Lactuca sativa L.) seeds undergo autohydrolysis. Release of carbohydrates is most rapid (0.4-0.6 [mu]g per endosperm) within the 1st h of incubation in buffer, but substantial autolysis is sustained for at least 10 h. Autolysis is temperature sensitive, and the optimum rate occurs at pH 5. The rate of autolysis increases markedly in the period just prior to radicle emergence. The cell-wall polysaccharide composition in micropylar and lateral endosperm regions differs significantly; the micropylar walls are rich in arabinose and glucose with substantially lower amounts of mannose. Although walls prepared from both micropylar and lateral regions undergo autolysis, micropylar walls release carbohydrates at a higher rate than lateral walls. Autolysis products elute as large polymers when subjected to size-exclusion chromatography, suggesting that endo-enzyme activity is responsible for release of fragments containing arabinose, galactose, mannose, and uronic acids. Arabinose, galactose, mannose, and glucose are also released as monomers. As a function of time, the ratio of polymers to monomers decreases, indicating that exo-enzyme activity is also present. Thermoinhibition or treatment with abscisic acid suppresses germination and reduces the rates of autolysis of walls isolated from the endosperm by about 25%. Treatments that alleviate thermoinhibition (kinetin and gibberellic acid) increase the rates of autolysis by 20 to 30% when compared to thermoinhibited controls.     

Characterization of polyphenol oxidase in coffee

Polyphenol oxidase (PPO) was characterized in partially purified extracts of leaves (PPO-L) and fruit endosperm (PPO-E) of coffee (Coffea arabica L.). PPO activity was higher in early developmental stages of both leaves and endosperm of fruits. Wounding or exposure of coffee leaves to methyl jasmonate increased PPO activity 1.5-4-fold. PPO was not latent and was not activated by protease treatment. PPO activity was stimulated 10-15% with sodium dodecyl sulphate (SDS) at 0.35-1.75 mM, but at higher concentrations activities were similar to the control samples, without detergent. Prolonged incubation of extracts with trypsin or proteinase K inhibited PPO activity but pepsin had no effect. Inhibition of PPO with proteinase K was increased in the presence of SDS. PPO activity from both tissues was optimal at pH 6-7 and at an assay temperature of 30 degrees C. Activity was highest with chlorogenic acid as substrate with a Km of 0.882 mM (PPO-L) and 2.27 mM (PPO-E). Hexadecyl trimethyl-ammonium bromide, polyvinylpyrrolidone 40. cinnamic acid and salicylhydroxamic acid inhibited PPO from both tissues. Both enzymes were inactivated by heat but the activity in endosperm extracts was more heat labile than that from leaves. The apparent Mr determined by gel filtration was 46 (PPO-L) and 50 kDa (PPO-E). Activity-stained SDS polyacrylamide gel electrophoresis (PAGE) gels and western blots probed with PPO antibodies suggested the existence of a 67 kDa PPO which is susceptible to proteolytic cleavage that generates a 45 kDa active form.     

Cloning and characterization of cDNAs coding for Vicia faba polyphenol oxidase

Three cDNA clones were isolated which code for the ubiquitous chloroplast enzyme, polyphenol oxidase (PPO), from Vicia faba. Analysis of the cloned DNA reveals that PPO is synthesized with an N-terminal extension of 92 amino acid residues, presumed to be a transit peptide. The mature protein is predicted to have a molecular mass of 58 kDa which is in close agreement to the molecular mass estimated for the in vivo protein upon SDS-PAGE. Differences in the DNA sequence of two full-length and one partial cDNA clones indicate that PPO is encoded by a gene family. Analysis of the deduced amino acid sequence shows that the chloroplast PPO shares homology with the 59 kDa PPOs in glandular trichomes of solanaceous species. A high degree of sequence conservation was found with the copper-binding domains of the 59 kDa tomato PPO as well as hemocyanins and tyrosinases from a wide diversity of taxa.     

Developing tomato seeds when removed from the fruit produce multiple forms of germinative and post-germinative endo-β-mannanase. Responses to desiccation, abscisic acid and osmoticum

Several isoforms of endo-1,4-D-mannanase (EC3.2.1.78) are produced in the endosperm and embryo of tomato (Lycopersicon esculentum Mill.) seed prior to the completion of germination. Other isoforms appear in the embryo and in the lateral endosperm following germination. This occurs in seeds removed from the fruit prior to completion of development at 45 d after pollination and placed directly on water, or following drying. Hence desiccation is not required to induce either germination- or post-germination-related mannanase activity. Incubating seeds in abscisic acid or osmoticum results in a reduction of both germination and total mannanase activity, but the isoforms that are produced in the embryo and micropylar region of the endosperm are identical to those produced in water-imbibed seeds prior to germination. Incubation of seeds in a high concentration of abscisic acid prevents all enzyme production. Only after the completion of germination does mannanase increase in the lateral regions of the endosperm. In contrast, mannanase is produced in the micropylar region regardless of whether the seed germinates or not. The isoforms produced in the two regions of the endosperm are different, those in the lateral endosperm being more similar to those produced in the cotyledons and axes of the embryo. Embryos and endosperms dissected prior to completion of germination and incubated separately produce far fewer isoforms than when these parts are together in the intact seed.Abbreviations ABA cis-abscisic acid - DAP days after pollination - GA gibberellin - IEF isoelectric focusing - PEG polyethyleneglycol - pI isoelectric point This work was supported by Natural Sciences and Engineering Council of Canada grant A2210. B.V. received a fellowship from the Deutscher Akademischer Austauschdienst for her research at the University of Guelph. We are grateful to Dr. H.W.M. Hilhorst, Wageningen, for his critical comments.     

Active, inactive and in vitro synthesized forms of polyphenoloxidase during leaf development

Polyphenoloxidase (PPO; EC 1.14.18.1) activity decreased 8-fold from young to mature Vicia faba L. Moensch (cv. Long pod) leaves. The K_m for catechol remained relatively constant from young to mature leaves. Electrophoretic separation and analysis showed that only one active form was present in extracts from various leaf sizes. The amount of this form appeared to decrease with leaf size/age. In extracts which had not tanned, electroblotting and immunostaining indicated that one enzyme form was present with a molecular mass of 45 kDa. Two leaf categories contained greater amounts of this immunological cross-reacting PPO than other leaf categories. When extracts were allowed to darken, immunoblotting detected three enzyme forms with molecular masses of 45, 59 and 63 kDa. The latter two immunological crossracting species had no detectable enzyme activity. Poly-A⁺ mRNA was isolated from six leaf sizes and translated in vitro. A product corresponding to PPO was present in all leaf categories. Greater amounts of this translation product were observed in medium-sized leaves than in very young or mature leaves. These results suggest that: (1) enzymatic assays for PPO are not reliable indicators of the total amount of PPO protein present in developing leaves, (2) immunoblotting can detect inactive enzyme forms, (3) only one active form of the enzyme is present at all developmental stages, and (4) mRNA corresponding to PPO is present at all developmental stages but appears to be more abundant in certain leaf sizes/ages.     

Embryo and Endosperm Development in Isatis Tinctoria L. and the Histochemical Study of Its Storage Reserve

The ovule is anatropous and bitegmic. The nuceIlar cells have disorganized except the chalazal proliferating tissue. The curved embryo sac comprises an egg apparatus and a central cell with two palar nuclei and wall ingrowths on its micropylar lateral wall. The antipodal cells disappear. Embryo development is of the Onagrad type. The filament suspensor grows to a length of 785 μm and degenerats at tarpedo embryo stage. The basal cell produces wall ingrowths on the micropylar end wall and lateral wall. The cells of mature embryo contain many globular protein bodies, 2.5–7.5 μm in diameter, composed of high concentration of protein and phytin, insoluble polysaccharide and lipid. The cells, except procambium, also contain many small starch grains. Some secretory cavities scattered in the ground tissue have liquidlike granules composed of protein, ploysacchaide and lipid. Endosperm development follows the nuclear pattern. At the late heart embryo stage, the endosperm around the embryo and the upper suspensor and the peripheral endosperm of the basal region of the U-shaped embryo sac becomes cellular. The endosperm at micropylar and chalazal ends remains free nuclear phase until the late bended cotyledon stage. Wall ingrowths at both micropylar and chalazal end wall and lateral wall of the embryo sac become more massive during endosperm development. Wall ingrowths also occur on the outer walls of the outer layer endosperm cells at both ends and lateral region of the embryo sac. When the embryo matures, many layers of chalazal endosperm ceils including 2–4 layers of transfer cells, a few of micropylar endosperm cells and 1–5 layers of peripheral endosperm cells are present. The nutrients of the embryo and endosperm at different stages of development are also discussed.     

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.     

西瓜胚乳吸器的发育及ATP酶的超微细胞化学定位

报道了西瓜（Citrullus lanatus)胚乳吸器发育过程,并对胚乳吸器细胞中的ATP酶进行了超微细胞化学定位,球形胚早期,胚囊合点端的壁伸长发育成一管状胚乳吸器,进而吸器靠近乳本体端膨大为囊状,球形胚晚期吸器自珠孔端向合点端逐渐细胞化,胚分化出子叶时,胚乳吸器自合点端向珠孔端退化,在刚形成的胚乳吸器细胞中,ATP酶活性反应主要分布在细胞的核膜,内质网上,胞间连丝和吸器细胞壁内的小球状物上也有较强的ATP酶活性反应;在开始退化的吸器细胞中,核膜上的ATP酶性的反应减弱较早,内质网稍晚,进一步退化的胚乳吸器细胞中,ATP酶主要集中分布在细胞壁,细胞间隙内,核上几乎没有ATP酶性反应,内质网上仅有微弱的ATP酶反应。