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.     

Variation in its C-terminal amino acids determines whether endo-beta-mannanase is active or inactive in ripening tomato fruits of different cultivars

Endo-beta-mannanase cDNAs were cloned and characterized from ripening tomato (Lycopersicon esculentum Mill. cv Trust) fruit, which produces an active enzyme, and from the tomato cv Walter, which produces an inactive enzyme. There is a two-nucleotide deletion in the gene from tomato cv Walter, which results in a frame shift and the deletion of four amino acids at the C terminus of the full-length protein. Other cultivars that produce either active or inactive enzyme show the same absence or presence of the two-nucleotide deletion. The endo-beta-mannanase enzyme protein was purified and characterized from ripe fruit to ensure that cDNA codes for the enzyme from fruit. Immunoblot analysis demonstrated that non-ripening mutants, which also fail to exhibit endo-beta-mannanase activity, do so because they fail to express the protein. In a two-way genetic cross between tomato cvs Walter and Trust, all F(1) progeny from both crosses produced fruit with active enzyme, suggesting that this form is dominant and homozygous in tomato cv Trust. Self-pollination of a plant from the heterozygous F(1) generation yielded F(2) plants that bear fruit with and without active enzyme at a ratio appropriate to Mendelian genetic segregation of alleles. Heterologous expression of the two endo-beta-mannanase genes in Escherichia coli resulted in active enzyme being produced from cultures containing the tomato cv Trust gene and inactive enzyme being produced from those containing the tomato cv Walter gene. Site-directed mutagenesis was used to establish key elements in the C terminus of the endo-beta-mannanase protein that are essential for full enzyme activity.     

Solid-phase tyrosine-specific protein kinase assay in multiwell substrate-immobilized polyacrylamide gel

Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser/Thr-specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compounded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and [tau-32P]ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. 32P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser/Thr-specific protein kinases, can also be analyzed by this technique.     

The perisperm-endosperm envelope in Cucumis: structure, proton diffusion and cell wall hydrolysing activity

BACKGROUND: and Aims The envelope surrounding the embryo in cucurbit seed, which consists of a single layer of live endosperm cells covered by lipid- and callose-rich layers, is reported to show semi-permeability and also to act as the primary barrier to radicle emergence. Structure, development and permeability of the envelope and activity of cell wall hydrolases during germination of cucumber and muskmelon seeds were investigated. METHODS: Sections of seeds were stained with aniline blue and Sudan III. Proton diffusion and endo-beta-mannanase activity were detected by tissue printing. A gel-diffusion assay was performed to quantify endo-beta-mannanase activity, while the activity of beta-glucanase was determined with laminarin as the substrate and glucose formation measured using the GOD-POD method. KEY RESULTS: The lipid layer differentiated during seed development in cucumber in the epidermis of a multilayered nucellus, whereas the callose layer appeared to develop outside the endosperm cell layer. Accordingly, the envelope has been called the perisperm-endosperm (PE) envelope. Chloroform treatment of seeds, which resulted in a substantial reduction in Sudan staining of the lipid layer, also enhanced the permeability of the PE envelope to 2,3,5-triphenyltetrazolium chloride. Proton diffusion occurred when the PE envelopes from seeds had their inner surface in contact with bromocresol purple-containing agarose gels, but not when their outer surface was in contact. Substantial endo-beta-mannanase activity was present in the caps of the PE envelopes, whereas a marked increase in beta-glucanase activity was observed in radicles prior to germination. CONCLUSIONS: The lipid layer seems to contribute to the semi-permeability of the PE envelope. The diffusion of protons might create an acidic environment conducive to the activity of cell wall hydrolases, namely endo-beta-mannanase (EC 3.2.1.78) and beta-glucanase [beta(1-->3)glucanohydrolase; EC 3.2.1.6], which, in turn, may play a role in the weakening of the PE envelope necessary for the protrusion of the radicle in cucumber and muskmelon seeds.     

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 Antisense Pectin Methylesterase Gene Alters Pectin Chemistry and Soluble Solids in Tomato Fruit

Pectin methylesterase (PME, EC 3.1.11) demethoxylates pectins and is believed to be involved in degradation of pectic cell wall components by polygalacturonase in ripening tomato fruit. We have introduced antisense and sense chimeric PME genes into tomato to elucidate the role of PME in fruit development and ripening. Fruits from transgenic plants expressing high levels of antisense PME RNA showed <10% of wild-type PME enzyme activity and undetectable levels of PME protein and mRNA. Lower PME enzyme activity in fruits from transgenic plants was associated with an increased molecular weight and methylesterification of pectins and decreased levels of total and chelator soluble polyuronides in cell walls. The fruits of transgenic plants also contained higher levels of soluble solids than wild-type fruits. This trait was maintained in subsequent generations and segregated in normal Mendelian fashion with the antisense PME gene. These results indicate that reduction in PME enzyme activity in ripening tomato fruits had a marked influence on fruit pectin metabolism and increased the soluble solids content of fruits, but did not interfere with the ripening process.     

A germination-specific endo-beta-mannanase gene is expressed in the micropylar endosperm cap of tomato seeds

Endo-beta-mannanase (EC 3.2.1.78) is involved in hydrolysis of the mannan-rich cell walls of the tomato (Lycopersicon esculentum Mill.) endosperm during germination and post-germinative seedling growth. Different electrophoretic isoforms of endo-beta-mannanase are expressed sequentially in different parts of the endosperm, initially in the micropylar endosperm cap covering the radicle tip and subsequently in the remaining lateral endosperm surrounding the rest of the embryo. We have isolated a cDNA from imbibed tomato seeds (LeMAN2) that shares 77% deduced amino acid sequence similarity with a post-germinative tomato mannanase (LeMAN1). When expressed in Escherichia coli, the protein encoded by LeMAN2 cDNA was recognized by anti-mannanase antibody and exhibited endo-beta-mannanase activity, confirming the identity of the gene. LeMAN2 was expressed exclusively in the endosperm cap tissue of tomato seeds prior to radicle emergence, whereas LeMAN1 was expressed only in the lateral endosperm after radicle emergence. LeMAN2 mRNA accumulation and mannanase activity were induced by gibberellin in gibberellin-deficient gib-1 mutant seeds but were not inhibited by abscisic acid in wild-type seeds. Distinct mannanases are involved in germination and post-germinative growth, with LeMAN2 being associated with endosperm cap weakening prior to radicle emergence, whereas LeMAN1 mobilizes galactomannan reserves in the lateral endosperm.     

Endopolygalacturonase from tomato fruit

A polygalacturonase was extracted from ripening tomato fruit. A four step procedure was developed producing a 44-fold increase in specific activity with 9% recovery. The enzyme was found to rapidly degrade pectic acid but not pectin. No transeliminase activity was detected. Viscosity and per cent hydrolysis studies formed a basis for suggesting that this enzyme cleaves its substrate in a random manner and is likely to be an endopolygalacturonase.     

Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined.     

Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells.

Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport. To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase). We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells. The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence. LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase. LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato. LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit. The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening.     

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation

An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated 'activator' (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of 'converting' the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.     

Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β-subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β-subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β-subunit protein. Cell walls prepared from mature-green fruit were employed for binding and assay of PG2. Walls were provided with rate-limiting quantities of purified PG2 and incubated at 100 mM KCl, pH 4.5, or 25 mM KCl, pH 6.0. Cell walls of both β-subunit antisense and wild-type fruit retained comparable quantities of added PG2. The release of pectin from PG2-loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β-Subunit-antisense cell walls released 2- to 3-fold higher levels of pectin in response to PG2 than did wild-type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2-mediated hydrolysis of β-subunit-antisense compared with wild-type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG-mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 mM CDTA (trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid), and were more extensive for the β-subunit-antisense cell walls compared with the wild-type walls. Pectic polymers derived from ethanol insoluble-powders showed molecular mass downshifts during ripening but differences between the β-subunit-antisense and wild-type fruits were not observed.     

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin.

A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a beta-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a [14C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.     

Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.     

Protein phosphorylation and membrane association of sucrose synthase in developing tomato fruit.

Calcium-dependent protein kinase (CDPK) activities were detected both in the soluble and the membrane fraction of various tomato (Lycopersicon esculentum Mill.) organs, using a synthetic peptide mimicking the serine 11 phosphorylation site of a tomato sucrose synthase (SS, EC 2.4.1.13) isoform as substrate. The levels of membrane and soluble Ser-CDPK activities were differentially regulated during fruit development. The membrane Ser-CDPK activity was maximal in young fruit but decreased as the fruit developed, suggesting a specific role during fruit growth. Using an in gel assay with purified tomato SS as substrate, we showed that partially purified soluble and membrane Ser-CDPK preparations both contained a SS-kinase polypeptide of 55 kDa. The membrane and soluble Ser-CDPK activities were largely inactivated in the absence of calcium or when MgCl(2) was replaced by MnCl(2). Both soluble and membrane Ser-CDPK activities were very sensitive to staurosporine. Using Fe(III)-immobilized metal chromatography to determine the apparent phosphorylation status of the enzyme in vivo, we showed that soluble SS was largely dephosphorylated in fruits fed EGTA or staurosporine, compared to fruits fed water or sucrose. Moreover, the level of SS increased by about two-fold in the membrane fraction of fruits fed the Ser-CDPK inhibitors, compared to the control. The level of SS protein in the membrane and soluble fractions of tomato fruit was developmentally regulated, the membrane form being specifically detected in actively growing fruits. Together, our results suggest that a mechanism involving protein phosphorylation/dephosphorylation and/or calcium would in part control the association of SS isoforms with membranes in developing tomato fruit.     

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits)

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.     

A novel endo-beta-mannanase gene in tomato LeMAN5 is associated with anther and pollen development

Endo-beta-mannanase (EC 3.2.1.78) is involved in cell wall disassembly and the weakening of plant tissues by degrading mannan polymers in the cell walls. Endo-beta-mannanase genes are expressed in tomato (Lycopersicon esculentum) seeds (LeMAN1 and LeMAN2) and fruits (LeMAN3 and LeMAN4). A novel endo-beta-mannanase gene (termed LeMAN5) was found in the tomato genome by genome-walking PCR and bacterial artificial chromosome library screening. The 5'-upstream region of this endo-beta-mannanase gene contained four copies of the pollen-specific cis-acting elements POLLEN1LELAT52 (AGAAA). A GUS-reporter gene driven with the putative LeMAN5 promoter (-543 to +38) was activated in anthers and pollen of transgenic Arabidopsis, with the highest beta-glucuronidase activity detected in pollen. beta-Glucuronidase expression was detected in mature pollen retained in sporangia, discharged pollen, and elongating pollen tubes in transgenic Arabidopsis. Consistently, expression of LeMAN5 mRNA and endo-beta-mannnanase activity was detected in tomato anthers and pollen. In anthers, the highest mRNA expression and endo-beta-mannanase activity were detected during late stages of anther development, when pollen maturation occurred. Endo-beta-mannanase activity was present in discharged pollen, which was easily eluted in a buffer, indicating that the enzyme proteins are probably secreted from, and deposited on, the surface of pollen. These data suggest that the LeMAN5 endo-beta-mannanase is associated with anther and pollen development.