Relationship of Endo-[beta]-D-Mannanase Activity and Cell Wall Hydrolysis in Tomato Endosperm to Germination Rates

The endosperm tissue enclosing the radicle tip (endosperm cap) governs radicle emergence in tomato (Lycopersicon esculentum Mill.) seeds. Weakening of the endosperm cap has been attributed to hydrolysis of its mannan-rich cell walls by endo-[beta]-D-mannanase. To test this hypothesis, we measured mannanase activity in tomato endosperm caps from seeds allowed to imbibe under conditions of varying germination rates. Over a range of suboptimal temperatures, mannanase activity prior to radicle emergence increased in accordance with accumulated thermal time. Reduced water potential delayed or prevented radicle emergence but enhanced mannanase activity in the endosperm caps. Abscisic acid did not prevent the initial increase in mannanase activity, although radicle emergence was markedly delayed. Sugar composition and percent mannose (Man) content of endosperm cap cell walls did not change prior to radicle emergence under any condition. Man, glucose, and other sugars were released into the incubation solution by endosperm caps isolated from intact seeds during imbibition. Pregerminative release of Man was suppressed and the release of glucose was enhanced when seeds were incubated in osmoticum or abscisic acid; the opposite occurred in the presence of gibberellin. Thus, whereas sugar release patterns were sensitive to environmental and hormonal factors affecting germination, neither assayable endo-[beta]-D-mannanase activity nor changes in cell wall sugar composition of endosperm caps correlated well with tomato seed germination rates under all conditions.     

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.     

Mechanism and control of Solanum lycocarpum seed germination

BACKGROUND AND AIMS: Solanaceae seed morphology and physiology have been widely studied but mainly in domesticated crops. The present study aimed to compare the seed morphology and the physiology of germination of Solanum lycocarpum, an important species native to the Brazilian Cerrado, with two species with endospermic seeds, tomato and coffee. METHODS: Morphological parameters of fruits and seeds were determined by microscopy. Germination was monitored for 40 d under different temperature regimes. Endosperm digestion and resistance, with endo-beta-mannanase activity and required force to puncture the endosperm cap as respective markers, were measured during germination in water and in abscisic acid. KEY RESULTS: Fruits of S. lycocarpum contain dormant seeds before natural dispersion. The best germination condition found was a 12-h alternating light/dark and high/low (20/30 degrees C) temperature cycle, which seemed to target properties of the endosperm cap. The endosperm cap contains 7-8 layers of elongated polygonal cells and is predestined to facilitate radicle protrusion. The force required to puncture the endosperm cap decreased in two stages during germination and showed a significant negative correlation with endo-beta-mannanase activity. As a result of the thick endosperm cap, the puncture force was significantly higher in S. lycocarpum than in tomato and coffee. Endo-beta-mannanase activity was detected in the endosperm cap prior to radicle protrusion. Abscisic acid inhibited germination, increase of embryo weight during imbibition, the second stage of weakening of the endosperm cap and of endo-beta-mannanase activity in the endosperm cap. CONCLUSIONS: The germination mechanism of S. lycocarpum bears resemblance to that of tomato and coffee seeds. However, quantitative differences were observed in embryo pressure potential, endo-beta-mannanase activity and endosperm cap resistance that were related to germination rates across the three species.     

Endo-[beta]-Mannanase Activity from Individual Tomato Endosperm Caps and Radicle Tips in Relation to Germination Rates

Endo-[beta]-mannanase is hypothesized to be a rate-limiting enzyme in endosperm weakening, which is a prerequisite for radicle emergence from tomato (Lycopersicon esculentum Mill.) seeds. Using a sensitive, single-seed assay, we have measured mannanase activity diffusing from excised tomato endosperm caps following treatments that alter the rate or percentage of radicle emergence. Most striking was the 100- to more than 10,000-fold range of mannanase activity detected among individual seeds of highly inbred tomato lines, which would not be detected in pooled samples. In some cases a threshold-type relationship between mannanase activity and radicle emergence was observed. However, when radicle emergence was delayed or prevented by osmoticum or abscisic acid, the initial increase in mannanase activity was unaffected or even enhanced. Partially dormant seed lots displayed a bimodal distribution of activity, with low activity apparently associated with dormant seeds in the population. Gibberellin- and abscisic acid-deficient mutant seeds exhibited a wide range of mannanase activity, consistent with their variation in hormonal sensitivity. Although the presence of mannanase activity in the endosperm cap is consistently associated with radicle emergence, it is not the sole or limiting factor under all conditions.     

The second step of the biphasic endosperm cap weakening that mediates tomato (Lycopersicon esculentum) seed germination is under control of ABA

The role of abscisic acid (ABA) in the weakening of the endosperm cap prior to radicle protrusion in tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds was studied. The endosperm cap weakened substantially in both water and ABA during the first 38 h of imbibition. After 38 h the force required for endosperm cap puncturing was arrested at 0.35 N in ABA, whereas in water a further decrease occurred until the radicle protruded. During the first 2 d of imbibition endo-beta-mannanase activity was correlated with the decrease in required puncture force and with the appearance of ice-crystal-induced porosity in the cell walls as observed by scanning electron microscopy. Prolonged incubation in ABA resulted in the loss of endo-beta-mannanase activity and the loss of ice-crystal-induced porosity, but not in a reversion of the required puncture force. ABA also had a distinct but minor effect on the growth potential of the embryo. However, endosperm cap resistance played the limiting role in the completion of germination. It was concluded that (a) endosperm cap weakening is a biphasic process and (b) inhibition of germination by ABA is through the second step in the endosperm cap weakening process.     

Class I chitinase and beta-1,3-glucanase are differentially regulated by wounding,methyl jasmonate,ethylene, and gibberellin in tomato seeds and leaves

Class I chitinase (Chi9) and beta-1,3-glucanase (GluB) genes are expressed in the micropylar endosperm cap of tomato (Lycopersicon esculentum) seeds just before radicle emergence through this tissue to complete germination. In gibberellin (GA)-deficient mutant (gib-1) seeds, expression of Chi9 and GluB mRNA and protein is dependent upon GA. However, as expression occurs relatively late in the germination process, we investigated whether the genes are induced indirectly in response to tissue wounding associated with endosperm cap weakening and radicle protrusion. Wounding and methyl jasmonate (MeJA) induced Chi9 expression, whereas ethylene, abscisic acid, sodium salicylate, fusicoccin, or beta-aminobutyric acid were without effect. Chi9 expression occurred only in the micropylar tissues when seeds were exposed to MeJA or were wounded at the chalazal end of the seed. Expression of Chi9, but not GluB, mRNA was reduced in germinating seeds of the jasmonate-deficient defenseless1 tomato mutant and could be restored by MeJA treatment. Chi9 expression during germination may be associated with "wounding" from cell wall hydrolysis and weakening in the endosperm cap leading to radicle protrusion, and jasmonate is involved in the signaling pathway for this response. Among these treatments and chemicals (other than GA), only MeJA and wounding induced a low level of GluB expression in gib-1 seeds. However, MeJA, wounding, and particularly ethylene induced both genes in leaves, whereas GA induced only Chi9 in leaves. Although normally expressed simultaneously during tomato seed germination, Chi9 and GluB genes are regulated distinctly and tissue specifically by hormones and wounding.     

A gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination.

Xyloglucan endotransglycosylases (XETs) modify xyloglucans, major components of primary cell walls in dicots. A cDNA encoding an XET (LeXET4) was isolated from a germinating tomato (Lycopersicon esculentum Mill.) seed cDNA library. DNA gel blot analysis showed that LeXET4 is a single-copy gene in the tomato genome. LeXET4 mRNA was strongly expressed in germinating seeds, was much less abundant in stems, and was not detected in roots, leaves or flower tissues. During germination, LeXET4 mRNA was detected in seeds within 12 h of imbibition with maximum mRNA abundance at 24 h. Tissue prints showed that LeXET4 mRNA was localized exclusively to the endosperm cap region. Expression of LeXET4 was dependent on exogenous gibberellin (GA) in GA-deficient (gib-1 mutant) tomato seeds, while abscisic acid, a seed germination inhibitor, had no effect on LeXET4 mRNA expression in wild-type seeds. LeXET4 mRNA disappeared after radicle emergence, even though degradation of the lateral endosperm cell walls continued. The temporal, spatial and hormonal regulation pattern of LeXET4 gene expression suggests that XET has a role in endosperm cap weakening, a key process regulating tomato seed germination.     

A Single-Seed Assay for Endo-[beta]-Mannanase Activity from Tomato Endosperm and Radicle Tissues

Completion of germination (radicle emergence) is an all-or-none developmental event for an individual seed. Variation in germination timing among seeds in a population therefore reflects variation among seeds in the rates or extents of physiological or biochemical processes prior to radicle emergence. For tomato (Lycopersicon esculentum Mill.) seeds, correlative evidence suggests that endo-[beta]-mannanase activity weakens the endosperm cap tissue opposite the radicle tip to permit radicle emergence. To test whether endo-[beta]-mannanase activity is causally related to germination rates, we have developed a sensitive assay suitable for use with individual radicle tips or endosperm caps. We show that endo-[beta]-mannanase activity varies at least 100-fold and often more than 1000-fold among individual inbred tomato seeds prior to radicle emergence. Other sources of variation (tissue size and experimental error) were evaluated and cannot account for this range of activity. Endo-[beta]-mannanase activity was generally 10-fold greater in leachates from endosperm caps than from radicle tips. Release of reducing sugars from individual endosperm caps also varied over a considerable (9-fold) range. These extreme biochemical differences among individual tomato seeds prior to radicle emergence indicate that results obtained from bulk samples could be misleading if it is assumed that all seeds exhibit the "average" behavior.     

Endo-[beta]-Mannanase Activity Present in Cell Wall Extracts of Lettuce Endosperm prior to Radicle Emergence

Lettuce (Lactuca sativa L.) endosperm cell walls isolated prior to radicle emergence underwent autohydrolysis, the rate of which was correlated with whether radicle emergence would subsequently occur. Extraction of endosperm cell walls with 6 M LiCl suppressed autohydrolysis, and the desalted extract possessed activity that was capable of hydrolyzing purified locust bean galactomannan but not arabinogalactan, carboxymethylcellulose, glucomannan, polygalacturonic acid, tomato galactomannan, or native lettuce endosperm cell walls. Some hydrolytic activity was detected on endosperm cell walls if they were modified by partial trifluoroacetic acid hydrolysis or pretreatment with guanidinium thiocyanate. In extended incubations the cell wall enzyme extract released only large molecular mass fragments from locust bean galactomannan, indicating primarily endo-activity. Galactomannan-hydrolyzing activity in the cell wall extract increased as a function of imbibition time and was greatest just prior to radicle emergence. Thermoinhibition (imbibition at 32[deg]C) or treatment with abscisic acid at a temperature optimal for germination (25[deg]C) suppressed both germination and endosperm cell wall mannanase activity, whereas alleviation of thermoinhibition with gibberellic acid was accompanied by significant enhancement of mannanase activity. We conclude that a cell wall-bound endo-[beta]-mannanase is expressed in lettuce endosperm prior to radicle emergence and is regulated by the same conditions that govern germination.     

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 GA(4+7). Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V(0) 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 V(1) 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.     

Expression of an expansin is associated with endosperm weakening during tomato seed germination

Expansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening. We have identified at least three expansin genes that are expressed in tomato (Lycopersicon esculentum Mill.) seeds during germination. Among these, LeEXP4 mRNA is specifically localized to the micropylar endosperm cap region, suggesting that the protein might contribute to tissue weakening that is required for radicle emergence. In gibberellin (GA)-deficient (gib-1) mutant seeds, which germinate only in the presence of exogenous GA, GA induces the expression of LeEXP4 within 12 hours of imbibition. When gib-1 seeds were imbibed in GA solution combined with 100 microM abscisic acid, the expression of LeEXP4 was not reduced, although radicle emergence was inhibited. In wild-type seeds, LeEXP4 mRNA accumulation was blocked by far-red light and decreased by low water potential but was not affected by abscisic acid. The presence of LeEXP4 mRNA during seed germination parallels endosperm cap weakening determined by puncture force analysis. We hypothesize that LeEXP4 is involved in the regulation of seed germination by contributing to cell wall disassembly associated with endosperm cap weakening.     

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.     

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.     

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 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.     

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.     

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.