Two tomato expansin genes show divergent expression and localization in embryos during seed development and germination.

Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth.

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.

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.

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.

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 a polygalacturonase associated with tomato seed germination.

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth.

A gel diffusion assay for quantification of pectin methylesterase activity.

Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 3.1.1.11) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log-linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.

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.

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 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.

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.

Development of Desiccation Tolerance during Embryogenesis in Rice (Oryza sativa) and Wild Rice (Zizania palustris) (Dehydrin Expression, Abscisic Acid Content, and Sucrose Accumulation).

The ability of seeds to withstand desiccation develops during embryogenesis and differs considerably among species. Paddy rice (Oryza sativa L.) grains readily survive dehydration to as low as 2% water content, whereas North American wild rice (Zizania palustris var interior [Fasset] Dore) grains are not tolerant of water contents below 6% and are sensitive to drying and imbibition conditions. During embryogenesis, dehydrin proteins, abscisic acid (ABA), and saccharides are synthesized, and all have been implicated in the development of desiccation tolerance. We examined the accumulation patterns of dehydrin protein, ABA, and soluble saccharides (sucrose and oligosaccharides) of rice embryos and wild rice axes in relation to the development of desiccation tolerance during embryogenesis. Dehydrin protein was detected immunologically with an antibody raised against a conserved dehydrin amino acid sequence. Both rice and wild rice embryos accumulated a 21-kD dehydrin protein during development, and an immunologically related 38-kD protein accumulated similarly in rice. Dehydrin protein synthesis was detected before desiccation tolerance had developed in both rice embryos and wild rice axes. However, the major accumulation of dehydrin occurred after most seeds of both species had become desiccation tolerant. ABA accumulated in wild rice axes to about twice the amount present in rice embryos. There were no obvious relationships between ABA and the temporal expression patterns of dehydrin protein in either rice or wild rice. Wild rice axes accumulated about twice as much sucrose as rice embryos. Oligosaccharides were present at only about one-tenth of the maximum sucrose concentrations in both rice and wild rice. We conclude that the desiccation sensitivity displayed by wild rice grains is not due to an inability to synthesize dehydrin proteins, ABA, or soluble carbohydrates.

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.

Germination and Dormancy of Abscisic Acid- and Gibberellin-Deficient Mutant Tomato (Lycopersicon esculentum) Seeds (Sensitivity of Germination to Abscisic Acid, Gibberellin, and Water Potential).

Germination responses of wild-type (MM), abscisic acid (ABA)-deficient (sitw), and gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds to ABA, GA4+7, reduced water potential ([psi]), and their combinations were analyzed using a population-based threshold model (B.R. Ni and K.J. Bradford [1992] Plant Physiol 98: 1057-1068). Among the three genotypes, sitw seeds germinated rapidly and completely in water, MM seeds germinated more slowly and were partially dormant, and gib-1 seeds did not germinate without exogenous GA4+7. Times to germination were inversely proportional to the differences between the external osmoticum, ABA, or GA4+7 concentrations and the corresponding threshold levels that would either prevent ([psi]b, log[ABAb]) or promote (log[GAb]) germination. The sensitivity of germination to ABA, GA4+7, and [psi] varied widely among individual seeds in the population, resulting in a distribution of germination times. The rapid germination rate of sitw seeds was attributable to their low mean [psi]b (-1.17 MPa). Postharvest dormancy in MM seeds was due to a high mean [psi]b (-0.35 MPa) and a distribution of [psi]b among seeds such that some seeds were unable to germinate even on water. GA4+7 (100 [mu]M) stimulated germination of MM and gib-1 seeds by lowering the mean [psi]b to -0.75 MPa, whereas ABA inhibited germination of MM and sitw seeds by increasing the mean [psi]b. The changes in [psi]b were not due to changes in embryo osmotic potential. Rather, hormonal effects on endosperm weakening opposite the radicle tip apparently determine the threshold [psi] for germination. The analysis demonstrates that ABA- and GA-dependent changes in seed dormancy and germination rates, whether due to endogenous or exogenous growth regulators, are based primarily upon corresponding shifts in the [psi] thresholds for radicle emergence. The [psi] thresholds, in turn, determine both the rate and final extent of germination within the seed population.

Expression of "Dehydrin-Like" Proteins in Embryos and Seedlings of Zizania palustris and Oryza sativa during Dehydration.

Proteins inducible by dehydration and abscisic acid (ABA), termed dehydrins or RAB (Responsive to ABA) proteins, have been identified in a number of species and have been suggested to play a role in desiccation tolerance, particularly during seed development. Seeds (caryopses) of North American wild rice (Zizania palustris var interior [Fassett] Dore) are tolerant of dehydration to <10% moisture content (fresh weight basis) only under restricted dehydration and rehydration conditions. In comparison, seeds of paddy rice (Oryza sativa L.) readily tolerate desiccation to <5% water content. Expression of "dehydrin-like" proteins in Zizania and Oryza seedlings and embryos was examined to investigate the relationship between the presence of such proteins and desiccation tolerance. [(35)S]Methionine labeling of newly synthesized proteins showed that seedlings (first leaf stage) of both Zizania and Oryza synthesized a novel "heat-stable" protein of apparent molecular weight = 20,000 when dehydrated to <75% of their initial fresh weight. ABA (100 micromolar) induced synthesis of a protein with similar electrophoretic mobility in both species. Western blots using antiserum raised against maize (Zea mays L.) dehydrin detected a protein band from dehydrated Zizania shoots and mature embryonic axes that comigrated with the labeled 20-kilodalton polypeptide. Northern blots using a cDNA for an ABA-responsive protein from Oryza (rab 16a) showed that both seedlings and excised embryonic axes of Zizania accumulated RNA similar in sequence to rab 16a in response to water loss. Zizania seedlings and embryonic axes were also capable of ABA accumulation during dehydration. The intolerance of Zizania seeds to dehydration at low temperature is apparently not due to an absence of dehydrin-like proteins or an inability to accumulate ABA.

Quantitative models characterizing seed germination responses to abscisic Acid and osmoticum.

Mathematical models were developed to characterize the physiological bases of the responses of tomato (Lycopersicon esculentum Mill. cv T5) seed germination to water potential (psi) and abscisic acid (ABA). Using probit analysis, three parameters were derived that can describe the germination time courses of a seed population at different psi or ABA levels. For the response of seed germination to reduced psi, these parameters are the mean base water potential ( psi(b), MPa), the standard deviation of the base water potential among seeds in the population (sigma(psib), MPa), and the "hydrotime constant" (theta(H), MPa.h). For the response to ABA, they are the log of the mean base ABA concentration ([unk]ABA(b), m), the standard deviation of the base ABA concentration among seeds in the population (sigma(ABA) (b), log[m]), and the "ABA-time constant" (theta(ABA), log[m].h). The values of psi(b) and [unk]ABA(b) provide quantitative estimates of the mean sensitivity of germination rate to psi or ABA, whereas sigma(psi) (b) and sigma(ABA) (b) account for the variation in sensitivity among seeds in the population. The time constants, theta(H) and theta(ABA), indicate the extent to which germination rate will be affected by a given change in psi or ABA. Using only these parameters, germination time courses can be predicted with reasonable accuracy at any medium psi according to the equation probit(g) = [psi - (theta(H)/t(g)) - psi(b)]/sigma(psib), or at any ABA concentration according to the equation probit(g) = [log[ABA] - (theta(ABA)/t(g)) - log[[unk]ABA(b)]]/sigma(ABA) (b), where t(g) is the time to radicle emergence of percentage g, and ABA is the ABA concentration (m) in the incubation solution. In the presence of both ABA and reduced psi, the same parameters can be used to predict seed germination time courses based upon strictly additive effects of psi and ABA in delaying the time of radicle emergence. Further analysis indicates that ABA and psi can act both independently and interactively to influence physiological processes preparatory for radicle growth, such as the accumulation of osmotic solutes in the embryo. The models provide quantitative values for the sensitivity of germination to ABA or psi, allow evaluation of independent and interactive effects of the two factors, and have implications for understanding how ABA and psi may regulate growth and development.

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 (Psi). An alternative approach is presented, based upon the timing of radicle emergence, to characterize the relationship of seed germination rates to Psi. Using only three parameters, a ;hydrotime constant' and the mean and standard deviation in minimum or base Psi among seeds in the population, germination time courses can be predicted at any Psi, 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 Psi or turgor.

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : III. Sensitivity of Germination to Water Potential and Abscisic Acid during Development.

Muskmelon (Cucumis melo L.) seeds are germinable 15 to 20 days before fruit maturity and are held at relatively high water content within the fruit, yet little precocious germination is observed. To investigate two possible factors preventing precocious germination, the inhibitory effects of abscisic acid and osmoticum on muskmelon seed germination were determined throughout development. Seeds were harvested at 5-day intervals from 30 to 65 days after anthesis (DAA) and incubated either fresh or after drying on factorial combinations of 0, 1, 3.3, 10, or 33 micromolar abscisic acid (ABA) and 0, -0.2, -0.4, -0.6, or -0.8 megapascals polyethylene glycol 8000 solutions at 30 degrees C. Radicle emergence was scored at 12-hour intervals for 10 days. In the absence of ABA, the water potential (Psi) required to inhibit fresh seed germination by 50% decreased from -0.3 to -0.8 megapascals between 30 and 60 DAA. The Psi inside developing fruits was from 0.4 to 1.4 megapascals lower than that required for germination at all stages of development, indicating that the fruit Psi is sufficiently low to prevent precocious germination. At 0 megapascal, the ABA concentration required to inhibit germination by 50% was approximately 10 micromolar up to 50 DAA and increased to >33 micromolar thereafter. Dehydration improved subsequent germination of immature seeds in ABA or low Psi. There was a linear additive interaction between ABA and Psi such that 10 micromolar ABA or -0.5 megapascal osmotic potential resulted in equivalent, and additive, reductions in germination rate and percentage of mature seeds. Abscisic acid had no effect on embryo solute potential or water content, but increased the apparent minimum turgor required for germination. ABA and osmoticum appear to influence germination rates and percentages by reducing the embryo growth potential (turgor in excess of a minimum threshold turgor) but via different mechanisms. Abscisic acid apparently increases the minimum turgor threshold, while low Psi reduces turgor by reducing seed water content.

Water Relations of Seed Development and Germination in Muskmelon (Cucumis melo L.) : IV. Characteristics of the Perisperm during Seed Development.

We previously reported that an apparent water potential disequilibrium is maintained late in muskmelon (Cucumis melo L.) seed development between the embryo and the surrounding fruit tissue (mesocarp). To further investigate the basis of this phenomenon, the permeability characteristics of the tissues surrounding muskmelon embryos (the mucilaginous endocarp, the testa, a 2- to 4-cell-layered perisperm and a single cell layer of endosperm) were examined from 20 to 65 days after anthesis (DAA). Water passes readily through the perisperm envelope (endosperm + perisperm), testa, and endocarp at all stages of development. Electrolyte leakage (conductivity of imbibition solutions) of individual intact seeds, decoated seeds (testa removed), and embryos (testa and perisperm envelope removed) was measured during imbibition of freshly harvested seeds. The testa accounted for up to 80% of the total electrolyte leakage. Leakage from decoated seeds fell by 8- to 10-fold between 25 and 45 DAA. Presence of the perisperm envelope prior to 40 DAA had little effect on leakage, while in more mature seeds, it reduced leakage by 2- to 3-fold. In mature seeds, freezing, soaking in methanol, autoclaving, accelerated aging, and other treatments which killed the embryos had little effect on leakage of intact or decoated seeds, but caused osmotic swelling of the perisperm envelope due to the leakage of solutes from the embryo into the space between the embryo and perisperm. The semipermeability of the perisperm envelope of mature seeds did not depend upon cellular viability or lipid membrane integrity. After maximum seed dry weight is attained (35-40 DAA), the perisperm envelope prevents the diffusion of solutes, but not of water, between the embryo and the surrounding testa, endocarp, and mesocarp tissue.