Mechanism of seed priming in circumventing thermodormancy in lettuce

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

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

     

Dual action of respiratory inhibitors: inhibition of germination and prevention of dormancy induction in lettuce seeds

`Grand Rapids' lettuce Lactuca sativa L. seeds germinate readily at 15°C but poorly at 25°C in darkness. When held in dark at 25°C for an extended period, the ungerminated seeds become dormant as shown by their inability to germinate or transfer to 15°C in darkness. Induction of dormancy at 25°C was prevented by exposure to CN<sup>−</sup>, azide, salicylhydroxamic acid (SHAM), dinitrophenol, and pure N<sub>2</sub> as determined by subsequent germination at 15°C on removal of inhibitors. The effectiveness of inhibitors to break dormancy declined as dormancy intensified. At relatively low levels, CN<sup>−</sup>, SHAM, and azide promoted dark germination at 25°C while at high levels they were inhibitory. Uptake of O<sub>2</sub> by seeds held at 25°C for 4 days in 1.0 millimolar KCN was inhibited by 67% but was promoted 61% when KCN was removed. Correspondingly greater inhibition (79%) and promotion (148%) occurred when 1.0 millimolar SHAM was added to KCN solution. When applied alone, SHAM had little effect on O<sub>2</sub> uptake. These data indicate that Cyt pathway of respiration plays a dominant role in the control of both dormancy induction and germination of lettuce seeds, and `alternative pathway' is effectively engaged in presence of CN⁻. The channeling of respiratory energy use for processes governing germination or dormancy is subject to control by physical and chemical factors.

A scheme is proposed that illustrates compensatory use of energy for processes controlling dormancy induction and germination. A block of germination, e.g. by low water potential polyethylene glycol solution or a supraoptimal temperature spares energy to be utilized for dormancy induction while a block of dormancy induction by low levels of CN⁻ (similar to GA and light effects) drives germination. Blocking both processes by inhibitors (e.g. CN⁻, CN⁻ + SHAM) presumably leads to accumulation of `reducing power' with consequent improvement in O₂ uptake and oxidation rates of processes controlling germination or dormancy induction upon removal of the inhibitors.

     

Early Actions of Gibberellic Acid on the Embryo and on the Endosperm of Avena fatua Seeds

Gibberellic acid at 0.1 μm stimulates amylase synthesis in dormant Avena fatua seeds without inducing germination; at 0.5 mm it enhances biosynthesis of proteins and RNA in both the embryo and the endosperm and utilization of the endosperm sugars by the embryo. These events occur in early hours (0-14th hour) and prior to germination, which begins 24 hours after gibberellic acid application. These observations are in agreemeent with the concept that in cereal grains gibberellic acid has two morphological sites of actions: the embryo and the endosperm, and that germination (radicle protrusion) is not caused by gibberellic acid-induced amylase synthesis in the endosperm.     

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

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

Effect of Water Stress, Seed Coat Restraint, and Abscisic Acid upon Different Germination Capabilities of Two Tomato Lines at Low Temperature

Two tomato (Lycopersicon esculentum Mill.) lines with greatly different capabilities to germinate at 10°C were compared with respect to sensitivity to experimental treatments which affect the water status of the embryo. Germination rates and final percentages could be drastically changed (at 25°C) by (a) removing the mechanical constraint from the radicle tip, (b) imposing water stress by an osmoticum, (c) stress hardening of the seeds through osmotic pretreatment, and (d) inhibiting embryo expansion by abscisic acid (ABA). All treatments showed a similar difference in germination vigor between the two lines indicating that cold sensitivity is in fact a matter of water relations rather than of phase transitions in membrane lipids. Inhibition of germination by ABA was completely abolished by removing the mechanical constraint from the radicle tip. Osmotic stress of −3 bar which quantitatively replaced this constraint in inhibiting germination also restored the sensitivity to ABA. It is concluded that all these treatments act on the balance between the hydraulic extension force of the embryo radicle and the opposing force of the seed layers covering the radicle tip. The difference in cold sensitivity between the two seed lines resides either in the osmotic potential or the pressure potential of the germinating embryo.     

Thermal thresholds as predictors of seed dormancy release and germination timing: altitude-related risks from climate warming for the wild grapevine Vitis vinifera subsp. sylvestris

Background and Aims

The importance of thermal thresholds for predicting seed dormancy release and germination timing under the present climate conditions and simulated climate change scenarios was investigated. In particular, Vitis vinifera subsp. sylvestris was investigated in four Sardinian populations over the full altitudinal range of the species (from approx. 100 to 800 m a.s.l).

Methods

Dried and fresh seeds from each population were incubated in the light at a range of temperatures (10–25 and 25/10 °C), without any pre-treatment and after a warm (3 months at 25 °C) or a cold (3 months at 5 °C) stratification. A thermal time approach was then applied to the germination results for dried seeds and the seed responses were modelled according to the present climate conditions and two simulated scenarios of the Intergovernmental Panel on Climate Change (IPCC): B1 (+1·8 °C) and A2 (+3·4 °C).

Key Results

Cold stratification released physiological dormancy, while very few seeds germinated without treatments or after warm stratification. Fresh, cold-stratified seeds germinated significantly better (>80 %) at temperatures ≥20 °C than at lower temperatures. A base temperature for germination (T_b) of 9·0–11·3 °C and a thermal time requirement for 50 % of germination (θ₅₀) ranging from 33·6 °Cd to 68·6 °Cd were identified for non-dormant cold-stratified seeds, depending on the populations. This complex combination of thermal requirements for dormancy release and germination allowed prediction of field emergence from March to May under the present climatic conditions for the investigated populations.

Conclusions

The thermal thresholds for seed germination identified in this study (T_b and θ₅₀) explained the differences in seed germination detected among populations. Under the two simulated IPCC scenarios, an altitude-related risk from climate warming is identified, with lowland populations being more threatened due to a compromised seed dormancy release and a narrowed seed germination window.     

Weakening of muskmelon perisperm envelope tissue 4

Muskmelon (Cucumis melo L.) embryos are enclosed in an envelopeof tissue consisting of a layer of endosperm and a multi-cell-layeredperisperm that the radicle must penetrate for germination tooccur. The force and energy required to penetrate the perispermenvelope tissue were measured using an Instron universal testingmachine at a crosshead speed of 5 mm min^–1 after 0, 10,15, 22, 23, and 25 h of imbibition at 25C. The cellular structureof perisperm envelope tissue surrounding the radicle was observedafter 10, 15, 20, 25, and 48 h of imbibition using scanningelectron microscopy. The force required to puncture 5-mm-long,micropylar seed pieces declined steadily from 1.65 N in driedseeds to 0.65 N after 21 h of imbibition. The penetration energydeclined from 3.0 N mm in dry seeds to 1.1 N mm at 21 h afterthe start of imbibition when the first seeds germinated. Theforce and energy required to penetrate germinated seed pieceswere 0.55 N and 0.9 N mm, respectively, so the net punctureforce and energy needed to rupture the micropylar region ofthe perisperm envelope was roughly 0.10 N and 0.2 N mm at radicleemergence, respectively. Instron measurements of penetrationforce and energy decreased dramatically at crosshead speedsless than the 5 mm min^–1. Crosshead speeds greater than5 mm min^–1 may overestimate the pressure needed to ruptureperisperm and endosperm tissues. Intracellular cracks were firstobserved in SEM images 15 h after the start of imbibition, andafter 20 h cracking was apparent throughout the micropylar regionof the perisperm envelope. The perisperm envelope ruptured inone of two ways, coincident with radicle emergence. In approximately85% of muskmelon seeds, a large crack formed in the perispermenvelope adjacent to the radicle, while in roughly 15 % a circulararea of the perisperm envelope detached during radicle emergence.In dead seeds, the penetration force remained constant from10–24 h after the start of imbibition, and there wereno visible signs of tissue degradation. Cellular degradationand weakening of the perisperm envelope tissue precedes radicleemergence in muskmelon seeds. Key words: Seed, Instron, turgor, cell wall, electron microscopy, Cucumis melo     

Effect of Temperature, Oxygen, and Gibberellic Acid on the Development of Photosensitivity in Oldenlandia corymbosa L. Seeds during Their Incubation in Darkness

Two successive phases can be distinguished in the development of the responsiveness to light in Oldenlandia corymbosa L. seeds during their incubation in darkness. During phase I, the responsiveness to light increases with time if there is sufficient O₂, and the higher the temperature, the faster the increase. This phase is stimulated by gibberellic acid. During the following phase (II), seeds remain responsive to light at 10 or 20°C, but lose their responsiveness at higher temperature (≥30°C). This second phase depends on O₂: loss of responsiveness is accelerated at lower O₂ concentration. Phase II is only slightly affected by gibberellic acid. The results are discussed in terms of variation of phytochrome and of a reaction along the transduction chain initiated by phototransformation of this pigment, which is finally expressed in germination.     

Seed dormancy,germination and storage behavior of Magnolia sinica,a plant species with extremely small populations of Magnoliaceae

Magnolia sinica is one of the most endangered Magnoliaceae species in China. Seed biology information concerning its long-term ex situ conservation and utilization is insufficient. This study investigated dormancy status, germination requirements and storage behavior of M. sinica. Freshly matured seeds germinated to ca. 86.5% at 25/15 °C but poorly at 30 °C; GA₃ and moist chilling promoted germination significantly at 20 °C. Embryos grew at temperatures (alternating or constant) between 20 °C and 25 °C, but not at 5 °C or 30 °C. Our results indicate that M. sinica seeds possibly have non-deep simple morphophysiological dormancy (MPD). Seeds survived desiccation to 9.27% and 4.85% moisture content (MC) as well as a further 6-month storage at −20 °C and in liquid nitrogen, including recovery in vitro as excised embryos. The established protocol ensured that at least 58% of seedlings were obtained after both cold storage and cryopreservation. These results indicate that both conventional seed banking and cryopreservation have potential as long-term ex situ conservation methods, although further optimized approaches are recommended for this critically endangered magnolia species.     

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (^·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ^·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By ³H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ^·OH in radicles and endosperm caps: the production and action of ^·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ^·OH attack on polysaccharides were evident. In vivo ^·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ^·OH is a controlled action of this type of reactive oxygen species.     

Control processes in the induction and relief of thermoinhibition of lettuce seed germination : actions of phytochrome and endogenous ethylene

Germination of lettuce seeds (Lactuca sativa L. cv Grand Rapids) in the dark was nearly 100% at 20°C but was inhibited at 27°C and higher temperatures (thermoinhibition). A single 5-minute exposure to red light completely overcame the inhibition at temperatures up to 28°C, above which the effectiveness of single light exposures gradually declined to reach a negligible level at 32°C. However, the promotive effect of light could be extended to 34°C by repeated irradiations. At any one temperature, increased frequency of irradiations increased germination percentage, and with each degree increase in temperature, increasingly frequent irradiations were necessary to elicit maximal germination. Loss of the effectiveness of single irradiations with increase in temperature may result either from acceleration of the thermal reversion of the far red-absorbing form of phytochrome or decrease in seed sensitivity toward a given percentage of the far red-absorbing form of phytochrome. Using continuous red light to induce germination, the role of endogenous C₂H₄ in germination at 32°C was studied. Ethylene evolution from irradiated seeds began to increase 2 hours prior to radicle protrusion, whereas the dark-incubated (nongerminating) seeds produced a low, constant amount of C₂H₄ throughout the 24 hour incubation period. Inhibition of C₂H₄ synthesis with 2-aminoethoxyvinyl glycine and/or inhibition of C₂H₄ action with 2,5-norbornadiene blocked the promotive effect of light. Exogenous C₂H₄ overcame these blockages. The results showed that participation by endogenous C₂H₄ was essential for the light-induced relief of thermoinhibition of lettuce seed germination. However, light did not act exclusively via C₂H₄ since exogenous C₂H₄ alone in darkness did not promote germination.     

Seed Germination in Chenopodium album L: Relationships between Nitrate and the Effects of Plant Hormones

Effects of ethylene, gibberellins, and kinetin on the germination of two lots of Chenopodium album L. seeds, collected from the field in 1982 and 1983, were studied in relation to the availability of nitrate. The experiments were conducted in darkness and at temperatures ranging from 12 to 32°C. Ethylene induced over 75% germination in the 1983 seed but had little effect on the 1982 seed. Nitrate was only slightly promotive in either of the two seed lots. A combination of ethylene and nitrate, however, acted synergistically on 1982 seed, resulting in as much germination as that induced in 1983 seed by ethylene alone. In 1983 seed, a combination of ethylene and nitrate was only marginally more effective than ethylene. A similar relationship was observed in the effects of gibberellic acid₄₊₇ (GA₄₊₇) and nitrate on seeds from the two lots. The 1982 seed, which responded synergistically to combinations of nitrate with ethylene or GA₄₊₇ was found to contain an extremely low endogenous level of nitrate as compared to 1983 seed. Thus, high levels of either endogenous or applied nitrate appeared to enhance the germination response to ethylene or GA₄₊₇.

Kinetin had no effect on 1982 seed and only a small promotive effect on 1983 seed. There was no synergism between kinetin and nitrate in either of the seed lots.

     

Action of Phytochrome During Prechilling of Amaranthus retroflexus L. Seeds

Dark germination of Amaranthus retroflexus L. seeds at 35° increased after several days of prechilling at 20° or lower. Irradiation with far-red light for short periods during the early hours of a prechilling period at 10° inhibited subsequent dark germination at 35°. The inhibition was completely reversible with red light. Far-red irradiation in the latter part of the prechilling period was less effective. Increased dark germination of A. retroflexus seeds following a prechilling period at 20° or less is attributed to action of preexistent P_FR, the far-red absorbing form of phytochrome, within the seeds. Inactivation of P_FR was found to proceed ca. 4 times more rapidly at 25° than at 20°. Failure of imbibition temperatures above 20° to increase dark germination of A. retroflexus seeds is attributed to the rapid thermal reversion of pre-existent P_FR. We suggest that the action of prechilling (layering) on many other seed kinds arises in a similar way.     

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds

Background and Aims

Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia.

Methods

Seeds were incubated in the light at a range of temperatures (10–25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (T_b), and thermal times for 50 % germination (θ₅₀) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated T_b and soil temperatures.

Key Results

Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. T_b was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ₅₀ was 2·6 log °Cd for untreated seeds and 2·17–2·19 log °Cd for stratified seeds. When θ₅₀ values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations.

Conclusions

T_b and θ₅₀ values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.     

Changes in the Strength of Lettuce Endosperm during Germination

The forces required to puncture intact lettuce (Lactuca sativa) seed and pericarp, endosperm and embryo were measured by the Instron Universal Testing Machine. It required about 0.6 newton to puncture the endosperm in seeds imbibed in the dark at 6, 12 and 24 hours. Endosperm of seeds imbibed in the light or in dark with gibberellic acid required about 4.2 newtons at 6 and at 12 hours and only about 0.15 newton at 24 hours. Forces required to puncture embryo at all treatments and times remained constant at about 0.3 newton. Changes in the strength of the endosperm do not appear to be related directly to protrusion of the radicle.     

Secretion of alpha-amylase by the aleurone layer and the scutellum of germinating barley grain

α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl₂, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl₂. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

     

Variation among Species in the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

The relationship between the thermal dependence of the reappearance of chlorophyll variable fluorescence following illumination and temperature dependence of the apparent Michaelis constant (K_m) of NADH hydroxypyruvate reductase for NADH was investigated in cool and warm season plant species. Brancker SF-20 and SF-30 fluorometers were used to evaluate induced fluorescence transients from detached leaves of wheat (Triticum aestivum L. cv TAM-101), cotton (Gossypium hirsutum L. cv Paymaster 145), tomato (Lycopersicon esculentum cv Del Oro), bell pepper (Capsicum annuum L. cv California Wonder), and petunia (Petunia hybrida cv. Red Sail). Following an illumination period at 25°C, the reappearance of variable fluorescence during a dark incubation was determined at 5°C intervals from 15°C to 45°C. Variable fluorescence recovery was normally distributed with the maximum recovery observed at 20°C in wheat, 30°C in cotton, 20°C to 25°C in tomato, 30 to 35°C in bell pepper and 25°C in petunia. Comparison of the thermal response of fluorescence recovery with the temperature sensitivity of the apparent K_m of hydroxypyruvate reductase for NADH showed that the range of temperatures providing fluorescence recovery corresponded with those temperatures providing the minimum apparent K_m values (viz. the thermal kinetic window).     

Oxygen-dependent aging of seeds

When seeds of soybeans (Glycine max Amsoy var.) or safflower were stored under high O₂ concentrations, their per cent germination declined rapidly. For example, soybean seeds stored under 7.7 atmospheres O₂ pressure at 25°C and 17% moisture lost all viability within 22 days, whereas under 7.7 atmospheres N₂, the per cent germination remained greater than 80%. Germination decreased continually in O₂ pressures ranging from 0 to 7.7 atmospheres. High levels of O₂, moisture, or temperature each acted independently to cause losses of germination, but when applied simultaneously, these factors acted synergistically. Soybean seeds were also aged under conditions of high temperature (44°C) and humidity (100% RH), which have been routinely used to accelerate aging. Under these conditions, no O₂ dependence of seed death was observable.

Increased lipid oxidation was not detected in seeds that had lost germination ability due to high O₂ treatment. Seeds of two safflower varieties that contained either high oleic or high linoleic fatty acid compositions were subjected to high O₂ treatment. Although the lipid of the high oleic variety is markedly more stable to oxidative degradation, we detected no significant difference in the O₂ tolerance of these seeds.

     

Analysis of the Effect of Light and Temperature on the Fluence Response Curves for Germination of Rumex obtusifolius

Both red light (10 minutes) and 35°C treatment (60 minutes) stimulate the germination of seeds of Rumex obtusifolius otherwise maintained in darkness at 25°C. Fluence response curves were determined for the effect of red light to stimulate germination of seeds with and without 35°C treatment. The endogenous far-red absorbing form (Pfr) level in the seeds was determined using short saturating fluences of wavelengths of light which maintain different proportions of phytochrome as Pfr at equilibrium. In the seed batches investigated, the endogenous Pfr level was found to be 4% or less of the total phytochrome. High dark germination after 35°C treatment does not result from an increase in sensitivity of the whole population to Pfr. Calculated fluence response curves for germination which best fit the experimental data suggest that seeds germinate in darkness after 35°C treatment because of a nonphytochrome-related process (overriding factor).     

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.