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     

Pepper seed germination assessed by combined X-radiography and computer-aided imaging analysis

A lot of pepper seeds having 87 % germination were subjected to X-ray inspection using a non lethal dose of radiation. Seeds with less than 2.7 % (on the basis of total seed area) of free space area, i.e. the spaces between embryo and endosperm, were classified as highly viable seeds (97–100 % germination) with the lowest level of abnormal seedlings. Seeds X-ray classified as good were subjected to a computerised image analysis to study seed imbibition and radicle elongation. The patterns of seed area increase, chosen as the most accurate indicator of seed swelling, resembled the triphasic curve of water uptake. The first phase was completed at 9 h followed by a second phase that varied widely in time until completion of germination between 52 and 96 h. The proportion of seeds with radicle protrusion between 52–56 h and 64–72 h assessed with the image analysis was significantly higher than that recorded using a conventional germination test. In addition, the rate of increase of seed area during the third phase of imbibition, mostly due to protrusion of the radicle tip and its growth, was highly correlated with the corresponding radicle elongation rate.     

Key Role for Endogenous Gibberellins in the Control of Seed Germination

The stimulative action of applied gibberellins (GA) on seedgermination has been reported for a large number of speciesin the past three decades. Applied GA often replaces the needfor environmental stimuli like specific temperature pretreatmentor light. Therefore, it has been suggested that endogenous GAsare essential intermediates in the stimulation of germination.Endogenous GAs have been identified in seeds of a limited numberof species, but most of the evidence for a regulatory role isentirely circumstantial. The use of isogenic mutants with lesionsin the GA biosynthesis has presented direct evidence of a keyrole for endogenous GAs in the regulation of germination. GAdeficiency absolutely prevents germination in seeds of Arabidopsisthaliana and tomato. Two different mechanisms of action exist The first one involves hydrolysis of reserve food. In seedsof tomato a factor, probably GA, diffuses prior to germinationfrom embryo to endosperm where it induces hydrolysis of gaJactomannan-richcell walls that are part of the mechanical resistance to theprotrusion of the radicle. A second mechanism of GA action consistsof a direct effect on the growth potential of the embryo. InArabidopsis the stimulation of germination by light dependson the ability of the seeds to synthesize GA, but light alsoenhances the sensitivity of the seeds to GA. Dry storage andpre-incubation at 2°C also increased the responsivenessof Arabidopsis seeds to GA     

Ascorbate and glutathione metabolism in embryo axes and cotyledons of germinating lupine seeds

Changes in ascorbate and glutathione contents and the activities and isoenzyme patterns of enzymes of the ascorbate-glutathione cycle were investigated in embryo axes and cotyledons of germinating lupine (Lupinus luteus L.) seeds. Ascorbate content was not significantly affected over the initial 12 h of imbibition in embryo axes, but afterwards increased, with the most rapid accumulation coinciding with radicle emergence. A somewhat similar trend was observed for glutathione with significant increase in embryo axes shortly before radicle protrusion followed by decline in the next hours. In cotyledons the ascorbate pool rose gradually during germination but the amount of glutathione showed fluctuations during a whole germination period. The activity of ascorbate peroxidase (APX) rose progressively in embryo axes, while activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) showed transient increase during germination. New isoforms of APX and GR were synthesized, suggesting that they play a relevant role during germination. All analyzed enzymes were already present in dry seeds which allowed them to be active immediately after imbibition.     

Activities of Antioxidant Systems During Germination of Chenopodium rubrum Seeds

The activities of superoxide-dismutase (SOD), catalase (CAT) and peroxidase (POD), and concentrations of glutathione and ascorbate have been studied during the first stages of germination in Chenopodium rubrum L. seeds. The highest CAT and SOD activity was found prior to radicle protrusion, while POD activity was maximal at the time of radicle protrusion and seedling development, new POD isozymes simultaneously appearing. The concentrations of total, reduced and oxidized glutathione showed similar changes during germination, the highest values being detected at the time of radicle protrusion. Ascorbic acid was present in the seeds in a detectable concentration only at the time preceding radicle protrusion, while its oxidized form dehydroascorbic acid was detected during the whole germination period studied. Gibberellic acid (GA₃, 160 M) had no effect on germination percentage, but in presence of GA₃, SOD and CAT activity notably increased prior to radicle protrusion, and oxidized glutathione concentration decreased in further germination.     

Phytochrome Control of Cellulase Activity in Datura ferox L. Seeds and its Relationship with Germination

The influence of phytochrome on endosperm softening and cellulaseactivity was studied on light-stimulated Datura ferox seeds.Endosperm softening preceded the earliest signs of radicle protrusion,and there was good correlation between the % of seeds with softendosperm at 48 h after R and germination at 96 h after R. Cellulaseactivity was stimulated by R and the increase in activity preceded,by more than 24h, radicle protrusion and endosperm softening.The effect of R was reversed by FR, but, by delaying the irradiationwith FR until cellulase activity had increased significantly,it was observed that removing Pfr did more than just stop anyfurther increase, the level of cellulase activity decreasedin about 24 h close to the dark controls. Cellulase activitywas decreased by a FR irradiation even when more than 60% germinationhad escaped from reversion. These results indicate that phytochromeinfluence on cellulase is not an indirect consequence of thestimulus of germination and that the continuous presence ofPfr is required for the cellulase activity to remain high. Thepossibility that cellulase and other degrading enzymes may bepart of the mechanism of light-induced germination is discussed. Key words: Phytochrome, germination, cellulase     

Proteomics of Arabidopsis seed germination. A comparative study of wild-type and gibberellin-deficient seeds

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component alpha-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for the ga1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a beta-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.     

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.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. II. Transport and Metabolism of 8[14C]t-Zeatin Applied to the Radicle

The application of 8[¹⁴C]t-zeatin to the radicle tips of germinatingPhaseolus vulgaris seeds revealed that cytokinins are transportedrapidly from the embryonic axis to the cotyledons, and are utilizedand metabolized extensively in these organs. The informationobtained on the transportation between the different parts ofthe embryo is consistent with the view that the mobilizationof food reserves from the cotyledons is controlled by cytokininswhich originate in the embryonic axis. Tentative identificationof the radioactive metabolites formed indicate that the appliedzeatin was altered by side-chain cleavage and by substitutionto the adenine ring. Phaseolus vulgaris, bean, germination, cytokinins, transport, radicle     

Dormancy and Impotency of Cocklebur Seeds. IX. Changes in ACC-Ethylene Conversion Activity and ACC Content of Dormant and Nondormant Seeds during Soaking

Differences in ethylene production between dormant (D) and nondormant(ND) lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.)were studied with respect to changes in the activity of conversionof 1-aminocyclopropane-l-carboxylic acid (ACC) to ethylene andin the contents of ACC and malonyl-ACC in their axial-tissuesduring soaking. Superior ethylene production in ND seeds ascompared to D seeds became evident during a soaking period rangingfrom 12–24 h, when the radicle protrusion in ND seedshad not yet occurred. Ethylene production in ND seeds increasedabruptly after the radicle protrusion. The inhibitors of ethyleneproduction, aminoethoxyvinyglycine, cobaltous ion and -aminoisobutyricacid, inhibited the germination of ND seeds, whereas ACC enabledD seeds to germinate. Activity of ACC-ethylene conversion was absent in dry axialtissues and developed with soaking. After 24 h, this activityin ND axes was superior to that in D axes. Under hypoxia, however,the difference in the ACC-conversion activity appeared before24 h. On the other hand, the contents of ACC in both D and NDaxes remained almost unchanged until 24 h of soaking. It isthus suggested that the inferior ethylene production in D seedsis associated mainly with their low activity of ACC-ethyleneconversion, though partly with their low activity of ACC supply. Activity of ACC-ethylene conversion in the axes of ND seedsincreased sharply after radicle protrusion which occurred after24 h of soaking. Correspondingly, the contents of both ACC andmalonyl-ACC increased in the axes of germinated ND seeds. Theseimply that the high ethylene production in the ND seeds in thepost-germination period comes from the increasing activitiesof ACC supply as well as ACC-ethylene conversion in their axes. Key words: Cocklebur seeds, Dormancy, Ethylene production, 1-aminocyclopropane-1-carboxylic acid, Germination, Xanthium     

Amounts of Nuclear DNA and Internal Morphology of Gibberellin- and Abscisic Acid-deficient Tomato (Lycopersicon esculentum Mill.) Seeds During Maturation, Imbibition and Germination

Using X-ray photography and flow cytometry, the internal morphologyand DNA replication activity of wild type (wt), GA- (gib-1 )and ABA-deficient (sit^w ) tomato (Lycopersicon esculentum Mill.cv. Moneymaker) mutant seeds were studied. During seed formation,from 30 to 45 d after pollination (DAP) the endosperm becomessolid and the seed starts to gain desiccation tolerance. Atthis time significant changes occur in the amounts of DNA inradicle tip cells. At 30 DAP, radicle tip cells of the threegenotypes manifest about 60% of 2C, 30% of 4C and 10% of 8Camounts of DNA. Upon maturation (45 DAP onwards), most cellsin the seeds of the three genotypes arrest in the G₁phase ofthe cell-cycle with 2C amounts of DNA. However, a relativelyhigh proportion of cells with 4C amounts of DNA was detectedin the radicle tip cells ofsit^w compared with wild type andgib-1. At the well-matured stage (60 DAP), there were about 2% ofseeds with free space in wild type andgib-1 , and about 13%insit^w . At the over-matured stage (75 DAP), even more seedswith free space were found insit^w , whereas no increase in theproportion of the seeds with free space was detected in theother two genotypes. In -1.0 MPa PEG-6000 with or without 10µM GA₄₊₇, no germination occurred in well-matured wildtype andgib-1 seeds, whether or not they were dried after harvest.However,sit^w seeds were able to germinate both in over-maturefruit and in -1.0 MPa PEG-6000. Priming of dried seeds in -1.0MPa PEG induced a large amount of free space in almost all seedsof the three genotypes, and nuclear DNA synthesis in the radicletip cells of wild type andsit^w seeds. However, PEG priming offresh (non-dried) seeds had no effect on the amount of freespace and 2C/4C DNA ratios in wild type orgib-1 seeds, but didinduce free space in about 20–25% ofsit^w seeds and provoked4C signals insit^w seeds. Removal of the endosperm and testaopposite the radicle tip of seeds resulted in root protrusion,the induction of free space and an increase of 4C DNA signalsin the three genotypes. It is concluded that ABA is crucialfor the efficient arrest of tomato embryo radicle tip cellsin G₁phase upon maturation, whereas GAs play an important rolein re-initiating 4C DNA levels upon germination. Dormancy; flow cytometry; free space; Lycopersicon esculentum ; maturation; priming; seed; tomato     

Mechanism and control of Genipa americana seed germination

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

Dissecting seed dormancy and germination in Aquilegia barbaricina,through thermal kinetics of embryo growth

• Threshold‐based thermal time models provide insight into the physiological switch from the dormant to the non‐dormant germinating seed.
• This approach was used to quantify the different growth responses of the embryo of seeds purported to have morphophysiological dormancy (MPD) through the complex phases of dormancy release and germination. Aquilegia barbaricina seeds were incubated at constant temperatures (10–25 °C) and 25/10 °C, without pre‐treatment, after warm+cold stratification (W+C) and GA₃ treatment. Embryo growth was assessed and the time of testa and endosperm rupture scored. Base temperatures (T_b) and thermal times for 50% (θ₅₀) of embryo growth and seed germination were calculated.
• W+C enabled slow embryo growth. W+C and GA₃ promoted rapid embryo growth and subsequent radicle emergence. The embryo internal growth base temperature (T_be) was ca. 5 °C for W+C and GA₃‐treated seeds. GA₃ treatment also resulted in similar T_b estimates for radicle emergence. The thermal times for embryo growth (θ_e50) and germination (θ_g50) were four‐ to six‐fold longer in the presence of GA₃ compared to W+C.
• A. barbaricina is characterised by a multi‐step seed germination. The slow embryo growth during W+C reflects continuation of the maternal programme of development, whilst the thermal kinetics of both embryo and radicle growth after the removal of physiological dormancy are distinctly different. The effects of W+C on the multiphasic germination response in MPD seeds are only partially mimicked by 250 mg·l^?1 GA₃. The thermal time approach could be a valid tool to model thermal kinetics of embryo growth and radicle protrusion.

     

Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth

Proteomics of Arabidopsis seeds revealed the differential accumulation during germination of two housekeeping enzymes. The first corresponded to methionine synthase that catalyses the last step in the plant methionine biosynthetic pathway. This protein was present at low level in dry mature seeds, and its level was increased strongly at 1-day imbibition, prior to radicle emergence. Its level was not increased further at 2-day imbibition, coincident with radicle emergence. However, its level in 1-day imbibed seeds strongly decreased upon subsequent drying of the imbibed seeds back to the original water content of the dry mature seeds. The second enzyme corresponded to S -adenosylmethionine synthetase that catalyses the synthesis of S -adenosylmethionine from methionine and ATP. In this case, this enzyme was detected in the form of two isozymes with different p I and M r. Both proteins were absent in dry mature seeds and in 1-day imbibed seeds, but specifically accumulated at the moment of radicle protrusion. Arabidopsis seed germination was strongly delayed in the presence of dl -propargylglycine, a specific inhibitor of methionine synthesis. Furthermore, this compound totally inhibited seedling growth. These phenotypic effects were largely alleviated upon methionine supplementation in the germination medium. The results indicated that methionine synthase and S -adenosylmethionine synthetase are fundamental components controlling metabolism in the transition from a quiescent to a highly active state during seed germination. Moreover, the observed temporal patterns of accumulation of these proteins are consistent with an essential role of endogenous ethylene in Arabidopsis only after radicle protrusion.     

Tegumento seminale e germinazione in Cercis siliquastrum L.

Abstract

Seminal integument and germination of Cercis siliquastrum L. – A histological study of the seminal integument of Cercis siliquastrum L. has been carried out, in order to explain whether the obstacle to the germination, removed by a cold-treatment, was caused by a mechanical action of the integument itself. We have noted that both the cold-treated seeds which germinate and the non-treated control seeds which don't germinate show in the long run the same modifications. We have therefore come to the conclusion that the inability to germinate found in the non-treated seeds is not due to the mechanical resistance of the integument and we think it necessary to return to further researches, also at a biochemical level.     

Seed Dormancy in Acer pseudoplatanus L.: the Role of the Covering Structures

The present studies with Acer pseudoplatanus L. suggest thatthe covering structures play an important and multiple rolein the dormancy of the fruit. Whole fruits and seeds with thetesta intact required a period of chilling at 5 °C beforedormancy was broken whereas bare embryos germinated immediatelyat 20 °C without pretreatment. This suggested that dormancywas coat-imposed and that the testa was responsible for thiseffect. Germination of dormant seeds was inhibited by lightwhereas the non-dormant bare embryos showed little response.Studies on the manner in which the testa imposed dormancy onthe embryo indicated that restriction on oxygen uptake, wateruptake, mechanical restriction to embryo enlargement, and thepresence of germination inhibitors in the testa were not limitingfactors at this stage of dormancy. Results from leaching experimentssuggest that dormancy was the result of the restriction by thetesta of the outward diffusion of a germination inhibitor(s)present in the embryo. In seeds that had nearly completed theirstratification requirements, the covering structures seemedto act in a manner other than by preventing the leaching ofan inhibitor from the embryo. At this point the physical propertiesof the covering structures seem to determine any further delaysin germination by the mechanical restriction of embryo enlargementby the testa and by restriction of oxygen uptake by the pericarp.     

Endo-β-mannanase isoforms are present in the endosperm and embryo of tomato seeds, but are not essentially linked to the completion of germination

A current hypothesis is that endo--mannanase activity in the endosperm cap of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds is induced by gibberellin (GA) and weakens the endosperm cap thus permitting radicle protrusion. We have tested this hypothesis. In isolated parts, the expression of endo--mannanase in the endosperm after germination is induced by GAs, but the expression of endo--mannanase in the endosperm cap prior to radicle protrusion is not induced by GAs. Also, abscisic acid (ABA) is incapable of inhibiting endo--mannanase activity in the endosperm cap, even though it strongly inhibits germination. However, ABA does inhibit enzyme activity in the endosperm and embryo after germination. There are several isoforms in the endosperm cap and embryo prior to radicle protrusion that are tissue-specific. Tissue prints showed that enzyme activity in the embryo spreads from the radicle tip to the cotyledons with time after the start of imbibition. The isoform and developmental patterns of enzyme activity on tissueprints are unaffected when seeds are incubated in ABA, even though germination is inhibited. We conclude that the presence of endo--mannanase activity in the endosperm cap is not in itself sufficient to permit tomato seeds to complete germination.Abbreviations ABA cis/trans-abscisic acid - GA(s) gibberellin(s) - IEF isoelectric focussing - pI(s) isoelectric point(s) We thank Dr. Bruce Downie for the seemingly endless but inspiring discussions.     

The Role of Germination Inhibitors and Oxygen in the Dormancy of the Light-densitive Seed of Betula spp.

Although unchilled, intact seeds of Betula pubescens and B.verrucosa require light for germination, isolated embryos germinateequally well in both light and darkness. An aqueous extract of these seeds has germination-inhibitoryproperties correlated with the presence of a non-fluorescent,single substance. The light requirement of isolated embryosis restored by the inhibitor. When intact seeds are leachedwith water to remove some inhibitor, it is found that the lightrequirement is reduced, short days and single light periodsthen eliciting greater germination than in unleached seeds. It has been found that scratching, pricking, and cutting theseed coat increases the germination of intact seeds in darkness,and that this is probably due to enhanced oxygen entry. Further,it has been found that germination in short days is increasedin oxygen-enriched atmospheres. It has been found that although the inhibitory effect of theseed coat in intact seeds is partially due to the reductionof the oxygen supply to the embryo, a low oxygen concentrationdoes not prevent germination of isolated embryos. Experimentalresults suggest that the inhibitor in the seed coat increasesthe oxygen requirement of the embryo.