Prolonged Imbibition Research Articles

A method to determine antifungal activity in seed exudates by nephelometry

BackgroundOne of the levers towards alternative solutions to pesticides is to improve seed defenses against pathogens, but a better understanding is needed on the type and regulation of existing pathways during germination. Dormant seeds are able to defend themselves against microorganisms during cycles of rehydration and dehydration in the soil. During imbibition, seeds leak copious amounts of exudates. Here, we developed a nephelometry method to assay antimicrobial activity (AA) in tomato seed exudates as a proxy to assess level of defenses.ResultsA protocol is described to determine the level of AA against the nonhost filamentous fungus Alternaria brassicicola in the exudates of tomato seeds and seedlings. The fungal and exudate concentrations can be adjusted to modulate the assay sensitivity, thereby providing a large window of AA detection. We established that AA in dormant seeds depends on the genotype. It ranged from very strong AA to complete absence of AA, even after prolonged imbibition. AA depends also on the stages of germination and seedling emergence. Exudates from germinated seeds and seedlings showed very strong AA, while those from dormant seeds exhibited less activity for the same imbibition time. The exudate AA did not impact the growth of a pathogenic fungus host of tomato, Alternaria alternata, illustrating the adaptation of this fungus to its host.ConclusionsWe demonstrate that our nephelometry method is a simple yet powerful bioassay to quantify AA in seed exudates. Different developmental stages from dormant seed to seedlings show different levels of AA in the exudate that vary between genotypes, highlighting a genetic diversity x developmental stage interaction in defense. These findings will be important to identify molecules in the exudates conferring antifungal properties and obtain a better understanding of the regulatory and biosynthetic pathways through the lifecycle of seeds, from dormant seeds until seedling emergence.

ROS Accumulation as a Hallmark of Dehydration Stress in Primed and Overprimed Medicago truncatula Seeds

Seed priming protocols implement incomplete imbibition phases, as well as physical, chemical or biological treatments, to activate pre-germinative metabolism and stress response, thus improving germination performances, seedling establishment and stress tolerance according to agricultural productivity requirements. The dehydration phase following priming treatments represents a critical variable, since an excessively prolonged imbibition (overpriming) impairs desiccation tolerance, compromising seed viability and seedling establishment. Priming protocols generally optimize imbibition-dehydration timing empirically to avoid overpriming. Hence, a better understanding of the dynamics underlying the loss of desiccation tolerance represents a promising route to test and develop efficient and cost-effective priming techniques. In the present work, priming and overpriming conditions were defined to explore the role of desiccation tolerance in seed priming efficiency in the model legume Medicago truncatula. The positive effects of hydropriming and kinetin-mediated hormopriming on germination parameters were screened in combination with conditions of short/prolonged priming and mild/severe overpriming. Biometric analyses highlighted contrasting responses in terms of germination performances and seedling development, while ROS (reactive oxygen species) levels measured during dehydration positively correlate with the loss of desiccation tolerance in early seedlings, suggesting possible applications to monitor priming progression and predict overpriming occurrence.

The transcriptomes of dormant leafy spurge seeds under alternating temperature are differentially affected by a germination-enhancing pretreatment

Seed dormancy is an important stage in the life cycle of many non-domesticated plants, often characterized by the temporary failure to germinate under conditions that normally favor the process. Pre-treating dormant imbibed seeds at a constant temperate accelerated germination of leafy spurge seeds under alternating temperatures. However, dormant seeds will also germinate without a pre-treatment, albeit at a much slower rate, which gives rise to longer periods of imbibition before germination. Transcriptome analyses on seeds exposed to prolonged imbibition highlighted pathways associated with phenylpropanoid biosynthesis and interacting networks of genes involved in plant defense. In addition to the many pathways associated with phenylpropanoid biosynthesis enriched with down-regulated genes upon germination, there were also numerous pathways enriched with up-regulated genes associated with energy metabolism, such as glycolysis. Transcriptome data further suggest that metabolism and signaling by the plant hormones ethylene, gibberellin, and abscisic acid are involved in the developmental transition from dormancy to germination. More specifically, sub-network enrichment analysis identified ABI3 as a central hub of a sub-network at germination including several down-regulated genes such as DELLA (i.e., RGL2), which represses gibberellin signaling processes required for germination. The 595-fold increase in the expression of ACC oxidase (ACO4) at germination also suggests an important role for ethylene biosynthesis in germinating leafy surge seeds. Furthermore, the 10–578-fold difference in expression of many genes such as HY5 and Histone H3 between two populations at germination, which were treated with and without a constant temperature germination-enhancing pretreatment, revealed disparate impacts on various biosynthetic, growth, signaling, and response processes. Overall, our results indicate a constant temperature pretreatment (20°C for 21d) is not required for germination of leafy spurge seeds at an alternating temperature. However, the presence or absence of the pretreatment does affect the rate of germination and the germination transcriptional programs.

Effect of Temperatures on Dormancy and Germination in Three Species in the Lamiaceae Occurring in Northern Wetlands

In the temperate region temperature is the main factor influencing the germination period of plant species. The purpose of this study was to examine effects of constant and fluctuating temperatures on dormancy and germination under laboratory and field conditions in the three wetland species Lycopus europaeus, Mentha aquatica and Stachys palustris. The results should give indications if the temperature-dependent regulation of dormancy and germination is phylogenetically constrained. Tests for germination requirements showed a minimum temperature for germination of 9 °C in Mentha and 12 °C in Lycopus and Stachys, and a maximum temperature of 33 °C for Lycopus and 36 °C for Mentha and Stachys. Fluctuating temperatures promoted germination in all three species but the amplitude required for high germination (>50%) differed: it was 8 °C in Mentha, 10 °C in Stachys and 14 °C in Lycopus (mean temperature 22 °C). The effect of temperatures on the level of dormancy was examined in the laboratory by imbibing seeds at temperatures between 3 °C and 18 °C for periods between 2 and 28 weeks, as well as by a 30-month burial period, followed by germination tests at various temperatures, in light and darkness. In the laboratory only low temperatures (≤12 °C) relieved primary dormancy in seeds of Lycopus, while in Mentha and Stachys also higher temperatures lead to an increase of germination. Dormancy was only induced in Lycopus seeds after prolonged imbibition at 12 °C in the laboratory. Buried seeds of all species exhibited annual dormancy cycles with lower germination in summer and higher germination from autumn to spring. Exhumed seeds, however, showed considerable differences in periods of germination success. Dormancy was relieved when ambient temperatures were below 12 °C. Ambient temperatures that caused an induction of dormancy varied depending on species and test condition, but even low temperatures (8 °C) were effective. At high test temperatures (25 °C) in light, exhumed seeds of all three species showed high germination throughout the year. The three species showed various differences in the effects of temperatures on dormancy and germination. Similarities in dormancy and germination found among the species are in common with other spring-germinating species occurring in wetlands, so it seems that the temperature dependent regulation of dormancy and germination are related to habitat and not to phylogenetic relatedness.

Ecophysiology of seed germination in Mediterranean geophytes. 1. Muscari spp

Ecophysiological aspects of seed germination were investigated in four Mediterranean geophytes of the genus Muscari (Liliaceae): M. comosum (tassel hyacinth), M. neglectum (common grape hyacinth), M. commutatum and M. weissii. Experiments were performed at constant temperatures in the dark and under temperature and light conditions simulating those prevailing in nature during November–January, i.e. well into the rainy season of the Mediterranean climate. In all species, no primary dormancy was revealed, and germination occurred in a rather narrow range of cool temperatures (optimum at 10 or 15°C) and at a remarkably slow rate; both germination characteristics seem to be associated with autumn/winter seed germination and seedling establishment. Such a postulated strategy is ecologically advantageous within an unpredictable rainfall regime, known to prevail during the start of the rainy period of the Mediterranean climate. This strategy may also explain the spread of germination of M. comosum seeds over two consecutive years, observed by Theophrastus. Far-red light, simulating light conditions under a dense canopy, resulted in only a slight delay of germination compared to dark controls. Diurnal white light, qualitatively simulating natural daylight, caused a significant decrease of the germination rate in all four species studied. Moreover, white light was found to suppress considerably final seed germination (photoinhibition) in M. weissii and M. neglectum; in the latter species, prolonged imbibition under white light also led to the induction of secondary dormancy.

Seed germination of Arabidopsis thaliana phyA/phyB double mutants is under phytochrome control.

We examined the photocontrol of seed germination in the phyA/phyB double mutants of Arabidopsis thaliana seeds. Dormant phyA/phyB seeds showed a red/far-red light (R/FR)-reversible induction of seed germination. This suggests the involvement of at least one other phytochrome, phyC, D, and/or E, in controlling seed germination. We designated this spectrally active phytochrome in phyA/phyB as phyX. The full reversibility of the R-induced germination by subsequent FR pulses, and the observation that the response is reversible by FR, even after a 3-h R treatment, indicates that this phyX response belongs to the low-fluence-response type. Thus, this phyX response is functionally related to phyB-mediated responses. However, in contrast to phyB-controlled seed germination, this phyX-mediated response needs a prolonged imbibition period and exhibits reversibility kinetics different from that needed for phyB. Furthermore, this phyX response requires a prolonged irradiation time and shows a fluence rate response dependency, showing a similarity to the high irradiance response of photomorphogenesis. Thus, phyX, with regard to its control of seed germination, is a functionally new phytochrome that shares some characteristics of both phyA- and phyB-mediated responses.

Induction of secondary dormancy in rape seeds ( Brassica napus L.) by prolonged imbibition under conditions of water stress or oxygen deficiency in darkness

Prolonged imbibition under conditions of water stress or oxygen deficiency can lead to the induction of secondary dormancy in rape seeds. During imbibition in darkness seeds develop light sensitivity. The percentage of seeds not germinating in the dark depends on various factors prevailing during and after the stress treatment, as well as on the intrinsic susceptibility of the seeds to these factors. In experiments on the effect of water stress it was found that the percentage of secondary dormant seeds could be increased in the range of 0–75%, by greater water suction and duration of imbibition of the seeds. Additionally, there were indications that increasing treatment temperature resulted in increasing percentages of dormant seeds. However, fewer secondary dormant seeds were produced when treatment temperature and test temperature differed greatly from each other. Experiments on the effect of oxygen deficiency also revealed a positive effect of treatment duration. The general degree of dormancy induction, however, in this series of experiments was low. At 20 °C almost no dormancy induction was observed, whilst at 12 °C there were between 0 and 30% dormant seeds. Genotypic differences previously assessed in model experiments with buried seeds were confirmed in both studies. Oilseed rape cultivars Bienvenu and Liglandor were shown to be able to build up high percentages of secondary dormant seeds, whereas cultivars Jet Neuf and Falcon had a rather low susceptibility towards the dormancy imposing factors tested. Cultivar Rubin was intermediate in response. There was often much variation between experiments with the same experimental design. The reasons for this are unclear.

Effects of Ageing on Amylase Activity and Scutellar Cell Structure during Imbibition in Wheat Seed

In wheat seed the scutellum plays an important role in the hydrolysis of stored substrate during germination. This layer is activated first, whilst the aleurone becomes activated later. A good correlation exists between the initiation of visible germination and the appearance of enzyme activity in the scutellum. Enzyme activity in the aleurone becomes apparent only when the germinating seedling reaches the rapid growth phase. Electron microscopic observations show that during the later stages of germination the scutellar cells develop finger like projections. These may serve to absorb endospermic reserves hydrolysed by aleurone amylase. The scutellum of aged non-germinating seeds showed no amylase activity and no finger like projections were produced even after prolonged imbibition.

Germination of the seeds of parasitic angiosperms

AbstractTotalling more than 3000 species, parasitic angiosperms are a most diverse group of organisms. Classified as either shoot or root parasites according to site of attachment, plant parasites are further classified, according to the degree of parasitism, into one of two divisions: holoparasitic or hemiparasitic. Within these divisions, angiosperm parasites are either obligate or facultative.Obligate parasites cannot survive to maturity without attaching to a host plant, thus the need to germinate in close proximity to a host first characterizes the specialized germination and seed dispersal strategies of these plants. However, seeds of all species of the rootholoparasitic Orobanchaceae and four genera within the Scrophulariaceae have evolved very specialized germination strategies. Once the seeds have undergone an after-ripening period, they require a prolonged imbibition period at temperatures of approx. 30°C before they acquire the potential to germinate. Minute seeds with very little reserves, they must germinate within millimetres of the host root for successful host attachment and continued survival. To this end, germination only occurs in responseto specific chemical germination triggers released from the host root. InStriga hermonthica(Del.) Benth., the perception of the host-derived trigger results in stimulation of endogenous ethylene production and it is this ethylene that initiates germination. It is proposed that the chemical triggers released from the host be consideredas ‘elicitors’ which function primarily as allelochemicals and that the parasite seeds have become adapted in such a way as to have an absolute requirement for external germination triggers.

Germination Responses of Lehmann Lovegrass to Light

Lebmann lovegrass (Eragrostis lehmanniana Nees.) is a perennial, warm-season bunchgrass that is native to South Africa and has been seeded and spread naturally in the southwestern United States. Germination of 4 seed lots of varying age was tested in relation to darkness and irradiance with red (R) and far-red (FR) light. Germination was low in continual darkness, but greatly increased after exposure to R. Irradiation with FR after exposure to R reduced germination, confirming phytochrome involvement. Exposure to R after prolonged imbibition in FR did not increase germination of 1-2-year-old seeds and only slightly increased germination of older seeds. An alternating temperature of 16 hours at 15 degrees C and 8 hours at 38 degrees C greatly increased germination of seeds exposed to fluorescent light and slightly increased germination of seeds in darkness compared to a constant temperature of 25 degrees C. Greater seedling emergence of Lehmann lovegrass when the canopy is opened by burning, mowing, or grazing is likely a function of red light stimulation of biologically active phytochrome and increased seedbed temperature fluctuations.

On the Mechanism of Skotodormancy Induction in Grand Rapids Lettuce ( Lactuca sativa L.) Seeds

Summary Prolonged imbibition of lettuce ( Lactuca sativa L. cv. Grand Rapids) seeds in darkness results in a gradual loss of responsiveness to a short Red irradiation. This induction of skotodormancy was clearly shown to be temperature dependent above a critical temperature between 15 and 17.5 °C. At lower temperatures (15 and 10 °C) skotodormancy was not imposed at all, at least during the 20-day incubation period tested. When lettuce seeds were kept under continuous Far Red, instead of being pretreated in darkness, the induction of dormancy did not attain the final level of the dark-incubated seeds. Moreover, the responsivity to R was eventually resumed after several days in cFR and the higher the fluence rate the earlier the recovery of responsivity. In fact, at the highest FR fluence rate used, no additional dormancy was imposed. However, in comparison to the untreated seeds, a marked delay of germination was observed for the FR pretreated ones. Both the induction of skotodormancy and the reverse effect produced by high fluence cFR are discussed in terms of phytochrome and its postulated reaction partner X.

Enhanced Phytochrome Sensitivity and Its Reversal in Amaranthus albus Seeds

Seed of Amaranthus alus L. develop an enhanced sensitivity to the farred absorbing form of phytochrome after prolonged imbibition at temperatures >32 degrees C. The enhanced sensitivity developed at 40 degrees C could be reversed by subsequent treatment at 20 degrees C and similarly reestablished by repeating a 40 degrees C treatment. It is concluded that relative sensitivity to the far-red absorbing form of phytochrome may be readily manipulated in seeds of A. albus.

Induction of Secondary Dormancy in Chenopodium bonus-henricus L. Seeds by Osmotic and High Temperature Treatments and Its Prevention by Light and Growth Regulators

Factors controlling the establishment and removal of secondary dormancy in Chenopodium bonus-henricus L. seeds were investigated. Unchilled seeds required light for germination. A moist-chilling treatment at 4 C for 28 to 30 days removed this primary dormancy. Chilled seeds now germinated in the dark. When chilled seeds were held in the dark in -8.6 bars polyethylene glycol 6000 solution at 15 C or in water at 29 C a secondary dormancy was induced which increased progressively with time as determined by subsequent germination. These seeds now failed to germinate under the condition (darkness) which previously allowed their germination. Continuous light or daily brief red light irradiations during prolonged imbibition in polyethylene glycol solution at 15 C or in water at 29 C prevented the establishment of the secondary dormancy and caused an advancement of subsequent germination. Far red irradiations immediately following red irradiation reestablished the secondary dormancy indicating phytochrome participation in "pregerminative" processes. The growth regulator combination, kinetin + ethephon + gibberellin A(4)+A(7) (GA(4+7)), and to a relatively lesser extent GA(4+7), was effective in preventing the establishment of the secondary dormancy and in advancing the germination or emergence time. Following the establishment of the secondary dormancy by osmotic or high temperature treatments the regulator combination was relatively more active than light or GA(4+7) in removing the dormancy. Prolonged dark treatment at 29 C seemed to induce changes that were partially independent of light or GA(4+7) control. The data presented here indicate that changes during germination preventing dark treatment determine whether the seed will germinate, show an advancement effect, or will become secondarily dormant. These changes appear to be modulated by light and hormones.

Ethylene Synthesis in Lettuce Seeds: Its Physiological Significance

The germination and pregermination ethylene production of Grand Rapids lettuce seeds (Lactuca sativa L.) incubated at 20 C after a red light treatment are inhibited if the seeds are first imbibed at 30 C for 36 hours. In this study, low concentrations of ethylene were found to enhance the germination of seeds pretreated at 30 C more than that of untreated controls. In the presence of high concentrations of ethylene, pretreated seeds and controls germinated at a similar rate. These results are consistent with the view that a prolonged imbibition at 30 C inhibits germination at a lower temperature through its effect on the ethylene production of the seeds. As a further test of the hypothesis, estimates were made of the pregermination ethylene content of untreated seeds and pretreated seeds incubated in the presence of sufficient ethylene to make them germinate as rapidly as untreated seeds. The values obtained were 0.65 and 0.74 nanoliter of ethylene per gram (dry weight) of seeds, respectively.

Inhibition of early steps in the gibberellin-activated synthesis of alpha-amylase.

Subsequent production of amylase is severely inhibited if barban [4-chloro-2-butynyl N-(3-chlorophenyl) carbamate] is added to embryo-free half seeds of barley within 4 to 5 hours after gibberellic acid treatment of these seeds. Thirty to 50 mg/L concentrations of barban are effective. Barban inhibition is non-competitive with respect to gibberellic acid. Addition of barban 7 hours or more after gibberellic acid treatment is almost without effect. The delay between gibberellic acid treatment and amylase formation tends to become shorter with more prolonged imbibition periods. Regardless of imbibition period, susceptibility to barban is lost within 7 hours after gibberellic acid treatment. Other herbicidally active phenylurethanes are also inhibitors, but none are as effective as barban. Phenethyl alcohol and 2 arylcarbamates can act as inhibitors.