Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.     

Germination and ROS detoxification in bell pepper (Capsicum annuum L.) under NaCl stress and treatment with microalgae extracts

We evaluated the salt tolerance of hybrids of pepper (Capsicum annuum L.) during germination. Treatments were applied at 0, 25, and 50 mM NaCl with preparations of supplemental extracts of the microalgae Dunaliella salina and Phaeodactylum tricornutum to determine the percentage germination rate as well as measured indicators of oxidative stress caused by the salt treatments during seed germination. We found that root growth was favorably influenced by the microalgae leading to increased germination rate. Tissues were analyzed in terms of superoxide radical production, lipid peroxidation, and activity of antioxidant enzymes viz. superoxide dismutase, catalase, and glutathione peroxidase. Our results suggest that application of microalgae extracts significantly reduced (p?<?0.05) superoxide radical production, as well as lower lipid peroxidation in comparison to plants without extracts of microalgae. The antioxidant enzymes increased in the presence of microalgae showing a significant difference (p?<?0.05). The results suggest differences in oxidative metabolism in response to the magnitude of salt stress and concentrations of microalgae help mitigate salt stress in plants during the germination process.     

Effects of CO(2) and O(2) on the Photosynthetic O(2) Evolution of Spirodela polyrrhiza Turions

Net photosynthetic rates of Spirodela polyrrhiza turions, at low O₂ levels, were 6.2 and 38.8 micromoles O₂ per gram fresh weight per hour at 1 millimolar HCO₃⁻ and CO₂ saturation, respectively, and much lower in a regular low-pH growth solution. Air equilibration O₂ concentrations decreased rates considerably, except at CO₂ saturation. The surfacing rate of turions in various inorganic carbon surroundings correlated positively with their photosynthetic rates, but were the same at high and low O₂ levels. The relevance of these findings in relation to environmental conditions conductive to germination of autotrophically growing turions is discussed.     

(2-Chloroethyl)phosphonic Acid Promotes Germination of Immature Spores of Ceratopteris richardii Brongn

Freshly collected spores of strain Hn-n of Ceratopteris richardii Brongn. require storage for several months before attaining maximum germination rate. Treatments using (2-chloroethyl)phosphonic acid increased germination rate in freshly collected spores and decreased germination rate in older spores.     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds

Aminopeptidases play important role in the mobilization of storage proteins at the cotyledon during seed germination. It is often referred as inducible component of defense against herbivore attack. However the role of aminopeptidase in response to pathogen attack in germinating seeds is remained to be unknown. An attempt was made to analyze change in the aminopeptidase (EC 3.4.11.1) activity during germination of pigeonpea (Cajanus cajan L.) seeds by infecting the seeds with fungi. Two aminopeptidase activity bands (AP1 and AP2) were detected in control as well as infected pigeonpea seeds. During latter stages of germination in control seeds, AP1 activity was replaced by AP2 activity. However AP1 activity was significantly induced in germinating seeds infected with Fusarium oxysporum f.sp. ciceri and Aspergillus niger var. niger. The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8 kDa respectively. The induced enzyme was purified up to 30 fold by gel filtration chromatography. The purified enzyme was preferentially cleaved leucine p-nitroanilide than alanine p-nitroanilide. The enzyme was strongly inhibited by bestatin and 1,10-phenanthroline. Almost 50% of enzyme activity was inhibited by ethylene diamine tetra acetate. The purified enzyme showed broad pH optima ranging from pH 6.0 to 9.0 and optimum at pH 8.5. The induction of aminopeptidase activity during pigeonpea seed germination and in response to pathogen attack indicates significant involvement of these enzymes in primary as well as secondary metabolism of the seeds. These findings could be helpful to further dissect defensive role of aminopeptidases in seed germination which is an important event in plant's life.     

Comparison of germination responses of Anigozanthos flavidus (Haemodoraceae), Gyrostemon racemiger and Gyrostemon ramulosus (Gyrostemonaceae) to smoke-water and the smoke-derived compounds karrikinolide (KAR1) and glyceronitrile

Background and Aims

A major germination-promoting chemical in smoke-water is 3-methyl-2H-furo[2,3-c]pyran-2-one (karrikinolide, KAR₁). However, not all species that germinate in response to smoke-water are responsive to KAR₁, such as Tersonia cyathiflora (Gyrostemonaceae). In this study, a test was made of whether two Gyrostemon species (Gyrostemonaceae) that have previously been shown to respond to smoke-water, respond to KAR₁. If not, then the smoke-derived chemical that stimulates germination of these species is currently unknown. Recently, glyceronitrile was isolated from smoke-water and promoted the germination of certain Anigozanthos species (Haemodoraceae). Whether this chemical promotes Gyrostemon racemiger germination is also examined. Furthermore, an investigation was carried out into whether these species germinate in response to smoke-water derived from burning cellulose alone.

Methods Gyrostemon racemiger

and G. ramulosus seeds were buried after collection and retrieved in autumn the following year when dormancy was alleviated and seeds had become responsive to smoke-water. Anigozanthos flavidus seeds were after-ripened at 35 °C to alleviate dormancy. Gyrostemon and Anigozanthos seeds were then tested with ‘Seed Starter’ smoke-water, KAR₁, glyceronitrile and cellulose-derived smoke-water.

Key Results

Although Gyrostemon racemiger, G. ramulosus and A. flavidus were all stimulated to germinate by ‘Seed Starter’ smoke-water, none of these species responded to KAR₁. Gyrostemon racemiger germination was not promoted by glyceronitrile. This is in contrast to A. flavidus, where glyceronitrile, at concentrations of 1–500 µm, promoted germination, although seedling growth was inhibited at ≥400 µm. Maximum A. flavidus germination occurred at glyceronitrile concentrations of 25–300 µm. Some Gyrostemon germination was promoted by cellulose-derived smoke-water.

Conclusions

KAR₁ and glyceronitrile, chemicals in smoke-water that are known to stimulate germination in other species, did not promote the germination of G. racemiger. This suggests that other chemical(s) which promote germination are present in smoke, and may be derived from burning cellulose alone.     

Nondeep simple morphophysiological dormancy in seeds of Ilex maximowicziana from northern (subtropical) and southern (tropical) Taiwan

In this study, we show that seeds of Ilex maximowicziana collected from northern and southern Taiwan differ in germination responses to temperature. Seeds produced by plants growing in the tropical environment of southern Taiwan were more responsive (in a positive way) to higher incubation temperatures than those produced by plants growing in the subtropical environment of northern Taiwan. On the other hand, seeds produced in northern Taiwan were more responsive (in a positive way) to low incubation temperatures and to cold stratification than those from southern Taiwan. Germination percentages and rates of seeds from northern Taiwan were higher at 20/10°C than at 30/20°C, reaching a plateau of >80% germination after 12 weeks incubation, whereas germination of seeds from southern Taiwan reached >80% at 30/20 and 25°C but not at 20/10°C. Gibberellic acid (GA₃) increased germination rate but not germination percentage of seeds from both southern and northern Taiwan. Freshly matured seeds of I. maximowicziana have rudimentary embryos. During dormancy break, embryo length increased 11.5- and 8.0-fold in northern and southern seeds, respectively, before radicle emergence. Thus, seeds of Ilex maximowicziana have nondeep simple morphophysiological dormancy. This is the first detailed study of the germination requirements of a subtropical/tropical species of the large cosmopolitan genus Ilex.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

Phosphorus-mediated changes in life history traits of the invasive New Zealand mudsnail (Potamopyrgus antipodarum)

The longer soil has to stay moist to allow germination the more likely that seedlings experience favourable moisture conditions. Since theory predicts that fitness variance-reducing traits will be negatively correlated, we tested the hypothesis that time to germination is negatively correlated with the ability of radicle growth to keep up with the drying front. We measured time to germination and root elongation rate (RER) in 14 Kalahari annuals. We controlled for habitat (canopy association and sand content), germinability, median base water potential for germination (ψ ₅₀), seed mass and seed shape as a persistence surrogate. For species and phylogenetically independent contrasts (PICs), we did not find a relationship between time to germination and RER. However, we found a negative relationship of time to germination with RER for PICs when controlling for sand content and ψ ₅₀. Seed shape increased with time to germination which can be explained by reduced opportunities for germination in slow-germinating species that select for persistence. We found a positive relationship between time to germination and ψ ₅₀, suggesting a continuum of risky to cautious germination. ψ ₅₀ was not correlated with RER suggesting that variation in ψ ₅₀ reflects different drought-adapted traits. Probably the relationship of time to germination with RER is not mediated by seed mass, which was not correlated with time to germination as found by others, though RER was positively correlated with seed mass. Instead of the seed size-seed number trade-off, a trade-off with resource capture may explain variation in RER: more root hairs or rootlets may increase resource capturing surface while reducing RER. For habitat, we found a (positive) relationship with time to germination only for canopy association. This may be explained by maximization of resource capture at the expense of RER being favoured by the higher nutrient and water availability under canopies. Future studies should clarify which trade-offs govern variation in time to germination, focussing on a possible resource capture-RER trade-off.     

Mycorrhizal association and symbiotic germination of the terrestrial orchid Bipinnula fimbriata (Poepp.) Johnst (Orchidaceae)

In this study mycorrhizal fungi were isolated from the roots of the endemic terrestrial orchid Bipinnula fimbriata. Seven isolates were previously identified as the form-genus Rhizoctonia, a polyphyletic group known to form mycorrhizal associations with Orchidaceae. Two other isolates were included in the study: #793 isolated from Chloraea crispa, and #1325 Rhizoctonia solani, isolated from potato. After morphological and molecular characterization of the nine isolates, they were divided into three groups, Ceratobasidium sp., Tulasnella calospora and Thanatephorus cucumeris, to determine the diversity between isolates. Consensus ITS sequences were used for a blast search on the GenBank database, which confirmed the results of the morphological observations. Once the isolates were identified, an in vitro germination test was done with four plates of oatmeal agar inoculated with each fungus, plus an asymbiotic control. The germination stages of the seeds were recorded 30 days after sowing. All isolates obtained from B. fimbriata, and the isolate #793 from Chloraea crispa, promoted seed germination. However, the isolate #1325 Rhizoctonia solani, which is known as both a pathogen and an orchid symbiont, did not promote germination. This shows that B. fimbriata is associated with more than one mycorrhizal fungus in its habitat and has a broader potential specificity in vitro. The results support the hypothesis that at least one fungal isolate promotes the germination of B. fimbriata, permitting the conservation of this species in ex situ conditions.     

Seed germination and seedling growth of Zostera marina L. (eelgrass) in the chesapeake bay

Seed germination and seedling growth of Zostera marina L. were monitored in the Chesapeake Bay in 1979 and 1980. Harvested seeds were placed in small acrylic tubes at several sites representing the salinity range of Z. marina distribution. Seed germination was observed first in late September and continued through May, with peaks in the fall and spring. The majority of seeds that germinated (66%) did so between December and March when water temperatures ranged from 0–10°C. There was no correlation between sites (different salinity regimes) and frequency of germination rates, indicating that salinity was not a major factor in the germination process in this study. Additional information on seed germination was available for seeds collected in 1977 and 1980 and subsequently monitored for germination at only one site. These data were similar to germination frequency recorded in 1979–1980.Seedling growth was measured from individuals collected from an existing Zostera marina bed. Seedlings were collected from November through May, at which time we could no longer distinguish seedlings from existing vegetative stock. Growth was characterized by the increased length of the primary shoot, number of leaves per shoot and numbers of shoots per plant. Seedling growth was slow during the winter months (water temperature ? 10°C) but rapidly increased in the spring (temperatures > 10°C). The size range of the harvested seedlings indicated that seed germination in the field probably occurred from October through April, corroborating evidence from the seed germination experiments.     

Arginine catabolism in the cotyledons of developing and germinating pea seeds

Arginine is the predominant free amino acid in the cotyledons of developing seeds of Pisum sativum L. cv Marzia. Breakdown of arginine was measured by injecting l-[guanido-¹⁴C]arginine into detached cotyledons. Cotyledons of developing seeds showed a low rate of ¹⁴CO₂ evolution whereas a much higher rate of ¹⁴CO₂ evolution was measured from cotyledons of seeds 4 days after the onset of germination. The activities of the catabolic enzymes arginase, urease, and ornithine aminotransferase were measured throughout development and germination. Arginase and ornithine aminotransferase were present at an early stage of development. Urease activity appeared later as the seeds started to desiccate. During germination, all three enzymes were present. The different course of activity of these enzymes indicates that they are controlled separately.     

Factors Affecting the Germination of Akinetes of Nodularia spumigena (Cyanobacteriaceae)

Nutritional and physical factors which influence the germination of akinetes of Nodularia spumigena (Cyanobacteriaceae) were examined. Low concentrations of phosphorus (<0.9 μM) were required for germination. Nitrate had no effect, but ammonia, at concentrations of >45 μM, inhibited germination. Salinities of >20‰ were inhibitory to germination. Optimum temperatures were 22°C or greater. Germination did not take place in the dark, but only very low light intensities (0.5 microeinstein m⁻² s⁻¹) were necessary to initiate germination. Red light (620 to 665 nm) was required. More than 24 h of continuous exposure to light was necessary for any significant germination to occur. The conditions for germination corresponded with conditions in the Peel-Harvey Estuary, Western Australia, 2 to 3 weeks before large summer Nodularia blooms.     

Activity of enzymes of arginine metabolism in the cotyledons of developing and germinating pea seeds

Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.

The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the following days. Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination.

     

Sterol conjugate interconversions during germination of white mustard (Sinapis alba)

Changes in the content of free sterols (FS), steryl esters (SE), steryl glucosides (SG) and acylated steryl glucosides (ASG) in germinating seeds of white mustard (Sinapis alba) were studied together with parrallel changes in specific activities of some enzymes involved in sterol conjugate transformation. It has been found that a distinct increase in the net SE content and a similar, but less pronounced, increase in SG content at the beginning of germination can be correlated with a distinctly earlier appearance of SE and SG synthesizing enzymes, i.e. triacylglycerol: sterol acyltransferase and UDPG: sterol glucosyltransferase in comparison with hydrolytic activities, i.e. SE hydrolase and SG hydrolase. Our results suggest that metabolism of SG and ASG takes place mainly in the cotyledons while SE metabolism takes place mainly in the roots.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.     

Ecology of seed dormancy and germination in sedges (Carex)

The genus Carex, with its wide distribution and large number of species yet with a rather uniform life history, is a very convenient group for comparative studies of germination ecology at the generic level. The combination of a strict or conditional primary dormancy, a light requirement for germination, low germination at constant temperatures, a positive response to diurnal temperature fluctuations and an induction of secondary dormancy in late spring by increasing environmental temperatures are attributes that were found to be characteristics shared by almost all the Carex species investigated, though there was variation between species in the degree to which these characters were expressed. In almost all species, dormancy was broken by stratification at low temperatures, though few species gained the ability to germinate at temperatures <10 °C. There is evidence that long-term physiological changes and the structure of seed coats can play a decisive role in delaying germination. High dormancy levels were found mainly in Carices with large seeds (>0.9 mg), probably due to a thicker seed coat and hence a higher resistance to germination. Differences in primary dormancy between sedges of various habitats could not be established. However, there was a tendency for temperature limits to be low in forest sedges. Many species of wetlands and open sites showed a greater capability to respond to fluctuating temperatures than species of dry sites. These dormancy and germination traits not only enable the accumulation of seeds in the soil, but also constitute seasonal seed regeneration strategies that rely on the high longevity of seeds and the formation of persistent seed banks. Temperate Carices are mainly adapted to exploit the temporally and spatially infrequent occurrence of canopy gaps that become available only in late spring or early summer, whereas the colonization of gaps at the beginning of the vegetation period is largely prevented by a high temperature requirement for germination. Many of the dormancy and germination characteristics of Carices are important in Cyperaceae generally. A greater diversity of germination responses, however, can be found in the related families, Juncaceae and Poaceae. Our present knowledge is not sufficient to determine unequivocally whether a phylogenetic component contributes significantly to the germination behaviour of the genus Carex, but certain tendencies are clearly indicated.     

Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.     

Seed ultrastructure and water absorption pathway of the root-parasitic plant Phelipanche aegyptiaca (Orobanchaceae)

Background and Aims

Obligate root parasitic plants of the Orobanchaceae do not germinate unless they chemically detect a host plant nearby. Members of this family, like Orobanche, Phelipanche and Striga, are noxious weeds that cause heavy damage to agriculture. In spite of their economic impact, only a few light microscopical studies of their minute seeds have been published, and there is no knowledge of their ultrastructure and of the role each tissue plays during the steps preceding germination. This paper describes the ultrastructure of Phelipanche seeds and contributes to our understanding of seed tissue function.

Methods

Seeds of P. aegyptiaca were examined under light, scanning electron, transmission electron and fluorescence microscopy following various fixations and staining protocols. The results were interpreted with physiological data regarding mode of water absorption and germination stimulation.

Key Results and Conclusions

The endothelium, which is the inner layer of the testa, rapidly absorbs water. Its interconnected cells are filled with mucilage and contain labyrinthine walls, facilitating water accumulation for germination that starts after receiving germination stimuli. Swelling of the endothelium leads to opening of the micropyle. The perisperm cells underneath this opening mediate between the rhizosphere and the embryo and are likely to be the location for the receptors of germination stimuli. The other perisperm cells are loaded with lipids and protein bodies, as are the endosperm and parts of the embryo. In the endosperm, the oil bodies fuse with each other while they are intact in the embryo and perisperm. Plasmodesmata connect the perisperm cells to each other, and the cells near the micropyle tightly surround the emerging seedling. These perisperm cells, and also the proximal embryo cells, have dense cytoplasmic contents, and they seem to represent the two seed components that are actively involved in transfer of reserve nutrients to the developing seedling during germination.