Molecular analysis of signals controlling dormancy and growth in underground adventitious buds of leafy spurge

Dormancy and subsequent regrowth of adventitious buds is a critical physiological process for many perennial plants. We have used the expression of hormone and cell cycle-responsive genes as markers to follow this process in leafy spurge (Euphorbia esula). In conjunction with earlier studies, we show that loss of mature leaves results in decreased sugar levels and increased gibberellin perception in underground adventitious buds. Gibberellin is sufficient for induction of S phase-specific but not M phase-specific gene expression. Loss of both apical and axillary buds or inhibition of polar auxin transport did not result in induction of S phase- or M phase-specific gene expression. Loss of polar auxin transport was necessary for continuation of the cell cycle and further bud development if the S phase was previously initiated.     

Changes in the expression of carbohydrate metabolism genes during three phases of bud dormancy in leafy spurge

Underground adventitious buds of leafy spurge (Euphorbia esula) undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy. In this study, relationships among genes involved in carbohydrate metabolism and bud dormancy were examined after paradormancy release (growth induction) by decapitation and in response to seasonal signals. Real-time PCR was used to determine the expression levels of carbohydrate metabolism genes at different phases of bud dormancy. Among differentially-regulated genes, expression of a specific Euphorbia esula β-amylase gene (Ee-BAM1) increased 100-fold after growth induction and 16,000-fold from July (paradormancy) to December (ecodormancy). Sequence data analysis indicated that two genes, Ee-BAM1 and Ee-BAM2, could encode this β-amylase. However, real-time PCR using gene-specific primer pairs only amplified Ee-BAM1, indicating that Ee-BAM2 is either specific to other organs or not abundant. The deduced amino acid sequences of these two genes are very similar at the N-terminal but differ at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29 kD (mature Ee-BAM1 after cleavage of the transit peptide) and a 35 kD (unprocessed EeBAM1) protein. Both 35 and 29 kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells at the shoot tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations suggests that Ee-BAM1 may have dual functions; serving as reserve protein in the cytosol and as a degrading enzyme at the surface of amyloplasts.     

Seasonal shifts in dormancy status, carbohydrate metabolism, and related gene expression in crown buds of leafy spurge

Crown buds of field-grown leafy spurge (Euphorbia esula L.) were examined to determine relationships between carbohydrate metabolism and gene expression throughout para-, endo-, and eco-dormancy during the transition from summer, autumn, and winter, respectively. The data indicates that endo-dormancy plays a role in preventing new shoot growth during the transition from autumn to winter. Cold temperature was involved in breaking endo-dormancy, inducing flowering competence, and inhibiting shoot growth. An inverse relationship developed between starch and soluble sugar (mainly sucrose) content in buds during the shift from para- to endo-dormancy, which continued through eco-dormancy. Unlike starch content, soluble sugars were lowest in crown buds during para-dormancy but increased over two- to three-fold during the transition to endo-dormancy. Several genes (AGPase, HK, SPS, SuSy, and UGPase) coding for proteins involved in sugar metabolism were differentially regulated in conjunction with well-defined phases of dormancy in crown buds. Marker genes for S-phase progression, cell wall biochemistry, or responsive to auxin were also differentially regulated during transition from para-, endo-, and eco-dormancy. The results were used to develop a model showing potential signalling pathways involved in regulating seasonal dormancy status in leafy spurge crown buds.     

The characterization of esulone C and chemotaxonomy of jatrophane diterpenes in leafy spurge

A new jatrophane diterpene, esulone C, was isolated and characterized from the ether extract of the roots of a North Dakota accession of the noxious weed leafy spurge. The ether extracts of North Dakota, Oregon, Montana and Austrian leafy spurge accessions were chromatographically compared for the occurrence of jatrophane diterpenes relative to the taxonomy and biological control of leafy spurge.     

Overcoming dormancy in seeds with ethanol and other anesthetics

Dormancy in fall panicum (Panicum dichotomiflorum Michx.) caryopses (seeds) is overcome by imbibition at 35° C in ethanol solutions. Whereas germination in the absence of ethanol depends on active phytochrome, the seeds may germinate in darkness after treatment with 0.2 to 0.5 M ethanol. Ethanol overcomes dormancy also in seeds of several other weedy grass species. Ethyl ether, chloroform, methanol, and acetone act similarly to ethanol. We suggest that this action depends on modifyng the properties of a membrane(s) in a manner related to the actions of other anesthetics.     

Kinetics of dormancy release and the high temperature germination response in Aesculus hippocastanum seeds

The kinetics of primary dormancy loss were investigated in seeds of horse chestnut (Aesculus hippocastanum L.) harvested in four different years. Freshly collected seeds from 1991 held for up to 1 year at temperatures between 2C and 42C exhibited two peaks in germination (radicle growth), representing a low temperature (2-8°C) and a high temperature response (31-36°C). Germination at 36°C generally occurred within 1 month of sowing, but was never fully expressed in the seedlots investigated. At low temperatures (2-8°C), germination started after around 4 months. Generally, very low levels of termination were observed at intermediate temperatures (11-26°C). Stratification at 6°C prior to germination at warmer temperatures increased the proportion of seeds that germinated, and the rate of germination for all seedlots. Within a harvest, germination percentage (on a probit scale) increased linearly with stratification time and this relationship was independent of germination temperature (16-26°C). However, inter-seasonal differences in the increases in germination capacity following chilling were observed, varying from 0.044 to 0.07 probits d^-1 of chilling at 6°C. Increased sensitivity to chilling was associated with warmer temperatures during the period of seed filling. The estimated base temperature for germination, Tb, for newly harvested seeds varied slightly between collection years but was close to 25°C. For all seedlots, Tb decreased by 1°C every 6 d of chilling at 6°C. This systematic reduction in Tb with chilling ultimately facilitated germination at 6°C after dormancy release.     

Sequential temperature control of multi-phasic dormancy release and germination of Paeonia corsica seeds

Aims The physiological responses during dormancy removal and multi-phasic germination were investigated in seeds of Paeonia corsica (Paeoniaceae).Methods Seeds of P. corsica were incubated in the light at a range of temperatures (10–25 and 25/10°C), without any pre-treatment, after W (3 months at 25°C), C (3 months at 5°C) and W + C (3 months at 25°C followed by 3 months at 5°C) stratification, and a GA 3 treatment (250 mg·l^-1 in the germination substrate). Embryo growth, time from testa to endosperm rupture and radicle emergence were assessed as separate phases. Epicotyl–plumule emergence was evaluated incubating the germinated seeds at 15°C for 2 weeks, at 5 and 25°C for 2 months on agar water before transplanting to the soil substrate at 10, 15 and 20°C and at 15°C for 2 months on the surface agar water with GA 3 .Important findings Embryo growth, testa rupture, endosperm rupture (radicle emergence) and growth of the epicotyl were identified as four sequential steps in seeds of P. corsica. Gibberellic acid alone and warm stratification followed by 15°C promoted embryo growth and subsequent seed germination. Cold stratification induced secondary dormancy, even when applied after warm stratification. After radicle emergence, epicotyl–plumule emergence was delayed for ca. 3 months. Mean time of epicotyl–plumule emergence was positively affected by cold stratification (2 months at 5°C) and GA 3. P. corsica seeds exhibited differential temperature sensitivity for the four sequential steps in the removal of dormancy and germination processes that resulted in the precise and optimal timing of seedling emergence.     

Clonal dynamics and evolution of dormancy in the leafy hepatic Lophozia silvicola

Dead shoots of colonies of a leafy hepatic species, Lophozia silvicola , are replaced by shoots developing from asexual propagules, the gemmae. Observations of two populations of L. silvicola showed a strong decreasing seasonal trend in germinability of the gemmae. We suggest that the non-germinating gemmae enter dormancy, and that the proportion of gemmae entering dormancy is season-specific. We assume that there are two types of gemmae, dormant and non-dormant and that only the dormant gemmae can survive during winter. Using a stochastic individual-based cellular automaton model, we investigated whether selection on season-specific dormancy fraction would lead to a decreasing proportion of germinating gemmae. Thus the germination schedule is the evolving trait in the model. Parameter estimates for the model were based on data collected from a population of L. silvicola in southern Finland over a three-year study period. In the simulations, the germination schedule shaped by evolutionary change was similar to the observed pattern. Thus the modelling results give support to the dormancy hypothesis. The qualitative pattern of decreasing germinability towards the end of the growing season is robust. Quantitative predictions are influenced by changes in parameters; for example, if winter mortality of shoots increases relative to mortality during the growing season, production of an increased fraction of dormant gemmae is favoured, especially at the end of the season.     

Organogenesis in cell cultures of leafy spurge (Euphorbiaceae) accessions from Europe and North America

Plants were regenerated from leafy spurge (Euphorbia esula L.) cell suspensions obtained from stem callus. A North Dakota accession was highly regenerable, but two accessions from Oregon and Austria formed only a few plantlets. Organogenesis occurred in media without growth regulators, under fluorescent lights (30 to 90 E m^–2 s^–1, 14 h photoperiod). Organogenesis was greatest in larger size clumps subcultured during maximum cell growth into media containing a reduced:oxidized nitrogen ratio of 33:67. Roots formed first and some clumps produced shoots. Organogenic suspension cultures also were initiated from hypocotyl and root segments of germinated seedlings, directly in liquid medium. Plantlets of the North Dakota accession formed in vitro adapted to greenhouse conditions. They were phenotypically similar to the parent plants.     

Hydrogen cyanide and embryonal dormancy in apple seeds

Embryos of apple ( Malus domestica Borh. cv. Antonówka) were treated with 1 m M gaseous HCN for 6 h and cultured under a 12 h photoperiod. HCN pretreatment stimulated germination, increased the length of hypocotyls, shortened the main root and decreased the percentages of seedlings with asymmetrically grown as well as with asymmetrically greened cotyledons. High activity of β-cyanoalanine synthase (EC 4.4.1.9) and a sharp increase in cyanogen content during embryo culture suggested very low levels of endogenous HCN. despite the activity of HCN releasing enzymes. The obtained data allow us to postulate an important role for cyanide in the regulatory complex controlling dormancy in apple seeds. Experiments with respiratory inhibitors indicated, however, that HCN pretreatment affected neither the alternative electron transport pathway nor residual respiration.     

The levels of GA3 and GA20 may be associated with dormancy release in Onopordum nervosum seeds

Endogenous levels of two gibberellins, GA₃ andGA₂₀, were quantified in unimbibed Onopordumnervosum seeds collected from two different populations, whichshoweddifferences in their germination capacity. After purifying the seed extracts,gibberellin levels were evaluated by gas chromatography mass spectrometry byusing selected ion monitoring (GC-MS-SIM) adding deuterated gibberellins asinternal standards. The intraspecific differences in germination capacity wereassociated with differences in the endogenous levels of both gibberellins. Thecontents of GA₃ and GA₂₀ in seeds with high germinationrate were twice and five times higher, respectively, than those from seeds witha low germination rate, indicating a possible role of gibberellins in dormancyrelease in this plant species.