Antagonistic effects of abscisic acid and gibberellic acid on the breaking of dormancy of Fagus sylvatica seeds

Study of the factors involved in the dormancy of Fagus sylvatica seeds shows that such dormancy is due partly to the seed coats and partly to endogenous factors. Seed coat removal accelerates both the release from dormancy and the effects of the other treatments that abolish it. The dormancy of these seeds is eliminated by cold treatment at 4°C over a period longer than 8 weeks, and exogenous application of abscisic acid (ABA) reverses the effects of low temperature, the seeds remaining in an ungerminated state. Additionally, ABA reduces protein synthesis but slightly increases RNA synthesis, which suggests its involvement in the synthesis of RNAs related to this process. In vitro translation of the RNAs isolated from these seeds shows that ABA delays the disappearance of at least 2 polypeptides (of ca 22 and 24 kDa), which are abundant in dormant seeds and under conditions that prevent the release from dormancy, but which disappear under treatments that abolish it. Exogenous application of gibberellic acid (GA₃) proved to be efficient in breaking the dormancy of these seeds and in substituting for cold treatment as well as in antagonizing the effects of ABA on the synthesis of both DNA and proteins. GA₃ also accelerates the disappearance of the two polypeptides abundant in dormant seeds and in ABA-treated seeds. These findings suggest that both ABA and GA₃ could be involved in the regulation of nucleic acid and protein metabolism during dormancy, acting antagonistically in these processes and, specifically, in the regulation of the synthesis of the two proteins that appear to play a role in the maintenance of dormancy in these seeds.     

Distribution of gibberellins and expressional analysis of GA 20-oxidase genes of morning glory during fruit maturation

Gibberellins A1/3 (GA1/3) and GA20 appeared earlier in surrounding tissues (pericarps/carpel/placenta) than in developing seeds of morning glory. The content of GA1/3 became higher in seeds than in the surrounding tissues at 9 days after anthesis (DAA), while that of GA20 stayed lower in seeds even at 12 DAA, suggesting the possibility that GA20 was translocated into seeds from the surrounding tissues and converted to GA1/3. The site of biosynthesis of GA20 in the fruits was determined by RNA-blotting and in situ hybridization of GA 20-oxidase genes (InGA20ox1, InGA20ox2). InGA20ox1 was not expressed in the surrounding tissues but in seeds, while no signal due to InGA20ox2 was detected in neither tissue. The expression of InGA20ox1 started in the seed coat near the hilum and spread in the seed coat like those of GA 3-oxidase and GA-inducible alpha-amylase genes. These observations suggest that GA biosynthesis is tissue-specifically and time-dependently regulated in the fruit of morning glory.     

GA2 and GA20-oxidase expressions are associated with the meristem position in Streptocarpus rexii (Gesneriaceae)

We examined genes involved in the regulatory pathway of gibberellin (GA) in meristems of Streptocarpus rexii. The plants do not possess a typical shoot apical meristem (SAM) and form unique meristems: the basal meristem extends the lamina area of one cotyledon to produce anisocotylous seedlings; the groove meristem forms new leaves at the base of the macrocotyledon. Exogenous application of GA significantly suppresses the basal meristem activity in developing cotyledons and the seedlings remain isocotyl. To examine the role of endogenous GA on these meristems in vivo, we isolated homologs of GA2-oxidase responsible for degrading active GAs (SrGA2ox), and GA20-oxidase regulating the rate limiting step of active GA synthesis (SrGA20ox). During embryogenesis, while first partly overlapping, the expression of SrGA2ox and SrGA20ox became more differentiated and mutually exclusive, ending with SrGA2ox being expressed solely in the adaxial–proximal domain of the embryo in regions with meristem activity, whereas SrGA20ox was restricted to the fork between the two cotyledons. The latter may be responsible for suppressing the formation of an embryonic SAM in S. rexii. In developing seedlings, SrGA2ox expression also followed the centers of meristem activity, where SrGA20ox expression was excluded. Our results suggest that low levels of GA are required in S. rexii meristems for their establishment and maintenance. Thus, the meristems in S. rexii share similar regulatory pathways suggested for the SAM in model plants, but that in S. rexii evolutionary modifications involving a lateral transfer of function, from shoot to leaves, is implicated in attaining the unusual morphology of the plants.     

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.     

Expression of gibberellin 20-oxidase1 (AtGA20ox1) in Arabidopsis seedlings with altered auxin status is regulated at multiple levels

Bioactive gibberellins (GAs) affect many biological processes including germination, stem growth, transition to flowering, and fruit development. The location, timing, and level of bioactive GA are finely tuned to ensure that optimal growth and development occur. The balance between GA biosynthesis and deactivation is controlled by external factors such as light and by internal factors that include auxin. The role of auxin transport inhibitors (ATIs) and auxins on GA homeostasis in intact light-grown Arabidopsis thaliana (L.) Heynh. seedlings was investigated. Two ATIs, 1-N-naphthylthalamic acid (NPA) and 1-naphthoxyacetic acid (NOA) caused elevated expression of the GA biosynthetic enzyme AtGA20-oxidase1 (AtGA20ox1) in shoot but not in root tissues, and only at certain developmental stages. It was investigated whether enhanced AtGA20ox1 gene expression was a consequence of altered flow through the GA biosynthetic pathway, or was due to impaired GA signalling that can lead to enhanced AtGA20ox1 expression and accumulation of a DELLA protein, Repressor of ga1-3 (RGA). Both ATIs promoted accumulation of GFP-fused RGA in shoots and roots, and this increase was counteracted by the application of GA(4). These results suggest that in ATI-treated seedlings the impediment to DELLA protein degradation may be a deficiency of bioactive GA at sites of GA response. It is proposed that the four different levels of AtGA20ox1 regulation observed here are imposed in a strict hierarchy: spatial (organ-, tissue-, cell-specific) > developmental > metabolic > auxin regulation. Thus results show that, in intact auxin- and auxin transport inhibitor-treated light-grown Arabidopsis seedlings, three other levels of regulation supersede the effects of auxin on AtGA20ox1.     

A unigene set for European beech (Fagus sylvatica L.) and its use to decipher the molecular mechanisms involved in dormancy regulation

Systematic sequencing is the method of choice for generating genomic resources for molecular marker development and candidate gene identification in nonmodel species. We generated 47 357 Sanger ESTs and 2.2M Roche‐454 reads from five cDNA libraries for European beech (Fagus sylvatica L.). This tree species of high ecological and economic value in Europe is among the most representative trees of deciduous broadleaf forests. The sequences generated were assembled into 21 057 contigs with MIRA software. Functional annotations were obtained for 85% of these contigs, from the proteomes of four plant species, Swissprot accessions and the Gene Ontology database. We were able to identify 28 079 in silico SNPs for future marker development. Moreover, RNAseq and qPCR approaches identified genes and gene networks regulated differentially between two critical phenological stages preceding vegetative bud burst (the quiescent and swelling buds stages). According to climatic model‐based projection, some European beech populations may be endangered, particularly at the southern and eastern edges of the European distribution range, which are strongly affected by current climate change. This first genomic resource for the genus Fagus should facilitate the identification of key genes for beech adaptation and management strategies for preserving beech adaptability.     

Endogenous gibberellins in immature seeds of Prunus persica L.: identification of GA(118), GA(119), GA(120), GA(121), GA(122) and GA(126)

The endogenous gibberellins in immature seeds of Prunus persica were analyzed by gas chromatography-mass spectrometry. Eleven known gibberellins, GA(3), GA(9), GA(17), GA(19), GA(30), GA(44), GA(61), GA(63), GA(87), GA(95) and GA(97) were identified. Additionally, several hitherto unknown gibberellins were detected and their putative structures were verified by synthesis of the authentic gibberellins. These gibberellins were then assigned trivial numbers, e.g. 1alpha-hydroxy GA(20) (GA(118)), 1alpha-hydroxy GA(9) (GA(119)), 1,2-didehydro GA(9) (GA(120)), 1,2-didehydro GA(70) (GA(121)), 1,2-didehydro GA(69) (GA(122)) and 1,2-didehydro GA(77) (GA(126)). GA(118) and GA(119) were the first 1alpha-hydroxy gibberellins identified from higher plants. The above profile of 1,2-didehydro gibberellins suggests that 1,2-dehydrogenation might occur prior to 3beta-hydroxylation in biosynthesis of GA(3), GA(30) and GA(87) in immature seeds of P. persica.     

Proteins and polyamines during dormancy breaking of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The studies were carried out on Fagus sylvatica seeds during stratification and their germination. After imbibition beechnuts were subjected to cold (3 °C — temperature which breaks dormancy) or warm (15 °C — temperature unable to break dormancy) stratification and alternatively were treated with polyamine synthesis inhibitors: canavanine and DFMO (difluoromethylornithine). After cold stratification in embryo axes we found (using 2-D electrophoresis) about 150 new proteins absent in dry seeds. Exogenous spermidine increased the protein synthesis, percent of germinated seeds and accelerated breaking of dormancy. In contrast, canavanine and DFMO decreased dynamic of protein synthesis, quantity of proteins probably synthesised de novo, and percent of germinated seeds. The maximum of polyamine content in embryo axes during cold stratification preceded such the maximum during warm stratification. Irrespective of the influence of PAs and inhibitors of PA synthesis, the comparison of electrophoregrams and autoradiograms showed that different groups synthesised de novo appeared after different periods of cold stratification. Probably the part of this protein is associated with Fagus sylvatica seeds dormancy breaking.     

Environmental regulation of dormancy loss in seeds of Lomatium dissectum (Apiaceae)

Background and Aims

Lomatium dissectum (Apiaceae) is a perennial, herbaceous plant of wide distribution in Western North America. At the time of dispersal, L. dissectum seeds are dormant and have under-developed embryos. The aims of this work were to determine the requirements for dormancy break and germination, to characterize the type of seed dormancy, and to determine the effect of dehydration after embryo growth on seed viability and secondary dormancy.

Methods

The temperature requirements for embryo growth and germination were investigated under growth chamber and field conditions. The effect of GA₃ on embryo growth was also analysed to determine the specific type of seed dormancy. The effect of dehydration on seed viability and induction of secondary dormancy were tested in seeds where embryos had elongated about 4-fold their initial length. Most experiments examining the nature of seed dormancy were conducted with seeds collected at one site in two different years. To characterize the degree of variation in dormancy-breaking requirements among seed populations, the stratification requirements of seeds collected at eight different sites were compared.

Key Results

Embryo growth prior to and during germination occurred at temperatures between 3 and 6 °C and was negligible at stratification temperatures of 0·5 and 9·1 °C. Seeds buried in the field and exposed to natural winter conditions showed similar trends. Interruption of the cold stratification period by 8 weeks of dehydration decreased seed viability by about 30 % and induced secondary dormancy in the remaining viable seeds. Comparison of the cold stratification requirements of different seed populations indicates that seeds collected from moist habitats have longer cold stratification requirements that those from semiarid environments.

Conclusions

Seeds of L. dissectum have deep complex morphophysiological dormancy. The requirements for dormancy break and germination reflect an adaptation to trigger germination in late winter.Key words: Apiaceae, cold stratification, Lomatium dissectum, morphophysiological dormancy, secondary dormancy, seed germination     

Production and scavenging of reactive oxygen species in Fagus sylvatica seeds during storage at varied temperature and humidity

The accumulation of reactive oxygen species (ROS) in seed tissues plays an important role in the loss of seed viability during storage. In the present study, we examined whether the loss of germination capacity and viability of beech (Fagus sylvatica L.) seeds during storage under different temperatures (4, 20 and 30 degrees C) and relative humidity levels (45% and 75% RH) is associated with: (1) an increase in the level of ROS, such as superoxide radical (O2*-), oxygen peroxide (H2O2); and, (2) changes in low molecular antioxidants (ascorbate and glutathione) and enzymatic scavengers such as ascorbate peroxidase dehydroascorbate reductase, glutathione reductase, catalase, superoxide dismutase and guaiacol peroxidase. Beech seeds progressively lost their ability to germinate during 9 weeks of storage under the above conditions. The deleterious effects of temperature treatments increased with growing seed moisture content at higher humidity. The loss of seed viability was correlated with the generation of ROS during storage, which was more intensive at higher temperatures and humidity levels. The ascorbate content significantly increased in seeds stored in all temperature and humidity variants, when the seeds lost the ability to germinate to a large degree. At the same time, glutathione content dramatically decreased, but it was possible to observe a defensive reaction in seeds stored at 20 degrees C. Activities of all scavenging enzymes, measured after slow imbibition of seeds, significantly increased in comparison to the non-treated control (8-9% MC, -10 degrees C). This increase was higher in embryo axes than in cotyledons. Our results suggest that the loss of viability of beech seeds during storage at different temperatures, above zero, and at different humidity levels is closely related to ROS production, and that the antioxidative system is not sufficient to protect them.     

Storage and remobilisation of sulphur in beech trees (Fagus sylvatica)

Three-year-old beech trees were fed ³⁵S-sulphate in August 1993 via a flap in a mature leaf of an upper branch. Harvest of beech trees was performed 24 h after feeding ³⁵S-sulphate, before leaf senescence, after leaf abscission, in early winter (January 1994). in late winter (March 1994). before bud break and after bud break. Twenty-four h after feeding ³⁵S-sulphate, 0.7 ± 0.5% of the ³⁵S-radioactivity taken up was exported out of the fed leaf. When trees were analysed 2 months later, i.e., before leaf senescence, this value had increased to 22 ± 7%. The exported ³⁵S-radioactivity was located in the branch containing the fed leaf (2.8 ± 13%). in basipetal parts of the trunk (41 ± 77%) and in the main rool (21 ± 6%). Leaves and apical parts of the trunk were no sink organs for the exported sulphur. Along the tree axis the main proportion of the radiolabel was located in the wood, predominantly in the acid soluble fraction. In the bark the greater portion of the radiolabel was found in the acid insoluble fraction. In both tissues the bulk of the ³⁵S of the soluble fraction was sulphate together with small amounts of glutathione. This pattern did not change until bud break. After bud break, basipetal parts of the trunk lost part of its ³⁵S-radioactivity. Of the ³⁵S-radioactivity which had been exported out of the fed leaf during the previous autumn, 16 ± 2% remained in the trunk, whereas 47 ± 7% of the ³⁵S was found in branches, mainly in the newly developed leaves. The present results show that sulphur, mainly in the form of sulphate, is stored along the tree axis in both bark and wood of beech trees and is re-mobilised during leaf development in spring.     

Release of heat pretreatment-induced dormancy in lettuce seeds by ethylene or cytokinin in relation to the production of ethylene and the synthesis of 1-aminocyclopropane-1-carboxylic acid during germination

The germination of lettuce (Lactuca sativa L.) seeds was greatly reduced when the seeds were heated at 97°C for 30 h prior to imbibition. This dormancy was effectively released when ethylene (1–100 ppm) or benzyladenine (BA) (0.005–0.05 mM) was applied during the imbibition period. Ethylene was not required during the early part of imbibition, but was essential during the period immediately prior to radicle protrusion. Treatment with 1-aminocyclopropane-1-carboxylic acid (ACC) (0.1–10 mM) stimulated germination, but was not as effective as ethylene or cytokinin treatment. During the germination of nondormant lettuce seeds, ethylene production increased rapidly and reached a peak at 24 h, which coincided with the emergence of the radicle, and then declined; the level of ACC increased as ethylene production rate increased, but remained at a high level after radicle protrusion. In heat-pretreated dormant lettuce seeds, the increases in percent germination, ethylene production, and ACC levels were all delayed and lower than those of nondormant seeds, and these increases were accelerated by treatment with ethylene or cytokinin.     

Polyamines and ethylene in the removal of embryonal dormancy in apple seeds

Putrescine (Put), spermidine (Spd) and spermine (Spm) were found in seeds of apple ( Malus domestica Borkh. cv. Antonovka), in amounts that increased in the order given. The levels slowly decreased during 30 days of stratification. Exogenous polyamines (PAs) affected germination of isolated embryos in a way dependent on the type of polyamine, its concentration, and the state of the embryo dormancy. The effect of Put and Spd on germination was stimulatory, while that of Spm was inhibitory. Stimulation of germination was also observed when embryos were treated with arginine, ornithine and methionine. Canavanine inhibited germination, and this effect was reversed by arginine or Put. Ethephon, aminooxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG) present during seed stratification had no effect on the levels of endogenous PAs. Put and Spd did not change ethylene production, neither during seed stratification nor during embryo germination, whereas Spm reduced ethylene evolution. The data suggest that Spm plays a role in the maintenance of dormancy by preventing ethylene production, while Put and Spd participate in dormancy removal, independently of ethylene.     

Structure and cytochemistry of the procambium in Salix buds during dormancy and dormancy breaking

This report presents a combined investigation of ultrastructural and enzymatic changes in the procambium from late winter to early spring. In January the procambial cells of dormant Salix buds have a convoluted plasma membrane with many plasmalemmasomes, numerous lipid bodies, large stacks of rough ER and plastids surrounded by smooth ER profiles. Several small lysosomes show activity of ATPase and acid phosphatases. In addition ER, nuclear envelopes, dictyosomes, and thylakoids have ATPase activity, and ER and plasmalemma, and nuclei also show acid phosphatase activity. In February metabolism seems to increase as indicated by lysosomes with membranous formations, dilated ER, nuclear envelopes, spiny vesicles, and polysomes. ATPase activity occurs in plasmalemma and vacuoles, and acid phosphatases in the middle lamella region of walls, in plasmalemma, vacuoles, ER, and nuclei. At the end of March, when growth starts inside the buds, but before they break, the stacks of rough ER disappear, and the vacuoles coalesce. Most of the lipid bodies have disappeared and the plastids have accumulated starch. Cell division and differentiation of procambial cells to protophloem and protoxylem have started. The distribution of ATPase increases; activity is found in walls and plasmalemma, and only a few small vacuoles still have ATPase and acid phosphatase activity. Notable is the appearance of ATPase in mitochondrial cristae and nucleoli and the occurrence of rather high levels also in endomembranes and dictyosomes.