Molecular mechanism of seed dormancy release induced by fluridone compared with cod stratification in Notopterygium incisum.

BACKGROUND: Notopterygium incisum is an important Chinese medicinal plant. Its mature seeds have underdeveloped embryos and are physiological dormant. We found the seeds with full developed embryos can germinate after treated by fluridone (FL), an inhibitor of abscisic acid (ABA). In order to understand the molecular mechanisms underlying seed dormancy release by FL, we compared the transcriptomic changes in dormancy release induced by two different methods, FL and cold stratification (CS) in N. incisum. We further analyzed the gene expression patterns involved in seed germination and dormancy using quantitative reverse-transcription PCR. RESULTS: RNA-sequence analysis revealed more dramatic changes in the transcriptomes of FL than those in CS, particularly for genes involved in the biosynthesis and regulation of gibberellins (GAs) and ABA. The down-regulation of ABA biosynthesis genes and the dramatic up-regulation of NiCYP707As, an ABA catabolic gene, contributed to the reduced ABA levels in FL. The increased GA3 levels in CS-treated seeds were due to the up-regulation of NiGA3OX. Both NiABI5 (a positive ABA regulator) and NiGAI (a negative regulator of GA) were down-regulated in FL and CS. The upregulation of strigolactones (SLs; the metabolites with the same precursor as ABA) biosynthesis and regulatory genes in both FL- and CS-treated seeds indicates that SLs contribute positively to seed dormancy release in N. incisum. CONCLUSIONS: Our results indicated that FL- and CS-seed dormancy release possibly depends on two totally different mechanisms: alleviation of the effects of ABA and potentiation of the effects of GA, respectively. However, NiABI5 and NiGAI probably function as common factors integrating the effects of ABA and GA on seed dormancy release.

Heat stress affects carbohydrate metabolism during cold-induced sweetening of potato (Solanum tuberosum L.).

MAIN CONCLUSION: Tolerance to heat stress for retention of low-temperature sweetening-resistant phenotype in potato is conferred by insensitivity of acid invertase activity to cold induction. Heat stress exacerbated cold sweetening (buildup of reducing sugars) of the LTS (low-temperature sweetening)-susceptible potato (Solanum tuberosum L.) cultivars, Ranger Russet and Russet Burbank, and completely abolished the resistance to cold sweetening in the LTS-resistant cultivars/clones, Sage Russet, GemStar Russet, POR06V12-3 and A02138-2. Payette Russet and EGA09702-2, however, demonstrated considerable tolerance to heat stress for retention of their LTS-resistant phenotype. Heat-primed Payette Russet and EGA09702-2 tubers accumulated fourfold more sucrose when subsequently stored at 4 °C, while reducing sugar concentrations also increased marginally but remained low relative to the non-heat-tolerant LTS-resistant clones, resulting in light-colored fries. By contrast, sucrose concentrations in heat-primed tubers of the non-heat-tolerant clones remained unchanged during LTS, but reducing sugars increased fivefold, resulting in darkening of processed fries. Acid invertase activity increased in the LTS-susceptible and non-heat-tolerant LTS-resistant cultivars/clones during cold storage. However, Payette Russet tubers maintained very low invertase activity regardless of heat stress and cold storage treatments, as was the case for Innate® Russet Burbank (W8) tubers, where silenced invertase conferred robust tolerance to heat stress for retention of LTS-resistant phenotype. Importantly, heat-stressed tubers of Payette Russet, EGA09702-2 and Innate® Russet Burbank (W8) demonstrated similar low reducing sugar and high sucrose-accumulating phenotypes when stored at 4 °C. Tolerance to heat stress for retention of LTS-resistant phenotype in Payette Russet and likely its maternal parent, EGA09702-2, is, therefore, conferred by the ability to maintain low invertase activity during cold storage of heat-stressed tubers.

Seed germination of medicinal plant, fennel (Foeniculum vulgare Mill), as affected by different priming techniques.

Reduced seed germination is among the most important factors adversely affecting crop stand and subsequent plant growth. Fennel (Foeniculum vulgare Mill) is an important medicinal plant with poor seed germination rate, occasionally. It is accordingly pertinent to find methods which can enhance fennel seed germination and remove the barriers of dormancy breaking. The present experiments studied the effects of two different priming (cold moist stratification and osmopriming) and 14 dormancy breaking techniques (hormonal, osmopriming, biopriming, chemical priming, and hydropriming) on the seed germination and seedling growth of two different fennel genotypes under growth chamber conditions. In the first and second experiment, the priming techniques including the time lengths of cold moist stratification (0, 15, 30, and 45 days) and the concentrations of polyethylene glycol 6000 (PEG6000, osmopriming at -0.99, -1.35, and -2.33 MPa) were used as the main plots. However, in both experiments, the dormancy breaking techniques and fennel genotypes were factorially combined and used as the subplots. Different seed- and seedling-related parameters including germination (%), plumule, radicle and seedling length, average germination time, rate and homogeneity of germination, and seed vigor index were determined. Both priming techniques were efficient on the enhancement of seed germination and seedling growth. Among the dormancy breaking techniques, Aminol Forte (biopriming), kadostim (biopriming), benzyl adenine + kinetin (biopriming), distilled water (hydropriming), gibberellin + kinetin (hormonal priming), and benzyl adenine + kinetin + gibberellin (biopriming) were the most effective ones. The related concentrations were equal to 100 mg/l, 10(-5) M, and 0.4 %. The fennel genotypes reacted significantly different under priming conditions. It is possible to enhance seed germination and seedling growth of fennel using priming and dormancy breaking techniques, which is useful for the increased production of fennel under different conditions. The results indicate that bio and hydropriming techniques were among the most effective ones, which significantly increased seed germination and seedling growth, and removed the seed dormancy barriers.

Electrophysiology of pumpkin seeds: Memristors in vivo.

Leon Chua, the discoverer of a memristor, theoretically predicted that voltage gated ion channels can be memristors. We recently found memristors in different plants such as the Venus flytrap, Mimosa pudica, Aloe vera, apple fruits, and in potato tubers. There are no publications in literature about the existence of memristors in seeds. The goal of this work was to discover if pumpkin seeds might have memristors. We selected Cucurbita pepo L., cv. Cinderella, Cucurbita maxima L. cv Warty Goblin, and Cucurbita maxima L., cv. Jarrahdale seeds for this analysis. In these seeds, we found the presence of resistors with memory. The analysis was based on cyclic voltammetry where a memristor should manifest itself as a nonlinear two-terminal electrical element, which exhibits a pinched hysteresis loop on a current-voltage plane for any bipolar cyclic voltage input signal. Dry dormant pumpkin seeds have very high electrical resistance without memristive properties. The electrostimulation by bipolar sinusoidal or triangular periodic waves induces electrical responses in imbibed pumpkin seeds with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, transforms a memristor to a resistor in pumpkin seeds. NPPB (5-Nitro-2-(3-phenylpropylamino)benzoic acid) inhibits the memristive properties of imbibed pumpkin seeds. The discovery of memristors in pumpkin seeds creates a new direction in the understanding of electrophysiological phenomena in seeds.

[Effects of fluridone, gibberellin acid and germination temperature on dormancy-breaking for Epimedium wushanense].

We introduced Epimedium wushanense seed which has been stratified for 90 days at 10/20 ℃ as experimental materials, with which we studied the effects of fluridone, gibberellin acid and temperature on E. wushanense germination. The results were suggested as shown below. ①Temperature, fluridone and gibberellin acid can both solely or jointly affect germination energy, germination rate significantly. Among those factors, fluridone affect germination rate and germination energy the most, followed by gibberellin acid and temperature. The highest germination rate under 4 ℃ and 10/20 ℃ stratification are 79.3%, 72.0% respectively, which resulted from treatment of F10GA300 and F20GA200 respectively. The highest germination energy under 4 ℃ and 10/20 ℃ stratification are 52.7%, 52.0%, respectively, which both resulted from F20GA200. ②Compared with 4 ℃ germination, seed could not germinate at 10/20 ℃ germination. Nontheless, application of fluridone can lead E. wushanense seeds to germinating.③The effects of gibberellin acid and interaction between gibberellin acid and fluridone significantly affect seed rotten rate during germination. In addition, soaking is another remarkable factor which increased seed rotten rate. As a result, it is feasible to promote E. wushanense dormancy releasing with gibberellin acid and fluridone associating with a proper germination temperature. Further, it is necessary taking actions to avoid seed rotten rate for saving E. wushanense nurseries'cost.

GA3-mediated dormancy alleviation in the reputed African potato, Hypoxis hemerocallidea.

BACKGROUND: Hypoxis hemerocallidea, arguably the most well-known medicinal species in South Africa, has been the subject of intensive harvesting from the wild leading to recent conservation concerns. The seeds of this species do not propagate easily and can lie dormant for up to twelve months. MATERIALS AND METHODS: In the in vitro germination experiments water, acid and chemical pre-sowing treatments were performed to determine the germination response of this species in both light and dark conditions. In the ex vitro experiment, intact seeds were sown and left to germinate in a potting soil mix under greenhouse conditions. RESULTS: Highest levels of germination (36.7-60.0% in the light and 36.7-46.7% in the dark) were achieved by treating mechanically scarified seeds with GA3 at various concentrations for 24 h. This was followed by scarified seeds soaked in water (26.7% in the light and 23.3% in the dark). Similar results (23.3 and 26.7%) were obtained in the 1% and 4% KNO3 treatments, respectively, under light conditions only. The fastest time to germinate was two days and was obtained in the 1200 ppm GA3 treatment in both light and dark conditions. Ex vitro germination of H. hemerocallidea seeds under greenhouse conditions was unsuccessful. CONCLUSION: H. hemerocallidea displays physical and non-deep physiological dormancy where pre-sowing treatments are required before the seeds will germinate.

Treatment of potato tubers with the synthetic cytokinin 1-(α-ethylbenzyl)-3-nitroguanidine results in rapid termination of endodormancy and induction of transcripts associated with cell proliferation and growth.

Perennial plants undergo repression of meristematic activity in a process called dormancy. Dormancy is a complex metabolic process with implications for plant breeding and crop yield. Endodormancy, a specific subclass of dormancy, is characteristic of internal physiological mechanisms resulting in growth suppression. In this study, we examine transcriptional changes associated with the natural cessation of endodormancy in potato tuber meristems and in endodormant tubers treated with the cytokinin analog 1-(α-ethylbenzyl)-3-niroguanidine (NG), which terminates dormancy. RNA-sequencing was used to examine transcriptome changes between endodormant and non-dormant meristems from four different harvest years. A total of 35,091 transcripts were detected with 2132 differentially expressed between endodormant and non-dormant tuber meristems. Endodormant potato tubers were treated with the synthetic cytokinin NG and transcriptome changes analyzed using RNA-seq after 1, 4, and 7 days following NG exposure. A comparison of natural cessation of dormancy and NG-treated tubers demonstrated that by 4 days after NG exposure, potato meristems exhibited transcriptional profiles similar to the non-dormant state with elevated expression of multiple histones, a variety of cyclins, and other genes associated with proliferation and cellular replication. Three homologues encoding for CYCD3 exhibited elevated expression in both non-dormant and NG-treated potato tissues. These results suggest that NG terminates dormancy and induces expression cell cycle-associated transcripts within 4 days of treatment.

Seed dormancy breaking diterpenoids, including novel brassicicenes J and K, from fungus Alternaria brassicicola, and their necrotic/apoptotic activities in HL-60 cells.

To find new metabolites similar to cotylenins and fusicoccins from the fungus Alternaria brassicicola, screening tests were carried out using the lettuce seed dormancy breaking assay. Activity-guided fractionation of the EtOAc extract from the culture using the assay afforded the isolation of two novel fusicoccane diterpenoids named brassicicenes J (1) and K (2), along with three known brassicicenes A (3), B (4), and F (5). Their structures were elucidated from extensive NMR spectral data and by comparison of these with those reported in the literature. Brassicicenes (1-5) exhibited weak to moderate seed dormancy breaking activities against lettuce seeds in the presence of abscisic acid. In addition, the necrotic/apoptotic activities of the brassicicenes (1-5), fusicoccin A (6) and cotylenin A (7) were evaluated by determining their cytotoxicity, cell viability and caspase-3/7 activation on the HL-60 cell line. Brassicicene K (2) exhibited similar cytostatic profiles to that of cotylenin A (7), and brassicicenes J (1), A (3), B (4), and F (5) exhibited necrotic activity. This is the first report of the seed dormancy breaking activity of brassicicenes in plants, and of necrotic/apoptotic activity in mammalian cells.

The role of the potato (Solanum tuberosum) CCD8 gene in stolon and tuber development.

· Strigolactones (SLs) are a class of phytohormones controlling shoot branching. In potato (Solanum tuberosum), tubers develop from underground stolons, diageotropic stems which originate from basal stem nodes. As the degree of stolon branching influences the number and size distribution of tubers, it was considered timely to investigate the effects of SL production on potato development and tuber life cycle. · Transgenic potato plants were generated in which the CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) gene, key in the SL biosynthetic pathway, was silenced by RNA interference (RNAi). · The resulting CCD8-RNAi potato plants showed significantly more lateral and main branches than control plants, reduced stolon formation, together with a dwarfing phenotype and a lack of flowering in the most severely affected lines. New tubers were formed from sessile buds of the mother tubers. The apical buds of newly formed transgenic tubers grew out as shoots when exposed to light. In addition, we found that CCD8 transcript levels were rapidly downregulated in tuber buds by the application of sprout-inducing treatments. · These results suggest that SLs could have an effect, solely or in combination with other phytohormones, in the morphology of potato plants and also in controlling stolon development and maintaining tuber dormancy.

[Study on seeds dormancy release and physiological change of Thesium chinense].

OBJECTIVE: To study the seeds dormancy release and physiological change of Thesium chinense. METHOD: To release dormancy, the seeds of T. chinense were treated with chemical reagent and stratification under 3-5 degrees C. RESULT: When washed with flowing water for 24 h, then soaked in 500 mg x L(-1) GA3 for 24 h, finally, treated with stratification method under 3-5 degrees C for 150 day, the split rate of T. chinense seeds reached 22%, crude fat decreased 50%, total sugar and dissolvability sugar increased 3-4 times, ABA decreased more than 90%, GA3 increased more than 5 times. CONCLUSION: The seeds dormancy of T. chinense can be released with the method of washing with flowing water for 24 h, then soaking in 500 mg x L(-1) GA3 for 24 h, finally, treated with stratification method under 3-5 degrees C for 150-180 day.

Alpha-tubulin (CsTUA) up-regulated during winter dormancy is a low temperature inducible gene in tea [Camellia sinensis (L.) O. Kuntze].

The present manuscript describes cloning and expression characterization of alpha-tubulin (CsTUA) gene in an evergreen tree tea [Camellia sinensis (L.) O. Kuntze] in response to winter dormancy (WD), abiotic stresses (sodium chloride, polyethylene glycol, and hydrogen peroxide) and plant growth regulators [abscisic acid (ABA), gibberellic acid (GA(3)), indole-3-butyric acid (IBA), and 6-benzylaminopurine (BA)]. CsTUA encoded a putative protein of 449 amino acids with a calculated molecular weight of 49.6 kDa and an isoelectric point (pI) of 5.09. CsTUA shared 76-84 and 90-95% identity at nucleotide and amino acid level, respectively with TUA genes from other plant species. During the period of active growth (PAG), CsTUA showed maximum expression in floral buds as compared to leaf, stem, fruit and root. Though the transcript was not detectable in the younger leaf tissue during the PAG, the expression was induced within 24 h of the low temperature (LT) treatment. The expression was not modulated by the plant growth regulators either in the tissue harvested during PAG or during WD. It was interesting to record that the expression of CsTUA was up-regulated in response to sodium chloride, polyethylene glycol, and hydrogen peroxide. Data has been discussed on the possible role of CsTUA in imparting tolerance to stresses including to LT so that the tea does not exhibit deciduous nature during winters.

Dose- and tissue-specific interaction of monoterpenes with the gibberellin-mediated release of potato tuber bud dormancy, sprout growth and induction of α-amylases and ß-amylases.

Gibberellins (GA) are involved in bud dormancy release in several species. We show here that GA-treatment released bud dormancy, initiated bud sprouting and promoted sprout growth of excised potato tuber bud discs ('eyes'). Monoterpenes from peppermint oil (PMO) and S-(+)-carvone (CAR) interact with the GA-mediated bud dormancy release in a hormesis-type response: low monoterpene concentrations enhance dormancy release and the initiation of bud sprouting, whereas high concentrations inhibit it. PMO and CAR did, however, not affect sprout growth rate after its onset. We further show that GA-induced dormancy release is associated with tissue-specific regulation of α- and ß-amylases. Molecular phylogenetic analysis shows that potato α-amylases cluster into two distinct groups: α-AMY1 and α-AMY2. GA-treatment induced transcript accumulation of members of both α-amylase groups, as well as α- and ß-amylase enzyme activity in sprout and 'sub-eye' tissues. In sprouts, CAR interacts with the GA-mediated accumulation of α-amylase transcripts in an α-AMY2-specific and dose-dependent manner. Low CAR concentrations enhance the accumulation of α-AMY2-type α-amylase transcripts, but do not affect the α-AMY1-type transcripts. Low CAR concentrations also enhance the accumulation of α- and ß-amylase enzyme activity in sprouts, but not in 'sub-eye' tissues. In contrast, high CAR concentrations have no appreciable effect in sprouts on the enzyme activities and the α-amylase transcript abundances of either group. The dose-dependent effects on the enzyme activities and the α-AMY2-type α-amylase transcripts in sprouts are specific for CAR but not for PMO. Different monoterpenes therefore may have specific targets for their interaction with hormone signalling pathways.

[Dormancy characteristics and breaking method of seeds from Epimedium wushanense].

OBJECTIVE: To research the mechanism of dormancy and find out the breaking method for the seeds of Epimedium wushanense. METHOD: The water permeability of seed coat was tested by weighing seeds. The germination inhibitor of the seeds were determined with biotic measurement. The development of embryos, germination rate and germination potential were determined after stratification. RESULT: The water permeability of seed coat was 41.86% after 5 h. The extracts of seeds had strong inhibition effects to the length growth of cabbage seedlings. The growth and development of embryos under the cold stratification (5 degrees C) were better than that under the other conditions. The embryo rate extended from 15.39% to 86.21% after 90 d. Germination rate and germination potential after stratification under 5 degrees C were significantly higher than that under other temperatures. CONCLUSION: The results showed that there was no obstacle of water permeability on the test of E. wushanense, after-ripening of embryogenesis and the germination inhibitor of the seed were the main reason for the seed dormancy. The cold stratification would be an effective way for breaking of the dormancy, which could significantly promote the seed embryogenesis and increase germination rate comparing to other methods.