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Seed germination is an important life-cycle transition because it determines subsequent plant survival and reproductive success. To detect optimal spatiotemporal conditions for germination, seeds act as sophisticated environmental sensors integrating information such as ambient temperature. Here we show that the DELAY OF GERMI-NATION 1 (DOG1) gene, known for providing dormancy adaptation to distinct environments, determines the optimal temperature for seed germination. By reciprocal gene-swapping experiments between Brassicaceae species we show that the DOG1-mediated dormancy mechanism is conserved. Biomechanical analyses show that this mechanism regulates the material properties of the endosperm, a seed tissue layer acting as germination barrier to control coat dormancy. We found that DOG1 inhibits the expression of gibberellin (GA)-regulated genes encoding cell-wall remodeling proteins in a temperaturedependent manner. Furthermore we demonstrate that DOG1 causes temperature-dependent alterations in the seed GA metabolism. These alterations in hormone metabolism are brought about by the temperature-dependent differential expression of genes encoding key enzymes of the GA biosynthetic pathway. These effects of DOG1 lead to a temperature-dependent control of endosperm weakening and determine the optimal temperature for germination. The conserved DOG1-mediated coat-dormancy mechanism provides a highly adaptable temperature-sensing mechanism to control the timing of germination.dormancy gene DOG1 | gibberellin metabolism | germination temperature | cell-wall remodelling | Lepidium sativum

A robust, reliable and high-throughput method for extraction and purification of gibberellins (GAs), a group of tetracyclic diterpenoid carboxylic acids that include endogenous growth hormones, from plant material was developed. The procedure consists of two solid-phase extraction steps (Oasis(®) MCX-HLB and Oasis(®) MAX) and gives selective enrichment and efficient clean-up of these compounds from complex plant extracts. The method was tested with plant extracts of Brassica napus and Arabidopsis thaliana, from which total recovery of internal standards of about 72% was achieved. A rapid baseline chromatographic separation of 20 non-derivatised GAs by ultra performance liquid chromatography is also presented where a reversed-phase chromatographic column Acquity CSH(®) and a mobile phase consisting of methanol and aqueous 10mM-ammonium formate is used. This method enables sensitive and precise quantitation of GAs by MS/MS in multiple-reaction monitoring mode (MRM) by a standard isotope dilution method. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. All studied GAs were determined as free acids giving dominant quasi-molecular ions of [M-H](-) with limits of detection ranging between 0.08 and 10 fmol and linear ranges over four orders of magnitude. Taking advantage of highly effective chromatographic separation of 20 GAs and very sensitive mass spectrometric detection, the presented bioanalytical method serves as a useful tool for plant biologists studying the physiological roles of these hormones in plant development.

Myrica gale L. (sweet gale) fruit leachate contains myrigalone A (MyA), a rare C-methylated dihydrochalcone and putative allelochemical, which is known to be a phytotoxin impeding seedling growth. We found that MyA inhibited Lepidium sativum L. seed germination in a dose-dependent manner. MyA did not affect testa rupture, but inhibited endosperm rupture and the transition to subsequent seedling growth. MyA inhibited micropylar endosperm cap (CAP) weakening and the increase in the growth potential of the radical/hypocotyl region (RAD) of the embryo, both being key processes required for endosperm rupture. We compared the contents of abscisic acid (ABA) and gibberellins in the tissues and found that the major bioactive forms of gibberellin in L. sativum seed tissues were GA(4) and GA(6), while GA(8) and GA(13) were abundant inactive metabolites. MyA did not appreciably affect the ABA contents, but severely interfered with gibberellin metabolism and signaling by inhibiting important steps catalyzed by GA3 oxidase, as well as by interfering with the GID1-type gibberellin signaling pathway. The hormonally and developmentally regulated formation of apoplastic superoxide radicals is important for embryo growth. Specific zones within the RAD were associated with accumulation of apoplastic superoxide radicals and endoreduplication indicative of embryo cell extension. MyA negatively affected both of these processes and acted as a scavenger of apoplastic reactive oxygen species. We propose that MyA is an allelochemical with a novel mode of action on seed germination.

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