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Ada Linkies, Kerstin Müller,
Karl Morris,
Veronika Turecková,
Meike Wenk,
Cassandra S C Cadman,
Françoise Corbineau,
Miroslav Strnad,
James R Lynn,
William E Finch-Savage,
Gerhard Leubner-Metzger
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Ada Linkies, Kerstin Müller,
Karl Morris,
Veronika Turecková,
Meike Wenk,
Cassandra S C Cadman,
Françoise Corbineau,
Miroslav Strnad,
James R Lynn,
William E Finch-Savage,
Gerhard Leubner-Metzger
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Ada Linkies, Kerstin Müller,
Karl Morris,
Veronika Turecková,
Meike Wenk,
Cassandra S C Cadman,
Françoise Corbineau,
Miroslav Strnad,
James R Lynn,
William E Finch-Savage,
Gerhard Leubner-Metzger
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Ada Linkies, Kerstin Müller,
Karl Morris,
Veronika Turecková,
Meike Wenk,
Cassandra S C Cadman,
Françoise Corbineau,
Miroslav Strnad,
James R Lynn,
William E Finch-Savage,
Gerhard Leubner-Metzger
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ABSTRACT: The methylesterification status of cell wall homogalacturonans, mediated through the action of pectin methylesterases (PMEs), influences the biophysical properties of plant cell walls such as elasticity and porosity, important parameters for cell elongation and water uptake. The completion of seed germination requires cell wall extensibility changes in both the radicle itself and in the micropylar tissues surrounding the radicle. In wild-type seeds of Arabidopsis thaliana ('Arabidopsis'), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We over-expressed an Arabidopsis PME-inhibitor (PMEI5) to investigate PME-involvement in seed germination. Seeds of the resultant lines showed a denser methylesterification status of their cell wall homogalacturonans but there were no changes in the neutral sugar- and uronic acid- composition of the cell walls. As compared to wild-type seeds, the PME activities of the over-expressing lines were greatly reduced throughout germination and the low steady-state levels neither increased nor decreased. The most striking phenotype was a significantly faster rate of germination, which was not connected to altered testa rupture morphology, but to alterations of the micropylar endosperm cells evident by environmental SEM. The transgenic seeds also exhibited an apparent reduced sensitivity to ABA with respect to its inhibitory effects on germination. We speculate that PME activity contributes to the temporal regulation of radicle emergence in endospermic seeds by altering the mechanical properties of the cell walls and thereby the balance between the two opposing forces of radicle elongation and mechanical resistance of the endosperm.
Plant physiology 11/2012; · 6.53 Impact Factor
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ABSTRACT: Reactive oxygen species are increasingly perceived as players in plant development and plant hormone signalling pathways. One of these species, superoxide, is produced in the apoplast by respiratory burst oxidase homologues (rbohs), a family of proteins that is conserved throughout the plant kingdom. Because of the availability of mutants, the focus of research into plant rbohs has been on Arabidopsis thaliana, mainly on AtrbohD and AtrbohF. This study investigates: (i) a different member of the Atrboh family, AtrbohB, and (ii) several rbohs from the close relative of A. thaliana, Lepidium sativum ('cress'). Five cress rbohs (Lesarbohs) were sequenced and it was found that their expression patterns were similar to their Arabidopsis orthologues throughout the life cycle. Cress plants in which LesarbohB expression was knocked down showed a strong seedling root phenotype that resembles phenotypes associated with defective auxin-related genes. These transgenic plants further displayed altered expression of auxin marker genes including those encoding the auxin responsive proteins 14 and 5 (IAA14 and IAA5), and LBD16 (LATERAL ORGAN BOUNDARIES DOMAIN16), an auxin-responsive protein implicated in lateral root initiation. It is speculated that ROS produced by rbohs play a role in root development via auxin signalling.
Journal of Experimental Botany 10/2012; · 5.36 Impact Factor
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Ada Linkies, Kerstin Müller,
Karl Morris,
Veronika Tureckova,
Meike Wenk,
Cassandra S C Cadman,
Françoise Corbineau,
Miroslav Strnad,
James R Lynn,
William E Finch-Savage,
Gerhard Leubner-Metzger
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ABSTRACT: Plants have a remarkable ability to react to seasonal changes by synchronizing life-cycle transitions with environmental conditions. We addressed the question of how transcriptional re-programming occurs in response to an environmental cue that triggers the major life cycle transition from seed dormancy to germination and seedling growth. We elucidated an important mechanistic aspect of this process by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3. Histone methylation patterns of major dormancy regulators changed during the transition to germination and seedling growth. We observed a switch from H3K4me3 and high transcription levels to silencing by the repressive H3K27me3 mark when dormancy was broken through exposure to moist chilling, underscoring that a functional PRC2 complex is necessary for this transition. Moreover, this reciprocal regulation by H3K4me3 and H3K27me3 is evolutionarily conserved from gymnosperms to angiosperms.
PLoS ONE 01/2012; 7(12):e51532. · 4.09 Impact Factor
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ABSTRACT: 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 β-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 β-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 β-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.
Planta 08/2011; 235(1):137-51. · 3.00 Impact Factor
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ABSTRACT: Germination of endospermic seeds is partly regulated by the micropylar endosperm, which acts as constraint to radicle protrusion. Gibberellin (GA) signalling pathways control coat-dormancy release, endosperm weakening, and organ expansion during seed germination. Three GIBBERELLIN INSENSITIVE DWARF1 (GID1) GA receptors are known in Arabidopsis thaliana: GID1a, GID1b, and GID1c. Molecular phylogenetic analysis of angiosperm GID1s reveals that they cluster into two eudicot (GID1ac, GID1b) groups and one monocot group. Eudicots have at least one gene from each of the two groups, indicating that the different GID1 receptors fulfil distinct roles during plant development. A comparative Brassicaceae approach was used, in which gid1 mutant and whole-seed transcript analyses in Arabidopsis were combined with seed-tissue-specific analyses of its close relative Lepidium sativum (garden cress), for which three GID1 orthologues were cloned. GA signalling via the GID1ac receptors is required for Arabidopsis seed germination, GID1b cannot compensate for the impaired germination of the gid1agid1c mutant. Transcript expression patterns differed temporarily, spatially, and hormonally, with GID1b being distinct from GID1ac in both species. Endosperm weakening is mediated, at least in part, through GA-induced genes encoding cell-wall-modifying proteins. A suppression subtraction hybridization (SSH) cDNA library enriched for sequences that are highly expressed during early germination in the micropylar endosperm contained expansins and xyloglucan endo-transglycosylases/hydrolases (XTHs). Their transcript expression patterns in both species strongly suggest that they are regulated by distinct GID1-mediated GA signalling pathways. The GID1ac and GID1b pathways seem to fulfil distinct regulatory roles during Brassicaceae seed germination and seem to control their downstream targets distinctly.
Journal of Experimental Botany 07/2011; 62(14):5131-47. · 5.36 Impact Factor
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ABSTRACT: Most plant seeds are dispersed in a dry, mature state. If these seeds are non-dormant and the environmental conditions are favourable, they will pass through the complex process of germination. In this review, recent progress made with state-of-the-art techniques including genome-wide gene expression analyses that provided deeper insight into the early phase of seed germination, which includes imbibition and the subsequent plateau phase of water uptake in which metabolism is reactivated, is summarized. The physiological state of a seed is determined, at least in part, by the stored mRNAs that are translated upon imbibition. Very early upon imbibition massive transcriptome changes occur, which are regulated by ambient temperature, light conditions, and plant hormones. The hormones abscisic acid and gibberellins play a major role in regulating early seed germination. The early germination phase of Arabidopsis thaliana culminates in testa rupture, which is followed by the late germination phase and endosperm rupture. An integrated view on the early phase of seed germination is provided and it is shown that it is characterized by dynamic biomechanical changes together with very early alterations in transcript, protein, and hormone levels that set the stage for the later events. Early seed germination thereby contributes to seed and seedling performance important for plant establishment in the natural and agricultural ecosystem.
Journal of Experimental Botany 03/2011; 62(10):3289-309. · 5.36 Impact Factor
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ABSTRACT: The completion of germination in Lepidium sativum and other endospermic seeds (e.g. Arabidopsis [Arabidopsis thaliana]) is regulated by two opposing forces, the growth potential of the radicle (RAD) and the resistance to this growth from the micropylar endosperm cap (CAP) surrounding it. We show by puncture force measurement that the CAP progressively weakens during germination, and we have conducted a time-course transcript analysis of RAD and CAP tissues throughout this process. We have also used specific inhibitors to investigate the importance of transcription, translation, and posttranslation levels of regulation of endosperm weakening in isolated CAPs. Although the impact of inhibiting translation is greater, both transcription and translation are required for the completion of endosperm weakening in the whole seed population. The majority of genes expressed during this process occur in both tissues, but where they are uniquely expressed, or significantly differentially expressed between tissues, this relates to the functions of the RAD as growing tissue and the CAP as a regulator of germination through weakening. More detailed analysis showed that putative orthologs of cell wall-remodeling genes are expressed in a complex manner during CAP weakening, suggesting distinct roles in the RAD and CAP. Expression patterns are also consistent with the CAP being a receptor for environmental signals influencing germination. Inhibitors of the aspartic, serine, and cysteine proteases reduced the number of isolated CAPs in which weakening developed, and inhibition of the 26S proteasome resulted in its complete cessation. This indicates that targeted protein degradation is a major control point for endosperm weakening.
Plant physiology 02/2011; 155(4):1851-70. · 6.53 Impact Factor
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ABSTRACT: Magnetic resonance imaging (MRI) is a superior noninvasive diagnostic tool widely used in clinical medicine, with more than 60 million MRI tests performed each year worldwide. More specialized high-resolution MRI systems capable of a resolution that is 100-1,000 times higher than standard MRI instruments are used primarily in materials science, but are used with increasing frequency in plant physiology. We have shown that high-resolution (1)H-nuclear magnetic resonance (NMR) microimaging can provide a wealth of information about the internal anatomy of plant seeds as small as 1 mm or even smaller. This chapter covers the methods associated with these imaging techniques in detail. We also discuss the application of (1)H-NMR microimaging to study in vivo seed imbibition, germination, and early seedling growth.
Methods in molecular biology (Clifton, N.J.) 01/2011; 773:319-27.
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Ada Linkies, Kerstin Müller,
Karl Morris,
Veronika Turecková,
Meike Wenk,
Cassandra S C Cadman,
Françoise Corbineau,
Miroslav Strnad,
James R Lynn,
William E Finch-Savage,
Gerhard Leubner-Metzger
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ABSTRACT: The micropylar endosperm cap covering the radicle in the mature seeds of most angiosperms acts as a constraint that regulates seed germination. Here, we report on a comparative seed biology study with the close Brassicaceae relatives Lepidium sativum and Arabidopsis thaliana showing that ethylene biosynthesis and signaling regulate seed germination by a mechanism that requires the coordinated action of the radicle and the endosperm cap. The larger seed size of Lepidium allows direct tissue-specific biomechanical, biochemical, and transcriptome analyses. We show that ethylene promotes endosperm cap weakening of Lepidium and endosperm rupture of both species and that it counteracts the inhibitory action of abscisic acid (ABA) on these two processes. Cross-species microarrays of the Lepidium micropylar endosperm cap and the radicle show that the ethylene-ABA antagonism involves both tissues and has the micropylar endosperm cap as a major target. Ethylene counteracts the ABA-induced inhibition without affecting seed ABA levels. The Arabidopsis loss-of-function mutants ACC oxidase2 (aco2; ethylene biosynthesis) and constitutive triple response1 (ethylene signaling) are impaired in the 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated reversion of the ABA-induced inhibition of seed germination. Ethylene production by the ACC oxidase orthologs Lepidium ACO2 and Arabidopsis ACO2 appears to be a key regulatory step. Endosperm cap weakening and rupture are promoted by ethylene and inhibited by ABA to regulate germination in a process conserved across the Brassicaceae.
The Plant Cell 12/2009; 21(12):3803-22. · 8.99 Impact Factor
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ABSTRACT: Seed dormancy is genetically determined with substantial environmental influence mediated, at least in part, by the plant hormone abscisic acid (ABA). The ABA-related transcription factor ABI3/VP1 (ABA INSENSITIVE3/VIVIPAROUS1) is widespread among green plants. Alternative splicing of its transcripts appears to be involved in regulating seed dormancy, but the role of ABI3/VP1 goes beyond seeds and dormancy. In contrast, DOG1 (DELAY OF GERMINATION 1), a major quantitative trait gene more specifically involved in seed dormancy, was so far only known from Arabidopsis thaliana (AtDOG1) and whether it also has roles during the germination of non-dormant seeds was not known. Seed germination of Lepidium sativum ('garden cress') is controlled by ABA and its antagonists gibberellins and ethylene and involves the production of apoplastic hydroxyl radicals. We found orthologs of AtDOG1 in the Brassicaceae relatives L. sativum (LesaDOG1) and Brassica rapa (BrDOG1) and compared their gene structure and the sequences of their transcripts expressed in seeds. Tissue-specific analysis of LesaDOG1 transcript levels in L. sativum seeds showed that they are degraded upon imbibition in the radicle and the micropylar endosperm. ABA inhibits germination in that it delays radicle protrusion and endosperm weakening and it increased LesaDOG1 transcript levels during early germination due to enhanced transcription and/or inhibited degradation. A reduced decrease in LesaDOG1 transcript levels upon ABA treatment is evident in the late germination phase in both tissues. This temporal and ABA-related transcript expression pattern suggests a role for LesaDOG1 in the control of germination timing of non-dormant L. sativum seeds. The possible involvement of the ABA-related transcription factors ABI3 and ABI5 in the regulation of DOG1 transcript expression is discussed. Other species of the monophyletic genus Lepidium showed coat or embryo dormancy and are therefore highly suited for comparative seed biology.
Plant Molecular Biology 12/2009; 73(1-2):67-87. · 4.15 Impact Factor
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ABSTRACT: Mature angiosperm seeds consist of an embryo surrounded by the endosperm and the testa. The endosperm cap that covers the radicle plays a regulatory role during germination and is a major target of abscisic acid-induced inhibition of germination. Cress (Lepidium sativum) is a close relative of the model plant Arabidopsis thaliana (Arabidopsis). Cress seeds offer the unique possibility of performing tissue-specific proteomics due to their larger size while benefiting the genomic tools available for Arabidopsis. This work provides the first description of endosperm cap proteomics during seed germination. An analysis of the proteome of the cress endosperm cap at key stages during germination and after radicle protrusion in the presence and absence of abscisic acid led to the identification of 144 proteins, which were clustered by the changes in their abundances and categorized by function. Proteins with a function in energy production, protein stability and stress response were overrepresented among the identified endosperm cap proteins. This strongly suggests that the cress endosperm cap is not a storage tissue as the cereal endosperm but a metabolically very active tissue regulating the rate of radicle protrusion.
Proteomics 11/2009; 10(3):406-16. · 4.43 Impact Factor
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ABSTRACT: The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed.
Journal of Experimental Botany 10/2009; 61(2):491-502. · 5.36 Impact Factor
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ABSTRACT: *Seeds can enter a state of dormancy, in which they do not germinate under optimal environmental conditions. Dormancy can be broken during seed after-ripening in the low-hydrated state. *By screening enhancer trap lines of Arabidopsis, we identified a role for the NADPH-oxidase AtrbohB in after-ripening. Semiquantitative PCR was used to investigate AtrbohB transcripts in seeds. These methods were complemented with a pharmacological approach using the inhibitor diphenylene iodonium chloride (DPI) and biomechanical measurements in the Brassicaceae seed model system cress (Lepidium sativum) as well as protein carbonylation assays. *atrbohB mutants fail to after-ripen and show reduced protein oxidation. AtrbohB pre-mRNA is alternatively spliced in seeds in a hormonally and developmentally regulated manner. AtrbohB is a major producer of superoxide in germinating Arabidopsis seeds, and inhibition of superoxide production by diphenylene iodonium (DPI) leads to a delay in Arabidopsis and cress seed germination and cress endosperm weakening. *Reactive oxygen species produced by AtrbohB during after-ripening could act via abscisic acid (ABA) signalling or post-translational protein modifications. Alternative splicing could be a general mechanism in after-ripening: by altered processing of stored pre-mRNAs seeds could react quickly to environmental changes.
New Phytologist 09/2009; 184(4):885-97. · 6.64 Impact Factor
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ABSTRACT: The endosperm is a barrier for radicle protrusion of many angiosperm seeds. Rupture of the testa (seed coat) and rupture of the endosperm are two sequential events during the germination of Lepidium sativum L. and Arabidopsis thaliana (L.) Heyhn. Abscisic acid (ABA) specifically inhibits the endosperm rupture of these two closely related Brassicaceae species. Lepidium seeds are large enough to allow the direct measurement of endosperm weakening by the puncture force method. We found that the endosperm weakens prior to endosperm rupture and that ABA delays the onset and decreases the rate of this weakening process in a dose-dependent manner. An early embryo signal is required and sufficient to induce endosperm weakening, which afterwards appears to be an organ-autonomous process. Gibberellins can replace this embryo signal; de novo gibberellin biosynthesis occurs in the endosperm and weakening is regulated by the gibberellin/ABA ratio. Our results suggest that the control of radicle protrusion during the germination of Brassicaceae seeds is mediated, at least in part, by endosperm weakening. We propose that Lepidium is an emerging Brassicaceae model system for endosperm weakening and that the complementary advantages of Lepidium and Arabidopsis can be used in parallel experiments to investigate the molecular mechanisms of endosperm weakening.
Plant and Cell Physiology 08/2006; 47(7):864-77. · 4.70 Impact Factor
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ABSTRACT: The regulation of water uptake of germinating tobacco (Nicotiana tabacum) seeds was studied spatially and temporally by in vivo (1)H-nuclear magnetic resonance (NMR) microimaging and (1)H-magic angle spinning NMR spectroscopy. These nondestructive state-of-the-art methods showed that water distribution in the water uptake phases II and III is inhomogeneous. The micropylar seed end is the major entry point of water. The micropylar endosperm and the radicle show the highest hydration. Germination of tobacco follows a distinct pattern of events: rupture of the testa is followed by rupture of the endosperm. Abscisic acid (ABA) specifically inhibits endosperm rupture and phase III water uptake, but does not alter the spatial and temporal pattern of phase I and II water uptake. Testa rupture was associated with an increase in water uptake due to initial embryo elongation, which was not inhibited by ABA. Overexpression of beta-1,3-glucanase in the seed-covering layers of transgenic tobacco seeds did not alter the moisture sorption isotherms or the spatial pattern of water uptake during imbibition, but partially reverted the ABA inhibition of phase III water uptake and of endosperm rupture. In vivo (13)C-magic angle spinning NMR spectroscopy showed that seed oil mobilization is not inhibited by ABA. ABA therefore does not inhibit germination by preventing oil mobilization or by decreasing the water-holding capacity of the micropylar endosperm and the radicle. Our results support the proposal that different seed tissues and organs hydrate at different extents and that the micropylar endosperm region of tobacco acts as a water reservoir for the embryo.
Plant physiology 08/2005; 138(3):1538-51. · 6.53 Impact Factor
