Kerstin Müller

First off the mark: early seed germination

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.

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By 3H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ·OH in radicles and endosperm caps: the production and action of ·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ·OH attack on polysaccharides were evident. In vivo ·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ·OH is a controlled action of this type of reactive oxygen species.

Ethylene Interacts with Abscisic Acid to Regulate Endosperm Rupture during Germination: A Comparative Approach Using Lepidium sativum and Arabidopsis thaliana

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.

Water Uptake and Distribution in Germinating Tobacco Seeds Investigated in Vivo by Nuclear Magnetic Resonance Imaging

The regulation of water uptake of germinating tobacco (Nicotiana tabacum) seeds was studied spatially and temporally by in vivo 1H-nuclear magnetic resonance (NMR) microimaging and 1H-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 β-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 13C-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.

The NADPH‐oxidase AtrbohB plays a role in Arabidopsis seed after‐ripening

*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.

Transcriptional Dynamics of Two Seed Compartments with Opposing Roles in Arabidopsis Seed Germination

Gene expression profiling in two seed compartments uncovers two transcriptional phases during seed germination that are separated by testa rupture. Seed germination is a critical stage in the plant life cycle and the first step toward successful plant establishment. Therefore, understanding germination is of important ecological and agronomical relevance. Previous research revealed that different seed compartments (testa, endosperm, and embryo) control germination, but little is known about the underlying spatial and temporal transcriptome changes that lead to seed germination. We analyzed genome-wide expression in germinating Arabidopsis (Arabidopsis thaliana) seeds with both temporal and spatial detail and provide Web-accessible visualizations of the data reported (vseed.nottingham.ac.uk). We show the potential of this high-resolution data set for the construction of meaningful coexpression networks, which provide insight into the genetic control of germination. The data set reveals two transcriptional phases during germination that are separated by testa rupture. The first phase is marked by large transcriptome changes as the seed switches from a dry, quiescent state to a hydrated and active state. At the end of this first transcriptional phase, the number of differentially expressed genes between consecutive time points drops. This increases again at testa rupture, the start of the second transcriptional phase. Transcriptome data indicate a role for mechano-induced signaling at this stage and subsequently highlight the fates of the endosperm and radicle: senescence and growth, respectively. Finally, using a phylotranscriptomic approach, we show that expression levels of evolutionarily young genes drop during the first transcriptional phase and increase during the second phase. Evolutionarily old genes show an opposite pattern, suggesting a more conserved transcriptome prior to the completion of germination.

Cross-species approaches to seed dormancy and germination : conservation and biodiversity of ABA-regulated mechanisms and the Brassicaceae DOG1 genes

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.

Members of the gibberellin receptor gene family GID1 (GIBBERELLIN INSENSITIVE DWARF1) play distinct roles during Lepidium sativum and Arabidopsis thaliana seed germination

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.

Demethylesterification of Cell Wall Pectins in Arabidopsis Plays a Role in Seed Germination

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 (Arabidopsis thaliana), PME activities peaked around the time of testa rupture but declined just before the completion of germination (endosperm weakening and rupture). We overexpressed an Arabidopsis PME inhibitor 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 with wild-type seeds, the PME activities of the overexpressing 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 scanning electron microscopy. The transgenic seeds also exhibited an apparent reduced sensitivity to abscisic acid 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.

Tuning of pectin methylesterification: consequences for cell wall biomechanics and development.

AbstractMain conclusionRecent publications have increased our knowledge of how pectin composition and the degree of homogalacturonan methylesterification impact the biochemical and biomechanical properties of plant cell walls, plant development, and plants’ interactions with their abiotic and biotic environments. Experimental observations have shown that the relationships between the DM, the pattern of de-methylesterificaton, its effect on cell wall elasticity, other biomechanical parameters, and growth are not straightforward. Working towards a detailed understanding of these relationships at single cell resolution is one of the big tasks of pectin research. Pectins are highly complex polysaccharides abundant in plant primary cell walls. New analytical and microscopy techniques are revealing the composition and mechanical properties of the cell wall and increasing our knowledge on the topic. Progress in plant physiological research supports a link between cell wall pectin modifications and plant development and interactions with the environment. Homogalacturonan pectins, which are major components of the primary cell wall, have a potential for modifications such as methylesterification, as well as an ability to form cross-linked structures with divalent cations. This contributes to changing the mechanical properties of the cell wall. This review aims to give a comprehensive overview of the pectin component homogalacturonan, including its synthesis, modification, regulation and role in the plant cell wall.