Distinct cell wall architectures in seed endosperms in representatives of the brassicaceae and solanaceae

In some species, a crucial role has been demonstrated for the seed endosperm during germination. The endosperm has been shown to integrate environmental cues with hormonal networks that underpin dormancy and seed germination, a process that involves the action of cell wall remodeling enzymes (CWREs). Here, we examine the cell wall architectures of the endosperms of two related Brassicaceae, Arabidopsis (Arabidopsis thaliana) and the close relative Lepidium (Lepidium sativum), and that of the Solanaceous species, tobacco (Nicotiana tabacum). The Brassicaceae species have a similar cell wall architecture that is rich in pectic homogalacturonan, arabinan, and xyloglucan. Distinctive features of the tobacco endosperm that are absent in the Brassicaceae representatives are major tissue asymmetries in cell wall structural components that reflect the future site of radicle emergence and abundant heteromannan. Cell wall architecture of the micropylar endosperm of tobacco seeds has structural components similar to those seen in Arabidopsis and Lepidium endosperms. In situ and biomechanical analyses were used to study changes in endosperms during seed germination and suggest a role for mannan degradation in tobacco. In the case of the Brassicaceae representatives, the structurally homogeneous cell walls of the endosperm can be acted on by spatially regulated CWRE expression. Genetic manipulations of cell wall components present in the Arabidopsis seed endosperm demonstrate the impact of cell wall architectural changes on germination kinetics

Sugar signalling during germination and early seedling establishment in Arabidopsis

Sugars have pronounced effects on many plant processes like gene expression, germination and early seedling development. Several screens for sugar insensitive mutants were performed to identify genes involved in sugar response pathways using the model plant Arabidopsis. These include sun, gin and sis screens explained earlier in this chapter. Both common mutants and dissimilar mutants were identified in the different screens. Interestingly, ABA and ABI4 seem to play an essential function in all these sugar response assays. To gain further insight in sugar signalling we studied sugar response pathways encompassing germination and early seedling establishment. We focussed our research on the sugar-regulated expression of PC in dark-grown seedlings, the glucose-induced delay of germination and the sugar-induced early seedling developmental block (gin and sis response). In Chapter 2 (and partly this Chapter) we describe that sugars delay Arabidopsis seed germination. Already low concentrations of glucose are able to delay seed germination and this delay is independent from osmotic signalling. Analysing mutants with a gin phenotype like ctr1, abi4 and abi5 shows that these mutants respond like wt to glucose with respect of the delay of germination. This indicates that the delay of germination and the gin response are two different processes. Since additional studies that show that stratification of seeds on sugar-free media suppresses the glucose-induced delay of germination but not the glucose-induced early seedling development arrest supports this conclusion. In Chapter 3 we propose a function for ABI3 (and ABI2) in sugar responses as well. We found that ABI3, ABI4 and ABI5 levels rise in seedlings that are challenged with glucose. abi3 mutants are insensitive for a range of sugar assays and abi3 mutants show a severely reduced induction of ABI4 and ABI5 after sugar treatment. These results suggests that ABA and sugar signalling (gin pathway) are more alike than assumed before. It has been hypothesized that elevated sugar concentrations induce ABA signalling by increasing ABA levels. Despite the large overlap in signalling components, analysis of a glucose and ABA insensitive mutant over expressing ABI4 (abi5-1/35S::ABI4) revealed that glucose and ABA are two distinct signals. In Chapter 4 we investigated to sun response pathway in more detail. Thus far, only one sun mutant, sun6/abi4, has been identified. We tested other sugar and ABA signalling mutants for their sun response. The other way around, sun mutants were tested for their gin response as well. Remarkably, ABA deficient mutants, which are strong gin mutants, do not show a significant sun phenotype. Thus, ABA is important for the gin response pathway but not in the sun pathway that regulates PC levels in response to sucrose. In Chapter 5 the results of the preceding chapters are summarized and discussed. The similarities and differences between the sugar response pathways studied are presented

Regulation of seed dormancy by abscisic acid and DELAY OF Germination 1

Physiological dormancy has been described as a physiological inhibiting mechanism that prevents radicle emergence. It can be caused by the embryo (embryo dormancy) as well as by the structures that cover the embryo. One of its functions is to time plant growth and reproduction to the most optimal season and therefore, in nature, dormancy is an important adaptive trait that is under selective pressure. Dormancy is a complex trait that is affected by many loci, as well as by an intricate web of plant hormone interactions. Moreover, it is strongly affected by a multitude of environmental factors. Its induction, maintenance, cycling and loss come down to the central paradigm, which is the balance between two key hormonal regulators, i.e. the plant hormone abscisic acid (ABA), which is required for dormancy induction, and gibberellins (GA), which are required for germination. In this review we will summarize recent developments in dormancy research (mainly) in the model plant Arabidopsis thaliana, focusing on two key players for dormancy induction, i.e. the plant hormone ABA and the DELAY OF GERMINATION 1 (DOG1) gene. We will address the role of ABA and DOG1 in relation to various aspects of seed dormancy, i.e. induction during seed maturation, loss during dry seed afterripening, the rehydrated state (including dormancy cycling) and the switch to germination

Visualization of molecular processes associated with seed dormancy and germination using MapMan

Seed dormancy and germination involve the concerted operation of molecular and biochemical programmes. It has become feasible to study these processes in great detail, using the current methods for transcriptome, proteome and metabolome analysis. Yet, the large amounts of data generated by these methods are often dazzling and demand efficient tools for data visualization. We have used the freely available PageMan/MapMan package (http://MapMan.gabipd.org) to visualize transcriptome and metabolome changes in Arabidopsis thaliana seeds during dormancy and germination. Using this package we developed two seed-specific MapMan pathways, which efficiently capture the most important molecular processes in seeds. The results demonstrated the usefulness of the PageMan/MapMan package for seed research

Abscisic acid (ABA) sensitivity regulates desiccation tolerance in germinated Arabidopsis seeds

During germination, orthodox seeds lose their desiccation tolerance (DT) and become sensitive to extreme drying. Yet, DT can be rescued, in a well-defined developmental window, by the application of a mild osmotic stress before dehydration. A role for abscisic acid (ABA) has been implicated in this stress response and in DT re-establishment. However, the path from the sensing of an osmotic cue and its signaling to DT re-establishment is still largely unknown. Analyses of DT, ABA sensitivity, ABA content and gene expression were performed in desiccation- sensitive (DS) and desiccation-tolerant Arabidopsis thaliana seeds. Furthermore, loss and re-establishment of DT in germinated Arabidopsis seeds was studied in ABA-deficient and ABA-insensitive mutants. We demonstrate that the developmental window in which DT can be re-established correlates strongly with the window in which ABA sensitivity is still present. Using ABA biosynthesis and signaling mutants, we show that this hormone plays a key role in DT re-establishment. Surprisingly, re-establishment of DT depends on the modulation of ABA sensitivity rather than enhanced ABA content. In addition, the evaluation of several ABA-insensitive mutants, which can still produce normal desiccation-tolerant seeds, but are impaired in the re-establishment of DT, shows that the acquisition of DT during seed development is genetically different from its re-establishment during germination

Galactinol as marker for seed longevity

Reduced seed longevity or storability is a major problem in seed storage and contributes to increasedcosts in crop production. Here we investigated whether seed galactinol contents could be predictive forseed storability behavior in Arabidopsis, cabbage and tomato. The analyses revealed a positive correla-tion between galactinol content and seed longevity in the three species tested, which indicates that thiscorrelation is conserved in the Brassicaceae and beyond. Quantitative trait loci (QTL) mapping in tomatorevealed a co-locating QTL for galactinol content and seed longevity on chromosome 2. A candidate for thisQTL is the GALACTINOL SYNTHASE gene (Solyc02g084980.2.1) that is located in the QTL interval. GALACTI-NOL SYNTHASE is a key enzyme of the raffinose family oligosaccharide (RFO) pathway. To investigate therole of enzymes in the RFO pathway in more detail, we applied a reverse genetics approach using T-DNAknock-out lines in genes encoding enzymes of this pathway (GALACTINOL SYNTHASE 1, GALACTINOL SYN-THASE 2, RAFFINOSE SYNTHASE, STACHYOSE SYNTHASE and ALPHA-GALACTOSIDASE) and overexpressorsof the cucumber GALACTINOL SYNTHASE 2 gene in Arabidopsis. The galactinol synthase 2 mutant and thegalactinol synthase 1 galactinol synthase 2 double mutant contained the lowest seed galactinol contentwhich coincided with lower seed longevity. These results show that galactinol content of mature dryseed can be used as a biomarker for seed longevity in Brassicaceae and tomato

Identification of reference genes for gene expression studies during seed germination and seedling establishment Ricinus communis L.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available

Identification of Reference Genes For RT-qPCR Expression Analysis In Arabidopsis And Tomato Seeds

Quantifying gene expression levels is an important research tool to understand biological systems. Reverse transcription–quantitative real-time PCR (RT–qPCR) is the preferred method for targeted gene expression measurements because of its sensitivity and reproducibility. However, normalization, necessary to correct for sample input and reverse transcriptase efficiency, is a crucial step to obtain reliable RT–qPCR results. Stably expressed genes (i.e. genes whose expression is not affected by the treatment or developmental stage under study) are indispensable for accurate normalization of RT–qPCR experiments. Lack of accurate normalization could affect the results and may lead to false conclusions. Since transcriptomes of seeds are different from other plant tissues, we aimed to identify reference genes specifically for RT–qPCR analyses in seeds of two important seed model species, i.e. Arabidopsis and tomato. We mined Arabidopsis seed microarray data to identify stably expressed genes and analyzed these together with putative reference genes from other sources. In total, the expression stability of 24 putative reference genes was validated by RT–qPCR in Arabidopsis seed samples. For tomato, we lacked transcriptome data sets of seeds and therefore we tested the tomato homologs of the reference genes found for Arabidopsis seeds. In conclusion, we identified 14 Arabidopsis and nine tomato reference genes. This provides a valuable resource for accurate normalization of gene expression experiments in seed research for two important seed model species

Transcriptional dynamics of two seed compartments with opposing roles in Arabidopsis seed germination

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