SeedTransNet: a directional translational network revealing regulatory patterns during seed maturation and germination.

Seed maturation is the developmental process that prepares the embryo for the desiccated waiting period before germination. It is associated with a series of physiological changes leading to the establishment of seed dormancy, seed longevity, and desiccation tolerance. We studied translational changes during seed maturation and observed a gradual reduction in global translation during seed maturation. Transcriptome and translatome profiling revealed specific reduction in the translation of thousands of genes. By including previously published data on germination and seedling establishment, a regulatory network based on polysome occupancy data was constructed: SeedTransNet. Network analysis predicted translational regulatory pathways involving hundreds of genes with distinct functions. The network identified specific transcript sequence features suggesting separate translational regulatory circuits. The network revealed several seed maturation-associated genes as central nodes, and this was confirmed by specific seed phenotypes of the respective mutants. One of the regulators identified, an AWPM19 family protein, PM19-Like1 (PM19L1), was shown to regulate seed dormancy and longevity. This putative RNA-binding protein also affects the translational regulation of its target mRNA, as identified by SeedTransNet. Our data show the usefulness of SeedTransNet in identifying regulatory pathways during seed phase transitions.

Plasticity of maternal environment-dependent expression-QTLs of tomato seeds.

Seeds are essential for plant reproduction, survival, and dispersal. Germination ability and successful establishment of young seedlings strongly depend on seed quality and on environmental factors such as nutrient availability. In tomato (Solanum lycopersicum) and many other species, seed quality and seedling establishment characteristics are determined by genetic variation, as well as the maternal environment in which the seeds develop and mature. The genetic contribution to variation in seed and seedling quality traits and environmental responsiveness can be estimated at transcriptome level in the dry seed by mapping genomic loci that affect gene expression (expression QTLs) in contrasting maternal environments. In this study, we applied RNA-sequencing to construct a linkage map and measure gene expression of seeds of a tomato recombinant inbred line (RIL) population derived from a cross between S. lycopersicum (cv. Moneymaker) and S. pimpinellifolium (G1.1554). The seeds matured on plants cultivated under different nutritional environments, i.e., on high phosphorus or low nitrogen. The obtained single-nucleotide polymorphisms (SNPs) were subsequently used to construct a genetic map. We show how the genetic landscape of plasticity in gene regulation in dry seeds is affected by the maternal nutrient environment. The combined information on natural genetic variation mediating (variation in) responsiveness to the environment may contribute to knowledge-based breeding programs aiming to develop crop cultivars that are resilient to stressful environments.

A Role for Allantoate Amidohydrolase (AtAAH) in the Germination of Arabidopsis thaliana Seeds.

Seed dormancy is a very complex trait controlled by interactions between genetic and environmental factors. Nitrate is inversely correlated with seed dormancy in Arabidopsis. This is explained by the fact that seed dry storage (after-ripening) reduces the need for nitrogen for germination. When nitrate is absorbed by plants, it is first reduced to nitrite and then to ammonium for incorporation into amino acids, nucleic acids and chlorophyll. Previously, we showed that ALLANTOATE AMIDOHYDROLASE (AtAAH) transcripts are up-regulated in imbibed dormant seeds compared with after-ripened seeds. AAH is an enzyme in the uric acid catabolic pathway which catalyzes the hydrolysis of allantoate to yield CO2, NH3 and S-ureidoglycine. This pathway is the final stage of purine catabolism, and functions in plants and some bacteria to provide nitrogen, particularly when other nitrogen sources are depleted. Ataah mutant seeds are more dormant and accumulate high levels of allantoate, allantoin and urea, whereas energy-related metabolites and several amino acids are lower upon seed imbibition in comparison with Columbia-0. AtAAH expression could be detected during the early stages of seed development, with a transient increase around 8 d after pollination. AtAAH expression is the highest in mature pollen. The application of exogenous potassium nitrate can partly complement the higher dormancy phenotype of the Ataah mutant seeds, whereas other nitrogen sources cannot. Our results indicate that potassium nitrate does not specifically overcome the alleviated dormancy levels in Ataah mutant seeds, but promotes germination in general. Possible pathways by which AtAAH affects seed germination are discussed.

The mRNA-binding proteome of a critical phase transition during Arabidopsis seed germination.

Arabidopsis thaliana seed germination is marked by extensive translational control at two critical phase transitions. The first transition refers to the start of hydration, the hydration translational shift. The second shift, the germination translational shift (GTS) is the phase between testa rupture and radicle protrusion at which the seed makes the all or nothing decision to germinate. The mechanism behind the translational regulation at these phase transitions is unknown. RNA binding proteins (RBPs) are versatile players in the post-transcriptional control of messenger RNAs (mRNAs) and as such candidates for regulating translation during seed germination. Here, we report the mRNA binding protein repertoire of seeds during the GTS. Thirty seed specific RBPs and 22 dynamic RBPs were identified during the GTS, like the putative RBP Vacuolar ATPase subunit A and RBP HSP101. Several stress granule markers were identified in this study, which suggests that seeds are prepared to quickly adapt the translation of specific mRNAs in response to changes in environmental conditions during the GTS. Taken together this study provides a detailed insight into the world of RBPs during seed germination and their possible regulatory role during this developmentally regulated process.

The membrane associated NAC transcription factors ANAC060 and ANAC040 are functionally redundant in the inhibition of seed dormancy in Arabidopsis thaliana.

The NAC family of transcription factors is involved in plant development and various biotic and abiotic stresses. The Arabidopsis thaliana ANAC genes ANAC060, ANAC040, and ANAC089 are highly homologous based on protein and nucleotide sequence similarity. These three genes are predicted to be membrane bound transcription factors (MTFs) containing a conserved NAC domain, but divergent C-terminal regions. The anac060 mutant shows increased dormancy when compared with the wild type. Mutations in ANAC040 lead to higher seed germination under salt stress, and a premature stop codon in ANAC089 Cvi allele results in seeds exhibiting insensitivity to high concentrations of fructose. Thus, these three homologous MTFs confer distinct functions, although all related to germination. To investigate whether the differences in function are caused by a differential spatial or temporal regulation, or by differences in the coding sequence (CDS), we performed swapping experiments in which the promoter and CDS of the three MTFs were exchanged. Seed dormancy and salt and fructose sensitivity analyses of transgenic swapping lines in mutant backgrounds showed that there is functional redundancy between ANAC060 and ANAC040, but not between ANAC060 and ANAC089.

DELAY OF GERMINATION 1-LIKE 4 acts as an inducer of seed reserve accumulation.

More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION 1-LIKE 4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.

Detection of QTLs for genotype × environment interactions in tomato seeds and seedlings.

Seed quality and seedling establishment are the most important factors affecting successful crop development. They depend on the genetic background and are acquired during seed maturation and therefor, affected by the maternal environment under which the seeds develop. There is little knowledge about the genetic and environmental factors that affect seed quality and seedling establishment. The aim of this study is to identify the loci and possible molecular mechanisms involved in acquisition of seed quality and how these are controlled by adverse maternal conditions. For this, we used a tomato recombinant inbred line (RIL) population consisting of 100 lines which were grown under two different nutritional environmental conditions, high phosphate and low nitrate. Most of the seed germination traits such as maximum germination percentage (Gmax ), germination rate (t50 ) and uniformity (U8416 ) showed ample variation between genotypes and under different germination conditions. This phenotypic variation leads to identification of quantitative trait loci (QTLs) which were dependent on genetic factors, but also on the interaction with the maternal environment (QTL × E). Further studies of these QTLs may ultimately help to predict the effect of different maternal environmental conditions on seed quality and seedling establishment which will be very useful to improve the production of high-performance seeds.

NADP-MALIC ENZYME 1 Affects Germination after Seed Storage in Arabidopsis thaliana.

Aging decreases the quality of seeds and results in agricultural and economic losses. The damage that occurs at the biochemical level can alter the seed physiological status. Although loss of viability has been investigated frequently, little information exists on the molecular and biochemical factors involved in seed deterioration and loss of viability. Oxidative stress has been implicated as a major contributor to seed deterioration, and several pathways are involved in protection against this. In this study, we show that seeds of Arabidopsis thaliana lacking a functional NADP-MALIC ENZYME 1 (NADP-ME1) have reduced seed viability relative to the wild type. Seeds of the NADP-ME1 loss-of-function mutant display higher levels of protein carbonylation than those of the wild type. NADP-ME1 catalyzes the oxidative decarboxylation of malate to pyruvate with the simultaneous production of CO2 and NADPH. Upon seed imbibition, malate and amino acids accumulate in embryos of aged seeds of the NADP-ME1 loss-of-function mutant compared with those of the wild type. NADP-ME1 expression is increased in imbibed aged as compared with non-aged seeds. NADP-ME1 activity at testa rupture promotes normal germination of aged seeds. In seedlings of aged seeds, NADP-ME1 is specifically active in the root meristematic zone. We propose that NADP-ME1 activity is required for protecting seeds against oxidation during seed dry storage.

The interaction between genotype and maternal nutritional environments affects tomato seed and seedling quality.

Seed and seedling traits are affected by the conditions of the maternal environment, such as light, temperature, and nutrient availability. In this study, we have investigated whether different maternally applied nitrate and phosphate concentrations affect the seed and seedling performance of two closely related tomato species: Solanum lycopersicum cv. Money maker and Solanum pimpinellifolium accession CGN14498. We observed large differences for seed and seedling traits between the two species. Additionally, we have shown that for nitrate most of the seed and seedling traits were significantly affected by genotype-environment interactions (G×E). The effect of the maternal environment was clearly visible in the primary metabolites of the dry seeds. For example, we could show that the amount of γ-aminobutyric acid (GABA) in Money maker seeds was affected by the differences in the maternal environments and was positively correlated with seed germination under high temperature. Overall, compared with phosphate, nitrate had a larger effect on seed and seedling performance in tomato. In general, the different responses to the maternal environments of the two tomato species showed a major role for G×E in shaping seed and seedling traits.

Arabidopsis Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure.

Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), by hydrolyzing the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), upon receptor stimulation. In plants, PLC's role is still unclear, especially because the primary targets of both second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, and because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes nine PLCs. We used a reversed-genetic approach to explore PLC's function in Arabidopsis, and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10-20%. Mutant seeds germinated more slowly but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter-ß-glucuronidase (GUS) analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most probably phloem companion cells, in roots, leaves and flowers. In vivo 32Pi labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plant's tolerance to drought. Our results provide genetic evidence for PLC's involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed.

Characterization of and genetic variation for tomato seed thermo-inhibition and thermo-dormancy.

BACKGROUND: Exposing imbibed seeds to high temperatures may lead to either thermo-inhibition of germination or thermo-dormancy responses. In thermo-inhibition, seed germination is inhibited but quickly resumed when temperatures are lowered. Upon prolonged exposure to elevated temperatures, thermo-dormancy may be induced and seeds are not able to germinate even at optimal temperatures. In order to explore underlying physiological and molecular aspects of thermo-induced secondary dormancy, we have investigated the physiological responses of tomato seeds to elevated temperatures and the molecular mechanisms that could explain the performance of tomato seeds at elevated temperature. RESULTS: In order to investigate how tomato seeds respond to high temperature we used two distinct tomato accessions: Solanum lycopersicum (cv. Moneymaker) (MM) and Solanum pimpinellifolium accession CGN14498 (PI). MM seeds did not germinate under high temperature conditions while seeds of PI reached a maximum germination of 80%. Despite the high germination percentage of PI, germinated seeds did not produce healthy seedling at 37 °C. By using a candidate gene approach we have tested if similar molecular pathways (abscisic acid (ABA) and gibberellic acid (GA)) present in lettuce and Arabidopsis, are regulating thermo-inhibition and thermo-dormancy responses in tomato. We showed that the ABA biosynthesis pathway genes NCED1 and NCED9 were upregulated whereas two of the GA-biosynthesis regulators (GA3ox1 and GA20ox1) were downregulated in tomato thermo-dormant seeds at elevated temperature. To identify novel regulators of tomato seed performance under high temperature, we screened a Recombinant Inbred Line (RIL) population derived from a cross between the two tomato accessions MM and PI for thermo-inhibition and dormancy induction. Several QTLs were detected, particularly for thermo-dormancy, which may be caused by new regulators of thermo-inhibition and thermo-dormancy in tomato. CONCLUSIONS: None of the genes studied in this research were co-locating with the detected QTLs. The new QTLs discovered in this study will therefore be useful to further elucidate the molecular mechanisms underlying the responses of tomato seeds to high temperature and eventually lead to identification of the causal genes regulating these responses.

The Arabidopsis DELAY OF GERMINATION 1 gene affects ABSCISIC ACID INSENSITIVE 5 (ABI5) expression and genetically interacts with ABI3 during Arabidopsis seed development.

The seed expressed gene DELAY OF GERMINATION (DOG) 1 is absolutely required for the induction of dormancy. Next to a non-dormant phenotype, the dog1-1 mutant is also characterized by a reduced seed longevity suggesting that DOG1 may affect additional seed processes as well. This aspect however, has been hardly studied and is poorly understood. To uncover additional roles of DOG1 in seeds we performed a detailed analysis of the dog1 mutant using both transcriptomics and metabolomics to investigate the molecular consequences of a dysfunctional DOG1 gene. Further, we used a genetic approach taking advantage of the weak aba insensitive (abi) 3-1 allele as a sensitized genetic background in a cross with dog1-1. DOG1 affects the expression of hundreds of genes including LATE EMBRYOGENESIS ABUNDANT and HEAT SHOCK PROTEIN genes which are affected by DOG1 partly via control of ABI5 expression. Furthermore, the content of a subset of primary metabolites, which normally accumulate during seed maturation, was found to be affected in the dog1-1 mutant. Surprisingly, the abi3-1 dog1-1 double mutant produced green seeds which are highly ABA insensitive, phenocopying severe abi3 mutants, indicating that dog1-1 acts as an enhancer of the weak abi3-1 allele and thus revealing a genetic interaction between both genes. Analysis of the dog1 and dog1 abi3 mutants revealed additional seed phenotypes and therefore we hypothesize that DOG1 function is not limited to dormancy but that it is required for multiple aspects of seed maturation, in part by interfering with ABA signalling components.

Effects of Parental Temperature and Nitrate on Seed Performance are Reflected by Partly Overlapping Genetic and Metabolic Pathways.

Seed performance is affected by the seed maturation environment, and previously we have shown that temperature, nitrate and light intensity were the most influential environmental factors affecting seed performance. Seeds developed in these environments were selected to assess the underlying metabolic pathways, using a combination of transcriptomics and metabolomics. These analyses revealed that the effects of the parental temperature and nitrate environments were reflected by partly overlapping genetic and metabolic networks, as indicated by similar changes in the expression levels of metabolites and transcripts. Nitrogen metabolism-related metabolites (asparagine, γ-aminobutyric acid and allantoin) were significantly decreased in both low temperature (15 °C) and low nitrate (N0) maturation environments. Correspondingly, nitrogen metabolism genes (ALLANTOINASE, NITRATE REDUCTASE 1, NITRITE REDUCTASE 1 and NITRILASE 4) were differentially regulated in the low temperature and nitrate maturation environments, as compared with control conditions. High light intensity during seed maturation increased galactinol content, and displayed a high correlation with seed longevity. Low light had a genotype-specific effect on cell surface-encoding genes in the DELAY OF GERMINATION 6-near isogenic line (NILDOG6). Overall, the integration of phenotypes, metabolites and transcripts led to new insights into the regulation of seed performance.

Altitudinal and climatic associations of seed dormancy and flowering traits evidence adaptation of annual life cycle timing in Arabidopsis thaliana.

The temporal control or timing of the life cycle of annual plants is presumed to provide adaptive strategies to escape harsh environments for survival and reproduction. This is mainly determined by the timing of germination, which is controlled by the level of seed dormancy, and of flowering initiation. However, the environmental factors driving the evolution of plant life cycles remain largely unknown. To address this question we have analysed nine quantitative life history traits, in a native regional collection of 300 wild accessions of Arabidopsis thaliana. Seed dormancy and flowering time were negatively correlated, indicating that these traits have coevolved. In addition, environmental-phenotypic analyses detected strong altitudinal and climatic clines for most life history traits. Overall, accessions showing life cycles with early flowering, small seeds, high seed dormancy and slow germination rate were associated with locations exposed to high temperature, low summer precipitation and high radiation. Furthermore, we analysed the expression level of the positive regulator of seed dormancy DELAY OF GERMINATION 1 (DOG1), finding similar but weaker altitudinal and climatic patterns than seed dormancy. Therefore, DOG1 regulatory mutations are likely to provide a quantitative molecular mechanism for the adaptation of A. thaliana life cycle to altitude and climate.

Effect of osmopriming on germination and initial growth of Physalis angulata L. under salt stress and on expression of associated genes.

This study aimed to evaluate the effects of priming on seed germination under salt stress and gene expression in seeds and seedlings of P. angulata L. After priming for 10 days, seed germination was tested in plastic trays containing 15 ml of water (0 dS m-1 - control) or 15 ml of NaCl solution (2, 4, 6, 8, 10, 12, 14 and 16 dS m-1). Fresh and dry weight of shoots and roots of seedlings were evaluated at 0, 2, 4, 6, 8 dS m-1. Total RNA was extracted from whole seeds and seedlings followed by RT-qPCR. The target genes selected for this study were: ascorbate peroxidase (APX), glutathione-S-transferase (GST), thioredoxin (TXN), high affinity potassium transporter protein 1 (HAK1) and salt overly sensitive 1 (SOS1). At an electroconductivity of 14 dS m-1 the primed seeds still germinated to 72%, in contrast with the non-primed seeds which did not germinate. The relative expression of APX was higher in primed seeds and this may have contributed to the maintenance of high germination in primed seeds at high salt concentrations. GST and TXN displayed increased transcript levels in shoots and roots of seedlings from primed seeds. Priming improved seed germination as well as salt tolerance and this is correlated with increased expression of APX in seeds and SOS1, GST and TXN in seedlings.

Identifying genotype-by-environment interactions in the metabolism of germinating arabidopsis seeds using generalized genetical genomics.

A complex phenotype such as seed germination is the result of several genetic and environmental cues and requires the concerted action of many genes. The use of well-structured recombinant inbred lines in combination with "omics" analysis can help to disentangle the genetic basis of such quantitative traits. This so-called genetical genomics approach can effectively capture both genetic and epistatic interactions. However, to understand how the environment interacts with genomic-encoded information, a better understanding of the perception and processing of environmental signals is needed. In a classical genetical genomics setup, this requires replication of the whole experiment in different environmental conditions. A novel generalized setup overcomes this limitation and includes environmental perturbation within a single experimental design. We developed a dedicated quantitative trait loci mapping procedure to implement this approach and used existing phenotypical data to demonstrate its power. In addition, we studied the genetic regulation of primary metabolism in dry and imbibed Arabidopsis (Arabidopsis thaliana) seeds. In the metabolome, many changes were observed that were under both environmental and genetic controls and their interaction. This concept offers unique reduction of experimental load with minimal compromise of statistical power and is of great potential in the field of systems genetics, which requires a broad understanding of both plasticity and dynamic regulation.

Visualizing the genetic landscape of Arabidopsis seed performance.

Perfect timing of germination is required to encounter optimal conditions for plant survival and is the result of a complex interaction between molecular processes, seed characteristics, and environmental cues. To detangle these processes, we made use of natural genetic variation present in an Arabidopsis (Arabidopsis thaliana) Bayreuth × Shahdara recombinant inbred line population. For a detailed analysis of the germination response, we characterized rate, uniformity, and maximum germination and discuss the added value of such precise measurements. The effects of after-ripening, stratification, and controlled deterioration as well as the effects of salt, mannitol, heat, cold, and abscisic acid (ABA) with and without cold stratification were analyzed for these germination characteristics. Seed morphology (size and length) of both dry and imbibed seeds was quantified by using image analysis. For the overwhelming amount of data produced in this study, we developed new approaches to perform and visualize high-throughput quantitative trait locus (QTL) analysis. We show correlation of trait data, (shared) QTL positions, and epistatic interactions. The detection of similar loci for different stresses indicates that, often, the molecular processes regulating environmental responses converge into similar pathways. Seven major QTL hotspots were confirmed using a heterogeneous inbred family approach. QTLs colocating with previously reported QTLs and well-characterized mutants are discussed. A new connection between dormancy, ABA, and a cripple mucilage formation due to a naturally occurring mutation in the MUCILAGE-MODIFIED2 gene is proposed, and this is an interesting lead for further research on the regulatory role of ABA in mucilage production and its multiple effects on germination parameters.

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.

Complex genetics controls natural variation among seed quality phenotypes in a recombinant inbred population of an interspecific cross between Solanum lycopersicum × Solanum pimpinellifolium.

Seed quality in tomato is associated with many complex physiological and genetic traits. While plant processes are frequently controlled by the action of small- to large-effect genes that follow classic Mendelian inheritance, our study suggests that seed quality is primarily quantitative and genetically complex. Using a recombinant inbred line population of Solanum lycopersicum × Solanum pimpinellifolium, we identified quantitative trait loci (QTLs) influencing seed quality phenotypes under non-stress, as well as salt, osmotic, cold, high-temperature and oxidative stress conditions. In total, 42 seed quality traits were analysed and 120 QTLs were identified for germination traits under different conditions. Significant phenotypic correlations were observed between germination traits under optimal conditions, as well as under different stress conditions. In conclusion, one or more QTLs were identified for each trait with some of these QTLs co-locating. Co-location of QTLs for different traits can be an indication that a locus has pleiotropic effects on multiple traits due to a common mechanistic basis. However, several QTLs also dissected seed quality in its separate components, suggesting different physiological mechanisms and signalling pathways for different seed quality attributes.

GERMINATOR: a software package for high-throughput scoring and curve fitting of Arabidopsis seed germination.

Over the past few decades seed physiology research has contributed to many important scientific discoveries and has provided valuable tools for the production of high quality seeds. An important instrument for this type of research is the accurate quantification of germination; however gathering cumulative germination data is a very laborious task that is often prohibitive to the execution of large experiments. In this paper we present the germinator package: a simple, highly cost-efficient and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The germinator package contains three modules: (i) design of experimental setup with various options to replicate and randomize samples; (ii) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; and (iii) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters. The curve-fitting module enables analysis of general cumulative germination data and can be used for all plant species. We show that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well. In this paper we show the accuracy, reproducibility and flexibility of the germinator package. We have successfully applied it to evaluate natural variation for salt tolerance in a large population of recombinant inbred lines and were able to identify several quantitative trait loci for salt tolerance. Germinator is a low-cost package that allows the monitoring of several thousands of germination tests, several times a day by a single person.