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 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.

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.

Evaluating the EPPO method for seed longevity analyses in Arabidopsis.

Seed longevity (storability) is an important seed quality trait. High seed quality is important in agriculture, for the industry, and for safeguarding biodiversity as many species are stored as seeds in genebanks. To ensure ex-situ seed survival, seeds are mostly stored at low relative humidity and low temperature. Oxidation is the main cause of seed deterioration in these dry storage conditions. The molecular mechanisms underlying dry seed survival remain poorly understood. Research on seed longevity is hampered by the lack of an experimental ageing method that mimics dry ageing well. Here, we propose the Elevated Partial Pressure of Oxygen (EPPO) method as the best available method to mimic and accelerate dry seed ageing. We have tested seed germination in Arabidopsis thaliana after EPPO storage at two different relative humidity (RH) conditions and confirm the large effect of oxygen and the seed moisture content on ageing during dry storage. Comparative Quantitative trait locus (QTL) analysis shows that EPPO at 55 % RH mimics dry ageing better than the commonly used Artificial Ageing and Controlled Deterioration tests at higher moisture levels.

Network Analysis Prioritizes DEWAX and ICE1 as the Candidate Genes for Major eQTL Hotspots in Seed Germination of Arabidopsis thaliana.

Seed germination is characterized by a constant change of gene expression across different time points. These changes are related to specific processes, which eventually determine the onset of seed germination. To get a better understanding on the regulation of gene expression during seed germination, we performed a quantitative trait locus mapping of gene expression (eQTL) at four important seed germination stages (primary dormant, after-ripened, six-hour after imbibition, and radicle protrusion stage) using Arabidopsis thaliana Bay x Sha recombinant inbred lines (RILs). The mapping displayed the distinctness of the eQTL landscape for each stage. We found several eQTL hotspots across stages associated with the regulation of expression of a large number of genes. Interestingly, an eQTL hotspot on chromosome five collocates with hotspots for phenotypic and metabolic QTL in the same population. Finally, we constructed a gene co-expression network to prioritize the regulatory genes for two major eQTL hotspots. The network analysis prioritizes transcription factors DEWAX and ICE1 as the most likely regulatory genes for the hotspot. Together, we have revealed that the genetic regulation of gene expression is dynamic along the course of seed germination.

Arabidopsis in the Wild-The Effect of Seasons on Seed Performance.

Climate changes play a central role in the adaptive life histories of organisms all over the world. In higher plants, these changes may impact seed performance, both during seed development and after dispersal. To examine the plasticity of seed performance as a response to environmental fluctuations, eight genotypes known to be affected in seed dormancy and longevity were grown in the field in all seasons of two years. Soil and air temperature, day length, precipitation, and sun hours per day were monitored. We show that seed performance depends on the season. Seeds produced by plants grown in the summer, when the days began to shorten and the temperature started to decrease, were smaller with deeper dormancy and lower seed longevity compared to the other seasons when seeds were matured at higher temperature over longer days. The performance of seeds developed in the different seasons was compared to seeds produced in controlled conditions. This revealed that plants grown in a controlled environment produced larger seeds with lower dormancy than those grown in the field. All together the results show that the effect of the environment largely overrules the genetic effects, and especially, differences in seed dormancy caused by the different seasons were larger than the differences between the genotypes.

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.

Reciprocal cybrids reveal how organellar genomes affect plant phenotypes.

Assessment of the impact of variation in chloroplast and mitochondrial DNA (collectively termed the plasmotype) on plant phenotypes is challenging due to the difficulty in separating their effect from nuclear-derived variation (the nucleotype). Haploid-inducer lines can be used as efficient plasmotype donors to generate new plasmotype-nucleotype combinations (cybrids)1. We generated a panel comprising all possible cybrids of seven Arabidopsis thaliana accessions and extensively phenotyped these lines for 1,859 phenotypes under both stable and fluctuating conditions. We show that natural variation in the plasmotype results in both additive and epistatic effects across all phenotypic categories. Plasmotypes that induce more additive phenotypic changes also cause more epistatic effects, suggesting a possible common basis for both additive and epistatic effects. On average, epistatic interactions explained twice as much of the variance in phenotypes as additive plasmotype effects. The impact of plasmotypic variation was also more pronounced under fluctuating and stressful environmental conditions. Thus, the phenotypic impact of variation in plasmotypes is the outcome of multi-level nucleotype-plasmotype-environment interactions and, as such, the plasmotype is likely to serve as a reservoir of variation that is predominantly exposed under certain conditions. The production of cybrids using haploid inducers is a rapid and precise method for assessment of the phenotypic effects of natural variation in organellar genomes. It will facilitate efficient screening of unique nucleotype-plasmotype combinations to both improve our understanding of natural variation in these combinations and identify favourable combinations to enhance plant performance.

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.

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.

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.

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.

Construction of a High-Density Genetic Map from RNA-Seq Data for an Arabidopsis Bay-0 × Shahdara RIL Population.

High-density genetic maps are essential for high resolution mapping of quantitative traits. Here, we present a new genetic map for an Arabidopsis Bayreuth × Shahdara recombinant inbred line (RIL) population, built on RNA-seq data. RNA-seq analysis on 160 RILs of this population identified 30,049 single-nucleotide polymorphisms (SNPs) covering the whole genome. Based on a 100-kbp window SNP binning method, 1059 bin-markers were identified, physically anchored on the genome. The total length of the RNA-seq genetic map spans 471.70 centimorgans (cM) with an average marker distance of 0.45 cM and a maximum marker distance of 4.81 cM. This high resolution genotyping revealed new recombination breakpoints in the population. To highlight the advantages of such high-density map, we compared it to two publicly available genetic maps for the same population, comprising 69 PCR-based markers and 497 gene expression markers derived from microarray data, respectively. In this study, we show that SNP markers can effectively be derived from RNA-seq data. The new RNA-seq map closes many existing gaps in marker coverage, saturating the previously available genetic maps. Quantitative trait locus (QTL) analysis for published phenotypes using the available genetic maps showed increased QTL mapping resolution and reduced QTL confidence interval using the RNA-seq map. The new high-density map is a valuable resource that facilitates the identification of candidate genes and map-based cloning approaches.

Metabolomic analysis of tomato seed germination.

INTRODUCTION: Seed germination is inherently related to seed metabolism, which changes throughout its maturation, desiccation and germination processes. The metabolite content of a seed and its ability to germinate are determined by underlying genetic architecture and environmental effects during development. OBJECTIVE: This study aimed to assess an integrative approach to explore genetics modulating seed metabolism in different developmental stages and the link between seed metabolic- and germination traits. METHODS: We have utilized gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) metabolite profiling to characterize tomato seeds during dry and imbibed stages. We describe, for the first time in tomato, the use of a so-called generalized genetical genomics (GGG) model to study the interaction between genetics, environment and seed metabolism using 100 tomato recombinant inbred lines (RILs) derived from a cross between Solanum lycopersicum and Solanum pimpinellifolium. RESULTS: QTLs were found for over two-thirds of the metabolites within several QTL hotspots. The transition from dry to 6 h imbibed seeds was associated with programmed metabolic switches. Significant correlations varied among individual metabolites and the obtained clusters were significantly enriched for metabolites involved in specific biochemical pathways. CONCLUSIONS: Extensive genetic variation in metabolite abundance was uncovered. Numerous identified genetic regions that coordinate groups of metabolites were detected and these will contain plausible candidate genes. The combined analysis of germination phenotypes and metabolite profiles provides a strong indication for the hypothesis that metabolic composition is related to germination phenotypes and thus to seed performance.

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.

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.

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.

Metabolite profiling of Ricinus communis germination at different temperatures provides new insights into thermo-mediated requirements for successful seedling establishment.

Ricinus communis seeds germinate to a high percentage and faster at 35 °C than at lower temperatures, but with compromised seedling establishment. However, seedlings are able to cope with high temperatures at later stages of seedling establishment if germination occurred at lower temperatures. Our objective was to assess the biochemical and molecular requirements of R. communis germination for successful seedling establishment at varying temperatures. For that, we performed metabolite profiling (GC-TOF-MS) and measured transcript levels of key genes involved in several energy-generating pathways, such as storage oil mobilization, ß-oxidation and gluconeogenesis of seeds germinated at three different temperatures. We identified a thermo-sensitive window during seed germination in which high temperatures compromise seedling development, most likely by down-regulating some energy-generating pathways. Overexpression of malate synthase (MLS) and glycerol kinase (GK) genes resulted in higher starch levels in Nicotiana benthamiana leaves, which highlights the importance of these genes in energy-generating pathways for seedling establishment. Additionally, we showed that GABA, which is a stress-responsive metabolite, accumulated in response to the water content of the seeds during the initial phase of imbibition. Herewith, we provide new insights into the molecular requirements for vigorous seedling growth of R. communis under different environmental conditions.