A commitment for life: Decades of unraveling the molecular mechanisms behind seed dormancy and germination.

Seeds are unique time capsules that can switch between 2 complex and highly interlinked stages: seed dormancy and germination. Dormancy contributes to the survival of plants because it allows to delay germination to optimal conditions. The switch between dormancy and germination occurs in response to developmental and environmental cues. In this review we provide a comprehensive overview of studies that have helped to unravel the molecular mechanisms underlying dormancy and germination over the last decades. Genetic and physiological studies provided a strong foundation for this field of research and revealed the critical role of the plant hormones abscisic acid and gibberellins in the regulation of dormancy and germination, and later natural variation studies together with quantitative genetics identified previously unknown genetic components that control these processes. Omics technologies like transcriptome, proteome, and translatomics analysis allowed us to mechanistically dissect these processes and identify new components in the regulation of seed dormancy and germination.

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.

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.

Dormancy cycling: translation-related transcripts are the main difference between dormant and non-dormant seeds in the field.

Primary seed dormancy is a mechanism that orchestrates the timing of seed germination in order to prevent out-of-season germination. Secondary dormancy can be induced in imbibed seeds when they encounter prolonged unfavourable conditions. Secondary dormancy is not induced during dry storage, and therefore the mechanisms underlying this process have remained largely unexplored. Here, a 2-year seed burial experiment in which dormancy cycling was studied at the physiological and transcriptional level is presented. For these analyses six different Arabidopsis thaliana genotypes were used: Landsberg erecta (Ler) and the dormancy associated DELAY OF GERMINATION (DOG) near-isogenic lines 1, 2, 3, 6 and 22 (NILDOG1, 2, 3, 6 and 22). The germination potential of seeds exhumed from the field showed that these seeds go through dormancy cycling and that the dynamics of this cycling is genotype dependent. RNA-seq analysis revealed large transcriptional changes during dormancy cycling, especially at the time points preceding shifts in dormancy status. Dormancy cycling is driven by soil temperature and the endosperm is important in the perception of the environment. Genes that are upregulated in the low- to non-dormant stages are enriched for genes involved in translation, indicating that the non-dormant seeds are prepared for rapid seed germination.

Seed-Stored mRNAs that Are Specifically Associated to Monosomes Are Translationally Regulated during Germination.

The life cycle of many organisms includes a quiescent stage, such as bacterial or fungal spores, insect larvae, or plant seeds. Common to these stages is their low water content and high survivability during harsh conditions. Upon rehydration, organisms need to reactivate metabolism and protein synthesis. Plant seeds contain many mRNAs that are transcribed during seed development. Translation of these mRNAs occurs during early seed germination, even before the requirement of transcription. Therefore, stored mRNAs are postulated to be important for germination. How these mRNAs are stored and protected during long-term storage is unknown. The aim of this study was to investigate how mRNAs are stored in dry seeds and whether they are indeed translated during seed germination. We investigated seed polysome profiles and the mRNAs and protein complexes that are associated with these ribosomes in seeds of the model organism Arabidopsis (Arabidopsis thaliana). We showed that most stored mRNAs are associated with monosomes in dry seeds; therefore, we focus on monosomes in this study. Seed ribosome complexes are associated with mRNA-binding proteins, stress granule, and P-body proteins, which suggests regulated packing of seed mRNAs. Interestingly, ∼17% of the mRNAs that are specifically associated with monosomes are translationally up-regulated during seed germination. These mRNAs are transcribed during seed maturation, suggesting a role for this developmental stage in determining the translational fate of mRNAs during early germination.

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.

Combined transcriptome and translatome analyses reveal a role for tryptophan-dependent auxin biosynthesis in the control of DOG1-dependent seed dormancy.

The importance of translational regulation during Arabidopsis seed germination has been shown previously. Here the role of transcriptional and translational regulation during seed imbibition of the very dormant DELAY OF GERMINATION 1 (DOG1) near-isogenic line was investigated. Polysome profiling was performed on dormant and after-ripened seeds imbibed for 6 and 24 h in water and in the transcription inhibitor cordycepin. Transcriptome and translatome changes were investigated. Ribosomal profiles of after-ripened seeds imbibed in cordycepin mimic those of dormant seeds. The polysome occupancy of mRNA species is not affected by germination inhibition, either as a result of seed dormancy or as a result of cordycepin treatment, indicating the importance of the regulation of transcript abundance. The expression of auxin metabolism genes is discriminative during the imbibition of after-ripened and dormant seeds, which is confirmed by altered concentrations of indole-3-acetic acid conjugates and precursors.

Seed dormancy release accelerated by elevated partial pressure of oxygen is associated with DOG loci.

Seed dormancy determines the timing of seed germination and may be released by dry storage, also referred to as after-ripening. Studies on dormancy-release mechanisms are often hampered by the long after-ripening requirements of seeds. After-ripening is thought to be mainly caused by oxidative processes during seed dry storage. These processes are also the main cause of seed ageing. Increasing partial oxygen pressure through the elevated partial pressure of oxygen (EPPO) system has been shown to mimic and accelerate dry seed ageing. In this study, we investigated whether the EPPO system may also release primary seed dormancy in Arabidopsis thaliana. EPPO mimics dry after-ripening at the genetic level, as quantitative trait locus (QTL) analysis after EPPO treatment identified the DELAY OF GERMINATION loci DOG1, DOG2, and DOG6 that were first described in a study using dry after-ripening to release seed dormancy. QTL analysis also showed that dormancy release by cold stratification (another common method to break seed dormancy) partly overlaps with release by after-ripening and EPPO treatment. We conclude that EPPO is an appropriate method to mimic and accelerate dormancy release and, as such, may have applications in both research and industry.

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.

Post-embryonic Hourglass Patterns Mark Ontogenetic Transitions in Plant Development.

The historic developmental hourglass concept depicts the convergence of animal embryos to a common form during the phylotypic period. Recently, it has been shown that a transcriptomic hourglass is associated with this morphological pattern, consistent with the idea of underlying selective constraints due to intense molecular interactions during body plan establishment. Although plants do not exhibit a morphological hourglass during embryogenesis, a transcriptomic hourglass has nevertheless been identified in the model plant Arabidopsis thaliana Here, we investigated whether plant hourglass patterns are also found postembryonically. We found that the two main phase changes during the life cycle of Arabidopsis, from embryonic to vegetative and from vegetative to reproductive development, are associated with transcriptomic hourglass patterns. In contrast, flower development, a process dominated by organ formation, is not. This suggests that plant hourglass patterns are decoupled from organogenesis and body plan establishment. Instead, they may reflect general transitions through organizational checkpoints.

Differentially expressed genes during the imbibition of dormant and after-ripened seeds - a reverse genetics approach.

BACKGROUND: Seed dormancy, defined as the incapability of a viable seed to germinate under favourable conditions, is an important trait in nature and agriculture. Despite extensive research on dormancy and germination, many questions about the molecular mechanisms controlling these traits remain unanswered, likely due to its genetic complexity and the large environmental effects which are characteristic of these quantitative traits. To boost research towards revealing mechanisms in the control of seed dormancy and germination we depend on the identification of genes controlling those traits. METHODS: We used transcriptome analysis combined with a reverse genetics approach to identify genes that are prominent for dormancy maintenance and germination in imbibed seeds of Arabidopsis thaliana. Comparative transcriptomics analysis was employed on freshly harvested (dormant) and after-ripened (AR; non-dormant) 24-h imbibed seeds of four different DELAY OF GERMINATION near isogenic lines (DOGNILs) and the Landsberg erecta (Ler) wild type with varying levels of primary dormancy. T-DNA knock-out lines of the identified genes were phenotypically investigated for their effect on dormancy and AR. RESULTS: We identified conserved sets of 46 and 25 genes which displayed higher expression in seeds of all dormant and all after-ripened DOGNILs and Ler, respectively. Knock-out mutants in these genes showed dormancy and germination related phenotypes. CONCLUSIONS: Most of the identified genes had not been implicated in seed dormancy or germination. This research will be useful to further decipher the molecular mechanisms by which these important ecological and commercial traits are regulated.

Extensive translational regulation during seed germination revealed by polysomal profiling.

This work investigates the extent of translational regulation during seed germination. The polysome occupancy of each gene is determined by genome-wide profiling of total mRNA and polysome-associated mRNA. This reveals extensive translational regulation during Arabidopsis thaliana seed germination. The polysome occupancy of thousands of individual mRNAs changes to a large extent during the germination process. Intriguingly, these changes are restricted to two temporal phases (shifts) during germination, seed hydration and germination. Sequence features, such as upstream open reading frame number, transcript length, mRNA stability, secondary structures, and the presence and location of specific motifs correlated with this translational regulation. These features differed significantly between the two shifts, indicating that independent mechanisms regulate translation during seed germination. This study reveals substantial translational dynamics during seed germination and identifies development-dependent sequence features and cis elements that correlate with the translation control, uncovering a novel and important layer of gene regulation during seed germination.

Reduced Dormancy5 encodes a protein phosphatase 2C that is required for seed dormancy in Arabidopsis.

Seed dormancy determines germination timing and contributes to crop production and the adaptation of natural populations to their environment. Our knowledge about its regulation is limited. In a mutagenesis screen of a highly dormant Arabidopsis thaliana line, the reduced dormancy5 (rdo5) mutant was isolated based on its strongly reduced seed dormancy. Cloning of RDO5 showed that it encodes a PP2C phosphatase. Several PP2C phosphatases belonging to clade A are involved in abscisic acid signaling and control seed dormancy. However, RDO5 does not cluster with clade A phosphatases, and abscisic acid levels and sensitivity are unaltered in the rdo5 mutant. RDO5 transcript could only be detected in seeds and was most abundant in dry seeds. RDO5 was found in cells throughout the embryo and is located in the nucleus. A transcriptome analysis revealed that several genes belonging to the conserved PUF family of RNA binding proteins, in particular Arabidopsis PUMILIO9 (APUM9) and APUM11, showed strongly enhanced transcript levels in rdo5 during seed imbibition. Further transgenic analyses indicated that APUM9 reduces seed dormancy. Interestingly, reduction of APUM transcripts by RNA interference complemented the reduced dormancy phenotype of rdo5, indicating that RDO5 functions by suppressing APUM transcript levels.

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.

Promotion of testa rupture during garden cress germination involves seed compartment-specific expression and activity of pectin methylesterases.

Pectin methylesterase (PME) controls the methylesterification status of pectins and thereby determines the biophysical properties of plant cell walls, which are important for tissue growth and weakening processes. We demonstrate here that tissue-specific and spatiotemporal alterations in cell wall pectin methylesterification occur during the germination of garden cress (Lepidium sativum). These cell wall changes are associated with characteristic expression patterns of PME genes and resultant enzyme activities in the key seed compartments CAP (micropylar endosperm) and RAD (radicle plus lower hypocotyl). Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis as well as PME enzyme activity measurements of separated seed compartments, including CAP and RAD, revealed distinct phases during germination. These were associated with hormonal and compartment-specific regulation of PME group 1, PME group 2, and PME inhibitor transcript expression and total PME activity. The regulatory patterns indicated a role for PME activity in testa rupture (TR). Consistent with a role for cell wall pectin methylesterification in TR, treatment of seeds with PME resulted in enhanced testa permeability and promoted TR. Mathematical modeling of transcript expression changes in germinating garden cress and Arabidopsis (Arabidopsis thaliana) seeds suggested that group 2 PMEs make a major contribution to the overall PME activity rather than acting as PME inhibitors. It is concluded that regulated changes in the degree of pectin methylesterification through CAP- and RAD-specific PME and PME inhibitor expression play a crucial role during Brassicaceae seed germination.

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.

Acquisition and loss of desiccation tolerance in seeds: from experimental model to biological relevance.

MAIN CONCLUSION: Besides being an important model to study desiccation tolerance, the induction of desiccation tolerance in germinated seeds may also play an ecological role in seedling establishment. Desiccation tolerance (DT) is the ability of certain organisms to survive extreme water losses without accumulation of lethal damage. This was a key feature in the conquering of dry land and is currently found in all taxa including bacteria, fungi, roundworms and plants. Not surprisingly, studies in various fields have been performed to unravel this intriguing phenomenon. In flowering plants, DT is rare in whole plants (vegetative tissues), yet is common in seeds. In this review, we present our current understanding of the evolution of DT in plants. We focus on the acquisition of DT in seeds and the subsequent loss during and after germination by highlighting and comparing research in two model plants Medicago truncatula and Arabidopsis thaliana. Finally, we discuss the ability of seeds to re-establish DT during post-germination, the possible ecological meaning of this phenomenon, and the hypothesis that DT, in combination with dormancy, optimizes seedling establishment.