The Arabidopsis pre-mRNA 3' end processing related protein FIP1 promotes seed dormancy via the DOG1 and ABA pathways.

Seed dormancy is an important adaptive trait to prevent germination occurring at an inappropriate time. The mechanisms governing seed dormancy and germination are complex. Here, we report that FACTOR INTERACTING WITH POLY(A) POLYMERASE 1 (FIP1), a component of the pre-mRNA 3' end processing machinery, is involved in seed dormancy and germination processes in Arabidopsis thaliana. FIP1 is mainly expressed in seeds and the knockout of FIP1 causes reduced seed dormancy, indicating that FIP1 positively influences seed dormancy. Meanwhile, fip1 mutants are insensitive to exogenous ABA during seed germination and early seedling establishment. The terms 'seed maturation' and 'response to ABA stimulus' are significantly enriched in a gene ontology analysis based on genes differentially expressed between fip1-1 and the wild type. Several of these genes, including ABI5, DOG1 and PYL12, show significantly decreased transcript levels in fip1. Genetic analysis showed that either cyp707a2 or dog1-5 partially, but in combination completely, represses the reduced seed dormancy of fip1, indicating that the double mutant cyp707a2 dog1-5 is epistatic to fip1. Moreover, FIP1 is required for CFIM59, another component of pre-mRNA 3' end processing machinery, to govern seed dormancy and germination. Overall, we identified FIP1 as a regulator of seed dormancy and germination that plays a crucial role in governing these processes through the DOG1 and ABA pathways.

Overexpression of Taetr1-1 promotes enhanced seed dormancy and ethylene insensitivity in wheat.

MAIN CONCLUSION: Taetr1-1 can promote enhanced seed dormancy and ethylene insensitivity in wheat, indicating a conserved function of ETR1 in regulating seed dormancy. Lots of wheat cultivars have weak dormant seed. Weak seed dormancy can cause pre-harvest sprouting (PHS) in grain which significantly reduces grain yield and quality. The mining of causal genes of PHS resistance will serve to enhance breeding selection and cultivar development. In a previous study in Arabidopsis, we identified reduced dormancy 3 as a loss-of-function mutant of the ethylene receptor 1 (ETR1), which can control seed dormancy through the ERF12-TPL-DOG1 pathway. However, it is unknown whether ETR1 also functions in the regulation of wheat seed dormancy. To identify the regulatory role of ETR1 in wheat, we cloned TaETR1 and overexpressed the gain-of-function mutant Taetr1-1. The result indicated that overexpression of Taetr1-1 can promote enhanced seed dormancy and ethylene insensitivity in wheat. This study contributed to our understanding of the molecular basis for the regulation of wheat PHS resistance.

REVERSAL OF RDO5 1, a Homolog of Rice Seed Dormancy4, Interacts with bHLH57 and Controls ABA Biosynthesis and Seed Dormancy in Arabidopsis.

The control of seed dormancy by abscisic acid (ABA) has been extensively studied, but the underlying mechanism is not fully understood. Here, we report the characterization of two ABA-related seed dormancy regulators in Arabidopsis (Arabidopsis thaliana): ODR1 (for reversal of rdo5), an ortholog of the rice (Oryza sativa) Seed dormancy4 (Sdr4), and the basic helix-loop-helix transcription factor bHLH57. ODR1, whose transcript levels are directly suppressed by the transcription factor ABA INSENSITIVE3 (ABI3), negatively regulates seed dormancy by affecting ABA biosynthesis and ABA signaling. By contrast, bHLH57 positively regulates seed dormancy by inducing the expression of the genes 9-CIS-EPOXYCAROTENOID DIOXYGENASE6 (NCED6) and NCED9, which encode ABA biosynthetic enzymes, and thus leads to higher ABA levels. ODR1 interacts with bHLH57 and inhibits bHLH57-modulated NCED6 and NCED9 expression in the nucleus. bhlh57 loss-of-function alleles can partially counteract the enhanced NCED6 and NCED9 expression seen in odr1 mutants and can therefore rescue their associated hyper-dormancy phenotype. Thus, we identified a novel ABI3-ODR1-bHLH57-NCED6/9 network that provides insights into the regulation of seed dormancy by ABA biosynthesis and signaling.

Ectopic expression of the Arabidopsis florigen gene FLOWERING LOCUS T in seeds enhances seed dormancy via the GA and DOG1 pathways.

Ectopic expression of specific genes in seeds could be a tool for molecular design of crops to alter seed dormancy and germination, thereby improving production. Here, a seed-specific vector, 12S-pLEELA, was applied to study the roles of genes in Arabidopsis seeds. Transgenic lines containing FLOWERING LOCUS T (FT) driven by the 12S promoter exhibited significantly increased seed dormancy and earlier flowering. Mutated FT(Y85H) and TERMINAL FLOWER1 (TFL1) transgenic lines also showed increased seed dormancy but without altered flowering time. FT(Y85H) and TFL1 caused weaker seed dormancy enhancement compared to FT. The FT and TFL1 transgenic lines showed hypersensitivity to paclobutrazol, but not to abscisic acid in seed germination. The levels of bioactive gibberellin 3 (GA3 ) and GA4 were significantly reduced, consistent with decreased expression of COPALYL DIPHOSPHATE SYNTHASE (CPS), KAURENE OXIDASE (KO), GIBBERELLIN 3-OXIDASE2 (GA3ox2), and GA20ox1 in p12S::FT lines. Exogenous GA4+7 could recover the germination ability of FT transgenic lines. These results revealed that FT regulates GA biosynthesis. A genetic analysis indicated that the GA signaling regulator SPINDLY (SPY) is epistatic to FT in GA-mediated seed germination. Furthermore, DELAY OF GERMINATION1 (DOG1) showed significantly higher transcript levels in p12S::FT lines. Seed dormancy analysis of dog1-2 spy-3 p12S::FT-2 indicated that the combination of SPY and DOG1 is epistatic to FT in the regulation of dormancy. Overall, we showed that ectopic expression of FT and TFL1 in seeds enhances dormancy through affecting GA and DOG1 pathways.

ETR1/RDO3 Regulates Seed Dormancy by Relieving the Inhibitory Effect of the ERF12-TPL Complex on DELAY OF GERMINATION1 Expression.

The control of seed dormancy by ethylene has been well studied, but the underlying molecular mechanisms are not fully understood. Here, we report the characterization of the Arabidopsis (Arabidopsis thaliana) mutant reduced dormancy 3 (rdo3) and the cloning of the underlying gene. We demonstrate that rdo3 is a loss-of-function mutant of the ethylene receptor ETHYLENE RESPONSE1 (ETR1). ETR1 controls seed dormancy partially through the DELAY OF GERMINATION1 (DOG1) pathway. Molecular and genetic analyses demonstrated that ETHYLENE RESPONSE FACTOR12 (ERF12) is involved in the regulation of seed dormancy downstream of ETR1. ERF12 interacts with TOPLESS (TPL) and genetically requires TPL to function. ERF12 and TPL repress the expression of DOG1 by occupying its promoter. Thus, we identified the dormancy pathway ETR1-ERF12-TPL-DOG1 and provide mechanistic insights into the regulation of seed dormancy by linking the ethylene and DOG1 pathways.

Dynamic hydrolase labelling as a marker for seed quality in Arabidopsis seeds.

Seed quality is affected by different constituents of the seed. In general, seed lots are considered to be of high quality when they exhibit fast and homogeneous germination. When seeds are stored, they undergo different degrees of damage that have detrimental effects on their quality. Therefore, accurate prediction of the seed quality and viability levels of a seed lot is of high importance in the seed-producing industry. Here, we describe the use of activity-based protein profiling of proteases to evaluate the quality of artificially and naturally aged seeds of Arabidopsis thaliana Using this approach, we have identified two protease activities with opposite behaviours in aged seeds of Arabidopsis that correlate with the quality status of the seeds. We show that vacuolar processing enzymes (VPEs) become more active during the ageing process, in both artificial and natural ageing treatments. Secondly, we demonstrate that serine hydrolases are active at the beginning of our artificial ageing treatment, but their labelling decreases along with seed viability. We present a list of candidate hydrolases active during seed germination and propose that these protease activities can be used in combination with VPEs to develop novel markers of seed quality.

Seed traits are pleiotropically regulated by the flowering time gene PERPETUAL FLOWERING 1 (PEP1) in the perennial Arabis alpina.

The life cycles of plants are characterized by two major life history transitions-germination and the initiation of flowering-the timing of which are important determinants of fitness. Unlike annuals, which make the transition from the vegetative to reproductive phase only once, perennials iterate reproduction in successive years. The floral repressor PERPETUAL FLOWERING 1 (PEP1), an ortholog of FLOWERING LOCUS C, in the alpine perennial Arabis alpina ensures the continuation of vegetative growth after flowering and thereby restricts the duration of the flowering episode. We performed greenhouse and garden experiments to compare flowering phenology, fecundity and seed traits between A. alpina accessions that have a functional PEP1 allele and flower seasonally and pep1 mutants and accessions that carry lesions in PEP1 and flower perpetually. In the garden, perpetual genotypes flower asynchronously and show higher winter mortality than seasonal ones. PEP1 also pleiotropically regulates seed dormancy and longevity in a way that is functionally divergent from FLC. Seeds from perpetual genotypes have shallow dormancy and reduced longevity regardless of whether they after-ripened in plants grown in the greenhouse or in the experimental garden. These results suggest that perpetual genotypes have higher mortality during winter but compensate by showing higher seedling establishment. Differences in seed traits between seasonal and perpetual genotypes are also coupled with differences in hormone sensitivity and expression of genes involved in hormonal pathways. Our study highlights the existence of pleiotropic regulation of seed traits by hub developmental regulators such as PEP1, suggesting that seed and flowering traits in perennial plants might be optimized in a coordinated fashion.

Seed Dormancy in Arabidopsis Requires Self-Binding Ability of DOG1 Protein and the Presence of Multiple Isoforms Generated by Alternative Splicing.

The Arabidopsis protein DELAY OF GERMINATION 1 (DOG1) is a key regulator of seed dormancy, which is a life history trait that determines the timing of seedling emergence. The amount of DOG1 protein in freshly harvested seeds determines their dormancy level. DOG1 has been identified as a major dormancy QTL and variation in DOG1 transcript levels between accessions contributes to natural variation for seed dormancy. The DOG1 gene is alternatively spliced. Alternative splicing increases the transcriptome and proteome diversity in higher eukaryotes by producing transcripts that encode for proteins with altered or lost function. It can also generate tissue specific transcripts or affect mRNA stability. Here we suggest a different role for alternative splicing of the DOG1 gene. DOG1 produces five transcript variants encoding three protein isoforms. Transgenic dog1 mutant seeds expressing single DOG1 transcript variants from the endogenous DOG1 promoter did not complement because they were non-dormant and lacked DOG1 protein. However, transgenic plants overexpressing single DOG1 variants from the 35S promoter could accumulate protein and showed complementation. Simultaneous expression of two or more DOG1 transcript variants from the endogenous DOG1 promoter also led to increased dormancy levels and accumulation of DOG1 protein. This suggests that single isoforms are functional, but require the presence of additional isoforms to prevent protein degradation. Subsequently, we found that the DOG1 protein can bind to itself and that this binding is required for DOG1 function but not for protein accumulation. Natural variation for DOG1 binding efficiency was observed among Arabidopsis accessions and contributes to variation in seed dormancy.

Sequence Polymorphisms at the REDUCED DORMANCY5 Pseudophosphatase Underlie Natural Variation in Arabidopsis Dormancy.

Seed dormancy controls the timing of germination, which regulates the adaptation of plants to their environment and influences agricultural production. The time of germination is under strong natural selection and shows variation within species due to local adaptation. The identification of genes underlying dormancy quantitative trait loci is a major scientific challenge, which is relevant for agricultural and ecological goals. In this study, we describe the identification of the DELAY OF GERMINATION18 (DOG18) quantitative trait locus, which was identified as a factor in natural variation for seed dormancy in Arabidopsis (Arabidopsis thaliana). DOG18 encodes a member of the clade A of the type 2C protein phosphatases family, which we previously identified as the REDUCED DORMANCY5 (RDO5) gene. DOG18/RDO5 shows a relatively high frequency of loss-of-function alleles in natural accessions restricted to northwestern Europe. The loss of dormancy in these loss-of-function alleles can be compensated for by genetic factors like DOG1 and DOG6, and by environmental factors such as low temperature. RDO5 does not have detectable phosphatase activity. Analysis of the phosphoproteome in dry and imbibed seeds revealed a general decrease in protein phosphorylation during seed imbibition that is enhanced in the rdo5 mutant. We conclude that RDO5 acts as a pseudophosphatase that inhibits dephosphorylation during seed imbibition.

Transcriptome and Degradome Sequencing Reveals Dormancy Mechanisms of Cunninghamia lanceolata Seeds.

Seeds with physiological dormancy usually experience primary and secondary dormancy in the nature; however, little is known about the differential regulation of primary and secondary dormancy. We combined multiple approaches to investigate cytological changes, hormonal levels, and gene expression dynamics in Cunninghamia lanceolata seeds during primary dormancy release and secondary dormancy induction. Light microscopy and transmission electron microscopy revealed that protein bodies in the embryo cells coalesced during primary dormancy release and then separated during secondary dormancy induction. Transcriptomic profiling demonstrated that expression of genes negatively regulating gibberellic acid (GA) sensitivity reduced specifically during primary dormancy release, whereas the expression of genes positively regulating abscisic acid (ABA) biosynthesis increased during secondary dormancy induction. Parallel analysis of RNA ends revealed uncapped transcripts for ∼55% of all unigenes. A negative correlation between fold changes in expression levels of uncapped versus capped mRNAs was observed during primary dormancy release. However, this correlation was loose during secondary dormancy induction. Our analyses suggest that the reversible changes in cytology and gene expression during dormancy release and induction are related to ABA/GA balance. Moreover, mRNA degradation functions as a critical posttranscriptional regulator during primary dormancy release. These findings provide a mechanistic framework for understanding physiological dormancy in seeds.

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.

Arabidopsis paired amphipathic helix proteins SNL1 and SNL2 redundantly regulate primary seed dormancy via abscisic acid-ethylene antagonism mediated by histone deacetylation.

Histone (de)acetylation is a highly conserved chromatin modification that is vital for development and growth. In this study, we identified a role in seed dormancy for two members of the histone deacetylation complex in Arabidopsis thaliana, SIN3-LIKE1 (SNL1) and SNL2. The double mutant snl1 snl2 shows reduced dormancy and hypersensitivity to the histone deacetylase inhibitors trichostatin A and diallyl disulfide compared with the wild type. SNL1 interacts with HISTONE DEACETYLASE19 in vitro and in planta, and loss-of-function mutants of SNL1 and SNL2 show increased acetylation levels of histone 3 lysine 9/18 (H3K9/18) and H3K14. Moreover, SNL1 and SNL2 regulate key genes involved in the ethylene and abscisic acid (ABA) pathways by decreasing their histone acetylation levels. Taken together, we showed that SNL1 and SNL2 regulate seed dormancy by mediating the ABA-ethylene antagonism in Arabidopsis. SNL1 and SNL2 could represent a cross-link point of the ABA and ethylene pathways in the regulation of seed dormancy.

Alternative splicing enhances transcriptome complexity in desiccating seeds.

Before being dispersed in the environment, mature seeds need to be dehydrated. The survival of seeds after dispersal depends on their low hydration in combination with high desiccation tolerance. These characteristics are established during seed maturation. Some key seed maturation genes have been reported to be regulated by alternative splicing (AS). However, so far AS was described only for single genes and a comprehensive analysis of AS during seed maturation has been lacking. We investigated gene expression and AS during Arabidopsis thaliana seed development at a global level, before and after desiccation. Bioinformatics tools were developed to identify differentially spliced regions within genes. Our data suggest the importance and shows the peculiar features of AS during seed desiccation. We identified AS in 34% of genes that are expressed at both timepoints before and after desiccation. Most of these AS transcript variants had not been found before in other tissues. Among the AS genes some seed master regulators could be found. Interestingly, 6% of all expressed transcripts were not transcriptionally regulated during desiccation, but only modified by AS. We propose that AS should be more routinely taken into account in the analysis of transcriptomic data to prevent overlooking potentially important regulators.

HISTONE DEACETYLASE 9 represses seedling traits in Arabidopsis thaliana dry seeds.

Plant life is characterized by major phase changes. We studied the role of histone deacetylase (HDAC) activity in the transition from seed to seedling in Arabidopsis. Pharmacological inhibition of HDAC stimulated germination of freshly harvested seeds. Subsequent analysis revealed that histone deacetylase 9 (hda9) mutant alleles displayed reduced seed dormancy and faster germination than wild-type plants. Transcriptome meta-analysis comparisons between the hda9 dry seed transcriptome and published datasets demonstrated that transcripts of genes that are induced during imbibition in wild-type prematurely accumulated in hda9-1 dry seeds. This included several genes associated with photosynthesis and photoautotrophic growth such as RuBisCO and RuBisCO activase (RCA). Chromatin immunoprecipitation experiments demonstrated enhanced histone acetylation levels at their loci in young hda9-1 seedlings. Our observations suggest that HDA9 negatively influences germination and is involved in the suppression of seedling traits in dry seeds, probably by transcriptional repression via histone deacetylation. Accordingly, HDA9 transcript is abundant in dry seeds and becomes reduced during imbibition in wild-type seeds. The proposed function of HDA9 is opposite to that of its homologous genes HDA6 and HDA19, which have been reported to repress embryonic properties in germinated seedlings.

The time required for dormancy release in Arabidopsis is determined by DELAY OF GERMINATION1 protein levels in freshly harvested seeds.

Seed dormancy controls the start of a plant's life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA.

Seed maturation in Arabidopsis thaliana is characterized by nuclear size reduction and increased chromatin condensation.

Most plant species rely on seeds for their dispersal and survival under unfavorable environmental conditions. Seeds are characterized by their low moisture content and significantly reduced metabolic activities. During the maturation phase, seeds accumulate storage reserves and become desiccation-tolerant and dormant. Growth is resumed after release of dormancy and the occurrence of favorable environmental conditions. Here we show that embryonic cotyledon nuclei of Arabidopsis thaliana seeds have a significantly reduced nuclear size, which is established at the beginning of seed maturation. In addition, the chromatin of embryonic cotyledon nuclei from mature seeds is highly condensed. Nuclei regain their size and chromatin condensation level during germination. The reduction in nuclear size is controlled by the seed maturation regulator ABSCISIC ACID-INSENSITIVE 3, and the increase during germination requires two predicted nuclear matrix proteins, LITTLE NUCLEI 1 and LITTLE NUCLEI 2. Our results suggest that the specific properties of nuclei in ripe seeds are an adaptation to desiccation, independent of dormancy. We conclude that the changes in nuclear size and chromatin condensation in seeds are independent, developmentally controlled processes.

The conserved splicing factor SUA controls alternative splicing of the developmental regulator ABI3 in Arabidopsis.

ABSCISIC ACID INSENSITIVE3 (ABI3) is a major regulator of seed maturation in Arabidopsis thaliana. We detected two ABI3 transcripts, ABI3-alpha and ABI3-beta, which encode full-length and truncated proteins, respectively. Alternative splicing of ABI3 is developmentally regulated, and the ABI3-beta transcript accumulates at the end of seed maturation. The two ABI3 transcripts differ by the presence of a cryptic intron in ABI3-alpha, which is spliced out in ABI3-beta. The suppressor of abi3-5 (sua) mutant consistently restores wild-type seed features in the frameshift mutant abi3-5 but does not suppress other abi3 mutant alleles. SUA is a conserved splicing factor, homologous to the human protein RBM5, and reduces splicing of the cryptic ABI3 intron, leading to a decrease in ABI3-beta transcript. In the abi3-5 mutant, ABI3-beta codes for a functional ABI3 protein due to frameshift restoration.

The MKK3-MPK7 cascade phosphorylates ERF4 and promotes its rapid degradation to release seed dormancy in Arabidopsis.

Seeds establish dormancy to delay germination until the arrival of a favorable growing season. In this study, we identify a fate switch comprised of the MKK3-MPK7 kinase cascade and the ethylene response factor ERF4 that is responsible for the seed state transition from dormancy to germination. We show that dormancy-breaking factors activate the MKK3-MPK7 module, which affects the expression of some α-EXPANSIN (EXPA) genes to control seed dormancy. Furthermore, we identify a direct downstream substrate of this module, ERF4, which suppresses the expression of these EXPAs by directly binding to the GCC boxes in their exon regions. The activated MKK3-MPK7 module phosphorylates ERF4, leading to its rapid degradation and thereby releasing its inhibitory effect on the expression of these EXPAs. Collectively, our work identifies a signaling chain consisting of protein phosphorylation, degradation, and gene transcription , by which the germination promoters within the embryo sense and are activated by germination signals from ambient conditions.

A novel role for histone methyltransferase KYP/SUVH4 in the control of Arabidopsis primary seed dormancy.

• Seed dormancy controls germination and plays a crucial role in the life cycle of plants. Chromatin modifications are involved in the regulation of seed dormancy; however, little is known about the underlying mechanism. • KYP/SUVH4 is required for histone H3 lysine 9 dimethylation. Mutations in this gene cause increased seed dormancy. KYP/SUVH4-overexpressing Arabidopsis plants show decreased dormancy. KYP/SUVH4 expression is regulated by abscisic acid (ABA) and gibberellins (GA). The sensitivity of seed germination to ABA and paclobutrazol (PAC) is enhanced slightly in kryptonite-2 (kyp-2) and suvh4-2/suvh5 mutants, but weakened in KYP/SUVH4-overexpressing plants. • In the kyp-2 mutant, several dormancy-related genes, including DOG1 and ABI3, show increased expression levels, in agreement with a negative role for KYP/SUVH4 in gene transcription. • Genetic analysis showed that DOG1 and HUB1 are epistatic to KYP/SUVH4, suggesting that these genes regulate seed dormancy in the same genetic pathway.