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International audience; Several pathways conferring environmental flowering responses in Arabidopsis (Arabidopsis thaliana) converge on developmental processes that mediate the floral transition in the shoot apical meristem. Many characterized mutations disrupt these environmental responses, but downstream developmental processes have been more refractory to mutagenesis. Here, we constructed a quintuple mutant impaired in several environmental pathways and showed that it possesses severely reduced flowering responses to changes in photoperiod and ambient temperature. RNA-sequencing (RNA-seq) analysis of the quintuple mutant showed that the expression of genes encoding gibberellin biosynthesis enzymes and transcription factors involved in the age pathway correlates with flowering. Mutagenesis of the quintuple mutant generated two late-flowering mutants, quintuple ems1 (qem1) and qem2. The mutated genes were identified by isogenic mapping and transgenic complementation. The qem1 mutant is an allele of the gibberellin 20-oxidase gene ga20ox2, confirming the importance of gibberellin for flowering in the absence of environmental responses. By contrast, qem2 is impaired in CHROMATIN REMODELING4 (CHR4), which has not been genetically implicated in floral induction. Using coimmunoprecipitation, RNA-seq, and chromatin immunoprecipitation sequencing, we show that CHR4 interacts with transcription factors involved in floral meristem identity and affects the expression of key floral regulators. Therefore, CHR4 mediates the response to endogenous flowering pathways in the inflorescence meristem to promote floral identity.

Several pathways conferring environmental flowering responses in Arabidopsis (

Single-molecule full-length complementary DNA (cDNA) sequencing can aid genome annotation by revealing transcript structure and alternative splice forms, yet current annotation pipelines do not incorporate such information. Here we present long-read annotation (LoReAn) software, an automated annotation pipeline utilizing short- and long-read cDNA sequencing, protein evidence, and ab initio prediction to generate accurate genome annotations. Based on annotations of two fungal genomes (Verticillium dahliae and Plicaturopsis crispa) and two plant genomes (Arabidopsis [Arabidopsis thaliana] and Oryza sativa), we show that LoReAn outperforms popular annotation pipelines by integrating single-molecule cDNA-sequencing data generated from either the Pacific Biosciences or MinION sequencing platforms, correctly predicting gene structure, and capturing genes missed by other annotation pipelines.

Flower development is controlled by the action of key regulatory transcription factors of the MADS-domain family. The function of these factors appears to be highly conserved among species based on mutant phenotypes. However, the conservation of their downstream processes is much less well understood, mostly because the evolutionary turnover and variation of their DNA-binding sites (BS) among plant species has not yet been experimentally determined.Here, we performed comparative ChIP-seq experiments of the MADS-domain transcription factor SEPALLATA3 (SEP3) in two closely related Arabidopsis species: A. thaliana and A. lyrata which have very similar floral organ morphology. We found that binding site conservation is associated with DNA sequence conservation, the presence of the CArG-box BS motif and on the relative position of the BS to its potential target gene. Differences in genome size and structure can explain that SEP3 BSs in A. lyrata can be located more distantly to their potential target genes than their counterparts in A. thaliana. In A. lyrata, we identified transposition as a mechanism to generate novel SEP3 binding locations in the genome. Comparative gene expression analysis shows that the loss/gain of BSs is associated with a change in gene expression. In summary, this study investigates the evolutionary dynamics of DNA BSs of a floral key-regulatory transcription factor, and explores factors affecting this phenomenon. ispartof: Molecular Biology and Evolution vol:33 issue:1 pages:185-200 ispartof: location:United States status: published

Nucleosomes are the basic repeating units of eukaryotic chromatin. They play important roles in chromatin compaction and gene regulation. Therefore, it is important to profile the in vivo locations of nucleosomes in the genome. Here we illustrate how to profile nucleosome occupancy at genome-wide scale using micrococcal nuclease (MNase) digestion combined with high throughput Illumina sequencing (MNase-seq). Nucleosome-associated DNA is relatively insensitive to digestion by micrococcal nuclease (MNase). Upon mild MNase treatment, the undigested nucleosomal DNA can be purified and sequenced allowing a precise localization of in vivo nucleosomes at a genome-wide level.

Single-molecule full-length cDNA sequencing can aid genome annotation by revealing transcript structure and alternative splice-forms, yet current annotation pipelines do not incorporate such information. Here we present LoReAn (LongReadAnnotation) software, an automated annotation pipeline utilizing short- and long-read cDNA sequencing, protein evidence, andab initioprediction to generate accurate genome annotations. Based on annotations of two fungal and two plant genomes, we show that LoReAn outperforms popular annotation pipelines by integrating single-molecule cDNA sequencing data generated from either the PacBio or MinION sequencing platforms, and correctly predicting gene structure and capturing genes missed by other annotation pipelines.

Nucleosomes are the basic repeating units of eukaryotic chromatin. They play important roles in chromatin compaction and gene regulation. Therefore, it is important to profile the in vivo locations of nucleosomes in the genome. Here we illustrate how to profile nucleosome occupancy at genome-wide scale using micrococcal nuclease (MNase) digestion combined with high throughput Illumina sequencing (MNase-seq). Nucleosome-associated DNA is relatively insensitive to digestion by micrococcal nuclease (MNase). Upon mild MNase treatment, the undigested nucleosomal DNA can be purified and sequenced allowing a precise localization of in vivo nucleosomes at a genome-wide level.

Plants integrate seasonal cues such as temperature and day length to optimally adjust their flowering time to the environment. Compared to the control of flowering before and after winter by the vernalization and day length pathways, mechanisms that delay or promote flowering during a transient cool or warm period, especially during spring, are less well understood. Due to global warming, understanding this ambient temperature pathway has gained increasing importance. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) is a critical flowering regulator of the ambient temperature pathway. FLM is alternatively spliced in a temperature-dependent manner and the two predominant splice variants, FLM-ß and FLM-δ, can repress and activate flowering in the genetic background of the A. thaliana reference accession Columbia-0. The relevance of this regulatory mechanism for the environmental adaptation across the entire range of the species is, however, unknown. Here, we identify insertion polymorphisms in the first intron of FLM as causative for accelerated flowering in many natural A. thaliana accessions, especially in cool (15°C) temperatures. We present evidence for a potential adaptive role of this structural variation and link it specifically to changes in the abundance of FLM-ß. Our results may allow predicting flowering in response to ambient temperatures in the Brassicaceae., Author Summary Plants control their flowering time in response to the temperatures of their environment, e.g. in response to the experience of winter or in response to cold and warm ambient temperatures experienced during spring. The knowledge about the evolutionary adaptation of plants to changing ambient temperatures is at present very limited. Understanding the latter is, however, becoming increasingly important due to the temperature changes associated with global warming and the anticipated changes in flowering time in ecosystems and agricultural systems. Here, we uncover an evolutionarily conserved molecular mechanism employed by Arabidopsis thaliana ecotypes for the adaptation of flowering time to cool temperatures. This structural change in the architecture of the gene FLOWERING LOCUS M can be found in multiple A. thaliana natural accessions and the knowledge gained in our study may be used to predict or modify flowering time in plants related to A. thaliana in the future.

Background Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in temperature promotes changes in plant morphology as well as the transition from the vegetative to the reproductive phase in Arabidopsis thaliana via changes in splicing of key regulatory genes. Here we investigate whether a particular histone modification affects ambient temperature-induced alternative splicing and flowering time. Results We use a genome-wide approach and perform RNA-sequencing (RNA-seq) analyses and histone H3 lysine 36 tri-methylation (H3K36me3) chromatin immunoprecipitation sequencing (ChIP-seq) in plants exposed to different ambient temperatures. Analysis and comparison of these datasets reveal that temperature-induced differentially spliced genes are enriched in H3K36me3. Moreover, we find that reduction of H3K36me3 deposition causes alteration in temperature-induced alternative splicing. We also show that plants with mutations in H3K36me3 writers, eraser, or readers have altered high ambient temperature-induced flowering. Conclusions Our results show a key role for the histone mark H3K36me3 in splicing regulation and plant plasticity to fluctuating ambient temperature. Our findings open new perspectives for the breeding of crops that can better cope with environmental changes due to climate change. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1235-x) contains supplementary material, which is available to authorized users.

Significance Shoot-branching patterns affect key aspects of plant life and are important targets for crop breeding. However, we are still ignorant of the genetic mechanisms controlling locally an important decision during branch development: whether the axillary bud grows out to give a lateral shoot or remains dormant. Here we show that the TEOSINTE BRANCHED1, CYCLOIDEA, PCF (TCP) transcriptional regulator BRANCHED1 (BRC1), which acts inside axillary buds, binds and activates three genes encoding Homeodomain leucine zipper (HD-ZIP) transcription factors. These factors, together with BRC1, trigger a cascade leading to local abscisic acid (ABA) accumulation and response, essential for bud dormancy under light-limiting conditions. This finding demonstrates a direct relationship between BRC1 and ABA signaling and places ABA downstream of BRC1 in the control of axillary bud dormancy.

Unlike animals, plants produce new organs throughout their life cycle using pools of stem cells that are organized in meristems. Although many key regulators of meristem and organ identities have been identified, it is still not well understood how they function at the molecular level and how they can switch an entire developmental programme in which thousands of genes are involved. Recent advances in the genome-wide identification of target genes controlled by key plant transcriptional regulators and their interactions with epigenetic factors provide new insights into general transcriptional regulatory mechanisms that control switches of developmental programmes and cell fates in complex organisms.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is a powerful technique for genome-wide identification of in vivo binding sites of DNA-binding proteins. The technique had been used to study many DNA-binding proteins in a broad variety of species. The basis of the ChIP-seq technique is the ability to covalently cross-link DNA and proteins that are located in very close proximity. This allows the use of an antibody against the (tagged) protein of interest to specifically enrich DNA-fragments bound by this protein. ChIP-seq can be performed using antibodies against the native protein or against tagged proteins. Using a specific antibody against a tag to immunoprecipitate tagged proteins eliminates the need for a specific antibody against the native protein and allows more experimental flexibility. In this chapter we present a complete workflow for experimental procedure and bioinformatic analysis that allows wet-lab biologists to perform and analyze ChIP-seq experiments.

Developmental plasticity enables plants to respond rapidly to changing environmental conditions, such as temperature fluctuations. Understanding how plants measure temperature and integrate this information into developmental programs at the molecular level will be essential to breed thermo-tolerant crop varieties. Recent studies identified alternative splicing (AS) as a possible ‘molecular thermometer’, allowing plants to quickly adjust the abundance of functional transcripts to environmental perturbations. In this review, recent advances regarding the effects of temperature-responsive AS on plant development will be discussed, with emphasis on the circadian clock and flowering time control. The challenge for the near future will be to understand the molecular mechanisms by which temperature can influence AS regulation.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is a powerful technique for genome-wide identification of in vivo binding sites of DNA-binding proteins. The technique had been used to study many DNA-binding proteins in a broad variety of species. The basis of the ChIP-seq technique is the ability to covalently cross-link DNA and proteins that are located in very close proximity. This allows the use of an antibody against the (tagged) protein of interest to specifically enrich DNA-fragments bound by this protein. ChIP-seq can be performed using antibodies against the native protein or against tagged proteins. Using a specific antibody against a tag to immunoprecipitate tagged proteins eliminates the need for a specific antibody against the native protein and allows more experimental flexibility. In this chapter we present a complete workflow for experimental procedure and bioinformatic analysis that allows wet-lab biologists to perform and analyze ChIP-seq experiments.

Successful plant reproduction relies on the perfect orchestration of singular processes that culminate in the product of reproduction: the seed. The floral transition, floral organ development, and fertilization are well-studied processes and the genetic regulation of the various steps is being increasingly unveiled. Initially, based predominantly on genetic studies, the regulatory pathways were considered to be linear, but recent genome-wide analyses, using high-throughput technologies, have begun to reveal a different scenario. Complex gene regulatory networks underlie these processes, including transcription factors, microRNAs, movable factors, hormones, and chromatin-modifying proteins. Here we review recent progress in understanding the networks that control the major steps in plant reproduction, showing how new advances in experimental and computational technologies have been instrumental. As these recent discoveries were obtained using the model species Arabidopsis thaliana, we will restrict this review to regulatory networks in this important model species. However, more fragmentary information obtained from other species reveals that both the developmental processes and the underlying regulatory networks are largely conserved, making this review also of interest to those studying other plant species.

Background Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood. Results We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes. Conclusions Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility., This work was supported by an NWO-VIDI grant to KK, and a Marie-Curie-ITN network grant SYSFLO (FP7/2007-2011, grant agreement no. 237909) to AP, PM, PK, and GCA. KK wishes to thank the Alexander-von-Humboldt foundation for support. This work was also supported by grants from Spanish Ministerio de Ciencia e Innovación (BFU2011-22734 to JLR). JTM and JJ were recipients of EMBO postdoctoral fellowships.

Reproductive organ development is one of the most important steps in the life cycle of plants. Studies using core eudicot species like thale cress (Arabidopsis thaliana) and snapdragon (Antirrhinum majus) have shown that MADS domain transcription factors belonging to the AGAMOUS (AG) subfamily regulate the identity of stamens, carpels, and ovules and that they are important for floral meristem determinacy. Here, we investigate the genetic interactions between the four rice (Oryza sativa) AG subfamily members, MADS3, MADS13, MADS21, and MADS58. Our data show that, in contrast with previous reports, MADS3 and MADS58 determine stamen and carpel identity and, together with MADS13, are important for floral meristem determinacy. In the mads3 mads58 double mutant, we observed a complete loss of reproductive organ identity and massive accumulation of lodicules in the third and fourth floral whorls. MADS21 is an AGL11 lineage gene whose expression is not restricted to ovules. Instead, its expression profile is similar to those of class C genes. However, our genetic analysis shows that MADS21 has no function in stamen, carpel, or ovule identity determination.