About
Richard M. Amasino is the Carlos O. Miler Professor of Biochemistry and a Howard Hughes Medical Institute Professor at the University of Wisconsin–Madison. His research focuses on developmental biology, gene expression, and RNA biology, with a particular emphasis on the regulation of plant development and mechanisms of floral induction. Amasino's work involves studying how environmental cues such as cold exposure influence flowering in plants, notably through the process of vernalization. He has identified key genetic components, such as the FLC gene in Arabidopsis thaliana, which prevents flowering unless the plant experiences cold, and has explored how cold promotes flowering via epigenetic switches. His research extends to studying vernalization in grasses like Brachypodium distachyon, which evolved independently of Arabidopsis, highlighting convergent evolution in plant cold response systems. Additionally, Amasino investigates biomass traits in Brachypodium and contributes to science education projects.
Research topics
- Biology
- Genetics
- Botany
- Business
- Mathematics
- Biotechnology
Selected publications
2024-05-15
peer-reviewOpen access1st authorCorrespondingSilencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1, suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.
2024-05-30
peer-reviewOpen access1st authorCorrespondingThe Plant Cell · 2024-04-23 · 12 citations
articleOpen accessSenior authorFlowering is a key developmental transition in the plant life cycle. In temperate climates, flowering often occurs in response to the perception of seasonal cues such as changes in day-length and temperature. However, the mechanisms that have evolved to control the timing of flowering in temperate grasses are not fully understood. We identified a Brachypodium distachyon mutant whose flowering is delayed under inductive long-day conditions due to a mutation in the JMJ1 gene, which encodes a Jumonji domain-containing protein. JMJ1 is a histone demethylase that mainly demethylates H3K4me2 and H3K4me3 in vitro and in vivo. Analysis of the genome-wide distribution of H3K4me1, H3K4me2, and H3K4me3 in wild-type plants by chromatin immunoprecipitation and sequencing combined with RNA sequencing revealed that H3K4m1 and H3K4me3 are positively associated with gene transcript levels, whereas H3K4me2 is negatively correlated with transcript levels. Furthermore, JMJ1 directly binds to the chromatin of the flowering regulator genes VRN1 and ID1 and affects their transcription by modifying their H3K4me2 and H3K4me3 levels. Genetic analyses indicated that JMJ1 promotes flowering by activating VRN1 expression. Our study reveals a role for JMJ1-mediated chromatin modification in the proper timing of flowering in B. distachyon.
Proceedings of the National Academy of Sciences · 2023-11-07 · 9 citations
articleOpen accessSenior authorThe transition to flowering is a major developmental switch in plants. In many temperate grasses, perception of indicators of seasonal change, such as changing day-length and temperature, leads to expression of FLOWERING LOCUS T1 ( FT1 ) and FT-Like ( FTL ) genes that are essential for promoting the transition to flowering. However, little is known about the upstream regulators of FT1 and FTL genes in temperate grasses. Here, we characterize the monocot-specific gene INDETERMINATE1 ( BdID1 ) in Brachypodium distachyon and demonstrate that BdID1 is a regulator of FT family genes. Mutations in ID1 impact the ability of the short-day (SD) vernalization, cold vernalization, and long-day (LD) photoperiod pathways to induce certain FTL genes. BdID1 is required for upregulation of FTL9 ( FT-LIKE9 ) expression by the SD vernalization pathway, and overexpression of FTL9 in an id1 background can partially restore the delayed flowering phenotype of id1 . We show that BdID1 binds in vitro to the promoter region of FTL genes suggesting that ID1 directly activates FTL expression. Transcriptome analysis shows that BdID1 is required for FT1 , FT2 , FTL12 , and FTL13 expression under inductive LD photoperiods, indicating that BdID1 is a regulator of the FT gene family. Moreover, overexpression of FT1 in the id1 background results in rapid flowering similar to overexpressing FT1 in the wild type, demonstrating that BdID1 is upstream of FT family genes. Interestingly, ID1 negatively regulates a previously uncharacterized FTL gene, FTL4, and we show that FTL4 is a repressor of flowering. Thus, BdID1 is critical for proper timing of flowering in temperate grasses.
Transgenic plants with altered senescence characteristics
OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-23 · 1 citations
articleOpen access1st authorCorrespondingThe identification of senescence-specific promoters from plants is described. Using information from the first senescence-specific promoter, SAG12 from Arabidopsis, other homologous promoters from another plant have been identified. Such promoters may be used to delay senescence in commercially important plants.
2023-09-12
peer-reviewOpen access1st authorCorrespondingSilencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase (Pol V). Loss of H1 resulted in heterochromatic TE enrichment by NRPE1. Increased NRPE1 binding was associated with increased chromatin accessibility in h1, suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 restricts the occupancy of the methylation reader and activator complex component, SUVH1, indicating that H1’s regulatory control of methylation pathways is not limited to RdDM. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.
2023-01-03
peer-reviewOpen access1st authorCorrespondingArticle Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract To synchronize flowering time with spring, many plants undergo vernalization, a floral-promotion process triggered by exposure to long-term winter cold. In Arabidopsis thaliana, this is achieved through cold-mediated epigenetic silencing of the floral repressor, FLOWERING LOCUS C (FLC). COOLAIR, a cold-induced antisense RNA transcribed from the FLC locus, has been proposed to facilitate FLC silencing. Here, we show that C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3′-end of FLC and CRT/DRE-binding factors (CBFs) are required for cold-mediated expression of COOLAIR. CBFs bind to CRT/DREs at the 3′-end of FLC, both in vitro and in vivo, and CBF levels increase gradually during vernalization. Cold-induced COOLAIR expression is severely impaired in cbfs mutants in which all CBF genes are knocked-out. Conversely, CBF-overexpressing plants show increased COOLAIR levels even at warm temperatures. We show that COOLAIR is induced by CBFs during early stages of vernalization but COOLAIR levels decrease in later phases as FLC chromatin transitions to an inactive state to which CBFs can no longer bind. We also demonstrate that cbfs and FLCΔCOOLAIR mutants exhibit a normal vernalization response despite their inability to activate COOLAIR expression during cold, revealing that COOLAIR is not required for the vernalization process. Editor's evaluation This important work advances our understanding of the systems that plants evolved to coordinate developmental processes such as the timing of flowering with seasonal change, particularly with respect to the regulation and role of long non-coding RNAs (lncRNAs) complementary to genes encoding proteins that regulate developmental switches. The evidence supporting the conclusions is solid. The work will be of interest to those interested plant development as well those interested in the role and regulation of lncRNAs. https://doi.org/10.7554/eLife.84594.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Long spells of cold winter weather may feel miserable, but they are often necessary for spring to blossom. Indeed, many plants need to face a prolonged period of low temperatures to be able to flower; this process is known as vernalization. While the molecular mechanisms which underpin vernalization are well-known, it is still unclear exactly how plants can 'sense' the difference between short and long periods of cold. Jeon, Jeong et al. set out to explore this question by focusing on COOLAIR, one of the rare genetic sequences identified as potentially being able to trigger vernalization. COOLAIR is a long noncoding RNA, a partial transcript of a gene that will not be 'read' by the cell to produce a protein but which instead regulates how and when certain genes are being switched on. COOLAIR emerges from the locus of the FLC gene, which is one of the main repressors of flowering, and it gradually accumulates in the plant when temperatures remain low for a long period. While some evidence suggests that COOLAIR may help to switch off FLC, other studies have raised some doubts about its involvement in vernalization. In response, Jeon, Jeong et al. examined the FLC gene in a range of plants closely related to A. thaliana, and in which COOLAIR also accumulates upon cold exposure. This helped them identify a class of proteins, known as CBFs, which could bind to sequences near the FLC gene to activate the production of COOLAIR when the plants were kept in cold conditions for a while. CBFs were already known to help plants adapt to short cold snaps, but these experiments confirmed that they could act as both short- and long-term cold sensors. This work allowed Jeon, Jeong et al. to propose a model in which CBF and therefore COOLAIR levels increase as the cold persists, until changes in the structure of the FLC gene prevent CBF from binding to it and COOLAIR production drops. Unexpectedly, examining the fate of mutants which could not produce COOLAIR revealed that these plants could still undergo vernalization, suggesting that the long noncoding RNA is in fact not necessary for this process. These results should prompt other scientists to further investigate the role of COOLAIR in vernalization; they also give insight into how coding and noncoding sequences may have evolved together in various members of the A. thaliana family to adapt to the environment. Introduction Appropriate timing of flowering provides evolutionary advantage of plant reproductive success. As sessile organisms, plants have evolved mechanisms through which seasonal cues coordinate the transition to flowering. One of the significant environmental factors affecting flowering time of plants adapted to temperate climates is the temperature changes during the seasons, and plants have evolved complex sensory mechanisms to monitor the surrounding temperature to properly control the timing of flowering and tolerate thermal stress (Went, 1953; Penfield, 2008; Ding and Yang, 2022). Cold acclimation and vernalization are two responses of plants to low temperatures. Cold acclimation is generally initiated by a short period of non-freezing cold exposure and increases the frost tolerance of plants (Weiser, 1970; Gilmour et al., 1988; Guy, 1990; Thomashow, 1999; Chinnusamy et al., 2007). The three C-REPEAT (CRT)/DEHYDRATION-RESPONSIVE ELEMENT (DRE) BINDING FACTORs (CBFs) and their encoding genes serve as signaling hubs for cold acclimation in Arabidopsis thaliana (Stockinger et al., 1997; Medina et al., 1999; Thomashow, 2010; Ding et al., 2019). When exposed to low temperatures, the transcription of CBFs is rapidly promoted by a group of cold-signal transducers, including the Ca2+/calmodulin-binding proteins, CALMODULIN-BINDING TRANSCRIPTION ACTIVATORs (CAMTAs) (Doherty et al., 2009; Kim et al., 2013; Kidokoro et al., 2017); the clock proteins, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE-ELONGATED HYPOCOTYL (LHY) (Dong et al., 2011); and the brassinosteroid-responsive proteins, BRASSINAZOLE-RESISTANT 1 (BZR1) and CESTA (CES) (Eremina et al., 2016; Li et al., 2017). In addition, cold also enhances the stability or activity of CBF proteins. For example, cold facilitates the interaction between CBFs and BASIC TRANSCRIPTION FACTOR 3s (BTF3s), which promotes CBF stability (Ding et al., 2018), and cold triggers degradation of co-repressor, HISTONE DEACETYLASE 2C (HD2C), thereby allowing CBFs to activate their targets (Park et al., 2018b). Furthermore, cold reduces oxidized CBFs, which increases active CBF monomers (Lee et al., 2021). Low-temperature-induced CBFs, in turn, activate the expression of cold-regulated (COR) genes by binding to CRT/DREs in their promoters (Stockinger et al., 1997; Medina et al., 1999). Diverse arrays of cryoprotective proteins encoded by COR genes allow plants to overcome freezing stress (Gilmour et al., 1988; Hajela et al., 1990; Thomashow et al., 1997; Shinozaki et al., 2003; Shi et al., 2018; Ding et al., 2019). In contrast to cold acclimation, vernalization, a floral-promotion process that occurs during winter, requires an extended cold period (Napp-Zinn, 1955; Chouard, 1960; Michaels and Amasino, 2000). This allows plants to synchronize the timing of flowering with favorable spring conditions. Vernalization in A. thaliana is mainly achieved by silencing the floral repressor gene, FLOWERING LOCUS C (FLC) (Michaels and Amasino, 1999; Sheldon et al., 1999; Sheldon et al., 2000; Michaels and Amasino, 2001). FLC encodes a MADS-box protein that represses the expression of floral activator genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and FLOWERING LOCUS T (FT, encoding florigen), by directly binding to their promoter regions (Lee et al., 2000; Michaels et al., 2005; Helliwell et al., 2006). In Arabidopsis winter annuals, such as San Feliu-2, Löv-1, and Sweden (SW) ecotypes, flowering is prevented by the high expression of FLC before exposure to winter cold (Lee et al., 1993; Shindo et al., 2005; Park et al., 2018a). This is caused by the strong transcriptional activation of FLC by the FRIGIDA (FRI) supercomplex, which recruits general transcription factors and several chromatin modifiers (Michaels and Amasino, 1999; Sheldon et al., 1999; Johanson et al., 2000; Choi et al., 2011; Li et al., 2018). Prior to vernalization, FLC chromatin is highly enriched with active histone marks such as histone H3 acetylation and trimethylation of Lys4 or Lys36 at H3 (H3K4me3/H3K36me3) (Bastow et al., 2004; Yang et al., 2014). In contrast, prolonged cold exposure results in gradual deacetylation of FLC chromatin and concomitant removal of H3K4me3 and H3K36me3 from the FLC (Bastow et al., 2004; Yang et al., 2014; Nishio et al., 2016). Additionally, VP1/ABI3-LIKE 1 (VAL1) and VAL2 recruit Polycomb Repressive Complex 2 (PRC2) onto FLC chromatin, thereby accumulating the repressive histone mark, H3 Lys27 trimethylation (H3K27me3), in the nucleation region around the first exon and intron of FLC (Sung and Amasino, 2004b; Wood et al., 2006; De Lucia et al., 2008; Angel et al., 2011; Nishio et al., 2016; Qüesta et al., 2016; Yuan et al., 2016). Subsequently, upon returning to warm temperatures, H3K27me3 marks are spread over the entire FLC chromatin region by LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), which ensures stable FLC suppression and renders plants competent to flower (Mylne et al., 2006; Sung et al., 2006; Yang et al., 2017). Several long-term cold-induced factors have been shown to play crucial roles in the epigenetic silencing of FLC. VERNALIZATION INSENSITIVE 3 (VIN3) family genes, which are upregulated by prolonged cold, encode plant homeodomain (PHD) proteins that recognize H3K9me2 enriched in FLC chromatin during vernalization (Sung and Amasino, 2004b; Kim and Sung, 2013; Kim and Sung, 2017a). These proteins mediate the recruitment of PRC2 and the subsequent deposition of H3K27me3 at the FLC nucleation region (Sung and Amasino, 2004b; De Lucia et al., 2008; Kim and Sung, 2013). In addition, vernalization-induced long noncoding RNAs (lncRNAs) are involved in such histone modifications. COLDAIR and COLDWRAP, the two lncRNAs transcribed from the first intron and promoter region of FLC, respectively, are required for H3K27me3 deposition in response to long-term cold (Heo and Sung, 2011; Kim and Sung, 2017b). COLDAIR and COLDWRAP are also thought to affect the formation of the intragenic chromatin loop at the FLC, which may be a part of the FLC silencing mechanism (Kim and Sung, 2017b). Unlike COLDAIR and COLDWRAP, which are transcribed in the sense direction of FLC, another lncRNA, COOLAIR, is an antisense transcript expressed from the 3′-end of FLC (Swiezewski et al., 2009). The gradual accumulation of COOLAIR reaches a maximum within a few weeks of cold exposure, whereas COLDAIR and COLDWRAP show peaks at a later phase of vernalization (Csorba et al., 2014; Kim and Sung, 2017b). COOLAIR was reported to remove active histone marks from FLC chromatin (Liu et al., 2007; Csorba et al., 2014; Fang et al., 2020; Xu et al., 2021a; Zhu et al., 2021). Particularly, in summer annuals, phase-separated RNA-processing complexes favor co-transcriptional proximal polyadenylation of COOLAIR (Marquardt et al., 2014; Wang et al., 2014; Fang et al., 2019; Wu et al., 2020; Xu et al., 2021b). The COOLAIR-processing machinery exhibits transient and dynamic interactions with an H3K4 demethylation complex, leading to FLC suppression at warm temperatures (Liu et al., 2007; Fang et al., 2020; Xu et al., 2021a). COOLAIR is also likely to be involved in reducing H3K36me3 at the FLC during vernalization process (Csorba et al., 2014). COOLAIR was reported to promote the sequestration of the FRI complex from the FLC promoter by condensing it into phase-separated nuclear bodies (Zhu et al., 2021). This has been suggested to cause the inactivation of FLC, which is probably accompanied by the silencing of FLC chromatin through the removal of H3K36me3. However, a previous study has raised the issue that COOLAIR appears not to be necessary for vernalization (Helliwell et al., 2011; Luo et al., 2019). Compared with the signaling pathway of cold acclimation, how a long-term cold signal is transduced to trigger the induction of VIN3 and lncRNAs is less well understood. It is marginally known that some chromatin modifiers, NAC WITH TRANSMEMBRANE MOTIF 1-LIKE 8 (NTL8), and CCA1/LHY act as positive regulators of VIN3 during the vernalization process (Kim et al., 2010; Jean Finnegan et al., 2011; Zhao et al., 2020; Kyung et al., 2022). However, little is known about the upstream regulators of COOLAIR required for cold-induction. Recent reports have shown that an NAC domain-containing protein, NTL8, and the WRKY transcription factor, WRKY63, can bind to the promoter of COOLAIR and activate its expression (Zhao et al., 2021; Hung et al., 2022). However, whether NTL8 and WRKY63 are necessary for the full extent of COOLAIR induction during vernalization has not been thoroughly addressed. In this study, we identified that CRT/DREs at the 3′-end of the FLC are required for the long-term cold response of COOLAIR. Additionally, we show that CBFs, which accumulate during the long-term winter cold, act as upstream regulators of COOLAIR during vernalization. Results A CRT/DRE-binding factor, CBF3, directly binds to CRT/DREs at the 3′-end of the FLC The proximal promoter region of COOLAIR is highly conserved among FLC orthologs from A. thaliana relatives (Castaings et al., 2014). Thus, we assumed that the cis-element conferring a long-term cold response would exist within that block. A comparison of the region near the transcriptional start site (TSS) of COOLAIR revealed the conservation of two CRT/DREs among six FLC orthologs from five related species of the Brassicaceae family (Figure 1A). CRT/DRE, the core sequence of which is CCGAC, is a regulatory element that imparts cold- or drought-responsive gene expression (Baker et al., 1994). CBF1, 2, and 3 encode APETALA 2 (AP2) domain-containing proteins that can bind to CRT/DRE and are usually present in the promoters of cold- and drought-responsive genes (Stockinger et al., 1997; Medina et al., 1999). Consistent with this, the Arabidopsis cistrome database and the genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) results from a previous study suggested that CBFs bind to the 3′-end sequence of FLC containing CRT/DREs (Figure 1—figure supplement 1; O'Malley et al., 2016; Song et al., 2021). Figure 1 with 2 supplements see all Download asset Open asset CBF3 directly binds to the CRT/DREs at the 3′-end of FLC. (A) Comparison of sequences around the 3′-end of the FLC orthologs from Arabidopsis relatives. The upper graphic presents the gene structure of AtFLC. The black bars, black lines, and white bars indicate exons, introns, and untranslated regions (UTRs), respectively. The blue line presents the region used for sequence comparison among six orthologous genes from five plant species. In the sequence alignment below, the gray line indicates the 3′-UTR of FLC orthologs, the blue arrow indicates the transcriptional start site (TSS) of AtCOOLAIR, and the red lines indicate CRT/DREs. At, Arabidopsis thaliana; Cr, Capsella rubella; Al, Arabidopsis lyrata; Aa, Arabis alpina; Th, Thellungiella halophila. (B) EMSA using one of the CRT/DREs located at the AtCOOLAIR promoter, DRE1, as a probe. In the upper graphic showing AtFLC gene structure, CRT/DRE-like sequences are marked as blue arrows and labeled as a, b, c, and d for CRT/DRE-like sequences and as 1 and 2 for CRT/DRE sequences. For the competition assay, these CRT/DRE-like sequences and mutant forms of DRE1 and DRE2 were used as competitors. A 100-fold molar excess of unlabeled competitors was added. No protein (−) or maltose-binding protein (MBP) were used as controls. FP, free probe. (C) ChIP assay result showing the enrichment of CBF3-myc protein on the AtFLC locus. Samples of NV, 10V, 20V, and 40V plants of pSuper:CBF3-myc were collected at zeitgeber time (ZT) 4 in an SD cycle. The CBF3-chromatin complex was immunoprecipitated (IP) with anti-myc antibodies (blue bars), and mouse IgG (black bars) was used as a control. Positions of qPCR amplicons used for ChIP-qPCR analysis are illustrated as P1, P2, and P3 in the upper graphic. The blue arrow in the graphic denotes the position of CRT/DREs on the AtCOOLAIR gene. ChIP-qPCR results have been represented as mean ± SEM of the three biological replicates in the lower panel. Open circles and squares represent each data point. Relative enrichments of the IP/5% input were normalized to that of pTUB2. The blue shadings indicate cold periods. Asterisks indicate a significant difference between IgG and anti-myc ChIP-qPCR results at each vernalization time point (*, p < 0.05; **, p < 0.01; unpaired Student's t-test). Figure 1—source data 1 Uncropped labeled gel image and the original image file for the EMSA result. https://cdn.elifesciences.org/articles/84594/elife-84594-fig1-data1-v3.zip Download elife-84594-fig1-data1-v3.zip We performed an electrophoretic mobility shift assay (EMSA) to confirm this binding using probes harboring CRT/DREs (named DRE1 or 2) from the COOLAIR promoter. The mobility of these two Cy5-labeled probes was retarded by maltose-binding protein (MBP)-fused CBF3 (Figure 1B, Figure 1—figure supplement 2). The band shift was competed out by adding an excess amount of unlabeled DRE1 or 2 oligonucleotides. In contrast, competitors containing mutant forms of DRE1 or 2 (DRE1m or 2m, respectively) failed to compete (Figure 1B). We also tested the binding of other CRT/DRE-like sequences in the FLC locus. Two (DREa and b) were present in the FLC promoter, while the other two (DREc and d), were present in the first and last exons, respectively (Figure 1—figure supplement 2). Only DREc competed with the band shift caused by the CBF3-DRE1 interaction (Figure 1B, Figure 1—figure supplement 2). The presence or absence of bases that determine the binding between CBFs and CRT/DRE could the in among CRT/DRE-like sequences at the FLC locus et al., Subsequently, a chromatin immunoprecipitation assay using a pSuper:CBF3-myc (Liu et al., was to determine whether CBF3 is with the FLC region containing CRT/DREs in For ChIP assay to show binding of CBF3 protein to the COOLAIR promoter during we used pSuper:CBF3-myc instead of CBF3 transcript is during vernalization as shown Consistent with the in vitro CBF3-myc protein was enriched at the 3′-end of FLC and the first exon DREc was located (Figure vernalization CBF3-myc was highly enriched at both the and of FLC and that CBF3 protein can activate FLC warm as reported et al., 2009). However, enrichment in the region rapidly during the vernalization period. In contrast, enrichment on P3 was until of vernalization and was This is with the expression of COOLAIR, which during the phase of vernalization (Csorba et al., 2014). COOLAIR is one of the CBF in Arabidopsis CBF genes are rapidly and induced upon exposure to cold et al., 1999). CBF which are genes, are or cold of a few (Gilmour et al., et al., and Thomashow, As COOLAIR in its promoter (Figure we whether COOLAIR CBF The expression of genes to the CBF is or by CBF warm conditions (Park et al., we that the transcript of COOLAIR increased in plants at temperature to that in the (Figure was mainly to the or COOLAIR As the levels of both proximal and of COOLAIR were in the CBF3 in the it is likely that COOLAIR instead of the is by CBF3 (Figure et al., 2010; et al., 2014). It has been reported that the targets of or 2 are not to those of CBF3, CBF genes show high sequence et al., 2007). Thus, we the expression levels of all COOLAIR in plants CBF1, 2, or 3 et al., 2009). all CBF-overexpressing plants increased levels of COOLAIR (Figure supplement However, each CBF-overexpressing plant a on COOLAIR The the of the proximal COOLAIR whereas the CBF3 the of COOLAIR. In contrast, the plants a increase in proximal COOLAIR levels and a increase in COOLAIR, to those in the results that the three CBFs regulate COOLAIR transcription Figure 2 with 1 supplement see all Download asset Open asset COOLAIR a expression with CBF (A) levels of COOLAIR and COOLAIR in the and plants The gene of FLC and COOLAIR are illustrated in the upper panel. The black arrows indicate the transcription start of FLC and COOLAIR. The black arrows indicate the of each COOLAIR The of proximal and and COOLAIR are The used for proximal and are marked in Figure supplement Relative transcript levels of COOLAIR and COOLAIR were normalized to that of and have been represented as mean ± SEM of three biological and squares represent each data point. Asterisks indicate a significant difference as to the (*, p < 0.05; **, p < 0.01; p < unpaired Student's t-test). (B) of COOLAIR 2, and cold were to 4 cold and at each time point. Relative transcript levels of COOLAIR to were normalized to that of The have been represented as mean ± SEM of three biological The blue indicates periods cold have been marked using p < 0.05; by (C) levels of COOLAIR and COOLAIR in and mutant before and a of 4 cold Relative levels of COOLAIR and COOLAIR were normalized to that of have been represented as mean ± SEM of three biological and squares indicate each data point. have been marked using p < 0.05; by of CBF is that their expression is by a of cold exposure (Park et al., Thus, we COOLAIR is also induced by cold exposure. We plants with 2, and of cold the levels of COOLAIR. The results that cold for less 3 failed to COOLAIR CBF a at 3 of cold exposure and Gilmour et al., However, of cold when CBF3 protein levels COOLAIR was induced and Thus, COOLAIR expression were to those of other that the expression was highly increased CBF transcript levels a upon cold exposure (Gilmour et al., and Thomashow, To confirm whether CBFs are for the cold response of COOLAIR, we examined COOLAIR induction a of cold in the and in which all three CBFs were out (Figure et al., 2016). as well as COOLAIR, failed to be induced by cold exposure in the cbfs a increase was in the Thus, these results that CBFs are required for COOLAIR even a short period of cold exposure. Figure 3 with 1 supplement see all Download asset Open asset levels of CBFs increase during the vernalization process. (A) levels of CBF1, 2, and 3 cold exposure. plants were to and of 4 cold Relative levels of CBF1, 2, and 3 were normalized to that of have been represented as mean ± SEM of three biological The blue shadings indicate cold periods. have been marked using p < 0.05; by (B) levels of CBF1, 2, and 3 and plants were with 4 vernalization an SD and collected at Relative levels of CBF1, 2, and 3 were normalized to that of have been represented as mean ± SEM of three biological The blue shadings periods cold. have been marked using p < 0.05; by (C) of CBF1, 2, and 3 transcript levels in or 40V plants an SD were collected 4 between and Relative transcript levels of CBF1, 2, and 3 were normalized to that of have been represented as mean ± SEM of three biological The white and black bars represent and respectively. Asterisks indicate a significant difference between and 40V p < of CBF3 protein cold exposure. The plants were to and of 4 cold CBF3 proteins were using anti-myc was the control. each band indicate signal to The mean of two biological replicates are of CBF3 protein during the vernalization process. The to 4 vernalization, were collected at of the time point. CBF3 proteins were using anti-myc was the control. each band indicate signal to The mean of three biological replicates are Figure data 1 Uncropped labeled and the original image for the Download CBFs accumulate during vernalization We whether CBFs are also for vernalization-induced COOLAIR It has been reported that COOLAIR is gradually upregulated as the cold period and peaks weeks of vernalization (Csorba et al., 2014). However, studies on CBF expression have been performed within a few the of CBFs has been in the of cold (Gilmour et al., et al., Medina et al., 1999; et al., we the expression of CBFs before and long-term cold exposure to determine the between the expression of CBFs and COOLAIR during vernalization. As the levels of all three CBFs within 3 of cold exposure and rapidly (Figure However, CBF levels increased as the cold period was prolonged suggesting that both and cold upregulated CBF expression (Figure CBFs exhibit (Dong et al., we also whether the expression of CBFs is by vernalization. plants were collected 4 a 8 both and 40V conditions. As shown in Figure the of each CBF was the transcript levels of all three CBFs were at 40V Consistent with the transcript the of CBF3 protein in plants was that in plants during all (Figure supplement To whether the increased transcription of CBF3 to protein we the of CBF3 at each time point of
2023-01-03
peer-reviewOpen access1st authorCorrespondingCBFs, the central regulators of low-temperature signaling, have a function to directly activate the expression of COOLAIR, an antisense RNA of FLC, during vernalization process, but COOLAIR is not required for the vernalization response.
PLoS Genetics · 2023-05-10 · 21 citations
articleOpen accessSenior authorCorrespondingDaylength sensing in many plants is critical for coordinating the timing of flowering with the appropriate season. Temperate climate-adapted grasses such as Brachypodium distachyon flower during the spring when days are becoming longer. The photoreceptor PHYTOCHROME C is essential for long-day (LD) flowering in B. distachyon. PHYC is required for the LD activation of a suite of genes in the photoperiod pathway including PHOTOPERIOD1 (PPD1) that, in turn, result in the activation of FLOWERING LOCUS T (FT1)/FLORIGEN, which causes flowering. Thus, B. distachyon phyC mutants are extremely delayed in flowering. Here we show that PHYC-mediated activation of PPD1 occurs via EARLY FLOWERING 3 (ELF3), a component of the evening complex in the circadian clock. The extreme delay of flowering of the phyC mutant disappears when combined with an elf3 loss-of-function mutation. Moreover, the dampened PPD1 expression in phyC mutant plants is elevated in phyC/elf3 mutant plants consistent with the rapid flowering of the double mutant. We show that loss of PPD1 function also results in reduced FT1 expression and extremely delayed flowering consistent with results from wheat and barley. Additionally, elf3 mutant plants have elevated expression levels of PPD1, and we show that overexpression of ELF3 results in delayed flowering associated with a reduction of PPD1 and FT1 expression, indicating that ELF3 represses PPD1 transcription consistent with previous studies showing that ELF3 binds to the PPD1 promoter. Indeed, PPD1 is the main target of ELF3-mediated flowering as elf3/ppd1 double mutant plants are delayed flowering. Our results indicate that ELF3 operates downstream from PHYC and acts as a repressor of PPD1 in the photoperiod flowering pathway of B. distachyon.
EARLY FLOWERING 3 and Photoperiod Sensing in Brachypodium distachyon
Frontiers in Plant Science · 2022 · 28 citations
- Biology
- Botany
- Genetics
under non-inductive photoperiods and that this acceleration of flowering is mediated by red light. Finally, we discuss advances and perspectives for research on the perception of photoperiod in temperate grasses.
Recent grants
NIH · $7k
Analysis of the Flowering Regulatory Network in the Grass Brachypodium distachyon
NSF · $663k · 2018–2023
Arabidopsis 2010: Gene Identification in the Photoperiod and Circadian Network of Flowering Control
NSF · $883k · 2002–2007
NIH · $1.1M · 2013
Analysis of the Autonomous Floral Induction Pathway
NSF · $450k · 2005–2010
Frequent coauthors
- 147 shared
Daniel P. Woods
University of California, San Diego
- 69 shared
Thomas S. Ream
Bayer (United States)
- 42 shared
Richard Sibout
Laboratoire D'étude des Résidus et Contaminants Dans les Aliments
- 39 shared
Mark R. Doyle
University of Wisconsin–Madison
- 38 shared
Yinxin Dong
- 37 shared
Scott D. Michaels
Indiana University Bloomington
- 35 shared
Seth J Davis
Henan University
- 35 shared
Frédéric Bouché
University of Liège
Education
- 1973
B.S., Botany
University of Wisconsin-Madison
- 1975
M.S., Botany
University of Wisconsin-Madison
- 1979
Ph.D., Botany
University of Wisconsin-Madison
Awards & honors
- Howard Hughes Medical Institute Professor
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