Franziska Turck

Regulation and identity of florigen: FLOWERING LOCUS T moves center stage.

The transition from vegetative to reproductive growth is controlled by day length in many plant species. Day length is perceived in leaves and induces a systemic signal, called florigen, that moves through the phloem to the shoot apex. At the shoot apical meristem (SAM), florigen causes changes in gene expression that reprogram the SAM to form flowers instead of leaves. Analysis of flowering of Arabidopsis thaliana placed the CONSTANS/FLOWERING LOCUS T (CO/FT) module at the core of a pathway that promotes flowering in response to changes in day length. We describe progress in defining the molecular mechanisms that activate this module in response to changing day length and the increasing evidence that FT protein is a major component of florigen. Finally, we discuss conservation of FT function in other species and how variation in its regulation could generate different flowering behaviors.

Arabidopsis TFL2/LHP1 Specifically Associates with Genes Marked by Trimethylation of Histone H3 Lysine 27

TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) is the only Arabidopsis protein with overall sequence similarity to the HETEROCHROMATIN PROTEIN 1 (HP1) family of metazoans and S. pombe. TFL2/LHP1 represses transcription of numerous genes, including the flowering-time genes FLOWERING LOCUS T (FT) and FLOWERING LOCUS C (FLC), as well as the floral organ identity genes AGAMOUS (AG) and APETALA 3 (AP3). These genes are also regulated by proteins of the Polycomb repressive complex 2 (PRC2), and it has been proposed that TFL2/LHP1 represents a potential stabilizing factor of PRC2 activity. Here we show by chromatin immunoprecipitation and hybridization to an Arabidopsis Chromosome 4 tiling array (ChIP-chip) that TFL2/LHP1 associates with hundreds of small domains, almost all of which correspond to genes located within euchromatin. We investigated the chromatin marks to which TFL2/LHP1 binds and show that, in vitro, TFL2/LHP1 binds to histone H3 di- or tri-methylated at lysine 9 (H3K9me2 or H3K9me3), the marks recognized by HP1, and to histone H3 trimethylated at lysine 27 (H3K27me3), the mark deposited by PRC2. However, in vivo TFL2/LHP1 association with chromatin occurs almost exclusively and co-extensively with domains marked by H3K27me3, but not H3K9me2 or -3. Moreover, the distribution of H3K27me3 is unaffected in lhp1 mutant plants, indicating that unlike PRC2 components, TFL2/LHP1 is not involved in the deposition of this mark. Rather, our data suggest that TFL2/LHP1 recognizes specifically H3K27me3 in vivo as part of a mechanism that represses the expression of many genes targeted by PRC2.

CONSTANS and the CCAAT Box Binding Complex Share a Functionally Important Domain and Interact to Regulate Flowering of Arabidopsis

The CCT (for CONSTANS, CONSTANS-LIKE, TOC1) domain is found in 45 Arabidopsis thaliana proteins involved in processes such as photoperiodic flowering, light signaling, and regulation of circadian rhythms. We show that this domain exhibits similarities to yeast HEME ACTIVATOR PROTEIN2 (HAP2), which is a subunit of the HAP2/HAP3/HAP5 trimeric complex that binds to CCAAT boxes in eukaryotic promoters. Moreover, we demonstrate that CONSTANS (CO), which promotes Arabidopsis flowering, interacts with At HAP3 and At HAP5 in yeast, in vitro, and in planta. Mutations in CO that delay flowering affect residues highly conserved between CCT and the DNA binding domain of HAP2. Taken together, these data suggest that CO might replace At HAP2 in the HAP complex to form a trimeric CO/At HAP3/At HAP5 complex. Flowering was delayed by overexpression of At HAP2 or At HAP3 throughout the plant or in phloem companion cells, where CO is expressed. This phenotype was correlated with reduced abundance of FLOWERING LOCUS T (FT) mRNA and no change in CO mRNA levels. At HAP2 or At HAP3 overexpression may therefore impair formation of a CO/At HAP3/At HAP5 complex leading to reduced expression of FT. During plant evolution, the number of genes encoding HAP proteins was greatly amplified, and these proteins may have acquired novel functions, such as mediating the effect of CCT domain proteins on gene expression.

PEP1 regulates perennial flowering in Arabis alpina.

Annual plants complete their life cycle in one year and initiate flowering only once, whereas perennials live for many years and flower repeatedly. How perennials undergo repeated cycles of vegetative growth and flowering that are synchronized to the changing seasons has not been extensively studied. Flowering is best understood in annual Arabidopsis thaliana, but many closely related species, such as Arabis alpina, are perennials. We identified the A. alpina mutant perpetual flowering 1 (pep1), and showed that PEP1 contributes to three perennial traits. It limits the duration of flowering, facilitating a return to vegetative development, prevents some branches from undergoing the floral transition allowing polycarpic growth habit, and confers a flowering response to winter temperatures that restricts flowering to spring. Here we show that PEP1 is the orthologue of the A. thaliana gene FLOWERING LOCUS C (FLC). The FLC transcription factor inhibits flowering until A. thaliana is exposed to winter temperatures, which trigger chromatin modifications that stably repress FLC transcription. In contrast, PEP1 is only transiently repressed by low temperatures, causing repeated seasonal cycles of repression and activation of PEP1 transcription that allow it to carry out functions characteristic of the cyclical life history of perennials. The patterns of chromatin modifications at FLC and PEP1 differ correlating with their distinct expression patterns. Thus we describe a critical mechanism by which flowering regulation differs between related perennial and annual species, and propose that differences in chromatin regulation contribute to this variation.

cis-Regulatory Elements and Chromatin State Coordinately Control Temporal and Spatial Expression of FLOWERING LOCUS T in Arabidopsis

Transcription of FLOWERING LOCUS T is tightly controlled by external and internal cues. This study reports that evolutionary conserved blocks of regulatory elements spaced at considerable distance within the promoter interact with factors regulating local chromatin structure to control transcription of the gene. Flowering time of summer annual Arabidopsis thaliana accessions is largely determined by the timing of FLOWERING LOCUS T (FT) expression in the leaf vasculature. To understand the complex interplay between activating and repressive inputs controlling flowering through FT, cis-regulatory sequences of FT were identified in this study. A proximal and an ∼5-kb upstream promoter region containing highly conserved sequence blocks were found to be essential for FT activation by CONSTANS (CO). Chromatin-associated protein complexes add another layer to FT regulation. In plants constitutively overexpressing CO, changes in chromatin status, such as a decrease in binding of LIKE HETEROCHROMATIN PROTEIN1 (LHP1) and increased acetylation of H3K9 and K14, were observed throughout the FT locus, although these changes appear to be a consequence of FT upregulation and not a prerequisite for activation. Binding of LHP1 was required to repress enhancer elements located between the CO-controlled regions. By contrast, the distal and proximal promoter sequences required for FT activation coincide with locally LHP1 and H3K27me3 depleted chromatin, indicating that chromatin status facilitates the accessibility of transcription factors to FT. Therefore, distant regulatory regions are required for FT transcription, reflecting the complexity of its control and differences in chromatin status delimit functionally important cis-regulatory regions.

VAL- and AtBMI1-Mediated H2Aub Initiate the Switch from Embryonic to Postgerminative Growth in Arabidopsis

Plant B3-domain transcription factors have an important role in regulating seed development, in particular seed maturation and germination. Among the B3 factors, the AFL (ABSCISIC ACID INSENSITIVE3 [ABI3], FUSCA3 [FUS3], and LEAFY COTYLEDON2 [LEC2]) proteins activate the seed maturation program in a complex network, while the VAL (VP1/ABI3-LIKE) 1/2/3 proteins suppress AFL action in order to initiate germination and vegetative development through an as yet unknown mechanism. In addition, the AFL genes and LEAFY COTYLEDON1 (LEC1), referred as seed maturation genes, are epigenetically repressed after germination by the Polycomb group (PcG) machinery via its histone-modifying activities: the histone H3 lysine 27 trimethyltransferase activity of the PcG repressive complex 2 (PRC2) and the E3 H2A monoubiquitin ligase activity of the PRC1. Both histone modifications are required for the repression; however, the underlying mechanism is far from clear, because the localization and the role of H2Aub marks are still unknown. In this work, we demonstrate that VAL proteins and AtBMI1-mediated H2Aub initiate repression of seed maturation genes. After the initial off switch, the repression is maintained by PRC2-mediated H3K27me3. Our results indicate that the regulation of seed maturation genes does not follow the classic hierarchical model proposed for animal PcG-mediated repression, since the PRC1 activity is required for the H3K27me3 modification of these genes. Furthermore, we show different mechanisms to achieve PcG repression in plants, as the repression of genes involved in other processes has different requirements for H2Aub and H3K27me3 marking.

Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors.

RADICAL-INDUCED CELL DEATH1 (RCD1) is an important regulator of stress and hormonal and developmental responses in Arabidopsis thaliana. Together with its closest homolog, SIMILAR TO RCD-ONE1 (SRO1), it is the only Arabidopsis protein containing the WWE domain, which is known to mediate protein-protein interactions in other organisms. Additionally, these two proteins contain the core catalytic region of poly-ADP-ribose transferases and a conserved C-terminal domain. Tissue and subcellular localization data indicate that RCD1 and SRO1 have partially overlapping functions in plant development. In contrast mutant data indicate that rcd1 has defects in plant development, whereas sro1 displays normal development. However, the rcd1 sro1 double mutant has severe growth defects, indicating that RCD1 and SRO1 exemplify an important genetic principle - unequal genetic redundancy. A large pair-wise interaction test against the REGIA transcription factor collection revealed that RCD1 interacts with a large number of transcription factors belonging to several protein families, such as AP2/ERF, NAC and basic helix-loop-helix (bHLH), and that SRO1 interacts with a smaller subset of these. Full genome array analysis indicated that in many cases targets of these transcription factors have altered expression in the rcd1 but not the sro1 mutant. Taken together RCD1 and SRO1 are required for proper plant development.

Phytohormones Participate in an S6 Kinase Signal Transduction Pathway in Arabidopsis

Addition of fresh medium to stationary cells of Arabidopsis suspension culture induces increased phosphorylation of the S6 ribosomal protein and activation of its cognate kinase, AtS6k. Analysis of the activation response revealed that medium constituents required for S6 kinase activation were the phytohormones 1-naphthylacetic acid (auxin) and kinetin. Pretreatment of cells with anti-auxin or PI3-kinase drugs inhibited this response. Consistent with these findings, LY294002, a PI3-kinase inhibitor, efficiently suppressed phytohormone-induced S6 phosphorylation and translational up-regulation of ribosomal protein S6 and S18A mRNAs without affecting global translation. These data indicate that (1) activation of AtS6k is regulated by phytohormones, at least in part, via a lipid kinase-dependent pathway, that (2) the translational regulation of ribosomal proteins appears to be conserved throughout the plant and animal kingdom, and that (3) these events are hallmarks of a growth-related signal transduction pathway novel in plants.

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k -mers

Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (needle in the k-stack), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in nonreference rice cultivars and to two mutants of the nonmodel species Arabis alpina. In both species, comparing pooled F2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, for any species amenable to mutagenesis, NIKS enables forward genetics without requiring segregating populations, genetic maps and reference sequences.

ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin‐immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G]CGT as ATAF1 consensus binding sequences. Co‐expression analysis across publicly available microarray experiments identified 25 genes co‐expressed with ATAF1. The promoter regions of ATAF1 co‐expressors were significantly enriched for ATAF1 binding sites, and TTGCGTA was identified in the promoter of the key abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP‐qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis.