PIF4 enhances DNA binding of CDF2 to co-regulate target gene expression and promote Arabidopsis hypocotyl cell elongation.

How specificity is conferred within gene regulatory networks is an important problem in biology. The basic helix-loop-helix PHYTOCHROME-INTERACTING FACTORs (PIFs) and single zinc-finger CYCLING DOF FACTORs (CDFs) mediate growth responses of Arabidopsis to light and temperature. We show that these two classes of transcription factor (TF) act cooperatively. CDF2 and PIF4 are temporally and spatially co-expressed, they interact to form a protein complex and act in the same genetic pathway to promote hypocotyl cell elongation. Furthermore, PIF4 substantially strengthens genome-wide occupancy of CDF2 at a subset of its target genes. One of these, YUCCA8, encodes an auxin biosynthesis enzyme whose transcription is increased by PIF4 and CDF2 to contribute to hypocotyl elongation. The binding sites of PIF4 and CDF2 in YUCCA8 are closely spaced, and in vitro PIF4 enhances binding of CDF2. We propose that this occurs by direct protein interaction and because PIF4 binding alters DNA conformation. Thus, we define mechanisms by which PIF and CDF TFs cooperate to achieve regulatory specificity and promote cell elongation in response to light.

Regulation of shoot meristem shape by photoperiodic signaling and phytohormones during floral induction of Arabidopsis.

Floral transition, the onset of plant reproduction, involves changes in shape and identity of the shoot apical meristem (SAM). The change in shape, termed doming, occurs early during floral transition when it is induced by environmental cues such as changes in day-length, but how it is regulated at the cellular level is unknown. We defined the morphological and cellular features of the SAM during floral transition of Arabidopsis thaliana. Both cell number and size increased during doming, and these changes were partially controlled by the gene regulatory network (GRN) that triggers flowering. Furthermore, dynamic modulation of expression of gibberellin (GA) biosynthesis and catabolism enzymes at the SAM contributed to doming. Expression of these enzymes was regulated by two MADS-domain transcription factors implicated in flowering. We provide a temporal and spatial framework for integrating the flowering GRN with cellular changes at the SAM and highlight the role of local regulation of GA.

Genetic and molecular basis of floral induction in Arabidopsis thaliana.

Many plants synchronize their life cycles in response to changing seasons and initiate flowering under favourable environmental conditions to ensure reproductive success. To confer a robust seasonal response, plants use diverse genetic programmes that integrate environmental and endogenous cues and converge on central floral regulatory hubs. Technological advances have allowed us to understand these complex processes more completely. Here, we review recent progress in our understanding of genetic and molecular mechanisms that control flowering in Arabidopsis thaliana.

Floral regulators FLC and SOC1 directly regulate expression of the B3-type transcription factor TARGET OF FLC AND SVP 1 at the Arabidopsis shoot apex via antagonistic chromatin modifications.

Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1), a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.

A regulatory circuit conferring varied flowering response to cold in annual and perennial plants.

The reproductive strategies of plants are highly variable. Short-lived annuals flower abundantly soon after germination, whereas longer-lived perennials postpone and spatially restrict flowering. We used CRISPR/Cas9 and interspecies gene transfer to understand divergence in reproductive patterns between annual and perennial crucifers. We show that in perennial Arabis alpina, flowering in response to winter cold depends on the floral integrator SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15), whose activity is limited to older shoots and branches during cold exposure. In annuals, this regulatory system is conserved, but cold-induced flowering occurs in young shoots, without requirement for SPL15, through the photoperiodic pathway when plants return to warm. By reconstructing the annual response in perennials, we conclude that characteristic patterns of reproduction in annuals and perennials are conferred through variation in dependency on distinct flowering pathways acting in parallel.

Automated distinction of shearing and distortion artefacts in structured illumination microscopy.

Any motion during an image acquisition leads to an artefact in the final image. Structured illumination microscopy (SIM) combines several raw images into one high-resolution image and is thus particularly prone to these motion artefacts. Their unpredictable shape cannot easily be distinguished from real high-resolution content. We previously implemented a motion detection specifically for SIM, which had two shortcomings which are solved here. First, the brightness dependency of the motion signal is removed. Second, the empirical threshold of the calculated motion signal was not a threshold at a maximum allowed artefact. Here we investigate which artefacts are still acceptable and which linear movement creates them. Thus, the motion signal is linked with the maximal strength of the expected artefact. A signal-to-noise analysis including classification successfully distinguishes between artefact-free imaging, shearing and distortion artefacts in biological specimens. A shearing, as in wide-field microscopy, is the dominant reconstruction artefact, while distortions arise not until surprisingly fast movements.

Divergence of regulatory networks governed by the orthologous transcription factors FLC and PEP1 in Brassicaceae species.

Genome-wide landscapes of transcription factor (TF) binding sites (BSs) diverge during evolution, conferring species-specific transcriptional patterns. The rate of divergence varies in different metazoan lineages but has not been widely studied in plants. We identified the BSs and assessed the effects on transcription of FLOWERING LOCUS C (FLC) and PERPETUAL FLOWERING 1 (PEP1), two orthologous MADS-box TFs that repress flowering and confer vernalization requirement in the Brassicaceae species Arabidopsis thaliana and Arabis alpina, respectively. We found that only 14% of their BSs were conserved in both species and that these contained a CArG-box that is recognized by MADS-box TFs. The CArG-box consensus at conserved BSs was extended compared with the core motif. By contrast, species-specific BSs usually lacked the CArG-box in the other species. Flowering-time genes were highly overrepresented among conserved targets, and their CArG-boxes were widely conserved among Brassicaceae species. Cold-regulated (COR) genes were also overrepresented among targets, but the cognate BSs and the identity of the regulated genes were usually different in each species. In cold, COR gene transcript levels were increased in flc and pep1-1 mutants compared with WT, and this correlated with reduced growth in pep1-1 Therefore, FLC orthologs regulate a set of conserved target genes mainly involved in reproductive development and were later independently recruited to modulate stress responses in different Brassicaceae lineages. Analysis of TF BSs in these lineages thus distinguishes widely conserved targets representing the core function of the TF from those that were recruited later in evolution.

PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length.

Seasonal reproduction in many organisms requires detection of day length. This is achieved by integrating information on the light environment with an internal photoperiodic time-keeping mechanism. Arabidopsis thaliana promotes flowering in response to long days (LDs), and CONSTANS (CO) transcription factor represents a photoperiodic timer whose stability is higher when plants are exposed to light under LDs. Here, we show that PSEUDO RESPONSE REGULATOR (PRR) proteins directly mediate this stabilization. PRRs interact with and stabilize CO at specific times during the day, thereby mediating its accumulation under LDs. PRR-mediated stabilization increases binding of CO to the promoter of FLOWERING LOCUS T (FT), leading to enhanced FT transcription and early flowering under these conditions. PRRs were previously reported to contribute to timekeeping by regulating CO transcription through their roles in the circadian clock. We propose an additional role for PRRs in which they act upon CO protein to promote flowering, directly coupling information on light exposure to the timekeeper and allowing recognition of LDs.

Multi-layered Regulation of SPL15 and Cooperation with SOC1 Integrate Endogenous Flowering Pathways at the Arabidopsis Shoot Meristem.

Flowering is initiated in response to environmental and internal cues that are integrated at the shoot apical meristem (SAM). We show that SPL15 coordinates the basal floral promotion pathways required for flowering of Arabidopsis in non-inductive environments. SPL15 directly activates transcription of the floral regulators FUL and miR172b in the SAM during floral induction, whereas its paralog SPL9 is expressed later on the flanks of the SAM. The capacity of SPL15 to promote flowering is regulated by age through miR156, which targets SPL15 mRNA, and gibberellin (GA), which releases SPL15 from DELLAs. Furthermore, SPL15 and the MADS-box protein SOC1 cooperate to promote transcription of their target genes. SPL15 recruits RNAPII and MED18, a Mediator complex component, in a GA-dependent manner, while SOC1 facilitates active chromatin formation with the histone demethylase REF6. Thus, we present a molecular basis for assimilation of flowering signals and transcriptional control at the SAM during flowering.

LLM-Domain B-GATA Transcription Factors Promote Stomatal Development Downstream of Light Signaling Pathways in Arabidopsis thaliana Hypocotyls.

Stomata are pores that regulate the gas and water exchange between the environment and aboveground plant tissues, including hypocotyls, leaves, and stems. Here, we show that mutants of Arabidopsis thaliana LLM-domain B-GATA genes are defective in stomata formation in hypocotyls. Conversely, stomata formation is strongly promoted by overexpression of various LLM-domain B-class GATA genes, most strikingly in hypocotyls but also in cotyledons. Genetic analyses indicate that these B-GATAs act upstream of the stomata formation regulators SPEECHLESS(SPCH), MUTE, and SCREAM/SCREAM2 and downstream or independent of the patterning regulators TOO MANY MOUTHS and STOMATAL DENSITY AND DISTRIBUTION1 The effects of the GATAs on stomata formation are light dependent but can be induced in dark-grown seedlings by red, far-red, or blue light treatments. PHYTOCHROME INTERACTING FACTOR(PIF) mutants form stomata in the dark, and in this genetic background, GATA expression is sufficient to induce stomata formation in the dark. Since the expression of the LLM-domain B-GATAs GNC(GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED) and GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1 as well as that of SPCH is red light induced but the induction of SPCH is compromised in a GATA gene mutant background, we hypothesize that PIF- and light-regulated stomata formation in hypocotyls is critically dependent on LLM-domain B-GATA genes.

Spatial Variation of Aerosol Chemical Composition and Organic Components Identified by Positive Matrix Factorization in the Barcelona Region.

The spatial distribution of PM1 components in the Barcelona metropolitan area was investigated using on-road mobile measurements of atmospheric particle- and gas-phase compounds during the DAURE campaign in March 2009. Positive matrix factorization (PMF) applied to organic aerosol (OA) data yielded 5 factors: hydrocarbon-like OA (HOA), cooking OA (COA), biomass burning OA (BBOA), and low volatility and semivolatile oxygenated OA (LV-OOA and SV-OOA). The area under investigation (∼500 km(2)) was divided into six zones (city center, harbor, industrial area, precoastal depression, 2 mountain ranges) for measurements and data analysis. Mean zonal OA concentrations are 4.9-9.5 µg m(-3). The area is heavily impacted by local primary emissions (HOA 14-38%, COA 10-18%, BBOA 10-12% of OA); concentrations of traffic-related components, especially black carbon, are biased high due to the on-road nature of the measurements. The formation of secondary OA adds more than half of the OA burden outside the city center (SV-OOA 14-40%, LV-OOA 17-42% of OA). A case study of one measurement drive from the shore to the precoastal mountain range furthest downwind of the city center indicates the importance of nonfossil over anthropogenic secondary OA based on OA/CO.

Combinatorial activities of SHORT VEGETATIVE PHASE and FLOWERING LOCUS C define distinct modes of flowering regulation in Arabidopsis.

BACKGROUND: The initiation of flowering is an important developmental transition as it marks the beginning of the reproductive phase in plants. The MADS-box transcription factors (TFs) FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) form a complex to repress the expression of genes that initiate flowering in Arabidopsis. Both TFs play a central role in the regulatory network by conferring seasonal patterns of flowering. However, their interdependence and biological relevance when acting as a complex have not been extensively studied. RESULTS: We characterized the effects of both TFs individually and as a complex on flowering initiation using transcriptome profiling and DNA-binding occupancy. We find four major clusters regulating transcriptional responses, and that DNA binding scenarios are highly affected by the presence of the cognate partner. Remarkably, we identify genes whose regulation depends exclusively on simultaneous action of both proteins, thus distinguishing between the specificity of the SVP:FLC complex and that of each TF acting individually. The downstream targets of the SVP:FLC complex include a higher proportion of genes regulating floral induction, whereas those bound by either TF independently are biased towards floral development. Many genes involved in gibberellin-related processes are bound by the SVP:FLC complex, suggesting that direct regulation of gibberellin metabolism by FLC and SVP contributes to their effects on flowering. CONCLUSIONS: The regulatory codes controlled by SVP and FLC were deciphered at the genome-wide level revealing substantial flexibility based on dependent and independent DNA binding that may contribute to variation and robustness in the regulation of flowering.

Convergence of auxin and gibberellin signaling on the regulation of the GATA transcription factors GNC and GNL in Arabidopsis thaliana.

Plant growth is regulated by a complex network of signaling events. Points of convergence for the signaling cross-talk between the phytohormones auxin and gibberellin (GA), which partly control overlapping processes during plant development, are largely unknown. At the cellular level, auxin responses are controlled by members of the AUXIN RESPONSE FACTOR (ARF) family of transcription factors as well as AUXIN/INDOLE-3-ACETIC ACID INDUCIBLE (AUX/IAA) proteins that repress the activity of at least a subset of ARFs. Here, we show that the two paralogous GATA transcription factors GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED (GNC) and GNC-LIKE (GNL)/CYTOKININ-RESPONSIVE GATA FACTOR1 (CGA1) are direct and critical transcription targets downstream from ARF2 in the control of greening, flowering time, and senescence. Mutants deficient in the synthesis or signaling of the phytohormone GA are also impaired in greening, flowering, and senescence, and interestingly, GNC and GNL were previously identified as important transcription targets of the GA signaling pathway. In line with a critical regulatory role for GNC and GNL downstream from both auxin and GA signaling, we show here that the constitutive activation of GA signaling is sufficient to suppress arf2 mutant phenotypes through repression of GNC and GNL. In addition, we show that GA promotes ARF2 protein abundance through a translation-dependent mechanism that could serve to override the autoinhibitory negative feedback regulation of ARF2 on its own transcription and thereby further promote GA signaling.

Cross-repressive interactions between SOC1 and the GATAs GNC and GNL/CGA1 in the control of greening, cold tolerance, and flowering time in Arabidopsis.

The paralogous and functionally redundant GATA transcription factors GNC (for GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED) and GNL/CGA1 (for GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1) from Arabidopsis (Arabidopsis thaliana) promote greening and repress flowering downstream from the phytohormone gibberellin. The target genes of GNC and GNL with regard to flowering time control have not been identified as yet. Here, we show by genetic and molecular analysis that the two GATA factors act upstream from the flowering time regulator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) to directly repress SOC1 expression and thereby repress flowering. Interestingly, this analysis inversely also reveals that the MADS box transcription factor SOC1 directly represses GNC and GNL expression to control cold tolerance and greening, two further physiological processes that are under the control of SOC1. In summary, these findings support the case of a cross-repressive interaction between the GATA factors GNC and GNL and the MADS box transcription factor SOC1 in flowering time control on the one side and greening and cold tolerance on the other that may be governed by the various signaling inputs that are integrated at the level of SOC1 expression.

Gibberellin regulates PIN-FORMED abundance and is required for auxin transport-dependent growth and development in Arabidopsis thaliana.

Plants integrate different regulatory signals to control their growth and development. Although a number of physiological observations suggest that there is crosstalk between the phytohormone gibberellin (GA) and auxin, as well as with auxin transport, the molecular basis for this hormonal crosstalk remains largely unexplained. Here, we show that auxin transport is reduced in the inflorescences of Arabidopsis thaliana mutants deficient in GA biosynthesis and signaling. We further show that this reduced auxin transport correlates with a reduction in the abundance of PIN-FORMED (PIN) auxin efflux facilitators in GA-deficient plants and that PIN protein levels recover to wild-type levels following GA treatment. We also demonstrate that the regulation of PIN protein levels cannot be explained by a transcriptional regulation of the PIN genes but that GA deficiency promotes, at least in the case of PIN2, the targeting of PIN proteins for vacuolar degradation. In genetic studies, we reveal that the reduced auxin transport of GA mutants correlates with an impairment in two PIN-dependent growth processes, namely, cotyledon differentiation and root gravitropic responses. Our study thus presents evidence for a role of GA in these growth responses and for a GA-dependent modulation of PIN turnover that may be causative for these differential growth responses.

MLN4924 is an efficient inhibitor of NEDD8 conjugation in plants.

The conjugation of the ubiquitin-like modifier NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWN-REGULATED PROTEIN8/RELATED TO UBIQUITIN1 (NEDD8/RUB1; neddylation) is best known as an important posttranslational modification of the cullin subunits of cullin-RING-type E3 ubiquitin ligases (CRLs). MLN4924 has recently been described as an inhibitor of NEDD8-ACTIVATING ENZYME1 (NAE1) in human. Here, we show that MLN4924 is also an effective and specific inhibitor of NAE1 enzymes from Arabidopsis (Arabidopsis thaliana) and other plant species. We found that MLN4924-treated wild-type seedlings have phenotypes that are highly similar to phenotypes of mutants with a partial defect in neddylation and that such neddylation-defective mutants are hypersensitive to MLN4924 treatment. We further found that MLN4924 efficiently blocks the neddylation of cullins in Arabidopsis and that MLN4924 thereby interferes with the degradation of CRL substrates and their downstream responses. MLN4924 treatments also induce characteristic phenotypes in tomato (Solanum lycopersicum), Cardamine hirsuta, and Brachypodium distachyon. Interestingly, MLN4924 also blocks the neddylation of a number of other NEDD8-modified proteins. In summary, we show that MLN4924 is a versatile and specific neddylation inhibitor that will be a useful tool to examine the role of NEDD8- and CRL-dependent processes in a wide range of plant species.

The GATA-type transcription factors GNC and GNL/CGA1 repress gibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS.

The phytohormone gibberellin (GA) regulates various developmental processes in plants such as germination, greening, elongation growth, and flowering time. DELLA proteins, which are degraded in response to GA, repress GA signaling by inhibitory interactions with PHYTOCHROME-INTERACTING FACTOR (PIF) family transcription factors. How GA signaling is controlled downstream from the DELLA and PIF regulators is, at present, unclear. Here, we characterize GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED) and GNL/CGA1 (GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1), two homologous GATA-type transcription factors from Arabidopsis thaliana that we initially identified as GA-regulated genes. Our genetic analyses of loss-of-function mutants and overexpression lines establish that GNC and GNL are functionally redundant regulators of germination, greening, elongation growth and flowering time. We further show by chromatin immunoprecipitation that both genes are potentially direct transcription targets of PIF transcription factors, and that their expression is up-regulated in pif mutant backgrounds. In line with a key role of GNC or GNL downstream from DELLA and PIF signaling, we find that their overexpression leads to gene expression changes that largely resemble those observed in a ga1 biosynthesis mutant or a pif quadruple mutant. These findings, together with the fact that gnc and gnl loss-of-function mutations suppress ga1 phenotypes, support the hypothesis that GNC and GNL are important repressors of GA signaling downstream from the DELLA and PIF regulators.

Minimally invasive rapid palatal expansion with an implant-supported hyrax screw.

Rapid palatal expansion is indicated in the orthopedic treatment of transverse maxillary deficiency for correction of severe crossbite. The fixation of the appliance at the tooth crowns leads to more tipping connected with resorptions at the buccal cortical bone, fenestrations and gingival retraction. The aim of the present study was focused on the improvement of bodily movement and optimization of the surgical osteotomy (Glassman's technique) in adult patients with application of the Dresden Distractor (DD). In 18 patients, the new method with a special mechanism of adaptation involving minimized surgical intervention and direct fixation of the hyrax screw by one implant and one bone screw was tested. The implants were loaded directly by activation of the hyrax screw two times per day. CT scans were taken before and 6 months after insertion of the DD. In the horizontal and vertical planes there was a V-shaped opening of the suture in anterior and cranial direction, corroborating previous studies. Dental arch also showed this V-shape, indicating tooth protection. DD is a suitable minimally invasive tooth-independent bone-borne expansion method, protecting teeth and causing skeletal as well as dental effect with tipping reduced by 10 degrees.