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1.  Regulation of leaf maturation by chromatin-mediated modulation of cytokinin responses 
Developmental cell  2013;24(4):438-445.
Plant shoots display indeterminate growth, while their evolutionary decedents, the leaves, are determinate. Determinate leaf growth is conditioned by the CIN-TCP transcription factors, which promote leaf maturation and which are negatively regulated by miR319 in leaf primordia. Here we show that CIN-TCPs reduce leaf sensitivity to cytokinin (CK), a phytohormone implicated in inhibition of differentiation in the shoot. We identify the SWI/SNF chromatin remodeling ATPase BRAHMA (BRM) as a genetic mediator of CIN-TCP activities and CK responses. An interactome screen further revealed that SWI/SNF complex components including BRM preferentially interacted with bHLH transcription factors and the bHLH-related CIN-TCPs. Indeed, TCP4 and BRM interacted in planta. Both TCP4 and BRM bound the promoter of an inhibitor of CK responses, ARR16, and induced its expression. Reconstituting ARR16 levels in leaves with reduced CIN-TCP activity restored normal growth. Thus, CIN-TCP and BRM together promote determinate leaf growth by stage-specific modification of CK responses.
PMCID: PMC3994294  PMID: 23449474
leaf maturation; CIN-TCP; chromatin remodeling; cytokinin; BRAHMA
2.  Mutations in two non-canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects 
SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. While metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well-studied BRAHMA and SPLAYED ATPases as well as two closely related non-canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here we show that while strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems and display few and aberrant cell division. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication-coupled chromatin assembly are discussed.
PMCID: PMC3561502  PMID: 23062007
stem cell maintenance; root meristem; shoot meristem; embryogenesis; chromatin remodeling; SWI2/SNF2 subgroup ATPases
3.  PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues 
The ability of proteins to associate with genomic DNA in the context of chromatin is critical for many nuclear processes including transcription, replication, recombination, and DNA repair. Chromatin immunoprecipication (ChIP) is a practical and useful technique for characterizing protein / DNA association in vivo. The procedure generally includes six steps: (1) crosslinking the protein to the DNA; (2) isolating the chromatin; (3) chromatin fragmentation; (4) imunoprecipitation with antibodies against the protein of interest; (5) DNA recovery; and (6) PCR identification of factor associated DNA sequences. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP in intact Arabidopsis tissues. This protocol has been used to study association of histone modifications, of chromatin remodeling ATPases, as well as of sequence-specific transcription factors with the genomic DNA in various Arabidopsis thaliana tissues. The protocol described focuses on ChIP-qPCR, but can readily be adapted for use in ChIP-chip or ChIP-seq experiments. The entire procedure can be completed within 3 days.
PMCID: PMC3952383  PMID: 24653666
4.  Glomerular Filtration Rate (GFR) determination via individual kinetics of the inulin-like polyfructosan sinistrin versus creatinine-based population-derived regression formulae 
BMC Nephrology  2013;14:159.
In renal patients estimation of GFR is routinely done by means of population-based formulae using serum creatinine levels. For GFR determination in the creatinine-blind regions or in cases of reno-hepatic syndrome as well as in critical cases of live kidney donors individualized measurements of GFR (mGFR) employing the kinetics of exogenous filtration markers such as the inulin-like polyfructosan sinistrin are necessary. The goal of this study is to compare mGFR values with the eGFR values gained by the Modification of Diet in Renal Disease (MDRD4) and Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) formulae.
In 170 subjects comprising persons with normal renal function or with various stages of kidney diseases (CKD 1-4) GFR was measured by application of intravenous bolus of sinistrin and assessment of temporal plasma concentration profiles by means of pharmacokinetic methods (mGFR). Comparisons of mGFR with MDRD4- and CKD-EPI-derived eGFR values were performed by means of linear regression and Bland-Altman analyses.
Reasonable agreement of mGFR and eGFR values was observed in patients with poor renal function [GFR below 60 (ml/min)/1.73 m2]. In cases of normal or mildly impaired renal function, GFR determination by MDRD4 or CKD-EPI tends to underestimate GFR. Notably, there is practically no difference between the two eGFR methods.
For routine purposes or for epidemiological studies in cases of poor renal function eGFR methods are generally reliable. But in creatinine-blind ranges [GFR above 60 (ml/min)/1.73 m2] eGFR values are unreliable and should be replaced by clinically and physiologically suitable methods for mGFR determination.
PMCID: PMC3726368  PMID: 23876053
eGFR; MDRD; CKD-EPI; mGFR; Sinistrin; Kinetics
5.  The stem cell - chromatin connection 
Stem cells self-renew and give rise to all differentiated cell types of the adult body. They are classified as toti-, pluri- or multi-potent based on the number of different cell types they can give rise to. Recently it has become apparent that chromatin regulation plays a critical role in determining the fate of stem cells and their descendants. In this review we will discuss the role of chromatin regulators in maintenance of stem cells and their ability to give rise to differentiating cells in both the animal and plant kingdom. We will highlight similarities and differences in chromatin-mediated control of stem cell fate in plants and animals. We will consider possible reasons why chromatin regulators play a central role in pluripotency in both kingdoms given that multicellularity evolved independently in each.
PMCID: PMC3407560  PMID: 19765665
Chromatin remodeling; Histone modification; Stem cells; Pluripotency; Differentiation
6.  Switching on Flowers: Transient LEAFY Induction Reveals Novel Aspects of the Regulation of Reproductive Development in Arabidopsis 
Developmental fate decisions in cell populations fundamentally depend on at least two parameters: a signal that is perceived by the cell and the intrinsic ability of the cell to respond to the signal. The same regulatory logic holds for phase transitions in the life cycle of an organism, for example the switch to reproductive development in flowering plants. Here we have tested the response of the monocarpic plant species Arabidopsis thaliana to a signal that directs flower formation, the plant-specific transcription factor LEAFY (LFY). Using transient steroid-dependent LEAFY (LFY) activation in lfy null mutant Arabidopsis plants, we show that the plant’s competence to respond to the LFY signal changes during development. Very early in the life cycle, the plant is not competent to respond to the signal. Subsequently, transient LFY activation can direct primordia at the flanks of the shoot apical meristem to adopt a floral fate. Finally, the plants acquire competence to initiate the flower-patterning program in response to transient LFY activation. Similar to a perennial life strategy, we did not observe reprogramming of all primordia after perception of the transient signal, instead only a small number of meristems responded, followed by reversion to the prior developmental program. The ability to initiate flower formation and to direct flower patterning in response to transient LFY upregulation was dependent on the known direct LFY target APETALA1 (AP1). Prolonged LFY or activation could alter the developmental gradient and bypass the requirement for AP1. Prolonged high AP1 levels, in turn, can also alter the plants’ competence. Our findings shed light on how plants can fine-tune important phase transitions and developmental responses.
PMCID: PMC3355602  PMID: 22639600
LEAFY; flower development; reproductive competence
7.  The microRNA regulated SBP-box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL, and APETALA1 
Developmental cell  2009;17(2):268-278.
When to form flowers is a developmental decision that profoundly impacts the fitness of flowering plants. In Arabidopsis this decision is ultimately controlled by the induction and subsequent activity of the transcription factors LEAFY (LFY), FRUITFULL (FUL) and APETALA1 (AP1). Despite their central importance, our current understanding of the regulation of LFY, FUL and AP1 expression is still incomplete. We show here that all three genes are directly activated by the microRNA targeted transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3). Our findings suggest that SPL3 acts together with other microRNA-regulated SPL transcription factors to control the timing of flower formation. Moreover, the identified SPL activity defines a distinct pathway in control of this vital developmental decision.
PMCID: PMC2908246  PMID: 19686687
8.  Vitamin D deficiency following Billroth II surgery - How much vitamin D is enough?: a case report 
Cases Journal  2010;3:12.
Vitamin D deficiency with all its consequences is a global health problem.
Case Presentation
We reported a 62-year-old Caucasian woman with alcohol-related liver cirrhosis (Child class A) and a medical history of Billroth II surgery. Although she has taken an oral dose of 16 800 IU vitamin D daily for six weeks to normalise her 25-hydroxyvitamin D level the raise was only moderate.
High-dose oral or parenteral vitamin D therapy is necessary to gain sufficient 25-hydroxyvitamin D serum levels in patients following gastric surgery.
PMCID: PMC2828992  PMID: 20180946
9.  The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways 
PLoS Pathogens  2008;4(12):e1000237.
Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks.
Author Summary
In eukaryotes, genomic DNA is organized into a complex DNA-protein structure termed chromatin. The organization of chromatin serves to compact DNA within the nucleus and plays a central role in regulating transcription by controlling the access of DNA to transcriptional machinery. Chromatin structure can be altered through several mechanisms, one of which is chromatin remodeling, a process that disrupts DNA–protein interactions resulting in altered accessibility of specific DNA regions to regulatory proteins in the transcriptional machinery. In this study, we investigated the biological role of chromatin remodeling in defense responses to biotic stresses using the model plant Arabidopsis. We found that a chromatin remodeling protein, SPLAYED, is required for gene expression within specific biotic stress signaling networks. Consistent with this observation, loss of SPLAYED chromatin-remodeling activity resulted in increased susceptibility to a fungal pathogen, Botrytis cinerea, but not to a bacterial pathogen, Pseudomonas syringae. These results demonstrate that reduced stress tolerance in a chromatin-remodeling mutant plant can be stress specific, and is not simply due to a decrease in overall fitness as a result of non-specific global mis-regulation of gene expression.
PMCID: PMC2588541  PMID: 19079584
10.  Histone modifications and dynamic regulation of genome accessibility in plants 
Current opinion in plant biology  2007;10(6):645-652.
In all eukaryotes chromatin physically restricts the accessibility of the genome to regulatory proteins such as transcription factors. Plant model systems have been instrumental in demonstrating that this restriction is dynamic and changes during development and in response to exogenous cues. Among the multiple epigenetic mechanisms that alter chromatin to regulate gene expression, histone modifications play a major role. Recent studies in Arabidopsis have provided the first genome-wide histone modification maps, revealed important biological roles for histone modifications, and advanced our understanding of stimulus-dependent changes in histone modifications.
PMCID: PMC2140274  PMID: 17884714
11.  O-GlcNAc-mediated interaction between VER2 and TaGRP2 elicits TaVRN1 mRNA accumulation during vernalization in winter wheat 
Nature Communications  2014;5:4572.
Vernalization, sensing of prolonged cold, is important for seasonal flowering in eudicots and monocots. While vernalization silences a repressor (FLC, MADS-box transcription factor) in eudicots, it induces an activator (TaVRN1, an AP1 clade MADS-box transcription factor) in monocots. The mechanism for TaVRN1 induction during vernalization is not well understood. Here we reveal a novel mechanism for controlling TaVRN1 mRNA accumulation in response to prolonged cold sensing in wheat. The carbohydrate-binding protein VER2, a jacalin lectin, promotes TaVRN1 upregulation by physically interacting with the RNA-binding protein TaGRP2. TaGRP2 binds to TaVRN1 pre-mRNA and inhibits TaVRN1 mRNA accumulation. The physical interaction between VER2 and TaGRP2 is controlled by TaGRP2 O-GlcNAc modification, which gradually increases during vernalization. The interaction between VER2 and O-GlcNAc-TaGRP2 reduces TaGRP2 protein accumulation in the nucleus and/or promotes TaGRP2 dissociation from TaVRN1, leading to TaVRN1 mRNA accumulation. Our data reveal a new mechanism for sensing prolonged cold in temperate cereals.
In temperate cereals such as winter wheat, prolonged periods of cold induce upregulation of TaVRN1 to control the timing of seasonal flowering. Xiao et al. show TaVRN1 mRNA accumulation is regulated by O-GlcNAc modification of the RNA-binding protein TaGRP2, which increases progressively in response to cold.
PMCID: PMC4143922  PMID: 25091017

Results 1-11 (11)