Passing the threshold gives a Giant output!

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Published on: February 23, 2017

A recent Arabidopsis Research Roundup included a paper from Adrienne Roeder’s lab in Cornell that includes James Locke and Henrik Jonsson from SLCU. The research focuses on the Arabidopsis sepal, which has been the central theme of the Roeder lab since it was set up a few years ago. On a personal level I recall seeing a talk on this topic maybe 10 years ago and it’s always struck me as a fantastically simplistic system that can be used to answer some fundamental questions about the processes that control cell patterning.

This latest paper is focused on the important question of how adjacent cells are set on different development paths, using the giant sepal cells as an excellent model system. This type of cell type specificity is thought to develop following mild stochastic fluctuations in gene expression that lead into feedback loops that accentuate these initial differences. However this has not yet been visualized in vivo until this new manuscript in Elife

The sepal is the outermost organ of the Arabidopsis flower and its correct shape relies on the formation of giant epidermal cells that can grow up to 20% of the entire organ length. These are necessary for the correct function of the organ (to facilitate flower opening) and they form in approximately equal numbers to non-giant cells. Prior to this paper the mechanism of this patterning remained opaque as giant cells can form either adjacent to or apart from each other. The ATML1 transcription factor plays an important role in general Arabidopsis epidermal patterning and has been shown to be required for the generation of giant sepal cells. Importantly the increased size of these giant cells is facilitated by rounds of endoreduplication that can result in 64C nuclei.


ATML1 is expressed in all sepal cells yet only a subset of these will become giant. By using ATML1-overexpression lines together with a simple genetic analysis, the authors show that gene dosage of the ATML1 gene determines the number of giant cells that form (constitutive ATML1 expression have all giant cells). The mechanism by which this dosage results in a mixed cell fate was unclear until they found (using a line containing a fluorescent ATML1-Citrine protein) that ATML1 expression fluctuated far more in the sepal cells than did other genes expressed in the same tissue.

The authors used some fantastic live imaging to show that there are high levels of ATML1 expression in cells destined for giant fate. Although this was not an absolute relationship (as some smaller cells also showed high ATML1 expression), they mathematically demonstrate that obtaining a high threshold of ATML1 correlated about 70% of the time with uptake of giant cell fate.

Finer detail was added to this picture when it became clear that obtaining this threshold at a particular phase of the cell cycle was much more strongly correlated with giant-cell fate. If this threshold is obtained when DNA content was 4C (occurring after DNA replication in G2 phase of the cell cycle) then in 80% of the time the cell became giant. As the authors state ‘a cell is competent to respond to high levels of ATML1 mainly during G2 to induce giant cell formation’.


Finally the authors used plants with a mutation in the LGO gene (LOSS OF GIANT CELLS FROM ORGANS), which have sepals with no giant cells, to determine whether there was feedback control of ATML1 once giant cell fate had been determined. The lgo mutant is epistatic to atml1 and consistent with this observation they show that ATML1 fluctuates normally in the lgo mutant but that this signal does not lead to endoreduplication and giant cell formation. Therefore there is no feedback loop that features endoreduplication and ATML1; rather there is a linear mechanism in which ATML1 fluctuations set in motion endoreduplication, which then continues independent of those ongoing fluctuations.

 

This data was then used to develop a model that could precisely predict the location of giant cell formation based on this information about rapid yet relatively small fluctuations in ATML1 levels.
  Overall this study is an outstanding example of using technological advances in live imaging in a simple experimental system to help develop an understanding of a complex regulatory system. It remains to be seen whether this type of threshold-fluctuation model is important for patterning in other tissues. However this case is an scientific tour of force, demonstrating what is possible when technical advances are put together with careful measurements and inspired experimental planning!

Arabidopsis Research Roundup: February 20th

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Published on: February 19, 2017

This weeks Arabidopsis Research Roundup begins with two papers that look at endogenous and exogenous causes of cell proliferation. Firstly Mike Bevan (JIC) leads a team that looks into the role of controlled protein degradation in this process whilst secondly, Peter Etchells from Durham is a co-author on a study that investigates how nematode pathogens stimulate cell proliferation at the site of infection.

Thirdly is work featuring Cyril Zipfel and colleagues from TSL that looks at how autophosphorylation controls the activity of calcium dependent protein kinases. Fourthly is a broad collaboration led by Richard Mott (UCL) that uses genomic structural variation to identify novel loci. Next Simon Turner from the University of Manchester phylogenetically defines the RALK peptide lineages across plant species. Finally researchers at the University of York conduct a structural analysis of the Arabidopsis AtGSTF2 glutathione transferase.


Dong H, Dumenil J, Lu FH, Na L, Vanhaeren H, Naumann C, Klecker M, Prior R, Smith C, McKenzie N, Saalbach G, Chen L, Xia T, Gonzalez N, Seguela M, Inze D, Dissmeyer N, Li Y, Bevan MW (2017) Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis.

Genes Dev. http:/​/​dx.​doi.​org/10.1101/gad.292235.116

Open Access


Mike Bevan (John Innes Centre) is the corresponding author of this study that also includes researchers from labs in Belgium, Germany and China. They investigate a fundamental determinant of organ shape, the pattern of cell proliferation that leads to final cell size. They show that two RING E3 ligases activate the DA1 peptidase that in turn affects the stabilization and activity of a range of other proteins including the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22. Overall this results in continued cell proliferation and repression of endoreduplication, which ultimately serves to regulate the timing of the transition from cell proliferation to organ differentiation.

Mike discusses the science surrounding this paper on the GARNet YouTube channel.


Guo X,, Wang J, Gardner M, Fukuda H, Kondo Y, Etchells JP, Wang X, Mitchum MG. Identification of cyst nematode B-type CLE peptides and modulation of the vascular stem cell pathway for feeding cell formation. PLoS Pathog. http:/​/​dx.​doi.​org/10.1371/journal.ppat.1006142

Open Access

Peter Etchells (University of Durham) is a co-author on this US-led study that looks at the effect of nematode-delivered CLE-like peptides on cell growth and how that impacts parasitism. This study has identified a new class of peptides from nematodes that are similar to the plant B-type CLE-like peptide TDIF (tracheary element differentiation inhibitory factor). They show that the nematodes alter the activity of the TDIF-TDR (TDIF receptor)-WOX4 signaling module during infection, whose endogenous function acts during procambial meristem cell proliferation. A variety of mutants involved in this process show reduced infection and leading to the hypothesis that WOX4 is a potential target for nematode CLEs. When exogenous nematode CLE peptides are added to Arabidopsis roots they cause massive cell proliferation. This demonstrates that this response is clearly important for the establishment of nematode infection, usually in cambial cell files.


Bender KW, Blackburn RK, Monaghan J, Derbyshire P, Menke FL, Zipfel C, Goshe MB, Zielinski RE, Huber SC (2017) Autophosphorylation-based calcium (Ca2+) sensitivity priming and Ca2+/Calmodulin inhibition of Arabidopsis thaliana Ca2+-dependent protein kinase 28 (CPK28) J Biol Chem.

http:/​/​dx.​doi.​org/10.1074/jbc.M116.763243

Cyril Zipfel (The Sainsbury Lab) features for a second consecutive week on the Arabidopsis research roundup, this time as a co-author in a study that investigates the role of autophosphorylation in the regulation of calcium (Ca2+) dependent protein kinases (CPKs). In addition they evaluated the role of Calmodulin (CaM) on the activity of CPKs, something that had been previously overlooked. Indeed they show that CPK28 is a CaM-binding protein and that autophosphorylation causes increased activity, especially in low Ca2+ concentrations. Therefore this research provides a mechanistic insight into how a cell might respond to low levels of Ca2+.


Imprialou M, Kahles A, Steffen JG, Osborne EJ, Gan X, Lempe J, Bhomra A, Belfield E, Visscher A, Greenhalgh R, Harberd NP, Goram R, Hein J, Robert-Seilaniantz A, Jones J, Stegle O, Kover P, Tsiantis M, Nordborg M, Rätsch G, Clark RM, Mott R Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits. Genetics. http:/​/​dx.​doi.​org/10.1534/genetics.116.192823

Open Access

Richard Mott (UCL) is corresponding author on this paper includes authors from throughout the UK, Europe and the US. They provide a new analysis of Arabidopsis populations that relies on the genome structural variation. They treat these structural variants as quantitative traits and subsequently map genetically in the same way as in a gene expression study. When a structural variant locus is linked to a genotype at a distant locus then it is designated as a site of transposition. Remarkably they show 25% of the structural variants can be assigned to the transposition events. This method of assessing structural variant loci is amendable to sequencing at low-coverage and this study identified loci that might be involved in germination and resistant to pathogens. Overall they find that genes within structural variants are more likely to be silenced and that this novel analysis technique is particularly useful when mapping transposition events.


Campbell L, Turner SR1(2017) A Comprehensive Analysis of RALF Proteins in Green Plants Suggests There Are Two Distinct Functional Groups. Front Plant Sci. http:/​/​dx.​doi.​org/10.3389/fpls.2017.00037

Open Access

This study from the lab of Simon Turner (University of Manchester) analyse Rapid Alkalinization Factor (RALFs) cysteine-rich peptides from across 51 plant species. They infer that these plant RALFs originate from four major clades in which the majority of the variation exists in the mature peptide sequence, indicative of clade-specific activities. Clade IV accounts for a third of the total peptides yet these lack a number of sequence features thought to be important for RALF function, which leads the authors to speculate that this clade should be thought of as containing RALF-related peptides instead of regular RALFs. Further experimental work is needed in order to define the true nature of the functional relationship between Clades I-III and Clade IV.


Ahmad L, Rylott EL, Bruce NC, Edwards R, Grogan G (2016) Structural evidence for Arabidopsis glutathione transferase AtGSTF2 functioning as a transporter of small organic ligands. FEBS Open Bio. http:/​/​dx.​doi.​org/10.1002/2211-5463.12168

Open Access

This paper links plant science and structural biology in a study that was undertaken at the University of York. Plant Glutathione transferases (GSTs) have multiple roles including in the detoxification of xenobiotics as well as in various non-catalytic roles. In this work the structure of the Arabidopsis AtGSTF2 is revealed in tandem with a variety of non-catalytic partners including indole-3-aldehyde, camalexin, the flavonoid quercetrin and its non-rhamnosylated analogue quercetin. These are thought to bind to AtGSTF2 by hydrophobic interactions at either one or two symmetrical binding sites. The authors speculate that this non-catalytic binding might have a possible role in ligand transport.

Arabidopsis Research Roundup: Feb 9th

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Published on: February 14, 2017

This weeks Arabidopsis Roundup again includes a broad selection of research topics. Firstly researchers at SLCU are involved in work that describes Arabidopsis sepal development. Secondly Cyril Zipfel from TSL leads a study that adds a layer of complexity to our knowledge of cellular pathogen perception. Thirdly the group of Reiner van der Hoorn from Oxford introduces the use of a novel set of inhibitors that reveals differential activity of proteosomal subunits during bacterial infection. Finally Hugh Pritchard from Kew Gardens is a co-author on a lipidomic study of the seed dessication-stress response.

Meyer HM, Teles J, Formosa-Jordan P, Refahi Y, San-Bento R, Ingram G, Jönsson H, Locke JC, Roeder AH (2017) Fluctuations of the transcription factor ATML1 generate the pattern of giant cells in the Arabidopsis sepal. Elife.

http:/​/​dx.​doi.​org/10.7554/eLife.19131

Open Access

James Locke and Henrik Jonsson (SLCU) are authors on this paper that is led by Adrienne Roeder at Cornell in the USA. The Roeder lab largely focused their research on development of the sepal. The SLCU researchers provided modeling support for this investigation into the critical role of the ATML1 gene in the differentiation of initially identical cells into giant or regular sized sepal cells. They show that there it is a threshold level of differential ATML1 expression that is key in determining cell fate. If this threshold is met during the G2 phase of the cell cycle the cells enter endoreduplication and become giant. If the threshold isn’t reached then the cells divide and remain at a ‘normal’ size. Ultimately they demonstrate a fluctuation-driven patterning mechanism that determines cell fate.

Stegmann M, Monaghan J, Smakowska-Luzan E, Rovenich H, Lehner A, Holton N, Belkhadir Y, Zipfel C (2017) The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling Science

http:/​/​dx.​doi.​org/10.1126/science.aal2541

Cyril Zipfel (The Sainsbury Lab, Norwich) is the lead author of this study that builds upon his labs work into mechanisms of pathogen perception by cell-surface receptor kinases. In this latest work they show that the SITE-1 PROTEASE (ST1P) cleaves endogenous RAPID ALKALINIZATION FACTOR (RALF) propeptides to inhibit plant immunity, a response mediated via the receptor kinase FERONIA (FER). The FER protein is also involved in the formation of other immune complexes. The authors have discovered a mechanism by which FER reglates RALK signaling, indicating that they might have uncovered a more general mechanism for this key control point of immune signaling.

Misas-Villamil JC,, van der Burgh AM, Grosse-Holz F, Bach-Pages M, Kovács J,, Kaschani F, Schilasky S, Emon AE, Ruben M, Kaiser M, Overkleeft HS, van der Hoorn RA (2017) Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections. Plant Journal

http:/​/​dx.​doi.​org/10.1111/tpj.13494

Reiner van der Hoorn (University of Oxford) lead this cross-Europe collaboration that introduces a range of inhibitors and probes that can discriminate between catalytic subunits of the proteasome. These tools were studied in both Arabidopsis and Nicotiana benthamiana and the authors used the plant-microbe interactions to further validate their specificity. They show that proteasomal subunits have separate paralogs that are differentiatially incorperated into the larger complex depending on an interaction with pathogenic bacteria. Aliquots of these probes are available on request from renier.vanderhoorn@plants.ox.ac.uk

The authors encourage their usage so as to increase the chance that they might become commercially available. More information from the Plant Chemetics lab.

Chen H, Yu X, Zhang X, Yang L, Huang X, Zhang J, Pritchard HW, Li W (2017) Phospholipase Dα1-mediated phosphatidic acid change is a key determinant of desiccation-induced viability loss in seeds. Plant Cell Environ.

http:/​/​dx.​doi.​org/10.1111/pce.12925

Hugh Pritchard (Kew Gardens) is a co-author on this Chinese-led study that investigates the role of phosphatidic acid (PA) on seed viability. Higher levels of PA correlated with lower seed viability after a desiccation stress. Using Arabidopsis seeds they showed that the enzyme phospholipase D α1 (PLD α1) localises to the plasma membrane following desiccation, where it produces PA. When PLD α1 was suppressed, seed recovery following desiccation improved. The authors used comparative lipidomics to compare PA levels in eight plant species and from their Arabidopsis work, they propose a new model for the mechanism by which seed desiccation effects germination rates.

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