Arabidopsis Research Roundup: Oct 28th

This latest Arabidopsis Research Roundup is rather GARNet-focused as members of the current Advisory Board lead three of the featured papers. Firstly we present a study into mechanisms that control meiotic recombination, which also includes a short audio-description from the lead author Dr Ian Henderson. Secondly we introduce a paper that identifies the function of a novel gene in the control of male fertility and thirdly, a study of a translation control-factor that is involved in regulation of cell size and ovule development. In addition we introduce some highly collaborative work that looks into the role of SUMO proteases in SA signaling. Finally there is a methods paper that presents a new protocol for measurement of cellulose content in Arabidopsis stems.

Yelina N, Lambing C, Hardcastle T, Zhao X, Santos B, Henderson I (2015) DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis Genes & Dev. 29: 2183-2202 http://dx.doi.org/10.1101/gad.270876.115

GARNet advisory board member Ian Henderson leads this study that assesses how methylation state influences the chromosomal regions that undergo meiotic recombination. It was previously known that highly-methylated regions, such as centromeres, do not often undergo recombination. This work naturally extends that knowledge by using RNA-directed DNA methylation to show that methylation of local euchromatic regions also have reduced recombination levels. Equally they show that global reductions in CG methylation, such as in met1 mutants, cause wide-scale alterations in recombination remodeling. Use of recombination mutants shows that these changes are due to the redistribution of interfering crossovers. Overall they confirm that DNA methylation is critical in establishing domains of meiotic recombination.

In this short audio file, Dr Henderson explains the main features of this paper.

Visscher AM, Belfield EJ, Vlad D, Irani N, Moore I, Harberd NP (2015) Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana. PLoS One.;10(10):e0140368. http://dx.doi.org/10.1371/journal.pone.0140368

Ian Moore and Nick Harberd (Oxford), who is also on the GARNet Advisory Board,  present this investigation of five unknown genes that had been previously identified from global expression studies as playing a role in multiple stress-responses. These are somewhat unimaginatively identified by their ‘At’ numbers and even though they are each responsive to multiple stresses, mutants with a T-DNA insertion in any of these genes have no change in phenotype compared to wildtype plants. In contrast, overexpression of At1g74450, but no other of the tested genes, resulted in stunted growth and reduced male fertility. As the stress-response is often manifested by alterations in male gametophyte development, this work introduces the function of a gene that may provide an important link between multiple environmental factors, fertility and plant growth. In future the authors hope to provide further insight into the function of At1g74450.

Bush M, Crowe N, Zheng T, Doonan J (2015) The RNA helicase, eIF4A-1, is required for ovule development and cell size homeostasis in Arabidopsis Plant J. http://dx.doi.org/10.1111/tpj.13062

John Doonan, another GARNet board member, leads this collaborative work between Aberystwyth and Norwich. They investigate the function of the RNA helicase/ATPase eIF4A-1 that is involved in the initiation of mRNA translation. Arabidopsis contains two isoforms of this genes and the knockdown eif4a-1 mutant displays a range of altered phenotypes that includes a reduction in the amount of mitotic cells in the root meristem. This change skews the relationship between cell size and cell cycle progression. Concomitantly several cell cycle-regulated genes have reduced expression in this mutant. Each of the eIF4A isoforms plays an important role in plant fertility as although single eif4a-1 mutants display some defects in ovule development, double eif4a1eif4a2 mutants cannot be isolated.

Bailey M, Srivastava A, Conti L, Nelis S, Zhang C, Florance H, Love A, Milner J, Napier R, Grant M, Sadanandom A (2015) Stability of small ubiquitin-like modifier (SUMO) proteases OVERLY TOLERANT TO SALT1 and -2 modulates salicylic acid signalling and SUMO1/2 conjugation in Arabidopsis thaliana J Exp Bot. http://dx.doi.org/10.1093/jxb/erv468

This study of the SUMO proteases OVERLY TOLERANT TO SALT1 and -2 (OTS) is a real pan-UK collaboration that features researchers from six institutions, led by Ari Sadanandom at Durham. The OTS proteins have been previously linked to salicylic acid (SA) signaling and this manuscript shows that in addition to containing higher level of SA, ots1ots2 double mutants are more resistant to virulent Pseudomonas syringae. This is in part linked to an upregulation of the SA biosynthetic gene ICS1. In wildtype plants SA promotes degradation of OTS1/2, which indicates that these proteins are involved in a positive feedback loop that ensures a higher SA response, which increases the efficacy of certain defence responses. However de novo synthesis of OTS1/2 will be antagonistic to SA biosynthesis and provides a brake to prevent the over-accumulation of SA-responses.

Kumar M, Turner S (2015) Protocol: a medium-throughput method for determination of cellulose content from single stem pieces of Arabidopsis thaliana Plant Methods. 11:46. http://dx.doi.org/10.1186/s13007-015-0090-6

Simon Turner (Manchester) is the lead author of this paper that presents a new method for determining cellulose content from Arabidopsis stems. This protocol is an adaptation of a previous method and uses aspiration rather than centrifugation for recovery of liquids throughout the procedure. This increases the throughout of the method and improves its potential usage as a screening protocol to identify mutants with altered cell wall composition.

Arabidopsis Research Roundup: October 20th

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Published on: October 20, 2015

There are just three research papers in this weeks Arabidopsis Roundup but they each represent important projects from established groups. Firstly is a significant output from the Edinburgh SynthSys Centre that documents their analysis of the Arabidopsis circadian clock. Secondly an international collaborative effort looks into the molecular signaling pathways that control the physiological response to increasing CO2 levels and thirdly a paper that uncovers a novel plant-specific molecular mechanism that controls the biogenesis of certain siRNAs. Finally we highlight a major review concerning the importance of Arabidopsis research over the past 50 years.

Flis A, Fernández AP, Zielinski T, Mengin V, Sulpice R, Stratford K, Hume A, Pokhilko A, Southern MM, Seaton DD, McWatters HG, Stitt M, Halliday KJ, Millar AJ (2015) Defining the robust behaviour of the plant clock gene circuit with absolute RNA timeseries and open infrastructure. Open Biol. 5(10). pii: 150042. http://dx.doi.org/10.1098/rsob.150042

This study of the Arabidopsis circadian clock, impressive in its breadth, is led by faculty members from the University of Edinburgh SynthSys Synthetic Biology Centre. The team measured RNA profiles of clock genes in plants grown with or without exogenous sucrose or from wildtype or mutant soil growth plants. They found surprisingly robust patterns of expression together with some novel genetic behaviours. In addition they discovered major differences in the absolute expression of certain clock genes, ranging from 50 up to 1500 copies/ cell. Importantly this information is freely-available within the BioDare repository and it is hoped that this will benefit future attempts at modeling the circadian clock.

Chater C, Peng K, Movahedi M, Dunn JA, Walker HJ, Liang YK, McLachlan DH, Casson S, Isner JC, Wilson I, Neill SJ, Hedrich R, Gray JE, Hetherington AM (2015) Elevated CO2-Induced Responses in Stomata Require ABA and ABA Signaling Curr Biol. pii: S0960-9822(15)01092-1. http://dx.doi.org/10.1016/j.cub.2015.09.013

This broad collaboration between UK, German and Chinese researchers is led by Alistair Hetherington (Bristol) and Julie Gray (Sheffield) and looks into the molecular events that respond to changing levels of CO2, specifically in guard cells. The new findings in this manuscript show that reduction in stomatal density in response to higher [CO2] relies on the production of reactive oxygen species (ROS), adding a new element to this signaling pathway. In addition they show that the ABA response pathway is also involved in this process and that, following genetic analysis, the CO2 response is mediated via this hormone pathway. However it is unclear whether this is due to ABA increasing CO2 sensitivity in this system or whether CO2 acts specifically in guard cells to increase ABA biosynthesis. A plants response to CO2 is ancestral in evolutionary terms so the authors suggest that this link with ABA signaling is similarly ancient.

CO2Pic

Zhai J, Bischof S, Wang H, Feng S, Lee TF, Teng C, Chen X, Park SY, Liu L, Gallego-Bartolome J, Liu W, Henderson IR, Meyers BC, Ausin I, Jacobsen SE (2015) A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis. Cell. 163(2):445-55 http://dx.doi.org/10.1016/j.cell.2015.09.032

GARNet Advisory Board member Ian Henderson is an author in this rare plant-focused paper in Cell, in work that results from his post-doc with Steve Jacobson at UCLA. The manuscript describes a novel mode of action surrounding the plant-specific RNA Polymerase IV (Pol IV). RNAs generated from the activity of Pol IV play an important role in RNA-directed DNA methylation. Intriguingly the authors found that Pol IV transcripts are surprisingly short, just 30 to 40 nt and are similarly adundant to the siRNAs that they subsequently form. The Pol IV RNAs exhibit transcriptional start points similar to those generated by Pol II, which might indicate there are similar mechanisms that control their activity. In addition they find that methylated DNA plays a role in locally reinforcing the silencing reaction. Overall this indicates that the transcripts produced by Pol IV go through a unique “one precursor, one siRNA” model, although the physiological significance of this remains opaque. Another paper on this topic is presented in ELife by the lab of Craig Pikaard.

PolIVpic

Provart et al (2015) 50 years of Arabidopsis research: highlights and future directions New Phytol. http://dx.doi.org/10.1111/nph.13687

Also worth noting this week is a Tansley Review in New Phytologist, which coincides with 50 years since the inaugural Arabidopsis Conference held in 1965. This review has been written by a number of senior Arabidopsis researchers, although no-one from the UK, to discuss the many important findings that have resulted from work on our favourite organism.

Variation in Agroinflitration

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Published on: October 19, 2015

Agroinflitration of Nicotiana benthamiana is an increasingly important technique that many plant scientists may use for transient expression of their gene of interest. These experiments might include localisation or interaction studies, monitoring of pathogen effects or simply for protein production where, in extreme cases, up to 50% of the total soluble protein will be expressed by the transgene.

As a previous user of this technique, I know that many aspects of early test experiments can be undertaken haphazardly, with little understanding or expectation regarding the optimum agrobacterium concentration, best leaf segment or inoculation timings to use. There also seems to be significant variation within the same inoculated leaves, between leafs on the same plant and especially so between leaves from different plants. Therefore it is satisfying to read a recent paper published in Plant Methods from the lab of Teemu Teeri (University of Helsinki) that attempts to provide some understanding as to the sources of variation in these infiltration experiments [1].

They conduct a simple set of experiments whereby they use an intron-containing luciferase gene as a reporter to test the factors that most influenced the infiltration efficiency. They inoculated the upper leaves of 6-week old tobacco plants with either luciferase driven by the constitutive 35S promoter or by an estradiol-inducible promoter. They showed that estradiol induced an eight-fold increase in enzyme production and that these induced levels were similar to those observed with the constitutive promoter.
In order to assess variation within experiments they compared samples from the same leaf, between leaves from the same plant and between leaves from different plants. Importantly they used a nested experimental design so that they could compare the amount of variation generated between hierarchical levels of infiltration, sampling or enzyme activity.
AgroInfliPic

Somewhat surprisingly they found that the largest component of variation in these experiments was between disks on the same leaf, whereas use of different leaves or different plants generated a 2.5x lower level of variation. Within disks from a single leaf, the amount of bacteria and protein showed little alteration, even though the luciferase enzyme activity was greatly different. The reason for this difference is unknown.

Therefore based on their results the authors make the following recommendations:

  1. Ensure tested plants are in the same physiological state.
  2. Combine the expression values from several sample disks on the same leaf, do not rely on one disk as a representative amount of expression on that leaf.
  3. Inflitrate more plants but less leaves on those plants. Sample more disks on each of the leaves that are used.
  4. Technical replicates (for luciferase assays) are not as important since these measurements are more reliable.

Good luck with your experiments!

1- Bashandy et al (2015) Plant Methods11:47 DOI: 10.1186/s13007-015-0091-5

Arabidopsis Research Roundup: October 12th

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Published on: October 11, 2015

The Arabidopsis Research Roundup is ‘defense-focused’ this week. We present three papers that highlight different aspects of plant immunity, two of which result from UK-US-China collaborations. Firstly a team from the Sainsbury Lab, Norwich looks at two molecular mechanisms that control stomatal closure. There are then two studies that involve University of Exeter researchers that investigate either the role of plant hormones in the response to bacterial pathogens or the role that the physical barrier of the cell wall plays in the prevention of infection. Next a group of JIC researchers present a Large Scale Biology investigation of microtubule interacting proteins. Finally a study from QMUL looks at the interaction between NPQ and photoinhibition in controlling the activity of Photosystem II.

Gou M, Zhang Z, Zhang N, Huang Q, Monaghan J, Yang H, Shi Z, Zipfel C, Hua J (2015) Opposing effects on two phases of defense responses from concerted actions of HSC70 and BON1 in Arabidopsis. Plant Physiol. http://dx.doi.org/10.1104/pp.15.00970

GARNet Advisory Board Member Cyril Zipfel is the UK lead on this US-China-UK collaboration that looks at two aspects of the plant immune response that are regulated by the same proteins, albeit in an antagonistic way. The heat shock protein HSC70 and the calcium binding protein BON1 both are involved in stomatal closure, the formers effect mediated by the SNC1 protein and the latter (BON1) via the activitation of SGT1 that in turn inhibits HSC70. These new functions demonstrate opposing roles for HSC80 and BON1 in the immune response and further highlight the complexity of the signaling pathways that ultimately feed into the gross phenotypic change of stomatal closure.

de Torres Zabala M, Zhai B, Jayaraman S, Eleftheriadou G, Winsbury R, Yang R, Truman W, Tang S, Smirnoff N, Grant M (2015) Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection New Phytol. http://dx.doi.org/10.1111/nph.13683

This is another UK-USA-China collaboration led by Murray Grant at the University of Exeter, in which the role of the plant hormones is assessed in the response to bacterial pathogens. The defence response is mediated by both the hormones salicylic acid (SA) and jasmonic acid (JA) which antagnise many of each others activity. Pathogens have been shown to produce a JA-mimic cornatine (COR) in order to stall SA-mediated effects. In this study the authors use a systems-biology based approach that involved targeted hormone profiling, high-temporal-resolution micro-array analysis, reverse genetics and mRNA-seq to introduce a complex network of regulation that involves JAZ proteins, which are repressors of the JA signal. In short they show that JAZ5 and JAZ10 specifically co-operate to inhibit pathogen growth by restricting COR cytotoxicity by novel mechanisms, which do not involve previously well-defined signaling proteins.

Marcos R, Izquierdo Y, Vellosillo T, Kulasekaran S, Cascón T, Hamberg M, Castresana C (2015) 9-Lipoxygenase-derived oxylipins activate brassinosteroid signaling to promote cell wall-based defense and limit pathogen infection Plant Physiol. http://dx.doi.org/10.1104/pp.15.00992

This work was performed in Madrid under the supervision of Carmen Castresana but includes the work of Satish Kulasekaran who is now at the Exeter. The focus of the work is the oxylipins, which are oxygenated lipid derivatives that regulate plant development and immunity. Using a variety of noxy mutants (non-responding to oxylipins) they show that the effect of the oxylipins is mediated via changes in the cell wall and this is signalled via the Brassinosteriod response pathway. Suspectibility to bacterial and fungal infection was enhanced in noxy mutants but plants were resistance when BR signalling was switched on. Therefore this manuscript introduces an important interaction between the oxylipins and BR signalling and helps to clarify their role in modulating plant defense.

Derbyshire P, Ménard D, Green P, Saalbach G, Buschmann H, Lloyd CW, Pesquet E (2015) Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis Plant Cell. http://dx.doi.org/10.1105/tpc.15.00314

The experiments in this Large Scale Biology paper were performed in the lab of Clive Lloyd (John Innes Centre) which included the work of Eduoard Pesquet, who now has his own lab in Sweden. They looked the microtubule patterning that defines the nature of tracheary element (TEs) thickening in plant vascular tissues. They used Arabidopsis cell suspension culture to isolate microtubule interacting proteins present during TE differentiation. One protein of interest was CELLULOSE SYNTHASE-INTERACTING PROTEIN1, associated with primary wall synthesis, which was enriched during secondary cell wall formation of TEs. The authors knocked-down the expression of some of their identified proteins and indeed showed that they were important for this differentiation. A take-home message is that the proteins that interact with microtubules and link them to different metabolic compartments do indeed specifically vary during TE differentiation, regulating different aspects of cell wall patterning.

Giovagnetti V, Ruban AV (2015) Discerning the effects of photoinhibition and photoprotection on the rate of oxygen evolution in Arabidopsis leaves J Photochem Photobiol B. http://dx.doi.org/10.1016/j.jphotobiol.2015.09.010

Arabidopsis Research Roundup Regular Alexandre Ruban (QMUL) again looks at the mechanisms that lessen the amount of photoinhibition (when photosystem II is damaged by being exposed to too much light). An opposing response is Non-photochemical quenching (NPQ) of chlorophyll a fluorescence which serves to protect PSII from high light conditions. In this study they confirm that a recently devised procedure that aims to discern between the effect of NPQ and photoinhibition works well as a measurement for the efficiency of PSII activity.

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