Arabidopsis Research Roundup: January 11th

The first Arabidopsis Research Roundup of 2017 includes a wide range of studies that use our favourite model organism.

Firstly Kerry Franklin (University of Bristol) is the corresponding author on a paper that describes the complex interaction between the responses to sunlight and heat. Secondly Paul Dupree (University of Cambridge) leads a study that defines the important structural relationship between xylan and cellulose. Thirdly members of Gos Micklem’s group in Cambridge are part of the Araport team that present their ThaleMine tool.

Richard Napier (University of Warwick) is a co-author on the fourth paper that introduces a new chemical tool for study of the auxin response. The penultimate paper includes Matthew Terry (University of Southampton) on a paper that investigates the role of a Fe-S-containing protein cluster in chlorophyll biosynthesis and finally there is a methods paper from Stefanie Rosa in Caroline Dean’s lab at the John Innes Centre that describes the use of FISH to detect single molecules of RNA.


Hayes S, Sharma A, Fraser DP, Trevisan M, Cragg-Barber CK, Tavridou E, Fankhauser C, Jenkins GI, Franklin KA (2016) UV-B Perceived by the UVR8 Photoreceptor Inhibits Plant Thermomorphogenesis. Current Biology http:/​/​dx.​doi.​org/10.1016/j.cub.2016.11.004

Open Access

This collaboration between the research groups of Kerry Franklin (University of Bristol) and Gareth Jenkins (University of Glasgow) looks at how the perception of UV-B light inhibits the morphological changes that occur in response to increased temperatures (thermomorphogenesis). This response includes induced hypocotyl elongation, which is mediated via PIF4 and various players in the auxin response. Interestingly the authors show that UV-B light perceived by UVR8 attenautes this response by preventing PIF4 abundance and by stabilising the the bHLH protein LONG HYPOCOTYL IN FAR RED (HFR1) protein. These results suggest that there exists a precise mechanism for fine-tuning the growth responses that occur in sunlight that would usually include both increased temperature and UV-B irradiation.
UVB_pic


Simmons TJ, Mortimer JC, Bernardinelli OD, Pöppler AC, Brown SP, deAzevedo ER, Dupree R, Dupree P (2016) Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR. Nat Commun.

http:/​/​dx.​doi.​org/10.1038/ncomms13902 Open Access
In this paper Paul Dupree (University Cambridge) collaborates both with colleagues in Spain and with his father Ray, who is a physicist at the University of Warwick. They use NMR to perform a structural analysis of xylan, which is the most prevalent non-cellulosic polysaccharide in the cell wall matrix and binds to cellulose microfibrils. Whereas in solution xylan forms a threefold helical screw, it flattens into a twofold helical screw ribbon to closely bind to cellulose when in the cell wall. They used the cellulose-deficient Arabidopsis irx3 mutant to show that the xylan two-fold screw confirmation breaks down when it cannot bind cellulose. The authors state that this finding has important implications in our understanding of the formation of the cell wall and perhaps more importantly how it might be broken down during attempts to maximise economic usages of plant biomass.

A local Cambridge newspaper reported that this finding could ‘pave the way for wooden skyscrapers’
XylanPic


Krishnakumar V, Contrino S, Cheng CY, Belyaeva I, Ferlanti ES, Miller JR, Vaughn MW, Micklem G, Town CD, Chan AP (2016) ThaleMine: A Warehouse for Arabidopsis Data Integration and Discovery. Plant Cell Physiol http:/​/​dx.​doi.​org/10.1093/pcp/pcw200 Open Access

This paper is presented by the Araport team, which is based in the USA but includes representatives from Gos Micklem’s lab in University of Cambridge. They outline the functionality of the ThaleMine data warehouse which is an important component of the tools included on Araport (https://www.araport.org/). ThaleMine collects a wide variety of data from public datasets and presents it in a easy-to-interrogate form, facilitating the experiments of both lab-based researchers or bioinformaticians. This tool is build upon the InterMine software framework, which has been widely adopted across other model organisms.

Chris Town and Sergio Contrino provided a hands-on workshop describing the tools on Araport in last year GARNet2016 meeting and their workshop materials can be downloaded here.


Steenackers WJ, Klíma P, Quareshy M, Cesarino I, Kumpf RP, Corneillie S, Araújo P, Viaene T, Goeminne G, Nowack MK, Ljung K, Friml J, Blakeslee JJ, Novák O, Zažímalová E, Napier RM, Boerjan WA, Vanholme B (2016) cis-cinnamic acid is a novel, natural auxin efflux inhibitor that promotes lateral root formation. Plant Physiol. http:/​/​dx.​doi.​org/pp.00943.2016 Open Access
cCApic
This pan-european collaboration includes members of Richard Napier’s lab at the University of Warwick. They outline the activity of a novel inhibitor of auxin efflux transport called cis-cinnamic acid (c-CA). When c-CA is applied to growth media plants appears to exhibit an auxin-response phenotype yet these experiments show that c-CA is neither an auxin or anti-auxin and in fact blocks local auxin efflux, thus causing buildup of cellular auxin. This effect does not occur with t-CA showing specificity for c-CA and it does not affect long distance auxin transport, which occurs through the phloem. Therefore this paper presents a new pharamolgical tool for the study of in planta auxin transport and homeostasis.


Hu X, Page MT, Sumida A, Tanaka A, Terry MJ, Tanaka R (2016) The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling. Plant J.

http:/​/​dx.​doi.​org/10.1111/tpj.13455

Matthew Terry and Mike Page (University of Southampton) are co-authors on this Japanese-led study that investigates the function of the SUFB subunit of the SUFBCD iron-sulfur cluster. These Fe-S protein clusters play roles in many metabolic processes and the SUFB mutant hmc1 exhibits a defect in chlorophyll biosynthesis due to an accumulation of Mg-containing biosynthetic intermediates. In addition both SUFC- and SUFD-deficient RNAi lines accumulated the same Mg intermediate indicating that the SUFBCD cluster is responsible for this step necessary for chlorophyll production.


Duncan S, Olsson TS, Hartley M, Dean C, Rosa S (2016) A method for detecting single mRNA molecules in Arabidopsis thaliana. Plant Methods. http:/​/​dx.​doi.​org/10.1186/s13007-016-0114-x

Open Access
This paper from is lead by Stefanie Rosa in Caroline Dean’s lab at the John Innes Centre describes a novel method for imaging single molecules of RNA by smFISH. They analyse the localisation of both nascent and mature mRNAs, allowing for analysis of the location of RNA processing and translation.<
RosaPic

Goldenbraid 2.0: A Standardised DNA Assembly Framework

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Published on: May 27, 2014

As promised in my posts about the Plant Engine meeting I attended a couple of weeks ago (1, 2), here is Diego Orzaez to explain his GoldenBraid cloning method and online DNA assembly framework. Diego co-leads the Plant Genomics and Biotechnology Lab at the Instituto de Biología Molecular y Celular de Plantas in Valencia, Spain. 

goldenbraid

GOLDENBRAID 2.0: A STANDARDIZED DNA ASSEMBLY FRAMEWORK FOR MULTIGENE ENGINEERING IN PLANTS

Engineering large multigenic constructs for Plant Synthetic Biology, such as complex metabolic pathways or intricate gene networks, requires efficient, flexible DNA synthesis and assembly technologies. Although custom gene synthesis is becoming increasingly affordable, the direct synthesis of large multigenic constructs remains prohibitive for most labs. Moreover, custom gene synthesis gives little room for combinatorial engineering, something that is highly valued by biotechnologists.

An alternative “building” strategy for multigene engineering is Modular Construction, that is, the fabrication of new devices by combination of prefabricated standard modules. Modular DNA Construction brings a number of advantages as speed, versatility, lab autonomy, combinatorial potential and often lower cost. As in any standardized methodology, the more users adopt the standard, the bigger the advantages.

GoldenBraid is a Modular DNA Construction method developed at the Plant Genomics and Biotechnology lab (IBMCP-Spain), especially designed for building exchangeable multigenic constructs for Plant Synthetic Biology. Routinely, 15-20 Kb constructs comprising 4-6 transcriptional units made of dozens of individual pre-fabricated modules (GBparts) can be created in few days. Longer constructs can be assembled with little additional effort.

To facilitate the process of genetic design using GoldenBraid, and to stimulate the exchange of genetic modules among laboratories, we have recently launched GoldenBraid2.0 (GB2.0), a web-based DNA assembly framework available at www.gbcloning.org site. The GB2.0 webpage hosts the public GB2.0 database, an increasingly populated collection of pre-made “GBparts” that conform to the GB standard. An embedded software tool named GBdomesticator provides users with personalized lab protocols for creating their own collection of standard genetic parts. Users can always combine their own parts with those deposited in the public GB2.0 database. Moreover, building new GB2.0 multigenic constructs is highly facilitated by the GB assembler tool, a software package that assists in the design of new multigenic constructs.

We believe that adopting common standards and creating of public repositories of exchangeable genetic parts will speed up progress in Plant Biotechnology. If you are interested in this field, we encourage you to explore the gbcloning.org webpages. The details of GB assembly system are described in the publications listed below, and there are tutorials online.

Comments on how to enhance community efforts towards the development of public repositories of standard DNA parts are most welcome, and can be addressed to goldenbraid@ibmcp.upv.es

 

References

Novel tools for reducing bias in Next Generation Sequencing of small RNAs

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Published on: April 15, 2014

Tamas Dalmay, Professor of RNA Biology at the University of East Anglia (Norwich), has developed a robust, simple method of profiling small RNAs using next generation sequencing. Here he explains his novel HD adapters and why they are more reliable than existing commercial adapters. 

Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).
Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).

Small RNAs (sRNAs) are key regulators of gene expression, and accurate representation of sRNA in sequencing experiments is critical to the interpretation of biological data. Next generation sequencing (NGS) is now the gold standard for profiling and discovering new sRNAs, so it is essential that the tools and protocols used in NGS generate accurate, reliable sequence data.

RNA ligases are essential in creating cDNA libraries prior to NGS sequencing. However, a number of recent publications reported that RNA ligases used in cDNA preparation actually mediate sequence specific ligation, so NGS approaches using these RNA ligases do not represent all sRNA present in biological samples. These publications highlighted the limitations associated with RNA ligases, questioning the reliability of currently widely used NGS approaches and the data generated from them.

Sequence specific ligation occurs because the ligases preferentially ligate ends that are more likely to be close to each other. This means that sRNAs that can efficiently anneal to the adapters have a higher chance of being ligated (Jayaprakash et al. 2011, Hafner et al. 2011 and Sorefan et al. 2012).

While identifying that cloning bias in sRNA libraries is RNA ligase dependent, our group at the School of Biological Sciences, University of East Anglia (Norwich), developed a novel, simple, robust solution to overcome this problem (Sorefan et al. 2012).

We developed a set of adapters (High Definition or HD adapters) that contain degenerated nucleotides, meaning they are a pool of many sequences instead of one fixed sequence. Consequently, many different sRNAs can form a stable duplex with them, leading to better coverage and more quantitative libraries. We have shown that using the HD adapters: (more…)

Plant science, by JoVE!

JoVE 2

Researchers from the University of Warwick published a methodology paper with a twist this week. The paper, published online by the Journal of Visualized Experiments (JoVE), gives step-by-step instructions and video demonstration of a method for purifying a protein and identifying proteins that perform its post-translational modifications.

Authors Sophie Piquerez, Alexi Balmuth, Jan Skenář, Alex JonesJohn Rathjen and Vardis Ntoukakis developed the method in order to characterize the interactions between nucleotide-binding leucine-rich-repeat proteins and the Prf/Pto complex in effector-triggered immunity. In principle the method could be applied to any protein – the protein of interest is epitope-tagged, immunoprecipitated and analysed by MS.

A video journal lends itself to new or improved methodology rather than high impact conclusions. As with a lot of JoVE articles, the scientifically significant results obtained using the protocol have already been published; in this case in Ntoukakis et al. 2013 (PLOS Pathogens, 10.1371/journal.ppat.1003123).

The authors conclude the abstract by saying the paper demonstrates:

  1. Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry;
  2. It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate;
  3. The immunoprecipitation step is essential to get enough protein to do the MS. (more…)

Testing heat tolerance in the field

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Published on: March 21, 2013

Global climate change and localised human impact, such as waste disposal or fertilizer use, has and will continue to have an effect on the world’s flora, both natural and agricultural. Predicting this effect can be difficult, but it is important. If land managers and farmers know which species will cope well with change, they will be better able to make a decision about the species which will struggle under certain conditions.

If a species is well-researched, it may be possible to look for QTL associated with resistance to heat, drought, flooding, or other abiotic stresses, but of course this does not predict real-world responses reliably and in any case is not an option in all cases. In the lab or greenhouse under controlled conditions, a simple observation experiment can tell you the effects of various conditions on a plant, but again this is not an indication of in situ viability.

Buchner et al. published a method of determining the heat tolerance of plants in the field in this month’s Plant Methods (vol. 9:7). Heat was the only imposed variable in their protocol, so any environmental factors are included in the experiment. The group, from Othmar Buchner’s group at Innsbruck, made their own Heat Tolerance Testing System (HTTS) from a number of pieces of technical equipment, including the customized exposure chambers seen in the image above (Figure 5B in the paper). (more…)

Arabidopsis hydroponics video method

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Published on: March 8, 2013

One of the most frustrating things in lab-based research is trying to learn a new method from a paper. In my short time in the lab, I sometimes had to follow a trail of breadcrumbs back through several papers to find details of a single step in a protocol, on one occasion digging around in the library archives for a paper from an out-of-publication journal. Once the various reagents had been rounded up, I’d interpreted the protocol (What kind of ‘mixing’? How slowly is ‘slowly add’?), and had failed to accurately measure a solution that dissolved my pipette tips, all I usually had to show for my pains was a questionable precipitate and a lot of washing up – at least for the first attempt.

Researchers from three Australian research centres had similar problems with hydroponic systems described in the literature. Conn et al. designed their own hydroponic system for Arabidopsis and published it in Plant Methods (2013, 9:4) along with the YouTube video above, demonstrating exactly how the protocol works in practice. Plant Methods is a friendly journal for the intrepid researcher attempting protocols new to their research group. This paper is typical and has a comprehensive list of necessary reagents and equipment, and clear step-by-step guide with critical points highlighted.

If you need to grow Arabidopsis in a controlled environment to look at the physiology of the whole plant and you are unhappy with your current growth facilities, take a look at this paper on DIY hydroponics. It is quite a work intensive set-up (drill-bits are mentioned) but most of the equipment is cheap and easily come by.

Video Credit: Matthew Gilliham, via YouTube.

New Methods and Resources (II)

Categories: methods
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Published on: February 12, 2013

As promised, here’s part two of my selection of recently published plant methods and resources.

Nisar et al. (2012; Plant Methods 8:50) present a method for easy inflorescence stem grafting in Arabidopsis. I can’t vouch for its ease, but the typical clear Plant Methods format provides plenty of description and comprehensive materials and methods section as well as a step-by-step guide to their customised wedge-cleft grafting technique. The authors even provide a table of technical tricks for each step in the protocol.

Abraham and Elbaum (2012; New Phyt. 197:1012-9) present a method of quantifying microfibril angle in secondary cell walls. The method is technical enough only to be of interest to researchers who need to know the angle of secondary cell wall microfibrils – this is not a look-see ‘Friday afternoon experiment,’ as my old supervisor used to say. To get a full picture, scanning electron microscopy, small-angle X-ray scattering, raman microspectroscopy should all be used in addition to the new technique, which is based on customised polarized light microscopy and LC-PolScope, an imaging software.

Cui et al. (2013; Plant Phys. 161: 36-47) demonstrate that the Tnt1 retrotransposon is a powerful tool for functional genomes in soybean. 62% of insertions from Agrobacterium-mediated transformations using a Tnt1 vector were into annotated genes, indicating the Tnt1 element preferentially inserts into protein-coding regions.  Multiple insertions occurred per transformation, and the transposons did not jump under normal growth conditions. The authors obtained the Tnt1 transposon from plasmid pHLV4909, which contains the entire sequence, and cloned it into the binary vector pZY101 for the transformations.

New Methods and Resources (I)

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Published on: February 5, 2013

On this blog, I highlight a new method or resource pretty regularly. I used to work in what I think is a fairly normal UK plant science lab, so I try to comment on aspects I would have found useful to know about, for example if the method requires a machine not every lab has, or if it is unclear about anything. However, there are many, probably excellent, new open software and techniques which I don’t highlight on the blog because I am completely unfamiliar with their background.

For today, here’s the first part of a round-up of plant methods and resources published over the last few months. If you have used them, feel free to let me know how they worked in the comments, or through email or Twitter. And if you would like to review a method or resource for this blog, please get in touch!

iRootHair is a free, online, curated, expandable database of root hair genomics. Kwasniewski et al. (2013; Plant Phys. 161:28-35) built the database, which currently includes information about 153 root-hair related genes. The majority of the genes are from Arabidopsis, but maize, rice, tomato, and barley genes are also included. There is a page showing figures of various root phenotypes, which users can click through to see the genes associated with a specific phenotype; and a similar one for root processes like tip growth. (more…)

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