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…)

The many advantages of chloroplasts

Chloroplasts are a major advantage to doing synthetic biology in plants. They produce starch and some amino acids as well as hosting photosynthesis, all fully separated from other cellular functions going on in the rest of the cell. Synthetic biology approaches could turn them into individualised micro-factories inside plant cells, synthesising whatever compound you fancy without poisoning the cell and with almost no risk of any transgenes escaping into other plants.

Stable plastid transformation was first achieved in tobacco in 1990.  Since then, chloroplast transformation has been successful in many plant species – a 2009 review by Huan-Hyan Wang et al. (JGG 36:387) contains a nice table summarizing the methods used in each species. Plastid-based biosynthesis of biopharmaceuticals has been researched for years, but synthetic biology technologies make it possible to consider moving beyond synthesis of antigens and relatively simple molecules (for examples see Daniell et al. 2009, Trends in Plant Sci 14:669) to more complex structures.

In today’s highlighted paper, Nielson et al. successfully built the P450-dependant dhurrin pathway into tobacco chloroplast cells. This in itself does not have a major benefit to science, as dhurrin has no real value, but as a proof of concept this is worthy of note. The three-step biosynthesis of dhurrin from L-tyrosine is normally based on the endoplasmic reticulum, and its rate is limited by low concentrations of NADPH. By building the pathway in a chloroplast, the authors have proven not only the feasibility of chloroplast pathway engineering, but also the potential of using reducing power from photosynthesis to run biosynthesis pathways.

For more information about chloroplast engineering, this 2011 paper reviews chloroplast transformation markers and this paper is another example of pathway engineering in chloroplasts.

More generally, to find out about synthetic biology approaches please register for our Synthetic Biology meeting, which aims to introduce synthetic biology to plant scientists. It is £250 for academics, and includes overnight accommodation and meals – there is a reduced rate for students and post-docs.

Highlighted paper: Agnieszka Zygadlo Nielsen, Bibi Ziersen, Kenneth Jensen, Lærke Münter Lassen, Carl Erik Olsen, Birger Lindberg Møller, and Poul Erik Jensen (2013) Redirecting Photosynthetic Reducing Power toward Bioactive Natural Product Synthesis. ACS Synthetic Biology DOI: 10.1021/sb300128r

Image credit: Martin Bahmann, via Wikimedia Commons.

What makes an invasive species?

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Published on: March 12, 2013
B. sylvaticum seeds

Brachypodium sylvaticum is a grass species known as slender false brome, and is native to Europe, Asia, and north Africa. It is a common sight in UK woodlands, and grows all over the country. In the USA though, its tufts don’t mark convenient picnic spots in woodland but are destroyed if seen in a new region because this invasive species has colonised miles of Oregon’s woodland floor. An Oregon-based research team has sequenced the B. sylvaticum transcriptome and hopes to use it as a mode for the evolution of invasive species.

Highlighted paper: Samuel E. Fox, Justin Preece, Jeffrey A. Kimbrel, Gina L. Marchini, Abigail Sage, Ken Youens-Clark, Mitchell B. Cruzan, and Pankaj Jaiswal 2013. Sequencing and De Novo Transcriptome Assembly of Brachypodium sylvaticum (Poaceae). Applications in Plant Sciences 1: 1200011

Slender false broom was widely planted in the in the mid part of the 20th century in an attempt to seed mountain rangelands in Utah, Wyoming, and Idaho. It was also planted in experimental gardens in two Oregon cities. The two attempts to establish the species were independent, but microsatellite analysis suggests the plants originated from the samestock of accessions. At some point in the late 20th century, some of these accessions crossed and the hybrids spread rapidly across Oregon’s forests (Rosenthal et al. 2008, Mol Ecol 17:4657). Today in Oregon this aggressive genotypes have formed thick monocultures that completely cover the forest floor at the expense of native flora, and have spread to California and Washington too. (more…)

Transpiration is perfectly in tune

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Published on: February 19, 2013

Plants contain a continuous water column from the roots, where water is absorbed, to the leaves, where water is lost through evaporation via the stomata. When a plant’s cells require water, opening stomata makes water potential in the xylem strongly negative and water is pulled from the soil into the roots and into the xylem strongly and quickly. However, the water potential gradient can be too steep, causing cavitation (bubbles) in the xylem, which slows down water transport. The optimum transpiration rate occurs when water potential and cavitation are balanced in the right way. According to research published recently in New Phytologist, plants are able to maintain a transpiration rate very close to the maximum theoretical transpiration potential, allowing partial cavitation but not letting it limit hydraulic conductivity.

Here, Manzoni et al. from Amilcare Porporato’s group at Duke University, compared the theoretical optimum transpiration rate with actual transpiration ates in a number of tree species (grouped into boreal, temperate, Mediterranean, tropical dry, and tropical moist species). Their parameters for calculating the theoretical optimum were extensive, including soil water potential, xylem hydraulic conductivity, and canopy height.

The actual maximum transpiration rate of these species was then collected from published papers, and sorted according to climate and the conditions under which the analysis was done. Only the data from well-irrigated systems was used. The average observed peak transpiration rate was close to the theoretical maximum transpiration rate, and both were fairly conserved among plant types of a similar size in a particular climate. (more…)

Valentine’s Volatiles

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Published on: February 14, 2013

Plenty of flowers are beautiful and expensive, but the lovely rose scent makes roses the perfect traditional gift to your Valentine. And since it is Valentine’s Day today, and this is a plant science blog, here’s a brief review of the science of floral scents and a recently published paper on the topic (roses not included).

Despite rose breeders managing to come up with flowers with stronger, subtle, or new scents from new rose varieties, the science of floral scents is not well understood. Floral scent can be under stronger natural phenotypic selection than flowers (Parachnowitsch et al. 2012; New Phyt. 195:667), but the agents of selection may be any number of organisms including pollinators and herbivores and the main influencing factor on scent evolution is not known (Theis and Adler 2012; Ecology 93:430). 

The molecular mechanism and regulation of biosynthesis of the volatile, low-molecular weight compounds that cause floral scent is also fairly uncharacterised. They are mainly products of the terpenoid, fatty acid, and phenylpropanoid pathways. Recently a group from the Hebrew University of Jerusalem characterised a regulation mechanism of the phenylpropanoid volatile biosynthesis pathway (Spitzer-Rimon et al. 2012; Plant Cell 24:5089).

All phenylpropanoids share the same precursor, Phe, which is biosynthesized via the shikimate pathway. A transcriptional regulator ODORANT1 (ODO1) regulates shikimate pathway enzymes and affects metabolic flow toward phenylpropanoid production. Another transcriptional regulator, EMISSION OF BENZENOIDS II (EOBII) directly regulates ODO1’s expression, indirectly affecting the shikimate pathway and the biosynthesis of phenylpropanoid volatiles. (Van Moerkercke et al. 2001, Plant J 67:917; Verdonk et al. 2005, Plant Cell 17:1612).

Working on petunia, Spitzer-Rimon et al. identified EOBI, another regulator of floral scent. (more…)

Ash trees and human health

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Published on: January 24, 2013
A lone ash tree in Worcestershire

I have to admit that as a plant scientist plugged into the major social media networks that when I was inundated with articles and posts about ash dieback (Chalara fraxineai) in December, I got a bit fed up with it. Of course I appreciate all species have intrinsic value and it will be sad if Britain loses its ash trees – but I have no emotional attachment to ash trees, and pathogens are a fact of life. British countryside is managed land, so with effective management other trees will fill the gaps. However, a paper published in the February issue of American Journal of Preventive Medicine (Donovan et al., 2013) suggests the health effects on humans of losing trees are significant, and that serious loss of ash trees in the UK could have consequences beyond the financial burden on the forestry industry and the short-term loss of trees. 

Adult emerald ash borer on a penny

The research paper is an analysis of the effects of emerald ash borer (Agrilus planipennis) damage to North American ash trees. Emerald ash borer is a green beetle native to Asia, and was introduced to North America in 2002. It causes significant damage to all North American ash species and an infestation can kill a mature tree within four years. For this study, Donovan et al. looked at human mortality data from 1296 counties across the 15 states where there were confirmed emerald ash borer infestations in 2010. (more…)

Investigating photosynthesis

In today’s highlighted article, the authors use traditional and far more modern biochemistry to uncover why photosynthesis is inhibited by Streptomyces spp., and characterise a previously unknown step in cyclic electron flow. This is also a good opportunity to point out these great photosynthesis outreach and education resources from Science and Plants for Schools. Admittedly, they don’t have anything on AA-sensitive CEF, because it’s unlikely anyone without a plant biochemistry PhD needs to know about that! But they have brilliant basic photosynthesis teaching resources, including these amazing algae-jelly-balls.

Photosynthesis background: Broadly speaking, photosynthesis is the process by which light energy from the sun is absorbed by Photosystems I and II (PSI and PSII), where it is channelled into electron transport chains and stored in ATP and NADPH. One electron carrier is ferredoxin (Fd).

There are two types of electron flow, cyclic and linear (CEF and LEF), which generate ATP. Though they are different processes, both CEF and LEF require PSI and PSII, two other thylakoid proteins, PGR5 and PGRL1, and electron carriers Fd, plastoquinone (PQ) and plastocyanin.

In CEF there are two processes by which electrons are transferred from Fd to PQ. One is a characterised NADH dehydrogenase-like compex dependent pathway, and all that is known about the other is that it is sensitive to antimycin A (AA), a product of Streptomyces spp. which inhibits CEF.

For background on AA-inhibition and the divergent electron transfer pathways in CEF, see Joët et al. (2001; Plant Phys. 24:1919). For more information on PGR5 and PGRL1, see DalCorso et al. (2008; Cell 132:273).

Gaps in knowledge of CEF:

  • What makes that electron transfer process from Fd to PQ sensitive to AA?
  • How do the electrons from photoreduced Fd get transferred to PQ and back into the electron transport chain?
  • What do functions do PGR5 and PGRL1 perform?

New from Hertle et al.: Titration and Western blotting experiments showed that PGR5 and PGRL1 dimerize to each other. Six cysteine residues were conserved in all PGRL1 proteins. Hertle et al. made mutant PGRL1 proteins in which one or more cysteine residues were substituted for serine, and tested the varients for their capacity to bind PGR5, iron, and to promote AA-sensitive CEF. They worked out that all six cysteines were essential for AA-sensitive CEF, while specific cysteines were involved in binding iron and PGR5.

In vitro assays demonstrated that PGRL1 is capable of transferring electrons between Fd and PQ analogue DMBQ in the presence of PGR5, and this reaction was inhibited in the presence of AA. Hertle et al. show clearly that PGRL1 is physically a fit for a ferredoxin-plastoquinone reductase and make a strong case for it being the mediator between Fd and PQ in CEF, and the AA-sensitive step in the cyclic electron flow.

Highlighted article: Alexander P. Hertle, Thomas Blunder, Tobias Wunder, Paolo Pesaresi, Mathias Pribil, Ute Armbruster, Dario Leister (2013) PGRL1 Is the Elusive Ferredoxin-Plastoquinone Reductase in Photosynthetic Cyclic Electron Flow. Molecular Cell – 03 January 2013, 10.1016/j.molcel.2012.11.030

Analysing phenotypes and measuring callose

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Published on: January 9, 2013
Part of Figure 7 from Green et al., showing the an example of Phenophyte output.

At the end of last year, you may have missed two useful publications from Plant Methods which use new free online tools to make your life easier.

Phenophyte can help you measure 2D areas quickly and accurately. It was described in November’s Plant Methods by Green et al., a team mainly from  Columbia, USA. Users chose if they want to analyse indivudial images, compare before/after images (as shown in the figure to the left), or analyse a timecourse. They then upload the images – the upload tool allows up to 2GB or 500 images, and sequential uploads are possible if required. The computational results can be previewed before submitting the job. When processing is complete, the user will be emailed a link to the results, which must be downloaded within a week. The manual provides detailed tips on how to take the photographs to upload, and the guidance is standard with the exception of the use of a colour/size checker (for example, this one), and the interface is straightforward and friendly.

Figure 5 from Zhou et al., showing the CalloseMeasurer interface and output.

A more specialized application is CalloseMeasurer, from the Robatzek group at The Sainsbury Laboratory. Zhou et al. describe a piece of software for quantifying callose deposition with enough accuracy to quantify the growth of filamentous pathogens within a plant by recognising the spreading network of callose deposition caused by the pathogen. The paper is heavy on technical detail, but guides readers through using CalloseMeasurer in the ‘Image Processing’ section of the paper. Users must have Acapella software installed, and they simply drag and drop the CalloseMeasurer script into the application window and start using the programme.

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