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.

Nanoscale plant cell wall architecture

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Published on: December 6, 2012

Highlighted article: Shi-You Ding, Yu-San Liu, Yining Zeng, Michael E. Himmel, John O. Baker, Edward A. Bayer (2012) How Does Plant Cell Wall Nanoscale Architecture Correlate with Enzymatic Digestibility? Science 23:1055-1060

cell walls stained with phloroglucinol, which stains lignin (not from paper)

I spent three years trying to uncover the various mysteries of plant cell wall architecture without ever considering using an imaging approach. Admittedly, I was a PhD student in a molecular biology group and the necessary microscopy equipment was not exactly under my nose, but Ding et al. (paper published in November’s issue of Science) make such good use of imaging for cell wall research, I am kicking myself for not being as inventive Shi-You Ding and his group at NREL in Colorado, USA.

The paper describes the use of bright-field microscopy, confocal laser scanning microscopy, two-colour stimulated Raman scattering microscopy, and atomic force microscopy to look at the structure of primary and secondary cell walls. The authors were able to follow degradation by bacterial cellulosomes and fungal cellulases of cell walls that were untreated or stripped of lignin.

As the authors say in the abstract, their main conclusions are in support of existing ideas. It has been reasonably well established that lignin is the main barrier to enzyme digestibility, but in my opinion this is the best evidence so far that this is the case. The second conclusion, the theory that leaving the polymers intact as much as possible during pretreatment because damaged micro- or macro-fibres are less effectively hydrolysed than structurally intact ones, is not demonstrated at all in this paper.

For me, there are two results in particular in this paper that are novel and useful. First of all, the atomic force microscopy images in this paper show that the acid chlorite delignification method is an efficient way of stripping away lignin with minimal polysaccharide damage.

Secondly, there is evidence that fungal cellulases use different mechanisms to bacterial cellulosomes, and act more quickly to hydrolyse de-lignified cell walls under the conditions used. Both pieces of information are valuable to cell wall researchers and biofuel producers, and projects like my PhD will run more smoothly because of them.

Image credit: Charis Cook

Synthetic enzyme reduces lignin content

Public domain image. Source: Glazer, A. W., and Nikaido, H. (1995). Microbial Biotechnology: fundamentals of applied microbiology. San Francisco: W. H. Freeman, p. 340. ISBN 0-71672608-4

Highlighted article: Kewwi Zhang, Mohammad-Wadud Bhuiya, Jorge Rencoret Pazo, Yuchen Miao, Hoon Kim, John Ralph, and Chang-Jun Liu (2012) An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis. Plant Cell Preview.

Zhang et al. reduce lignin content by introducing an artificial enzyme to the cell wall biosynthesis pathway. This is the first time synthetic biology has been used to change cell wall structure, which is usually modified by changing the expression of endogenous enzymes or introducing a protein from another organism. In fact at the moment, synthetic biology is not a common method of manipulating any plant pathway.

Relevant background

public domain image, courtesy of Chino

Lignin is one of three components of secondary cell walls. It is the part which makes extracting sugar from the cell wall, for example for second generation biofuel production, difficult.

Lignin is made up of three monolignols: coniferyl, sinapyl, and p-coumaryl.

They are synthesised in the cytosol and transported to the cell wall. At the cell wall, the monolignols are oxidised, causing their phenol group to become radicalised. The phenoxy radicals polymerise to form the lignin macromolecule.

The Liu lab had the idea of preventing monolignol oxidation by methylation of the phenol group so that the phenoxy radicals were prevented from forming. Their first attempt was to synthesise a selection of monolignol 4-O-methyltransferases (MOMTs). The artificial MOMTS were fusions of two naturally occurring enzymes: lignin biosynthesis pathway methyltransferase COMT, which does not have any 4-O-methyltransferase activity; and fairy fan enzyme isoeugenol O-methyltransferase, which catalyzes 4-O-methylation of isoeugenol and eugenol, but doesn’t affect monolignols. Although several of these artificial enzymes were able to 4-O-methylate monolignols as expected in vitro, they had no activity in vivo.


Zhang et al. used MOMT3, a promising enzyme from their earlier work, as a starting point. (more…)

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