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Page history last edited by Kolby Jardine 1 year, 10 months ago



Oxford Growth Facility 

Currently 6 California poplar trees established (commercial source, Plants of the Wild, WA, USA). http://www.plantsofthewild.com/



We have decided to primarily work with natural genetic variants in cell wall metabolism for both greenhouse and field experiments. The one exception to this is the Reduced Wall Acetylation (RWA) line at ORNL.


The Arabidopsis genes and in some cases the corresponding poplar genes that we are interested in studying are listed below. 

If feasible, we could consider primarily working with the RWA transgenic poplar (are quadruple mutants available?) and an ESKIMO1 type mutant in Ptrichocarpa if available (please see the text below for description of genes in Ptrichocarpa). This is because mutation of this single TBL/AXY gene in Arabidopsis dramatically alters xyloglucan acetylation and has a constitutive drought type phenotype with very low transpiraiton rates, partially collapsed xylem vessels, etc. This would be fantastic to also demonstrate in Ptrichocarpa with  PtrXOAT1-12, xylan-specific acetyltransferase genes (potentially with a focus on PtrXOAT1-3 due to their ability to rescue the ESK1 mutant in Arabidopsis). Another target genes to look for in natural in Ptrichocarpa mutants are PAE1: a pectin o-acetyl esterase and PtxtPME1, A xylem‐abundant pectin methyl esterase discovered in aspen. As described below, other genes identified in rice and Arabidopsis would also be good to look for including the BS1 gene in rice with is a O-xylan acetyl esterase. However, also note that compensatory upregulation of RWA genes following deacetylation of cell walls by a xylan acetyl esterase expressed using the 35Spromoter has been reported (https://onlinelibrary.wiley.com/doi/full/10.1111/pbi.12232).  Although Arabidopsis gene OS1/QUA2/TSD2 has been identified as a likely pectin methyltransferase, we need to look for such a gene in P. trichocarpa. PME6 in Arabidopsis is a pectin methyl esterase found to be highly expressed in guard cells, but it is not clear if P. trichocarpa also has such a gene.


A. Reduced Wall Acetylation (RWA): RWA1/2/3/4 in Arabidopsis, Transport of acetyl donors across the Golgi apparatus membrane (acetyl-donor translocators, Lee et al., 2011). 

**In a poplar hybrid (Populus tremula x tremuloides) by P. Pawar et al., 2017, https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14489), RWA-A,B,C,D. When all four RWA genes were suppressed together using a wood-specific promoter, wood acetylation was reduced by 25%. While not Ptrichocarpa, Wellington indicated that he has created transgenic lines and is maintaining them at ORNL. 


B. Trichome birefrengence-like (TBL)/Altered xylogugan (AXY): Acetylation of xylan  The main putative xylan acetyl transferase has been identified in Arabidopsis as AtTBL-29/ESKIMO1 (Xiong et al., 2013). Under normal growth conditions, esk1 plants have a reduced size and accumulate high levels of the hormone abscisic acid as if under drought (Bouchabke-Coussa et al., 2008). The transcriptome of esk1 is similar to drought-stressed wild-type (WT) plants (Bouchabke-Coussa et al., 2008) indicating that a lack of ESK1 mimics these stress conditions. Furthermore, esk1 stems exhibits irregular xylem phenotype with partitially collapsed xylem vessels and altered plant hydraulics inlcluding environemtnal responses (Lefebvre et al., 2011). TBL29/ESK1 (transcriptome of esk1 is similar to drought-stressed wild-type (WT) plants (Bouchabke-Coussa et al., 2008) indicating that a lack of ESK1 mimics these stress conditions. Furthermore, esk1 stems exhibit an irregular xylem phenotype (Lefebvre et al., 2011). TBL32TBL33 and ESK1 in Arabidopsis resulted in a severe reduction in xylan acetyl level down to 15% that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth (Yuan et al., 2016). Arabidopsis plants with defective ESK1 enzymes (xylan-specific acetyltransferase) have a constitutive drought syndrome and collapsed xylem vessels, low hydraulic conductivity along with low acetylation levels in xylan and mannan, low transpiration rates, high water use efficiency, and dwarfism (Lefebvre et al., 2011, Ramirez et al., 2018).**In Ptrichocarpa, acetylation of xylan has been attributed to PtrXOAT1-12 (Zhong et al 2018, https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194532). When expressed in the Arabidopsis eskimo1 mutant, PtrXOAT1, PtrXAOT2 and PtrXOAT3 were able to rescue the defects in xylan acetylation. 
C. Pectin methyl transferase (PMT): Methylation of pectin. Arabidopsis gene OS1/QUA2/TSD2 (pectin methyltransferase, Qu et al. 2016, Krupkova et al., 2007Mouille et al., 2007). OSU1/QUA2/TSD2-encoded putative methyltransferase is required for normal C/N nutrient balance response in Arabidopsis. (Gao et al., 2008, https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001387).  **Need to search what the PMT genes are in Ptrichocarpa.
D. Pectin methyl esterase (PME): Methyl ester hydrolysis of pectin. ***A xylem‐abundant pectin methyl esterase was discovered in aspen, PtxtPME1. Homogalacturonans and their methylation patterns were found to influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties via activity of PtxtPME1 (Allario et al., 2018, https://onlinelibrary.wiley.com/doi/abs/10.1111/ppl.12702). PME6 in Arabidopsis is a pectin methyl esterase found to be highly expressed in guard cells and required for stomatal function and response to environmental variables, Amsbury et al., 2016, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5106435/).E. Pectin methyl esterase inhibitor (PMEI): Inhibitor of PME (need to research) 
F. **Pectin Acetyl Esterase (PAE): PAE1, o-acetyl hydrolysis of pectin, Ptrichocarpa (Gou et al. 2012, http://www.plantcell.org/content/24/1/50.short), 10 putative PAE genes were deduced in the P. trichocarpa genome (Geisler-Lee et al., 2006) 
G. **Xylan Acetyl Esterase (XAE): BS1 gene in rice catalyzes the O-acetyl hydrolysis of xylan and a natural mutant brittle leaf sheath1 (bs1) has fragile leaf sheaths and stunted growth phenotype and was demonstrated with enhanced acetylation levels of xylan, Zhang et al. 2017. https://www.nature.com/articles/nplants201717. Note that this paper is also a very good reference for anlytical techniques involved in cell wall acetyation analysis. Also see commentary in Nature by Scheller from LBNL, https://www.nature.com/articles/nplants201724

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