Modification of plant cell wall chemistry impacts metabolome and microbiome composition in Populus PdKOR1 RNAi plants

Plant Soil https://doi.org/10.1007/s11104-018-3692-8 REGULAR ARTICLE Modification of plant cell wall chemistry impacts metabolome and microbiome composition in Populus PdKOR1 RNAi plants Allison M. Veach & Daniel Yip & Nancy L. Engle & Zamin K. Yang & Amber Bible & Jennifer Morrell-Falvey & Timothy J. Tschaplinski & Udaya C. Kalluri & Christopher W. Schadt Received: 10 October 2017 / Accepted: 18 May 2018 # The Author(s) 2018 Abstract Aims We examined the effect of downregulating PdKOR1 gene, an endo-β-1,4-glucanase gene family member previously characterized to affect cellulose biosynthesis and cell wall composition in Populus, on the secondary metabolome and microbiome of field-grown Populus deltoides. Methods We revealed differences in metabolite profiles of PdKOR1 RNAi and control roots using gas chromatography-mass spectrometry, and microbiome identification via Illumina MiSeq 16S and ITS2 rRNA sequencing in root endospheres and rhizospheres. Results PdKOR1 RNAi root metabolites differed from control plants: free amino acids (valine, isoleucine, alanine) were reduced while caffeoyl-shikimates, salicylicacid derivatives, and flavonoid metabolites increased in PdKOR1 RNAi roots. The Actinobacterial family Micromonosporaceae were more abundant in RNAi root endospheres, whereas Nitrospirae was reduced in PdKOR1 RNAi rhizospheres. Ascomycota were lower and Basidiomycota greater in PdKOR1 rhizospheres. Bacterial and fungal community composition, as measured by Bray-Curtis dissimilarity, differed between PdKOR1 RNAi and control rhizospheres and endospheres. Conclusions These results indicate that modification of plant cell walls via downregulation of PdKOR1 gene in Populus impacts carbon metabolism in roots and concomitant alterations in root-associated microbial communities. Such an understanding of functional and ecological implications of biomass chemistry improvement efforts is critical to address the goals of sustainable bioenergy crop production and management. Keywords Cellulose . Cell wall alteration . Endo-1,4-ßglucanase . Microbiome . Metabolome . Populus Introduction Responsible Editor: Birgit Mitter. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11104-018-3692-8) contains supplementary material, which is available to authorized users. A. M. Veach : D. Yip : N. L. Engle : Z. K. Yang : A. Bible : J. Morrell-Falvey : T. J. Tschaplinski : U. C. Kalluri (*) : C. W. Schadt (*) Biosciences Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA e-mail: ; e-mail: Cellulose synthesis and cell wall formation are important processes which regulate plant structure and function. Differences in cell wall composition results in physiological changes, such as altered stress responses (Zhao and Dixon 2014), water transport (Liang et al. 2010), and growth rates (Thomas et al. 2013). As a consequence, cell wall structure strongly regulates plant development and overall health. Optimization of plant cell wall and lignocellulosic biomass composition, via Plant Soil genetic modification or selection of cell wall pathway or carbon metabolism genes, are broadly-used strategies in advanced bioenergy feedstock improvement efforts (Fu et al. 2011; Kalluri et al. 2014; Li et al. 2016; Loqué et al. 2015; Petersen et al. 2012; Yang et al. 2013). Such strategies have been demonstrated to achieve shifts in lignin composition (Baxter and Stewart Jr 2013; Furtado et al. 2014), cellulose and hemicellulose content (Hernández-Blanco et al. 2007; Kalluri et al. 2016), and greater hexose:pentose ratios within wood tissue (Loqué et al. 2015) with the specific target of improving saccharification of lignocellulosic biomass. For the long-range goal of sustainable production of bioenergy crops, research efforts must co-optimize plants for biomass amount and composition as well as sustainable field performance characteristics such as enhanced water and nutrient-use efficiency, resistance to microbial pathogens, and interactions with beneficial microbes. KORRIGAN (KOR) is a membrane bound endo-ß1,4-glucanase (EGase) in the glycosyl hydrolase family 9 (GH9) (Urbanowicz et al. 2007; Vain et al. 2014) shown to be associated with the cellulose synthase complex (Darley et al. 2001; Joshi and Mansfield 2007). Previous characterization of KOR homologs in Populus deltoides has shown that RNAi-mediated downregulation of KOR (PdKOR1 and PdKOR2) impacts an array of plant properties, including reductions in cellulose, lignin and soluble sugar content, increase in cellulose crystallinity and phenolic conjugates, and reduced stem and root growth (Bali et al. 2016; Kalluri et al. 2016). The altered carbon allocation and secondary metabolome of PdKOR1 RNAi plants has been previously shown to increase colonization rates of the ectomycorrhizal fungal symbiont, Laccaria bicolor under greenhouse conditions (Kalluri et al. 2016). A broader understanding of plant – microbe interactions under field conditions and potential impacts on beneficial root endophytic and rhizospheric bacteria and fungi, in the context of modifying cellulose biosynthesis and secondary metabolism, is relatively unexplored. Microbial establishment with plant hosts can confer benefits, such as antagonistic suppression of pathogens, enhanced nutrient acquisition, enhanced growth, and/or increased stress resiliency (Mendes et al. 2013) and plant hosts may also select for specific groups of beneficial microorganisms. For example, it has been reported that plants growing in contaminated soils show a preferential colonization by root endophytic microbes carrying toxin degradation genes (Siciliano et al. 2001; Thijs et al. 2016). Downregulation of a lignin biosynthesis gene and higher levels of phenolic metabolites in field-grown Populus tremula x Populus alba was also shown to alter bacterial community structure and metabolic capabilities in root endospheres via selection of the metabolically diverse Pseudomonas putida (Beckers et al. 2016). That study (Beckers et al. 2016) provided a novel insight that regulation of an enzyme integral for lignin production can have direct (i.e., modified lignin content and metabolite production) as well as indirect (i.e., large changes in overall plant phenotype) implications for host selection of microbial partners of transgenic tree species. Previous work also indicated PdKOR1 RNAi plants have cascading effects on multiple phenotypic traits in Populus (Kalluri et al. 2016; Maloney and Mansfield 2010) resulting in significant impacts on interactions with a fungal mutualist (Kalluri et al. 2016). Within this context, here we studied the impact of PdKOR1 gene downregulation on the root secondary metabolome and the overall bacterial and fungal microbiomes within the rhizosphere and root endospheres in field-grown Populus deltoides. Our goals were to first examine how PdKOR1 RNAi plants differed in secondary metabolites within fieldgr (...truncated)