Regulation of the leaf proteome by inoculation of Populus × canescens with two Paxillus involutus isolates differing in root colonization rates

Abstract

During ectomycorrhizal symbioses, up to 30% of the carbon produced in leaves may be translocated to the fungal partner. Given that the leaf response to root colonization is largely unknown, we performed a leaf proteome analysis of Populus × canescens inoculated in vitro with two isolates of Paxillus involutus significantly differing in root colonization rates (65 ± 7% vs 14 ± 7%), together with plant growth and leaf biochemistry analyses to determine the response of plant leaves to ectomycorrhizal root colonization. The isolate that more efficiently colonized roots (isolate H) affected 9.1% of the leaf proteome compared with control plants. Simultaneously, ectomycorrhiza in isolate H-inoculated plants led to improved plant growth and an increased abundance of leaf proteins involved in protein turnover, stress response, carbohydrate metabolism, and photosynthesis. The protein increment was also correlated with increases in chlorophyll, foliar carbon, and carbohydrate contents. Although inoculation of P. × canescens roots with the other P. involutus isolate (isolate L, characterized by a low root colonization ratio) affected 6.8% of the leaf proteome compared with control plants, most proteins were downregulated. The proteomic signals of increased carbohydrate biosynthesis were not detected, and carbohydrate, carbon, and leaf pigment levels and plant biomass did not differ from the noninoculated plants. Our results revealed that the upregulation of the photosynthetic protein abundance and levels of leaf carbohydrate are positively related to rates of root colonization. Upregulation of photosynthetic proteins, chlorophyll, and leaf carbohydrate levels in ectomycorrhizal plants was positively related to root colonization rates and resulted in increased carbon translocation and sequestration underground. Keywords Ectomycorrhiza. Root colonization rate. Plant biometrics. Protein turnover. Stress response. Leaf carbohydrates Abbreviations 2DE Two-dimensional gel electrophoresis Chl a Chlorophyll a Chl b Chlorophyll b DW Dry weight FW Fresh weight ECM Ectomycorrhizal Rca RuBisCo activase SC Soluble carbohydrates SEM Scanning electron microscopy TNC Total non-structural carbohydrates Electronic supplementary material The online version of this article (

Integrated proteomic and metabolomic analyses revealed molecular adjustments in Populus × canescens colonized with the ectomycorrhizal fungus Paxillus involutus, which limited plant host growth

“…Based on root tip morphology, root tips were divided into three categories in accordance with the methods of Szuba et al . (2017, 2019): non‐colonized root tips (those without any visible signs of fungal cell presence) and two stages of root tip colonization, changed root tips (root tips devoid of root hairs and covered with loose fungal hyphae) and fully mycorrhized root tips (those that are swollen and short, with a very well‐developed mantle). Root tip morphotype percentages were determined using ImageJ v1.48 software (Wayne Rasband, Bethesda, MD, USA) in conjunction with high‐resolution images captured during harvest.…”

“…Non‐structural soluble carbohydrate (SC) and starch concentrations were assessed in dry leaves according to the methods of Szuba et al . (2019). For trials that were originally performed with dried samples, the concentration normalized to the FW was estimated according to the following equation: …”

“…The leaf proteome was analysed with two‐dimensional electrophoresis (2DE) coupled with the MS/MS approach to obtain basic leaf physiology information and to determine the experimental MW/pI values of particular proteins (especially Rubisco activase, which is known to be affected by ECMs; Szuba et al ., 2019). The proteins were isolated from 500 mg of leaf tissue and 100 mg of root tissue ( n = 5; whole root systems were used for the preparation of the samples) according to the modified phenol extraction method described by Szuba et al .…”

“…The proteins were isolated from 500 mg of leaf tissue and 100 mg of root tissue ( n = 5; whole root systems were used for the preparation of the samples) according to the modified phenol extraction method described by Szuba et al . (2019). The proteins were resuspended in buffer [5 M urea, 2 M thiourea, 2% CHAPS, 2% carrier ampholyte (Pharmalyte 4–7, GE Healthcare LS, Uppsala, Sweden) for leaf proteins as well as 4 M urea and 50 mM ammonium bicarbonate for root proteins].…”

“…S2). Selected spots were cut off and digested using a standard in‐gel digestion procedure (Szuba et al ., 2019). Peptide solutions were analysed with a MALDI‐TOF/TOF UltrafleXtreme mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany).…”