Oak protein profile alterations upon root colonization by an ectomycorrhizal fungus

An increased knowledge on the real impacts of ectomycorrhizal symbiosis in forest species is needed to optimize forest sustainable productivity and thus to improve forest services and their capacity to act as carbon sinks. In this study, we investigated the response of an oak species to ectomycorrhizae formation using a proteomics approach complemented by biochemical analysis of carbohydrate levels. Comparative proteome analysis between mycorrhizal and nonmycorrhizal cork oak plants revealed no differences at the foliar level. However, the protein profile of 34 unique oak proteins was altered in the roots. Consistent with the results of the biochemical analysis, the proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose for the metabolic activity of mycorrhizal roots which is consistent with an increased allocation of carbohydrates from the plant to the fungus in order to sustain the symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of defense reactions, increased nutrient mobilization from the plant-fungus interface (N and P), as well as cytoskeleton rearrangements and induction of plant cell wall loosening for fungal root accommodation in colonized roots are also suggested by the results. The suggested improvement in root capacity to take up nutrients accompanied by an increase of root biomass without apparent changes in aboveground biomass strongly re-enforces the potential of mycorrhizal inoculation to improve cork oak forest resistance capacity to cope with coming climate change.


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The ectomycorrhizal (ECM) symbiosis is a mutualistic association between the fine 63 roots of trees and soil inhabiting fungi, typically found in temperate and boreal forests. 64 The intimate contact between the two partners that occurs in ectomycorrhizae results in 2-DE and mass spectrometry, and the development of genomic sequence databases 95 for peptide mass matches made it possible to achieve a high throughput of plant protein identifying proteins with confidence by using mass spectrometry. Differential in gel 101 electrophoresis (DIGE) is a method that can be used to accurately quantify protein 102 accumulation differences under various conditions. Using the DIGE technology, 103 proteome analysis can be carried out similarly to a microarray experiment in that two 104 samples are compared on one gel by analyzing the ratio of two fluorescent labels 105 between two samples for each protein (Unlu et al. 1997). In this work we investigated 106 the differences in the protein profiles between mycorrhizal and non-mycorrhizal cork  (CaCl2) 5.5-6.5, organic matter > 70%). After germination, three months old plantlets 127 were transferred to 1,5 L pots containing soil and simultaneously inoculated with P. 128 tinctorius peat-vermiculite inoculum (3 months old), according to Sebastiana et al. 129 (2013a). Control plants were treated with a non-inoculated peat-vermiculite mixture. 130 Plants were grown in pots in a greenhouse and watered once a week with 500 mL of tap 131 water. No fertilization was applied.    155 The experiment included two different comparisons: (1) mycorhizal roots versus non-156 mycorrhizal roots and (2) "mycorrhizal" leaves versus "non-mycorrhizal" leaves. 157 Before electrophoresis protein samples were labelled with the CyDye DIGE Fluors 158 (Cy5, Cy3 and Cy2; GE Healthcare). Before the labelling reaction, the pH of the 159 extracted protein solution was adjusted to 8. 5  with 2% (w/v) DTE, followed by alkylation with 3% (w/v) iodoacetamide in the same 178 buffer. Next, SDS-PAGE was performed using 12.5% polyacrylamide gels using the 179 EttanDALTtwelve system (GE Healthcare). Separation was performed overnight at 180 20°C with 1 st step at 80 V, 10 mA/gel and 1 W/gel, and 2 nd step at 100 V, 17mA/gel and 181 1.5 W/gel. 2D-DIGE gels were scanned using low-fluorescence glass plates at a           (Table 1). This is probably related to the early    Gene ontology (GO) annotation of the biological processes affected by the interaction of 444 cork oak roots with P. tinctorius is shown in Figure 2. As expected, "metabolic process" 445 and "cellular process" were the most abundant categories. More than 65% of the 446 proteins were assigned with the GO annotations "carbohydrate metabolic process", 447 "protein metabolic process" and "transport". Another relevant category was "response 448 to stress".

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Functional analysis and database searches revealed a putative involvement of the 450 identified proteins in several cellular pathways such as, carbon and energy metabolism, 451 protein folding, stability and degradation, stress and defense, nutrient acquisition, lipid 452 transport/metabolism, cell wall remodelling and cytoskeleton.

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In order to better characterize the mycorrhizal responsive proteins identified in our 454 experiment we analyzed their sequences for sub-cellular location prediction (Table 2).

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Proteins predicted to be cytoplasm-located were mostly found to be involved in carbon  In the next sections we will discuss the possible role of the identified proteins in the 471 context of ECM symbiosis.  (Table 1).

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It is commonly accepted that in ectomycorrhizae established with basidiomycotic fungi 499 like P. tinctorius, plant derived sucrose in the plant-fungus interface is hydrolyzed by 500 plant cell wall invertases into hexoses, from which glucose seems to be preferred by the 501 mycobiont (Nehls et al. 2010). This carbon drain to the fungal partner, suggested by our 502 results, does not seem to result in biomass loss since foliar biomass was unaltered and 503 root biomass was even increased by the inoculation with P. tinctorius (Table 1). This is 504 suggestive of a fully functional symbiotic relationship between Q. suber and P.  (Table 1). In fact, starch levels were identical in mycorrhizal and non-mycorrhizal 514 roots, suggesting that the plant is not mobilizing stored sugar pools for transfer to the 515 symbiotic fungus. Besides its role in sucrose metabolism, this enzyme is also involved 516 in the synthesis of UDPG for cell wall polysaccharide synthesis (Kleczkowski et al.              (Table 2) 1130