NO is an important regulatory molecule in eukaryotes. Much of its effect is ascribed to the action of NO as a signalling molecule. However, NO can also directly modify proteins thus affecting their activities. Although the signalling functions of NO are relatively well recognized in plants, very little is known about its potential influence on the structural integrity of plant cells. In this study, the reorganization of the actin cytoskeleton, and the recycling of wall polysaccharides in plants via the endocytic pathway in the presence of NO or NO-modulating substances were analysed. The actin cytoskeleton and endocytosis in maize (Zea mays) root apices were visualized with fluorescence immunocytochemistry. The organization of the actin cytoskeleton is modulated via NO levels and the extent of such modulation is cell-type specific. In endodermis cells, actin cables change their orientation from longitudinal to oblique and cellular cross-wall domains become actin-depleted/depolymerized. The reaction is reversible and depends on the type of NO donor. Actin-dependent vesicle trafficking is also affected. This was demonstrated through the analysis of recycled wall material transported to newly-formed cell plates and BFA compartments. Therefore, it is concluded that, in plant cells, NO affects the functioning of the actin cytoskeleton and actin-dependent processes. Mechanisms for the reorganization of the actin cytoskeleton are cell-type specific, and such rearrangements might selectively impinge on the functioning of various cellular domains. Thus, the dynamic actin cytoskeleton could be considered as a downstream effector of NO signalling in cells of root apices.

Aims Ectomycorrhizal fungi can improve poplar growth and tolerance to heavy metal stress, and may be useful during the afforestation and phytoremediation of polluted regions with poplar trees. In this study, we determined the effects of the symbiotic interaction between Populus × canescens trees and Paxillus involutus strains different in their tolerance to lead. Methods In vitro inoculated and non-inoculated plants were treated with 0.75 mM Pb(NO 3 ) 2 . The root colonization rate of the two fungal strains, as well as their impacts on poplar health and lead accumulation were examined. Results Based on the colonization level, the roots were classified into one of three categories: non-mycorrhized, changed (ie, fungal cells were present on the root surface, but the Hartig net did not fully develop), and fully mycorrhized. The lead-tolerant P. involutus strain colonized roots better than the non-tolerant strain (ie, changed and fully mycorrhized roots). Moreover, plants inoculated with the tolerant fungal strain grew better than the control plants (217 % increase in dry weight over the controls), and accumulated lead in the roots and stems.Conclusions Inoculation of P. × canescens trees with a Pb-tolerant strain of P. involutus improves host plant growth and may increase Pb phytostabilization potential.

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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 (

It is believed that resource exchange, which is responsible for intensified growth of ectomycorrhizal plants, occurs in the fungus-plant interface. However, increasing evidence indicates that such intensified plant growth, especially root growth promotion, may be independent of root colonization. Nevertheless, the molecular adjustments in low-colonized plants remain poorly understood. Here, we analyzed the metabolome of Populus × canescens microcuttings characterized by significantly increased growth triggered by inoculation with Paxillus involutus, which successfully colonized only 2.1 ± 0.3% of root tips. High-throughput metabolomic analyses of leaves, stems and roots of Populus × canescens microcuttings supplemented with leaf proteome data were performed to determine ECM-triggered changes in N-, P- and C-compounds. The molecular adjustments were relatively low in low-colonized (M) plants. Nevertheless, the levels of foliar phenolic compounds were significantly increased in M plants. Increases of total soluble carbohydrates, starch as well as P concentrations were also observed in M leaves along with the increased abundance of the majority of glycerophosphocholines detected in M roots. However, compared with the leaves of the non-inoculated controls, M leaves presented lower concentrations of both N and most photosynthesis-related proteins and all individual mono- and disaccharides. In M stems, only a few compounds with different abundances were detected, including a decrease in carbohydrates, which was also detected in M roots. Thus, these results suggest that the growth improvement of low-colonized poplar trees is independent of an increased photosynthesis rate, massively increased resource (C:N) exchange and delivery of most nutrients to leaves. The mechanism responsible for poplar growth promotion remains unknown but may be related to increased P uptake, subtle leaf pigment changes, the abundance of certain photosynthetic proteins, slight increases in stem and root amino acid levels and the increase in flavonoids (increasing the antioxidant capacity in poplar), all of which improve the fitness of low-colonized poplars.

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High-quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, “Eridano,” “Villafranca,” “NE-42,” and “Luisa Avanzo,” which have not yet been used for the proteomic studies.

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