A significant <a href="http://wiki.gis.com/wiki/index.php?title=Strain,_and_mixed_infections_inside_person_cells_in_a_single_person,_do">Strain,
and mixed infections inside individual cells in 1 person, do</a> outcome of our study is that the primary P starvation responses can be disentangled from down-stream gene regulation related to changes in other nutrient elements.Materials and methods Plant material, growth conditions and harvestGrowth of Populus canescens (INRA717 1-B4) with three different P availabilities has been reported before . Three biological replicates (each consisting of two pooled tissue samples) were analyzed per treatment and tissue (fine roots and uppermost leaves).Statistical analyses of microarraysMicroarray raw data were analyzed using the free statistic software R (version 2.14.2; ) after the protocol described by Janz et al. . The R package "affy" was used for normalization of the array probes ("rma" function) using Bioconductor . The log2 expression value of transcripts that were present ("mas5calls" function) on all replicate chips of at leas.And identified gene modules that were correlated with nutrient element concentrations. The network showed a hierarchical structure with a top module related to P concentrations that was connected with two main secondary modules, one related to C and N and the other to Mg, S and biomass. The secondary modules were connected by co-expression to tertiary modules. The identified gene modules and their putative functions in the poplar P starvation response are discussed. As the consequence of P starvation the tissue concentrations of other nutrient elements also show massive modification. A significant outcome of our study is that the primary P starvation responses can be disentangled from down-stream gene regulation related to changes in other nutrient elements.Materials and methods Plant material, growth conditions and harvestGrowth of Populus canescens (INRA717 1-B4) with three different P availabilities has been reported before . Briefly, the plants were grown in sand culture and irrigated with one ofPLOS ONE | DOI:10.1371/journal.pone.0171958 February 21,2 /Nutrient-related co-expression networks in poplarthree nutrient solutions. Long Ashton nutrient solution  with 641 M P was used for control plants (high phosphate P). For intermediate phosphate (MP) availability, the phosphate concentration was reduced to 6.41 M and potassium added as KCl (675.8 M), for low phosphate (LP) availability to 0.0641 M (additional 682.5 M KCl). After two months of growth with different P supplies, the poplars were harvested. Aliquots of the plant tissues (leaves, stem, coarse roots, fine roots < 2 mm diameter) were dried at 60 for seven days for determination of biomass (n = 10 per treatment): tissue otal tissue biomass dry mass of aliquot mass of aliquot fresh mass . fresh The first three leaves from the top (> 2 cm length) and aliquots of fine roots (< 2 mm diameter) were immediately shock frozen in liquid nitrogen and stored at -80 for RNAextraction.Element concentrationsDry tissues of 2-month-old HP-, MP- and LP-poplars (n = 4 per treatment) were milled (Retsch, type MM2, Haan, Germany) and 10 to 45 mg of plant powder was pressure-extracted in HNO3 . Element concentrations were measured using an inductively coupled plasma optical emission spectrometer (ICP-OES; Optima 5300 DV, PerkinElmer Life and Analytical Sciences, Rodgau, Germany). For determination of carbon and nitrogen concentrations, 0.7 to 0.9 mg dry plant powder (2-month-old HP-, MP- and LP-poplars, n = 5 per treatment) was weighed into tin capsules (Hekatech, Wegberg, Germany) and analyzed in duplicates in an Elemental Analyzer EA1108 (Carlo Erba Strumentazione, Rodano, Italy).