Phosphorus nutrition of poplar

Phosphorus (P) is a major plant nutrient required for the biosynthesis of nucleic acids, nucleotides, membrane lipids and metabolites such as organic esters. P further plays a role in enzyme regulation by phosphorylation. Plants take up P in its inorganic form H2PO4- (Pi). Pi is present only in low concentrations in the soil solution and therefore has to be replenished all the time to ensure sufficient plant nutrition. In plants different strategies can be distinguished in response to P starvation: (i) P conservation by re-use of P from P containing compounds e.g. membrane lipids and avoidance of P requiring metabolic pathways. This results in growth reduction. (ii) Enhanced acquisition of P from the soil. For this purpose plants secrete purple acid phosphatases (PAPs) to mobilize Pi from organic sources and increase the activity of phosphate transporters (PHTs) for enhanced uptake capacity. Only little is known for woody plants about the molecular responses to low P availability because to date most of our knowledge stems from the model plant Arabidopsis. The main aims of this thesis were to characterize molecular changes at the whole-transcriptome level in leaves and roots of Populus × canescens in response to P deprivation and to relate these changes to poplar growth, Pi acquisition and Pi uptake. The following questions were addressed: (a) How does P deficiency affect the nutrient states of the plant and which genes are involved in the response to P limitation in poplar? (b) How is the poplar Pi uptake kinetics affected by decreasing P availabilities and how are PHTs transcriptionally regulated by P deficiency? (c) How does low P availability affect the expression profile of PAPs and which members of the large PAP family are released into the medium upon P starvation? To address these questions transcriptome analyses for poplars (Populus × canescens) grown under reduced phosphorus availabilities were conducted using microarray technology. Plant nutrient concentrations were determined by inductively coupled plasma optical emission spectrometry and uptake by use of radioactive P. The secreted proteins were determined by proteome analyses using liquid-chromatography electrospray-ionization mass spectrometry. (a) Poplars exhibited strong growth reduction and increased P use efficiency in response to lower P availabilities. P starvation resulted also in changes of most other elements (S, N, K, Mg, Ca, Fe, Zn, Mn, Al) studied. A high number of genes (12068 in total) was differently expressed upon P starvation. These genes were clustered in eleven co-expression modules of which seven were correlated with distinct elements in the plant tissues. One module with 565 genes (4.7 % of all differentially expressed genes) was correlated with changes in the P concentration in the plant tissues and with the biomass. In this module, PAPs but no PHTs were identified among the highly upregulated P-related genes. The functional category “galactolipid synthesis” was enriched among the P-related genes. Galactolipids substitute phospholipids in membranes under P limitation. Two modules, one correlated with C and N and the other with biomass, S and Mg, were connected with the P-related module by co-expression. In these modules GO terms indicating DNA modification and cell division as well as defense (ethylene, respiratory burst) and RNA modification and signaling were enriched. In conclusion, most differentially expressed genes were not directly related to the tissue P concentrations and were, therefore, most likely regulated by downstream events of P starvation. (b) Whole-plant P uptake kinetics and expression profiles of members of the phosphate transporter families were studied under high, intermediate and low P availability in relation to plant performance. The maximum P uptake rate was more than 13-times higher in P-starved than in well-supplied poplars. The Km-values ranged between 20 µM and 26 µM for P starved poplars. The minimum concentration for net P uptake from the nutrient solution was 1 µM P. Among the PHT subfamilies, all PHTs of family 1 (PHT1) studied showed significant up-regulation upon P starvation and were higher expressed in roots than leaves, with the exception of PtPHT1;3. The transcript abundance of PtPHT1;3 was high in leaves under high P supply and increased further under P starvation. PtPHT1;1 and PtPHT1;2 showed root- and P-starvation specific expression. Expression profiles of distinct members of the PHT subfamilies, especially those of PHT1 were linked with changes in P uptake and allocation at whole-plant level. The regulation was tissue-specific with lower P-responsiveness in leaves than in roots. Because the Km for P uptake was higher than typical soil concentrations of Pi, non-mycorrhizal poplars are expected to suffer from P limitations in most environments. (c) To study the purple acid phosphatases, transcriptome and proteome analyses were combined with phosphatase activity assays. The family of PAPs was annotated showing 33 poplar PAPs that formed three main subfamilies. Among these PAPs, 23 had a probe set on the microarray and showed significant transcript abundances. Ten PAPs were transcriptionally upregulated in roots and leaves of P-starved poplars. The P-starved poplars further showed higher phosphatase activity on the roots than the well P-supplied plants. The protein PtaPAP4 was secreted by poplar roots under high and low P conditions, whereas PtaPAP1 was secreted only under low P conditions. These results suggest that increased P acquisition from organic P sources under low P conditions is mediated in roots by a specific PAP enzyme. Overall, the results of this thesis support that enhanced phosphate transporter and phosphatase activity can improve P uptake efficiency. Since poplar plantations for biomass production are often established on marginal sites where nutrients are limited, the present findings suggest that the selection of natural genotypes or molecular breeding can be used to improve tree P nutrition

Data from: Dissecting nutrient-related co-expression networks in phosphate starved poplars

Phosphorus (P) is an essential plant nutrient, but its availability is often limited in soil. Here, we studied changes in the transcriptome and in nutrient element concentrations in leaves and roots of poplars (Populus × canescens) in response to P deficiency. P starvation resulted in decreased concentrations of S and major cations (K, Mg, Ca), in increased concentrations of N, Zn and Al, while C, Fe and Mn were only little affected. In roots and leaves >4,000 and >9,000 genes were differently expressed upon P starvation. These genes clustered in eleven co-expression modules of which seven were correlated with distinct elements in the plant tissues. One module (4.7% of all differentially expressed genes) was strongly correlated with changes in the P concentration in the plant. In this module the GO term “response to P starvation” was enriched with phosphoenolpyruvate carboxylase kinases, phosphatases and pyrophosphatases as well as regulatory domains such as SPX, but no phosphate transporters. The P-related module was also enriched in genes of the functional category “galactolipid synthesis”. Galactolipids substitute phospholipids in membranes under P limitation. Two modules, one correlated with C and N and the other with biomass, S and Mg, were connected with the P-related module by co-expression. In these modules GO terms indicating “DNA modification” and “cell division” as well as “defense” and “RNA modification” and “signaling” were enriched; they contained phosphate transporters. Bark storage proteins were among the most strongly upregulated genes in the growth-related module suggesting that N, which could not be used for growth, accumulated in typical storage compounds. In conclusion, weighted gene coexpression network analysis revealed a hierarchical structure of gene clusters, which separated phosphate starvation responses correlated with P tissue concentrations from other gene modules, which most likely represented transcriptional adjustments related to down-stream nutritional changes and stress

Data from: Dissecting nutrient-related co-expression networks in phosphate starved poplars

Phosphorus (P) is an essential plant nutrient, but its availability is often limited in soil. Here, we studied changes in the transcriptome and in nutrient element concentrations in leaves and roots of poplars (Populus × canescens) in response to P deficiency. P starvation resulted in decreased concentrations of S and major cations (K, Mg, Ca), in increased concentrations of N, Zn and Al, while C, Fe and Mn were only little affected. In roots and leaves >4,000 and >9,000 genes were differently expressed upon P starvation. These genes clustered in eleven co-expression modules of which seven were correlated with distinct elements in the plant tissues. One module (4.7% of all differentially expressed genes) was strongly correlated with changes in the P concentration in the plant. In this module the GO term “response to P starvation” was enriched with phosphoenolpyruvate carboxylase kinases, phosphatases and pyrophosphatases as well as regulatory domains such as SPX, but no phosphate transporters. The P-related module was also enriched in genes of the functional category “galactolipid synthesis”. Galactolipids substitute phospholipids in membranes under P limitation. Two modules, one correlated with C and N and the other with biomass, S and Mg, were connected with the P-related module by co-expression. In these modules GO terms indicating “DNA modification” and “cell division” as well as “defense” and “RNA modification” and “signaling” were enriched; they contained phosphate transporters. Bark storage proteins were among the most strongly upregulated genes in the growth-related module suggesting that N, which could not be used for growth, accumulated in typical storage compounds. In conclusion, weighted gene coexpression network analysis revealed a hierarchical structure of gene clusters, which separated phosphate starvation responses correlated with P tissue concentrations from other gene modules, which most likely represented transcriptional adjustments related to down-stream nutritional changes and stress

Correlation analysis of element concentrations.

<p>Correlation analysis of element concentrations.</p

Element concentrations in leaves, stem, coarse roots and fine roots of poplars.

<p><i>P</i>. × <i>canescens</i> was grown with high (HP, 641 μM, black), intermediate (MP, 6.4 μM, dark grey) or low (LP, 0.064 μM, light grey) P supply. Different letters indicate significant differences (p ≤ 0.05, Two-Way-ANOVA and Tukey's honest significance test, mean±SE, n = 4–10. P concentration and biomass were taken from Kavka and Polle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171958#pone.0171958.ref007" target="_blank">7</a>]. S: sulfur, C: carbon, N: nitrogen, K: potassium, Mg: magnesium, Ca: calcium, Fe: iron, Zn: zinc, Mn: manganese, Al: aluminum.</p

Hierarchy of enriched GO-terms of Biological Process in modules “Green”, “Blue” and “Brown”.

<p>Enriched GO terms are colored with module color, white: GO terms not enriched in modules. GO term hierarchy was drawn with GOPathDrawer. To enable presentation of all GO terms, the figure was split in three parts a), b) and c) and the connections between these parts were omitted.</p

Additional file 1: of Phosphate uptake kinetics and tissue-specific transporter expression profiles in poplar (Populus × canescens) at different phosphorus availabilities

Table S1. In silico analyses of putative poplar phosphate transporters; Table S2. Primers used for qRT PCR of putative P transporter genes; Table S3. Transcript abundances of phosphate transporter genes; Figure S1. Biomass and performance of poplar grown with five different P concentrations; Figure S2. Neighbor-Joining tree of the amino acid sequences for inorganic phosphate transporters in poplar; Figure S3. Correlations of absolute microarray expression data (log2-value) and qRT PCR relative expression values (log2) for PtPHTs. (PDF 1255 kb

ANOVA results for the main factors “Treatment” and “Tissue”.

<p>ANOVA results for the main factors “Treatment” and “Tissue”.</p

Co-expression within and between modules of DEGs.

<p>Due to computing power (about 12,000 nodes (= DEGs); about 45,000 edges (= co-expression between two DEGs) merely for module “Green”), only DEGs with p ≤ 0.00001 (limma) were drawn. Using a fold-change cut-off resulted in a similar network picture (not shown). Nodes (= DEGs of modules) are shown by their colors. Co-expression (Weighted Gene Co-expression Network Analysis) between two nodes is represented by grey line (edge). Higher adjacency between two DEGs is indicated by darker line color (adjacency threshold for edge drawing: 0.25). The reduced number of DEGs used to draw the network is indicated below module’s name.</p

Dissecting nutrient-related co-expression networks in phosphate starved poplars - Fig 6

<p><b>Change in transcript abundance of P-responsive genes in roots relative to that in leaves for the modules “Green” (a), “Brown” (b) and “Blue” (c).</b> Log₂-fold change of MP/HP and LP/HP DEGs in roots were plotted to the corresponding log₂-fold changes in leaves. Abbreviations of the genes names are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171958#pone.0171958.s004" target="_blank">S3 Table</a>.</p