The nitrate transporter (NRT) gene family in poplar

Abstract

Nitrate is an important nutrient required for plant growth. It also acts as a signal regulating plant development. Nitrate is actively taken up and transported by nitrate transporters (NRT), which form a large family with many members and distinct functions. In contrast to Arabidopsis and rice there is little information about the NRT family in woody plants such as Populus. In this study, a comprehensive analysis of the Populus NRT family was performed. Sixty-eight PtNRT1/PTR, 6 PtNRT2, and 5 PtNRT3 genes were identified in the P. trichocarpa genome. Phylogenetic analysis confirmed that the genes of the NRT family are divided into three clades: NRT1/PTR with four subclades, NRT2, and NRT3. Topological analysis indicated that all members of PtNRT1/PTR and PtNRT2 have 8 to 12 trans-membrane domains, whereas the PtNRT3 proteins have no or up to two trans-membrane domains. Four PtNRT3 members were predicted as secreted proteins. Microarray analyses revealed tissue-specific expression patterns of PtNRT genes with distinct clusters of NRTs for roots, for the elongation zone of the apical stem segment and the developing xylem and a further cluster for leaves, bark and wood. A comparison of different poplar species (P. trichocarpa, P. tremula, P. euphratica, P. fremontii x P. angustifolia, and P. x canescens) showed that the tissue-specific patterns of the NRT genes varied to some extent with species. Bioinformatic analysis of putative cis-regulatory elements in the promoter regions of PtNRT family retrieved motifs suggesting the regulation of the NRT genes by N metabolism, by energy and carbon metabolism, and by phytohormones and stress. Multivariate analysis suggested that the combination and abundance of motifs in distinct promoters may lead to tissue-specificity. Our genome wide analysis of the PtNRT genes provides a valuable basis for functional analysis towards understanding the role of nitrate transporters for tree growth.

Figure 1. Overview on the phylogeny of the NRT families in P. trichocarpa and A. thaliana.

Detailed information of each clade is presented in figure 2. ClustalX2 was used to perform the alignment of the amino acids and to calculate the final tree with the neighbor-joining method and a bootstrap value of n = 1,000. The tree was displayed using MEGA 5.

Figure 2. Expanded view of the phylogenetic relationships of the genes in the NRT families of P. trichocarpa and A. thaliana.

A. subclade NRT1a. B. subclade NRT1b. C. subclade NRT1c. D. subclade NRT1d. E. clade NRT2 and NRT3. A–D represent together clade NRT1/PTR. ClustalX2 was used for the alignment of the amino acids and to calculate the final tree with the neighbor-joining method and a bootstrap value of n = 1,000. The tree was displayed using MEGA 5.

Figure 3. Expression profiles of NRT family genes in Populus across different tissues.

The heatmap represents the hierarchical clustering of average log₂(signal intensity) of Populus NRT genes in various tissues. T, stem top; DX, developing xylem; L, leaves; W, wood; B, bark; R, roots. The numbers refer to different experiments compiled in Table S2. The color of the gene name represents the clade in the phylogenetic tree (Figure 1). The Affymetrix microarray data were obtained from ArrayExpression database (http://www.ebi.ac.uk/arrayexpress) and their accession number are shown in Table S2.

Figure 4. Comparison of tissue-specific expression patterns of NRT genes in P. trichocarpa (Ptri) and P. tremula (Ptre).

The heatmap represents the hierarchical clustering of average log₂(relative expression) of NRT genes in stem top (T), bark (B), developing xylem (DX), leaves (L), wood (W), and roots (R). The color of the gene name represents the clade in the phylogenetic tree (Figure 1).

Figure 5. cis-regulatory elements of NRT genes in P. trichocarpa.

A. Overlapping CREs of NRT genes in leaves, bark, wood, and roots. The diagram shows the number of CREs in PtNRT genes expressed in specific tissues. The Venn diagram was drawn using VENNY (http://bioinfogp.cnb.csic.es/tools/venny/). B. Nonmetric multidimensional scaling (NMDS) ordination of CREs abundance and tissue specific expression of PtNRT genes. T, stem top; DX, developing xylem; L, leaves; W, wood; B, bark; R, roots. The data matrix used for the NMDS analyses is shown in Table S3.