Identification of phloem-specific proteinaSEOus structure heterogeneity in sieve element of Populus trichocarpa

Abstract

Phloem, an exceptional plant vascular tissue, facilitates the transport of photoassimilates, RNAs, and other signaling substances from the leaves to the roots throughout the plant. Among the specialized phloem cells are the conductive sieve elements (SEs), which are unique in that they remain alive despite lacking several cell organelles, including the nucleus, plastids, and most mitochondria. These SEs contain a specific proteinaceous structure composed of phloem-specific proteins (P-proteins), whose function is not yet fully understood. Various P-proteins have been characterized in broad range of model species, including Arabidopsis thaliana, and reported in Fabaceae and Cucurbitaceae plants. To date, only one P-protein has been identified in the model tree species Populus trichocarpa. Given the presence of multiple P-protein encoding genes across numerous plant species, we hypothesized the existence of multiple such genes in the Populus genome. Our genomic analysis uncovered 12 genes being potential orthologues to one of A. thaliana P-protein - SEOR (sieve element occlusion-related) genes, which may contribute to the proteinaceous structures observed in differentiating sieve elements. Our transcriptomic and proteomic analyses confirmed the expression of at least seven of these genes, indicating that the protein structure visible in mature sieve elements in P. trichocarpa may be heterogeneous.

Keywords: Populus trichocarpa; P-protein; Phloem; SEO; SEOR; Sieve element.

Fig. 1

Illustrative overview of the primary vascular tissue developmental stages in the pioneer root of P. trichocarpa: A – tangenial section; B transverse sections. The phloem and xylem are represented in a gradient of green and red shades respectively, and not drawn to scale. I – meristematic tissue, II – phloem precursors, III – maturing phloem and xylem precursors, IV – mature phloem and maturing xylem

Fig. 2

Sieve elements and P-proteins in the pioneer root of P. trichocarpa. An undifferentiated sieve element with electron-dense cytoplasm containing all organelles (corresponds to stadium II in Fig. 1). B Mature sieve element with electron-translucent cytoplasm, devoid of the nucleus and most other organelles (corresponds to stadium IV in Fig. 1). C-D P-proteins in different cross sections of the same sieve element (corresponds to stadium III in Fig. 1). E–F Regularly arranged bundles of P-protein filaments (corresponds to stadium III in Fig. 1). N – Nucleus, CW – Cell Wall, GA – Golgi Apparatus, scale bars = 500 nm

Fig. 3

Chromosomes of P. trichocarpa showing the locations of all identified SEOR encoding genes. Chromosomes harboring at least one SEOR gene are colored purple. Additionally, position of SEOR-encoding genes identified on chromosome 1 (pink) and 10 (green)

Fig. 4

Phylogenetic tree of SEO/SEOR genes from model species: P. trichocarpa (Potri), A. thaliana (At), Medicago truncatula (Medtr), Vicia faba (Vf), Pisum sativum (Ps), Cucumis sativus (Cucsa), and Malus domestica (MD) with identified conserved peptide motifs. Peptide motifs identified as fragments of SEO_N and SEO_C domains are fully colored and framed with a solid and dashed black line, respectively. Unidentified motifs are presented as partially transparent

Fig. 5

A UMAP visualization of 24 cell clusters in the P. trichocarpa root. Dots represent single nuclei with colors denoting clusters. B Expression pattern of known sieve element marker genes. C Expression pattern of identified SEOR genes across all clusters. Dot diameter indicates the proportion of cluster nuclei expressing the gene, and color denotes the average relative expression of a particular gene in each cluster

Fig. 6

Heatmap visualization of relative expression of P. trichocarpa SEOR genes in four root developmental stages (I-IV) using six pairs of primers specific for one or overlapping sequences of 2/6 genes, as detailed in Tab. 1. Relative quantification for each primer pair-reaction was normalized according to actin and ubiquitin gene expression. Pink indicates up-regulation relative to stage I, which represents the apical meristem (with no differentiating phloem cells), and green indicates down-regulation. Color intensity represents expression levels

Fig. 7

Normalized total spectral counts for identified SEOR proteins across root developmental stages I-IV, from three biological replicates, showing mean values ± standard error. Statistical analysis: one-way ANOVA with Tukey post-hoc test, p-value < 0.05