A transcriptomic network underlies microstructural and physiological responses to cadmium in Populus x canescens

Abstract

Bark tissue of Populus × canescens can hyperaccumulate cadmium, but microstructural, transcriptomic, and physiological response mechanisms are poorly understood. Histochemical assays, transmission electron microscopic observations, energy-dispersive x-ray microanalysis, and transcriptomic and physiological analyses have been performed to enhance our understanding of cadmium accumulation and detoxification in P. × canescens. Cadmium was allocated to the phloem of the bark, and subcellular cadmium compartmentalization occurred mainly in vacuoles of phloem cells. Transcripts involved in microstructural alteration, changes in nutrition and primary metabolism, and stimulation of stress responses showed significantly differential expression in the bark of P. × canescens exposed to cadmium. About 48% of the differentially regulated transcripts formed a coregulation network in which 43 hub genes played a central role both in cross talk among distinct biological processes and in coordinating the transcriptomic regulation in the bark of P. × canescens in response to cadmium. The cadmium transcriptome in the bark of P. × canescens was mirrored by physiological readouts. Cadmium accumulation led to decreased total nitrogen, phosphorus, and calcium and increased sulfur in the bark. Cadmium inhibited photosynthesis, resulting in decreased carbohydrate levels. Cadmium induced oxidative stress and antioxidants, including free proline, soluble phenolics, ascorbate, and thiol compounds. These results suggest that orchestrated microstructural, transcriptomic, and physiological regulation may sustain cadmium hyperaccumulation in P. × canescens bark and provide new insights into engineering woody plants for phytoremediation.

Figure 1.

Cadmium concentrations in the bark, root, and leaf tissues of P. × canescens exposed to 0 (blank bars) or 200 µm (filled bars) CdSO₄ for 20 d. Bars indicate means ± se (n = 6). Asterisks on the bars for the same tissue indicate significant differences between treatments. [See online article for color version of this figure.]

Figure 2.

Cadmium localization in the bark (A and B), root (C–F), and leaf tissues (G and H) of P. × canescens exposed to 0 (A, C, E, and G) or 200 µm (B, D, F, and H) CdSO₄ for 20 d. Arrows point to precipitates of cadmium-dithizone. B, Cadmium-dithizone precipitates in the bark. D, Cadmium-dithizone precipitates in epidermal cells of fine roots. F, Cadmium-dithizone precipitates in intercellular spaces, cell walls, and cytoplasm of cortical cells. H, Cadmium-dithizone precipitates in leaf veins and in intercellular spaces of mesophyll cells. col, Collenchyma; cor, cortex; pf, phloem fiber; pr, phloem ray; ph, phloem; cam, cambium; v, vessel; xf, xylem fiber; xyl, xylem; cc, cortical cells; lv, leaf vein; m, mesophyll cells. [See online article for color version of this figure.]

Figure 3.

Transmission electron microscopic micrographs of granular deposits in vacuoles of phloem cells (A and B) and companion cells (C–H) in the bark of P. × canescens exposed to 0 (A, C, E, and G) or 200 µm (B, D, F, and H) CdSO₄ for 20 d. The insert in B highlights a granular deposit in a vacuole. Arrows point to cadmium deposits. Cadmium concentrations in granular deposits are shown in Figure 4A. The EDX spectra of vacuoles (A) and granular deposits in vacuoles (B) are shown in Supplemental Figure S2. CW, Cell wall; PM, plasma membrane; N, nucleus; V, vacuole; Ch, chloroplast; Gr, granum; SG, starch grain; M, mitochondrion; ER, endoplasmic reticulum.

Figure 4.

EDX analysis determined cadmium concentrations (A) and the correlation between cadmium and sulfur (B) in granules of different cell compartments of P. × canescens exposed to 0 (blank bars) or 200 µm (filled bars) CdSO₄ for 20 d. Bars indicate means ± se (n = 3). Asterisks on bars for the same cell compartment indicate significant differences between treatments. The data for correlation analysis were based on cadmium and sulfur concentrations in granules of all examined subcellular compartments. Transmission electron microscopic micrographs of cadmium-deposited granules in subcellular compartments of roots and leaves are shown in Supplemental Figure S3. pc.vac, Vacuoles of phloem cells; epi.ito, inside tonoplast of epidermal cells; cc.cw, cell walls of cortical cells; cc.vac, vacuoles of cortical cells; mc.vac, vacuoles of mesophyll cells. [See online article for color version of this figure.]

Figure 5.

Number of genes assigned to each category by MapMan analysis. Detailed information for each category is presented in Supplemental Table S3. [See online article for color version of this figure.]

Figure 6.

Transcripts involved in stress (A) and metabolism (B) assigned by MapMan in the bark of P. × canescens exposed to 0 or 200 µm CdSO₄ for 20 d. Positive fold change values (red) indicate up-regulation, whereas negative fold change values (blue) denote down-regulation. Color saturates at 5-fold change. Each square represents a differentially expressed transcript. [See online article for color version of this figure.]

Figure 7.

The coexpression network of significantly differentially expressed genes (A), the subnetwork of hub genes (B), and the GO term enrichment of hub genes (C) in the bark of P. × canescens exposed to 0 or 200 μm CdSO₄ for 20 d. The network was generated by setting a cutoff value of 0.15 for Kolmogorov-Smirnov quality-control statistics and an r² value of 0.36, which gives reliable results of coexpressed genes in CressExpress as suggested by Wei et al. (2006). An edge indicates the coexpression between two genes. Triangle nodes represent genes from the category of RNA regulation, diamond nodes represent genes from the signaling category, and circle nodes represent genes from other categories. Red and blue nodes represent up- and down-regulated genes, respectively. The presence of each node in the network and the hub genes (≥20 edges) are indicated in Supplemental Table S3. [See online article for color version of this figure.]

Figure 8.

PCA plots of nutrients (A), soluble sugars and sugar alcohols (B), and oxidants and antioxidants (C) in the bark, fine roots, and leaves of P. × canescens exposed to 0 or 200 μm CdSO₄. Pink indicates 0 μm CdSO₄, and blue indicates 200 μm CdSO₄. PCA was conducted based on data (both values were averaged in the same tissue with the same treatment) presented in Supplemental Table S5 (A), Supplemental Table S6 (B), and Supplemental Figures S4 to S6 (C). [See online article for color version of this figure.]