Effects of Cadmium on Root Morpho-Physiology of Durum Wheat

Abstract

Durum wheat [Triticum turgidum L. subsp. durum (Desf.) Husn.] can accumulate a high level of Cd in grains with a significant variability depending on cultivars. Understanding how this toxic element is distributed in cereal tissues and grains is essential to improve the nutritional quality of cereal-based products. The main objective of this work was to investigate roots of durum wheat plants (cv. Iride) exposed to different Cd concentrations (0.5 and 5.0 μM) to identify the mechanisms involved in Cd management. Results showed that the root morphology was altered by Cd treatment both at macroscopic (increased number of tips and primary root length) and ultrastructural levels (cell membrane system damaged, cell walls thickened and enriched in suberin). On the other side, Cd was localized in vesicles and in cell walls, and the metal colocalized with the phytosiderophore nicotianamine (NA). Overall, data suggest that Cd is chelated by NA and then compartmentalized, through vesicular trafficking, in the root thickened walls reducing Cd translocation to the aerial organs of the plant.

Keywords: Triticum durum; cadmium; heavy metal; phytosiderophore; vesicular trafficking.

Figure 1

Effect of exogenous Cd on the morphology of roots from seedlings grown in hydroponics for 15 days (starting from sprouting) in the presence of Cd 0.5 and 5.0 μM. Panel (A) Representative pictures of roots grown in absence or presence of Cd; panel (B) the same root systems of panel A tracked by the image analysis tool Root System Analyzer; panel (C) tips total number; panel (D) primary root length; and panel (E) root average diameter. T-test p < 0.01**.

Figure 2

Scanning electron microscope (SEM) microphotographs of root cross-sections obtained from plants grown for 15 days (starting from sprouting) in absence or presence of Cd (0.5 or 5.0 μM). Panels (A) and (B) Root cross-sections in the control roots; panels (C) and (D) root cross-sections of plants grown in the presence of Cd 0.5 μM; panels (E) and (F) root cross-sections of plants grown in the presence of Cd 5.0 μM. en: endodermis; pe: pericycle; xy: xylem. Red arrows indicate pericycle and xylem cell wall thickening. (G), cell wall thickness in the xylem vessels. Eight independent roots were assessed for each treatment (12 cross-sections for root). T-test p < 0.001***.

Figure 3

Cadmium in grains, shoots and roots of seedlings grown for 15 days (starting from sprouting) in control conditions and in the presence of Cd 0.5 and 5.0 μM. Panels (A) and (B) Cd concentrations in μg/g DW (dry weight) of grains, shoots and roots. To appreciate the differences between the two treatments (Cd 0.5 and 5.0 μM) in grains, a third panel was added: panel (C) data of Cd concentration in grains.

Figure 4

Mineral elements in roots and shoots of wheat seedlings grown for 15 days (starting from sprouting) in control conditions and in presence of Cd 0.5 and 5.0 μM. Panel (A) Mn, Fe, Zn, and Cu concentrations expressed as μg/g DW of root. Panel (B) Mn, Fe, Zn, and Cu concentrations as μg/g DW of shoot. T-test p < 0.05*, <0.01**, and <0.001***.

Figure 5

Cd accumulation in roots of the wheat (15 days from sprouting and from the start of Cd treatments) visualized by fluorescent imaging using Leadmium Green AM dye. Green fluorescence represents the binding of the dye to Cd. Panel (A) Control roots, a faint but not bright signal is present. Panel (B) Schematic overview of wheat root anatomy. Panel (C) Roots grown in presence of Cd 0.5 μM, fluorescence is localized in rhizodermis and endodermis (white arrows). Panel (D) Detail at higher magnification of the rhizodermis area corresponding to red circle of panel C. Panel (E) Roots grown in presence of Cd 5.0 μM, fluorescence is also distributed in the cell wall of the parenchyma cells of the cortical region (white arrows). Panel (F) Detail at higher magnification of the corresponding to the red circle of panel E.

Figure 6

Transmission electron micrographs showing toxic effects of Cd on ultrastructure of roots (15 days from sprouting and from the start of Cd treatments). Panel (A) Control roots; panels (B–D) toxic effects observed in most of root cortical cells treated with Cd. CW, cell wall; G, Golgi apparatus; N, nucleus; ER, endoplasmic reticulum; V, vacuole; and M, mitochondria.

Figure 7

Transmission electron micrographs of root cortical cells (15 days from sprouting and from the start of Cd treatments). Panel (A) Control roots; panel (B) roots treated with Cd 0.5 μM; panel (C) roots treated with Cd 5.0 μM.

Figure 8

Immuno-localization of nicotianamine (NA) in roots of wheat plants grown for 15 days (starting from sprouting) in control conditions and with Cd 0.5 μM and 5.0 μM; Auto: root tissues autofluorescence; LG: root cross-sections stained with Leadmium Green AM; Anti_NA: root cross-sections labeled with antibodies directed against NA/fluorescent secondary antibodies; merge: images of green (LG) and red (Anti_NA) channels merged; white arrows indicated points of the red and green pixels colocalization. Scale bars = 50 μm.

Figure 9

Colocalization of Cd (labeled with LG dye) and nicotianamine (labeled with anti_NA and fluorescent secondary antibodies) in root cross-sections (15 days from sprouting and from the start of Cd treatments). Panel (A) Representative “crosshair” scatterplots of green (LG) and red (Anti_NA) pixels intensity and colocalization in rhizodermis, parenchyma cells and xylem. Panel (B) Evaluation of green (LG) and red (Anti_NA) pixels colocalization by the Mander’s overlap coefficient. Data were analyzed by ANOVA followed by Tukey’s post hoc test. Different letters correspond to statistically different means.

Figure 10

Expression analysis of genes coding for transporters in roots (15 days from sprouting and from the start of Cd treatments). ZIF1 (zinc-induced facilitator 1), ZIFL1 (zinc-induced facilitator-like 1), ZIFL2 (zinc-induced facilitator-like 2), ZTP29 (zinc transporter 29), IREG2 (iron regulated 2), ABC27 (ATP-binding cassette 27), VIT (vacuolar iron transporter), HMA5 (heavy metal atpase 5), NRAMP (Natural resistance-associated macrophage protein metal ion transporter 2), YSL1 (yellow stripe like 1), YSL2 (yellow stripe like 2), HMT1 (heavy metal tolerance 1). Red lines highlight the fold changes 2 and − 2 to graphically identify the genes differentially expressed.

Figure 11

Expression analysis of genes coding for enzymes involved in suberin biosynthesis in Iride roots (15 days from sprouting and from the start of Cd treatments). GPAT5 (glycerol-3-phosphate acyl transferase 5), CYP861 (cytochrome P450), DAISY (docosanoic acid synthase), GSO2 (LRR receptor-like serine/threonine-protein kinase), ABCG1 (ATP Binding Cassette Subfamily G Member 1), ASFT (Aliphatic Suberin Feruloyl Transferase). Red lines highlight the fold changes 2 and − 2 to graphically identify the genes differentially expressed.

Figure 12

Expression analysis of genes coding for nicotianamine synthases (NASs) and enzymes involved in the pathway of Yang Cycle in roots (15 days from sprouting and from the start of Cd treatments). NAS2 (nicotianamine synthase 2), NAS3 (nicotianamine synthase 3) and NAS4 (nicotianamine synthase 4), ARD1 (acireductone dioxygenase 1), TAT (tyrosine transaminase 1), MAT (S-adenosyl-L-methionine synthase), MTN2 (methylthioadenosine nucleosidase 2), MTK (S-methyl-5-thioribose kinase), MTI (5-methylthioribose kinase 1), DEP (dehydratase/enolase/phosphatase), NAAT (nicotianamine aminotransferase). Red lines highlight the fold changes 2 and − 2 to graphically identify the genes differentially expressed.

Figure 13

Expression analysis of genes coding for proteins involved in the vesicular trafficking in roots (15 days from sprouting and from the start of Cd treatments). RAB1 (Ras-related in brain 1), RAB2 (Ras-related in brain 2), COPß1 (coat protein ß1), COPϒ (coat protein ϒ), EXO70F1 (exocyst component of 70 kDa F1), EXO70E1 (exocyst component of 70 kDa E1), EXO70H6 (exocyst component of 70 kDa H6), SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor), SNARE13 (soluble N-ethylmaleimide– sensitive factor attachment receptor 13), DYN1A (dynamin 1A), DYN1C (dynamin 1C), DYN2 (dynamin 2), DYN1 (dynamin 1), DYN2b (dynamin 2b), ENA1 (nicotianamine exporter 1). Red lines highlight the fold changes 2 and − 2 to graphically identify the genes differentially expressed.

Figure 14

Suberin in roots visualized by fluorescent imaging (staining with Fluorol Yellow 088). Panel (A) Yellow fluorescence in the images represents the binding of the dye to suberin; control: a faint but not bright signal was observed; Cd 0.5 μM: fluorescence is localized in rhizodermis, endodermis and vascular cylinder (red arrows); Cd 5.0 μM: fluorescence is also localized in the cell wall of the parenchyma cells of the cortical region (red arrows). Panel (B) Quantification of the relative intensity of the fluorescence signals. T-test value of p < 0.001***.