Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis

Abstract

Phytochelatin synthases (PCS) mediate cellular heavy-metal resistance in plants, fungi, and worms. However, phytochelatins (PCs) are generally considered to function as intracellular heavy-metal detoxification mechanisms, and whether long-distance transport of PCs occurs during heavy-metal detoxification remains unknown. Here, wheat TaPCS1 cDNA expression was either targeted to Arabidopsis roots with the Arabidopsis alcohol dehydrogenase (Adh) promoter (Adh::TaPCS1/cad1-3) or ectopically expressed with the cauliflower mosaic virus 35S promoter (35S::TaPCS1/cad1-3) in the PC-deficient mutant cad1-3. Adh::TaPCS1/cad1-3 and 35S::TaPCS1/cad1-3 complemented the cadmium, mercury, and arsenic sensitivities of the cad1-3 mutant. Northern blot, RT-PCR, and Western blot analyses showed Adh promoter-driven TaPCS1 expression only in roots and thus demonstrated lack of long-distance TaPCS1 mRNA and protein transport in plants. Fluorescence HPLC analyses showed that under Cd2+ stress, no PCs were detectable in cad1-3. However, in Adh::TaPCS1/cad1-3 plants, PCs were detected in roots and in rosette leaves and stems. Inductively coupled plasma atomic emission spectrometer analyses showed that either root-specific or ectopic expression of TaPCS1 significantly enhanced long-distance Cd2+ transport into stems and rosette leaves. Unexpectedly, transgenic expression of TaPCS1 reduced Cd2+ accumulation in roots compared with cad1-3. The reduced Cd2+ accumulation in roots and enhanced root-to-shoot Cd2+ transport in transgenic plants were abrogated by l-buthionine sulfoximine. The presented findings show that (i) transgenic expression of TaPCS1 suppresses the heavy-metal sensitivity of cad1-3, (ii) PCs can be transported from roots to shoots, and (iii) transgenic expression of the TaPCS1 gene increases long-distance root-to-shoot Cd2+ transport and reduces Cd2+ accumulation in roots.

Fig. 1.

TaPCS1 mRNA and protein are not translocated from roots to shoots. (A) Northern blots probing AtPCS1 expression in WT (Col-0) (row 1) and TaPCS1 expression using the root promoter in Adh/cad1-3 (Adh::TaPCS1/cad1-3) (row 2) and the ectopic promoter in 35S/cad1-3 (35S::TaPCS1/cad1-3) (row 3). 18S rRNA was used as loading control (row 4). (B) RT-PCR was performed with TaPCS1::myc fusion-specific primers. Actin was used as control. (C) Protein was extracted from stems (S), rosette leaves (L), and roots (R) of WT (Col-0), Adh/cad1-3 (Adh::TaPCS1/cad1-3), and 35S/cad1-3 (35S::TaPCS1/cad1-3) plants. Western blot analyses were performed as described in Materials and Methods. WT protein extracts were used as negative controls.

Fig. 2.

TaPCS1 complements Cd²⁺, Hg²⁺, and As sensitivity in cad1-3 at seedling stage. For all panels: Upper Left, cad1-3; Upper Right, Adh/cad1-3 (Adh::TaPCS1/cad1-3); Lower Left, 35S/cad1-3 (35S::TaPCS1/cad1-3); and Lower Right, WT (Col-0). Seeds were plated and horizontally germinated on one-quarter-strength Murashige and Skoog medium with no added heavy metals (A), 80 μM KH₂AsO₄ (B), 10 μM HgCl₂ (C), and 40 μM CdCl₂ (D). Photographs were taken after 21 days.

Fig. 3.

Heavy-metal tolerance in cad1-3 was restored completely or partially by ectopic or root-specific expression of TaPCS1. Four-week-old hydroponically grown cad1-3 mutant, Adh/cad1-3 (Adh::TaPCS1/cad1-3), 35S/cad1-3 (35S::TaPCS1/cad1-3), and WT plants were exposed to 20 μM CdCl₂ for 3 days. Phenotypes were assessed for roots and rosette leaves (A) and stems (B and C). (B) Blue arrows show sites sensitive to Cd²⁺ stress in Adh::TaPCS1/cad1-3.

Fig. 4.

PCs can be transported from roots to shoots. Four-week-old plants were exposed to 20 μM CdCl₂ for 3 days in hydroponic media, and PCs were extracted from root, leaf (rosette), and stem tissues of cad1-3 (A), WT Col-0 (B), 35S/cad1-3 (35S::TaPCS1/cad1-3)(C), and Adh/cad1-3 (Adh::TaPCS1/cad1-3)(D). PC levels were measured by fluorescence HPLC. (E) Synthesized PC₂, PC₃, and PC₄ (2 μM each) PC standards were measured, and three copies of standard measurements are illustrated for easy comparison to the measured samples. PC₂, PC₃, and PC₄ peaks are indicated by arrows.

Fig. 5.

Enhanced long-distance Cd²⁺ transport and reduced Cd²⁺ accumulation in roots by transgenic expression of TaPCS1. Four-week-old 35S/cad1-3 (35S::TaPCS1/cad1-3), Adh/cad1-3 (Adh::TaPCS1/cad1-3), WT (WT Col-0), and cad1-3 plants grown in hydroponic solution were exposed to 20 μM CdCl₂. Samples were harvested at timed intervals after Cd²⁺ exposure as indicated. Cd²⁺ accumulation in stems (A), rosette leaves (B), and roots (C) was determined by ICP-AES. Data show mean values ± SE, n = 6 plants.

Fig. 6.

Short-term BSO exposure mimics the effect of AtPCS1 gene disruption in cad1-3 with respect to Cd²⁺ accumulation in roots and shoots. Four-week-old cad1-3, Adh/cad1-3 (Adh::TaPCS1/cad1-3), 35S/cad1-3 (35S::TaPCS1/cad1-3), and WT plants grown in hydroponic solution were pretreated with 0 mM/1 mM BSO for 12 h; 20 μM CdCl₂ was then applied to either the untreated plants (A) or the 1 mM BSO pretreated plants with 0.5 mM BSO for 54 h (B). Cd²⁺ accumulation in stems (S), rosette leaves (L), and roots (R) was determined by ICP-AES. Data show mean values ± SE, n = 6 and 3 plants in A and B, respectively.