An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis

Abstract

Phytochelatins (PCs) are peptides that function in heavy-metal chelation and detoxification in plants and fungi. A recent study showed that PCs have the ability to undergo long-distance transport in a root-to-shoot direction in transgenic Arabidopsis (Arabidopsis thaliana). To determine whether long-distance transport of PCs can occur in the opposite direction, from shoots to roots, the wheat (Triticum aestivum) PC synthase (TaPCS1) gene was expressed under the control of a shoot-specific promoter (CAB2) in an Arabidopsis PC-deficient mutant, cad1-3 (CAB2TaPCS1/cad1-3). Analyses demonstrated that TaPCS1 is expressed only in shoots and that CAB2TaPCS1/cad1-3 lines complement the cadmium (Cd) and arsenic metal sensitivity of cad1-3 shoots. CAB2TaPCS1/cad1-3 plants exhibited higher Cd accumulation in roots and lower Cd accumulation in shoots compared to wild type. Fluorescence HPLC coupled to mass spectrometry analyses directly detected PC2 in the roots of CAB2:TaPCS1/cad1-3 but not in cad1-3 controls, suggesting that PC2 is transported over long distances in the shoot-to-root direction. In addition, wild-type shoot tissues were grafted onto PC synthase cad1-3 atpcs2-1 double loss-of-function mutant root tissues. An Arabidopsis grafting technique for mature plants was modified to obtain an 84% success rate, significantly greater than a previous rate of approximately 11%. Fluorescence HPLC-mass spectrometry showed the presence of PC2, PC3, and PC4 in the root tissue of grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots, demonstrating that PCs are transported over long distances from shoots to roots in Arabidopsis.

Figure 1.

Expression of TaPCS1 mRNA is targeted to the shoots of CAB2∷TaPCS1/cad1-3 plants. A, Northern blots probing TaPCS1 expression in wild type (Col-0), three independent lines of CAB2∷TaPCS1/cad1-3 (C-1, C-2, C-3), and 35S∷TaPCS1/cad1-3 (35s). Actin was used as a loading control (n = 2). B, Fifty cycles of RT-PCR show lack of any TaPCS1 expression in roots and strong TaPCS1 expression in shoots. RT-PCR was performed with TaPCS1∷c-myc fusion-specific primers. Actin7 primers were used as a loading control (n = 2).

Figure 2.

CAB2∷TaPCS1 expression complements the Cd and As sensitivity of cad1-3 in shoots. For each image, seeds of four different lines were germinated as diagrammed in A: top left, cad1-3 (atpcs1); top right, CAB2∷TaPCS1 (CAB2∷TaPCS1/cad1-3); bottom left, 35S∷TaPCS1(35S∷TaPCS1/cad1-3); bottom right, wild type (Col-0 ecotype). All seeds were germinated and grown on 25% Murashige and Skoog medium for 14 d on medium containing no heavy metal as a control (B), 80 μm As (C), or 40 μm Cd (D).

Figure 3.

CAB2∷TaPCS1 expression in shoots of cad1-3 does not recover the wild-type Cd-sensitive root growth. Wild type (WT), three CAB2∷TaPCS1/cad1-3 lines (C-1, C-2, C-3), and cad1-3 seedlings were germinated on 25% Murashige and Skoog medium with no added Cd for 5 d and then transferred to plates containing 20 μm CdCl₂ for 3 d. Root length presented is a measure of new root growth after the transfer of seedlings to plates containing 20 μm CdCl₂. Data show mean values ± sem; n = 60 plants per plant line.

Figure 4.

PCs detected in shoot tissue and PC2 in root tissues of CAB2∷TaPCS1/cad1-3 plants. Four-week-old plants grown in hydroponic conditions were exposed to 20 μm CdCl₂, and PCs in shoot and root tissue extracts were labeled with monobromobimane (shoot tissue extracts left column [A, C, and E]; root tissue extracts right column [B, D, and F]). PC2, PC3, and PC4, indicated by arrows, were detected by fluorescence HPLC and compared to synthesized standards (G and H). Note that the positions of arrows indicating PC peaks were calibrated using PC standard control experiments after every fifth sample. The slight shifts in PC peak retention times from experiment to experiment are due to the normal changes in the properties of the HPLC column over time. PC standards in G and H are identical and shown twice for visual analysis of all traces. E and F, cad1-3 served as negative controls with a magnified y axis, and wild type (Col-0; A and B) served as positive control. n = 13 to 14 plants were analyzed for each of the three CAB2∷TaPCS1/cad1-3 lines.

Figure 5.

MS run concurrently with fluorescence HPLC confirms HPLC peaks as PCs in root tissues. A to D, Data from root samples. PC2 conjugated with two monobromobimane molecules showed a value of 920 m/z (z = ion charge) as indicated by arrows. A, Synthesized PC2 standard. B, Root sample from wild type (Col-0). C, Root sample from CAB2∷TaPCS1/cad1-3. D, Root sample from cad1-3.

Figure 6.

Cd overaccumulation in roots and reduced accumulation in shoots of CAB2∷TaPCS1/cad1-3 and cad1-3 plants. Wild type (WT; Col-0), three independent lines of CAB2∷TaPCS1/cad1-3 (C-1, C-2, C-3), and cad1-3 were grown under hydroponic conditions and exposed to 20 μm CdCl₂ for 4 d. Cd²⁺ accumulation in shoot and root tissues was determined by ICP-OES. Data show mean values ± sem; n = 9 plants per line.

Figure 7.

Grafting of mature Arabidopsis plants. A, Diagram of grafting technique. The top portion illustrates preparation of a portion of the graft, labeled as scion, not containing the root system in which a horizontal transverse cut was made in the stem directly above the cotyledons (see “Materials and Methods”). The bottom portion illustrates preparation of the stock, the portion of the graft containing the root system in which a transverse cut was made in the stem directly above the first set of rosette leaves and the subsequent removal of the cotyledons and lowest rosette leaves. Scion and stock were secured together with a steel pin. B, Grafted mature Arabidopsis plant at day 1. Arrow points to grafting junction between the shoot and root tissue. Graft junction did not come into direct contact with hydroponic sponge (bottom). See “Materials and Methods” for details.

Figure 8.

PCs are transported from shoots to roots in grafts between wild-type shoots and cad1-3 atpcs2-1 double-mutant roots. Plant were grown under hydroponic conditions and tissues extracted for PC analyses prepared 7 d postgrafting, which included a 3-d exposure to 20 μm CdCl₂ (shoot tissue extracts left column [A, C, E, and G]; root tissue extracts right column [B, D, F, and H]). PCs were labeled with monobromobimane and detected by fluorescence HPLC. A and B, Grafts between wild-type (Ws × Col-0 F2) shoots and roots (n = 7). C and D, Grafts between wild-type (Ws × Col-0 F2 individuals) shoots and cad1-3 atpcs2-1 roots (n = 12). Note that the y axes are not identical and amplified in D and F. E and F, Grafts between cad1-3 atpcs2-1 shoots and roots (n = 9). G and H, Synthesized PC standards. Note that the positions of arrows indicating PC peaks were calibrated using PC standard control experiments after every fifth sample. The slight shifts in PC peak retention times from experiment to experiment are due to the normal changes in the properties of the HPLC column over time. G and H are identical traces and shown twice to facilitate visualization above fluorescence HPLC traces. y axes represent millivolts (mV), and x axes represent retention time (min).

Figure 9.

MS run concurrently with fluorescence HPLC confirms PCs in root samples. A to F, Data from root samples. A, B, and C, Raw MS data from root samples from plants with wild-type shoots grafted to cad1-3 atpcs2-1 roots. D to F, Data from root samples from plants with cad1-3 atpcs2-1 shoots grafted to cad1-3 atpcs2-1 roots. G to I, PC standards. PC2 at the +1 ion state was 920 m/z (z = ion charge), PC3 at the +2 ion state was 672 m/z, and PC4 at the +2 ion state was 883 m/z as indicated by arrows. y axis represents relative abundance percentage and x axis represents mass per ion charge (m/z) as labeled.