PIN FORMED 2 Modulates the Transport of Arsenite in Arabidopsis thaliana

Abstract

Arsenic contamination is a major environmental issue, as it may lead to serious health hazard. The reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals in Arabidopsis thaliana is strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Root transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and inductively coupled plasma mass spectrometry analysis revealed that pin2 plants accumulate higher concentrations of arsenite in roots compared with the wild-type. At the cellular level, arsenite specifically targets intracellular sorting of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 function results in arsenite hypersensitivity in roots. XFI coupled with XAS further revealed that loss of PIN2 function results in specific accumulation of arsenical species, but not the other metals such as iron, zinc, or calcium in the root tip. Collectively, these results suggest that PIN2 likely functions as an arsenite efflux transporter for the distribution of arsenical species in planta.

Keywords: PIN2; arsenite; auxin; trafficking; transport.

Figure 1

Effect of Arsenite on Root Elongation Response. Five-day-old light-grown wild-type or mutant seedlings were transferred to new agar plates supplemented with or without arsenite and incubated for various lengths of time under continuous light. (A) Time course of arsenite-induced inhibition of root elongation in wild type. (B) Dose response of arsenite for root elongation in wild-type after 3-day incubation. Approximately 50% inhibition of root growth was observed at 10 μM arsenite. (C) Representative images of root phenotype of wild-type, pin mutants, and pin2 complemented line after 10 μM arsenite treatment for 3 days. Scale Bar represents 0.5 cm. (D)pin2/eir1-4 mutant shows hypersensitive response to arsenite-induced root growth inhibition. Five-day-old Arabidopsis seedlings were subjected to arsenite treatment for 3 days. Compared with wild-type, eir1-4 showed hypersensitive response to arsenite-induced inhibition of root elongation at all concentrations we tested as judged by Student's t-test (P < 0.0001), while complemented line of pin2, eir1-4-PIN2:PIN2 show wild-type-like response to arsenite-induced root growth inhibition. For data shown in (A), (B), and (D), vertical bars represent mean ± SE of the experimental means from at least five independent experiments (n = 5 or more), where experimental means were obtained from 8–10 seedlings per experiment.

Figure 2

Arsenite Inhibits Root Gravity, Alters Intracellular Auxin Response, and Inhibits Auxin Transport. (A) Effect of Arsenite on root gravity response. For assaying gravitropism, 5-day-old light-grown seedlings were transferred to arsenite and gravistimuated. Data for root tip orientation were collected for 2, 4, 6, and 8 h. Vertical bars represent mean ± SE of the experimental means from at least five independent experiments (n = 5 or more), where experimental means were obtained from 8–10 seedlings per experiment. Arsenite-induced inhibition of root gravity response was significant at all time points as judged by Student's t-test (P < 0.0001). (B) Effect of arsenite on root elongation during the gravity assay. Arsenite-induced root growth inhibition was insignificant at all time points as judged by Student's t-test. (C) Arsenite alters the intracellular auxin response. Five-day-old light-grown IAA2-GUS seedlings were treated with 10 μM arsenite for 1 h and 2 h. After the arsenite treatment, GUS staining was performed by incubating the seedlings in GUS staining buffer for 1 h at 37°C. Images are representative of 15–20 roots obtained from at least three independent experiments. Scale bar represents 100 μm. (D) Quantification of GUS activity obtained from experiment in (C). Vertical bars represent mean ± SE. Compared with the control treatment, arsenite-induced increase in GUS activity was highly significant as judged by Student's t-test (P < 0.0001) at both time points as judged by Student's -test. (E) Effect of arsenite on shootward auxin transport. Five-day-old light-grown seedlings were transferred to new agar plates and subjected to arsenite treatment before transport of [³H]IAA over 2 h was measured as described in Methods. The experiments were conducted using at least three biological replicates. For each biological replicate, three technical replicates were assayed. (Col-control, n = 57; Col-arsenite, n = 52). Asterisks represent the statistical significance between treatment as judged by Student's t-test (***P < 0.0001). Vertical bars represent mean ± SE of the experimental means.

Figure 3

Arsenite Specifically Affects the Intracellular Dynamic Cycling of PIN2. Five-day-old PIN2::PIN2-GFP, PIN1::PIN1-GFP, and EGFP-LTI6b transgenic seedlings were treated with arsenite for 2 h. After the incubation, seedlings were treated with 20 μM BFA for 40 min. The images were captured using same confocal setting and are representative of 15–20 roots obtained from at least four independent experiments. (A) Effect of arsenite on PIN2 trafficking. Scale bar represents 10 μm. (B) Quantitative analysis of formation of PIN2-BFA body in the transition zone of PIN2::PIN2-GFP transgenic plants in presence or absence of arsenite. Total number of BFA bodies and number of cells were counted in the imaged area. Bar graph represents the average number of BFA bodies formed per cell. Vertical bars represent mean ± SE of the experimental means (n = 4 or more). Asterisks represent the statistical significance between treatments as judged by Student's t-test (***P < 0.0001). (C and D) Effect of arsenite on PIN1 (C) and LTI6b (D) trafficking. Note that BFA bodies are formed in the presence of arsenite. Arrowheads indicate BFA bodies. Scale bar represents 10 μm.

Figure 4

pin2/eir1-1 Shows Altered Transport and Accumulation of Arsenite. (A and B) Allocation of ^74,73As in Col-0 and eir1-1(A). Five-day-old Col-0 and eir1-1 roots were incubated in 0.1 μM and 10 μM ^74,73As for 2 h. ^74,73As radiation of the whole root was captured by an imaging plate. Images are representative of three independent experiments. (B) Quantification of As allocation in the whole root from the experiment in (A). The data were obtained from three independent experiments with 10 seedling roots in each treatment. Vertical bars mean ± SE. Asterisks represent the statistical significance between treatments as judged by Student's t-test: **P < 0.001. (C) Scintillation counting of ^74,73As activity in Col-0 and eir1-1. Five-day-old Col-0 and eir1-1 seedlings were incubated for 2 h at 0.1, 10 and 100 μM ^74,73 As. Whole root was collected after the incubation. ^74,73 As activity of the whole root was measured by liquid scintillation counting. The data were obtained from 10 individual roots for each treatment. Vertical bars represent mean ± SD. Asterisks represent the statistical significance between treatments as judged by Student's t-test: ***P < 0.0001. (D) Arsenic content in Col-0 and eir1-1. Five-day-old light-grown Col-0 and eir1-1 seedlings were transferred to 10 μM and 100 μM arsenite solution and incubated for 2 h. A 5-mm root tip of 20 seedlings for each treatment was used to measure As by ICP-MS. The data were obtained from three independent experiments. Vertical bars represent mean ± SE. Asterisks represent the statistical significance between treatments as judged by Student's t-test: **P < 0.001 and ***P < 0.0001.

Figure 5

As Accumulation and Distribution in Arabidopsis Roots Exposed to 10 μM Arsenite are Influenced by PIN2. (A) Combined XFI As and potassium (K) elemental distributions in whole Arabidopsis plants. As is denoted by red and K by green, with brighter colors corresponding to higher concentrations. As (and K) intensities are on a common scale for two specimens. The samples were scanned with 35-μm steps at beamline 10-2 (SSRL). Spatial scale bar represents 3.5 mm. (B) The images of roots shown in (A) are magnified by 1.4 times to show differences in the As distribution in the apical part in the root meristem in Col-0 and eir1-1/pin2. (C) High-resolution As XFI maps of the apical root meristem demonstrate higher accumulation of As in eir1-1/pin2 and less concentrated and more dispersed distribution in Col-0. Arsenic intensity is on a common scale for two specimens. Brighter colors correspond to higher concentrations. The circular markers denote spatial points that were selected for collection of As micro-XAS in roots. The samples were scanned with 2-μm steps at beamline 2-3 (SSRL). Spatial scale bar represents 100 μm. (D) Micro-XAS As-K near-edge spectra collected at the points of the apical root meristem in Col-0 (marked by thin dotted lines) and eir1-1/pin2 roots as labeled in (C). The spectra are normalized by intensity of the incident radiation but otherwise are not processed. Apart from eir1-1/pin2 #7 (marked by small red empty circles), micro-XAS As-K spectra in eir1-1/pin2 are much more intense compared with Col-0. (E) Same near-edge XAS As-K spectra as in (D), collected in the root points shown in (C), with background removed, normalized by the intensity of the incident radiation and the edge jump. All these spectra show a high similarity to As(III)-thiolated species, best represented by As(GS)3 standard. (F) High-resolution XFI As areal density distributions in hydrated root specimens of Arabidopsis collected at beamline 2-ID-E (APS) with spatial resolution 1 μm × 1 μm.