Volume 8, Issue 5, Pages 722-733 (May 2015) Arabidopsis NIP3;1 Plays an Important Role in Arsenic Uptake and Root-to-Shoot Translocation under Arsenite Stress Conditions  Wenzhong Xu, Wentao Dai, Huili Yan, Sheng Li, Hongling Shen, Yanshan Chen, Hua Xu, Yangyang Sun, Zhenyan He, Mi Ma  Molecular Plant  Volume 8, Issue 5, Pages 722-733 (May 2015) DOI: 10.1016/j.molp.2015.01.005 Copyright © 2015 The Author Terms and Conditions

Figure 1 Enhanced Aboveground Tolerance to Arsenite of NIP3;1 T-DNA-Insertion Lines. (A) Structure of the NIP3;1 genomic region and schematic representation of the T-DNA insertion sites (nip3;1-1, nip3;1-2, nip3;1-3, nip3;1-4, and nip3;1-5). Boxes and lines represent exons and introns, respectively. The positions of the T-DNA inserts are represented by triangles. (B) RT–PCR did not detect intact NIP3;1 mRNA in nip3;1-1, nip3;1-2, nip3;1-3, nip3;1-4, or nip3;1-5 mutant plants. (C and D) Phenotype (C) and shoot fresh weight (D) of Ws-4 wild-type and nip3;1-1 mutant plants grown for 2 weeks on half-strength MS agar plates without or with various arsenite concentrations. (E and F) Phenotype (E) and shoot fresh weight (F) of the Col-0 wild-type, nip3;1-2, nip3;1-3, nip3;1-4, and nip3;1-5 plants grown for 2 weeks on half-strength MS agar plates without or with various arsenite concentrations. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). Molecular Plant 2015 8, 722-733DOI: (10.1016/j.molp.2015.01.005) Copyright © 2015 The Author Terms and Conditions

Figure 2 Expression of NIP3;1 Under the NIP1;1 Promoter in nip1;1 Mutants Restores Arsenite Sensitivity. (A) Phenotypes of Col-0 wild-type plants, and representative transgenic lines of NIP1;1pro::NIP3;1-5, -8 and the NIP1;1-knockout mutant nip1;1 in nip1;1 plants grown on half-strength MS agar plates without (left) or with 20 μM arsenite (right) for 7 d. (B) Root length was measured after transfer to plates without or with 20 μM arsenite on the seventh day. (C) RT–PCR analysis of the full-length NIP1;1 mRNA or the fused mRNA of NIP1;1 first exon and NIP3;1 in wild-type Col-0, NIP1;1pro::NIP3;1 transformants (L5 and L8), and nip1;1. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). Molecular Plant 2015 8, 722-733DOI: (10.1016/j.molp.2015.01.005) Copyright © 2015 The Author Terms and Conditions

Figure 3 The nip3;1 Insertion Lines Show Decreased As Translocation to the Shoots. (A and B) As contents in shoots (A) and roots (B) of nip3;1-1 and corresponding wild-type Ws-4 plants under arsenite conditions. (C and D) As contents in shoots (C) and roots (D) of nip3;1-2, nip3;1-3, nip3;1-4, nip3;1-5, and corresponding wild-type Col-0 plants under arsenite conditions. Plants were grown on half-strength MS agar plates for 14 d and transferred to filter paper soaked with 40 μM arsenite solution for 24–48 h. Shoots (A and C) and roots (B and D) were harvested at 24 or 48 h as indicated, and As concentrations in the tissues were determined. Data are means ± SEs of n = 6 (>50 plants per sample). Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). FW, fresh weight. Molecular Plant 2015 8, 722-733DOI: (10.1016/j.molp.2015.01.005) Copyright © 2015 The Author Terms and Conditions

Figure 4 NIP3;1 Expression Pattern at the Tissue Level. (A) RT–PCR analysis of NIP3;1 mRNA levels in the wild-type plants under normal growth conditions. Wild-type plants were grown on half-strength MS agar plates for 2 weeks, and total RNAs isolated from shoot and root tissues were analyzed using 30 cycles of RT–PCR for NIP3;1. The bottom panel shows the ACTIN2 control (25 cycles). (B–I) GUS staining in NIP3;1 promoter-GUS transgenic plants grown on half-strength MS agar plates for 7 d (B–G, and I) and 14 d (H). Whole plant (B and H), root tip (C and G), root–hypocotyl junction (D and F), leaf primordia (E), and a cross section of the root hair zone (I) are shown. For the high-magnification views of tissues (E–G and I), GUS staining seedlings were observed with a differential interference discrepancy contrast microscope system. Molecular Plant 2015 8, 722-733DOI: (10.1016/j.molp.2015.01.005) Copyright © 2015 The Author Terms and Conditions

Figure 5 nip3;1 nip1;1 Double Mutant Exhibited Multiple Phenotypes for Arsenite Tolerance. (A) RT–PCR did not detect intact NIP3;1 mRNA or NIP1;1 mRNA in nip3;1 nip1;1 double-knockout mutants or their corresponding single-knockout mutants. (B–E) Wild-type (Col-0), nip3;1 single-knockout mutant (nip3;1-4), nip3;1 nip1;1 double-knockout mutant, and nip1;1 single-knockout mutant (nip1;1) plants grown on half-strength MS agar plates in the presence or absence of various added arsenite concentrations for 7–14 d. The experiment was performed in triplicate, and all results showed a similar pattern. The representative phenotype (B) and root length (C) of the plants was determined after 7 d. The representative phenotype (D) and fresh weight (E) of plants was determined after 14 d. For (C) and (E), values are the means ± SEs of nine plants (n = 3). Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). Molecular Plant 2015 8, 722-733DOI: (10.1016/j.molp.2015.01.005) Copyright © 2015 The Author Terms and Conditions

Figure 6 As Accumulation in Double and Single Mutants Under Arsenite Conditions. (A and B) As contents in shoots (A) and roots (B) of Col-0 (wild-type), nip3;1-4, nip3;1 nip1;1, and nip1;1 plants. Two-week-old plants grown on half-strength MS agar plates were transferred to filter paper soaked with 40 μM arsenite solution for 24–48 h. Shoots (A) and roots (B) were harvested at 24 or 48 h as indicated, and the As content in the tissues was determined. Mean values with different letters denote significant differences according to multiple comparisons (P < 0.05). FW, fresh weight. Molecular Plant 2015 8, 722-733DOI: (10.1016/j.molp.2015.01.005) Copyright © 2015 The Author Terms and Conditions

Figure 7 Functional Analysis of NIP3;1 Expression in Yeast. (A) Growth of the Δfps1 yeast mutant expressing either the empty vector (EV), full-length NIP3;1 (NIP3;1), or N-/C-terminally truncated versions of NIP3;1 (NIP3;1Δ2–29, NIP3;1Δ266–323, or NIP3;1Δ2–29, Δ266–323). Δfps1 has high tolerance toward arsenite due to the lack of arsenite influx aquaglyceroporin protein Fps1p. (B) The Δfps1 mutant transformed with the empty vector (EV), or plasmids expressing the NIP3;1 versions were cultured in SG liquid medium for 2 d and then exposed to 500 μM sodium arsenite after 30 and 60 min. The As contents in yeast cells were measured. The values shown represent the means ± SEs of at least four independent experiments. DW, dry weight. Asterisks indicate significant differences compared with the empty vector transformants under 500 μM arsenite exposure at the indicated time points according to t-test (P < 0.05). (C) Growth of the Δacr3 yeast mutant expressing either the empty vector or plasmids expressing the indicated proteins. After entering yeast cells via phosphate transporters, arsenate is rapidly reduced to arsenite, and Δacr3 is sensitive to arsenate due to the lack of the main arsenite-exfflux transporter ACR3 in yeast. Yeast was grown on SG-agar medium with various concentrations of As(III) (A) or As(V) (C) for 5 d. Molecular Plant 2015 8, 722-733DOI: (10.1016/j.molp.2015.01.005) Copyright © 2015 The Author Terms and Conditions