Volume 4, Issue 4, Pages 663-678 (July 2011) COPPER AMINE OXIDASE1 (CuAO1) of Arabidopsis thaliana Contributes to Abscisic Acid-and Polyamine-Induced Nitric Oxide Biosynthesis and Abscisic Acid Signal Transduction  Wimalasekera Rinukshi , Villar Corina , Begum Tahmina , Scherer Günther F.E.   Molecular Plant  Volume 4, Issue 4, Pages 663-678 (July 2011) DOI: 10.1093/mp/ssr023 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 Organ-Specific Expression Pattern of CuAO1. (A) Transcript abundance of CuAO1 in Columbia (Col) wild-type (WT) 3-day-old whole seedlings, 12-day-old whole seedlings, and in roots, leaves, and cotyledons of 12-day-old seedlings analyzed by real-time RT–PCR are presented as relative fold expression relative to 18S ribosomal RNA as internal standard. Data shown are means ± SE from three biological replications and three technical repeats in each. Significant difference P < 0.001 (***) based on Student's t-test with respect to 12-day-old whole seedlings. (B) Transcript abundance of CuAO1 in roots and shoot of Col (WT) 6-day-old seedlings. Expression levels of CuAO1 analyzed by real-time RT–PCR are presented as relative fold expression relative to 18S ribosomal RNA as internal standard. Data shown are means ± SE from three biological replications and three technical repeats in each. Significant difference P < 0.001 (***) based on Student's t-test with respect to roots of 6-day-old seedlings. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Identification of cuao1-1 and cuao1-2 T-DNA Insertional Knockouts. (A) Schematic representation of the positions of T-DNA insertions in cuao1-1 (SALK_019030.48.25.x) and cuao1-2 (RATM12-5261–1) knockouts. (B-I) Transcription analysis of CuAO1 in Col (WT), cuao1-1, No (WT), and cuao1-2 by RT–PCR using primers TZ1 and TZ2 specific for CuAO1 CDS. (B-II) Analysis of Col (WT), cuao1-1, No (WT), and cuao1-2 cDNA for the amplification of Actin 9/7 by RT–PCR as positive controls. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 PA-Induced NO Production in Col (WT), cuao1-1, No (WT), and cuao1-2 Seedlings. Quantification of putrescine (put) and spermidine (spd)-induced NO release in (A) Col (WT) and cuao1-1 (B) No (WT), and cuao1-2 by fluorometry. Relative changes in NO release were quantified using 2.5 μM DAF-2 in 1 mM put or 1 mM spd in incubation buffer for 30 min and mock-treated (control) 6-day-old Col (WT), cuao1-1, No (WT), and cuao1-2 seedlings. Changes in fluorescence by treatment with put and spd was calculated relative to fluorescence values of mock-treated (control) seedlings that were set as 100%. Means ± SE in three individual experiments are shown. Significant difference P < 0.001 (***) based on Student's t-test with respect to the relevant WT in each treatment. (C) Quantification of NO release induced by 1 mM put or spd from 6-day-old Col (WT) pre-treated by 200 μM cPTIO for 2 h (+cPTIO). Means ± SE in three individual experiments are shown. Significant differences determined with respect to the cPTIO-untreated values (–cPTIO) in each treatment P < 0.001 (***) based on Student's t-test. (D) Fluorescence microscopic images of PA-treated Col (WT), cuao1-1, No (WT), and cuao1-2 primary root tips. Seedlings of 6 d were pre-treated with 5 μM DAR-4M AM and then treated with 1 mM of put, spd for 3 h, or mock-treated and imaged by fluorescence microscope with identical settings for each image to be compared. Scale = 100 μM. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 Detection of S-Nitrosylated Proteins in Arabidopsis Seedling Extracts.(A) Protein extracts from Col (WT) seedlings mock-treated (control), treated with 500 μM GSNO (GSNO), 10 mM DTT (DTT), or protein extracts from seedlings mock-treated (control), treated with 1 mM putrescine (put), 1 mM spermidine (spd), and 1 mM spermine (spm) were subjected to the biotin switch assay, separated by SDS–PAGE, and immunoblotted with an anti-biotin antibody. Protein loading was verified by Ponceau S staining of the membrane. The relative masses of protein standards are shown (MW). (B) Protein extracts from Col (WT) seedlings mock-treated (control), pre-treated with 200 μM cPTIO, and mock-treated (control + cPTIO), treated with 1 mM spermidine (spd), pre-treated with 200 μM cPTIO, and treated with 1 mM spermidine (spd +cPTIO) were subjected to the biotin switch assay, separated by SDS–PAGE and immunoblotted with an anti-biotin antibody. Protein loading was verified by Ponceau S staining of the membrane. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 ABA-Induced NO Production in Col (WT), cuao1-1, No (WT), and cuao1-2 Seedlings. Quantification of ABA-induced NO release in (A) Col (WT) and cuao1-1 (B) No (WT), and cuao1-2 by fluorometry. Six-day-old seedlings were treated with 5 and 50 μM ABA for 30 min in incubation buffer and NO was quantified using DAF-2 (2.5 μM). Change in fluorescence by ABA treatment was calculated relative to fluorescence values of ABA mock-treated (control) seedlings, which were set as 100%. Means ± SE in three individual experiments are shown. Significant differences determined with respect to the relevant WT in each treatment P < 0.001 (***) based on Student's t-test. (C) Quantification of 50 μM ABA for 30 min induced NO release from 200 μM cPTIO pre-treated 6-day-old Col (WT). Means ± SE in three individual experiments are shown. Significant differences determined with respect to the cPTIO-untreated (–cPTIO) values in each treatment P < 0.001 (***) based on Student's t-test. (D) Fluorescence microscopic images of 50 μM ABA for 30 min treated or mock-treated (control) primary root tips of 6-day-old Col (WT), cuao1-1, No (WT), and cuao1-2, and imaged by fluorescence microscopy with identical settings for each image to be compared. Scale = 100 μM. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 ABA-Induced Expression of CuAO1. Transcript abundance of CuAO1 in 6-day-old (A) Columbia (Col) and (B) Nossen (No) WT seedlings treated with 50 μM ABA for 0, 1, 3, 6, and 8 h in 0.5 MS liquid medium. Fold expression levels of transcription analyzed by real-time RT–PCR are presented as relative to 18S ribosomal RNA and normalized with respect to the expression levels of ABA-untreated samples at time 0 h. Data shown are means ± SE from three biological replications and three technical repeats in each. Significant differences determined with respect to ABA-untreated seedlings at 0 h. P < 0.001(***) based on Student's t-test. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 PA-Induced H2O2 Production in Col (WT), cuao1-1, No (WT), and cuao1-2 Seedlings. Quantification of putrescine (put) and spermidine (spd)-induced H2O2 release in (A) Col (WT) and cuao1-1 (B) No (WT) and cuao1-2 by fluorometry. Relative changes in H2O2 release was quantified using Amplex Red, in 1 mM put or 1 mM spd in incubation buffer for 30 min and mock-treated (control) 6-day-old Col (WT), cuao1-1, No (WT), and cuao1-2 seedlings. Changes in fluorescence by put and spd treatment was calculated relative to fluorescence values of mock-treated (control) seedlings, which were set as 100%. Means ± SE in three individual experiments are shown. Significant difference P < 0.001(***) based on Student's t-test with respect to the relevant WT in each treatment. (C) Quantification of 1 mM put and 1 mM spd-induced H2O2 release from 200 units ml−1 catalase pre-treated Col (WT). Means ± SE in three individual experiments are shown. Significant differences determined with respect to the catalase-untreated (–catalase) values in each treatment. P < 0.01 (**) and P < 0.001 (***) based on Student's t-test. Fluorescence microscopic images of (D) 1 mM put-treated Col (WT), cuao1-1, No (WT), and cuao1-2 primary root tips. (E) 1 mM spd-treated Col (WT), cuao1-1, No (WT), and cuao1-2 primary root tips. Seedlings of 6 d were pre-treated with 40 μM DCFA-DA and then treated with 1 mM of put for 3 h and 1 mM spd for 1 h. Scale = 200 μM. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 ABA-Induced H2O2 Production in Col (WT), cuao1-1, No (WT), and cuao1-2 Seedlings. Quantification of ABA-induced H2O2 release by fluorometry. (A) Col(WT) and cuao1-1 (B) No(WT) and cuao1-2 6-day-old seedlings were treated with 5 and 50 μM ABA for 30 min in incubation buffer and H2O2 was quantified using Amplex red. Change in fluorescence by ABA treatment was calculated relative to fluorescence values of ABA mock-treated (control) seedlings, which were set as 100%. Means ± SE in three individual experiments are shown. Significant differences determined with respect to the relevant WT in each treatment. P < 0.05 (*) based on Student's t-test. (C) Quantification of 5 and 50 μM ABA 30 min induced H2O2 release from 200 units ml−1 catalase pre-treated Col (WT). Means ± SE in three individual experiments are shown. Significant differences determined with respect to the catalase-untreated (–catalase) values in each treatment. P < 0.05 (*) and P < 0.001 (***) based on Student's t-test. (D) Fluorescence microscopic images of primary root tips of 50 μM ABA-treated Col (WT), cuao1-1, No (WT), and cuao1-2 primary root tips. Col(WT) seedlings of 6 d were pre-treated with 40 μM DCFA-DA and then treated with 50 μM ABA for 3 h and imaged by fluorescence microscopy with identical settings for each image to be compared. Scale = 200 μM. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 9 Germination and Post-Germination Growth of Col (WT), cuao1-1, No (WT), and cuao1-2 Seedlings in ABA-Supplemented Growth Medium. (A) Percentage germination scored as radical emergence at day 4. (B) Percentage of seeds that developed green expanded cotyledons scored at day 12 of growth in 0.5 MS (1% agar) medium supplemented with 0, 0.5, 1.0, 1.5, 2.0, and 2.5 μM ABA. The values are the mean ± standard error in three independent experiments. (C) Post-germination growth at day 12 in MS medium supplemented with the indicated ABA concentrations. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 10 Root Phenotype of Col (WT), cuao1-1, No (WT), and cuao1-2 in ABA and Mannitol-Supplemented Growth Medium. Seedlings pre-grown for 3–4 d in B5 medium were transferred and grown for a further 8 d in 1:50 minimal B5 medium supplemented with 0.01, 0.1, and 0.5 μM ABA or transferred and grown for 12 d in 1:50 minimal B5 medium supplemented with 250 mM mannitol. Quantification of (A) primary root lengths, (B) lateral root numbers, (C) root phenotype of Col (WT), cuao1-1, No (WT), and cuao1-2 seedlings in 0, 0.01, 0.1, and 0.5 μM ABA after 8 d of growth. Quantification of (D) primary root lengths, (E) lateral root numbers in 250 mM mannitol-supplemented minimal B5 medium after 12 d of growth. (F) Root phenotype of No (WT) and cuao1-2 seedlings grown in minimal B5 medium supplemented with 250 mM mannitol. Mean values of 20–30 seedlings in two experiments and error bars represent ± SE. Significant differences determined relative to the respective WT and each treatment P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***) based on Student's t-test. Scale = 10 mm. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 11 Expression Profiles of ABA Biosynthetic and Stress-Responsive Genes in Col (WT), cuao1-1, No (WT), and cuao1-2 in Response to Exogenous ABA. Transcript abundance of (A) ABA1, (B) RD29A, and (C) ADH1 in 6-day-old seedlings grown in 0.5 MS liquid medium and mock-treated (–ABA) or with 50 μM ABA (+ABA) for 3 h were analyzed by real-time RT–PCR. Fold expression levels of transcription are presented as relative values that are normalized with respect to the expression in mock-treated plants using levels of 18S ribosomal RNA as internal standard. Data shown are means ± SE from three biological replications and three technical repeats each. Significant differences determined relative to the respective WT and each treatment P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***) based on Student's t-test. Molecular Plant 2011 4, 663-678DOI: (10.1093/mp/ssr023) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions