Volume 3, Issue 3, Pages 610-625 (May 2010) Plant Phosphatidylcholine-Hydrolyzing Phospholipases C NPC3 and NPC4 with Roles in Root Development and Brassinolide Signaling in Arabidopsis thaliana  Wimalasekera Rinukshi , Pejchar Přemysl , Holk André , Martinec Jan , Scherer Günther F.E.   Molecular Plant  Volume 3, Issue 3, Pages 610-625 (May 2010) DOI: 10.1093/mp/ssq005 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Phylogenetic Relationship, Gene Structure, and Amino Acid Sequence Alignment of Putative PC-PLC (NPC) in Arabidopsis. (A) Phylogenetic tree of bacterial PC-PLC proteins of Mycobacterium tuberculosis (accession no. P95246), Burkholderia pseudomallei (accession no. Q9RGS8), Burkholderia thailandensis (accession no. YP_439303), Pseudomonas aeruginosa (accession no. P15713), and Xanthomonas campestris (accession no. YP365022), a fungal hypothetical protein related to phosphoesterase family Aspergillus nidulans (accession no. XP_681815), a phosphoesterase family protein of a protozoan Tetrahymena thermophilia (accession no. EAR87012), phosphoesterase family proteins of Oryza sativa NP1 (accession no. ABF99945), NP2 (accession no. XP_463753), NP3 (accession no. BAB92134), and NP4 (accession no. ABA94565), and putative PC-PLC proteins of Arabidopsis NPC1 (accession no. NP_172203), NPC2 (accession no. NP_180255), NPC3 (accession no. NP_187002), NPC4 (accession no. NP_566206), NPC5 (accession no. NP_566207), and NPC6 (accession no. NP_190430). The phylogenetic tree was produced by the program ClustalW and displayed by the program TREEVIEW. (B) Gene structure of the putative PC-PLC family (NPC) in Arabidopsis. NPC1 (At1g07230), NPC2 (At2g26870), NPC3 (At3g03520), NPC4 (At3g03530), NPC5 (At3g03540), and NPC6 (At3g48610). Exons are symbolized by white boxes and introns by black lines. Gray boxes indicate the 3’ and 5’ untranslated regions. (C) Sequence alignment of N-termini of PC-PLC proteins from bacteria (Burkholderia pseudomallei, Pseudomonas aeruginosa, Mycobacterium tuberculosis) and putative PC-PLC (NPC) of Arabidopsis NPC1, NPC2, NPC3, NPC4, NPC5, and NPC6. Color code: blue, basic; red, acidic; green, polar; black, apolar amino acids: light blue, cystein. Predicted cleavage sites of the signal peptides (http://www.cbs.dtu.dk/services/SignalP-3.0/) in NPC1, NPC2, and NPC6 are indicated by black arrows. (D) Sequence alignment of C-termini of NPC proteins of Arabidopsis. The amino acids forming potential zinc finger-like motifs (C and/or H) are underlined. Color code: blue, basic; red, acidic; green, polar; black, apolar amino acids. Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 RT–PCR Analysis of NPC Expression in Arabidopsis thaliana Col. (WT). (A) Organ-specific expression of NPC genes. Total RNA isolated from root, stem, leaf, flower, and silique of 30-day-old in vitro-grown plants reverse transcribed and PCR (35 cycles) was performed with NPC gene-specific oligonucleotides. Actin was used as a standard. (B) NPC expression in response to exogenous IAA, BL, and zeatin application. Twelve-day-old seedlings were treated with 10 μM IAA and 0.1 μM BL for 24 h. Seedlings were grown in 5 μM zeatin-supplemented AM for 14 d. Total RNA was isolated from whole seedlings, reverse transcribed, and PCR was performed with NPC gene-specific and actin oligonucleotides in the same PCR (for IAA and BL treatments, 30 cycles; for zeatin treatment NPC1, 2, 6–30 cycles; for NPC 3, 4, 5–32 cycles). (C) NPC4 expression in response to different auxin types. Twelve-day-old seedlings were treated with 10 μM of IAA, 1-NAA, and 2,4-D for 0, 1, 3, 6, and 24 h. Total RNA was isolated from whole seedlings, reverse transcribed, and PCR was done with NPC4-specific and actin oligonucleotides together in the same PCR (30 cycles). (D) NPC4 expression in response to zeatin. Twelve-day-old seedlings were treated with 5 μM zeatin for 1 and 6 h. Total RNA was isolated from whole seedlings, reverse transcribed, and PCR was done with NPC4-specific and ACTIN2/7 oligonucleotides in the same PCR (32 cycles). (E) NPC expression in response to phosphate deficiency. Seedlings were grown for 10 d in 0.5 AM either lacking Pi (–Pi) or containing 1.25 mM Pi (+Pi). Total RNA was isolated from whole plants, reverse transcribed, and PCR was done with NPC-specific and ACTIN2/7 oligonucleotides in the same PCR (30 cycles). (F) NPC4 expression in response to phosphate deficiency. Seedlings were grown as in (E) and total RNA was isolated from shoot and root separately. PCR was done with NPC4-specific and ACTIN2/7 oligonucleotides in the same PCR (30 cycles). Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Histochemical Analysis of PNPC3:GUS and PNPC4:GUS Expression during Development of Arabidopsis thaliana. a PNPC3:GUS 30-day-old adult plant. b PNPC4:GUS 30-day-old adult plant. c PNPC3:GUS leaf of 30-day-old plant. d PNPC4:GUS leaf of 30-day-old plant. e PNPC3:GUS primary root tip of a 14-day-old seedling. f PNPC4:GUS primary root tip of a 14-day-old seedling. g PNPC3:GUS young flower. h PNPC4:GUS young flower. i PNPC3:GUS mature flower. j PNPC4:GUS mature flower. k PNPC3:GUS young anther. l PNPC4:GUS young anther. m PNPC4:GUS pollen sac tissues of young anther. n PNPC3:GUS mature anther. o PNPC4:GUS mature anther p PNPC4:GUS pollen grains. q PNPC4:GUS silique r PNPC4:GUS developing seed. GUS assay was done by incubating the plants in 2 mM X-Gluc buffer at 37°C for 16 h. Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Histochemical GUS Assay of Induced and Non-Induced PNPC3:GUS and PNPC4:GUS Seedlings of Arabidopsis thaliana. (A) PNPC3:GUS 12-day-old seedlings treated with or without 10 μM 1-NAA for 24 h. Aa: primary root tip, Ac: lateral root tip, Ae: hypocotyl, Ag: apical region and young leaves of 0 μM 1-NAA controls. Ab: primary root tip, Ad: lateral root tip, Af: hypocotyl, Ah: apical region and young leaves treated with 10 μM 1-NAA. (B) PNPC4:GUS 12-day-old seedlings were treated with or without 10 μM 1-NAA for 24 h. Ba: primary root tip, Bc: lateral root tip, Be: hypocotyl, Bg: apical region and young leaves of 0 μM 1-NAA controls. Bb: primary root tip, Bd: lateral root tip, Bf: hypocotyl, Bh: apical region and young leaves treated with 10 μM 1-NAA. (C) PNPC4:GUS 12-day-old seedlings were treated with or without 5 μM zeatin for 48 h. Ca: primary root tip, Cc: lateral root tip, Ce: apical region and young leaves of 0 μM zeatin controls. Cb: primary root tip, Cd: lateral root tip, Cf: apical region and young leaves treated with 5 μM zeatin. (D) PNPC3:GUS 12-day-old seedlings were treated with or without 0.1 μM BL for 48 h. Da: primary root tip, Dc: cotyledon, De: leaf, Dg: apical region and young leaves of 0 μM BL controls. Db: primary root tip, Dd: cotyledon, Df: leaf, Dh: apical region and young leaves treated with 0.1 μM BL. (E) PNPC4:GUS 12-day-old seedlings were treated with or without 0.1 μM BL for 48 h. Ea: primary root tip, Ec: cotyledon, Ee: leaf, Eg: apical region and young leaves of 0 μM BL controls. Eb: primary root tip, Ed: cotyledon, Ef: leaf, Eh: apical region and young leaves treated with 0.1 μM BL. (F) PNPC4:GUS seedlings were grown for 10 d in AM either lacking Pi (–Pi) or containing 1.25 mM Pi (+Pi). Fa: primary root tip, Fc: lateral root primodium, Fe and Fg: developing lateral roots of +Pi-grown plants. Fb: primary root tip, Fd: lateral root primodium, Ff and Fh: developing lateral roots of –Pi-grown plants. GUS assays for all the treatments were done by incubating the seedlings in 2 mM X-Gluc buffer at 37°C for 16 h. Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Effect of Phosphate Deficiency, 1-NAA and BL on Root Phenotype of npc3-1 and npc3-2 Knockouts. Arabidopsis thaliana Col. wild-type (WT), npc3-1 and npc3-2 seedlings pre-grown for 3 d in Pi-containing (+Pi) Arabidopsis medium (AM) were transferred and grown for a further 7 d in a, b: +Pi AM, c, d: Pi lacking (–Pi) AM, e, f: +Pi AM supplemented with 0.03 μM of 1-NAA, g, h: +Pi AM supplemented with 0.05 μM BL. Quantification of i: primary root lengths and j: lateral root densities in +Pi AM and –Pi AM, k: primary root lengths and l: lateral root densities in 0, 0.01, 0.03, 0.1, 0.3, and 1.0 μM 1-NAA-supplemented +Pi AM, m: primary root lengths and n: lateral root densities in 0, 0.001, 0.005, 0.01, 0.05, and 1.0 μM BL-supplemented +Pi AM. Root quantification was done after 7 d of growth. Columns indicate mean quantification of 30–60 seedlings of two to three experiments and error bars represent ± SEM. Asterisks indicate a statistically significant (P < 0.05) difference in comparison to WT of similar treatment according to Student's t-test. Scale bars = 5.0 mm. Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 Effect of Phosphate Deficiency, 1-NAA, and BL on Root Phenotype of npc4-1 and npc4-2 Knockouts. Arabidopsis thaliana Col. wild-type (WT), npc4-1, and npc4-2 seedlings pre-grown for 3 d in Pi-containing (+Pi) Arabidopsis medium (AM) were transferred and grown for a further 7 d in a, b: +Pi AM, c, d: Pi-lacking (–Pi) AM, e, f: +Pi AM supplemented with 0.03 μM of 1-NAA, g, h: +Pi AM supplemented with 0.05 μM BL. Quantification of i: primary root lengths and j: lateral root densities in +Pi AM and –Pi AM, k: primary root lengths and l: lateral root densities in 0, 0.01, 0.03, 0.1, 0.3, and 1.0 μM 1-NAA-supplemented +Pi AM, m: primary root lengths and n: lateral root densities in 0, 0.001, 0.005, 0.01, 0.05, and 1.0 μM BL-supplemented +Pi AM. Root quantification was done after 7 d of growth. Columns indicate mean quantification of 30–60 seedlings of two to three experiments and error bars represent ± SEM. Asterisks indicate a statistically significant (P < 0.05) difference in comparison to WT of similar treatment according to Student's t-test. Scale bars = 5.0 mm. Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 7 Transcription of 1-NAA-Activated and BL-Activated Genes in WT, npc3-1, and npc4-1. Transcript abundance of (A) IAA11, (B) IAA19, and (C) IAA20. Eight-day-old WT, npc3-1, and npc4-1 seedlings were treated with no 1-NAA (-NAA) or 1 μM 1-NAA for 1 h (+NAA). Transcript abundance of (D) CPD (CYP90A1), (E) CYP90D, (F) TCH4 (xyloglucan endotransglycosylase4), (G) LRX2 (LEUCINE RICH REPEAT/EXTENSIN1). Eight-day-old WT, npc3-1, and npc4-1 seedlings were treated with no BL (–BL) or 50 nM BL for 6 h (+BL). Transcript abundance was determined by real time RT–PCR. Fold expression levels of transcription are presented as relative values that are normalized with respect to the levels of 18S ribosomal RNA. Data shown are means ± SE from three biological replications and three technical repeats each (Livak and Schmittgen, 2001). In (A), (D), (E), (F), and (G), –NAA and –BL treatments ± SE bars are smaller to be noticeable. Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 8 Concentration-Dependent and Time-Dependent Stimulation of Diacylglycerol (DAG) Accumulation in BL-Treated Tobacco BY-2 Cells. (A) Thin layer plate showing increased accumulation of DAG in response to 1, 10, and 100 nM BL treatment for 24 h. (B) Quantification of accumulation of BODIPY-labeled DAG in tobacco BY-2 cells treated with 1, 10, and 100 nm BL for 24 h. (C) Quantification of accumulation of BODIPY-labeled DAG in 1 μM BL-treated and BL-untreated tobacco BY-2 cells after 15, 30, and 60 min. Molecular Plant 2010 3, 610-625DOI: (10.1093/mp/ssq005) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions