Supplemental Data. Wang et al. (2017). Plant Cell /tpc NIP1;2 NIP7;1

Transcription

1 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc NIP1;1 NIP1;2 NIP2;1 NIP3;1 NIP4;1 NIP5;1 NIP6;1 NIP7;1 Supplemental Figure 1. Distinct Localization of NIPs in Root Epidermal Cells. (Supports Figure 1.) rabidopsis NIPs N-terminally tagged with GFP were expressed in root epidermal cells under the control of the NIP5;1 promoter in a nip5;1-1 background. Epidermal cells in longitudinal optical sections of roots are shown. Bar = 2 µm. 1

2 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc YFP and mcherry-npsn12 YFP mcherry Merged stele-side soil-side YFP/mCherry soil-side YFP/mCherry stele-side = 1.15 ±.13 (Mean ± SD, n = 3 epidermal cells from 3 roots.) B a mcherry-npsn12 b Fluorescence intensity Soil-side Profile plot of line a Stele-side Soil-side Profile plot of line b Stele-side C PIP2;1-mCherry a b Fluorescence intensity Soil-side Stele-side Soil-side Stele-side Supplemental Figure 2. Weakly Polar Localization of NPSN12 and PIP2;1 in Root Epidermal Cells. (Supports Figure 1.) () n optical longitudinal section of a root transition zone of a transgenic plant harboing ProUBQ1:YFP-NPSN12 and ProUBQ1:mCherry-NPSN12 (a F1 plant of WVE131Y x WVE131R, Geldner et al., 29). Bar = 2 µm. (B) and (C) Cross sections of roots of ProUBQ1:mCherry-NPSN12 (B) and Pro35S:PIP2;1-mCherry (C) plants. Profile plots along the lines indicated in the image are shown. Note that the peaks at the stele-side of the epidermal cells include the signal at the soil-side of the cortical cells. Excitation and detection wavelengths were 488 nm and nm for YFP and 552 nm and nm for mcherry. Bars = 5 µm. 2

3 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc t-nip7; MNGERSRV--VDQEGS--TPSTLRDEDHPSRQR LFGCLPYDIDLN 43 t-nip5;1 MPPEEVGVMVMPPTPGTPGT-PGGPLITGMRVDSMS----FDHRKPTPRCKCLPVMGSTWGQHDT----CFTDFPSPDVS 75 t-nip6; MDHEEIPSTPSTPTTPGT-PGPLFGGFEGKRNGHNGRYTPKSLLKSCKCFSVD-NEWLEDGRLPPVTCSLPPPNVS 77 t-nip4; MSS HSDEI-E-EEQISRIEKGKGKDCQG------G------IETVICTSPSIVC 4 t-nip3; MEISDITT---QTQTVVLDIENY QS--IDDSR SSDLSPLVSVS 4 t-nip2; MDDISVSKS--NHGNVVVLNIKSS-L-----DTSLPSNK HESSSPPLLSVH 45 t-nip1; MDISGNGYGNREEVVMVNLKDEV-EHQQEMEDIHNPRPL KKQDSLLSVSVP 52 t-nip1; MEISGNG-GDRDGVVVNLKEED-EQQQQQQIH--KPL KKQDSLLSISVP 49 : Nt t-nip7;1 PLRIVMELVGTFILMFSVCGVISSTQLSGGHVGLLEYVTGLSVVVVVYSIGHISGHLNPSITIFVFGGFPWSQVPLYI 127 t-nip5;1 LTRKLGEFVGTFILIFTTGPIVNQKYDGETLIGNCGLVMIIILSTGHISGHLNPSLTIFLRHFPWHVPYI 159 t-nip6;1 LYRKLGEFVGTLILIFGTTIVNQKTDGETLIGCSGLVMIVILSTGHISGHLNPVTIFLKHFPWKHVPVYI 161 t-nip4;1 LTQKLIEMIGTYFIVFSGCGVVVVNVLYGGTITFPGICVTWGLIVMVMIYSTGHISGHFNPVTVTFIFRRFPWHQVPLYI 124 t-nip3;1 FVQKLIGEFVGTFTMIFGCSIVVNETYGKPVTLPGILVWGLVVTVMIYSIGHVSGHFNPVSIFSSKKFPFNQVPGYI 124 t-nip2;1 FLQKLLELVGTYYLIFGCIVNQHNHVVTLVGIVVWGIVIMVLVYCLGHLS-HFNPVTLLSSQRFPLNQVPYI 128 t-nip1;1 FLQKLIEFLGTYFLVFTGCSVVVNMQNDNVVTLPGIIVWGLTIMVLIYSLGHISGHINPVTIFSCGRFPLKQVPYV 136 t-nip1;2 FLQKLMEVLGTYFLIFGCVVNTQHDKVTLPGIIVWGLTVMVLVYSLGHISGHFNPVTIFSCGRFPLKQVPYV 133 : :.*.:** ::*:.. :. *: : :::. **:* **:**::::::* ** :** *: t-nip7;1 TQTLGTTLVGVSVYGVNDI MTKPLSCVSFFVELITSIVVFLSLHCGPHQNLGNLTGFVIGTVISL 22 t-nip5;1 QVSSICSFLKGVFHPFMS---G-----GVTIPSVSLGQFLEFIITFILLFVVTVT-DTRVGELGIVGTVML 234 t-nip6;1 GQVMSVSFLKVFEPTMS---G-----GVTVPTVGLSQFLEFIISFNLMFVVTVT-DTRVGELGIVGTVML 236 t-nip4;1 GQFGSLLSLTLRLMFKVTP EFFGTTPDSPRLVEIIISFLLMFVISGVT-DNRVGELGIVGMTIMV 2 t-nip3;1 QLLGSTLVLRLVFHLDDDVCSLKGDVYVGTYPSNSNTTSFVMEFITFNLMFVISVT-DKRTGSFGIIGTIVL 24 t-nip2;1 TVQVIGSTLSTLRLLFDLNNDVCSKKHDVFLGSSPSGSDLQFVMEFIITGFLMLVVCVTT-TKRTTEELEGLIIGTVTL 211 t-nip1;1 ISQVIGSTLTLRLLFGLDHDVCSGKHDVFIGSSPVGSDLQFTMEFIVTFYLMFIISGVT-DNRIGELGLIGSTVLL 219 t-nip1;2 ISQVIGSTLTLRLLFGLDQDVCSGKHDVFVGTLPSGSNLQSFVIEFIITFYLMFVISGVT-DNRIGELGLVGSTVLL 216 *.: *:. :: : *. :: *:* : ::::.: :.: *: :*.: : t-nip7;1 GVLITGPISGGSMNPRSLGPVVWDFEDLWIYMTPVIGIIGVLTYRSISLKTRPCPSPVSPSVSSLLR t-nip5;1 NILVGPSTGGSMNPVRTLGPVSGNYRSLWVYLVPTLGISGVYTGVKLNDSVTDPPRPVR--SFRR t-nip6;1 NILIGPTSSMNPVRTLGPINNYRIWVYLTPILGLIGGTYTIVKLPEEDE-PKERR--SFRR t-nip4;1 NVFVGPISGSMNPRSLGPLVMGVYKHIWVYIVGPVLGVISGGFVYNLIRFTDKPLRELTKS--SFLRVSPSHKGSSSK 282 t-nip3;1 DILFSGPISGSMNPRSLGPLIWGCYKDLWLYIVSPVIGLSGWTYGLLRSTKKSYSEIIRPN--CNKVSSRDRQESQDE 289 t-nip2;1 NVIFGEVSGSMNPRSIGPLVWGCYKGIWIYLLPTLGVSGLIHKMLPSIQNEPEFSKTG--SSHKRVTDLPL t-nip1;1 NVLIPVSSSMNPGRSLGPLVYGCYKGIWIYLVPTLGIGWVYNTVRYTDKPLREITKSG--SFLKTVRIGST t-nip1;2 NVIIGPVSGSMNPGRSLGPMVYSCYRGLWIYIVSPIVGVSGWVYNMVRYTDKPLREITKSG--SFLKTVRNGSSR ::.:. :..**** *::***: :. :*:*:.* :*.: * : :. t-nip7; t-nip5; t-nip6; t-nip4;1 T t-nip3;1 ICVLRVVDPNQNYFICSSPTDINGKCNVTCKL 323 t-nip2; t-nip1; t-nip1; B t-nip5;1 MPPEEVG----VMVMPPTPG-TPGTPGG 27 t-nip6;1 MDHE EIPSTPSTPTTPGTPG 22 Os-NIP3;1 MEMPNGGGGMSSPVNGSPTPGTP- 31 Zm-NIP3;1 MEPG STPPNG-SPTPGTP- 25 Ct Supplemental Figure 3. lignment of NIP Homolog Sequences. (Supports Figures 1B and 2.) () Sequence alignment of NIP homologs from rabidopsis using Clustal Omega ( msa/-clustalo/). The predicted N- and C-terminal regions (Nt and Ct) are indicated by the magenta and green lines below the sequences, respectively. The prediction was based on the consensus prediction in the ramemnon database (Schwacke et al., al. 23; Genes and locus IDs: t-nip7;1, t3g61.1; t-nip6;1, t1g876.1; t-nip5;1, t4g138.1; t-nip4;1: t5g3781.1; t-nip3;1, t1g ; t-nip2;1, t2g3439.1; t-nip1;2, t4g1891.1; t-nip1;1, t4g Magenta color with asterisk (*), identical residues; green color with colon (:), residues with strongly similar properties; black color with period (.), residues with weakly similar properties. (B) Sequence alignment of N-terminal regions of boric acid channels identified in rabidopsis, rice, and maize. t-nip5;1, t4g138.1; t-nip6;1, t1g876.1; Os-NIP3;1, LOC_Os1g ; Zm-NIP3;1, GRMZM2G ll the boric acid channels identified share the TPGTP sequence (magenta) in the N-terminal region. t, rabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays. 3

4 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc WT B ΔNt1 17 C ΔNt1 26 F Nt1 26 Dark treatment D WT E Nt1 17 Supplemental Figure 4. Deletion of the N-Terminal Sequence Induces Vacuolar Trafficking of NIP5;1. (Supports Figure 2B.) () to (C) Confocal images of GFP-NIP5;1 ΔNt1 17 and ΔNt1 26 in the root epidermal cells. Intracellular punctate structures are indicated by white arrowheads. (D) to (F) GFP signals derived from GFP-NIP5;1 variants under dark conditions for 8 h. Bar = 2 µm. 4

5 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc GFP FM4-64 Merged GFP (Cross section) WT T15 T16 T19 T15 T T19 B D NIP6;1 PIs Longitudinal T15 T16 T19 C BC B T15 T16 T C NIP6;1 PIs Cross T15 T16 T19 B B T15 T16 B T19 Supplemental Figure 5. Conserved Threonine Residues in the N-Terminal Region re Essential for Polar Localization of NIP6;1. (Supports Figure 2B.) () Confocal images of GFP-NIP6;1 wild-type and the T15 T16 T19, T15, T16 and T19 variants in the epidermal cells. FM4-64 was used to stain the PM and shown in magenta. Bar = 2 µm in the optical longitudinal sections and 5 µm in the cross sections. (B) Polarity indexes in the optical longitudinal sec-tions (PI Longitudinal ) and in the cross sections (PI Cross ). Means ± SDs are shown. n = total 3 5 epidermal cells from 3 5 independent transgenic T1 plants for PI Longitudinal and n = total 3 6 epidermal cells from 5 1 independent transgenic lines for PI Cross. Different letters above each bar indicate a significant difference (one-way NOV with the LSD- Duncan s test; P <.1). Dashed lines indicate the polarity index = 1. 5

6 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc h 1.5 h 3 h 6 h 12 h 18 h 24 h GFP-NIP5;1 WT GFP+FM4-64 GFP-NIP5;1 T18 T21 T24 GFP+FM4-64 B GFP-NIP5;1 WT enhanced images of 1.5 h T18 T21 T24 C Signal intensity 24 h (WT) 18 h 12 h 6 h 3 h 1.5 h h Stele-side Soil-side Signal intensity (T18 T21 T24) Stele-side Soil-side Supplemental Figure 6. Time-Course Imaging of Newly-Synthesized GFP-NIP5;1. (Supports Figure 2.) () Wild-type ProNIP5;1:GFP-NIP5;1(genomic) wild-type and T18 T21 T24/nip5;1-1 plants were grown on 1 µm B medium for 4 days and then shifted to low-b (.3 µm) medium. fter 1.5, 3, 6, 12, 18, and 24 h, GFP-NIP5;1 in the root epidermal cells was imaged in optical longitudinal sections. FM4-64 was used to stain the PM and is shown in magenta. Bars = 2 µm. (B) GFP signals in the transverse PM were enhanced by the ImageJ software to indicate the polar localization. Bar = 2 µm. (C) GFP intensity in the transverse PM as plotted along the line indicated in (). 6

7 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc ProNIP5;1: GFP-NIP5;1(genomic) in nip5;1-1 1 µm B 1 µm B GFP intensity in root tip (.U.) B.3 µm B 9 min 1 µm B.3 µm B +CHX 9 min µm B.3 µm B.3 µm B 9 min +CHX,9 min Supplemental Figure 7. CHX Inhibits the ppearance of GFP-NIP5;1. (Supports Figures 3 and 4.) () Plants (3 4 days old) of ProNIP5;1:GFP-NIP5;1(genomic)/nip5;1-1 plants were shifted from high (1 µm) to low B (.3 µm) medium for 9 min. Low-B media supplemented with or without CHX (5 µm) was used for comparison. Bar = 5 µm. (B) GFP intensity in root tips (ROI: 9 x 38 µm). n = 3 or 4 roots. Means ± SDs are shown. 7

8 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc nip5;1-1 ap2m-1 ap2m-2 GFP-NIP5;1 GFP+FM4-64 GFP-NIP5;1 GFP+FM4-64 GFP-NIP5;1 GFP+FM4-64 Supplemental Figure 8. P2M Is Required for Polar Localization of GFP-NIP5;1 in the PM. (Supports Figure 5.) GFP-NIP5;1 in the root tips of ProNIP5;1:GFP-NIP5;1/ap2m plants. Plants were grown for 3 4 days on solid medium supplemented with 1 µm B. FM4-64 was used to stain the PM and is shown in magenta. Bar = 5 µm. 8

9 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc coen GFP-NIP5;1 Wild-Type B C F D E G ep co en H en co ep GFP-NIP5;1 T18 T21 I J M K L N ep co en 255 en co ep Supplemental Figure 9. Polar Localization of GFP-NIP5;1 Wild-Type and Weakly Polar Localization of the GFP-NIP5;1 T18.T21.T24 Variant in ProNIP5;1:GFP-NIP5;1(genomic)/nip5;1-1 Plants. (Supports Figures 2F, 7 and 7B.) Confocal images of GFP-NIP5;1 in ProNIP5;1: GFP-NIP5;1(genomic)/nip5;1-1 plants ( G) and ProNIP5;1: GFP-NIP5;1(genomic) T18 T21 T24/nip5;1-1 plants(h N). Root tip (, H), epidermal cells in transition zone (B, C, I and J), and epidermal and endodermal cells in the root hair zone (D, E, K and L) in longitudinal optical sections and transition zone (F, M) and root hair zone (G, N) in cross sections are shown. GFP signals in the cross sections (F, G, M and N) are shown in green-fire-blue color-coded by the ImageJ software to indicate the relative abundance. Plants were grown on solid medium supplemented with 1 µm B for 4 days. FM4-64 (4 µm) was used to stain the PM and is shown in magenta (C, E, J and L). Bars = 2 µm in B, C, I and J, 5 µm in, D G, H, and K-N. ep: epidermis; en: endodermis and co: cortex. 9

10 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc Col- nip5;1-1 ProNIP5;1:GFP-NIP5;1(genomic)/nip5;1-1 WT T18 T21 T24 #3 #6 #7 #5 #1 #1 #12.3 µm B.1 µm B 1. µm B 3. µm B Supplemental Figure 1. Growth Characterization of the nip5;1-1 Lines with Wild-Type GFP-NIP5;1 and the T18 T21 T24 Variant under Boron-Limited Conditions. (Supports Figure 7C and 7D). T3 homozygous plants of the nip5;1-1 mutant wild-type carrying ProNIP5;1:GFP-NIP5;1:GFP-NIP5;1 (genomic) wild-type and T variant constructs were grown for 7 8 days on solid medium with.3 3 µm boric acid. Bars = 1. cm. 1

11 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc ProNIP5;1:GFP-NIP5;1(genomic) in nip5;1-1 #1 #2 #3 #6 #7 #18 #9 #1 ProNIP5;1:GFP-NIP5;1(genomic) (T18 T21 T24) in nip5;1-1 #1 #2 #4 #5 #8 #1 #11 # Supplemental Figure 11. Quantification of GFP-NIP5;1 Protein Levels in Root Tips by Fluorescent Imaging. (Supports Figure 7B.) Plants (3 or 4 d old) grown on solid medium supplemented with 1 µm B were shifted to solid medium supplementedwith.3 µm B for 24 h. Z-stack images were taken at 2.5-µm intervals for 22 slices and the sum image was projected with ImageJ software in Rainbow RGB color code. Bar = 15 µm. 11

12 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc Supplemental Table 1. One-Way NOV Data. (Supports Figure 7E.) Plants (a) Plants (b) Mean Differences (a-b) Col- nip5; * WT # WT # Mutant #5.1 Mutant #1.175 nip5;1-1 WT #3 WT #6 Mutant #5 Mutant #1 Col- WT #3 WT #6 Mutant #5 Mutant #1 Col- nip5;1-1 WT #6 Mutant #5 Mutant #1 Col- nip5;1-1 WT #3 Mutant #5 Mutant #1 Col- nip5;1-1 WT #3 WT #6 Mutant #1 Col- nip5;1-1 WT #3 WT #6 Mutant # * * * *.9.275*.215* * * * Std. Error * The mean difference is significat at the.5 level. Significance 95% Confidence Interval Lower Bound Upper Bound Numbers in the table indicate different plants, 1: Col-; 2: nip5;1-1; WT #3: GFP-NIP5;1genomic #3; WT #6: GFP-NIP5;1genomic #6; Mutant #5: GFP-NIP5;1genomic(T18 T21 T24) #5; Mutant #1: GFP-NIP5;1genomic(T18 T21 T24) #1. 12

13 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc Supplemental Table 2. Primers used in This Study Primers Sequences 5` to 3` Primers for vector construction NIP1;1-F NIP1;1-R CCCTGGCGGTTCTCGGGC TCGTGCTCCGTTCTCCGGTC NIP1;2-F CCCTGGCGGGTCTCGGG NIP1;2-R TTCGGGCTCCGTTTCGCC NIP2;1-F CCCTGGTGCTTCGTGG NIP2;1-R TCCGGGGTCGGTCTC NIP3;1-F CCCTGGCTGGTTTCTGTTTCTC NIP3;1-R TCCGCCCTTCTGTCTTCTTTG NIP4;1-F CCCTGTCTTCGCTGTGTGT NIP4;1-R TTGTCTTGCTGCCCTTGTG NIP5;1-F CCCTGGCTCCCCGGGGCT NIP5;1-R TTCGCGGCTCCTCCGGCG NIP6;1-F CCCTGGTCTGGGTTCC TCTCTTCTGGCTCCTCCTCTC NIP7;1-F CCCTGTGGTGGGCCGGT NIP7;1-R TTCGTGTGGCGGGGG pronip5;1-f CCCGCTTCGGCGCTTC pronip5;1-r GTCTGGGGCTTTGGTTTTT PIP2;1-F CCCTGGCGGTGTGGGC PIP2;1-R NPSN12-F TTGCGTTGGCGCCTTCTG GCGGCTGTCGTGGCGTCTGTTGCCGT NPSN12-R LTI6a-F CGTCTGCGCCGCTTTCTCTGGTCGG GCGGCTGTCGTGGTCGCTCTTTCGT LTI6a-R CGTCTGCGCCGTCTTTGGTGGGCTTG NIP5;1genomic-F TGGCGGCTGTCGTGGCTCCCCGGGGCTG NIP5;1genomic-R CTGTTCGTTCGTGTGGTCGGCTCC pronip5;1:gfpgenomic-f TGTCGCTCGTCCTGCCGTG pronip5;1:gfpgenomic-r GTTCGTTCTGGTCTGCTGTTTCCTGT PCR2-F GCGGCTGTCGTGGGCTCCCCTTC PCR2-R CGTCTGCGCCGTTGCGGGTCTGCCGCCTT NIP6;1-R Primers for chimera construction Nt5;1-PCR2-F Nt5;1-PCR2-R Nt1;2-NIP5;1-F Nt1;2-NIP5;1-R Nt5;1-NIP1;2-F Nt5;1-NIP1;2-R Nt5;1-TMD1;2-Nt5;1-F Nt5;1-TMD1;2-Nt5;1-R NIP5;1-Ct1;2-F NIP5;1-Ct1;2-R Nt5;1-PIP2;1-F Nt5;1-PIP2;1-R GTCTCCCTCCTCGCTGGGCTCCCCTTC TTGCGGGTCTGCCGCCTTCGTCTGCGCCG GTCCCTTTCTTCGCTTGGGCCGGTTCG CTCGGCTCCGCTTTTGTGGGGCGGTGGG GTCTCCCTCCTCGCGTTGTGGCGGGGTTTTG CTCCGCCTCCTTGCGGTGGGGGCTCG GGTGCGTGGGTTTTCGGTGTTGCTTCGTGCG GCTTCCCTGTTCCCCGCCCCGC GGTGCGCGGTCTCCTGGTTCGTTCTGTGCC GTCCCTTTCTTCGCTTGGGCCGGTTCG GCGTTTCGCCGGTTCGTG CTCTGCGTCTGCGCGGTGGGGGCTCG 13

14 Supplemental Data. Wang et al. (217). Plant Cell 1.115/tpc Primers Sequences 5` to 3` Primers for mutation construction a NIP5;1 N17-F GCGGCTGTCGCGCCGGGGCCCGGGC NIP5;1 N26-F GCGGCTGTCGGGCCGTTGTCCGGGGTG NIP5;1 T18,21,24-F CCGGGGGCCCGGGGCCCGGGGGCCGTTGTC NIP5;1 T18,21,24-R TCCCGGTGCCCCCGGCGCTGGGGGCCTCCCTTC NIP5;1 T18,21,24D-F CCGGGGGTCCGGGGTCCGGGGGCCGTTGTCC NIP5;1 T18,21,24D-R TCCCGGTCCCCCGGTCTGGGGGCCTCCCTTC NIP5;1 T18-F TCCTCCGCGCCGGGGCCCGGGCC NIP5;1 T18-R CCCGGCGCTGGGGGCCTCCCTTC NIP5;1 T21-F GCCGGGGGCCCGGGCCCGGGGGC NIP5;1 T21-R CCCGGTGCCCCCGGCGTTGGGGGCCTC NIP5;1 T24-F CCGGGGCCCGGGGGCCGTTGTC NIP5;1 T24-R CCCGGTGCTCCCGGTGTCCCCGGCGTTG NIP6;1 T15,16,19-F GCGCCCGGGGGCTCCGGGCGCCGCTCTTTGG NIP6;1 T15,16,19-R GCCCCCGGTGCTGCCGCCGGCGTTGGGGCGTGG NIP6;1 T16-F GGCGCGCCCCGGGGCTCCGGGCG NIP6;1 T16-R CCCGGGGCTGTCGCCGGCGTTGGGGCG NIP6;1 T19-F CCCGGGGGCTCCGGGCGCCGCTCTTTG NIP6;1 T19-R CCTGGGCCCCCGGGGTTGTCGCCGGCG Primers for construction of in vitro synthesized complementary RN (crn) sgfp-nip5;1-f sgfp-nip5;1-r sgfp-nip5;1 T18,21,24-F sgfp-nip5;1 T18,21,24-R sgfp-nip1;2-f sgfp-nip1;2-r Primers for genotyping CCCGGTCC TGGTGGCGG GGTCC TTCGCGGCTCCTC CCCGGTCC TGGTGGCGG GGTCC TTCGCGGCTCCTC CCCGGTCC TGGTGGCGG GGTCC TTCGGGCTCCGTTTCG ap2m-1-lp ap2m-1-rp ap2m-2-lp ap2m-2-rp Primers for quantitative real-time RN GCCGGTCGTGGC GCTGCTTGTTGCTTGTG GGGGGTTTTCTCCTTCC TTTTCCCGCTTGGTTTG NIP5;1-F NIP5;1-R UBQ1-F UBQ1-R CCCGTTTTCCCTCCTGT GCTGCGCGTTCCGTT GTGGTGCTGGGGG TCGCTTCCTCCTTCTT a The nucleotides altered to produce amino acid substitutions are underlined. 14