Dirigent Protein-Like Genes Expression in Response to Mechanical and Biological Wounding

SUPPLEMENTAL MATERIALS AND METHODS

1 Microarray Hybridizations and Microarray Analysis

2 The construction of the spruce 16.7K array and further details of the microarray

experiment will be described elsewhere (Ralph et al., in prep.). Briefly,

hybridizations were performed using the Genisphere Array350 kit (Genisphere,

Hatfield, USA) following manufacturer’s instructions. Forty µg total RNA was

reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo

d(T18) primers with a 5’ unique sequence overhang specific to either the Cy3 or

Cy5 labeling reactions. The RNA strand of the resulting cDNA:RNA hybrid was

hydrolyzed in 0.075 M NaOH / 0.0075 M EDTA at 65°C for 15 min followed by

neutralization in 0.175 M Tris-HCl (pH 8.0). Following pooling of the appropriate

cDNAs, samples were precipitated with linear acrylamide and resuspended in a 45

µL hybridization solution consisting of 0.25 M NaPO4, 1 SSC, 0.5% SDS, 2

Denhardt’s solution, 1 mM EDTA, 4.0 µL LDNA d(T) blocker, 0.5 µg/µL sheared

salmon testes DNA (Invitrogen) and 0.3 µL of Cy5-labeled GFP cDNA (Cy5-

dUTP and Ready-To-Go labeling beads, Amersham Pharmacia Biotech).

Immediately prior to use, arrays were pre-washed 3 in 0.1% SDS at room

temperature for 5 min each, followed by two washes in MilliQ-H2O for 2 min each,

3 min at 95°C in MilliQ-H2O, and dried by centrifugation (5 minutes at 2000 rpm

in an IEC Centra CL2 centrifuge with rotor IEC 2367-00 in 50 mL conical tube).

The cDNA probe was heat denatured at 80°C for 10 min, then maintained at 65°C

prior to adding to a microarray slide heated to 55°C, covered with a 22  60  1.5

mm glass coverslip (Fisher Scientific), and incubated for 16 h at 60°C. Arrays

were washed in 2 SSC, 0.2% SDS at room temperature for 5 min to remove the

coverslip, followed by 15 min at 65°C in the same solution, then three washes of 5

3 min in 2 SSC at room temperature, and three washes of 5 min in 0.2 SSC at

room temperature, and dried by centrifugation. The Cy3 and Cy5 3DNA capture

reagent (Genisphere) were then hybridized to the bound cDNA on the microarray in

a 45 µL volume consisting of 0.25 M NaPO4, 1 SSC, 0.5% SDS, 2 Denhardt’s

solution, 1 mM EDTA, 2.5 µL Cy3 capture reagent and 2.5 µL Cy5 capture

reagent. The 3DNA capture reagent is bound to its complementary cDNA capture

sequence on the Cy3 or Cy5 oligo d(T) primers. The second hybridization was

performed for 3 h at 60°C, then washed and dried as before. Fluorescent images of

hybridized arrays were acquired by using ScanArray Express (Perkin Elmer, Foster

City, USA). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm,

respectively. All scans were performed at the same laser power (90%), but with the

photomultiplier tube settings for the two channels adjusted such that the ratio of the

mean signal intensities was ~1, and the percentage of saturated array elements was

< 0.5% but > 0%, while minimizing background fluorescence. Fluorescent

intensity data were extracted by using the ImaGene 5.5 software (Biodiscovery, El

Segundo, USA).

Before normalization, the lowest 10% of median foreground intensities was subtracted from the median foreground intensities to correct for background intensity.

After quantification of the signal intensities, data were normalized to compensate for nonlinearity of intensity distributions using the vsn method (Huber et al., 2002). A linear model was applied to obtain a single estimate and standard error of the ΔH difference statistic of expression for each parameter examined (i.e. mechanical wounding versus

4 untreated control at 2 h) (Smyth, 2004). The ratio of the estimate to the standard error was used to calculate a t statistic, from which a P value was obtained.

5 Supplemental Table V. Gymnosperm and angiosperm DIR and DIR-like genes represented in phylogenetic analyses. Species DIR FLcDNA Genomic ORF Nomenclature Accession Accession/AGI Number Number Gymnosperms Picea glauca (white spruce) PDIR1 DQ395241 n.a. PDIR7 DQ395247 n.a. PDIR10 DQ395250 n.a. PDIR12 DQ395252 n.a. PDIR18 DQ395258 n.a. Picea sitchensis (Sitka spruce) PDIR11 DQ395251 n.a. PDIR15 DQ395255 n.a. PDIR16 DQ395256 n.a. PDIR17 DQ395257 n.a. PDIR19 DQ395259 n.a. Picea glauca x engelmannii (interior spruce) PDIR2 DQ395242 n.a. PDIR3 DQ395243 n.a. PDIR4 DQ395244 n.a. PDIR5 DQ395245 n.a. PDIR6 DQ395246 n.a. PDIR8 DQ395248 n.a. PDIR9 DQ395249 n.a. PDIR13 DQ395253 n.a. PDIR14 DQ395254 n.a. Tsuga heterophylla (western hemlock) ThDIR1 AF210071 n.a. ThDIR2 AF210072 n.a. Thuja plicata (western red cedar) TpDIR1 AF210063 n.a. TpDIR2 AF210064 n.a. TpDIR3 AF210065 n.a. TpDIR4 AF210066 n.a. TpDIR5 AF210067 n.a. TpDIR6 AF210068 n.a. TpDIR7 AF210069 n.a. TpDIR8 AF210070 n.a. TpDIR9 AF487405 n.a. Angiosperms Arabidopsis thaliana (thale cress) AtDIR1 n.a. At5g42510 AtDIR2 AY093095 At5g42500 AtDIR3 n.a. At5g49040 AtDIR4 n.a. At2g21110 AtDIR5 AK175255 At1g64160 AtDIR6 BT002439 At4g23690 AtDIR7 AK118030 At3g13650 AtDIR8 n.a. At3g13662 AtDIR9 BT010722 At2g39430 AtDIR10 BT002889 At2g28670 AtDIR11 AK176442 At1g22900 AtDIR12 BT004016 At4g11180 AtDIR13 BT009718 At4g11190 AtDIR14 n.a. At4g11210 AtDIR15 CNS0A3JZ At4g38700 AtDIR16 BT008336 At3g24020 AtDIR17 n.a. CAB67637 AtDIR18 AY081267 At4g13580 AtDIR19 AK117899 At1g58170 AtDIR20 AY128336 At1g55210 AtDIR21 n.a. At1g65870 AtDIR22 BT015420 At3g13660 AtDIR23 BT005788 At2g21100 AtDIR24 CNS0A5KQ At3g55230 AtDIR25 n.a. At1g07730 Oryza sativa (Japonica) (rice) OsDIR1 AK109288 NM_186138 OsDIR2 n.a. NM_186143 OsDIR3 AK065027 AP006186 OsDIR4 AK108186 XM_482075 OsDIR5 AK108922 NM_196355 OsDIR6 n.a. NM_189817 OsDIR7 n.a. NM_189810 OsDIR8 n.a. NM_184035 OsDIR9 AK108101 NM_194246 OsDIR10 n.a. NM_184603 OsDIR11 AK106022 XM_479255 OsDIR12 n.a. XM_479260 OsDIR13 n.a. XM_479267

6 OsDIR14 n.a. NM_188847 OsDIR15 AK108983 XM_479273 OsDIR16 n.a. XM_479258 OsDIR17 n.a. NM_195796 OsDIR18 n.a. XM_450817 OsDIR19 AK121408 AC135595 OsDIR20 n.a. AC119796 OsDIR21 n.a. NM_184427 OsDIR22 n.a. AP003856 OsDIR23 n.a. NM_196360 OsDIR24 n.a. AP003369 OsDIR25 n.a. NM_195790 OsDIR26 n.a. NM_195802 OsDIR27 n.a. XM_481885 OsDIR28 n.a. XM_481843 OsDIR29 n.a. XM_481834 OsDIR30 n.a. XM_481890 Hordeum vulgare (barley) HvDIR1 U43497 n.a. HvDIR2 U43496 n.a. HvDIR3 AF021258 n.a. Triticum aestivum (wheat) TaDIR1 U32427 n.a. TaDIR2 AF483596 n.a. TaDIR3 AB012103 n.a. Saccharum officinarum (sugarcane) SoDIR1 AY421731 n.a. SoDIR2 AJ626722 n.a. SoDIR3 AY781903 n.a. Pisum sativum (pea) PsDIR1 AF115574 n.a. PsDIR2 U11716 n.a. Forsythia intermedia (shrub) FiDIR1 AF210061 n.a. FiDIR2 AF210062 n.a. Podophyllum peltatum (mayapple) PpDIR1 AF352736 n.a. Sorghum bicolor (sorghum) SbDIR1 AF527807 n.a. Gossypium barbadense (cotton) GbDIR1 AY560544 n.a. Zea mays (corn) ZmDIR1 AF232008 n.a.

7 Supplemental Table VI. Oligonucleotides used in real-time PCR.

Isoform Primer name Sequence Description1 PDir2 Dir2F 5’-TGTTTCATTCTCATGCAGATTTG-3’ +93 to +116 Dir2R 5’-TTATTTCACTCTACGCTAGCTG-3’ +142 to +164 PDir5 Dir5F 5’-TCCTTCTGCTCTCCATTTCTATG-3’ +30 to +52 Dir5R 5’-CATTATTTCACTGCACGCTACAG-3’ +104 to +127 PDir6 Dir6F 5’-CTTTTGCAAATCTGCATTGGTAG-3’ +101 to +124 Dir6R 5’-TTCAATCAATCATGTGAGGAGC-3’ +190 to +213 PDir8 Dir8F 5’-GCGTGCAGTGAAATAATGTCTAC-3’ +88 to +111 Dir8R 5’-GAGAACGATTCTGACAAAATCTC-3’ +182 to +205 PDir13 Dir14F 5’-ACTGCTTATCACATGAGTTGCG-3’ +12 to +34 Dir14R 5’-GGACCTTCATGGAGATCCCTC-3’ +104 to +125 PDir19 Dir15F 5’-TCTGCCACCGTAAGCAAGGACC-3’ +1 to +23 Dir15R 5’-GATGAGCATAGCTCCACACAAATC-3’ +74 to +95 PDir1 Dir1F 5’-GCTGTGGGTAGGTATTCTGATC-3’ +70 to +91 Dir1R 5’-TATGCACAGAAGACACGGCCATG-3’ +136 to +158 Dir10F 5’-TGCCCGGCAGTGGTATATACATTG-3’ +46 to + 69 PDir10 Dir10R 5’-GCAAGCAGCATTGGATACTGTCAC-3’ +119 to +142 5’-TGTAGAGGTGAAATCAAGGCTCAGTC- Dir16F +79 to +103 PDir12 3’ Dir16R 5’-TGGTCATGATTAGACTACCCTTTG-3’ +161 to +184 ß-Actin ActinF 5’-GGTATCCATGAGACTACATAC-3’ ActinR 5’-CAGGAAACATGGTAGAACCAC-3’ 1Primer position from stop codon.