1 2 3 4 5 6 7 8 9 10 11 12 Figure S1. Characterization of the irx9l-1 mutant. (A) Diagram of the Arabidopsis IRX9L gene drawn based on information from TAIR (the Arabidopsis Information Research). Exons are blue boxes, 5 and 3 untranslated regions are white boxes, and introns are lines. The T-DNA insertion site is indicated by a blue triangle and arrows represent the locations of primer annealing sites used for genotyping. (B) RT-PCR analysis of IRX9L expression in flower buds of wild-type Col-0 and irx9l-1. No transcripts were found in the mutant sample. ACTIN primers were used as the positive control, reacion was carried out for 32 cycles (C) Vegetative growth phenotype of wild-type Col-0 and irx9l-1. No morphological differences between the mutant and the wild-type Col-0 were observed. 1
13 14 15 16 17 18 19 20 21 22 23 Figure S2. UPEX gene structure and expression. (A) Diagram of the Arabidopsis UPEX gene (based on The Arabidopsis Information Research database). Exons are solid boxes, 5 and 3 untranslated regions are white boxes and introns are lines. T-DNA insertion sites are indicated by triangles. (B) RT-PCR analysis of UPEX expression. UPEX is expressed specifically in closed flower buds (wild-type buds) and no transcript was detectable in the buds of upex-1. ACTIN2 expressions are shown as controls. 28 cycles of PCR reactions were run. (C) Vegetative growth of wild-type Col-0 and upex-1. No morphological differences between the mutant and the wild-type Col-0 were observed. 2
24 25 26 27 28 29 30 31 32 Figure S3. UPEX.1 is the major variant expressed in plants and upex has normal vegetative growth. (A) UPEX protein coding gene model drawn based on TAIR. Arrows represent the UPEX.1 and UPEX.2 specific primer annealing sites. The UPEX.2 reverse primer contains extra nucleotide sequences not found in the UPEX.1 primer. The reverse primer used for UPEX.1 spans both exons 3 and 4. (B) 27 cycles RT-PCR using wild-type closed flower bud cdna as template shows UPEX.1 is the major variant. 3
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure S4. Quantification of LM10 immuno-gold signal in wild-type, upex1, and irx9l microspore walls. Gold particles were counted in immuno-gold labeled TEM sections taken from early-uninucleate stage anthers. Gold particles were counted in three randomly chosen pollen wall sectors from TEM micrographs from each genotype. Stars indicate significant difference from Col-0 (Student s t-test p<0.05). 4
48 49 50 51 52 53 54 55 56 Figure S5. Scanning electron micrographs of pollen derived from upex1/upex1 and irx9l IRX9L plants. (A), representative pollen from a upex1/upex1 plant. (B), representative pollen from an irx9l IRX9L plant. Scale bars = 20μm 5
57 58 59 60 61 62 63 64 Figure S6. Comparison of exine structure in wild-type, irx9l, upex1, and irx9l upex1 pollen. (A) to (D), confocal micrographs of mature pollen grains taken from the genotypes indicated and stained with the fluorescent dye Auramine O (Aur-O). (E) to (H), SEM micrographs of pollen grains taken from the same genotypes. Pollen from the irx9l upex1 double mutant appeared to have an additive phenotype, with both spongy exine typical of irx9l (arrow, panel H) and apparently detached exine typical of upex1 (arrow, panel D). Bars=5μm 6
65 66 68 Table S1. Fertility of irx91 and upex1 mutants genotype Average seeds/silique 67 1 Col-0 54±15 irx9l 50±8 upex1 48±14 1 n=29-32 69 7
70 Table S2. Primers used for genotyping Arabidopsis mutant lines Primer Name Primer sequence 5' to 3' UPEX1 CDS RP WITH STOP TAA ATT CAT GAC TCT TAA TTG upex1 SK_091466 LP TTT ACT GCT TGG TGA GTT CTC G upex1 SK_091466 RP AAT GGC AGT GCA AAA CAT AGC irx9l SK_037323 LP GAC TAG TTG AGT CGC CTG TGC irx9l SK_037323 RP CTT CCA TCT CGC TTT CAT CAG UPEX1 FP native pro TTG TTG TGT AAG TGT AAC CAG UPEX1 RP native pro GAC GAA GAA GCG AAA ATT AGC UPEX1 RT fp TGT GTC GTT AGG AGC ATG GAT UPEX1 CDS cloning FP ATG AGG GCG AAA GCT GCT TCG UPEX1 SAIL_544_C02 RP GAG TTA GCA GCA GCA CGT ACC UPEX1 SAIL_544_C02 LP AGT GTC TCC TTC AGC ACA AGC IRX9L pro fp CCA TCT TTT TTC CTT IRX9L pro rp CCG GAT TGA CGC CAT CAG AT IRX9L cds rp TTT CAT AGT TAT GAG AGC CTG IRX9Lcds fp ATG GCG TCA ATC CGG CGA ACT irx14 SK066961rp ATC GAT GTA CGG TGT GAG GAG irx14 SK066961lp CTT GCT CTT CGA CAC TCT TGG irx14l Sk038212lp AAC GAC ACG TGT ACC TCC TTG irx14l Sk038212rp AAC ATC ACA ATC CCA TCA AGC UPEX1 pro rp2 CGC CCT CAT CTC CGC GCC T UPEX1 pro rp CTC CGC GCC TCC TTA GTG CTA UPEX1 pro fp CTA ATT AAG TAT GGT AGA CCA UPEXspl var size fp CTC CTG GTG TTG CAC TGT GT UPEX1 spl var size longrp CAG CAG CAA GAA AAG ACG TG UPEX1 spl var short rp TAT CAC AAA CCT TAC AAC AAT AT5G41890 SALK_070701 LP TGT TAC TGC AAT GTC TGC AGG AT5G41890 SALK_070701 RP TGG TCC TCC ATT TAA CCA CAC AT5G41890 GK-509B03 LP1 TGA CTT GTT CGG AGC ATT TTC AT5G41890 GK-509B03 RP1 AAA CCG TTT CTG AAT TGG TCC AT4G41890 GK-509B03 LP2 GGC AAA TTC CAT AAA TGC AAC AT4G41890 GK-509B03 RP2 ACG CAA TTG ACA CCG ATT TAG AT5G41890 SALK_070701 RT LP AGC AGC TTG TGA GGA CAG GT AT5G41890 SALK_070701 RT RP AGC CTT TGA GCG AGT GAT TG wat4g28395 SALK_038995 RT RP TTC TCA ACG TCG GGA TTC TT AT4G28395 SALK_038995 ATG ATG CAT AAT AAA CTC CAC C AT4G28395 SALK_038995 STOP TTA ATT TAA TTT CTC AAC GTC G AT4G28390 LP (RT) GAC GTC AGG TGA AGC AGT GA AT4G28390 RP (RT) CCA CCA CCA GAG CCA TAC TT AT4G28395 GK-288A12 LP TGG CTT TGA AAG TGT TGG ATC AT5G41890 SK_148701 RP AGG ATT CAT TCA GCT GGT GAC AT5G41890 SK_148701 LP TCC CCA CTG ATG TCC TAC AAG AT4G29250 SK_147254 LP TTC TGT GTA ATC CAA TCG TTG C AT4G29250 SK_147254 RP AAC CAA ACC AAA ATA AAC CGG AT3G23840 GK180G04 LP GACTCATCAAGTTCTGCCCTG 8
71 72 AT3G23840 GK180G04 RP AT3G23840 SK_079871 LP AT3G23840 SK_079871 RP AT3G23840 SK_126074 LP AT3G23840 SK_126074 RP AT1G27600 SK_089553 LP AT1G27600 SK_089553 RP AT1G27600 GK_742B12 LP AT1G27600 GK_742B12 RP GAT CTA CTG CCC CAA AAC CTC ACA GCA CGT GAC TTG ACT GTG CAA TCT CAT CGA TCA CAA CCC GTC ACT TCG CAA AAA GCA CTC AGA TTG TGA AAT CGA TCG GTG ACC TCT ACG CGA GCT TTT AGG TGA AAC TAG ATA TCT GCT TGT ACC G ATG GTT CAG CTC CAT GTG TTC TGT CCG TTT GTT ATC GAT TCC 9
73 10