Figure S1 (Chen) (a) 1xABRC321:GUS 1xABRC321 2xABRC321:GUS 2xABRC321 3xABRC321:GUS 3xABRC321 GUS 0xABRC321:GUS Amy64 mini PHVA22 In1-Ex2-In2 HVA22 3 HVA1 3xABRC321:HVA1 Amy64 mini P Nos 3 HVA22 In1-Ex2-In2 3xABRC321 (c) (b) Figure S1. The synthetic promoter 3xABRC321 has low-background but is highly inducible by ABA in transgenic rice. (a) Constructs contain 0-3 tandem repeats of ABRC321 controlling GUS expression. The lengths of DNA fragments constituting the expression cassette are: ABRC321, 56 bp; Amy64 minimal promoter (mini P), 99 bp; intron 1-exon 2-intron 2 (In1-Ex2-In2) of HVA22, 240 bp; GUS cDNA, 2 kb; HVA22 terminator (3), 120 bp. (b) Constructs contain 3 tandem repeats of ABRC321 for HVA1 expression. Lengths of HVA1 cDNA and Nos 3 are 642 and 300 bp, respectively. (c) Constructs shown in (a) were introduced into the genome Tainung 67. Leaves and roots of 10-day-old T2 seedlings of four transgenic lines of each construct were incubated with (+) or without (-) 10 M ABA for 16 h, and GUS activity was analyzed. Numbers above bars indicate fold induction of GUS activity by ABA. Error bars represent SD (n = 3) GUS activity (110 4 ) xABRC321:GUS1xABRC321:GUS2xABRC321:GUS3xABRC321:GUS Line ABA + ABA ABA + ABA Root Leaf Figure S2 (Chen) Seminal root Crown root Lateral root Fig. S2. Morphology of various branch roots in rice. A 10-day-old seeding of rice cultivar Tainung 67. Figure S3 (Chen) Oct Aug Sep Nov Dec Temperature (C) Rainfall (mm) 2011 Irrigated field Non-irrigated field (a)(b) Figure S3. 3xABRC321:HVA1 transgenic lines grown in fields. (a) Kitaake transgenic lines expressing 3xABRC321:HVA1 were grown in two separated irrigated and non-irrigated fields. Both fields were subject to natural rainfall occasionally. Plants were photographed one month after transplanting. (b) Records of temperature and rain fall for the non- irrigated field trial in 2011-Fall. The grain yield is shown in the upper panel in Figure 6b. Red dotted line indicates daily temperature, red line indicates daily mean temperature, and green bar indicates daily rainfall (mm). The weather information was obtained from Figure S4 (Chen) (b) Figure S4. 3xABRC321:HVA1 has similar tissue-specific and ABA-inducible expression patterns to the endogenous LEA3 in transgenic rice roots. (a) Ten-day-old seedlings of 3xABRC321:HVA1 transgenic line (T-33) were treated with or without 10 M ABA for 16 h, and roots were sectioned for immunohistological fluorescence assay of Lea3 and HVA1. Quantification of fluorescence was performed by ZEN microscopy and image software for the Carl Zeiss confocal microscope. F: image of fluorescence field. C: composite images of fluorescence and transmission fields. P: pericycle. Scale bar: 20 m. (b) ABRC321 shares similar cis-acting elements to the rice LEA3 promoter. DNA motifs in the LEA3 promoter similar to the three cis-acting elements in ABRC321 are aligned. -1 indicates one bp upstream of the transcription start site. 3xABRC321: HVA1 Fluorescence image Quantification of fluorescence WT (a) WT 3xABRC321:HVA1 -ABA +ABA -ABA +ABA -ABA +ABA Figure S5 (Chen) WT3xABRC321:HVA1 Fluorescence enhanced Fluorescence enhanced (a)(b) F F+TF Fluorescence enhanced LRP En/P Ex * LRP En/P Ex * LRP En/P Ex * LRP En/P Ex * LRP En/P Ex * LRP En/P * Ex LRP En/P Ex * C C C C C C C C C Cross section Longitudinal section Figure S5. LEA3 and 3xABRC321:HVA1 are expressed in cortical and exodermal cells overlaying the developing LRP in transgenic rice. Ten-day-old seedlings were treated with 10 M ABA for 16 h, and root elongation zones were sectioned for immunohistological fluorescence assay. (a) Localization of LEA3 in WT (b) Localization of LEA3 and HVA1 in transgenic line 3xABRC321:HVA1 (T-33). Fluorescence images in blue boxes have been enhanced, and in both blue and green boxes are shown in Figure 7b. LEA3 and HVA1 in WT and transgenic lines, respectively, are also expressed in cortex and exodermal cells overlaying the LRP as indicated by the asterisk. F: image of fluorescence field. F+T: composite images of fluorescence and transmission fields. Scale bar: 20 m. Abbreviation: C: cortex; En: endodermis; Ex: exodermis; P, pericycle. Figure S6 (Chen) +ABA-ABA a1 DR5:GUSHVA1 DR5:GUSDR5:GUSHVA1 DR5:GUS a2b2 (a) (b) b1 Figure S6. 3xABRC321:HVA1 and ABA enhances asymmetrical localization of auxin maxima in LRP founder cells and in RAM in transgenic rice. Three-day-old seedlings of transgenic lines DR5:GUS and HVA1 DR5:GUS were transferred to MS medium with or without 0.2 M ABA for 11 days. Crown roots were stained for GUS expression, treated with ethanol and examined under a microscope. Arrowhead indicates LRP. Enlarged photos of LRP are shown next to the boxed root segment and are also shown in Figure 9a,b. (a) Without ABA treatment. (b) With ABA treatment. (a1) and (b1) are transgenic line DR5:GUS. (a2) and (b2) are transgenic line HVA1 DR5:GUS. Scale bar: 500 m. DR5:GUS -ABA+ABA-ABA+ABA -ABA+ABA-ABA+ABA HVA1 DR5:GUS LRP RAM Figure S7 (Chen) Figure 7. 3xABRC321:HVA1 and ABA enhance asymmetrical localization of auxin in LRP founder cells and auxin accumulation in RAM in transgenic rice. Three-day-old seedlings of transgenic lines DR5:GUS and HVA1 DR5:GUS were transferred to MS medium with (+) or without (-) 0.2 M ABA for 11 days, and roots were stained for GUS expression in LR founder cells and RAM. (a) GUS expression in line DR5:GUS was suppressed by ABA. (b) GUS expression in line HVA1 DR5:GUS was enhanced or maintained with ABA treatment. Scale bar: 100 m. (a) (b) + ABA- ABA DR5:GUS + ABA- ABA HVA1 DR5:GUS Figure S8. 3xABRC321:HVA1 and ABA enhances asymmetrical localization of auxin in LRP founder cells. Three-day-old seedlings of transgenic lines DR5:GUS and HVA1 DR5:GUS were transferred to MS medium with or without 0.2 M ABA for 11 days, and roots were sectioned and stained for GUS expression. Asymmetrical localization of GUS was suppressed in line DR5:GUS but enhanced in line HVA1 DR5:GUS with ABA. Scale bar: 50 m. Abbreviation: C: cortex; Cc: companion cells; En: endodermis; P: pericycle. LR En/P C C CC C C C C Cc Figure S8 (Chen)