supplementalfigure1.morphologicalphenotypesof int173 snc1 · 3/20/2012 · 22°c 28°c col-0...

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Supplemental Data. Mang et al. Plant Cell. (2012). 10.1105/tpc.112.096198

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Supplemental Figure 1. Morphological phenotypes of int173 snc1Growth was measured by diameters of rosettes as well as length,width, and petiole length of the 4th leaf. The wild‐type Col‐0, snc1‐1,int173, and int173 snc1 plants were grown under constant light for 3weeks at 22°C or 28°C. Error bars represent standard deviation (SD)(n=30). Asterisks indicate a significant difference of 28°C grown plantsfrom the 22°C grown plants of the same genotype with Student’s t test(p ≤ 0.05).

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Supplemental Figure 2. The int173mutant is a new loss‐of‐function allele of ABA2.(A) Seed germination of the wild‐type Col‐0 (left panels) and int173 (right panels) in the presence of0.2 M NaCl (upper panels) and 0.33 M sucrose (lower panels). Photographs were taken at 7 daysafter sowing. Inset in upper panel is a magnified view of a representative seedling.(B) The aba2‐1 snc1‐1 double mutants were more resistant to Pseudomonas syringae pv tomato (Pst)DC3000 at 28°C than the wild type or the single mutants. Shown is bacterial growth at 0 day postinoculation (dpi) or 3 dpi. Error bars represent standard deviations (n=3). Asterisks indicate asignificant difference from the wild type Col‐0. Please also define the error bars and give the n.

Supplemental Figure 3. Categorization of subcellular distribution of fusion proteinsof SNC1 and GFP expressed in Arabidopsis protoplasts.Protoplasts were isolated from the wild‐type Col‐0 plants and transformed withp35S::SNC1WT:GFP. Shown are images of green fluorescent signals (from GFP), redfluorescent signals (from chlorophyll), and merged signals of green and redfluorescence from two representative protoplasts. Pictures were taken with ZEISSAXIO. The intensity of GFP signals was determined by subtracting the red signals fromthe green signals using the software Image J. A protoplast (such as one shown in theupper panel) was categorized as having a strong nuclear GFP signals (named as 2N) ifthe nucleus/cytosol signal ratio was greater than or equal to 10, whereas a protoplast(such as the one shown in the lower panel) was categorized as having a weak nuclearsignal (named 1N) if the nucleus/cytosol signal ratio was less than 10.

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Supplemental Figure 4. Growth phenotypes of pSNC1:SNC1‐1:GFPtransgenic lines in Col‐0 and aba2‐21Shown are transgenic plants of pSNC1::SNC1‐1:GFP in the wild‐type Col‐0 or aba2‐21 background as well as the wild‐type Col‐0, snc1‐1, aba2‐21,and aba2‐21 snc1‐1 plants grown at 22°C or 28°C before bolting. Thetransgenic plants of pSNC1::SNC1‐1:GFP in both Col‐0 and aba2‐21displayed a dwarf phenotype at 22°C while only aba2‐21 expressingpSNC1::SNC1‐1:GFP displayed a dwarf phenotype at 28°C.

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Supplemental Figure 5. Expression of ABA biosynthesis genes at 22°C and 28°CShown are quantitative RT‐PCR of expression of ABA biosynthesis genes ABA1 (A),NCED5 (B), NCED9 (C), AAO3 (D), and ABA2 (E). Total RNAs were isolated from leaves of3‐week‐old plants grown under constant light at 22°C or 28°C. The TUBULIN gene wasused as an internal control. Expression in Col‐0 at 22°C was normalized to 1. Valuesindicate means ± SD (n=5).

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Supplemental Figure 6. ABA deficiency enhances nuclear accumulation and celldeath‐inducing activity of SNC1WT:GFP in N. benthamiana.The SNC1WT:GFP fusion protein was transiently expressed under the 35S promoterusing Agro‐infiltration in N. benthamiana leaves at 22°C and 28°C. Infiltrated leaveswere treated with either buffer (mock) or the ABA biosynthesis inhibitor NDGA.GFP signals were monitored using confocal microscope (upper panels) and celldeath was visualized after tissue clearing (lower panels). Red circles indicateinfiltration sites and pink arrows point to some but not all areas of cell death.


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Supplemental Figure 7. Expression levels of RPS4:GFP fusion proteins in N. benthamiana.The upper panel shows expression levels of RPS4:GFP in N.benthamiana treated with buffer (mock) or NDGA at 22°C or28°C (as in Figure 6) detected by immunoblotting using α‐GFP antibodies. The lower panel shows Ponceau S‐stainedproteins as a loading control.


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Supplemental Figure 8. RPS2‐ and RPM1‐mediated diseaseresistances in ABA mutants.Growth of Pst DC3000 avrRpt2 (A) and Pst DC3000 avrRpm1 (B) inwild type Col‐0 and various ABA mutants at 22°C and 28°C at 0 and3 days post inoculation (dpi). The aba1‐6, aba2‐1, and aba2‐21mutants are ABA deficient, while abi4‐1 is ABA insensitive. Thesnc1‐4, rps2, and rpm1 mutants were used as controls. Valuesrepresent mean ± SD (n=3). The asterisks indicate a significantdifference from the wild type plants as determined by Student’s ttest (P ≤ 0.05). Similar results were obtained in two independentexperiments.

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Supplemental Figure 9. Differential distributionof the SNC1 and GFP fusion proteins inepidermal and mesophyll cells of N.benthamiana and Arabidopsis(A) Nuclear fractionation of whole leaves of N.benthamiana expressing SNC1WT:GFP or SNC1‐1:GFP. The two proteins were predominantlyfound in the nuclei‐depleted fraction with orwithout the treatment with 10 μM NDGA. Shownare immunoblots of total protein, nuclei‐depleted fraction, and nuclear‐enriched fraction.PEPC and histone were used as markers fornuclei‐depleted and nuclear‐enriched fractions,respectively. The nuclear‐enriched fraction wasloaded at 20 times higher concentrationcompared to the nuclei‐depleted fraction.(B) Expression of SNC1‐1:GFP and GFP under thecontrol of the CaMV35S promoter in epidermaland mesophyll cells of N. benthamiana visualizedby confocal microscope. Images were taken at 2days after Agro‐infiltration.(C) Expression of pSNC1::SNC1‐1:GFP (upperpanels) and p35S::GFP (lower panels) intransgenic Arabidopsis plants visualized byconfocal microscopes. Leaves were detachedfrom 3 individual T3 plants grown on soil for 4weeks and representative areas werephotographed using a confocal microscope. Barsrepresent 25 μm.


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Gene Name Primer Name Primer Sequences

ABA1 ABA1-F GAACGTACTATAAAGGGAGAATGG

ABA1 ABA1-R CTGAGACGAAGGGATCACAAT

ABA2 ABA2-F GATAGACATGATAAATTGGCGG

ABA2 ABA2-R TCTCTTGTGTTCTTGATGTGACAG

ICS1 ICS1-F GGGGATAAGGGGTTCTCACAA

ICS1 ICS1-R GCCCTAGTTACAACCCGAAAAG

NCED5 NCED5-F CCTCCGTTAGTTTCACCAACACT

NCED5 NCED5-R GGTGTGTCGGAGACGGAGTT

NCED9 NCED9-F GGAAAACGCCATGATCTCACA

NCED9 NCED9-R AGGATCCGCCGTTTTAGGAT

PR1 PR1-F CTTTGTAGCTCTTGTAGGTGCTCT

PR1 PR1-R TCCTGCATATGATGCTCCTTATTGA

SNC1 ColB1 ATATGGAGATAGCTTCTTCTTCTG

SNC1 ColB4 AAGATCATATCCGGCCATAACTG

Supplemental Figure 10: Sequences of primers used in RNA expression analyses.Listed are primers and their sequences used in RNA blot and RT‐PCR analyses. F stands for forward (sense direction), and R stands for reverse (antisense direction).

supplementalfigure1.morphologicalphenotypesof int173 snc1 · 3/20/2012 · 22°c 28°c col-0...

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