Supplemental Data. Ondzighi-Assoume et al. Plant Cell (2016) /tpc G 60

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1 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc ABA/Alexa 488 Merge Bright-field luorescence intensity A G 6 Average of fluorescence intensity of endodermis B Z 4 a a RAM 2 c Supplemental igure. In-situ Localization of ABA Accumulation in the ndodermis of the Growing Root Tip (A) to () istriution of ABA in 7-d-old primary roots of Araidopsis ol- grown on ½x MS. (A) Low magnification of confocal image of root stained with the anti-aba/alexa luor 488. (B) Bright-field micrograph. () Merged image of anti-aba/alexa luor 488 and right-field. (), () and () High magnification micrographs of area outlined with white dashed oxes in (A), (B) and () respectively. Green arrowheads indicate the endodermal layer, which strongly laels with the anti-aba antiody. White arrowhead points to the eginning of the elongation zone (Z). (G) luorescence intensity of ABA in the endodermal layer as oserved in M, N, O and P. These results show that LU significantly decreases the immunoreactivity of ABA in Araidopsis roots. rror ars represent the mean ± Standard rror (S) of n=4. ifferent letters denote a statistically significant difference among means at p-value <.5 (Tukey's test). RAM, root apical meristem. Scale ars: (A) to () = 5 µm.

2 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc A B Supplemental igure 2. In-situ Localization of ABA in the Quiescent enter of the Growing Root Tip (A) to () istriution of ABA in chemically fixed 7-d-old primary root tips of Araidopsis ol- grown on ½x MS. (A) Low magnification of confocal image of root stained with the anti-aba/alexa luor 488. (B) Same root stained with propidium iodide (PI). () Merged image of anti-aba/alexa luor 488 and PI. () High magnification micrographs of eight 7-µm slices of the area outlined with a white dashed ox in (). Green arrowheads indicate the endodermal layer, which strongly laels with the anti-aba antiody. White arrows and arrowheads point to the cortical/endodermal initial cells and quiescent center area, respectively. These results show a strong immunoreactivity of ABA in the cortical/endodermal initial cells and light staining in the quiescent center area. Scale ars: (A) to () = 5 µm and () = 2 µm.

3 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc ontrol ABA/Alexa 488 Bright-field Merge A I B J G K ABA+KNO 3 ABA KNO 3 H L Supplemental igure 3. NO 3 is Ale to Stimulate Active ABA Pools in the Growing Root Tip in the Araidopsis aa2- Mutant (A) to (L) onfocal micrographs showing the spatial localization of ABA in 7-d-old primary roots in the aa2- mutant. (A) to () Spatial localization of ABA in the endodermal layer of growing root tips stained with the anti-aba/alexa luor 488. () to (H) Bright-field micrographs showing the morphology of roots. (I) to (L) Merged images of ABA/Alexa luor 488 localization and Bright-field. (A), () and (I) ontrol (½x MS) roots. (B), () and (J) ABA-treated roots (2d). (), (G) and (K) KNO 3 -treated roots (2d). (), (H) and (L) represent roots co-treated with oth ABA and KNO 3 (2d). (B) and () Root tips exhiit an increased ABA pool in the presence of exogenous ABA. () The root tip exhiits an increase of ABA accumulation in response to KNO 3 as compared to (A). These findings indicate that NO 3 still stimulates ABA accumulation in the asence of ABA synthesis, indicating that NO 3 induces ABA accumulation independent of de novo ABA synthesis in the growing root tip. Treatment concentrations: mm Kl, mm KNO 3 (for a final concentration of 3 mm NO 3 ), µm ABA. Scale ars: (A) to (L) = 5 µm.

4 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc A B Xy Pe n o p R PI ergp Merge G H I Supplemental igure 4. ffect of Nitrate and ABA on the Pattern of ProSR:erGP Activity in the Growing Root Tip of Araidopsis (A) to (I) onfocal micrographs of Araidopsis transgenic roots carrying the ProSR:erGP construct. (A) Root stained with propidium iodide (PI) showing the cellular organization of root tissue. (B) ergp fluorescence showing the spatial pattern of the ProSR:erGP expression in the endodermis. () Merged image of PI and ProSR:erGP. () to (I) Merged micrographs of PI and ProSR:erGP and corresponding transverse line scans in untreated and treated 2d ProSR:erGP roots. () ontrol root (½x MS). () Kl-treated root. () KNO 3 -treated root. (G) LU-treated root. (H) ABA-treated root. (I) Root co-treated with oth ABA and KNO 3. Green arrowheads indicate peaks of relative fluorescence intensity of ProSR:erGP expression in the endodermal layer. Inset are transverse line scans of the relative fluorescence intensity (y-axis) across the image at the position (distance in pixels [x-axis]) indicated y green arrowheads. Green arrowheads in inset point to peaks of ergp fluorescence intensity of ProSR:erGP expression. Treatment concentrations: mm Kl, mm KNO 3 (for a final concentration of 3 mm NO 3 ) µm ABA, 5 µm LU. o, ortex, p, epidermis, n, endodermis, Pe, pericycle, R, root cap, Xy, xylem. Scale ars: (A) to (I) = 5 µm.

5 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc KNO 3 Kl ontrol RI RI RI ABA/Alexa 555 ProSR-erGP A G Merge Transverse line scan J 5 B H K 5 istance (pixels) 5 I 5 istance (pixels) L 5 5 istance (pixels) Supplemental igure 5. The Peak of ABA Staining olocalizes with Root Tip SR xpression in Nitrate-treated Araidopsis ProSR:erGP Roots (A) to (I) onfocal micrographs showing ABA localization and ProSR:erGP expression in 7-dold treated 2d and untreated primary roots. (A) to () Spatial localization of ABA in ol- wildtype roots stained with the anti-aba/alexa luor 555 (magenta). () to () rgp fluorescence micrographs showing the localization pattern of promoter SARROW expression in the endodermal layer. (G) to (I) Merged images of ABA/Alexa 555 fluorescence and ProSR:erGP fusion protein. (A), () and (G) ontrol (½x MS). (B), () and (H) Kl-treated roots. (), () and (I) KNO 3 -treated roots. () and (I) exhiit increased ABA immunofluorescence in the presence of an additional mm KNO 3. (J), (K) and (L) Plots of line scans showing ABA/Alexa luor 555 and ProSR:erGP fluorescence intensities taken transversally at a distance of 75 µm as indicated y the white arrowheads in (A), (B) and () respectively. Yellow arrowheads point to peaks of highest fluorescence intensity of ABA and ProSR:erGP oserved in the endodermal layer. All plots display a strong correlation etween the spatial localization of ABA and ProSR:erGP expression. RI, relative intensity. Scale ars: (A) to (I) = 5 µm.

6 Wt gna ait- ait-2 acg25- acg25-2 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc ait- (A) AIT (Atg6985) ait-2 SALK_72696 SALK_ UTR 3 -UTR ATG P RP acg25-2 Stop SALK_28873 (B) ABG25 (Atg796) acg25- SALK_ UTR 3 -UTR ATG P RP Stop ol- AIT ABG25 Wt H ait- L ait-2 acg25- P acg25-2 T P/RP RP/La. () ol- (Wt) AIT ABG25 ABA KNO 3 Kl ontrol G I J K M N O Q R S U V W Supplemental igure 6. ABA Transporter Genes Structure, Genotypes and Immunofluorescence In-situ of ABA in the Growing Root Tip in Araidopsis ABA Transporter Mutant Lines (A) and (B) Schematic representation of the AIT and ABG25 genes map and T-NA insertion sites. (A) The AIT gene contains five exons (numered lack oxes) separated y four introns (lack lines) with two T-NA insertional sites SALK_72696 (ait-2) and SALK_4643 (ait-) located in intron four and exon four respectively. (B) The ABG25 gene contains four exons (numered lack oxes) separated y three introns (lack lines) and has two T-NA insertional sites SALK_74738 (acg25-) and SALK_28873 (acg25-2) located in intron two and exon three, respectively. () PR analysis of the four T-NA insertion lines and ol- wild type genomic NAs. T-NA insertion sites in the gene were confirmed y PR using the left order primer (LBa.) and a gene specific primer (RP, reverse primer). The homozygous lines were confirmed for SALK_4643 (ait-) and SALK_72696 (ait-2) for the AIT gene and SALK_74738 (acg25-) and SALK_28873 (acg25-) for the ABG25 gene using gene-specific primes. () to (W) onfocal micrographs showing the spatial localization of ABA in 7-d-old primary roots stained with the anti- ABA/Alexa luor 488 in ol-o wild-type vs. oth ait and acg25 mutants. Spatial localization of ABA in the growing root tip of ol- wild-type (Wt) () to (G), ait- (H) to (K), ait-2 (L) to (O), acg25- (P) to (S) and acg25-2 (T) to (W). (), (H), (L), (P) and (T) ontrol (½x MS) roots. (), (I), (M), (Q) and (U) Kl-treated roots (2d). (), (J), (N), (R) and (V) KNO 3 -treated roots (2d). (G), (K), (O), (S) and (W) ABA-treated roots (2d). These results show that NO 3 is ale to stimulate ABA accumulation in the growing root tip of oth ait and acg25 mutants suggesting that NO 3 regulates ABA accumulation independent of the transport of ABA y AIT or ABG25. Treatment concentrations: mm Kl, mm KNO 3 (for a final concentration of 3 mm NO 3 ), µm ABA. Scale ars: () to (W) = 5 µm.

7 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc LBa. LBa. (A) BG/BGL (Atg524.) SALK_22533 ( g-) SALK_7573 ( g-2) 5 -UTR UTR ATG P RP SALK_24344 Stop (3 p) ( g-3) (B) () S-22LP/RP S-22RP/LBa. S-75LP/RP S-75RP/LBa. S-24LP/RP S-24RP/LBa. T-NA insertion lines ol- (Wt) p - ~778 - ~ ~ ~75 LBa. SALK_22533 (g-) SALK_7573 (g-2) SALK_24344 (g-3) 4 p AtBG f- () ol- (Wt) g- g-2 g-3 Supplemental igure 7. Araidopsis β-gluosias (BG) Gene Structure, Genotypes and Phenotypes of g Mutant Lines (A) Schematic representation of the BG gene map (locus numer AA443) and T-NA insertion sites. The BG gene has 3 ase pairs (p), contains twelve exons (numered lack oxes) separated y eleven introns (lack lines) and three T-NA insertional sites: SALK_22533 (g-) located in intron nine, SALK_24344 (g-3) inserted in exon eleven and SALK_7573 (g-2) inserted in exon twelve. (B) PR analysis of the three T-NA insertion lines and wild-type genomic NAs. T-NA insertion sites in the gene were confirmed y PR using the left order primer (LBa.) and a gene specific primer (RP, Reverse primer), which produced 695-p, 89-p, and 75-p, fragments for g-, g-2 and g-3 respectively. As a control, BG-specific products were amplified y PR with forward (R) and reverse (RP) primers, which produced 778-p, 95-p and 83-p fragments specific to g-, g-2 and g-3 respectively. () RT-PR analysis of the three T-NA insertion lines shows that lanes 4 and 5 (g-2) and 6 and 7 (g-) gave no detectale mrna, which confirms the PR results, indicating that these two lines are knockout mutants. However, RT- PR in lanes 2 and 3 (g-3) gave smaller ands, suggesting cryptic transcription yielding a truncated mrna. () Phenotypes of mature ol- wild-type and g mutant plants. Both mutants g- and g-2 exhiit decreased plant iomass compared to either ol- wild-type (Wt) or g-3. These findings show that oth g- and g-2 are loss-of-function mutants of the BG gene whereas the T-NA insertion line g-3 displays a truncated mrna. We used the g- and g-2 mutants for the immunofluorescence and qrt-pr analyses. Nucleotide sequences of primers used for these experiments are listed in supplemental Tale.

8 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc Lateral root numer per plant Lateral root density Primary root length (cm) Lateral root numer per plant Primary root length (cm) Lateral root density Nitrate 2d treatment Nitrate 7d treatment A B ontrol Kl KNO3 a a a ol- (Wt) g-2 g- ontrol Kl KNO3 a a a a a a a a ontrol Kl KNO3 a a a c ol- (Wt) g-2 g- ontrol a Kl KNO3 a cd cd cd cd cd cd ol- (Wt) g-2 g- ol- (Wt) g-2 g ontrol Kl KNO3 a a a a a a a a ol- (Wt) g-2 g ontrol Kl KNO3 a a c c c c cc ol- (Wt) g-2 g- Supplemental igure 8. The ABA-deficient g Mutant is Insensitive to the ffects of NO 3 on Root Architecture ol- wild-type and g mutants were sujected to either a transient, 2d nitrate treatment, or a continuous 7d nitrate treatment. or the transient nitrate treatment, seedlings were grown on ½x MS control medium for 5d and then transferred to either ½x MS control medium or ½x MS supplemented with either Kl or KNO 3 and grown for an additional 2d. or the continuous nitrate treatment, seedlings were grown continuously on either ½x MS control medium or ½x MS supplemented with either Kl or KNO 3 for 7d. (A) and () Primary root length of untreated and treated ol- wild-type and g-2 and g- roots for 2d and 7d respectively. (B) and () Lateral root numer of untreated and treated ol- wild-type and g-2 and g- roots for 2d and 7d respectively. () and () Lateral root density of untreated and treated ol- wildtype and g-2 and g- roots for 2d and 7d respectively. rror ars represent the mean ± S of three iological replicates (-5 roots recorded per treatment and per genotype), and different letters denote statistically significant difference among means at p-value <.5 according to two-way ANOVA (Tukey's test). Treatment concentrations: mm Kl and mm KNO 3 (for a final concentration of 3 mm NO 3 ).

9 Kl ontrol ABA Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc longation zone of primary roots ProRAB8:GP PI Merge A B merging lateral roots ProRAB8:GP PI Merge G H I J K L M N O P Q R KNO 3 S T U V W X Supplemental igure 9. NO 3 Strongly Stimulates ProRAB8:GP Activity in Primary and Lateral Roots (A) to (X) onfocal micrographs of untreated and treated transgenic roots carrying the ProRAB8:GP construct. (A), (G), (M) and (S) GP fluorescence showing promoter RAB8 activity in the elongation zone of primary roots. (B), (H), (N) and (T) ProRAB8:GP roots stained with propidium iodide (PI). (), (I), (O) and (U) Merged micrographs of PI and ProRAB8:GP. (), (J), (P) and (V) GP fluorescence showing promoter RAB8 activity in lateral roots. (), (K), (Q) and (W) ProRAB8:GP lateral roots stained with PI. (), (L), (R) and (X) Merged micrographs of PI and ProRAB8:GP. Seedlings were grown on ½x MS control medium for 5d and then transferred to ½x MS supplemented with the compound of interest, and grown for an additional 2d. (A) to () ontrol roots (½x MS). (G) to (L) Kl-treated roots. (M) to (R) KNO 3 - treated roots. (S) to (X) ABA-treated roots. Treatment concentrations: mm Kl, mm KNO 3 (for a final concentration of 3 mm NO 3 ), µm ABA. Scale ars: (B) to (X) = 5 µm.

10 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc Primary root length (cm) ontrol Kl KNO 3 ABA ABA+KNO 3 A B LU LU KNO 3 LU KNO 3 +LU LU ABA LU ABA+KNO 3 K G H I a ac a c J.5.5 Supplemental igure. ffect of NO 3, ABA, and luridone on Root Growth in Araidopsis Seedlings ol- wild-type seedlings were grown on ½x MS control medium for 5d and then transferred to ½x MS supplemented with the compound of interest, such as Kl, KNO 3, ABA, LU, or ABA+KNO 3, and grown for an additional 2d. LU-treated roots were first treated with LU for d, and then followed y d of secondary treatment, as indicated y the arrow. (A) to (J) Photographs of 7-d-old seedlings grown with the indicated treatments. (K) Total primary root length of untreated and treated ol- roots. rror ars indicate the mean ± S of three iological replicates (5 roots recorded per replicate and per treatment). ifferent letters indicate a statistically significant difference in the total length of roots compared to the untreated control or to KNO 3 - treated roots (p-value <.5) (Tukey s test). Treatment concentrations: mm Kl, mm KNO 3 (for a final concentration of 3 mm NO 3 ), µm ABA, 5 µm LU. Scale ars: (A) to (J) =.5 cm.

11 P Alexa-luor 555 Alexa-luor 488 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc A Bright-field Merge A G B H I J ontrol M KNO 3 Mt N - A K R R ontrol - A N V Pl KNO 3 V W Pre-immune L Mt ontrol Pre-immune O Mt Mt R KNO 3 V Pl W V G µm cis(±)aba µm cis(±)aba Supplemental igure. Immunological ontrols for ABA Antiody Specificity in the Growing Root Tip at the Tissue and ellular Level (A) and (B) Immunofluorescence laeling controls using anti-rait IgG secondary antiody tagged either with Alexa-luor 488 (A 488) or Alexa-luor 555 (A 555) respectively, oth with omission of the primary antiody anti- ABA (-A). () and () Bright-field micrographs corresponding to fluorescence images (A) and (B) respectively, and the corresponding merged images (G) and (H). () Auto-fluorescence micrograph of the growing root tip viewed under a cyan fluorescent protein (P) filter set. () Bright-field micrograph corresponding to () and the resulting merged image (I). (J) to (O) Immunogold laeling control experiments performed on control (½x MS) and KNO 3 - treated roots with or without the primary antiody anti-aba (-A). (J) and (M) lectron micrographs of control and KNO 3 -treated roots chemically fixed and laeled with anti-rait IgG tagged gold particle without the primary antiody anti-aba in endodermis cells showing cytoplasmic space and nucleus in endodermis cells respectively. (K) and (N) ontrol and KNO 3 -treated roots High Pressure reezing-fixed and chemically fixed respectively, and laeled with a sustituted pre-immune rait serum as a primary antiody and anti-rait IgG tagged gold particle as a secondary antiody. (L) and (O) ontrol and KNO 3 -treated roots High Pressure reezing-fixed and chemically fixed respectively, and laeled with a primary antiody anti-aba pre-incuated with µm (±)ABA overnight at 4 and then lotted with an anti-rait IgG tagged gold particle as a secondary antiody. In all control experiments, no fluorescence or gold particles were oserved. These controls confirm the specificity of the primary and secondary antiodies for the root tip cells studied. W, cell wall;, cytoplasm; R, endoplasmic reticulum; Mt, mitochondria; G, Golgi; N, nucleus; Pl, plastid; V, vacuole. Scale Bars: (A) to (I) = 5 µm. (J) to (O) =.5 µm.

12 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc Supplemental Tale : Primer Sequences Used for PR Genotyping AGI code Atg796 Atg6985 Atg524 Gene name ABG25 AIT BG T-NA insertion line # SALK_74738 (acg25-) SALK_28873 (acg25-2) SALK_4643 (ait-) SALK_72696 (ait-2) SALK_22533 (g-) SALK_7573 (g-2) SALK_24344 (g-3) Primer sequences LP: 5'-AATGAGAAAAAGGATG-3' RP: 5'-TTTTAAAAGTGGTTATG-3' LP2: 5'-TGTGGAAAGTATTTAT-3' RP2: 5'-AAGAAAGATTGGTGATT-3' LP: 5'-AAAGTTTTTTGAGTGG-3' RP: 5'-AATGGTGTTAGTAGTG-3' LP2: 5'-TTTTTTAGTTTGG-3' RP2:5'-TGAAAGGAGATTATAAGTTAAGTAG-3' LP: 5'-TGTTGAGATTAGAAATG-3' RP: 5'-TTTATTAAAATGGGTG-3' LP2: 5'-TTTGGTAAAATTATGG-3' RP2: 5'-AGGTAGATGAAAAGAT-3' LP3: 5'-TTTGGTAAAATTATGG-3' RP3: 5'-AGGTAGATGAAAAGAT-3'

13 Supplemental ata. Ondzighi-Assoume et al. Plant ell (26).5/tpc Supplemental Tale 2: Primer Sequences Used for RT-qPR xperiments AGI code Gene name Primer sequences At3g878 Actin2 W: 5'-TATGAATTAGATGGGAAG-3' RV: 5'-TGGAAAAGATTTGGGAT-3' Atg524 BG W: 5'-TTATATATTAGTGTTTGAAAAG-3' RV: 5'-TAGAGTTTTTAGTTG-3' At2g3286 BG2 W: 5'- AATGGGATGGAGAGATGA -3' RV: 5'- GTAATGTTAGTAGTTGTTA -3' Atg5234 ABA2 W: 5'-ATGTAAGAAATGAATTTTT-3' RV: 5'-GATGTTGGAAAAATAAG-3' At2g3627 ABI5 W: 5'-ATGGGAAGGAGAATAA-3' RV: 5'-AGTGTGTGTTGTTGTT-3' At5g664 RAB8 W: 5'-AAGATAAGGAGAAGTTGAGG-3' RV: 5'-GTAAAAAAAATGAGGAG-3' At5g523 R29A W: 5'-GATATGAAAGGATGTGG-3' RV: 5'-GTATAGGTTTTTG-3' Atg7776 NIA W: 5'-ATGTAAAGAAAGAAGT-3' RV: 5'-AGGAGATGGATGAGTT-3' Atg373 NIA2 W: 5'-GGTTAGATATTGGAG-3' RV: 5'-ATGAGAAAGAAT-3' At2g562 NIR W: 5'-ATGGGATGTTAAAGAG-3' RV: 5'-AATGGAAAATGTGA-3' References (Xu et al., 22) (Xu et al., 22) (Xu et al., 22) (Lee et al., 26) (ui et al., 22) (uan et al., 23) (Xu et al., 22) (Wang et al., 24) (Wang et al., 24) (Konishi and Yanagisawa, 2)