A AT3G50310 B MS+ABA. Intensity. aik1-1

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Amanda Hicks
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1 Intensity T3G531 + Supplemental Figure 1. Screening for mutant., IK1 expression analysis by tgenexpress Visualization Tool., Screening for mutant under medium containing 5 μm by root-bending assay. 5-d-old seedlings were transferred to medium supplemented with 5 μm. Pictures were taken 3 d later. The wild-type and aik1 mutant were marked with red arrows and white lines.

IK1/CTIN IK1/CTIN GFP Transmission Chloroplast Merged GFP-IK1 GFP

The IK1 protein localization in cells and gene expression in

, Localization of GFP signals from IK1 fused with GFP.

merged images of rabidopsis mesophyll protoplasts transfected

, Expression of IK1 promoter-gus in wild type plants.

GUS staining of transgenic plants shows IK1 expressed in

from 1- d-old seedlings, the stems, flowers, rosette leaves and

2 IK1/CTIN IK1/CTIN GFP Transmission Chloroplast Merged GFP-IK1 GFP a b c d e f g C D Time (h) Supplemental Figure. The IK1 protein localization in cells and gene expression in different tissues., Localization of GFP signals from IK1 fused with GFP. Epifluorescence, chloroplast autofluorescence, bright field and merged images of rabidopsis mesophyll protoplasts transfected with constructs expressing different fusion proteins, bars = 5 μm., Expression of IK1 promoter-gus in wild type plants. GUS reporter gene was under the control of the IK1 promoter. GUS staining of transgenic plants shows IK1 expressed in seedlings ([a] to [c]), root [d], leaf [e], flowers [f] and silique [g]. C, IK1 expression in root, stem, flower, leaf, stem-leaf and cotyledon. The mrn of cotyledons were from 7-d-old seedlings, the roots were from 1- d-old seedlings, the stems, flowers, rosette leaves and stem leaves were from -d-old plants. The expression of IK1 was examined by qrt-pcr. Values were normalized to the expression of CTIN. Error bars represent ± SD of three replicates. D, The IK1 expression in 1-d-old wild-type seedlings with 1 μm treatment monitored by qrt-pcr.

IK1/CTIN IK1/CTIN aik1- Promoter MKKK C aik1- aik1- IK1 CTIN D 1.5 1..9..3 aik1- E 1 18 15 1 9 3 aik1- - + Supplemental Figure 3. Screening and identification of the IK1 mutants.

3 IK1/CTIN IK1/CTIN aik1- Promoter MKKK C aik1- aik1- IK1 CTIN D aik1- E aik Supplemental Figure 3. Screening and identification of the IK1 mutants., The T-DN insertion mutants of and aik1- were examined by PCR analysis., The map of IK1 T- DN insertion mutants. C, The semi-quantative RT-PCR analysis of IK1 expression in the and aik1 mutant plants. D, nalysis of the IK1 expression in and mutants by qrt-pcr showed that and aik1- were knockdown mutants. E, The qrt-pcr results showed the IK1 expression in and mutants, with or without, the seedlings were exposed to 1 μm.

4 Relative root growth (%) Root length (mm) 8 aik aik (μm) Supplemental Figure. aik1 plants were insensitive to in root growth with a dose-dependent manner., aik1 showed insensitive root growth to different concentration of. -d-old seedlings were treated with for 3 d. Root length was measured 3 d after the seedlings were transferred to -containing medium. Values are the means of approximately seedlings (± SD) from three independent experiments., Root growth of the and aik1 mutants is affected by. Relative root growth is root length in the presence of expressed as a percentage of root length in the absence of. Values are the means of approximately seedlings (± SD) from three independent experiments. sterisks indicate significant differences from the at P <.5 and P <.1 by Student s t-test.

IK1/CTIN 1 1 1 8 OE1 OE OE1 OE MOCK 5 μm 5 μm Supplemental Figure 5.

5 IK1/CTIN OE1 OE OE1 OE MOCK 5 μm 5 μm Supplemental Figure 5. Over-expression of IK1 showed sensitive to in the inhibition of plant growth and development., The expression of IK1 was measured by qrt-pcr in IK1 overexpression transgenic plants., 5-d-old seedlings of and IK1 overexpression seedlings (OE1, OE) were transferred to medium with or without. The medium contained the same volume of ethanol as MOCK. Pictures were taken at 5 d after the seedlings transferring.

Root length (μm) EZ length (μm) aik1- + a b a b a b a b a b a b C 5 7 aik1- aik1-8 8 3 + 1 +

, Roots with or without were stained by 1 μm FM-.

green arrowheads indicate the approximate position of the maturation zone and white lines

For pictures a, bars = 5 μm; for pictures b, bars = 1 μm.

treated for. Values are means ± SD from groups of five seedlings.

6 Root length (μm) EZ length (μm) aik1- + a b a b a b a b a b a b C 5 7 aik1- aik Supplemental Figure. IK1 negatively regulates root cell division and cell elongation., Roots with or without were stained by 1 μm FM-. White arrowheads indicate the approximate position where cells began to elongate noticeably, green arrowheads indicate the approximate position of the maturation zone and white lines indicate the length of the elongation zone (EZ). For pictures a, bars = 5 μm; for pictures b, bars = 1 μm., Root lengths from the tip to the elongation zone of seedlings with or without (5 μm) treated for. Values are means ± SD from groups of five seedlings. sterisks indicate significant differences from the at P <.1 by Student s t-test. C, Root lengths of the elongation zone. Values are means ± SD from groups of five seedlings. sterisks indicate significant differences from the at P <.1 by Student s t-test.

7 Stomatal conductance (mmol m - s -1 ) 3 Light intensity (μmol m - s -1 ) Time (min) Supplemental Figure 7. Stomatal conductance in response to light and dark in wild-type and aik1 mutant plants. Values are the means of ± SD (n = 3) from about 5-d-old plants in three independent experiments. sterisks indicate significant differences from the at P <.5 by Student s t-test.

8 Total cell numbers per mm aik1- Supplemental Figure 8. Total cell numbers of the leaf epidermis in wild-type and aik1 mutants. The numbers of guard cells and pavement cells per mm of the leaf epidermis, ± SD determined from leaves of at least 15 individual wild-type and mutant plants. Five independent counts were performed on each leaf.

9 Query Sequence M313 M7 IK1 K3M Interpro Protein kinase-like domain TP binding site ctive site Position Lys3 (K3) 3 Supplemental Figure 9. The sequence analysis of IK1 protein by InterPro ( and sketch map of the expressed proteins of IK1. IK1 is a classical protein kinase, the whole sequence of IK1 include protein kinase-like domain, TP binding site and active site. The sketch map shows two truncated segments of IK1, M313 (1 313 ) and M7 (1 7 ), and an inactive form of IK1 (IK1 K3M ).

Root length (mm) MKK5/CTIN MKK5/CTIN MKK/CTIN mkk5-1 mkk5- Promoter UTR MKK5 UTR mkk

Decreased sensitivity of root growth to in aik1 and mkk5 mutants, but not mkk.

() The schematic diagram of MKK and MKK5 T-DN insertion mutants.

sterisks indicate significant differences from the at P <.5 and P <.1 by Student s t-test.

10 Root length (mm) MKK5/CTIN MKK5/CTIN MKK/CTIN mkk5-1 mkk5- Promoter UTR MKK5 UTR mkk Promoter UTR MKK UTR C 1. mkk mkk5-1 mkk5-.9. E mkk mkk5-1 D.3 1. mkk mkk5-1 mkk5- F mkk mkk5-1 G Supplemental Figure 1. Decreased sensitivity of root growth to in aik1 and mkk5 mutants, but not mkk. () PCR analysis screen of mkks mutants. () The schematic diagram of MKK and MKK5 T-DN insertion mutants. (C, D) The qrt-pcr results showed the expression level of MKK and MKK5 in, mkk and mkk5. sterisks indicate significant differences from the at P <.5 and P <.1 by Student s t-test. (E, F) 5-d-old seedlings were transferred to medium supplemented with 5 μm. Pictures were taken 3 d later. Values are the means of approximately seedlings. sterisks indicate significant differences from the at P <.1 by Student s t-test. (G) The qrt-pcr results showed induced the expression level of MKK5 in.

11 Root length (mm) EZ length (μm) Root length (μm) Water loss (% of FW) MKK9/CTIN Root length (mm) MKK5/CTIN C aik1- MKK5 DD D (- DEX) aik1- E + (- DEX) F G (- DEX) + H I K Time (min) MOCK L 1 + (- DEX) Time (h) W: nti-flg Protein stain (+ DEX) Rubisco C J (+ DEX) (+ DEX) 5 μm. μm DEX 5 μm +. μm DEX

12 Supplemental Figure 11. Decreased sensitivity to in root growth and water loss in plants. and, The qrt- PCR results showed the decreased expression of MKK5 in. sterisks indicate significant differences from the at P <.5 by Student s t-test. C, The decreased sensitivity of root growth to in plants (without DEX, - DEX). 5-dold seedlings were transferred to medium supplemented with 5 μm. Pictures were taken 3 d later. D, 5-d-old seedlings were treated with for 3 d, and the elongated root length was as shown. Values are the means of approximately seedlings. sterisks indicate significant differences from the at P <.1 by Student s t-test. E, Roots were stained with FM-. White arrowheads indicate the approximate position where cells began to elongate noticeably, green arrowheads indicate the approximate position of the maturation zone and white lines indicate the length of the elongation zone (EZ). ars = μm. F, Root lengths (-DEX) from the tip to the elongation zone. Values are means ± SD from groups of five seedlings. sterisks indicate significant differences from the at P <.1 by Student s t-test. G, Root lengths (-DEX) of the elongation zone. Values are means ± SD from groups of five seedlings. sterisks indicate significant differences from the at P <.1 by Student s t-test. H, nalysis of transpirational water loss. bout four-week-old rosette leaves were detached and allowed to dry at room temperature for the indicated periods. Water loss is presented as a percentage of initial fresh weight (n = 5/per sample). I and J, 5-d-old seedlings of and were transferred to. μm DEX-containing medium for d to activate MKK5 in seedlings, and then the plants (with DEX, + DEX) were transferred to medium supplemented with 5 μm and. μm DEX. Pictures were taken and the elongated root length was determined 3 d later. Values are the means of approximately seedlings. K, 5-d-old plants and seedlings were transferred to medium, or medium with. μm DEX/5 μm, or medium both contained DEX and. The medium contained the same volume of ethanol as MOCK. The pictures were taken 1 d later. L, DEX induced the expression of in seedlings. 5-d-old seedlings of were transferred to. μm DEX-containing medium for d (time point as h), and then the plants were transferred to. μm DEX-containing medium for 7 h. Total protein was extracted from rabidopsis seedlings, expression of the transgene was monitored by western blot (W) analysis. Equal amounts of the samples were resolved on SDS-PGE and stained with C. The large subunit (LSU) of ribulose-1,5- bis-phosphate carboxylase/oxygenase (Rubisco) is shown as the loading control for equal protein amounts in seedlings.

Relative root growth (%) Root length (mm) MPK3/CTIN MPK/CTIN YFP Transmission Chloroplast

G D 1 1 1 8 55 kda 3 kda mpk-1 mpk3 5 5 C 1 1 1 8 - + - + (μm ) E 15 1 9 3 mpk3 mpk-1 5 5

Decreased sensitivity of root growth to in mpk3 and mpk mutants.

protoplasts co-transfected with constructs expressing different fusion proteins.

and C, The qrt-pcr results showed the expression level of MPK3 and MPK in and plants with

Pictures were taken at 3 d after transferring.

sterisks indicate significant differences from the at P <.1 by Student s t-test.

For relative root growth, the root length of was considered as 1, the relative root length

13 Relative root growth (%) Root length (mm) MPK3/CTIN MPK/CTIN YFP Transmission Chloroplast Merged F MKK5-YFP C +MPK3-YFP N MKK5-YFP C +MPK-YFP N MKK5-YFP C +YFP N mpk-1 mpk- + G D kda 3 kda mpk-1 mpk3 5 5 C (μm ) E mpk3 mpk (μm ) + /NaCl min MPK Supplemental Figure 1. Decreased sensitivity of root growth to in mpk3 and mpk mutants., nalysis of MKK5 interactions with MPK3 and MPK by ifc, with rabidopsis mesophyll protoplasts co-transfected with constructs expressing different fusion proteins. Vectors expressing the MKK5-YFP C and YFP N were used as negative controls. and C, The qrt-pcr results showed the expression level of MPK3 and MPK in and plants with or without. D and E, 5-d-old seedlings were transferred to medium supplemented with 5 μm. Pictures were taken at 3 d after transferring. Values are the means of approximately seedlings. sterisks indicate significant differences from the at P <.1 by Student s t-test. F, Root growth of the and mpk mutants as affected by. For relative root growth, the root length of was considered as 1, the relative root length of mpk was statistical analyzed. Values are the means of at least 3 seedlings (± SD) from three independent experiments. sterisks indicate significant differences from the at P <.1 by Student s t-test. G, In-gel assay for MPK activity in -treated and seedlings. The NaCl-treated mpk3 and mpk seedlings was as control.

() Cotyledon phenotype of wild type, aik1-1, mkk5, mpk, mkk5 and mpk plants.

14 Length of cotyledon (cm) + mkk5 mpk mpk mkk5 mkk5 mpk mkk5 mpk + Supplemental Figure 13. Comparative analysis of, mkk5 and mpk single and double mutants in response to in terms of seedling growth. () Cotyledon phenotype of wild type, aik1-1, mkk5, mpk, mkk5 and mpk plants. The cotyledons were detached and measured after the seedlings were transferred to 5 μm -contained medium for d. ar =.5 cm. () Statistical data of the cotyledon length. Data are shown as means ± SD (n = 1 plants), asterisks indicate significant differences from the and mutants at p<.5 and p<.1 by Student t-test.

Laplaze et al. (2007). Cytokinins act directly on lateral root founder cells to inhibit root initiation.

Laplaze et al. (2007). Cytokinins act directly on lateral root founder cells to inhibit root initiation. Laplaze et al. (27). Cytokinins act directly on lateral root founder cells to inhibit root initiation. A Root length (cm) 5 4,5 4,5 2,5 2,5,5 DAG B Lateral root density (LR/cm) 4,5 N.T. 2,5-8M 2BA -7M,5