Nature - USA (2020-01-02)

86 | Nature | Vol 577 | 2 January 2020

Article

24 h Mock 24 h RGF1

Mock Col-0 RGF1 Col-0 Mock rgfr123 RGF1 rgfr123

a

e

b

gh

c

d

f

Mock RGF1 Mock RGF1

Col-0

Col-0

rgfr123

rgfr123

24 h Mock

24 h RGF1

To tal NBT intensity (AU)

8,0007,000 Mock RGF1

6,0005,000

4,0003,000

2,0001,000

0

Mock

RGF1

Col rgfr 123

Average

BES-H

O 2

-Ac intensity 2

4,000

3,000

2,000

1000

0

Mock 20 nM RGF1

Mock 20 nM RGF1

40,000

30,000

20,000

10,000

0

To tal NBTintensity (AU)

Average intensityof BES-H

O 2

-Ac 2

(^7060)
50
(^4030)
(^2010)
0

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  Fig. 1 | Distribution of ROS levels upon RGF1 treatment. a, Confocal images of
  roots 24 h after mock treatment or treatment with 20 nM RGF1. Propidium
  iodide (PI) staining is in red; H 2 O 2 -BES-Ac f luorescence is in green. b, Roots
  stained with NBT 24 h after mock treatment or treatment with 20 nM RGF1.
  c, Quantification of H 2 O 2 -BES-Ac intensity in the meristematic zone (n = 6
  independent roots; P < 0.003). d, Quantification of NBT staining intensity (in
  arbitrary units, AU) in the meristematic zone (n = 7 independent roots;
  P = 3.16 × 10−5). e, Confocal images of roots 24 h after mock treatment or
  treatment with 20 nM RGF1 in wild-type roots (Columbia-0 (Col-0)
  background) or rg fr 1/2/3 mutants. Staining as in a. f, Quantification of H 2 O 2 -
  BES-Ac staining intensity in the meristematic zone in wild-type and rg fr 1/2/3
  roots (n = 5 independent roots; P < 0.025). g, Wild-type or rg fr 1/2/3 roots
  stained with NBT 24 h after mock treatment or treatment with 20 nM RGF1.
  h, Quantification of NBT staining intensity in the meristematic zone of wild-
  type or rg fr 1/2/3 roots (n = 5 independent roots; P = 1 .65 × 10−6). White and blue
  arrowheads indicate the junction between the meristematic and elongation
  zones. Scale bar, 50 μm. Bar graphs show means. Error bars show ± s.d. Dots
  indicate each data point. P values are calculated by two-sided Student’s t-test.
  ab c
  e
  h
  ij k
  d
  fg
  Expression of
  RITF1
  Expression of
  RITF1
  Relative expression
  1,000
  800
  600
  400
  200
  (^0) 1 h Mock 1 h 100 nM RGF1 Meristematiczone Elongationzone Differentiationzone
  50
  40
  30
  20
  10
  0
  1 h RGF1 6 h RGF1 24 h RGF1
  20 nM RGF1
  Mock
  4.0
  3.0
  3.5
  2.5
  1.5
  0.5
  2.0
  1.0
  0
  Col-0
  rgfr123
  Col-0
  Mock Mock
  rgfr123
  RGF1 RGF1
  To tal
  pRITF1–GFP
  intensity (AU)
  Col-0 rgfr123
  16,000
  14,000
  12,000
  10,000
  8,000
  6,000
  4,000
  2,000
  0
  Col-0 XVE–RITF1 Col-0 XVE–RITF1
  Mock Oestradiol MockOestradiol MockOestradiol Mock Oestradiol
  Col-0 XVE–RITF1
  Mock OestradiolMockOestradiol
  Col-0 XVE–RITF1 Col-0 XVE–RITF1
  Mock1 0 μm
  Oestradiol Mock^10 Oestradiolμm Mock 10 μm
  Oestradiol
  Number of cells inmeristematic zone
  80
  70
  60
  50
  40
  30
  20
  10
  0
  Average
  BES-H
  O 22
  -Ac intensity
  Average
  NBT intensity
  Col-0 XVE–RITF1
  (^180160)
  (^140120)
  (^10080)
  (^6040)
  (^200)
  140
  120
  100
  80
  60
  40
  20
  0

  
  


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  Fig. 2 | Expression of RITF1 and phenotype of RITF1 overexpression line.
  a, Expression of RITF1 in the meristematic zone 1 h after treatment with 100 nM
  RGF1, measured by RNA-seq (CPM, counts per million mapped reads; n = 3
  independent experiments; P < 0.01. b, Expression of RITF1 in developmental
  zones as measured by RNA-seq (FPKM, fragments per kilobase of transcript per
  million mapped reads). c, Expression of RITF1 in the meristematic zone of wild-
  type and rg fr1/2/3 roots upon treatment with RGF1, measured by quantitative
  R T– P C R (n = 3 independent experiments; P < 0.001, P < 0.002, P < 0.02).
  d, Confocal images of pRITF1- GFP expression and PI staining in wild-type and
  rg fr1/2/3 roots after RGF1 treatment. e, Total intensity of pRITF1- GFP
  expression in wild-type and rg fr1/2/3 roots 24 h after RGF1 treatment (n = 5
  independent roots; P < 0.001). f, g, Confocal images of roots stained with PI (f)
  and H 2 O 2 -BES-Ac (g) in Col-0 and X VE–R ITF1 roots after mock or oestradiol
  treatment. h, Light microscope images of NBT-stained roots after mock or
  oestradiol treatment. i, Number of cells in the meristematic zone in Col-0 and
  X VE–R ITF1 roots after mock or oestradiol treatment (n = 6 independent roots;
  P < 0.001). j, Average intensity of BES-H 2 O 2 -Ac in the differentiation zone after
  mock or oestradiol treatment (n = 6 independent roots; P < 0.001). k, Average
  intensity of NBT staining in the differentiation zone after mock or oestradiol
  treatment (n = 7 independent roots; P < 0.001). Scale bar, 50 μm. White and
  blue arrowheads throughout indicate the junctions between the meristematic
  and elongation zones and between the elongation and differentiation zones.
  Bar graphs show means. Error bars are ± s.d. Dots indicate each data point.
  P values are calculated by two-sided Student’s t-test.