Science - USA (2021-12-24)

Roszaket al.,Science 374 , eaba5531 (2021) 24 December 2021 6of9

pAPL(3kb)::erVenus

0

1

2

3

4

5

6

7

8

GFP

BSA

2818265124722370220420751939181816261439131012201050911708576571471405253135

*

*

*

*

*

* *

Fold enrichment

*p<0.01 Student’s t-test

*

*

pAPL::erRFP

pNAC86::erVenus

pPEAR1::XVE>>PEAR1-mTurq

PSE

MSE

procambium

stem

cells

0

1

2

3

4

5

0

1

2

3

4

5

PLT1 PEAR1^6

0

-5

5

Z-score

PEAR2

TMO6

DOF6

PEAR1

AB C

Col-0 pear sextuple

D E F 10/10 6/8 3/5

G

H

(mock) (Ind 30h)

WT pear

sextuple

(Ind 45h)

APL

NAC086NAC045NEN4

100

120

80

(^6080)
40
20
0
wild type (Col-0)
pear sextuple
pZAT14::3xYFP
pPEAR1::XVE>>
ZAT14-3xYFP
induced (21h)
J K L
pZAT14-LIKE::
3xYFP
pPEAR1::XVE>>
ZAT14-LIKE
-3xYFP
NAC86
NAC45
ZAT14
NAC10
HD2C
TMO6
HD2A
ZRF1B
HAM3
bZIP7
GATA17L
WHY2
BPE
AT4G13620
PEAR2
PEAR1
DOF6
GATA19
AT1G69580
APL
Upregulated
Down regulated
induced (21h)
pear
sextuple
pear
sextuple
HD2AGATA17LZRF1BHD2CWHY2PEAR2PEAR1TMO6DOF6GATA19APLAT4G13620BPEHAM3AT1G69580NAC10bZIP7NAC86NAC45ZAT14
0 0.5 1
**

• 
  
  pPEAR1::
  H2B-YFP
  Readcounts

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  (a)
  (c)
  (d)
  (b)
  20
  15
  10
  5
  pAPL 3kb
  n=35
  pAPL 3kb
  DOF(I)
  n=67
  pAPL 3kb
  DOF(II)
  n=55
  pAPL 3kb
  DOF(I+II)
  pAPL n=61
  3kb
  DOF(I)
  ::erVenus
  pAPL
  3kb
  DOF(II)
  ::erVenus
  pAPL
  3kb
  DOF(I+II)
  ::erVenus
  pAPL(2039bp)::erVenus
  DOF (I)
  DOF (II)
  Distance from the QC [cells]
  I
  Distance to ORF
  pPEAR1::
  erVenus
  8/8 6/8 11/11 11/11 10/10
  Fig. 4. PEARs orchestrate phloem differentiation.(A) Force-directed clustering
  of 272 single-cell transcriptomes obtained using thepPEAR1D::erVenusreporter.
  Plotted is the expression of stem cell–abundantPLT1.Arrows indicate cellular
  trajectories inferred from known gene expression patterns (fig. S6). (B) Strong
  enrichment ofPEAR1expression in protophloem sieve element and metaphloem
  sieve element trajectories confirmed by thepPEAR1::erVenusreporter line.
  White arrowheads indicate the protophloem sieve element and red arrowheads
  the metaphloem sieve element. (C) Expression heatmap. PEAR genes are
  among the earliest phloem-specific TFs. (D) Lack of protophloem sieve element
  differentiation in the mature part of thepearsextuple mutant root. Arrowheads
  indicate the protophloem sieve element position. (E) Lack ofAPLpathway
  activation in the roots ofpearsextuple mutant based on RNA-seq analysis.
  (F) Inducible expression ofPEAR1-mTurqis sufficient to activate transcription of
  APLandNAC86reporters in thepearsextuple mutant background. (G) ChIP-qPCR
  of PEAR1-YFP shows a direct interaction of PEAR1 withAPLpromoter at
  multiple positions. Two prominent PEAR1-binding sites are indicated with red
  dashed rectangles. (H) Expression patterns of modifiedpAPLreporter lines.
  Length of the“3kb”promoter equals 2962 bp. DOF(I) and DOF(II) correspond
  to the two enhancer elements indicated in (G). Details of this modification are
  provided in fig. S7C. (I) Quantification of the onset ofpAPLexpression after
  RESEARCH | RESEARCH ARTICLE