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
(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
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