Science - USA (2021-12-24)

that loss of PLETHORA function in its native
domain allows precocious expression of mid-
to late-stage protophloem sieve element differ-
entiation regulators (Fig. 3F and fig. S5, F to H).
We sought to further test whether PLT2 di-
rectly regulates the protophloem sieve element–
specific differentiation program because we
found AP2 (a member of the PLETHORA fam-
ily) family–binding sites in theAPLpromoter
region, as determined by the DAP-seq technique
( 26 ). Indeed, we confirmed the direct bind-
ing of PLT2 to several regions of theAPLpro-
moter by chromatin immunoprecipitation
(ChIP) followed by quantitative PCR (qPCR)
(Fig. 3G). Furthermore, along with AP2 sites,
theAPLpromoter is also enriched for binding

sites of HANABA TANARU (HAN), a GATA

TF. In turn,HANis a PLETHORA target ( 33 )

and, accordingly, upon ectopic PLT2 expres-

sion we detectedHANtranscripts expressed in

late protophloem sieve element development

(fig.S5,CandI).EctopicHANexpression

underpNAC86:XVEled to a delay in enuclea-

tion (fig. S5J), similar to PLT2 overexpression

in the same domain. We conclude that the

PLETHORA gradient directly (and possibly in

a feedforward manner with HAN) orchestrates

protophloem sieve element differentiation by

cell autonomously repressing transcription of

the phloem regulatorAPL. Overall, our results

show how the PLETHORA gradient first pro-

motes cell proliferation in the protophloem

sieve element lineage and then helps to time

the later stages of cellular maturation.

PEARs promoteAPLto orchestrate

phloem differentiation

Given the results above, we reasoned that an

early phloem-specific TF must activateAPL

expression. To identify genes that could fill

that role, we first generated a list of sieve ele-

ment genes enriched in our bulk-sorted cells

from that tissue compared with published

data profiling other tissue types of the root

meristem [( 37 ); fig. S6A and table S4]. We fur-

ther narrowed the list by intersecting it with

sieve element–enriched genes identified in the

cluster analysis of single-cell RNA-sequencing

Roszaket al.,Science 374 , eaba5531 (2021) 24 December 2021 5of9

Fig. 3. PLT2 inhibits phloem
differentiation by directly
repressing APL expression.
(A) Quantification of the
fluorescent intensity of PLT2-YFP
in protophloem sieve element
cells of nine roots indicated
with dots of different colors.
Percentage of roots expressing
APLin a given protophloem
sieve element cell is indicated as
a red line (n= 9). Onset ofAPL
expression coincides with
diminishing level of PLT2 protein.
Arrowhead indicates the onset
ofAPLexpression in the
protophloem sieve element.
(B) Ectopic expression ofPLT2
under thepNAC86::XVEpromoter
delays protophloem sieve
element enucleation. Square
brackets indicate extended
expression domain ofpCALS7::
H2B-RFP, a reporter used for
monitoring enucleation. (C) Native
expression profile of PLT2 targets
in protophloem sieve element
cells ordered in pseudotime.
Genes up-regulated after 6 hours
of induction of the line shown
in (B) are plotted. Upper panel
shows gradually diminishing
expression of target genes,
which reflects the PLT2 protein
gradient. Lower panel shows PLT2
up-regulated cell cycle genes
with an oscillatory expression
pattern. (D) In situ hybridization
ofAPLbefore and 6 hours
after ectopic expression of PLT2-
3xYFP. Arrowheads indicate the
positions of protophloem sieve
element enucleation beyond which pointAPLis expressed in the phloem pole pericycle, companion cells, and the metaphloem sieve element (fig. S5E). Brackets
indicate thepNAC086activity domain. (E) Time course of transcriptional repression ofAPLin cells ectopically expressing PLT2-RFP under the induciblepPEAR1::XVE
promoter. (F) Early activation ofAPLexpression 48 hours after phloem-specific knockout ofPLT2.(G) ChIP-qPCR of PLT2-3xYFP on theAPLpromoter revealing
the PLETHORA-binding region–2204 to–1439 bp upstream of theAPLORF. All scale bars, 25mm.

pNAC86:XVE>>gPLT2-3YFP

pCALS7::H2B-RFP

pNAC86::XVE>>gPLT2-3YFP

APL 16/16 9/13

AB

CD

non-induced

APL

F

induced (6h)

pAPL::erTurq plt1 plt2; pPLT2::PLT2-3YFP G

non-induced induced (48h)

* *

non-induced induced (24h)

pPEAR1:XVE>>Cas9_sgRNA>PLT2

11/11 6/7

GFP

BSA

** p<0.01 Student’s t-test

Fold enrichment

* p<0.05 Student’s t-test

0

2

4

6

8

10

12

265124722370220420751959181816021439131012201050907576571471253135

*

*

**

*

**

**

Distance to ORF

non-ind ind (9h) ind (25h)

7/7 3/3 11/11

pPEAR1:XVE>>gPLT2-RFP

pAPL::3xYFP

pAPL::er-mTurq

pPLT2::gPLT2-YFP

E

Cell distance from QC

APL expression (%)

Fluorescent intensity of PLT2-YFP

1 5 10 15 20

10

20

30

40

50

60

70

0.2

0.4

0.6

0.8

1.0

0 0.0

PLT protein

PLT2-up

PLT- up (S) phase

(M)

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