Science - 16.08.2019

(Fig. 3D and fig. S13C) showed that LG1 could
bind the GTAC sites in theZmRAVL1promoter
in vitro. Further, ChIP-qPCR identified enrich-
ment in the fragment spanning the GTAC sites
in theZmRAVL1promoter (Fig. 3E). Taken to-

gether, these results indicated that LG1 regulates

ZmRAVL1expression.

OurresultsshowedthatLG1couldbind

ZmRAVL1promoter, whereas DRL1 could bind

the sequence surrounding S2 atUPA2,adistant

cis-regulatory variant located 9.5 kb upstream of

ZmRAVL1. To further determine how this circuit

was regulated, we tested for an interaction be-

tween the LG1 and DRL1 proteins. We performed

the yeast two-hybrid assay (Y2H) (Fig. 4A) and

Tianet al.,Science 365 , 658–664 (2019) 16 August 2019 3of7

Fig. 2.ZmRAVL1regulates maize leaf angle.(A)ZmRAVL1-knockout
lines (ZmRAVL1-KO#1 andZmRAVL1-KO#2) exhibited reduced leaf angle
in lower, middle, and upper leaves. (B)ZmRAVL1RNAi lines (ZmRAVL1-
RNAi#1 andZmRAVL1-RNAi#2) showed reduced leaf angle in lower,

middle, and upper leaves. (C)ZmRAVL1overexpression lines (ZmRAVL1-

OE#1 andZmRAVL1-OE#2) exhibited increased leaf angle in lower, middle,

and upper leaves. WT, wild-type. Values are means ± SD. **P<0.01

(Student’sttest).

Fig. 3. DRL1 bindsUPA2and LG1 binds
ZmRAVL1promoter.(A) Relative locations
ofUPA2andZmRAVL1.S2(TG/–) flanks a
putative C2C2-binding motif (red box).
(B) EMSA shows that S2 was associated
with differential binding by DRL1. The
biotin-labeled probes are indicated in (A).
(C) ChIP-qPCR indicates that DRL1
could bind the sequence surrounding
S2 in vivo. The fragments used in ChIP-qPCR
are indicated in (A). The F3 fragment
contains the putative C2C2-binding motif
surrounding S2, whereas the F1 and F2
fragments contain no putative C2C2-binding
motif. (D) EMSA indicates that LG1 could
bind the GTAC sites in theZmRAVL1promoter.
Biotin-labeled probes and mutant probes
are indicated in (A). (E) ChIP-qPCR identified
significant enrichment in the fragment
containing the GTAC sites in theZmRAVL1
promoter. The fragments used in ChIP-qPCR are
indicated in (A). The F4 fragment contains the
putative SBP-binding motifs, whereas the F5 and
F6 fragments contain no GTAC sequences. Fold
enrichments in (C) and (E) were calculated
relative to input. Values are means ± SD
(n= 3 repeats) in (C) and (E). **P<0.01
(Student’sttest). N.S., not significant.

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