the split firefly luciferase complementation as-
say in tobacco leaf epidermal cells (Fig. 4B). Both
assays showed that LG1 and DRL1 interact in
vitro and in vivo.
To investigate how DRL1 and LG1 together
regulateZmRAVL1expression, we performed
transient expression assays in maize protoplasts,
in which the coding sequence of DRL1 and LG1
driven by the 35Spromoter were used as effec-
tors, and the UPA2 fragment (240 bp) from W22
or 8759 fused into the upstream of the luciferase
(LUC) gene driven by a 1.7-kbZmRAVL1promot-
er was used as the reporter (designated W22
and 8759 reporter, respectively) (Fig. 4C). When
cotransforming with an empty effector con-
struct, the 8759 reporter exhibited lower LUC
activity than did the W22 reporter (Fig. 4D). Over-
expressing LG1 alone induced the LUC activities
of both the 8759 and W22 reporters, whereas over-
expressing DRL1 alone repressed the LUC activ-
ities of the two reporters (Fig. 4D). Coexpression
of LG1 with DRL1 repressed LG1-activated LUC
activity (Fig. 4D). In these three cases, the 8759
reporter displayed lower LUC activity than did
the W22 reporter (Fig. 4D). Thus, DRL1 physi-
cally interacts with LG1 to attenuate its effect on
the transcription ofZmRAVL1.
Brassinosteroid C-6 oxidase
underliesUPA1
We also performed fine mapping for the second-
largest leaf angle QTL,UPA1on chromosome
1 (Fig. 1A and table S1). Different fromUPA2,the
teosinte allele atUPA1exhibited increased leaf
anglerelativetothemaizeallele(fig.S14).Pheno-
typic analysis of the two NILs forUPA1showed
thatUPA1also has a canopy-wide effect in alter-
ing leaf angles in different leaves (Fig. 5, A and B).
Thedifferenceinleafangleisduetothealteration
in auricle size and the number of sclerenchyma
cell layers on the adaxial side (fig. S15). Through
fine mapping,UPA1was delimited to a 223-kb
physical region containing a single gene (Fig. 5, C
to E),GRMZM2G103773,encodingbrassinosteroid
C-6 oxidase(brd1), which catalyzes the final
steps of brassinosteroid synthesis ( 19 ). We com-
pared thebrd1coding sequence between W22
and 8759 and identified seven SNPs, including
two nonsynonymous SNPs, but not in the con-
served region (fig. S16A). We comparedbrd1
expression in the two NILs forUPA1.UPA1-NIL^8759
with large leaf angle exhibited higherbrd1ex-
pression thanUPA1-NILW22with small leaf angle
in developing leaves (fig. S16B). Indeed, abrd1-null
mutant exhibits dwarfism and aberration in the
blade–auricle border ( 19 ). We overexpressedbrd1
and found that thebrd1overexpression lines ex-
hibited normal ligular development but increased
leaf angle (fig. S17) because of the enlarged auricle
and the decreased number of sclerenchyma cells
on the adaxial side (fig. S18). These results indi-
cate thatbrd1is involved in the regulation of maize
leaf angle.
InZmRAVL1-knockout plants,brd1expression
was down-regulated (fig. S19A), whereas inbrd1-
overexpressed lines,ZmRAVL1exhibited no change
(fig.S19B).ThetwoNILsforUPA2differed inbrd1
expression (fig. S19C), whereasZmRAVL1showed
similar expression in the two NILs forUPA1(fig.
S19D). These results suggested thatbrd1functions
downstream ofZmRAVL1. We next tested whether
ZmRAVL1could regulatebrd1expression. B3 tran-
scription factors bind E-box motif (CANNTG) ( 16 ).
We identified five putative E-box elements in the
brd1promoter (Fig. 5F). Y1H, EMSA, and ChIP-
qPCR showed that ZmRAVL1 could bind thebrd1
promoter in vitro and in vivo (Fig. 5, G and H, andfig. S20). Transient expression assay in maize pro-
toplasts confirmed thatZmRAVL1activatesbrd1
transcription (Fig. 5, I and J).
Of the various phytosteroids, only brassino-
lide and its immediate precursor, castasterone,
possess biological activity in planta ( 20 ).brd1en-
codes a C-6 oxidase involving the conversion of
6-deoxocastasterone to castasterone ( 20 ). We mea-
sured endogenous brassinosteroid accumula-
tion in the developing ligular region of thebrd1Tianet al.,Science 365 , 658–664 (2019) 16 August 2019 4of7
Fig. 4. DRL1 and LG1 together regulateZmRAVL1expression.(A) Y2H indicates that LG1 and
DRL1 interact in vitro. (B) The split firefly luciferase complementation assay shows that LG1 and
DRL1 interact in vivo. (C) Schematic diagram shows the constructs used in the transient
transcriptional activity assays. (D) DRL1 represses LG1 to activate the expression ofZmRAVL1.
Values are means ± SD (n= 4 repeats). Different letters denote significant differences (P< 0.05)
from a Duncan’s multiple-range test.RESEARCH | RESEARCH ARTICLE