overexpression andZmRAVL1-knockout lines.
Brassinolide was not detected in our samples.
Plants withbrd1overexpression exhibited in-
creased castasterone content (fig. S21).ZmRAVL1-
knockout plants that showed reducedbrd1
expression had less castasterone than did wild-
type plants (fig. S21). These results suggested
thatZmRAVL1affects endogenous brassinoste-
roid content.
UPA2-UPA1–associated regulatory
model for leaf angle
We have developed a model for how the leaf
angle difference betweenUPA2-NIL^8759 andUPA2-
NILW22is regulated (fig. S22). We suggest that
UPA2is controlled by a 2-bp sequence variant
(TG/–) located 9.5 kb upstream ofZmRAVL1.
Compared with the cultivated W22 allele, the
wild 8759 allele containing the TG nucleotidesatUPA2is associated with a higher binding af-
finity for DRL1. DRL1 physically interacts with
LG1, which attenuates the transcriptional acti-
vation function of LG1 onZmRAVL1expression.
LowZmRAVL1expression inUPA2-NIL^8759 re-
sults in less expression ofbrd1, consequently
decreasing endogenous brassinosteroid in the
ligular region, reducing auricle expansion, and
leading to smaller leaf angle. InUPA2-NILW22,Tianet al.,Science 365 , 658–664 (2019) 16 August 2019 5of7
Fig. 5. Positional cloning ofUPA1.(A) Gross morphologies of
UPA1-NILW22andUPA1-NIL^8759. The white arrows indicate the lower,
middle, and upper leaves in which leaf angle was scored. Scale bar,
20 cm. (B) Comparison of leaf angle in lower, middle, and upper leaves
betweenUPA1-NILW22andUPA1-NIL^8759 .(C) Location ofUPA1on maize
chromosome 1. CEN, centromere. (D) Fine mapping ofUPA1using an
NIL population (n= 2040). The number of recombinants between
adjacent markers is indicated below the linkage map. (E) Progeny
testing of recombinants delimitedUPA1to a 223-kb physical region
containing only one annotated gene,GRMZM2G103773(brd1). The
graphical genotypes of the five representative recombinants are shown on
the left. White, black, and gray segments indicate regions homozygous for
W22, regions homozygous for 8759, and heterozygous regions, respec-
tively. The bar graphs on the right compare middle leaf angle between
homozygous recombinants and homozygous nonrecombinants within
each recombinant-derived F 3 family. (F) Schematic diagram of the
brd1promoter. Red dots indicate the putative E-box motif. (G) EMSA
shows that ZmRAVL1 binds the putative E-box motif in thebrd1promoter.
The biotin-labeled probe (P2) is indicated in (F). Unlabeled probes were
used in the competition assay. (H) ChIP–qPCR identified significant
enrichment in the fragment containing the E-box motif in theZmRAVL1
promoter. The fragment (F7) used in ChIP-qPCR is indicated in (F).
Fold enrichments were calculated relative to input. (I) Schematic diagram
shows the effectors and reporter used in the transient transcriptional
activity assays. (J)ZmRAVL1activatesbrd1expression. Values are
means ± SD in (B), (H), and (J). **P< 0.01 (Student’sttest).RESEARCH | RESEARCH ARTICLE