Science - 16.08.2019

was detected in the fragment containing the pu-
tative C2C2-binding motif around S2 (Fig. 3C). These
results indicated that the S2 variant inUPA2was
associated with differential binding by DRL1.
lg1andlg2genes establish the blade–sheath
boundary, withlg2functioning beforelg1(10, 13).
To determine the regulatory relationship of
ZmRAVL1withlg1andlg2, we examined their

expression in the developing ligular region in

their null mutants (fig. S12). Inlg1andlg2mu-

tants,ZmRAVL1expression was repressed (fig.

S12, A and B), whereas in theZmRAVL1-knockout

line, the expression oflg1andlg2exhibited no al-

teration (fig. S12C). These results, together with the

finding thatZmRAVL1-knockout lines exhibited

normal ligular development and only affected

auricle expansion, suggested thatZmRAVL1func-

tions downstream oflg1andlg2. We next tested

whether LG1 could regulateZmRAVL1expression.

Previous studies have shown that SBP-domain

proteins recognize and bind GTAC motifs ( 18 ).

Motif analysis identified two putative GTAC sites

in theZmRAVL1promoter (Fig. 3A and fig. S13A).

Yeast one-hybrid assay (Y1H) (fig. S13B) and EMSA

Tianet al.,Science 365 , 658–664 (2019) 16 August 2019 2of7

Fig. 1. Positional cloning ofUPA2.(A) QTL mapping for middle leaf
angle in the maize–teosinte BC 2 S 3 population. LOD, logarithm of odds.
UPA2andUPA1are the largest- and second-largest leaf angle QTL,
respectively. The dashed gray line at LOD of 5 indicates the threshold of
claiming significant QTLs. (B) Gross morphologies ofUPA2-NILW22and
UPA2-NIL^8759. The white arrows indicate the lower, middle, and upper
leaves in which leaf angle was scored. Scale bars, 20 cm. (C) Comparison
of leaf angle in lower, middle, and upper leaves betweenUPA2-NILW22
andUPA2-NIL^8759 .(D) Scanning electron microscopy analysis of the
ligular region ofUPA2-NILW22andUPA2-NIL^8759. The ligular band and
the mature auricle region are indicated by white dashed lines. Scale bars,
3 mm (top) and 500mm (bottom). (E) Comparison of the width of the
ligular band and auricle margin betweenUPA2-NILW22andUPA2-NIL^8759.
(F) Cross-sections of the mature ligular region ofUPA2-NILW22and
UPA2-NIL^8759. Top shows the abaxial side and bottom shows the adaxial
side. Scale bars, 100mm. (G) Comparison of number of the abaxial
sclerenchyma cell layers (left) and the adaxial sclerenchyma cell layers

(right) betweenUPA2-NILW22andUPA2-NIL^8759 .(H) Location ofUPA2

on maize chromosome 2. CEN, centromere. (I) Fine mapping ofUPA2

using an NIL population (n= 3180). The number of recombinants

between adjacent markers is indicated below the linkage map. (J) Progeny

testing of recombinants delimitedUPA2to a 240-bp noncoding region

(red lines). The graphical genotypes of the five critical recombinants

are shown on the left. White, black, and gray segments indicate regions

homozygous for W22, regions homozygous for 8759, and heterozygous

regions, respectively. The bar graphs on the right compare middle

leaf angle between homozygous recombinants and homozygous non-

recombinants within each recombinant-derived F 3 family. Black and

white bars represent homozygous progenies that inherited the 8759

and W22 chromosome from the parental recombinant, respectively. The

240-bp region ofUPA2is located 9540 bp upstream of the start codon

(ATG) ofGRMZM2G102059(ZmRAVL1). Pink and gray regions indicate

the exon and untranslated regions (UTR), respectively. Values are

means ± SD. **P< 0.01 (Student’sttest). N.S., not significant.

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