RESEARCH ARTICLE
◥PLANT SCIENCE
Teosinte ligule allele narrows
plant architecture and enhances
high-density maize yields
Jinge Tian, Chenglong Wang, Jinliang Xia, Lishuan Wu, Guanghui Xu, Weihao Wu,
Dan Li, Wenchao Qin, Xu Han, Qiuyue Chen, Weiwei Jin, Feng Tian†
Increased planting densities have boosted maize yields. Upright plant architecture
facilitates dense planting. Here, we clonedUPA1(Upright Plant Architecture1) andUPA2,
two quantitative trait loci conferring upright plant architecture.UPA2is controlled by a
two-base sequence polymorphism regulating the expression of a B3-domain transcription
factor (ZmRAVL1) located 9.5 kilobases downstream.UPA2exhibits differential binding by
DRL1 (DROOPING LEAF1), and DRL1 physically interacts with LG1 (LIGULELESS1) and
represses LG1 activation ofZmRAVL1.ZmRAVL1regulatesbrd1(brassinosteroid C-6
oxidase1), which underliesUPA1, altering endogenous brassinosteroid content and leaf
angle. TheUPA2allele that reduces leaf angle originated from teosinte, the wild ancestor of
maize, and has been lost during maize domestication. Introgressing the wildUPA2allele
into modern hybrids and editingZmRAVL1enhance high-density maize yields.
F
eeding the ever-increasing world population
requires increased crop yield from limited
arable lands ( 1 ). One solution to this chal-
lenge is to grow more plants per unit area
to achieve higher productivity. However,
dense planting imposes competition for water,
nutrients, and light. Breeders of the cereal crop
maize (Zea maysssp.mays) have addressed this
challenge by adapting plant architecture to dense
planting. Plant architecture with more upright
leaves (i.e., smaller leaf angle) decreases mutual
shading and sustains light capture for photo-
synthesis despite increased plant density, thus
improving the accumulation of leaf nitrogen
for grain filling and increasing grain yield ( 2 – 5 ).
With such adaptations, the planting density of
maize has increased from 30,000 plants per hectare
in the 1930s to >80,000 plants per hectare in the
2010s ( 6 , 7 ).
The ligular region, between the maize blade
and sheath, consists of the ligule and auricle ( 8 )
and establishes leaf angle, which determines the
plant’s overall architecture. Mutant studies in maize
have identified genes essential for development
of the ligular region ( 9 – 13 ).lg1andlg2mutants
exhibit erect leaf architecture because of defects
in ligule and auricle development ( 10 , 13 ). Such
erect leaf architecture facilitates dense plant-
ing ( 3 , 4 ). However, leaves inlgmutant plants
aretooerecttobeusefulincommercialhybrids.
We searched for natural alleles that optimize plant
architecture and leaf angle for dense planting.Upright Plant Architecture2regulates
leaf angle throughZmRAVL1
We mapped 12 quantitative trait loci (QTLs) for
leaf angle in a population of 866 maize–teosinte
BC 2 S 3 recombinant inbred lines derived from a
cross between the maize inbred line W22 and
the teosinte accession CIMMYT 8759 (Z. maysssp.
parviglumis, hereafter referred to as 8759) (Fig. 1A
and table S1). The QTL with the largest effect,
UPA2(Upright Plant Architecture2), located on
chromosome 2, was selected for positional clon-
ing. The teosinte allele exhibited reduced leaf
angle relative to the maize allele (fig. S1A). To
verify the allelic effect, we developed a pair of near-
isogenic lines (NILs) forUPA2(UPA2-NILW22and
UPA2-NIL^8759 ) (Fig. 1B) from a recombinant in-
bred line that was heterozygous in the target
region (fig. S1B). The two NILs differed in leaf
angle in upper, middle, and lower leaves (Fig. 1C),
indicating a canopy-wide effect ofUPA2in alter-
ing plant architecture. We performed scanning
electron microscopy and histological analyses for
UPA2-NIL^8759 andUPA2-NILW22, which exhibited
similar size of auricle cells (fig. S2) but differed in
auricle expansion. Compared withUPA2-NILW22,
UPA2-NIL^8759 with upright leaf angle had a nar-
rower ligular band, resulting in reduced auricle
size at maturity (Fig. 1, D and E, and fig. S3).
Sclerenchymal layers in the ligular region pro-
vide mechanical strength for the blade.UPA2-
NIL^8759 contained more layers of sclerenchyma
cells on the adaxial side than didUPA2-NILW22
(Fig. 1, F and G).To identify the genetic factor controllingUPA2,
we generated a NIL population (n= 3180) and
performed fine mapping following previously
described methods ( 14 , 15 ).UPA2was narrowed
down to a 240–base pair (bp) noncoding region
according to the maize reference sequence (Fig.
1, H to J).GRMZM2G102059,encodingaB3
domain–containing protein homologous to RAVL1
in rice ( 16 )(fig.S4),islocated9540bpdownstream
of the 240-bp region ofUPA2(Fig. 1, H to J). We
thus namedGRMZM2G102059“ZmRAVL1.”The
240-bp region ofUPA2may function as a distant
cis element to regulateZmRAVL1expression. Con-
sistent with this hypothesis,UPA2-NIL^8759 with
small leaf angle showed lowerZmRAVL1expres-
sion thanUPA2-NILW22with large leaf angle in
developing leaves (fig. S5A). ZmRAVL1 is local-
ized in the nucleus (fig. S5B). To validate the func-
tion ofZmRAVL1,wedown-regulatedZmRAVL1
expression by RNA interference (RNAi) and knocked
outZmRAVL1using CRISPR-Cas9 ( 17 ). In the T1
family ofZmRAVL1-KO#1andZmRAVL1-KO# 2
carrying homozygous-null mutations (fig. S6A),
Cas9-free plants were identified and propagated
for phenotypic analysis and field trials. We found
that bothZmRAVL1RNAi and knockout lines
exhibited smaller leaf angle in lower, middle, and
upper leaves compared with wild-type plants
(Fig.2,AandB,andfig.S6,BtoD)becauseofde-
creased auricle size and increased adaxial scleren-
chyma cells in the ligular region (figs. S7 and S8).
By contrast, overexpressingZmRAVL1led to lar-
ger leaf angle in lower, middle, and upper leaves
compared with wild type (Fig. 2C and fig. S9).
These results indicate thatZmRAVL1functions
as a positive regulator of maize leaf angle.
To identify the causative sequence variant in
the 240-bp region ofUPA2, we sequenced the
240-bp region in W22 and 8759 and identified
four sequence differences, including one single-
nucleotide polymorphism (SNP) and three one-
or two-base sequence polymorphisms, designated
S1 to S4, respectively (fig. S10). We investigated
whether these four sequence variants were as-
sociated with alteration of conserved regulatory
sequences that may cause differential regulation
by an upstream regulator. Among the four se-
quence variants, only S2 (TG/–) flanks a putative
C2C2-binding motif (AGTGTG) (Fig. 3A and fig.
S10). Maize YABBY genesdrl1(drooping leaf1)
anddrl2contain a C2C2 zinc-finger domain at
the N terminus ( 12 ). Their null mutants ex-
hibited increased leaf angle and displayed a
dropping-leaf phenotype ( 12 ). We tested whether
DRL proteins could bind to the sequence sur-
rounding S2 atUPA2. Because of the high se-
quence similarity between the DRL1 and DRL2
proteins ( 12 ), we selected DRL1 for further analysis.
Electrophoretic mobility shift assay (EMSA) de-
tected band shifts, and the probe from 8759 con-
taining the TG nucleotides at S2 exhibited a
stronger binding affinity for DRL1 compared with
the probe from W22 lacking TG nucleotides (Fig.
3B and fig. S11). We also performed chromatin
immunoprecipitation–quantitative polymerase
chain reaction (ChiP-qPCR) using flag antibody
against flag-tagged DRL1 protein. EnrichmentRESEARCH
Tianet al.,Science 365 , 658–664 (2019) 16 August 2019 1of7
State Key Laboratory of Plant Physiology and Biochemistry,
National Maize Improvement Center, Key Laboratory of
Biology and Genetic Improvement of Maize (MOA), Beijing
Key Laboratory of Crop Genetic Improvement, China
Agricultural University, Beijing 100193, China.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected]