ORIGINAL RESEARCH
published: 10 January 2017
doi: 10.3389/fpls.2016.02041
Frontiers in Plant Science | www.frontiersin.org 1 January 2017 | Volume 7 | Article 2041
Edited by:
Hua Lu,
University of Maryland, Baltimore
County, USA
Reviewed by:
Xiping Wang,
Northwest A&F University, China
Zonghua Wang,
Fujian Agriculture and Forestry
University, China
*Correspondence:
Jiafeng Wang
bcjfwang@gmail.com
Zhiqiang Chen
chenlin@scau.edu.cn
Specialty section:
This article was submitted to
Plant Biotic Interactions,
a section of the journal
Frontiers in Plant Science
Received: 02 September 2016
Accepted: 20 December 2016
Published: 10 January 2017
Citation:
Liu H, Guo Z, Gu F, Ke S, Sun D,
Dong S, Liu W, Huang M, Xiao W,
Yang G, Liu Y, Guo T, Wang H, Wang J
and Chen Z (2017) 4-Coumarate-CoA
Ligase-Like Gene OsAAE3 Negatively
Mediates the Rice Blast Resistance,
Floret Development and Lignin
Biosynthesis. Front. Plant Sci. 7:2041.
doi: 10.3389/fpls.2016.02041
4-Coumarate-CoA Ligase-Like Gene
OsAAE3 Negatively Mediates the
Rice Blast Resistance, Floret
Development and Lignin Biosynthesis
Hao Liu
1
, Zhenhua Guo
2
, Fengwei Gu
1
, Shanwen Ke
3
, Dayuan Sun
4
, Shuangyu Dong
1
,
Wei Liu
1
, Ming Huang
1
, Wuming Xiao
1
, Guili Yang
1
, Yongzhu Liu
1
, Tao Guo
1
, Hui Wang
1
,
Jiafeng Wang
1
*
and Zhiqiang Chen
1
*
1
National Engineering Research Center of Plant Space Breeding, South China Agricultural University, Guangzhou, China,
2
Department of Rice Breeding, Jiamusi Rice Research Institute of Heilongjiang Academy of Agricultural Sciences, Jiamusi,
China,
3
Department of Plant Breeding, College of Agricultural, South China Agricultural University, Guangzhou, China,
4
Plant
Protection Research Institute Guangdong Academy of Agricultural Sciences/Guangdong Provincial key Laboratory of High
Technology for Plant Protection, Guangzhou, China
Although adenosine monophosphate (AMP) binding domain is widely distributed in
multiple plant species, detailed molecular functions of AMP binding proteins (AMPBPs) in
plant development and plant-pathogen interaction remain unclear. In the present study,
we identified an AMPBP OsAAE3 from a previous analysis of early responsive genes in
rice during Magnaporthe oryzae infection. OsAAE3 is a homolog of Arabidopsis AAE3
in rice, which encodes a 4-coumarate-Co-A ligase (4CL) like protein. A phylogenetic
analysis showed that OsAAE3 was most likely 4CL-like 10 in an independent group.
OsAAE3 was localized to cytoplasm, and it could be expressed in various tissues.
Histochemical staining of transgenic plants carrying OsAAE3 promoter-driven GUS
(β-glucuronidase) reporter gene suggested that OsAAE3 was expressed in all tissues
of rice. Furthermore, OsAAE3-OX plants showed increased susceptibility to M. Oryzae,
and this finding was attributable to decreased expression of pathogen-related 1a
(PR1) and low level of peroxidase (POD) activity. Moreover, OsAAE3 over-expression
resulted in increased content of H
2
O
2
, leading to programmed cell-death induced by
reactive oxygen species (ROS). In addition, OsAAE3 over-expression repressed the floret
development, exhibiting dramatically twisted glume and decreased fertility rate of anther.
Meanwhile, the expressions of lignin biosynthesis genes were significantly decreased in
OsAAE3-OX plants, thereby leading to reduced lignin content. Taken together, OsAAE3
functioned as a negative regulator in rice blast resistance, floret development, and lignin
biosynthesis. Our findings further expanded the knowledge in functions of AMBPs in
plant floret development and the regulation of rice-fungus interaction.
Keywords: 4CL-like, Rice blast, AMPBP, Lignin, ROS
INTRODUCTION
Adenosine monophosphate (AMP) binding domain-containing proteins widely exist in various
plant species, and this family consists of members with diverse functions, including luciferases,
peptide antibiotic synthetases, acetyl-CoA synthetases (ACSs), acyl-CoA synthetases, 4-coumarate-
CoA ligases (4CLs), and various other closely-related synthetases (
Shockey et al., 2000;
Liu et al. OsAAE3 Regulates Lignin Biosynthesis
Stremmel et al., 2001; Shockey and Browse, 2011). Members of
this superfamily catalyze the initial adenylation of a carboxylate
to form an acyl-AMP intermediate, followed by a secondary
partial reaction, most commonly the formation of a thioester
(
Can et al., 2014). ACSs play a crucial role in both de novo
synthesis and modification of existing lipids, and the resulting
products also participate in the regulation of plant growth and
development (Sasaki and Nagano, 2004; Souza et al., 2008).
AMP-binding domain-containing 4CLs are critical enzymes in
the phenylpropanoid metabolism pathway, which drive the
carbon flow from primary metabolism to different branches
of secondary metabolism in plant. 4CLs in Arabidopsis have
overlapping and distinct roles in phenylpropanoid metabolism.
4CL1 accounts for the majority of the total 4CL activity, and
loss of 4CL1 leads to reduced lignin content without growth
defect (Soltani et al., 2006). 4CL3 is expressed in a broad range
of cell types, and it probably has an extra function in flavonoid
metabolism. In addition, free fatty acids released from the
plastids become metabolically available when they are converted
to their corresponding Co-A thioesters (
Li et al., 2015). This
activation is induced by long-chain acyl-coenzyme asynthetases
(LACSs). LACS4 and LACS9 double-mutants have shown to
strongly reduce biosynthesis of endoplasmic reticulum-derived
lipid precursors, which are necessary substrates for glycolipid
synthesis in the plastids (Jessen et al., 2011). The expression of
rice OsBIABP1 is activated by M. oryzae infection, which may be a
defense-related AMP-binding protein (AMPBP) that is involved
in the regulation of defense response through SA and/or JA/ET
signaling pathways. However, functions of t hose genes remain
unexplored in rice (Zhang et al., 2009).
As a polyphenolic polymer, lignin is accumulated and
deposited in cell wall, and this accumulation enhances the
ability of th e cell wall and provides mechanized protection
for the plasma membrane-wrapped protoplasm (Zhao, 2016).
However, the lignin deposition is highly dependent on the cell
type, tissue, developmental stage and plant species. As part of
the normal differentiation and function of specific cell types,
lignification also serves as an integral feature of restriction to
plant non-woody tissues (Barros et al., 2015). Lignin biosynthesis
can be triggered as a response to various biotic and abiotic
stresses in cells. Evidence has clearly illustrated that lignin
biosynthesis genes play crucial roles in basa l defense and normal
growth of plants (
Wang and Balint-Kurti, 2016). PALs can
catalyze the lignin precursor phenylalanine and transform it
into cinnamic acid in lignin biosynthesis pathway (Pascual
et al., 2016). OsPAL4 is able t o improve broad-spectrum disease
resistance in rice by increasing the expression of OsPAL2 and
repressing the expression of unlinked OsPAL6 (Tonnessen et al.,
2015). Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last
step of monolignol biosynthesis. OsCAD2 is largely responsible
for monolignol biosynthesis in rice stem, while mutant plant
exhibits drastically reduced CAD activity and undetectable
sinapyl alcohol dehydrogenase activity (Zhang et al., 2006;
Hirano et al., 2012
). 4CL mediates the activation of a number
of hydroxycinnamic acids for the biosynthesis of monolignols
and other phenolic secondary metabolites in higher plants.
Suppression of Os4CL3 expression results in significant lignin
reduction, impaired plant growth, decreased panicle fertility and
other morphological changes (
Gui et al., 2011).
Rice blast is caused by the ascomycetous fungus Magnaporthe
oryzae, which is one of the most serious and devastating epiphytic
diseases in rice production worldwide (
Ashkani et al., 2015).
Currently, more than 24 major R genes conferring resistance
to M. oryzae have been identified in rice, including Pi-ta
(Jia et al., 2016), Pi-k (Wu et al., 2014), and Pb1 (Inoue
et al., 2013), and modulation of these R genes significantly
maintains and improves the grain yield and quality in
modern rice cultivars. These major R genes mainly encode the
nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins
that recognize diverse effectors (Avirulence proteins, Avr) and
activate the downstream immunity response (DeYoung and
Innes, 2006; Marone et al., 2013). Meanwhile, the rice-blast
system has been developed as a model t o study the mechanism
of pathogen-associated molecular pattern-triggered immunity
(PTI) and effector-triggered immunity (ETI) in plant-fungus
interaction (
Andolfo and Ercolano, 2015; Stael et al., 2015). To
date, the underlying molecular mechanism of rice resistance
to diseases has been illustrated in multiple levels, including
transcriptome, proteome, post-transcriptional modification and
epigenetic regulation (Miah et al., 2013; Xu et al., 2015; Li
et al., 2016; Sharma et al., 2016). Various regulatory factors
mediating the blast resistance in rice have been identified by
a combination of biochemical, genetic and hig h -throughput
sequencing approaches. However, functional roles of single gene
in complex defense network of rice blast still need to be further
elucidated.
In the present study, we identified and characterized an
Arabidopsis AAE3 (
Foster et al., 2012) homolog in rice.
Arabidopsis AAE3 encodes a specific cytoplasmic oxalyl-
CoA synthetase containing the conserved AMP-binding
domain, which is required for oxalate degrad ation, normal
seed development and defense against an oxalate-producing
fungal pathogen. Here, the cytoplasmic 4CL like protein
OsAAE3 (LOC_Os04g58710) was identified from an analysis
of transcriptome and proteome profile. In leaf tissue, increased
OsAAE3 activity was significantly correlated with decreased
resistance to rice blast and reduced lignin content. Furthermore,
our results also showed that OsAAE3 possessed multiple potential
roles in metabolism and plant anther development.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
Oryzae sativa japonic a cultivar Pik-H4 NILs was used as the
wild-type rice strain in this study. Pik-H4 NIL contains the Pik-
H4 resistance gene (an allele of Pik locus) in the sus cepti ble
cultivar LTH background. The M. oryzae race GDYJ7, one of the
primary M. oryzae races found in Guangdong Province, China, is
incompatible with Pik-H4.
Sixth-leaf-stage rice seedlings were used in the present study,
which were grown under natural light in a greenhouse at 26
◦
C for
inoculation of the rice blast fungus. Freshly prepared M. oryzae
spores (1 × 10
5
conidia/mL 0.02% v/v gelatin) were sprayed onto
the rice leaves using an air sprayer. Inoculated plants were kept in
Frontiers in Plant Science | www.frontiersin.org 2 January 2017 | Volume 7 | Article 2041
Liu et al. OsAAE3 Regulates Lignin Biosynthesis
a humidity chamber at 28
◦
C, and rice leaves were harvested for
RNA extraction at 0, 12, 24, 36, and 48 h after inoculation.
Subcellular Localization Analysis
The full-length OsAAE3 cDNA insert without a stop codon
was amplified by PCR. Amplified fragments were digested with
XbaI/BamHI and cloned between the cauliflower mosaic virus
(CaMV) 35S promoter and the GFP gene. Rice protoplast was
isolated from 12-day-old rice seedling stem and sheath. Briefly,
30 rice seedlings were cut into approximately 0.5 mm strips, and
then incubated in an enzyme solution (1.5% Cellulase RS, 0 .75%
Macerozyme R-10, 0.6 M mannitol, 10 mM MES at pH 5.7, 10
mM CaCl
2,
and 0.1% BSA) for 4–5 h in the dark with gentle
shaking (60–80 rpm). After washing twice with W5 solution
(154 mM NaCl, 125 mM CaCl
2
, 5 mM KCl, and 2 mM MES
at pH 5.7), last protoplast was resuspended in MMG solution
(0.4 M mannitol, 15 mM MgCl
2,
and 4 mM MES at pH 5.7).
The resulting OsAAE3-GFP fusion construct and empty GFP
vector were transiently co-expressed in rice protoplasts by 40%
PEG induction. Fluorescence was examined using laser-scanning
confocal microscope (
Zhang et al., 2011) (Model LSM 780; Carl
Zeiss, Jena, Germany).
GUS Assay
We first cloned about 2 kb promoter region of OsAAE3 from
rice genomic DNA. The amplified sequence was inserted into
the NcoI/BamHI sites of pCAMBIA1305 vector. The resulting
construct was introduced into agrobacterium strain EHA105
and transformed to wild-type (Pik-H4 NIL) calli as described
previously. The tissues of the transgenic plants were washed three
times with 100 mM NaPO
4
buffer (pH 7.0), and incubated with
a staining solution [100 mM NaPO
4
(pH 7.0), 10 mM EDTA, 2
mM 5-bromo-4-chloro-3-indolyl-b-GlcA, 5 mM K
4
Fe(CN)
6
, 5
mM K
3
Fe(CN)
6
, and 0.2% Triton X-100] for 20 min to 24 h a t
37
◦
C (
Jefferson et al., 1987).
Total RNA Extraction and Real-Time PCR
Analysis
Total RNA was extracted from 100 mg of fourth-leaf-stage
rice seedling with Trizol Reagent (Invitrogen, Beijing, China),
and purified RNA was reversely transcribed into cDNA using
PrimeScript RT reagent Kit (Takara, Dalian, China) according
to the manufacturer’s instructions. The cDNA was quantified by
real-time PCR using a 20 µL reaction system by SYBR Premix
ExTaq
TM
(TaKaRa, Dalian, China) on an ABI StepOne Plus
system. Table S3 lists the primer sequences used for PCR analysis.
Differences in gene expression were expressed as fold change
relative to control and calculated using the 2
−11CT
method. Each
measurement was carried out in triplicate, and the error bars
represent SE of the mean of fold changes for three biological
replicates.
Generation of the OsAAE3-OX Transgenic
Plants
The full-length of OsAAE3 cDNA was isolated by RT-PCR
from the le aves of fourth-leaf-stage rice plants using the cDNA
F/R primers (Table S3) encompassing the translation start and
stop codons. This cDNA insert was digested with BamHI and
cloned between the maize ubiquitin promoter and the Nos
terminator in the plant expression vector pOX containing the
hygromycin resist ance gene as a selection maker. Prof. Yaoguang
Liu (South China Agricultural University, Guangzhou 510642,
China) provided the plant binary vector pOX. pOX-OsAAE3
was then introduced into agrobacterium strain EHA105 and
then transformed to wild-type (Pik-H4 NIL) calli as described
previously (
Hiei et al., 1994). Transgenic rice plants were
regenerated from the transformed calli on selection medium
containing 50 mg/L hygromycin and 250 mg/L cefotaxime.
OsAAE3 levels in the transgenic ric e plants were further
confirmed with rea l-time PCR.
Measure POD A ctiviti es and H
2
O
2
Content
in Fresh Leaves
The enzyme extracts of POD were prepared following t h e method
of
Cai et al. (2008) with some modifications. Briefly, 300 mg fresh
leaves were frozen and ground in liquid N
2
, and the powder was
mixed with 4 mL 0.05 M PBS (pH 7.8) and transferred into 5-ml
tube. After thawing, the tubes were centrifuged at 8000 rpm/min
for 15 min, and the supernatant containing the total peroxidase
(POD) was collected. The POD activity was measured as the
rate of decomposition of H
2
O
2
by POD, with guaiacol as the
hydrogen donor, by spectrophotometrically measuring the rate
of color development at 436 nm due to guaiacol oxidation (
Cai
et al., 2008).
Hydrogen peroxide was performed using the Ferric Xylenol
Orange method as des cribed previously. Actually, fresh leaf tissue
was ground in cold acetone and filtered to remove cellular debris.
The supernatants were extracted with CCl4-CHCl3 solution.
Then the extract was transferred into a new tube containing 250
µM ferrous ammonium sulfate, 100 µM sorbitol and 100 µM
xylenol orange in 25 mM H
2
SO4. The mixture reacted 30 min in
the dark at room temperature, and the absorb ance was detected
at 560 nm (
Gay et al., 1999).
Lignin Content Assay
Briefly, 1 g of fresh leaves was homogenized in 5 mL cold 95%
ethanol and centrifuged at 5000 rpm/min for 30 min, and the
precipitate was washed by ethanol-hexane solution (1:2, V/V) for
three times. After thoroughly dried, the washed precipitat e was
placed in a glass reaction vial (15 mL) with 5 mL of 25% (v/v)
acetyl bromide in acetic acid, sealed with Teflon lined caps, and
heated at 70
◦
C for 30 min. After digestion, the vial’s contents were
quantitatively transferred to a 10-mL volumetric flask containing
0.9 mL of 2 M NaOH, 5 mL of acetic acid and 0.1 mL of 7.5
M hydroxylamine, and the flask was filled to 10 mL wit h acetic
acid. After reaction solution was centrifuged at 1000 g for 7 min,
the absorption values of supernatant were determined at 280 nm.
According to the standard curve, lignin contents were calculated
(
Xie et al., 2011).
RESULTS
OsAAE3 Expression Indu ced by M. oryzae
We have previously compared the global gene expression
in resistance line Pik-H4 NILs wit h the susceptible cultivar
LTH after M. oryzae inoculation via a transcriptome-proteome
Frontiers in Plant Science | www.frontiersin.org 3 January 2017 | Volume 7 | Article 2041
Liu et al. OsAAE3 Regulates Lignin Biosynthesis
analysis. We identified 61 and 69 genes that were up-regulated
and down-regulated in Pik-H4 NILs line, respectively (Table
S1). Based on our transcriptome-proteome analysis in Pik-
H4 NILs line, Pik-H4 modulates multiple genes involved in
diverse biological processes, including defense-related hormone
biosynthesis, disease resistance, response stress, photosynthesis,
and signal transduction(Figure 1A).
An AMPBP3 (4CL-like protein), named OsAAE3
(LOC_Os04g58710), was identified from the transcriptome
and proteome analysis of early responsive genes in rice during
M. oryzae infection (Table S1). We then examined the expression
pattern of OsAAE3 over a time course of 48 h after inoculation
with M. oryzae by quantitative RT-PCR (qRT-PCR). The OsAAE3
expression at the mRNA level was significantly decreased at 12 h
and reached its lowest level at 24 h, and then it was maintained
at a relatively low level from 24 to 48 h after inoculation with M.
oryzae in wild-type plants (Figure 1B). In contrast, the OsAAE3
expression presented circadian rhythmicity pattern during the
whole stage after spraying of water.
Identification and Characterization of
OsAAE3
The genome sequence and the cDNA fragment encoding of
OsAAE3 were isolated from rice using gene-specific primers
based on the sequence (LOC_Os04g58710) of the rice genome
database (Rice Genome Annotation Project). The genome
sequence of OsAAE3 was 2221 bp, full-length coding sequence
(CDS) was 1557 bp, which harbored two exons and one intron,
and it encoded a protein of 519 amino acid residues with a
deduced molecular weight of 54.47 kDa. This result was similar
to our DNA sequence analysis (Figures 2A–C).
According to the prediction from Pfam database and
comparison with other AMPBPs, amino acid sequence analysis
showed that an AMP-binding domain was located from 34 to
439 aa in the OsAAE3 sequence. Furthermore, we identified
orthologous protein sequence of OsAAE3 from several plant
models, including rice, Arabidopsis, maize, sorghum and
soybean. A phylogenetic analysis based on those sequences
showed that OsAAE3 was most likely 4CL-like 10 in an
independent group (Figure 2D). As indicated from the sequence
alignment, there were 90% similarities between OsAAE3 and
4CL-like 10 (Figure S1). Therefore, it is of great interest to verify
whether OsAAE3 was indeed involved in monolignol catabolism
in t he same way as its homolog in Arabidopsis.
OsAAE3 Is Localized to the Cytosol
The AMP-binding domain of OsAAE3 suggested that it was
probably localized to the cytosol like its homolog AAE3
in Arabidopsis. Meanwhile, the PSORT database revealed
the multi-organelle localization of OsAAE3, including the
mitochondrial inner membrane, plasma membrane, Golgi body,
and mitochondrial intermembrane space, but the available
prediction scores were unreliable. To further investigate the
subcellular localization of OsAAE3, we constructed an OsAAE3-
GFP fusion protein driven by the CaMV 35S promoter,
and the empty GFP was used as the negative control. The
resulting vectors 35S:OsAAE3-GFP and GFP were transiently co-
transformed into rice protoplast cells with the PEG-mediated
procedure. Interestingly, the OsAAE3-GFP fusion protein
exhibited similar pattern to the empty GFP control, and the
OsAAE3-GFP signal was strongly detected in the cytoplasm
of rice protoplast cells (Figure 3). Therefore, the transient
expression assay indicated that OsAAE3 encoded a cytoplasmic
synthetase like 4CL that could possibly catalyze the lignin
degradation.
Expression Pattern Analysis of OsAAE3
To evaluate the expression pattern of OsAAE3 in different
tissues, total RNA was extracted from root, stem, leaf sheath,
leaf blade, young panicle and glume of rice at the heading
stage. Semi-quantitative PCR and real-time PCR (RT-PCR) were
performed to determine the relative expression of OsAAE3. Semi-
quantitative PCR results suggested that the full-length coding
sequence of OsAAE3 could be easily amplified from all of ti ssues
without tissue specificity (Figure 4B). OsAAE3 was constitutively
expressed in various types of tissues, but its highest expression
was detected in leaf blade, followed by root, young panicle, glume,
stem, and leaf sheath. Such finding was confirmed by RT-PCR
experiment (Figure 4C).
We furt her cloned the promoter sequence of OsAAE3
into GUS (β-glucuronidase) reporter system, and then the
constructed promoter (OsAAE3)-GUS fusion vector was
transformed into rice callus to assess the promoter activity.
GUS staining analysis indicated that the OsAAE3 promoter
activity was detected in the root, epidermis cells of leaves, stem
vascular cells, glume vascular cells and anther (Figure 4A).
Taken toget h er, OsAAE3 was expressed in all tissues of the plant.
Generation of the OsAAE3-OX Transgenic
Plants
To determine the detailed molecular roles of OsAAE3 in rice
physiological and biochemical reactions, we constructed the
transgenic rice plants over-expressing OsAAE3 (OsAAE3-OX)
under the control of ubiquitin promoter (Figure 5B). The
OsAAE3 expression at the mRNA level was significantly increased
in transgenic plants (OsAAE3-OX) compared with wild-type
plants, and these data were validated using qRT-PCR and semi-
quantitative PCR (Figures 5C,D). Over-expression of OsAAE3
repressed the plant growt h, showing dwarfing, rolling, and
narrow leaves as well as abnormal glumes (Figure 5A and Figure
S2). Therefore, based on the observed abnormal phenotypes
of OsAAE3-OX plants, we believed that OsAAE3 served as a
negative regulator in the regulation of plant development and
growth.
Over-Expression of OsAAE3 Reduces the
Rice Blast Resistance
We next examined resistance of the transgenic plants to M.
oryzae in order to elucidate the molecular basis of OsAAE3
in disease resistance in rice. Disease symptoms in plants were
quantified at 7 days after inoculation, and the OsAAE3-OX
plants exhibited reduced resistance to M. oryzae compared with
Frontiers in Plant Science | www.frontiersin.org 4 January 2017 | Volume 7 | Article 2041
Liu et al. OsAAE3 Regulates Lignin Biosynthesis
FIGURE 1 | OsAAE3 expression was down-regulated by M. oryzae. (A) GO annotation of differential genes expression in Pik-H4 NILs by
transcription-proteomics analysis. (B) OsAAE3 expression induced by M. oryzae after 48 h, Sterile H
2
O was used as controls. Values shown are means ± SD from
three independent experiments, and asterisks indicate a significant difference according to the t-test (P < 0.05) compared with control group.
FIGURE 2 | Identification and characterization of OsAAE3. (A) Schematic gene structure of OsAAE3 in rice genome. (B,C) The OsAAE3 DNA and CDS
fragments were examined by agarose gel electrophoresis. (D) Comparative phylogenetic analysis of the OsAAE3 proteins in plants. Sequences were aligned using
ClustalX. The evolutionary history was inferred using a Neighbor-Joining phylogenetic tree generated with the software MEGA6. The percentage of replicate trees in
which the associated taxa clustered together in the bootstrap test (2000 replicates) is shown next to each branch. Putative OsAAE3 (Os04g58710) members in A.
thaliana (AT2G17650, AT3G16910, AT3G48990), O. sativa (Os07g17970, Os08g04770, Os02g02700, Os02g0177600, Os03g04130, Os01g24030, Os03g19240,
Os03g38350, Os08g0245200, Os04g24530, Os06g0656500), Sorghum bicolor (Sb06g033410), Zea mays (GRMZM2G074759, GRMZM2G333861), Setaria italica
(Si021736 mg).
Frontiers in Plant Science | www.frontiersin.org 5 January 2017 | Volume 7 | Article 2041