Maize and Arabidopsis ARGOS Proteins Interact with
Ethylene Receptor Signaling Complex, Supporting a
Regulatory Role for ARGOS in Ethylene
Signal Transduction[OPEN]
Jinrui Shi, Bruce J. Drummond, Hongyu Wang, Rayeann L. Archibald, and Jeffrey E. Habben
DuPont Pioneer, Johnston, Iowa 50131-1004
ORCID IDs: 0000-0002-1754-7593 (B.J.D.); 0000-0001-7163-4244 (R.L.A.).
The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS
genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea
mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the
ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of
ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the
Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE
SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The
protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we
found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with
maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when
overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide
fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved
among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain
is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical
association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role
for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize.
The phytohormone ethylene regulates diverse aspects of plant growth and development as well as plant
response to environmental stress. Reducing ethylene
biosynthesis by silencing aminocyclopropane-1-carboxylic
acid synthase6 can improve maize (Zea mays) grain
yield in water-limiting environments (Habben et al.,
2014). Lowering plant sensitivity to ethylene by overexpressing maize ARGOS8 also enhances plant tolerance
to drought and increases maize yield under water-deficit
conditions (Shi et al., 2015). ZmARGOS8 is a member of
the AUXIN-REGULATED GENE INVOLVED IN ORGAN
SIZE (ARGOS) gene family (Hu et al., 2003), which encode
integral membrane proteins localized to the endoplasmic
reticulum (ER) and Golgi (Rai et al., 2015; Shi et al., 2015).
Arabidopsis ARGOS and homologs from other species
*Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Jinrui Shi ([email protected]).
J.S. and J.E.H. designed the research; B.J.D., H.W., R.L.A., and J.S.
performed the research and analyzed the data; J.S. wrote the article
with contributions of all the authors.
[OPEN]
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promote cell division and/or expansion when overexpressed, affecting organ size in transgenic plants (Hu et al.,
2003, 2006; Wang et al., 2009; Kuluev et al., 2011; Feng
et al., 2011; Guo et al., 2014). Initially, it was hypothesized
that ARGOS may transduce auxin signals downstream of
AUXIN RESISTANCE1 to regulate cell proliferation and
organ growth through AINTEGUMENTA (Hu et al., 2003).
However, two recent studies have shown that ARGOS
overexpressing transgenic Arabidopsis plants have reduced response to ethylene (Shi et al., 2015; Rai et al., 2015).
The ER- and Golgi-localized ZmARGOS1 is suggested to
negatively regulate ethylene signal transduction by affecting ethylene perception or the earlier stages of ethylene
signaling (Shi et al., 2015).
In Arabidopsis (Arabidopsis thaliana), ethylene is perceived by a family of five receptors that are related to
prokaryotic two-component sensor His kinases and reside in the ER and Golgi membrane (Chang et al., 1993;
Hua and Meyerowitz, 1998). CONSTITUTIVE TRIPLE
RESPONSE1 (CTR1), a Raf-like kinase physically interacting with the receptors (Kieber et al., 1993; Clark et al.,
1998), transduces ethylene signals from the receptors to
the ER-tethered ETHYLENE-INSENSITIVE2 (EIN2) by
modifying its phosphorylation status. EIN2 then relays
the ethylene signal to nuclei to activate the master transcription factors EIN3 and ETHYLENE-INSENSITIVE3
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LIKE1 (Chao et al., 1997; Solano et al., 1998) via its
cleavable C-terminal domain (Qiao et al., 2012; Ju et al.,
2012). In this signal transduction pathway, CTR1 is a
negative regulator and represses downstream signaling in
the absence of ethylene (Kieber et al., 1993; Roman et al.,
1995). Genetic studies suggest that ZmARGOS1 modulates ethylene signal transduction by acting at or upstream of CTR1 because overexpressed ZmARGOS1 was
not able to suppress the constitutive ethylene response
phenotype of the loss-of-function mutant ctr1-1 (Shi et al.,
2015). The ethylene receptors interact with each other and
may function as a heterogeneous cluster (Gao et al., 2008;
Grefen et al., 2008; Chen et al., 2010). CTR1 and the
ethylene receptors form a signaling complex through
protein-protein interactions at ER membranes (Clark
et al., 1998; Gao et al., 2003). The His kinase domain of
the receptor ETHYLENE RESPONSE1 (ETR1) and
ETHYLENE RESPONSE SENSOR1 interact with the
CTR1 N-terminal domain (Clark et al., 1998), and this
interaction is required for signaling from ethylene perception to the regulation of CTR1 activity (Huang et al.,
2003; Gao et al., 2003). ETR1 also physically interacts
with another membrane protein in the signaling complex, which is encoded by REVERSION-TO-ETHYLENE
SENSITIVITY1 (RTE1; Resnick et al., 2006; Dong et al.,
2008, 2010). RTE1 was identified in a genetic screen for
suppressors of the etr1-2 mutation (Resnick et al., 2006).
It is thought that RTE1 may cause a conformational
change in the ethylene binding domain of ETR1 through
protein-protein interactions, therefore regulating the activity of ETR1 (Resnick et al., 2008). Genetic analysis
suggested that a normal interaction between RTE1 and
ETR1 is likely required for ZmARGOS1 reducing ethylene sensitivity in Arabidopsis, as the etr1-7 rte1-2 double
mutation partially suppresses the reduced ethylene
sensitivity phenotype in ZmARGOS1 overexpression
plants while ZmARGOS1 functions normally in the single mutant backgrounds (Shi et al., 2015).
Arabidopsis RTE1 reduces ethylene responsiveness
in an ETR1-dependent manner when overexpressed in
Arabidopsis (Resnick et al., 2006; Zhou et al., 2007).
Similarly, overexpression of RTE1 tomato (Solanum
lycopersicum) homologs GREEN-RIPE (GR) and GREENRIPE LIKE1 (GRL1), as well as ectopic expression of the
dominant gain-of-function mutant allele Green-ripe (Gr),
decreases ethylene response in tomato plants, as shown in
seedlings, fruit, abscission zones, and petioles (Barry and
Giovannoni, 2006; Ma et al., 2012). Transgenic rice (Oryza
sativa) plants overexpressing rice REVERSION-TOETHYLENE SENSITIVITY1 HOMOLOG1 also showed
reduced ethylene responsiveness across a range of biological processes, such as leaf senescence, seedling leaf
elongation and development, and coleoptile elongation
as well as adventitious root development (Zhang et al.,
2012). In the RTE1 gene family, some members, for
example, Arabidopsis REVERSION-TO-ETHYLENE
SENSITIVITY1 HOMOLOG (RTH) and tomato GRL2,
do not appear to play the same role as RTE1 in ethylene
signaling (Resnick et al., 2006; Ma et al., 2012). Rice
RTH2 and RTH3 were not able to complement the
Arabidopsis rte1-2 mutant when their overexpression
was driven by the cauliflower mosaic virus 35S promoter (Zhang et al., 2012), but whether they modulate
ethylene sensitivity in rice was not determined.
Colocalization of ARGOS proteins and the ethylene
receptor signaling complex in ER membranes supports
the genetic evidence for ARGOS modulating ethylene
perception or the early steps of ethylene signal transduction (Shi et al., 2015). However, the mechanisms for
the regulatory function of ARGOS are unknown. In this
study, we first investigate the basic structural need to
confer reduced ethylene sensitivity for the ARGOS
protein family, among whose members overall amino
acid sequence similarity is quite low (for example, 29–
36% amino acid identity between ZmARGOS1 and
Arabidopsis ARGOS proteins). We then test the proteinprotein interactions of ZmARGOS1 and Arabidopsis
ARGOS homolog ORGAN SIZE RELATED1 (OSR1)
with AtRTE1 in Arabidopsis, as well as with maize RTE1
homolog REVERSION-TO-ETHYLENE SENSITIVITY1LIKE (RTL) proteins in a yeast model system. In addition,
to determine the function of ARGOS-interacting proteins
in maize, the maize RTL genes are characterized using
transgenic maize plants. Our results support direct association of ARGOS proteins with the ethylene receptor
signaling complex through RTE1 in Arabidopsis. Detection of a physical interaction between the maize ARGOS
and RTL proteins lends insight into how overexpressed
ZmARGOS1 and ZmARGOS8 reduces ethylene sensitivity in transgenic maize plants and improves grain yield.
RESULTS
The TM1-PRM-TM2 Domain of ARGOS Proteins Is
Necessary and Sufficient to Confer Reduced Ethylene
Sensitivity in Arabidopsis
Members of the ARGOS protein family contain a
conserved Pro-rich motif (PRM), Pro-Pro-Leu-Pro-ProPro-Pro-X, where X denotes a variable amino acid residue. Although the similarity of amino acid sequences
declines rapidly with increasing distance in either direction from the PRM, two transmembrane helices
(TM1 and TM2), which immediately flank the PRM, are
well conserved among ARGOS family members, possibly determining the topology of the protein in the ER
and Golgi members with both the N and C termini
predicted (PRODIV-TMHMM; Viklund and Elofsson,
2004) to be exposed to the cytosolic side and the PRM
exposed to the luminal side (Fig. 1A). The TM1-PRM-TM2
domain (TPT) represents a common feature in the secondary structure of ARGOS proteins. Previously, we have
shown that substitution of a Leu with Asp in the PRM of
ZmARGOS1 and ZmARGOS8 inactivated these proteins
(Shi et al., 2015). To assess the importance of Pro in the
ZmARGOS1 PRM, each Pro was replaced with Asp, and
the ZmARGOS1 gene variants then were overexpressed in
Arabidopsis under control of the cauliflower mosaic virus
35S promoter (35S). Transgenic seeds (T1) were selected
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Association of ARGOS and RTE1 in Ethylene Signaling
Figure 1. Functional analysis of truncated and mutated ZmARGOS1 in transgenic Arabidopsis. A, Schematic representation of
predicted ARGOS orientation in membranes. PRM, Pro-rich motif consisting of Pro-Pro-Leu-Pro-Pro-Pro-Pro-X, where X represents an unconserved amino acid residual; TM1 and TM2, predicted transmembrane a-helix; N-term, N-terminal region;
C-term, C-terminal region. B, Ethylene triple responses of 35S:ZmARGOS1 transgenic plants (ARGOS1) and wild-type control to
10 mM ACC. Composite image of representative 3-d-old etiolated seedlings. Bar = 2 mm. C, Hypocotyl and root lengths of
transgenic Arabidopsis seedlings. The plants overexpressing a wild-type ZmARGOS1 and the ZmARGOS1 variants with amino
acid substitution in the PRM were grown in the dark in the presence or absence of 10 mM ACC for 3 d. Data shown are means 6 SD
of 20 independent transgenic lines randomly selected for each construct based on YFP marker expression in T1 seeds. Vector,
empty vector controls. Significant differences of the amino acid substitution mutants from the non-mutant ZmARGOS1 are
denoted by asterisks (**P , 0.01, ANOVA, Tukey HSD). D, Schematic representation of ZmARGOS1 variants with truncation and
amino acid substitution in TMs. Truncation in the N- and C-terminal regions of a full-length ZmARGOS1 produced TR-n (amino
acids 62–144) and TR-c (1–134), respectively. TR-nc, amino acids 62–134. TM1m, Ala-84, and Leu-85 substitution with Asp.
TM2, Leu-120, 121, and 122 substitution with Asp. E. Hypocotyl and root lengths of etiolated Arabidopsis seedlings. Transgenic
plants overexpressing the full-length ZmARGOS1 protein as well as truncated and mutated ZmARGOS1 were grown in the dark
in the presence or absence of 10 mM ACC for 3 d. Data are means 6 SD of 25 independent transgenic lines randomly selected for
each construct based on YFP marker expression in T1 seeds. Significant differences of the truncation and amino acid substitution
mutants from the full-length ZmARGOS1 are denoted by asterisks (**P , 0.01, ANOVA, Tukey HSD). FL, full length.
based on expression of visible marker yellow fluorescence
protein (YFP). Coexpression of the transgene of interest
with transformation selection marker bialaphos resistance
gene and the visible marker YFP was confirmed by reverse transcription (RT) PCR. The ethylene triple response
assay (Bleecker et al., 1988) was used to evaluate the activity of overexpressed ARGOS proteins in conferring
reduced ethylene sensitivity in Arabidopsis (Shi et al.,
2015). The 35S:ZmARGOS1 plants had reduced ethylene
responses relative to wild type controls (Fig. 1B). Unlike
the non-mutant ZmARGOS1 transgenic plants, the plants
overexpressing the mutated version ZmARGOS1(P106D)
and ZmARGOS1(P107D) exhibited ethylene responses
similar to wild type. Root and hypocotyl growth in the
35S:ZmARGOS1(P106D) and 35S:ZmARGOS1(P107D)
plants was inhibited by the ethylene precursor
aminocyclopropane-1-carboxylic acid (ACC; Fig. 1C).
These phenotypes are similar to that displayed in the
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Shi et al.
transgenic plants overexpressing the loss-of-function
ZmARGOS1 variant ZmARGOS1(L104D) (Shi et al., 2015;
Fig. 1C), suggesting that the Pro-106 and Pro-107 are required for ZmARGOS1 function. For the Pro-108, Asp substitution also had a significant effect on ZmARGOS1 activity.
Both the root and hypocotyl of the 35S:ZmARGOS1(P108D)
plants were shorter than that in the non-mutant
ZmARGOS1 plants (Fig. 1C) although the Pro-108 substitution affected root growth to a lesser extent relative to
the Pro-106 and Pro-107. In contrast, substitution of Pro102, Pro-103, and Pro-105 with Asp had no effect on
ZmARGOS1 function in terms of root responses to ACC.
The hypocotyl was slightly shorter than that in the nonmutant ZmARGOS1 plants (Fig. 1C), but longer than the
empty vector control (P , 0.01, Tukey HSD), indicating
that the ZmARGOS1(P102D), ZmARGOS1(P103D), and
ZmARGOS1(P105D) mutant proteins can reduce ethylene responses when overexpressed. Taken together, these
results suggested that the PRM between the two transmembrane helices is important for the ZmARGOS1 protein to confer reduced ethylene sensitivity in Arabidopsis.
Mutations were also introduced into the two transmembrane helices of ZmARGOS1 to determine their
role in ARGOS activity. Substitution of Ala-84 and Leu-85
with Asp in TM1 abolished the capability of ZmARGOS1
in reducing ethylene responses (Fig. 1, D and E; Supplemental
Fig. S1). A similar effect was obtained when TM2 was disrupted by substituting Leu-120, Leu-121, and Leu-122 with
Asp in the helix region. These results suggested that
the transmembrane domains are required for ARGOS
to modulate ethylene sensitivity in Arabidopsis. Because the N- and C-terminal regions of ARGOS proteins
are not conserved among the family members, we
next tested the deletion variants of Zm-ARGOS1 in
these regions.
Truncated versions of ZmARGOS1 were generated
by deletion in the N- and C-terminal regions (Fig. 1D).
The truncation variants, TR-n and TR-c, were active,
similar to the full-length ZmARGOS1, as revealed in
the ethylene triple response assay of the transgenic
Arabidopsis overexpressing the truncation variants
(Fig. 1E), indicating that the first 60 amino acids in the N
terminus of ZmARGOS1 (amino acids 2–61; GenBank,
JN252297) and the last 10 amino acids in the C terminus (amino acids 135–144) are dispensable. Transgenic
plants overexpressing ZmARGOS1(TR-nc), a truncated
ZmARGOS1 that is composed of 74 amino acids and
contains only the TPT domain, displayed the same phenotype of reduced ethylene response as that of the fulllength ZmARGOS1 in etiolated seedling triple response
assay (Fig. 1, D and E; Supplemental Fig. S1), suggesting
that the TPT domain is sufficient for ZmARGOS1 negatively regulating ethylene signal transduction.
A Truncated ZmARGOS8 Can Reduce Ethylene Sensitivity
in Maize
A previous study has shown that ZmARGOS8 reduces ethylene sensitivity when overexpressed in
Arabidopsis and maize though the phenotype is
weaker than that in ZmARGOS1 (Shi et al., 2015). A
truncated version of ZmARGOS8, containing the TPT
domain, generated by deleting 35 and 12 amino acids
from the N and C termini, respectively, and thereafter
referred to as ZmARGOS8(TR) (amino acids 36–106;
GenBank: JN252302), is able to significantly reduce
ethylene response in Arabidopsis, producing a strong
phenotype relative to the full-length ZmARGOS8, as
exhibited by the triple response assay of etiolated
transgenic seedlings (Fig. 2, A and B). In growth conditions under light, the roots of 35S:ZmARGOS8(TR)
Arabidopsis plants also showed reduced response to
0.2 and 0.5 mM ACC (Fig. 2, C and D). The transcript
levels of the ethylene-inducible ERF1 gene were lower
in the 35S:ZmARGOS8(TR) plants than that in the wildtype control treated with 10 mL L21 exogenously supplied ethylene (Fig. 2E).
The ZmARGOS8 gene has improved functionality
over ZmARGOS1 in enhancing maize grain yield in
both drought-stressed and well-watered environments
(Shi et al., 2015). To take advantage of the strong phenotype of the ZmARGOS8(TR) transgenic plants for
studying mode of action of ZmARGOS8, we first determine whether ZmARGOS8(TR) can modulate ethylene response in maize as well. Therefore, ZmARGOS8
(TR), along with the full-length ZmARGOS8, was overexpressed in maize plants under control of the maize
UBIQUITIN1 promoter (UBI1). An ethylene-responsive
root elongation assay (Fig. 3A), using etiolated seedlings and 100 mM ACC (Shi et al., 2015), showed that the
UBI1:ZmARGOS8(TR) transgenic plants had reduced
responses to ethylene in roots (Fig. 3, A and B). In the
same assay, the reduced ethylene response phenotypes
were not detected in transgenic lines overexpressing the
full-length ZmARGOS8 driven by the UBI1 promoter
(Fig. 3, B and C). Instead, a stronger promoter, the banana (Musa) streak virus promoter, was needed to
produce the phenotype in maize (Shi et al., 2015). These
results demonstrated that the truncation variant
ZmARGOS8(TR) can negatively regulate ethylene signaling in maize, similar to that in Arabidopsis.
Maize and Arabidopsis ARGOS Proteins Interact with
AtRTE1 in Arabidopsis
Genetic analysis suggested that ZmARGOS1 targets
the ethylene signaling pathway between the ethylene receptors and CTR1 (Shi et al., 2015). To test if ZmARGOS1
physically interacts with Arabidopsis RTE1, bimolecular
fluorescence complementation assay (BiFC; Hu et al.,
2002; Walter et al., 2004) was used. The sequence encoding the N- and C-terminal halves of split Aequorea
coerulescens GFP (nGFP and cGFP) was fused in frame
to AtRTE1 at the N terminus and ZmARGOS1 at the C
terminus, respectively. The fusion genes were transformed
individually into Arabidopsis to generate transgenic lines.
The ER-localized membrane protein AtRTE1 has a cytosolic N terminus (Dong et al., 2010). Both the N and C
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Association of ARGOS and RTE1 in Ethylene Signaling
Figure 2. Overexpressing a truncation variant ZmARGOS8(TR) reduces ethylene response in Arabidopsis. A, Western-blot analysis of
transgenic Arabidopsis. Three 35S:ZmARGOS8 lines (2E1, 2E2, and 2E3) and three 35S:ZmARGOS8(TR) lines (E1, E2, and E3) are shown for
protein expression in leaves of 18-d-old plants. Std, 0.625 ng of purified recombinant ARGOS8 protein; trans, transgenic maize overexpressing ZmARGOS8 as positive controls; null, nontransgenic maize. B, Root and hypocotyl lengths of etiolated Arabidopsis seedlings.
The transgenic plants overexpressing ZmARGOS8(TR) (two lines, E2 and E3) and the wild-type control were grown in the dark in the
presence of ACC at indicated concentrations for 4 d. A representative full-length ZmARGOS8 transgenic line (2E3) is included for comparison. Data are means 6 SD (n = 20). Significant differences of the transgenic plants from the wild-type control are denoted by asterisks
(**P , 0.01, ANOVA, Tukey HSD). C, Root phenotypes in 5-d-old 35S:ZmARGOS8(TR) (line E2) and 35S:ZmARGOS8 (line 2E3) transgenic
Arabidopsis plants and wild-type controls. Plants were grown in agar that contained one-half-strength Murashige and Skoog medium with 0,
0.2, or 0.5 mM ACC and were set vertically in a growth chamber under a regime of 16 h of light at 24˚C and 8 h of darkness at 23˚C. D, Root
lengths of 5-d-old light-grown Arabidopsis seedlings. The 35S:ZmARGOS8(TR) and 35S:ZmARGOS8 transgenic plants as well as wild-type
controls were grown in the presence of 0, 0.2, or 0.5 mM ACC. Data are means 6 SD (n = 20). Significant differences of the transgenic plants
from the wild-type control are denoted by asterisks (**P , 0.01, ANOVA, Tukey HSD). E, Relative expression levels of the ethylene-inducible
gene ERF1 in wild-type and 35S:ZmARGOS8(TR) transgenic plants. Five-d-old light-grown seedlings were exposed to ethylene gas at
10 mL L21 for 5 h. The ERF1 transcripts were measured using qRT-PCR and data are means 6 SD (n = 4). Significant differences of the transgenic
plants from the wild-type control are denoted by asterisks (*P , 0.05, **P , 0.01, ANOVA, Tukey HSD). WT, wild-type Arabidopsis.
termini of ZmARGOS1 are predicted (PRODIV-TMHMM;
Viklund and Elofsson, 2004) to be exposed to the cytosol.
Overexpression of the ZmARGOS1-cGFP transgene reduced ethylene response in Arabidopsis (data not shown),
as did nGFP-AtRTE1 (data not shown), as previously
reported by Dong et al. (2010), indicating that the split
GFP-tagged proteins retain their function. The nGFPAtRTE1 transgenic plants did not show green florescence,
nor those overexpressing the ZmARGOS1-cGFP (Fig. 4A),
as expected. When the two constructs were brought together by crossing the transgenic plants, both ZmARGOS1cGFP and nGFP-AtRTE1 fusion proteins were detectable in
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Figure 3. Reduced ethylene response in transgenic maize plants overexpressing ZmARGOS8(TR). A, Responses of maize roots to
ethylene precursor ACC treatments. The UBI1:ZmARGOS8(TR) transgenic and null seeds were germinated in filter paper rolls set
vertically in the dark in the presence or absence of ACC for 5 d. Representative seedlings are shown for reduced ethylene response
in the UBI1:ZmARGOS8(TR) transgenic plants. Bar = 5 cm. B, Root lengths of etiolated seedlings of transgenic maize. The UBI1:
ZmARGOS8(TR) and UBI1:ZmARGOS8 plants were grown in the dark in the presence of 0 or 100 mM ACC for 5 d. The primary
roots of 12 seedlings per transgenic line per treatment were measured. Data show means 6 SD of three transgenic lines. Student’s
t test was performed to compare the transgenic plants with nulls. **P , 0.01. C, Relative expression levels of the transgene in the
UBI1:ZmARGOS8(TR) and UBI1:ZmARGOS8 plants. Leaf samples were taken from V4 seedlings and transcript abundance was
measured using a quantitative reverse transcription PCR with the primers and probe derived from the rice UBIQUITIN terminator
of the constructs. Data are means 6 SD of three transgenic lines for each construct.
F1 plants via western blotting (Fig. 4C) and GFP-positive
fluorescence signals was observed in hypocotyl cells of
etiolated seedlings (Fig. 4, A and B) as well as in epidermal
cells of leaves of plants grown under light (Fig. 4D), indicating protein-protein interactions between ZmARGOS1
and AtRTE1. BiFC signals in hypocotyl cells were associated with small bodies and interconnected threads in the
cytoplasm (Fig. 4B), consistent with the subcellular localization of the ARGOS and AtRTE1 proteins in the ER and
Golgi (Dong et al., 2008; Feng et al., 2011; Rai et al., 2015; Shi
et al., 2015). Strong BiFC signals were detected in the vascular tissues (Fig. 5) where the DMMV promoter-driven
expression was relatively higher than other leaf tissues
(Dey and Maiti, 1999).
Using this BiFC assay, the Arabidopsis ARGOS homolog OSR1 and ZmARGOS8(TR) were found to also interact with AtRTE1. In leaves, BiFC signals associated with
the vascular tissues were detectable under a fluorescence
stereo microscope in the DMMV:nGFP-AtRTE1 plants
coexpressing AtOSR1-cGFP or ZmARGOS8(TR)-cGFP
(Fig. 5, A and B). However, no BiFC signals were observed in plants coexpressing ZmARGOS8-cGFP and
nGFP-AtRTE1. AtRTE1 was reported to interact with
the ER-localized cytochrome b5 (AtCb5; Chang et al.,
2014). Therefore, we used Arabidopsis Cb5 isoform D
(AtCb5D) as a positive control for the BiFC assay, and a
similar pattern of fluorescence signals was found in the
vascular tissues (Fig. 5) and the epidermal cells (Fig. 4E).
For negative controls, F1 plants were derived from crosses
of plants overexpressing the nGFP-tagged Arabidopsis
endomembrane cation/H+ exchanger CHX20 (Padmanaban
et al., 2007) and ZmARGOS1-cGFP. No BiFC signals were
detected in the negative control (Fig. 5A).
Interaction of ARGOS with Arabidopsis RTE1 and Maize
Homologs in a Yeast Model System
To confirm the protein-protein interactions of
ZmARGOS1 and AtOSR1 with AtRTE1, a mating-based
split-ubiquitin yeast two-hybrid system (Obrdlik et al., 2004)
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Association of ARGOS and RTE1 in Ethylene Signaling
Figure 4. Visualization of protein-protein interactions of maize ARGOS1 with AtRTE1 in Arabidopsis using BiFC analysis. A,
Fluorescence image of the hypocotyl-root junction region of a 3-d-old etiolated Arabidopsis F1 seedling coexpressing nGFPAtRTE1 and ZmARGOS1-cGFP (left) showing reconstituted GFP signals due to protein-protein interactions between ZmARGOS1
and AtRTE1. Seedlings expressing nGFP-AtRTE1 and ZmARGOS1-cGFP alone did not show fluorescence (middle and right).
Bar = 500 mm. B, Representative fluorescence images of hypocotyl cells from a 3-d-old etiolated Arabidopsis seedling coexpressing nGFP-AtRTE1 and ZmARGOS1-cGFP showing GFP fluorescence as small punctate bodies and interconnected threads
within each cell. The highly vacuolated nature of these cells crowds the GFP-positive bodies toward inner perimeter of the cells. A
laser-scanning confocal microscope was used to capture GFP and autofluorescence signals at 500 to 530 nm (excitation 488 nm)
and 430 to 480 nm (excitation: 408 nm), respectively. A merged image of GFP (green) and autofluorescence (blue) is shown at
right. Bar = 25 mm. C, Western-blotting analysis of ZmARGOS1-cGFP and nGFP-AtRTE1 fusion protein expression in Arabidopsis
plants. Proteins were extracted from 18-d-old F1 plants. Anti-HA antibodies were used to detect the fusion proteins, which
contain the HA epitope. D, Representative fluorescence images of epidermal cells in a leaf of a 14-d-old plant coexpressing
nGFP-AtRTE1 and ZmARGOS1-cGFP showing reconstituted GFP fluorescence (left) captured using a laser-scanning confocal
microscope with a GFP filter (500–530 nm, excitation: 488 nm). An autofluorescence image (middle) was captured using a DAPI
band-pass filter (430–480 nm, excitation 408 nm). At right is the merged image of GFP (green) and autofluorescence (blue). Bar =
25 mm. E, Representative fluorescence images of epidermal cells in a leaf of a 14-d-old plant coexpressing nGFP-AtRTE1 and
cGFP-AtCb5D showing reconstituted GFP fluorescence, as positive controls for the BiFC assay. Bar = 25 mm.
was employed. The coding sequences of ZmARGOS1
(prey) and AtOSR1 (prey) were fused in frame to the
N-terminal half of a mutated ubiquitin (NubG; containing mutation Ile-13Gly). AtRTE1 (bait) was cloned
as a translational fusion to the C-terminal half of ubiquitin (Cub) followed by a synthetic transcription factor,
PLV (protease A-LexA-VP16). The NubG with reduced
affinity to the Cub moiety is unable to reconstitute
functional ubiquitin. Only when the bait and prey
proteins interact at the ER membranes is the NubG
brought into the vicinity of the Cub domain in the cytosol side of the ER membrane, forming a functional
ubiquitin. Endogenous ubiquitin-specific proteases then
release the transcription factor, which diffuses into
the nucleus where it activates the transcription of reporter genes (HIS3, ADE2, and lacZ). Expression of the
AtRTE1-Cub-PLV fusion protein, as well as cleavage
and function of the LexA-VP16 transcription factor, was
verified by pairing the bait construct with a prey construct containing the N-terminal domain of wild-type
ubiquitin (NubWT; Fig. 6, A and B). The NubWT interacts with Cub independent of the prey-bait association,
reconstituting ubiquitin and activating reporters. An
empty NubG vector, which expresses a soluble NubG,
serves as controls to eliminate the possibility of selfactivation of the AtRTE1-Cub-PLV fusion protein (Fig. 6).
Yeast diploid cells produced by mating yeast strains
containing the ZmARGOS1 and AtOSR1 prey constructs
with the strain containing the AtRTE1 bait construct can
grow on the synthetic complete (SC) -Leu-Trp-His-Ade
medium (Fig. 6A), indicating protein-protein interactions between the bait and prey. Lack of red pigment
accumulation in the diploid cells grown on the SC-LeuTrp medium indicates that the transcription of the reporter gene ADE2 was also activated (Fig. 6A), consistent
with the results from the HIS3-dependent growth assay.
Interactions of ZmARGOS1 and AtOSR1 with AtRTE1
were further verified with the b-galactosidase (b-Gal)
assay (Fig. 6B), which measures the activity of the LacZ
reporter. As a negative control, Arabidopsis CHX20 was
fused in frame to the Cub-PLV to produce a bait construct
that was used in mating experiments with the ARGOS
prey constructs (Fig. 6C). No apparent growth under His
selective conditions were observed in these matings,
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Figure 5. Visualization of the interaction
of maize and Arabidopsis ARGOS proteins with AtRTE1 in Arabidopsis using
BiFC analysis. A, Fluorescence images of
the lower side of leaves in Arabidopsis
transgenic plants overexpressing nGFPAtRTE1 and cGFP-tagged maize and
Arabidopsis ARGOS proteins. Reconstituted green fluorescence associated
with the vascular tissues is shown for
representative leaves of 18-d-old F1
plants. F1 plants derived from the nGFPAtRTE1 and cGFP-AtCb5D crosses and
the AtCHX20-nGFP and ZmARGOS1cGFP crosses serve as positive and negative controls, respectively. All images
were captured using a fluorescence stereo microscope with the same setting
(1-s exposure time). Bar = 500 mm. B,
Fluorescence images of a section of a
leaf in 14-d-old Arabidopsis transgenic
plants overexpressing nGFP-AtRTE1 and
cGFP-tagged maize and Arabidopsis
ARGOS proteins. A confocal laser scanning microscope was used to capture
reconstituted GFP fluorescence (500–
530 nm, excitation: 488 nm), which is
associated with vascular tissues. Autofluorescence signals were captured at
430–480 nm (excitation: 408 nm).
Merged images of GFP (green) and
autofluorescence (blue) are shown.
Bar = 25 mm.
neither was b-Gal activity detected (Fig. 6D). The diploid
cells grown on the SC-Leu-Trp medium accumulated a
red pigment (Fig. 6C), indicating that the reporter gene
ADE2 was inactive as well. Taken together, the data suggested that ZmARGOS1 and AtOSR1 physically interact
with the ethylene receptor regulator AtRTE1 in yeast,
corroborating the BiFC results obtained in Arabidopsis.
Using the same yeast system, we found that Arabidopsis ARGOS (AT3G59900) and ARGOS-LIKE (ARL;
AT2G44080) also interact with AtRTE1 (Fig. 6). Yeast
diploid cells expressing ZmARGOS8-NubG grew less
than the cells expressing other ARGOS-NubG fusion
proteins in the interaction-dependent growth assay
(Fig. 6A) and concomitantly showed lower b-Gal
activity (Fig. 6B), indicating a weak interaction
between ZmARGOS8 and AtRTE1. The truncation
variant ZmARGOS8(TR), however, displayed a strong
interaction with AtRTE1 (Fig. 6, A and B), consistent
with the results of the BiFC assay in Arabidopsis and its
high activity in reducing ethylene sensitivity in Arabidopsis and maize plants. With the establishment of interactions between AtRTE1 and various ARGOS proteins,
we next tested ARGOS interaction with maize RTE1
homologs.
A BLAST search with the AtRTE1 protein sequence
revealed four homologous genes in the maize genome
(Supplemental Fig. S2). We designated these genes
as REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE1
(RTL1), RTL2, RTL3, and RTL4. Their amino acid sequences are 52, 53, 50, and 42% identical to AtRTE1,
respectively, in a pairwise comparison. The RTL genes
express broadly across various tissues in maize
(Supplemental Fig. S3) and their functions are unknown. Phylogenetic analysis indicate that ZmRTL1,
ZmRTL2, and ZmRTL3 belong to the AtRTE1/SlGR
clade of the RTE1 gene family (Barry and Giovannoni,
2006; Ma et al., 2012) while ZmRTL4 is more closely
related to the AtRTH/SlGRL2 clade (Supplemental Fig.
S4) and therefore, we elected to determine the function
of ZmRTL2 and ZmRTL4. The coding region was cloned
into the bait construct that was paired with the ARGOS
prey for testing protein-protein interactions in the yeast
split-ubiquitin two-hybrid system. Data presented in
Figure 7 revealed that ZmRTL4 interacts with ZmARGOS1,
ZmARGOS8(TR), and three Arabidopsis ARGOS proteins.
The growth assay of diploid cells on SC-Leu-Trp-HisAde selective medium indicated a very weak interaction between ZmARGOS8 and ZmRTL4, but b-Gal
activity was not significantly different from the empty
vector control (Fig. 7). With the same assay, ZmRTL2
was found to interact with ZmARGOS8(TR) and
AtOSR1 (Fig. 7, C and D). A weak interaction with
ZmARGOS1, AtARGOS, and AtARL also is evident
(Fig. 7, C and D).
To verify the protein-protein interaction of maize
ARGOS and RTL, BiFC was performed using stably
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Association of ARGOS and RTE1 in Ethylene Signaling
Figure 6. Interactions of Arabidopsis and maize ARGOS with AtRTE1 as revealed with the yeast split ubiquitin system. A, Growth
of yeast diploid cells on selective SC dropout media to show protein-protein interactions. Diploid cells were generated by mating
haploid strain THY.AP4 containing the bait construct of AtRTE1-Cub-PLV with THY.AP5 carrying the prey constructs of Arabidopsis or maize ARGOS and NubG fusions. Empty vector NubG serves as a control for autoactivation of the bait AtRTE1-Cub-PLV.
The prey construct AtARGOS-NubWT is included to show that the AtRTE1-Cub-PLV fusion protein is properly expressed and
cleaved PLV is functional (also see C). Serial dilutions of liquid cultures were spotted on the indicated plates and incubated at 28˚C
for 4 d. Top panel shows growth and accumulation of red pigments in yeast cells on adenine and His-supplemented SC dropout
medium. Bottom panel shows growth on His selective medium reporting interactions of AtRTE1 with various ARGOS proteins. B,
b-Gal assay for yeast diploid cells expressing AtRTE1-Cub-PLV and various ARGOS-NubG fusion proteins. Yeast cells were
cultured in SC-Leu-Trp liquid medium. The b-Gal activity assay uses O-nitrophenylglucoside as a substrate. Data are means 6 SD
(n = 6). Significant differences of the ARGOS constructs from the empty vector control are denoted by asterisks (**P , 0.01,
ANOVA, Tukey HSD). C, Growth of yeast diploid cells from the mating of haploid strains containing ARGOS-NubG constructs
and negative control AtCHX20-Cub-LPV construct. The plates were incubated at 28˚C for 4 d. D, Liquid b-Gal assay for yeast
diploid cells expressing negative control AtCHX20-Cub-PLV and various ARGOS-NubG fusion proteins.
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Figure 7. Interactions of Arabidopsis and maize ARGOS with ZmRTL4 and ZmRTL2 in the yeast split-ubiquitin assay. A, Growth
of yeast diploid cells on SC dropout media to show protein-protein interaction of ZmRTL4 with Arabidopsis and maize ARGOS.
The SC-Leu-Trp and SC-Leu-Trp-His-Ade media contain 134 and 375 mM Met, respectively. The plates were incubated at 28˚C for
4 d. B, b-Gal assay for yeast diploid cells expressing ZmRTL4-Cub-PLV and various ARGOS-NubG fusion proteins. Data are
means 6 SD (n = 6). Significant differences of the ARGOS constructs from the empty vector control are denoted by asterisks (**P ,
0.01, ANOVA, Tukey HSD). C, Growth of yeast diploid cells on SC dropout media to show protein-protein interaction of ZmRTL2
with Arabidopsis and maize ARGOS. The SC-Leu-Trp and SC-Leu-Trp-His-Ade media contain 134 mM Met. The plates were
incubated at 28˚C for 5 d. D, b-Gal assay for yeast diploid cells expressing ZmRTL2-Cub-PLV and various ARGOS-NubG fusion
proteins. Data are means 6 SD (n = 6). Significant differences of the ARGOS constructs from the empty vector control are denoted
by asterisks (**P , 0.01, ANOVA, Tukey HSD).
transformed lines of Arabidopsis as described above.
Reconstituted green fluorescence was observed in
leaves of F1 plants derived from crosses of the
ZmARGOS1-cGFP and nGFP-ZmRTL4 transgenic plants
(Fig. 8). When nGFP-ZmRTL4 was brought together with
cGFP-tagged ZmARGOS8(TR) or ZmARGOS8, BiFC
signals were detected in both, but the signal from the
ZmARGOS8(TR) combination was stronger than the full-
length ZmARGOS8 (Fig. 8). In the ZmARGOS8-cGFP/nGFPZmRTL4 plants, green fluorescence was visible only in
the vascular tissues where the DMMV promoter-driven
expression was relatively higher than other leaf tissues
(Dey and Maiti, 1999). ZmRTL2 also interacts with
ZmARGOS1 and ZmARGOS8(TR), but no BiFC signals
were detected in F1 plants of the ZmARGOS8-cGFP
and nGFP-ZmRTL2 crosses (Fig. 8).
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Association of ARGOS and RTE1 in Ethylene Signaling
Overexpression of Maize RTL Genes Reduces Ethylene
Responses in Maize
Arabidopsis RTE1 reduces ethylene sensitivity when
overexpressed in Arabidopsis (Resnick et al., 2006). To
determine the effect of maize AtRTE1 homologs on
ethylene response, ZmRTL2 and ZmRTL4 were overexpressed in transgenic maize plants under control of
the maize UBI1 promoter. Multiple single-copy transgenic lines were generated for each construct in an inbred background, ZmRTL transgene expression was
confirmed by RT-PCR, and hybrid seeds were produced by top-crossing the transformants to a tester inbred. The ethylene responsiveness of the transgenic
plants was assessed by measuring primary root lengths
in etiolated seedlings in the presence of 100 mM ACC.
Data presented in Figure 9A show that overexpression
of ZmRTL2 and ZmRTL4 alleviates the inhibitory effect
of ACC on root growth, indicating reduced ethylene
response in the transgenic plants.
It was suggested that petunia (Petunia hybrida)
AtRTE1 homolog GREEN RIPE LIKE2 (PhGRL2) may
negatively regulate ethylene biosynthesis by inhibiting
ACC oxidase activity (Tan et al., 2014). Therefore, we
determined the effect of overexpressing ZmRTL2 and
ZmRTL4 on ethylene biosynthesis in maize. The ethylene emission rate in one ZmRTL2 and three ZmRTL4
transgenic lines was not significantly different from
that in nontransgenic controls (Fig. 9B). One ZmRTL2
line emitted significantly more ethylene than the
null control (Fig. 9B). These results indicated that
ZmRTL2 and ZmRTL4 may not play the same role
in maize as PhGRL2 does in petunia for ethylene
biosynthesis.
DISCUSSION
Maize and Arabidopsis ARGOS genes are negative
regulators of ethylene responses recently identified
using transgenic plants (Rai et al., 2015; Shi et al., 2015).
ARGOS reduces plant sensitivity to ethylene when
overexpressed. ARGOS proteins are small integral
membrane proteins and the conserved TPT domain is
sufficient to confer reduced ethylene response. Protein
sequence analysis did not reveal any catalytic sites in
ARGOS proteins. There is no evidence indicating that
ARGOS is one of the signaling cascade steps to relay the
ethylene signal. We hypothesized that ARGOS may
directly, or indirectly, modify the ethylene signaling
components (Shi et al., 2015). Genetic analysis suggested that overexpressed ZmARGOS1 protein targets
signaling components at or upstream of CTR1 in the
ethylene signaling pathway in Arabidopsis, regulating
ethylene perception, or the early steps of the ethylene
signal transduction (Shi et al., 2015). Using the BiFC
assay and the yeast split-ubiquitin two-hybrid system,
we found that maize ARGOS1 and ARGOS8 as well as
Arabidopsis OSR1 physically interact with Arabidopsis
RTE1 in vivo. AtRTE1 is a constituent of the ethylene
receptor signaling complex; through protein-protein
interaction, AtRTE1 stabilizes or promotes the active
conformation of the ethylene receptor ETR1 in Arabidopsis (Dong et al., 2008, 2010). The molecular association of ARGOS proteins with AtRTE1 is consistent
with their colocalization in the ER and Golgi membranes (Rai et al., 2015; Shi et al., 2015; Dong et al., 2008).
This finding is also consistent with the genetic data indicating that a normal interaction between ETR1 and
RTE1 is required for ZmARGOS1 to reduce ethylene
Figure 8. Visualization of the interaction of maize
ARGOS and RTL proteins in Arabidopsis using the
BiFC assay. A, Representative fluorescence images
are shown for leaves of 14-d-old F1 Arabidopsis
transgenic plants overexpressing nGFP-tagged
ZmRTL and cGFP-tagged ZmARGOS fusion
proteins. All images were captured using a
fluorescence stereo microscope with the same
setting (1-s exposure time). Bar = 500 mm. B,
Representative fluorescence images of a section of a leaf in Arabidopsis transgenic plants
overexpressing nGFP-tagged ZmRTL and cGFPtagged ZmARGOS proteins. A confocal laser
scanning microscope was used to capture
reconstituted GFP fluorescence and autofluorescence signals in vascular tissues at
500 to 530 nm (excitation: 488 nm) and 430 to
480 nm (excitation: 408 nm), respectively.
Merged images of GFP (green) and autofluorescence (blue) are shown. Bar = 50 mm.
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Figure 9. Overexpression of maize RTL2 and RTL4 reduces ethylene responses in maize plants. A, Root lengths of etiolated
seedlings of transgenic maize. The UBI1:ZmRTL2 and UBI1:ZmRTL4 plants were grown in a filter-paper roll set vertically in the
dark in the presence of 0 or 100 mM ACC for 5 d. The primary roots of 15 seedlings per transgenic line per treatment were
measured. Data show means 6 SD of five lines. Student’s t test was performed to compare the transgenic plants with nulls. *P ,
0.05. B, Ethylene emission from leaves of UBI1:ZmRTL2 and UBI1:ZmRTL4 transgenic lines and nontransgenic (Null) control.
Leaf discs were taken from the seventh leaf of V8 plants grown in greenhouse. Ethylene was collected for a period of 22 to 24 h and
subsequently measured using a gas chromatograph. Error bars indicate SD; n = 35 for nulls and 9 for each transgenic line. One-way
ANOVA with post-hoc Tukey HSD test was performed to compare the transgenic lines with nulls. **P , 0.01.
sensitivity in Arabidopsis (Shi et al., 2015). Overall,
these results suggest a physical association between
ARGOS and the ethylene receptor signaling complex
via AtRTE1, supporting a role for ARGOS in regulating
ethylene perception and signaling in Arabidopsis.
A similar mechanism is likely operational in maize as
well. Maize has four RTE1 homologous genes: RTL1,
RTL2, RTL3, and RTL4. Like AtRTE1 in Arabidopsis,
ZmRTL2 and ZmRTL4 reduce ethylene responses when
overexpressed in maize; the primary roots of etiolated
seedlings of transgenic maize are less responsive to
exogenously supplied ACC relative to nontransgenic
controls. In addition, ZmRTL4 and, to a lesser extent,
ZmRTL2 physically interact with maize ARGOS1 and
ARGOS8 as well as Arabidopsis ARGOS proteins, as
revealed in the yeast split-ubiquitin two-hybrid assay.
In maize, there are at least eight ARGOS genes;
ZmRTLs may have binding preference to different
ARGOS family members. Both ZmRTL4 and ZmRTL2
showed strong interaction with the truncation variant
ZmARGOS8(TR). The physical interaction of maize
RTL and ARGOS proteins was confirmed with the BiFC
assay in Arabidopsis. Furthermore, the functional and
structural conservation of the ethylene receptors between maize and Arabidopsis is evident (Chen and
Gallie, 2010). Collectively, these findings suggest a
similar mode of action for ARGOS modulating ethylene
signaling in maize as in Arabidopsis.
Despite the similarity between ZmRTL4 and AtRTE1
in interacting with ARGOS proteins and in regulating
ethylene signaling, the maize homolog also possesses
some distinct features. Previously, phylogenetic analysis suggested that members of the RTE1 gene family
from different species form two clades, AtRTE1/SlGR
and AtRTH/SIGRL2 (Barry and Giovannoni, 2006; Ma
et al., 2012). The AtRTE1/SlGR clade consists of the
RTE1 homologs that regulate ethylene signal transduction, such as Arabidopsis RTE1, tomato GR and
GRL1, and rice RTH1 (Zhang et al., 2012). The AtRTH/
SIGRL2 clade includes AtRTH and SlGRL2, which have
no apparent role in ethylene signaling (Resnick et al.,
2006; Ma et al., 2012); petunia PhGRL2, which was
proposed to regulate ethylene biosynthesis (Tan et al.,
2014), and rice RTH3, whose function is unknown
(Zhang et al., 2012). ZmRTL4 is more closely related to
the AtRTH/SlGRL2 clade, while ZmRTL2 apparently
belongs to the AtRTE1/SlGR clade (Supplemental Fig.
S4). However, both ZmRTL2 and ZmRTL4 can reduce
ethylene responses when overexpressed in maize. In
addition, ZmRTL4 was not able to complement the
Arabidopsis rte1-2 mutant when expressed under control of 35S or to reduce ethylene responses in Arabidopsis, as measured in the ethylene triple response
assay and a root growth assay using light-grown
seedlings (J. Shi, R.L. Archibald, H. Wang, and B.J.
Drummond, unpublished data). The finding of UBI1:
ZmRTL4 reducing ethylene responses in maize suggests
that the function of regulating ethylene signaling is not
limited to the members of the AtRTE1/SlGR clade.
Consistent with this hypothesis, AtRTH was reported
to physically interact with Arabidopsis ARL and the
cytochrome b5 isoform D (Cb5D) in the yeast splitubiquitin two-hybrid assay (Jones et al., 2014; Chang
et al., 2014). Both ARL and Cb5D negatively regulate
ethylene responses when overexpressed in Arabidopsis
(Rai et al., 2015; Chang et al., 2014) and also interact
with AtRTE1 (Fig. 6). The ER-localized Cb5 isoforms
promote ETR1-mediated repression of ethylene signaling (Chang et al., 2014).
From a structural perspective, questions remain regarding the binding determinants for the molecular
association of ARGOS with RTE1 proteins. The N- and
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Association of ARGOS and RTE1 in Ethylene Signaling
C-terminal regions are not required for the activity of
reducing ethylene sensitivity and the binding of AtRTE1
or maize RTL proteins, as revealed by the truncation
variant ZmARGOS1(TR-nc) and ZmARGOS8(TR).
However, TM1, TM2, and PRM are necessary for
ARGOS activity. The TPT domain alone is sufficient to
reduce ethylene responses in Arabidopsis and maize
when overexpressed, and can bind to AtRTE1 and
ZmRTL, as shown in the BiFC assay and the yeast splitubiquitin two-hybrid assay. Given the membrane localization of the protein-protein interaction, it is possible that
the two transmembrane helices of ARGOS proteins are
responsible for the association with AtRTE1 that is predicted to contain two or four transmembrane domains. In
this scenario, the PRM may function as a connector to
properly position the two transmembrane helices, forging
a functional conformation for ARGOS. Substitution of
Leu or Pro in this region would disturb the relative position of the two transmembrane helices, inactivating
ARGOS, as observed in the mutation analysis. Alternatively, the PRM, predicted to be exposed to the luminal
side of the ER, may function as a determinant for ARGOS
in protein-protein interactions. The Pro-rich regions in
proteins preferentially adopt a poly-Pro type II helical
conformation that facilitates transient intermolecular interactions (Williamson, 1994). Six distinct families of the
PRM binding domains are known to be present in various
proteins (Ball et al., 2005). However, it remains to be determined whether RTE1 contains one of such PRM
binding modules and what role the PRM of ARGOS may
play in the interaction with RTE1.
In view of the highly variable N- and C-terminal regions among ARGOS protein family members and lack
of a cytosolic N-terminal domain in AtOSR2 that possesses ARGOS activity (Shi et al., 2015), it is not surprising to find that the conserved TPT domain alone can
reduce ethylene sensitivity. The strong phenotype of
Arabidopsis and maize plants overexpressing the truncation variant ZmARGOS8(TR) suggests that the cytosolic N- and C-terminal domains may interact with the
TPT domain, inhibiting ARGOS8 activity. However, it
is also possible that the truncated protein is more stable
than the full-length ARGOS8 in the transgenic plants.
Nevertheless, the truncated version interacts with
AtRTE1 and ZmRTL in BiFC and the yeast splitubiquitin two-hybrid assay more strongly than the
full-length ZmARGOS8. The correlation in the reduced
ethylene response phenotype and protein-protein interaction strength is consistent with the concept that
ARGOS proteins regulate ethylene signaling via
protein-protein interactions.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
The Arabidopsis (Arabidopsis thaliana) ecotype Col-0 was used as the wild
type. Plants were grown under fluorescent lamps supplemented with incandescent lights (;120 mE m22 s21) in growth chambers with 16 h light period at
24°C and 8 h dark period at 23°C and 50% relative humidity. For the
Arabidopsis triple-response assay (Bleecker et al., 1988), surface-sterilized and
stratified seeds were germinated in dark conditions on medium (one-halfstrength Murashige and Skoog salts with 1% [w/v] Suc and 0.8% [w/v] agar)
containing ACC (Calbiochem) at the stated concentrations. Hypocotyl and root
length of etiolated seedlings were measured by photographing the seedlings
under a dissection microscope with a digital camera and using the image
analysis software ImageJ (National Institutes of Health). For assaying root response of light-grown seedlings to ethylene (Ruzicka et al., 2007), Arabidopsis
plants were grown for 5 d in agar that contained one-half-strength Murashige
and Skoog salts and 1% [w/v] Suc supplemented with 0, 0.2, or 0.5 mM ACC
and were set vertically in a growth chamber under a regime of 16 h of light
(;120 mE m22 s21) at 24°C and 8 h of darkness at 23°C. For ethylene treatments,
5-d-old light-grown seedlings were exposed to ethylene gas at 10 mL L21 for 5 h.
For assaying the maize (Zea mays) seedling response to ethylene, 15 seeds
were placed in a row between two layers of filter paper wetted in an ACC
aqueous solution at stated concentrations. The filter paper was rolled up with a
piece of waxed paper on the outside and set vertically in a beaker with a depth
of 2.5 cm of the same solution. The beaker was covered with plastic wrap to
prevent excessive evaporation and placed at 24°C in the dark. Seedlings were
photographed with a digital camera 5 d after seeding, and the length of primary
roots was measured in these images using the software ImageJ.
Transgene Constructs and Plant Transformation
The 35S:ZmARGOS1 and 35S:ZmARGOS8 constructs were assembled and
transgenic Arabidopsis plants generated as described by Shi et al. (2015). Mutated versions of ZmARGOS1 were created by PCR using primers containing
desired mutations. Truncation variants of ARGOS genes were PCR-amplified
from the full-length cDNA using gene-specific primer pairs containing an ATG
start codon in the forward primer and a stop codon in the reverse primer. PCR
products were cloned into the pENTRY/D-TOPO vector (Invitrogen), confirmed by DNA sequencing and mobilized into the binary vector pBC.Yellow
(de la Luz Gutiérrez-Nava et al., 2008), which contains the cauliflower mosaic
virus 35S promoter (35S) and the phaseolin terminator, using the Gateway recombination system (Invitrogen). The resultant constructs were transferred into
Agrobacterium tumefaciens strain GV3101 and used to transform Arabidopsis
Col-0 by the floral-dip method (Clough and Bent, 1998). Transformants were
selected based on expression of the visible marker yellow fluorescent protein,
driven by the desiccation-responsive AtRd29 promoter, in seeds or by spraying
seedlings with the herbicide Finale (active ingredient glufosinate; Bayer
CropScience) at 1 and 2 weeks after germination.
To generate constructs overexpressing maize ARGOS8, ZmRTL2, and
ZmRTL4 in maize plants, the coding sequences were PCR-amplified, verified by
DNA sequencing and integrated between the maize UBIQUITIN1 promoter
and the rice (Oryza sativa) UBIQUITIN terminator in a Gateway-modified derivative of pSB11 (Ishida et al., 1996) using the Gateway system. The T-DNA region
contains the selectable marker bialaphos resistance gene, phosphinothricin-Nacetyl-transferase, for transgenic plant selection and a visible marker red fluorescent protein for seed sorting. These plasmids were then cointegrated into the super
binary pSB1 vector in Agrobacterium strain LBA4404 (Komari et al., 1996) by electroporation. Maize transformants were produced by using Agrobacteriummediated transformation as described (Cho et al., 2014). Single-copy T-DNA
integration lines that expressed the transgene were selected and advanced for
crosses to wild-type plants and further characterization.
Yeast Split-Ubiquitin Assay
The mating-based split-ubiquitin system (mbSUS) was employed to test
membrane protein-protein interactions in yeast as described by Obrdlik et al.
(2004). Saccharomyces cerevisiae strains THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1
lexA::HIS3 lexA::ADE2) and THY.AP5 (MATa URA3 leu2 trp1 his3 loxP::ade2) as
well as the mbSUS Gateway (GW) version vectors pMetYC_GW, pNX33_GW,
pXN22_GW, pNubWT-X_GW, and pX-NubWT_GW were obtained from the
Arabidopsis Biological Resource Center (OH State University). The coding sequences of maize ARGOS and RTL genes were codon-optimized for appropriate expression in S. cerevisiae, synthesized by commercial vendors, and
cloned into the vectors containing the N- and C-terminal halves of split
ubiquitin (Nub and Cub), respectively, using the Gateway system. Arabidopsis
ARGOS genes RTE1, RTH, and CHX20 were synthesized without codon optimization, and cloned into the mbSUS vectors in the same way as the maize
genes. The two yeast strains were transformed with respective vectors using the
lithium acetate method according to the manufacturer’s instructions (Clontech).
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Transformants were selected on synthetic complete (SC) media lacking Trp and
Leu for Nub and Cub fusions, respectively. Multiple colonies from each THY.
AP5 (Nub) and THY.AP4 (Cub) transformation were bulked and cultured in
liquid SC media to the stationary phase for making glycerol stocks and for
subsequent protein-protein interaction assays. Mating was carried out by
mixing equal amounts of THY.AP5 (Nub) and THY.AP4 (Cub) cells from the
stationary cultures, harvesting the cells by centrifugation, re-suspending with
1 mL 23 yeast extract/peptone/dextrose/adenine (YPDA) medium, transferring to the 12-well cell culture plates, and incubating overnight on a rotating
platform at 28°C. The overnight cultures then were centrifuged and cells
resuspended in 350 mL of 0.53 YPDA medium. For growth assay, serial dilutions of diploid cells were plated out on the media SC-Trp-Leu and the SC-TrpLeu-His-Ade with 134 mM Met (Clontech), and the plates were incubated at
28°C for 2 to 5 d. The expression of bait fusion protein is under control of the
Met-25 promoter; increasing Met concentrations in media reduces the bait
protein level and increase selection stringency. For b-galactosidase assay
(b-Gal), diploid cells were used to inoculate SC-Trp-Leu media, cultured at
30°C for 6 h. b-Gal activities were measured using the yeast b-Gal assay kit (no.
75768; Thermo Fisher Scientific) according to the manufacturer’s instructions. In
brief, 100 mL of yeast cell cultures for each sample with six replications were
placed into wells of a 96-well plate and OD660 was measured. One-hundred
microliters of a freshly made working solution (a mixture of equal volume of 23
b-Gal assay buffer and Y-PER reagent) were added and a timer was used to
monitor the reaction. When the yellow color appeared, the plate was centrifuged
and 150 mL of supernatants were transferred to a plate for measuring OD420.
Bimolecular Fluorescence Complementation
in Arabidopsis
For the BiFC assay (Hu et al., 2002; Walter et al., 2004), the Gateway system
was used to construct vectors expressing split GFP-tagged proteins for generating stably transformed Arabidopsis lines. The pENTRY plasmid (Invitrogen)
was modified by replacing the DNA fragment between the ATTL3/4 recombination sites with an expression cassette composed of the mirabilis mosaic
caulimovirus promoter with double enhancer domains (DMMV; Dey and
Maiti, 1999), the N-terminal half of Aequorea coerulescens GFP (nGFP; encoding
amino acids 1–155), a linker sequence coding for GGGGSGGG, the hemagglutinin (HA) epitope tag (YPYDVPDYA), two SfiI restriction sites for directional cloning, and the Solanum tuberosum proteinase inhibitor II terminator. The
above DNA fragment was synthesized and cloned into pENTRY. The resultant
plasmid was verified by DNA sequencing. Synthesized AtRTE1 and maize
homologous genes were then cloned into the intermediate vector using the SfiI
restriction sites to generate nGFP-AtRTE1 fusions. Using the same strategy, the
C terminus of Arabidopsis and maize ARGOS genes was translationally fused
to the C-terminal half of GFP (cGFP; encoding amino acids 156–239). ZmARGOS8,
ZmRTL2, and ZmRTL4 were codon-optimized for appropriate expression in Arabidopsis. The recombinant genes were integrated into a binary vector containing
the T-DNA left border, the selectable marker bialaphos resistance gene, the
ATTR3/4 recombination sites, and the T-DNA right border using the Gateway
system. Agrobacterium-mediated Arabidopsis transformation was performed as
described above. For the BiFC assay, the nGFP- and cGFP-tagged proteins were
brought together by crossing respective Arabidopsis lines. Two independent lines
were used for each construct. Seedlings and leaves of plants were examined for
fluorescence signals under a model no. MZ10F stereo microscope (Leica Microsystems) equipped with a filter set ET GFP (excitation: ET470:40 nm; emission
ET525:50 nm; Leica Microsystems). A confocal laser scanning microscope (CLMS
DM5500Q; Leica Microsystems) also was used to capture GFP green fluorescence
and autofluorescence signals at 500 to 530 nm (excitation: 488 nm) and 430 to
480 nm (excitation: 408 nm), respectively.
Transgene Expression Analysis
To detect overexpressed proteins, extracts were prepared from leaf tissues,
proteins separated by SDS-PAGE, blotted to a nitrocellulose membrane, and
probed with monoclonal anti-HA antibodies (Thermo Fisher Scientific) for the
HA epitope-tagged fusion proteins. The primary antibodies were detected with
the Fast Western-Blot Kit (Pierce), ECL Substrate (Thermo Fisher Scientific).
ARGOS8 proteins were detected with a monoclonal anti-ARGOS8 antibody.
To determine mRNA expression of the transgene in Arabidopsis and maize
with reverse transcription PCR, cDNA was synthesized with oligo(dT) primers
using SuperScript II RNase H- reverse transcriptase (Invitrogen). PCR was
conducted using the Advantage-GC 2 PCR kit (Clontech). Quantitative RT-PCR
experiments were performed using TaqMan Universal Master Mix (Applied
Biosystems) and the TaqMan probe (Applied Biosystems). Relative quantitation
values were determined using the difference in Ct from the target gene and an
internal control. ZmUBIQUITIN5 and AtPP2A were used as internal controls
for maize and Arabidopsis, respectively.
Ethylene Emission Analysis
Ethylene measurements were conducted on leaf disks taken from plants
grown in the greenhouse. Thirty leaf disks (1-cm diameter) were allowed to
release wound-generated ethylene for 2 h, placed in 9.77-mL volume amber glass
vials containing a filter-paper disc wetted with 50 mL distilled water and then
sealed with aluminum crimp seals. After a 22 to 24 h incubation period, 1-mL
samples were taken from the headspace of each sealed vial. The ethylene content was quantified by gas chromatography, as previously described in Habben
et al. (2014). Ethylene production rate was expressed as pmol per h per milligram of dry weight.
Accession Numbers
Sequence data for the genes described in this article can be found in the
Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: ZmARGOS1 (JN252297), ZmARGOS8 (JN252302),
ZmRTL1 (NM_001151994), ZmRTL2 (BT036282), ZmRTL3 (ACN37097),
ZmRTL4 (NM_001150599), AtARGOS (At3G59900), AtARL (AT2G44080),
AtOSR1 (At2g41230), and AtRTE1 (At2g26070).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Hypocotyl and root lengths of transgenic Arabidopsis seedlings overexpressing ZmARGOS1.
Supplemental Figure S2. Sequence analysis of Arabidopsis and maize
RTE1 proteins.
Supplemental Figure S3. Maize RTL gene expression in B73 inbred.
Supplemental Figure S4. Phylogenetic relationship of maize RTL proteins
and other RTE1 homologs.
ACKNOWLEDGMENTS
We thank Kimberly Glassman, Terry Hu, and Mary Trimnell for producing
transgenic maize plants; Kathleen Schellin, Karen Kratky, Katherine Thilges,
Mark Chamberlin, Jennifer Hanks, Matthew Hanson, and Zhenglin Hou for
technical assistance; and Karen Broglie for providing BiFC vectors. We are
grateful to Wuyi Wang for critical reading of the manuscript and helpful
comments. We acknowledge Mei Guo for her research on ARGOS at DuPont
Pioneer. We also thank Tom Greene, Mark Cooper, and Dave Warner for their
organizational leadership and helpful input.
Received March 2, 2016; accepted June 6, 2016; published June 7, 2016.
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