Fruit ripening specific expression of β-D-N-acetylhexosaminidase (β-Hex) gene in tomato is transcriptionally regulated by Ethylene Response Factor SlERF.E4

Abstract

N-glycans and N-glycan processing enzymes are key players in regulating the ripening of tomato (Solanum lycopersicum) fruits, a model for fleshy fruit ripening. β-D-N-acetylhexosaminidase (β-Hex) is a N-glycan processing enzyme involved in fruit ripening. The suppression of β-Hex results in enhanced fruit shelf life and firmness in both climacteric and non-climacteric fruits. Previously, we have shown that ripening specific expression of β-Hex is regulated by RIPENING INHIBITOR (RIN), ABSCISIC ACID STRESS RIPENING 1 (SlASR1) and ethylene. However, the precise mechanism of ethylene-mediated regulation of β-Hex remains elusive. To gain insights into this, we have performed 5’ deletion mapping of tomato β-Hex promoter and a shorter promoter fragment (pD-200, 200 bp upstream to translational start site) is identified, which was found critical for spatio-temporal transcriptional regulation of β-Hex. Further, site specific mutagenesis in RIN and ASR1 binding sites in pD-200 provides key insights into ripening specific promoter activity. Furthermore, induction of GUS activity by ethylene, yeast one hybrid assay and EMSA identify Ethylene Response Factor SlERF.E4 as a positive regulator of β-Hex. Taken together, our study suggest that SlERF.E4 together with RIN and SlASR1 transcriptionally regulates β-Hex and all these three proteins are essential for fruit ripening specific expression of β-Hex in tomato.

Full text

Plant Science 323 (2022) 111380
Available online 14 July 2022
0168-9452/Published by Elsevier B.V.
Fruit ripening specic expression of β-D-N-acetylhexosaminidase (β-Hex)
gene in tomato is transcriptionally regulated by ethylene response
factor SlERF.E4
Mohammad Irfan
a
,
b
,
**
,
1
, Pankaj Kumar
a
,
c
,
2
, Vinay Kumar
a
,
d
,
3
, Asis Datta
a
,
*
,
4
a
National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India
b
Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York, USA
c
Department of Biotechnology, Dr. Y.S. Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India
d
Department of Physiology and Cell Biology, Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA
ARTICLE INFO
Keywords:
β-Hex
Ethylene, Fruit Ripening
Promoter
RIN
SlASR1
SlERF.E4
ABSTRACT
N-glycans and N-glycan processing enzymes are key players in regulating the ripening of tomato (Solanum
lycopersicum) fruits, a model for eshy fruit ripening. β-D-N-acetylhexosaminidase (β-Hex) is a N-glycan pro-
cessing enzyme involved in fruit ripening. The suppression of β-Hex results in enhanced fruit shelf life and
rmness in both climacteric and non-climacteric fruits. Previously, we have shown that ripening specic
expression of β-Hex is regulated by RIPENING INHIBITOR (RIN), ABSCISIC ACID STRESS RIPENING 1 (SlASR1)
and ethylene. However, the precise mechanism of ethylene-mediated regulation of β-Hex remains elusive. To
gain insights into this, we have performed 5’ deletion mapping of tomato β-Hex promoter and a shorter promoter
fragment (pD-200, 200 bp upstream to translational start site) is identied, which was found critical for spatio-
temporal transcriptional regulation of β-Hex. Further, site specic mutagenesis in RIN and ASR1 binding sites in
pD-200 provides key insights into ripening specic promoter activity. Furthermore, induction of GUS activity by
ethylene, yeast one hybrid assay and EMSA identify Ethylene Response Factor SlERF.E4 as a positive regulator of
β-Hex. Taken together, our study suggest that SlERF.E4 together with RIN and SlASR1 transcriptionally regulates
β-Hex and all these three proteins are essential for fruit ripening specic expression of β-Hex in tomato.
1. Introduction
Tomato (Solanum lycopersicum) is an economically important fruit
crop grown worldwide (FAOSTAT, 2019) and is one of the most
researched crops to date due to its genomics accessibility (Giovannoni
et al., 2017; Wang et al., 2020). The perishable nature of tomato fruits
and excessive fruit softening during ripening results in signicant
post-harvest losses not only of tomato but also other fruits and vegeta-
bles (Ghosh et al., 2011; Kumari et al., 2022; Meli et al., 2010; Shipman
et al., 2021). Fruit ripening is a complex and well-coordinated process
involving numerous metabolic changes mediated by genetic regulators,
transcription factors and endogenous factors, etc (Chen et al., 2020;
Giovannoni et al., 2017; Tayal et al., 2022). Increased expression of
cell-wall degradation enzymes is one of the main factors leading to fruit
softening (Chen et al., 2020; Uluisik et al., 2016; Wang et al., 2020).
The cell wall of plants contains signicant amount of N-glycopro-
teins, which have key roles in a variety of physiological processes such as
plant stress response, plant development including seed development
and fruit ripening (De Coninck et al., 2021; Kang et al., 2008; Kaul-
fürst-Soboll et al., 2021; Lannoo and Van Damme, 2015; Nagashima
et al., 2018). It has also been reported that during tomato fruit ripening,
the level of free N-glycans, which are present as precursors to glyco-
sylation or products of glycoprotein proteolysis, is substantially
increased (Ghosh et al., 2011; Maeda and Kimura, 2014; Nakamura
* Corresponding author.
** Corresponding author at: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India.
E-mail addresses: mi239@cornell.edu (M. Irfan), asis_datta@rediffmail.com (A. Datta).
1
0000-0002-7877-5530
2
0000-0002-2275-9144
3
0000-0002-6751-5741
4
0000-0002-1391-4982
Contents lists available at ScienceDirect
Plant Science
journal homepage: www.elsevier.com/locate/plantsci
https://doi.org/10.1016/j.plantsci.2022.111380
Received 26 February 2022; Received in revised form 8 June 2022; Accepted 12 July 2022

Plant Science 323 (2022) 111380
2
et al., 2008; Priem et al., 1993). Several reports suggest that cell wall
located N-glycan processing enzymes such as
α
-mannosidase (
α
-Man)
and β-D- N-acetylhexosaminidase (β-Hex) are critically involved in fruit
ripening and their activity particularly increases during fruit ripening
(Cao et al., 2014; Ghosh et al., 2011; Hossain et al., 2009; Hossain and
Roslan, 2014; Jagadeesh et al., 2004; Jagadeesh and Prabha, 2002; Meli
et al., 2010). Our earlier ndings provide detailed mechanistic insights
into role of
α
-Man and β-Hex in climactric and nonclimactric fruit
ripening (Ghosh et al., 2011; Irfan et al., 2016, 2014; Meli et al., 2010).
RNAi-mediated silencing of
α
-Man and β-Hex in tomato and capsicum
results in enhanced fruit rmness and shelf life suggesting a common
strategy for reducing post-harvest loss of both climacteric and
non-climacteric fruits (Ghosh et al., 2011; Meli et al., 2010). More
recently, several other reports suggest that β-Hex enzymes are involved
in ripening-associated softening in strawberry and peach fruits (Bose
et al., 2021; Wang et al., 2021).
β-Hex (EC 3.2.1.52), a glycosyl hydrolase family 20 enzyme, breaks
the terminal N-acetyl-β-D-glucosamine (β-D-GlcNAc) and N-acetyl-β-D-
galactosamine (β-D-GalNAc) residues of N-acetyl-β-D-hexosaminides
and generates paucimannosidic N-Glycans (Ghosh et al., 2011; Hossain
and Roslan, 2014; Liebminger et al., 2011; Meli et al., 2010; Strasser
et al., 2007). In our prior study, we have investigated the transcriptional
regulation of β-Hex in tomato and showed that promoter of β-Hex gene is
fruit ripening specic (Irfan et al., 2014). β-Hex is transcriptionally
regulated by a MADS-box protein RIPENING INHIBITOR (RIN), the
master regulator of fruit ripening and ethylene, the key ripening hor-
mone (Irfan et al., 2014; Kumar et al., 2016). Moreover, in rin mutant,
β-Hex expression represses signicantly and promoter activation of
β-Hex was reported compromised in rin mutant fruit (Irfan et al., 2014;
Meli et al., 2010). RIN specically binds to RIN binding sites or CArG
boxes [C(T/A/C)(A/T)
6
(A/T/G)G] of β-Hex promoter to regulate the
β-Hex directly (Irfan et al., 2014). RIN also regulates β-Hex indirectly
through ABSCISIC ACID STRESS RIPENING 1 (SlASR1) via binding to
SlASR1 promoter (Irfan et al., 2014). Moreover, SlASR1 also directly
binds to ASR1 binding sites (C
2–3
(C/G)A) of β-Hex promoter and acts as
positive transcriptional regulator of β-Hex. The expression of β-Hex was
also reported to decreased in VIGS-suppressed SlASR1 fruits (Irfan et al.,
2014).
Previously, Irfan et. al. (2014) also identied Ethylene Response
Factor 6 (SlERF6, other name of SlERF.E4) as a putative transcription
factor of β-Hex through yeast one hybrid screening and its expression is
upregulated at the onset of ripening. Another study also suggest that
expression of SlERF6 increases during ripening and SlERF6 regulates
fruit ripening by integrating ethylene and carotenoid biosynthesis
pathways (Lee et al., 2012). All these ndings strengthen the hypothesis
that SlERF.E4 might be involved in the transcriptional regulation of
β-Hex during fruit ripening. In the present study, we provide key insights
into SlERF.E4 (Solyc01g065980.2.1) mediated transcriptional regula-
tion of β-Hex (Solyc01g081610.2.1) during tomato fruit ripening. For
this, 5’ deletion mapping of β-Hex promoter with GUS fusion using
agroinjection based approach is performed. Further, mutagenesis in RIN
and ASR1 binding sites of β-Hex promoter, developing promoter::GUS
fusion constructs and stable transgenic lines were carried out to get a
more clear picture. Moreover, ethylene inducibilty and SlERF.E4
mediated regulation of β-Hex using DNA-protein interaction approaches
has also been studied.
2. Materials and methods
2.1. Plant material and growth conditions
The seeds of tomato (Solanum lycopersicum cv. pusa ruby), procured
from National Seeds Corporation Ltd., New Delhi, India, were germi-
nated in pre-sterilized soil in greenhouse at 25 ◦C with 70 % humidity
and 14 h light (~250
μ
mol m
−2
s
−1
) and 10 h dark cycles. Fruits were
tagged at anthesis and ripening stages were dened as mature green (40
days after anthesis), breaker (mature green +3 days), pink (breaker +2
days) and red ripe (pink +3 days) according to Ghosh et al. (2013).
2.2. Construction of deletion vectors
Deletion vectors of β-Hex promoter were generated in pBI121 vector
by substituting CaMV35S promoter with β-Hex promoter. The PCR
amplication was carried out for β-Hex promoter (1001 bp) and its 5’
deletion fragments (−200 bp, −400 bp, −600 bp, and −800 bp upstream
to β-Hex translational start site) using AccuPrime Pfx high delity DNA
Polymerase (Thermo Fisher Scientic, USA), cloned upstream to GUS
and were named as HP-1001, D-800, D-600, D-400, and D-200,
respectively. Before agroinjection, PCR conrmation of deletion vectors
in the Agrobacterium cultures was carried out using deletion fragment
specic forward and a common reverse primer to ensure only desired
deletion fragment is present in the respective culture.
2.3. Site-directed mutagenesis
Site-directed mutagenesis of the conserved bases of RIN and ASR1
binding sites was performed with In-Fusion HD Cloning kit (Takara Bio
Inc., Japan) using manufacturer’s protocol. Mutated plasmids i.e.
mCArG_D-200::GUS, mASR1_D-200::GUS and mCArG/mASR1_D-200
along with D-200::GUS (as a control, without mutation) were used to
generate stable transgenic lines of tomato.
2.4. Agrobacterium-based transient and stable transformation
Agrobacterium-based transient assay or agroinjection was performed
as explained by Orzaez et al. (2006) and Irfan et al. (2016). Agro-
bacterium tumifaciens EHA 105 cultures containing deletion or control
vectors were grown in YEP media at 28 ◦C until the optical densities of
0.8–1.0. The cells were then resuspended in inltration medium (10 mM
MgCl2, 10 mM MES, and 200 mM acetosyringone, pH 5.6), and incu-
bated at room temperature for 2 h with gentle agitation (20 rpm). Fruits
at mature green, breaker and pink ripening stages were injected with
1 ml needle through stylar apex and harvested at pink, breaker and red
ripe stages respectively. For each sample, 6 fruits were injected and
included in the study. The stable transgenic plants of tomato were
generated using Agrobacterium tumifaciens EHA 105 as a standard pro-
tocol reported earlier (Irfan et al., 2014).
2.5. GUS histochemical and uorometric assay
The GUS assays were performed as per the optimized protocol re-
ported earlier (Irfan et al., 2014). 35 S::GUS was used as positive control
and WT or fruits injected with Agrobacterium culture without any vector
were used as negative control. For histochemical assay, the fruits were
cut into transverse sections and dipped in GUS-staining solution
(100 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA, 0.5 mM K3Fe
(CN)6, 0.5 mM K4Fe(CN)6.3H2O, 0.1 % Triton-X100, 20 % methanol,
and 1 mM X-Gluc) followed by vacuum inltration. Samples were
incubated in dark at 37 ◦C for overnight and destained with 75 %
ethanol.
The quantication of GUS activity was performed using the synthesis
of 4-methylumbelliferone (4-MU) as pmol 4-MU mg
–1
min
–1
using the
standard curve shown in the Fig. S3. Fruits were homogenized in 400
μ
l
GUS extraction buffer (50 mM sodium phosphate buffer, pH 7.0, 10 mM
DTT, 10 mM EDTA, 0.1 % sodium lauryl sarcosine, and 0.1 % Triton
X100). The supernatant (50
μ
l) was mixed with 450
μ
l of GUS extraction
buffer containing 10 mM MUG and incubated for 1 h at 37 ◦C. The re-
action was stopped with 900
μ
l of 0.2 M Na
2
CO
3
to 100
μ
l of above ali-
quots. The uorescence was measured using a uorometer (Cary Eclipse,
Varian) with excitation at 380 nm and emission at 454 nm.
M. Irfan et al.

Plant Science 323 (2022) 111380
3
2.6. RNA isolation and quantitative RT-PCR
RNA was extracted and puried using the RNeasy Mini Kit (Qiagen,
USA) as described previously (Irfan et al., 2021). The cDNA was syn-
thesized from 5 µg of puried RNA SuperScrip III First-Strand Synthesis
System (Thermo Fisher Scientic, USA). qRT-PCR was carried out using
One-Step Real-Time RT PCR (Applied Biosystems) using SYBR Green
dye. All qRT-PCR analyses were performed with three biological repli-
cates. The tomato actin gene was used as an internal reference gene and
analysis was performed using the 2
−ΔΔCt
method (Irfan et al., 2021).
2.7. Yeast one hybrid assay
The yeast one hybrid assay was performed with Yeast one-hybrid
system - Matchmaker Gold (Takara Bio Inc., Japan) following manu-
facturer’s protocol. D-200 promoter fragment was cloned upstream to
HIS3 reporter in pHIS2.1 vector, while SlERF.E4 was cloned upstream of
GAL4 activation domain of pGADT7-Rec2 vector. To evaluate HIS3 re-
porter gene activation, the pHIS-D-200 and pGADT7-SlERF.E4 plasmids
were co-transformed into Saccharomyces cerevisiae strain Y187 and
cultured on medium SD/-Leu/-Trp/-His/+5 mM 3-AT (TDO +3-AT)
plates. The empty vector pGADT7 co-transformed with pHIS2.1-D-200
along with untransformed yeast strain were used as controls.
2.8. Protein purication and EMSA
For protein purication, full length SlERF.E4 was cloned in pGEX4T-
2 expression vector with N-terminal GST tag and transformed in E. coli
BL21 cells. Induction of SlERF.E4 has been tested at different tempera-
ture ranges after the addition of 0.6 mM IPTG. Induced proteins were
then puried by afnity chromatography using Glutathione Sepharose
(GE Healthcare, USA). EMSA was performed to check the binding of
SlERF.E4 proteins D-200 promoter probe as per the method reported
earlier (Irfan et al., 2016). Briey, 1
μ
g of SlERF.E4 protein was mixed
with radioactively labeled D-200 β-hex promoter in EMSA binding
buffer (20 mM HEPES, pH 7.5, 20 % glycerol, 0.05
μ
g poly (dIdC): poly
(dIdC), 10 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT and 25 mM NaCl) at
room temperature for 30 min. The reaction was run on 6 % native PAGE
with a constant current (10 mA) using 0.5X TBE as running buffer and
visualized by autoradiography.
2.9. ACC treatment
Fruits from transgenic and WT plants were harvested at mature green
stage and treated with 100 µM ACC (Sigma–Aldrich) as described by Su
et al. (2015). For each group, 6 fruits were included for the treatment.
Briey, fruits were inlterated with a solution of 10 mM MES (pH 5.6),
sorbitol (3 % w/v) and 100
μ
M of ACC using styler apex using 1 ml sy-
ringe. The controls were inlterated with above solution without ACC.
The treated fruits were incubated at 26 ◦C under 16 h light/8 h until pink
stage of ripening (~72 h). The GUS histochemical assay of fruit slices
was performed immidiatedly and slices were ash frozen and stored at
−80 for qRT-PCR and MUG assay.
2.10. Dual luciferase assay
The reporter vector was constructed in pGreenII 0800-LUC by clon-
ing pD-200 promoter upsteam to LUC using In-Fusion HD Cloning kit
(Takara Bio Inc., Japan). For effector vectors, coding sequence of RIN,
SlASR1 and SlERF.E4 were cloned into pEAQ vector under CaMV35S
promoter. For transient expression, protoplasts were isolated from
Arabidopsis leaf mesophyll cells as described previously (Yoo et al.,
2007). Effector and reporter vectors were transfected together at a ratio
of 2:1 into freshly isolated protoplasts using poly-ethylene glycol
PEG4000 (Yoo et al., 2007). The transfected protoplasts were cultured at
22 ◦C for 2 days and then lysed using passive lysis buffer of Dual-Glo®
Luciferase Assay System (Promega, USA). The ratio of rey luciferase
(LUC) to Renilla luciferase (REN) activity was analyzed using manu-
facturer’s protocol to determine the transcriptional efciency of pD-200
promoter.
Fig. 1. 5’ Deletion mapping of β-Hex promoter
of tomato. (A) Schematic representation of
deletion mapping of tomato β-Hex promoter.
200 bp (D-200), 400 bp (D-400), 600 bp (D-
600), 800 bp (D-800) and 1001 bp (HP, full
length β-Hex promoter) upstream of trans-
lational start site were cloned by replacing
CaMV35S promoter in pBI121 binary vector.
(B) PCR conrmation of deletion vectors using
deletion fragment specic primers. Lane 1, 2, 3,
4, 5 represent PCR reactions using a common
reverse and HP-1001, D-800, D-600, D-400, D-
200 fragment specic forward primers using
HP-1001, D-800, D-600, D-400, D-200 plasmids
respectively as template. PCR conrmed that in
D-200 sample only pD-200 is present whereas
HP-1001 contain all fragments and so on.
M. Irfan et al.

Plant Science 323 (2022) 111380
4
2.11. Statistical analysis
All values are presented as the means of atleast three biological
replicates (SE). For each biological sample, three technical replicates
were considered and used to calculate one average value per biological
sample. The comparison among different samples was made using Stu-
dent’s t tests at 95 % signicance level (p ≤0.05).
3. Results
3.1. Deletion mapping of β-Hex promoter of tomato
To gain insights into transcriptional regulation of β-Hex, a series of
promoter deletions of β-Hex followed by promoter-driven expression of
GUS activity in tomato plants were aimed. To do this, deletion fragments
of β-Hex were selected and labelled as D-800, D-600, D-400 and D-200
and HP-1001 for 800, 600, 400, 200 and 1001 bp from translational start
site (Fig. 1A, Fig. S1). These fragments were cloned upstream to GUS
into a binary vector pBI121 by replacing CaMV35S promoter to study
the promoter mediated GUS expression. Successful integration of all
promoter fragments into respective vectors was conrmed by PCR
(Fig. 1B). The agroinjection harboring these constructs was performed in
tomato fruits at different ripening stages as mentioned in materials and
methods. After three days of agoinjection, harvested fruits were
sectioned to examine the GUS activity by histochemical assay and
uorometric MUG assay (Fig. 2). 35S::GUS construct and Agrobacterium
culture without any vector were used as positive and negative controls
respectively. GUS activity driven by all β-Hex promoter deletion frag-
ments (D-800, D-600, D-400 and D-200) at breaker and red ripe stages of
ripening was nearly comparable to β-Hex promoter activity (HP-1001)
(Fig. 2). However, there was a reduction of GUS activity at pink stage in
D-800 and D-600 (Fig. 2). Interestingly, there was no siginicat decrease
Fig. 2. Agrobacterium-mediated transient assay and GUS histochemical and uorometric analysis. The mature green, breaker and pink stage fruits were injected with
Agrobacterium cultures harboring deletion vectors and harvested at breaker, pink and red ripe stages respectively. 35 S::GUS was used as positive control and
Agrobacterium culture without any vector was used as negative control. The GUS activity (A) of all deletion fragments of β-Hex promoter at breaker and red ripening
stages was almost similar to the full-length β-Hex promoter suggesting D-200 is equally active for ripening specic expression as HP-1001 promoter. Fluorometric
quantication of GUS activity (B) also demonstrated no decrease in reported gene activity conferred by D-200 fragment. Data are presented as the mean ( ±standard
error) of GUS activity from three independent determinations. **P < 0.01 displays signicant differences (Student’s t-test), n.s., not signicantly different.
M. Irfan et al.

Plant Science 323 (2022) 111380
5
in GUS activity in D4–200 as HP-1001 at all three stages of ripening
suggesting that D4–200 is equal active as HP-1001 promoter for regu-
lating ripening specic expression of β-Hex gene.
3.2. Generation and analysis of D4 promoter::GUS fusion transgenic lines
of tomato
In order to conrm the D-200 promoter mediated GUS expression
and its comparison to HP-1001 promoter for fruit ripening-specic
activation of β-Hex gene in tomato fruit, the stable transgenic lines of
D-200::GUS were generated (Fig. 3A). The screening of transgenic plants
was carried out using PCR (Fig. S2) and a total 8 independent transgenic
events developed with D-200::GUS fusion construct were advanced to
T2 generation and T2 transgenic events with single transgene copy
insertion were chosen for further analysis. Our previous studies (Meli
et al., 2010; Irfan et al., 2014) suggest that expression of β-Hex reaches
peak at pink stage, therefore pink stage of fruit ripening was selected for
GUS staining. The GUS histochemical assay (Fig. 3B) suggests that in
D-200::GUS transgenic fruits, GUS activity is equally intense to HP::GUS
transgenic fruits. To validate this result, GUS relative transcript level
using qRT-PCR and GUS uorometric activity through uorometric
4-methylumbelliferone glucuronide (MUG) assay of transgenic tomato
fruits were performed (Fig. 3C). This data also suggests that D-200
promoter is equally active as HP-1001 and is sufcient for ripening
specic expression. Moreover, to conrm the fruit ripening specic
expesion of D-200, the GUS activity assay was also performed with
leaves and owers. The data suggests that no GUS activity was detected
in leaves and ower petals of D-200::GUS lines, however, slight GUS
activity was noticed in sepals (Fig. 4A) which is similar to HP-1001 lines.
Similar pattern was also observed in GUS uorometric quantication
assay (Fig. 4B). All this data suggest that D-200 fragment of β-Hex
promoter is sufcient for fruit ripening specic expression and is similar
to HP-1001 trigerred ripening specic expression.
3.3. RIN and ASR1 mediated transcriptional regulation of β-Hex
During fruit ripening, a MADS-box transcription factor RIN plays a
crucial role and it binds to CArG boxes of promoters of many ripening
associated genes including β-Hex (Fujisawa et al., 2013; Irfan et al.,
2014). Additionally, SlASR1, was also characterized as transcriptional
regulators of β-Hex via its binding to conserved motif C
2–3
(C/G)A (Irfan
et al., 2014) of β-Hex promoter. Here, we further investigated the as-
sociation of RIN and SlASR1 in regulation of β-Hex particularly in the
promoter region of D4–200 using site directed mutation strategies. The
conserved bases of ASR1 binding sites [C
2–3
(C/G)A] and RIN binding
sites (CArG) were mutated in D-200 promoter fragment indivually and
together as shown in the Fig. 5A and mutated promoter::GUS fusion
vectors were constructed, which were used to generate stable transgenic
lines. The fruits from these transgenic lines were harvested at pink stage
of ripening and were used for GUS qRT-PCR, histochemical and
Fig. 3. Comparative GUS analysis of D-200::
GUS stable transgenic lines of tomato. (A)
Schematic representation of D-200::GUS
construct used to generate transgenic plants.
(B) GUS histochemical assay of D-200::GUS
transgenic fruits at pink stage and its compari-
son with HP::GUS and 35 S:GUS transgenic
fruits. (C) GUS transcript and uorometric
analysis of transgenic fruits. The data suggest
that D-200 fragment of promoter is sufcient
for ripening specic expression. Data are pre-
sented as the mean ( ±standard error) of GUS
activity from three independent de-
terminations. **P < 0.01 displays signicant
differences (Student’s t-test), n.s., not signi-
cantly different.
M. Irfan et al.

Plant Science 323 (2022) 111380
6
quatitative assay (Fig. 5B and C). We observed reduction in GUS tran-
script accumulation and GUS activity (histochemically and quatita-
tively) in CArG box mutated (mCArG_D-200::GUS) and ASR1 mutated
(mASR1_D-200::GUS) D-200 promoter::GUS transgenic fruits (Fig. 5B
and C). To explore the idea that what would happen when both CArG
box and ASR1 binding sites are mutated together, the stable transgenic
plants are raised with mutation in both sites (mCArG/mASR1_D-200::
GUS). Intrestingly, we found that GUS activity was reduced signicantly
in the mCArG/mASR1_D-200::GUS transgenic tomato fruits as compared
to fruits of individual mutation lines of ASR1 binding site and CarG box
of D4–200 promoter i.e mCArG_D-200::GUS and mASR1_D-200::GUS
respectively. These ndings are in line with our previous study (Irfan
et al., 2014) that ASR1 and RIN specically bind to CArG box and ASR1
binding sites of the D-200 fragment of β-Hex promoter in order to
regulate β-Hex expression during fruit ripening.
3.4. Ethylene and SlERF.E4-mediated transcriptional regulation of β-Hex
Although, after mutating the ASR1 and RIN binding sites in D-200
promoter fragment, the GUS activity was signicantly reduced, however
slight activity was also reported even after above mutations (Fig. 5B). In
our previous study, it was shown that β-Hex gene is also regulated by
ethylene, therefore it was our profound interest that this remaining GUS
activity in the mCArG/mASR1_D-200::GUS transgenic tomato fruits
could be due to ethylene mediated regulation. To test this hypothesis,
mCArG/mASR1_D-200::GUS transgenic tomato fruits were treated with
1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of
ethylene. Intrestingly, GUS activity in native D-200 transgenic fruit and
mCArG/mASR1_D-200::GUS transgenic fruits was signicantly upregu-
lated by ACC as compared to untreated fruits (Fig. 6A). This GUS his-
tochemical data is also supported by GUS transcript level pattern and
MUG quantitative assay (Fig. 6B). Therefore, it is quite possible that
remaining GUS activity in the mCArG/mASR1_D-200::GUS transgenic
fruits could be due to ethylene.
To further clarify this, yeast one hybrid assay was carried out to test
the interaction between D-200 promoter fragment and SlERF.E4, which
was reported as putative transcriptional regulator of β-Hex by Irfan et al.
(2014). For this, D-200 fragment of β-Hex promoter was cloned up-
stream to HIS3 reporter to make pHIS-D-200 vector whereas SlERF.E4
was cloned upstream to GAL4 activation domain of pGADT7 vector
(Fig. 7A). After co-transformation into Saccharomyces cerevisiae strain
Y187 followed by growth on SD selection medium, positive interaction
between D-200 promoter fragment and SlERF.E4 was observed (Fig. 7B)
suggesting that SlERF.E4 interacts with D-200 promoter fragment of
β-Hex. To further validate this interaction, electrophoretic mobility shift
assay (EMSA) was performed using N-terminal GST tagged recombinant
protein SlERF.E4 (GST-SlERF.E4), puried from E. coli BL21 cells after
optimizing the protein induction by IPTG (Fig. 7C). D-200 fragment of
β-Hex promoter which may contain a putative SlERF.E4 binding site was
used as a probe in EMSA. As shown in the Fig. 7D, a specic interaction
of SlERF.E4 with D-200 β-Hex promoter was observed. GST alone did not
show any binding activity and surplus unlabelled D-200 promoter
fragment was capable to compete for the binding activity of GST-SlERF.
E4 whereas nonspecic DNA fragments were incapable to compete
(Fig. 7D). All these ndings further strengthen the specic binding of
SlERF.E4 to the D-200 β-Hex promoter.
3.5. RIN, SlASR1 and SlERF.E4 functions are complementary in β-Hex
regulation
Our data and previous studies suggest that transcriptional regulation
of β-Hex is quite complex and all three transcription factors RIN, SlASR1
and SlERF.E4 bind to pD-200 β-Hex promoter. To investigate the rela-
tionship among multiple regulatory mechanisms by these transcriotion
factors, dual luciferase assays were performed using RIN, SlASR1 and
SlERF.E4 as effectors and D-200 β-Hex promoter fused with rey
luciferase (LUC) as reporter (Fig. 8A). Renilla luciferase (REN) gene
driven by 35 S promoter was used for normalization. The effector and
reporter vectors were transfected together in Arabidopsis leaf mesophyll
protoplasts. Signicant increase in relative LUC/REN activity by RIN,
SlASR1 and SlERF.E4 individually as compared to empty vector (35 S::
Empty) was observed which corroborates our results and previous
studies that RIN, SlASR1 and SlERF.E4 are transcriptional activators of
D-200 β-Hex promoter (Fig. 8B). When a combination of effectors such
as RIN and SlASR1, or RIN and SlERF.E4 or SlASR1 and SlERF.E4 were
used, the activation of D-200 β-Hex promoter was cumulative (Fig. 8B).
Interestingly, strong activation of D-200 β-Hex promoter was detected
after simultaneous cotransfection of RIN, SlASR1 and SlERF.E4 together
with pD-200 reporter (Fig. 8B). This suggests that RIN, SlASR1 and
Fig. 4. Analysis of GUS activity in leaf and ower of different promoter::GUS
transgenic plants driven by 35 S, HP and D-200 promoters. GUS histochemical
assay (A) and uorometric assay (B) detected the constitutive expression of GUS
in leaf and ower of 35S::GUS transgenic plants. GUS activity was not observed
in HP::GUS and D4–200::GUS transgenic leaf, however ower of these trans-
genic plants showed little GUS activity. Data are presented as the mean
(±standard error) of GUS activity from three independent determinations.
**P < 0.01 displays signicant differences (Student’s t-test), n.s., not signi-
cantly different.
M. Irfan et al.

Plant Science 323 (2022) 111380
7
SlERF.E4 works in a complemetary manner in order to regulate the
expression of β-Hex during fruit ripening.
4. Discussion
The physiological and molecular mechanism of β-Hex functions in
the ripening-related fruit softening has been elucidated previously (Bose
et al., 2021; Cao et al., 2014; Ghosh et al., 2011; Jagadeesh et al., 2004;
Jagadeesh and Prabha, 2002; Meli et al., 2010; Wang et al., 2021). Our
prior study on transcriptional regulation of β-Hex reveals that RIN and
SlASR1 positively regulates its transcription during fruit ripening (Irfan
et al., 2014). Ethylene also induces the expression of β-Hex (Irfan et al.,
2014; Meli et al., 2010), however, the precise mechanism of ethylene
mediated action is not clear. This study was designed to gain insights
into ethylene mediated regulation of β-Hex during fruit ripening. To
identify the promoter region of β-Hex associated with ethylene mediated
regulation, 5’ deletion mapping of β-Hex was carried out. The data
revealed that truncated promoters still exhibited high activity in fruits
even when −200 bp of 5’ to ATG (pD-200) was used as promoter driven
GUS expression through agroinjection based transcient assay (Fig. 2).
Therefore, it is quite possible that pD4–200 contains all the necessary
cis-acting elements required for the ripening specic expression of β-Hex
Fig. 5. : GUS analysis of mutated D-200::GUS transgenic lines of tomato. Left panel shows schematic representation of site-directed mutation strategies. ASR1 and
RIN binding sites were mutated as shown in the gure and the mutated plasmids were used to generate stable transgenic lines. The fruits of pink stages were used for
GUS activity. Mutated CArG box and mutated ASR1 binding sites showed reduction in GUS activity in fruits both histochemically (B) and quantitatively (C). When
both transcription factor binding sites were mutated together, the GUS activity was decreased signicantly as compared to individual mutation of ASR1 binding site
and CArG box. Data are presented as the mean ( ±standard error) of GUS activity from three independent determinations. **P < 0.01 displays signicant differences
(Student’s t-test), n.s., not signicantly different.
M. Irfan et al.

Plant Science 323 (2022) 111380
8
gene. In the previous study, Irfan et al. (2014) identied that one CArG
box and two SlASR1 binding sites exist in this pD-200 promoter region
and RIN and ASR1 specically binds these sites of D-200 promoter
suggesting that D-200 promoter is critical for transcriptional regulation
and is indispensable for the spatio-temporal regulation of the endoge-
nous β-Hex gene.
Although agroinjection based deletion mapping provided encouraging
results, however it holds several disadvantages such as unequal injection and
massive presence of Agrobacterium cells in the fruit can also induce side ef-
fects (Orzaez et al., 2006). Therefore, to validate the pD-200 mediated
ripening specic expression, stable pD-200::GUS fusion transgenic lines
were developed. Interestingly, even in the pD-200::GUS fusion transgenic
lines, pD-200 promoter was equally active as full length promoter, revealed
by GUS transcript accumulation, histochemical and uorometric assays
Fig. 6. ACC treatment enhanced GUS activity in
D-200::GUS and mCArG/mASR1_D-200::GUS
transgenic fruits. The transgenic fruits were
treated with ACC, and GUS assays were performed
after 2–3 days of treatment. ACC increased the GUS
activity in fruits of both types of transgenic lines D-
200::GUS and mCArG/mASR1_D-200::GUS sug-
gesting ethylene dependent and ASR1 and RIN
independent regulation of β-Hex. Data are pre-
sented as the mean ( ±standard error) of GUS
activity from three independent determinations.
**P < 0.01 displays signicant differences (Stu-
dent’s t-test).
M. Irfan et al.

Plant Science 323 (2022) 111380
9
Fig. 7. SlERF.E4 is a transcriptional regulator of β-Hex. (A) and (B) Yeast one hybrid assay to identify SlERF.E4 as a potential interacting partner of D-200 promoter
fragment. Y1H vectors construction strategy is represented in A. D-200 promoter fragment of β-Hex was cloned upstream to HIS3 reporter in pHIS2.1 vector and
SlERF.E4 was cloned upstream to GAL4 activation domain in pGADT7-Rec2 vector as shown in A. These Y1H vectors were co-transformed into Saccharomyces
cerevisiae strain Y187 and grown on SD medium lacking Trp, Leu, and His, but containing 5 mM 3AT to determine HIS3 reporter gene activation. Empty pGADT7
vector co-transformed with pHIS2.1-D-200 and untransformed strain (Y187) served as the controls (B). (C) Purication of SlERF.E4 from E. coli. SlERF.E4 was cloned
in pGEX4T-2 expression vector with N-terminal GST tag and transformed in E. coli BL21 cells. Maximum induction of SlERF.E4 was observed at 28
◦C after adding
0.6 mM IPTG. Induced proteins were puried by afnity chromatography by using Glutathione sepharose 4B. (D) Electrophoretic mobility shift assay (EMSA)
showing binding of SlERF.E4 proteins to D-200. EMSA was carried out with 1
μ
g of SlERF.E4 protein with radioactively labelled D-200 β-Hex promoter. The reactions
were carried out at 25 ◦C and then resolved on 6 % native polyacrylamide gels using 0.5X TBE buffer and visualized by autoradiography. The shift was competed out
by 100-fold excess of specic and nonspecic competitors.
M. Irfan et al.

Plant Science 323 (2022) 111380
10
(Fig. 3). The deceased activity of pD-800 and pD-600 at pink stages could be
due to epigenetic regulation of β-Hex during ripening progression. It is re-
ported that β-Hex promoter shows differential methylation patterns in fruit
developmental and ripening stages (Zhong et al., 2013). Moreover, CArG
boxes are frequently localized in the demethylated promoter region of β-Hex,
and binding of RIN occurs in concert with demethylation (Zhong et al.,
2013). The presence of several RIN binding sites in −800 to −1001 bp re-
gion (Fig. S1) and binding of RIN protein to those sites results in highest
activity. In D-600 and D-800 promoters, the methylation pattern might affect
transcription of these fragments at pink stage probably because of hyper-
methylation of this region. Further, no methylation in −1 bp to −400 bp
promoter was identied, therefore the activity of these fragments is mainly
due to D-200 promoter and transcription factors’ binding to this region
during fruit ripening. The fruit ripening specic expression of pD-200 is also
supported by nil or little GUS activity in the other plants parts of pD-200::GUS
fusion transgenic plants and pD-200 is not activated in leaves and ower
parts except sepals (Fig. 4). The little GUS activity in the sepals indicate that
β-Hex gene might be involved in sepal development and may have other
physiological functions in the sepals, however, Meli et al. (2010) did not
report impairment in sepal development in tomato RNAi lines of β-Hex.
The RIN and ASR1 specically binds to CArG box and ASR1 binding
sites of pD-200, therefore several mutations were created in CArG box
and ASR1 binding sites of this region and mutated promoter::GUS stable
transgenic plants were raised. The analysis of GUS activity and tran-
script accumulation in either mCArG-D-200::GUS or mASR1-D-200::
GUS transgenic fruits revealed a signicant reduction in promoter ac-
tivity (Fig. 5). The D-200 promoter even becomes lesser active when
both CArG box and ASR1 binding sites are mutated together as
compared to either mutated CArG or mutated ASR1 binding sites
(Fig. 5). This conrms that RIN and ASR1 specically binds to CArG box
and ASR1 binding sites of pD-200 as reported by Irfan et al. (2014)
previously. Although the D-200 promoter activity was drastically
reduced in mCArG/mASR1-D-200::GUS transgenic fruits, however it
was still little active.
In order to identify the cause of this remaining GUS activity, we
hypothesise that this could be due to ethylene which has been reported
to induce the expression of β-Hex in ripening fruits (Irfan et al., 2014;
Meli et al., 2010). Interestingly, ACC, a precursor of ethylene induces the
mCArG/mASR1-D4–200 promoter suggesting the potential role of
ethylene in β-Hex regulation in ripening fruits. Moreover, the expression
of β-Hex is repressed in the ripening impaired Nr (Never Ripe) mutant
(Meli et al., 2010). Ethylene has long been associated to ripening con-
trols and ERFs are considered to be the primary actors in mediating
responses to this hormone (Gao et al., 2020). There are total 77 ERFs
have been identied in the tomato genome and most ERFs exhibit a
ripening-related expression (Liu et al., 2016). For instance, SlERF.E4, or
SlERF6 plays a critical role in regulating ethylene synthesis thus
affecting the changes in fruit quality traits including carotenoid accu-
mulation (Lee et al., 2012). Moreover, signicant downregualtion of
SlERF.E4 in Nr mutant suggest its ethylene mediated regulation (Lee
et al., 2012). Interestingly, it has been reported that RIN binds to the
CArG boxes of SlERF.E4 promoter and expression of SlERF.E4 was also
found repressed in rin mutant (Lee et al., 2012; Liu et al., 2016).
Irfan et al. (2014) found that SlERF.E4 is expressed in a similar way
as β-Hex expression and it interacts with β-Hex promoter (HP-1001)
suggesting its involvement in the regulation of β-Hex. Here, we revealed
that SlERF.E4 interacts with D4–200 fragment of β-Hex promoter in
yeast one hybrid assay (Fig. 7). This interaction was further validated
through EMSA using SlERF.E4 protein and D-200 β-Hex promoter as
probe (Fig. 7). Typically, ERF family transcription factors bind to the
GCC box (AGCCGCC), GCC-like box (TCCGCC), and CCGAC box (Hao
et al., 2002; Ohme-Takagi and Shinshi, 1995). However, ERFs are also
found active on promoters lacking the canonical GCC box (Chakravarthy
et al., 2003; Liu et al., 2016; Pirrello et al., 2012). Moreover, ERFs from
subclass A, B and E (SlERF.E4 belongs to subclass E) are weak activators
on the GCC box (Pirrello et al., 2012). Although β-Hex promoter does not
contain GCC and GCC-like box, however, it is quite possible that SlERF.
E4 binds to non-GCC motif of D-200 β-Hex promoter in order to regulate
the expression of β-Hex during fruit ripening. It is also reported that
suppression of β-Hex results in inhibition of ethylene biosynthesis genes
and related transcription factors including ERFs possibly because of
signaling roles of N-glycans generated by β-Hex (Meli et al., 2010). In
one of the previous studies, it has been demonstrated that injection of
N-glycans in fruits promotes ethylene biosynthesis (Priem and Gross,
1992).
Taken together, these results suggest that all three proteins RIN,
SlASR1 and SlERF.E4 are transcriptional activators of β-Hex expression
and specically bind to D-200 region of β-Hex, the essential promoter
region required for ripening specic expresion of β-Hex. This is further
supported by increased LUC/REN activity of pD-200 by RIN, SlASR1 and
SlERF.E4 (Fig. 8). All these regulatory mechanisms work in a com-
plemantary manner to regulate the expression of β-Hex during ripening
(Fig. 9).
Fig. 8. Dual luciferase reporter assay in Arabidopsis protoplasts for activation of
D-200 β-Hex promoter by RIN, SlASR1 and SlERF.E4. (A) Schematic represen-
tation of effectors and reporter constructs used for dual luciferase assays. For
the reporter, expression of LUC and REN genes was driven by pD-200 and
pCaMV35S promoters respectively. For effectors, RIN, SlASR1, SlERF.E4 were
expressed under pCaMV35S promoter. The effectors and reporter constructs
were co-transfected in Arabidopsis protoplasts and relative LUC/REN activity
derived from pD-200:LUC was analyzed. All the three effectors RIN, SlASR1,
SlERF.E4 increased the pD-200 activity when expressed individually. RIN,
SlASR1, and SlERF.E4 increased maximum activity when expressed all together
suggesting that RIN, SlASR1, and SlERF.E4 work in a complementary manner to
regulate the expression of β-Hex gene. Data indicate the mean ( ±SE) of three
biological replicates. **P < 0.01 displays signicant differences (t-test)
compared to empty vector.
M. Irfan et al.

Plant Science 323 (2022) 111380
11
5. Conclusions
In this study, we have unidentied and functionally characterized
the smaller fragment of β-Hex promoter (D-200) to asses its potential in
ripening specic expression using promoter::GUS fusion transgenic
lines. The data suggest that D-200 β-Hex promoter is critical for tran-
scriptional regulation of β-Hex and is indispensable for spatio-temporal
regulation of endogenous β-Hex gene. In accordance with previous
study, we found that RIN and SlASR1 acts as positive transcriptional
regulator of D-200 β-Hex promoter as revealed by site directed muata-
genesis of RIN and SlASR1 binding sites. We further provided insights
into ethylene mediated regulation of β-Hex and shown SlERF.E4 acts as a
transcriptional regulator of β-Hex. In conclusion, the expression of β-Hex
is positively regulated by RIN, SlASR1 and SlERF.E4 via direct binding to
D-200 β-Hex promoter fragment during tomato fruit ripening. The D-200
β-Hex is sufcient for ripening specic expression of β-Hex and can be
used in fruit ripening specic expresion studies with a view to improve
fruit quality traits.
Author contribution statement
AD and MI conceptualized and designed the experiments. AD coor-
dinated the project. MI, PK and VK performed the experiments, gener-
ated and analyzed the data. MI wrote the manuscript with inputs from
PK and VK. AD edited the draft. All authors have read and approved the
nal manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgements
This study was funded by core research grant from National Institute
of Plant Genome Research and a research grant from Department of
Biotechnology, Govt. of India. MI acknowledges CSIR, India for Senior
Research Associateship (CSIR Pool Number: 8862-A).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.plantsci.2022.111380.
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