Plant Biotechnology Journal (2023), pp. 1–15 doi: 10.1111/pbi.

14228

The tomato EAR-motif repressor, SlERF36, accelerates

growth transitions and reduces plant life cycle by
regulating GA levels and responses
Rashmi Garg1,2 , Hrishikesh Mahato1,2, Upasana Choudhury1,2, Ravindra S. Thakur2,3, Pratima Debnath1,2,
Nasreen G. Ansari2,3, Vidhu A. Sane1,2 and Aniruddha P. Sane1,2,*
1
Plant Gene Expression Lab, CSIR-National Botanical Research Institute (Council of Scientific and Industrial Research), Lucknow, India
2
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
3
Analytical Chemistry Laboratory, Regulatory Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, India

Received 31 January 2023; Summary

revised 6 October 2023;
accepted 27 October 2023.
*Correspondence (Tel +91 522 2297959;
fax +91 522 2205836; email ap.sane@nbri.
res.in)
Keywords: early maturity, flowering
time, GA2 oxidase, AP2-ERF domain,
DELLA, gibberellins.
Faster vegetative growth and early maturity/harvest reduce plant life cycle time and are
important agricultural traits facilitating early crop rotation. GA is a key hormone governing
developmental transitions that determine growth speed in plants. An EAR-motif repressor,
SlERF36 that regulates various growth transitions, partly through regulation of the GA pathway
and GA levels, was identified in tomato. Suppression of SlERF36 delayed germination, slowed
down organ growth and delayed the onset of flowering time, fruit harvest and whole-plant
senescence by 10–15 days. Its over-expression promoted faster growth by accelerating all these
transitions besides increasing organ expansion and plant height substantially. The plant life cycle
and fruit harvest were completed 20–30 days earlier than control without affecting yield, in
glasshouse as well as net-house conditions, across seasons and generations. These changes in life
cycle were associated with reciprocal changes in expression of GA pathway genes and basal GA
levels between suppression and over-expression lines. SlERF36 interacted with the promoters of
two GA2 oxidase genes, SlGA2ox3 and SlGA2ox4, and the DELLA gene, SlDELLA, reducing their
transcription and causing a 3–5-fold increase in basal GA3/GA4 levels. Its suppression increased
SlGA2ox3/4 transcript levels and reduced GA3/GA4 levels by 30%–50%. SlERF36 is conserved
across families making it an important candidate in agricultural and horticultural crops for
manipulation of plant growth and developmental transitions to reduce life cycles for faster
harvest.

Introduction
Plant growth is marked by multiple growth cycles and transitions
leading from seed germination to organ expansion, adult growth,
onset of flowering, fruit formation and whole-plant senescence.
These growth transitions occur within a defined period primarily
in response to developmental cues but are also strongly
influenced by changes in the external environment. The duration
of the growth cycles in plant life is important in agriculture since
short durations can allow farmers to grow multiple crops in a
year, reduce water/nutrient losses by early cropping and minimize
exposure of the plant to vagaries of the external environment.
The onset of growth transitions is regulated by a balance between
different hormones and ensures completion of the life cycle in a
given time frame. Any change in hormonal balance, brought
about by a change in expression of regulatory genes, can alter the
speed of transitions. GA is one of the key hormones that controls
these transitions (Castro-Camba et al., 2022; Weiss and
Ori, 2007; Yamaguchi, 2008). Its interaction with other hormones
governs the outcome of developmental changes leading to seed
germination (Finch-Savage and Leubner-Metzger, 2006; Li et al.,
2019b; Liu and Hou, 2018), bud break (Liu and Sherif, 2019; Wen
et al., 2016), organ expansion (Achard et al., 2006; Fu and
Harberd, 2003; Li et al., 2012), stem and tuber development
(Chen et al., 2022), flowering (Bauerle, 2022; Blazquez
et al., 1998; Izawa, 2020), fruit set (Serrani et al., 2008; Shinozaki
et al., 2015) and adaptation (Dubois et al., 2013; Kuroha et al.,
2018; Shohat et al., 2020, 2021a, 2021b). Changes in basal
GA levels and/or responses change plant growth characteristics
as well as stress responses and have had far-reaching effects in
agriculture including development of varieties that led to the
Green Revolution in the sixties (Hedden, 2003; Khush, 1999).
GA levels are regulated in different tissues through fine control
over GA biosynthesis by a family of GA20/GA3 oxidases while
reduction in levels of active GAs is brought about by a family of
GA2 oxidases. Multiple genes encoding these enzymes are
differentially transcribed and collectively regulate stem elonga-
tion, leaf expansion, branching, flowering initiation, fruit set,
trichome development, stresses etc. (Castro-Camba et al., 2022;
Chen et al., 2016; Illouz-Eliaz et al., 2019, 2020; Kozaki and
Aoyanagi, 2022; Li et al., 2012, 2019a; Magome et al., 2008;
Martınez-Bello et al., 2015; Matıas-Hern andez et al., 2016;

Please cite this article as: Garg, R., Mahato, H., Choudhury, U., Thakur, R.S., Debnath, P., Ansari, N.G., Sane, V.A. and Sane, A.P. (2023) The tomato EAR-motif
repressor, SlERF36, accelerates growth transitions and reduces plant life cycle by regulating GA levels and responses. Plant Biotechnol. J., https://doi.org/10.1111/
pbi.14228.

ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. 1
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2 Rashmi Garg et al.
Monna et al., 2002; Shohat et al., 2020, 2021b; Yanai
et al., 2011). A great deal of regulation is also exerted through
GA signalling by GA receptors that facilitate GA action (Illouz-
Eliaz et al., 2019; Sun, 2010) and by DELLA proteins that suppress
GA action, inhibit growth and thereby allow the plant to respond
to environmental challenges (Achard et al., 2006; Colebrook
et al., 2014; Sun, 2010). Despite considerable studies on how GA
pathway components affect growth in plants like Arabidopsis,
rice and tomato, the regulation of individual components of the
pathway across development remain largely un-deciphered.
The AP2 domain family of transcription factors is large family
governing various aspects of development and adaptation in
plants. It is characterized by the presence of a conserved 58–59
amino acid DNA-binding AP2 domain with further functional sub-
groups based on distinctions within the domains and the
presence of additional domains flanking these (Nakano
et al., 2006; Pirrello et al., 2012). While most AP2 domain family
members function as activators of transcription, a small sub-
group (with 8 members in Arabidopsis and 7 in tomato) is
characterized by the presence of a short C-terminal repressor
motif (L/F)DLN(L/F)xP designated as the ERF-associated amphi-
philic repression (EAR) motif (Ohta et al., 2001). EAR-motif
proteins function as dominant repressors of transcription in
combination with co-repressors and histone deacetylases
(HDACs) and are responsible for chromatin modification (Kagale
and Rozwadowski, 2011). Detailed studies on some of these have
demonstrated roles in governing sensitivity to hormones like ABA,
ethylene, JA in processes like germination, fruit ripening, stomatal
responses, abiotic stresses and herbivory (Deng et al., 2022;
Licausi et al., 2013; Lu et al., 2011; McGrath et al., 2005; Plant
et al., 2021; Song et al., 2005; Upadhyay et al., 2013; Yang
et al., 2005). Nevertheless, detailed analysis of these genes
remains a challenge in most plants.
In this paper, we show that SlERF36, an AP2 domain EAR-motif
suppressor in tomato, controls the timing of developmental
transitions in tomato, partly by regulating GA levels and GA
signalling by reducing the expression of the GA2 oxidase genes,
GA2ox3 and GA2ox4, and the sole DELLA gene in tomato,
SlDELLA. Changes brought about by SlERF36 alter plant growth
cycle reproducibly by 30–40 days under glasshouse as well as
nethouse conditions demonstrating the robustness of its regula-
tion and usefulness of the gene under field conditions.
Results
SlERF36 is an EAR-motif repressor and its levels regulate
germination and organ expansion during juvenile/adult
vegetative growth
SlERF36 was previously identified in our lab as an EAR-motif ERF
that accelerated flowering time when ectopically expressed in
tobacco and Arabidopsis (Upadhyay et al., 2013, 2014). It is
expressed ubiquitously across tissues at varying levels (Figure S1a).
In order to elucidate its function in tomato, transgenic lines
expressing SlERF36 in the sense and antisense orientations under
the CaMV35S promoter were generated and monitored for
various phenotypic changes ranging from seed germination to
senescence. Three SlERF36 over-expression lines, OEx1, OEx3 and
OEx4 (with 3–5-fold higher transcript levels in seedlings and 4–6-
fold higher transcript levels in leaves), and three suppression lines,
Sup3, Sup6, and Sup7 (with transcript levels reduced to 36%–
44% in seedlings and leaves), were chosen from ten lines
generated for the study (Figure S1b). The expression of a closely
ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–15
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related gene, SlERF37, was first monitored to ensure that its
expression did not undergo a change in the transgenic lines
(Figure S1c).
Detailed phenotypic analysis of transgenic SlERF36 lines revealed
clear changes in the timing of growth transitions and the speed of
plant growth, starting with germination. Compared to control
seeds which germinated between days 2–5, seeds of all the over-
expression lines germinated from day 1 itself and completed
germination by day 4. In contrast, the seeds of all suppression lines
began germinating between days 3 (lines 6 and 7) and 6 (line 3)
and reached completion only by days 9–11 (Figure 1a). Even at day
5, when control and OEx lines had completely germinated,
suppression lines showed less than 20% germination. The most
prominent differences were observed on day 2, with nearly 80%
germination in over-expression lines, 55% germination in control
seeds, but no germination in suppression lines.
Following seedling establishment, further growth differences
were noted in subsequent stages of plant growth in tissues such as
roots, leaves and stem between the SlERF36 OEx and suppression
lines. All the OEx lines grew faster while the suppression lines grew
slower as evident from the 34%–36% increase in root length of 1-
month-old transgenic SlERF36 OEx plants and a reduction of 12%–
16% in suppression lines over control roots (Figure S2a). The
differences were even more apparent in aerial tissues with the OEx
lines showing an increase in plant height ranging from 25% to
69% at 30 days and 50%–130% at 75–90 days, compared
to control (Figure 1b). As the OEx plants attained their final height,
the growth increase slowed down, although plants remained taller
by 20%–24% than controls even at day 150. In comparison,
suppression lines were slow-growing and stayed shorter than
controls by 10%–20% up to 30 days and by 15%–25% thereafter.
At harvest, the plants were shorter by 20%–30% and never
attained the height of the control. These large reciprocal differences
between OEx and suppression lines were indicative of dose-
dependent changes in levels of SlERF36.
Leaf growth was also substantially affected in transgenic lines,
with all three suppression lines showing a reduction in leaf area
compared to control plants (Figure 1c). The reduction ranged
from 12% to 27% up to 45 days and 25%–35% between 60–
75 days. Even at harvest, leaves of suppression lines were smaller
by 11%–25% compared to control. In contrast, OEx lines showed
a much greater change in leaf area with an increase ranging from
45% to 85% by 45 days, 70%–112% by 60 days and 100–
150% by 90 days. At 5 months, OEx plants had leaves that were
~two-fold larger (70%–130% increase) than controls and >2.5-
fold larger than leaves of suppression lines.
A true indication of faster growth is an increase in dry weight
of plants. To verify this, the fresh weight and dry weight of roots
as well as aerial tissues was estimated in 30-day-old plants. All the
suppression lines showed a 25%–40% reduction in the root fresh
weight and 40%–50% reduction in root dry weight, compared to
control (Figure 1d; Figure S2b). A similar change was observed for
shoots with a 50%–55% reduction in fresh weight and a 47%–
52% reduction in dry weight (Figure 1d; Figure S2b). Strikingly,
OEx lines showed an increase of 50%–86% in root fresh weight
and 15%–18% in dry weight while shoot fresh weight increased
by 13%–21% and dry weight by 60%–100% (compared to
control) suggesting a greater resource allocation to aerial parts
upon SlERF36 over-expression.
The effects of SlERF36 expression were not restricted to just
organ expansion and growth but also affected leaf bud initiation
with a 30%–38% reduction in leaf number in all suppression lines
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SlERF36 controls GA and growth transitions 3

Figure 1 Manipulation of SlERF36 levels alters plant growth and the timing of developmental transitions in tomato. (a) Germination percentage of seeds
of control and SlERF36 over-expression and suppression lines on ½ MS medium. Germination was monitored based on radical emergence. The number of
days excludes the two-day stratification (n = 30). (b) Graphical representation of height of control and transgenic SlERF36 over-expression and suppression
lines during the course of their growth. Measurements were made in nethouse-grown plants on days 15, 30, 45, 60, 75, 90 120 and 150. Values are
averages ( SD) of ten plants per line. The inset shows height of 5-month-old plants of control and SlERF36 transgenic lines. (c) Graphical representation of
changes in leaf area of control and SlERF36 transgenic lines. Measurements were made in nethouse-grown plants on days 15, 30, 45, 60, 75, 90 120 and
150 using the 4th leaf from bottom. The values represent the mean ( SD) of leaves of five plants. The inset shows leaves of 5-month-old plants of control
and SlERF36 transgenic lines. (d) Dry weight of the entire aerial part (inclusive of stem and leaves) and roots of 30-day-old plants. The values represent
average  SD of five replicates. (e) Graphical analysis of differences in the leaf number in control and transgenic SlERF36 over-expression and suppression
plants during the course of their development under nethouse conditions. The values are average  SD of twenty plants per line.

and a 32%–62% increase in OEx lines compared to controls at
the final stage (Figure 1e). Thus, OEx lines not only had more
leaves but also larger leaves while suppression lines had fewer
and smaller leaves. Internodal distance was greater by 1.5–2-fold
in over-expression lines but reduced by 20%–40% in suppression
lines (Figure S2c).
Manipulation of SlERF36 expression alters the timing of
flowering, senescence and fruit harvest without
affecting yield
The changes in vegetative growth observed in SlERF36-altered
lines had a marked effect on the transition from the vegetative to
reproductive phase as observed by the onset of flowering. The
first flower buds were observed around 90.6  1.1 days in
control but were seen at least 6–7 days earlier at
84.6  1.2 days, 84.4  1 days, and 83.4  0.9 days in the
SlERF36 OEx lines OEx1, OEx3 and OEx4, respectively (Figure 2a).
In contrast, suppression lines showed a delay of 6–8 days with
initiation after 98.2  1, 97.4  0.96 and 96.4  1.3 days in
lines Sup3, Sup6 and Sup7, respectively. Thus, flowering time
differed by 13–14 days between OEx and suppression lines.
Not unexpectedly, the onset of whole-plant senescence post-
flowering and fruiting was also early in all the SlERF36 over-
expression lines, but delayed in the suppression lines. Senescence,
examined as the time when plants had more than 50% senescent
leaves (wilted and brown), was at least a week earlier in OEx lines
but delayed by about 7–10 days in suppression lines compared to
control (Figure 2b). The loss of chlorophyll, an indicator of

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4 Rashmi Garg et al.

Figure 2 Manipulation of SlERF36 levels alters the timing of flowering, senescence and fruit harvest in tomato. (a) Graphical analysis of differences in the
flowering time (time of appearance of the first bud on the plant) in control and transgenic SlERF36 over-expression and suppression plants grown in the
nethouse. The values are average  SD of twenty plants per line. (b) Representative picture showing progression of whole-plant senescence in nethouse-
grown plants of control and transgenic SlERF36 lines in the 6th month. (c) Graphical analysis of yield (total fruit weight per plant) in control and transgenic
SlERF36 OEx and suppression line plants. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (Student’s t-test).

senescence, was examined in detached leaves after every 12 h

using a PAM fluorescence meter and occurred earlier within 1.5–
2 days in OEx lines compared to 4 days in control and 5–9 days in
suppression lines (Figure S2d).
Finally, manipulation of SlERF36 expression did not affect final
yield (Figure 2c). However, the time taken to harvest at least 80%
of the total fruits was considerably altered with OEx lines showing
early harvest by 20–30 days compared to control while suppres-
sion lines showed delayed harvest by ~12–14 days compared to
control (Table 1).
These results showed that SlERF36 expression levels influenced
whole-plant growth considerably by altering the timings of
different growth transitions and whole-plant growth. The studies
were repeated across different seasons in experiments carried out
over a span of 4 years in T3 to T6 generation plants in glasshouse
as well as nethouse (Table 1). These showed that the harvest of
~80% of fruits on the plants was completed in a range of 165–
183 days in control, 132–153 days in OEx lines and 180–
195 days in suppression lines in glasshouse as well as nethouse
with slight differences. The studies indicated the robustness of
regulation by SlERF36.
SlERF36-mediated changes in growth in stem and leaves
are dependent on GA levels as well as GA signalling.
Growth changes associated with developmental transitions are
marked by changes in hormone homeostasis with GA being one
of the key hormones responsible for many transitions (Castro-
Camba et al., 2022). Although most of the changes in traits
observed in SlERF36-altered lines (germination, height, leaf area,
flowering time) are controlled by multiple factors, GA is a
common regulatory factor in all these, making it a likely candidate
for regulation by SlERF36. To test this, control and transgenic
SlERF36 OEx and suppression line plants were grown with regular
sprays of either water (as control), 100 lM GA3 or with 100 lM
paclobutrazol (a GA biosynthesis inhibitor) at 3-day intervals for
3 months to check whether differences in plant height and leaf
area could be overcome by these. As shown in Figure 3a
(Figure S3a), the OEx lines showed a significant increase in height
that ranged from 20% to 40% over the control plants from 14 to
28 days under untreated conditions. This increased to 80%–
130% from days 42 to 70 and reduced to 25%–30% by day 84.
A similar quantum of increase was also observed in the leaf area

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SlERF36 controls GA and growth transitions 5

Table 1 Time taken (days) for various transitions (including harvest) in control and SlERF36 transgenic lines under glasshouse/nethouse conditions
in different seasons and generations

Flowering time initiation (Days) Total time for >80% fruit harvest (days)
Germination
Line (Days) GH1 GH2 NH1 NH2 GH1 GH2 NH1 NH2

Control 5 89.8  1.1 86.4  1.1 90.6  1.1 87.9  1.7 168–182 165–180 170–183 166–180
OEx 1 4 83.5  1.2 78.7  1.8 84.6  1.2 80.1  1.7 138–152 132–150 141–153 135–150
OEx 2 4 82.8  1 78.1  1 84.4  1.1 79.2  1.1 138–152 132–150 141–153 135–150
OEx 3 4 81.9  1.1 77.5  1.7 83.4  1 78.9  1.1 138–152 132–150 140–153 135–150
Sup 1 11 97.7  1 96.1  1 98.2  1.1 96.9  1 182–194 180–192 183–195 180–192
Sup 2 10 96.8  1 95  1.2 97.4  1 95.9  1.3 182–194 180–192 183–195 180–192
Sup 3 9 95.9  1.1 94.3  1 96.4  1.3 95  1.1 182–194 180–192 183–195 180–192

GH1 = Glasshouse (Aug 2017-Feb 2018), T3 generation, n = 25–30 plants (plants/line).

GH2 = Glasshouse (Dec 2018-Jun 2019), T4 generation, n = 15–20 plants (plants/line).
NH1 = Net house (Aug 2019-Feb 2020), T5 generation, n = 10–12 (plants/line).
NH2 = Net house (Oct 2020-Apr 2021), T6 generation, n = 10–12 plants (plants/line).

Figure 3 Sensitivity of control and transgenic SlERF36 altered lines to exogenous GA3 and paclobutrazol. Graphical representation of plant height (a) and
leaf area (b) in control and transgenic SlERF36 plants upon exogenous application of GA3 and paclobutrazol. Control and transgenic SlERF36 plants were
sprayed with either water (untreated) or with 100 lM GA3 or 100 lM paclobutrazol in three independent sets at three-day intervals for 3 months and
measurements for height and leaf area taken at 14, 28, 42, 56, 70 and 84 days post-germination. Bars represent value ( SD) averaged over 10–15
measurements per line. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001 (Student’s t-test).

of OEx lines over the controls at the corresponding stages
(Figure 3b; Figure S3b). Suppression lines showed a 25–37%
decrease in height compared to control at all stages from 14 to
70 days and a 15%–20% decrease by day 84 under untreated
conditions with similar differences in leaf area at corresponding
stages. Treatment with 100 lM GA increased the plant height in
all the lines. However, the differences in height between control
and SlERF36 OEx lines were largely reduced with significant
differences of only 5%–10% from days 14 to 28. Thereafter, no
differences were observed in heights between OEx and control
plants. Although leaf area showed significant differences over the
control at the corresponding stages, these too were only in
the range of 4%–11%, except at day 14 where the difference
was higher. Suppression lines also grew taller following GA
treatment. Surprisingly, they continued to remain shorter than
controls by 25%–35% from 14 to 56 days and by 10%–20% at

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6 Rashmi Garg et al.

Figure 4 Manipulation of SlERF36 affects the expression of GA pathway genes in different tissues. (a) qRT-PCR analysis showing relative changes in
transcript levels of GA biosynthesis genes (SlGA20ox1, SlGA20ox3 and SlGA3ox1), GA catabolism genes (SlGA2ox3 and SlGA2ox4) and SlDELLA in control
and transgenic SlERF36-altered lines in germinating seeds (3DPI) (upper panel) and in 2-month-old leaves (lower panel). The values were normalized against
SlCAC and represent means of three biological replicates. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001
(Student’s t-test). (b) Real-time PCR analysis of expression of SlERF36 in leaves of the procera mutant.

days 70 and 84 with similar differences in leaf area at
corresponding stages. When the plants were treated with
paclobutrazol, there was marked reduction in the heights of all
the lines at all stages. The differences between control and OEx
lines were much reduced compared to untreated plants and were
in the range of 8%–22% in different lines from days 14 to 70.
There was nevertheless an increase of 25%–54% in heights in
OEx lines at day 84 over the control. An increase of 10%–20%
was also seen in leaf area of OEx lines from 14 to 42 days with a
much higher increase of 25%–50% at days 70 and 84. In
contrast, suppression lines showed a much greater reduction
in height compared to controls with the decrease ranging from
21% to 33% from days 14 to 70 and little lower at 84 days. The
leaf area of suppression lines also showed a much larger
reduction in the range of 25%–37% compared to control at all
stages. Thus, treatment with GA as well as paclobutrazol largely
reduced differences in growth (height and leaf area) between
OEx lines and controls but not as much between suppression lines
and controls.
The results indicated that GA levels as well as GA signalling (or
GA-independent processes) were, in part, responsible for the
SlERF36-mediated growth changes.
Changes in plant growth are associated with SlERF36-
mediated control of expression of GA biosynthesis/
degradation and signalling
Since GA appeared to be a strong candidate that may influence
the changes in SlERF36-mediated plant growth, the GA pathway
(biosynthesis, metabolism and signalling) was studied in greater
detail. Real-time PCR analysis of the GA pathway genes was
carried out in SlERF36 over-expression, suppression and control
plants at two developmental stages namely, seed germination (at
3DPI) and in leaves of 2-month-old plants, when differences were
most prominent between these lines. Clear reciprocal changes
were observed in expression of several GA pathway genes
between OEx and suppression lines. Transcript levels of the GA
biosynthesis genes SlGA20ox1 and SlGA20ox3 increased by
2.5–3-fold and 4–6-fold respectively in all the OEx line seeds
but were reduced to 50%–55% (SlGA20ox1) and just 20%–35%
(SlGA20ox3) in all the suppression line seeds (Figure 4a).
Expression of SlGA3ox1 increased 2–3-fold in OEx lines but was
reduced to only 20%–40% in suppression lines. In contrast, the
expression profiles of the GA2ox genes (responsible for GA
degradation) showed opposite effects with the most significant
reduction in transcript levels of SlGA2ox3 and SlGA2ox4. These
decreased by 30%–40% in OEx lines and increased 1.5-fold in
suppression lines compared to control. Other GA2oxs such as
SlGA2ox1, SlGA2ox2 and SlGA2oxs 5–10 also showed a
reduction in OEx lines and an increase in suppression lines
although these were not as significant (Figure S4a).
The differences in expression of GA pathway genes were more
prominent in leaves of 2-month-old plants although SlERF36
transcript levels were in the range as seen in seedlings (4–5-fold
higher in OEx lines and 50%–70% reduced in suppression lines).
As shown (Figure 4a), transcript levels of the GA biosynthesis
genes were up-regulated by 3.5–5-fold (SlGA20ox1), 3–7.5-fold
(SlGA20ox3) and 4–8-fold (SlGA3ox1) in all OEx lines but reduced
by 20%–50% (SlGA20ox1), 50%–70% (SlGA20ox3) and ~20%
(SlGA3ox1) in all the suppression lines compared to the control. In
contrast, transcript levels of SlGA2ox3 and SlGA2ox4 (but not
other GA2ox genes; Figure S4b) were reduced by 65%–70% and
55%–70%, respectively, in over-expression lines and increased
3–5-fold and 2–2.4-fold in all the suppression lines.
Besides GA biosynthesis and degradation, transcript levels of
the DELLA gene, SlDELLA, involved in GA signalling, also showed
a significant 20%–40% reduction in seedlings and 35%–50%
reduction in leaves of all the OEx lines but a slight increase in
suppression lines. Interestingly, the expression of SlERF36 was
about twofold higher in leaves of the SlDELLA mutant, procera
(Figure 4b). No differences were observed in transcript levels of
the three GID genes in the transgenic lines (data not shown).
Changes in SlERF36-mediated plant growth are
associated with changes in GA3 and GA4 levels in stem
and leaves
In view of the correlation between the expression of the GA
pathway genes and the large-scale differences in height and leaf
area of SlERF36-altered lines, an estimation of endogenous levels

ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–15

of GA3 and GA4 was carried out in stem and leaves of 60-day-old
plants of all the SlERF36 transgenic lines and control. A marked
change in the basal GA content of 60-day-old leaf tissues was
observed between controls and transgenic lines (Figure 5a). The
levels of GA3 and GA4 in controls (5.5  0.4 and
28.1  0.7 ng g 1 FW, respectively) increased by 21, 93
and 135% for GA3 in OEx lines 1, 3 and 4, respectively and by
22%–35% for GA4 in leaves of all OEx lines. In contrast, leaves of
60-day-old suppression lines showed a significant decrease of
31%–47% for GA3 and 60%–66% for GA4.
The levels for GA3 and GA4 in corresponding stems of 60-day-
old control plants were 6.3  1.6 and 58  7 ng g 1 FW,
respectively. These increased by 75%–118% for GA3 and by
33%–62% for GA4 in stems of the OEx lines. In contrast, stems of
60-day-old suppression lines showed a slight decrease of 20%–
22% for lines 3 and 6 for GA3 that was not significant but
showed a significant decrease of 24%–38% for GA4 in all the
lines.
An estimation of GA levels in leaves of 120-day-old plants and
stems of 75-day-old plants (Figure 5b) revealed even more
prominent differences. GA3 levels in control and transgenic OEx
lines increased almost 3.5-fold in leaves of 120-day-old plants
over 60-day-old plants while GA4 levels shot up ~sixfold in control
but 9–10-fold in transgenic OEx lines. Compared to 120-day-old
control plants, these were associated with ~twofold higher levels
of GA3 and 2.5-fold higher levels of GA4 in the OEx lines but a
reduction by 50%–70% (GA3) and >70% (GA4) in suppression
lines. GA levels also increased in 75-day-old stem by about 1.8-
fold for GA3 and GA4 over the 60-day stem while they were
significantly reduced in suppression lines. These were associated
with an increase of 13%–56% in OEx lines (GA3) and 27%–62%
(GA4) but a reduction by 26%–40% for GA3 and 14%–36% for
GA4 for suppression lines, compared to controls. The results
confirmed that GA levels were partly to substantially responsible
for changes in the growth phenotypes of leaves and stem of
SlERF36 transgenic lines.
SlERF36 functions as a repressor and binds the promoters
of SlGA2ox3, SlGA2ox4 as well as SlDELLA
EAR-motif proteins are well known to function as repressors of
transcription (Kagale and Rozwadowski, 2011; Ohta et al., 2001).
In order to confirm whether SlERF36 functioned as a repressor, a
protoplast transfection assay was performed with 35S-GAL4-DBD
and 35S-GAL4DBD-SlERF36 as effector constructs, and GAL4
(4X)-CaMV35S-GUS as reporter construct as described (Singh
et al., 2019; Wang et al., 2007). The effector construct expressing
GAL4DBD under the CaMV35S promoter could drive GUS activity
from the reporter in absence of SlERF36. However, in presence of
SlERF36 (under the control of the CaMV35S double promoter),
there was 60% reduction in GUS activity (Figure 6a). Having
established the ability of SlERF36 to function as a repressor, and
given the observations of strong suppression of SlGA2ox3,
SlGA2ox4 as well as SlDELLA in SlERF36 OEx lines by real-time
analysis (Figure 4a), yeast one-hybrid assays were carried out to
study whether SlERF36 could directly bind their promoters
to regulate them. A region of the promoters comprising
1931 bp (SlGA2ox3), 2498 bp (SlGA2ox4) and 1983 bp
(SlDELLA), upstream of the initiation codons, was chosen for
study and showed clear interactions with SlERF36 (Figure 6b).
These interactions were further confirmed in vivo using luciferase
assays. As shown (Figure 6c), expression of luciferase driven by
the promoters of SlGA2ox3, SlGA2ox4 and SlDELLA was
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SlERF36 controls GA and growth transitions 7
observed as fluorescence in tobacco leaves agro-infiltrated with
constructs lacking the SlERF36 construct. However, co-expression
of these promoters with SlERF36 strongly reduced promoter-
driven luciferase activity and fluorescence to low/undetectable
levels indicating the suppression of SlGA2ox3, SlGA2ox4 and
SlDELLA promoters by SlERF36. Further confirmation of SlERF36
function was obtained at least for SlGA2ox3 through EMSA
studies wherein binding of the SlERF36-His tagged protein to the
GCC-box cis element at position 1343 in the SlGA2ox3
promoter led to a shift in the band that was competed out by
5x unlabelled probe (Figure 6d). No binding was observed when
the cis element was mutated. Collectively, the studies highlighted
how regulation of multiple growth transitions could be effectively
controlled by SlERF36 at least partly through manipulation of GA
levels and responses through suppression of SlGA2ox3, SlGA2ox4
and SlDELLA.
Discussion
GA is one of the primary hormones associated with multiple
growth cycles that mark transitions in a plant and can impact
important agricultural traits such as maturity, harvest time and
crop duration (Castro-Camba et al., 2022; Hedden, 2003). The
GA pathway needs to be tightly controlled as misregulation of GA
levels/signalling can markedly affect plant growth and fitness. We
have identified the tomato EAR-motif repressor, SlERF36, as a
global regulator of the GA pathway that alters GA levels and
signalling across tissues, affecting multiple transitions and plant
growth duration.
Manipulation of SlERF36 levels influences the timing of
developmental events such that higher SlERF36 expression
accelerates germination (Figure 1a), increases the speed of organ
growth (root length, leaf area, internodal distance, height and dry
weight) (Figure 1b–d), accelerates flowering time (in days)
(Figure 2a) and whole-plant senescence (Figure 2b). Its suppres-
sion has opposite effects that retard growth and delay transitions.
These changes lead to a 6–8-day difference in germination, 15-
day difference in flowering time and a difference of about 30–
42 days in completion of the fruit harvest between SlERF36 OEx
and suppression lines. The results are indicative of dose-
dependent effects of SlERF36 that alter the speed of plant
growth.
Although each of these traits is influenced by a combination of
hormones and different tissue-specific regulators (Blazquez
et al., 1998; Castro-Camba et al., 2022; Finch-Savage and
Leubner-Metzger, 2006; Fu and Harberd, 2003), GA plays a
decisive role in determining how these changes progress and is
strongly and reciprocally altered in SlERF36 OEx and suppression
lines across tissues like germinating seeds, leaves and stem
(Figures 4 and 5). SlERF36 indirectly activates transcription of the
GA biosynthesis genes SlGA20ox1/3 and SlGA3ox1 (Figure 4a)
and simultaneously reduces GA degradation by directly binding to
the promoters of GA2 oxidase genes, SlGA2ox3 and SlGA2ox4,
and the GA response inhibitor SlDELLA, reducing their transcrip-
tion in seeds as well as leaves (Figures 4a and 6A–D). Its action
increases basal GA3 and GA4 levels in stem and leaves throughout
development with 3–5-fold higher levels in SlERF36 OEx lines and
30%–70% reduction in suppression lines (Figure 5a,b). Although
GA levels change across tissues, the changes are greater for GA4
compared to GA3 in leaves as well as stems indicating that the
GA2ox3/4 enzymes may preferentially target GA4 than GA3.
The greater differences in GA4 levels in stems suggest that it may
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8 Rashmi Garg et al.

Figure 5 SlERF36 manipulation alters GA levels in stem and leaves. Box plots of liquid chromatography-mass spectrometry estimation of GA3 and GA4 in
(a) leaves and stems of 60-day-old plants of control and transgenic SlERF36 over-expression and suppression lines and (b) leaves of 120-day-old plants and
stem of 75-day-old plants. The values are average ( SD) of three replicates. The error bars represent SE, * indicates P < 0.05, ** indicates P < 0.01, ***
indicates P < 0.001 (Student’s t-test).

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SlERF36 controls GA and growth transitions 9

Figure 6 SlERF36 functions as a transcriptional repressor and physically interacts with the SlGA2ox3/SlGA2ox4/SlDELLA promoters. (a) SlERF36 functions
as a repressor of GAL4 in protoplast transfection assays. The effectors GAL4DBD and GAL4DBD-SlERF36 under the CaMV35 double promoter and the
GAL4(4X)::GUS reporter constructs were co-transfected into protoplasts extracted from 3 to 4 week-old Arabidopsis Col-0 plants. GUS activity was studied
after 16 h of transfection in dark. Graph represents the mean  standard error of three replicates. (b) Yeast one-hybrid studies showing interactions
between SlERF36 and the promoters of the GA catabolism genes PSlGA2ox3 ( 1931 bp, second row), PSlGA2ox4 ( 2498 bp, third row) and the GA signalling
inhibitor PSlDELLA ( 1983 bp, fourth row) in different serial dilutions. The positive and negative controls are present in the first and last rows, respectively.
The minimal inhibitory concentration of the aureobasidin-A (AbA) antibiotic for selection of positive bait-prey interactions was optimized to 100 ng/mL and
selection performed on SD/AbA/-Ura agar plates. (c) Luciferase assays depicting suppression of the above promoters of PSlGA2ox3, PSlGA2ox4 and PSlDELLA by
SlERF36 (driven by the CaMV35S promoter) in tobacco leaves. The scheme showing the agro-injection of constructs in different regions of the leaves
(empty vectors at top; promoter-LUC constructs without SlERF36 on the right; and promoter-LUC constructs with SlERF36 on the left) is provided in the
extreme right panel. The fluorescence in the right part of the leaf indicates basal promoter activity in absence of SlERF36. (d) Electrophoretic mobility shift
assays showing binding of SlERF36 to the biotin-labelled primers containing the GCC box cis element at 1343 nt (upstream of the initiation codon) in the
promoter of SlGA2ox3. Biotin-labelled primer probes (double-stranded) were electrophoresed with SlERF36 protein on a 6% native polyacrylamide gel as
described in methods. Interactions were checked with biotinylated mutated probe and by competition with excess cold probe. The arrow shows the band
of shifted probe bound to SlERF36.

contribute more to differences in height. The changes in GA levels
and signalling seem to have a cascading effect on overall plant
growth and transitions leading to marked changes in flowering
time, fruit harvest and senescence. Tomato organ growth is
known to be affected by changes in GA biosynthesis or
degradation (Olimpieri et al., 2011; Schrager-Lavelle
et al., 2019; Shohat et al., 2021b; Yanai et al., 2011) and by
GA signalling (Jasinski et al., 2008; Liu et al., 2016; Livne
et al., 2015; Nir et al., 2017; Ramon et al., 2021). A reduction in
GA levels by suppression of GA20ox1 reduces internodal distance
and height in tomato plants and affects leaf morphology and
floral tissues (Olimpieri et al., 2011; Shohat et al., 2021a) while
over-expression of CcGA20ox1 in tomato affects stem length and
fruit set (Garcıa-Hurtado et al., 2012). Alterations in GA levels are
also caused by inactivation of SlGA2ox7 which affects GA2
oxidase activity leading to increased internode length in the
tomato internode elongated-1 mutant (Schrager-Lavelle
et al., 2019). The lanceolate mutation la in tomato and its semi-
dominant form La-2 (which positively affects GA responses) affect
leaf lamina growth by changing GA20ox1 and GA2ox4 expres-
sion in the margins, thereby affecting leaf complexity (Yanai
et al., 2011). Mutations in the procera mutant, affecting the only
DELLA gene in tomato, increase GA responses leading to
elongated, thin stems and simpler leaves (Jasinski et al., 2008;
Livne et al., 2015) while a gain-of-function mutant lacking DELLA
domain is GA-insensitive and severely reduces growth (Nir
et al., 2017). SlDELLA expression also regulates stomatal
responses and reduces stomatal opening in drought (Nir
et al., 2017; Shohat et al., 2020, 2021a). SlERF36 seems to
simultaneously control many of the above genes, and the GA
pathway as a whole, through its action. It also affects growth
independently of GA levels since treatment with exogenous GA
or paclobutrazol (Figure 3a,b) does not completely overcome the
growth phenotypes in transgenic lines. These effects, which are
more prominent in SlERF36 suppression lines under GA-limiting
conditions (paclobutrazol treatment), could partly be mediated by
regulating GA signalling through increased SlDELLA expression
and its stability (due to high GA oxidase activity) but also through
GA-independent regulators (as evident from an ongoing tran-
scriptomic study). The changes in SlDELLA stability in SlERF36-
manipulated lines could selectively affect its interactions with GID
receptors in different tissues thereby regulating growth differen-
tially, as previously shown (Illouz-Eliaz et al., 2019). This aspect,
along with GA pathway-independent regulation, is currently
under study. SlERF36 effects on germination and organ growth/
expansion can be explained by SlERF36-mediated changes in the

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10 Rashmi Garg et al.

Figure 7 Model showing the function of SlERF36 in regulating developmental transitions through its action on the GA pathway and through putative GA-
independent regulators. SlERF36, through co-repressors and HDACs, suppresses the GA2 oxidases, SlGA2ox3 and SlGA2ox4 and the GA action inhibitor,
SlDELLA, to increase the GA pathway output which in turn activates intermediate components (some known, some yet to be identified) that activate
various transitions. It also regulates these through suppression of GA-independent regulators that suppress the transitions through other intermediaries.

GA pathway and an increase in basal GA levels and responses.
Although flowering has also been shown to be promoted by GA
in Arabidopsis (Blazquez et al., 1998; Yamaguchi et al., 2014) and
some other plants, higher GA levels or higher GA responses (as in
the procera mutant) have actually been shown to delay flowering
in tomato (Carrera et al., 2012; Garcıa-Hurtado et al., 2012).
Interestingly, while SlERF36 over-expression increases leaf num-
ber (an indication of delay in flowering, Figure 1e), flowering time
is actually accelerated by ~7 days compared to control and
delayed in suppression lines (Figure 2a). This is unlike the procera
mutant, where leaf number is higher and the time to anthesis of
the first flower is also delayed (Carrera et al., 2012). Early
flowering was also observed upon ectopic expression of SlERF36
in tobacco and Arabidopsis (Upadhyay et al., 2013, 2014). The
disparity between increased leaf number and reduced flowering
time in SlERF36-altered lines could stem from faster overall
growth and morphogenetic development in OEx lines and growth
retardation in suppression lines. The observations suggest that
SlERF36 effects on flowering may be more complex and
dependent on additional targets that differentially affect flower-
ing time and plant growth, partly through GA-independent
effects. GA application is also known to delay senescence (Yu
et al., 2009) although its effects on senescence are conflicting
since DELLA function can also delay senescence (Zhang
et al., 2018). It is likely that whole-plant senescence that is
activated in annuals post-flowering, may be regulated differently
by GA/DELLA than that of individual organs alone (although
SlERF36 OEx leaf discs also show early senescence symptoms).
Conversely, it may be regulated by SlERF36 differently through
GA-independent targets. A model explaining the possible mode
of action of SlERF36 on whole-plant development through GA-
dependent and independent pathways is shown (Figure 7). That
SlERF36 action may also involve additional GA-independent
targets is implied through its growth phenotypes which unlike
procera (which is tall and thin) cause plants to be sturdy because
of an increase in stem girth (data not shown). The highly
expanded leaves, that develop early on, increase the canopy area
and allow greater capture of light and could aid in photo-
assimilation of carbon dioxide and would explain the faster
increase in fresh and dry weight in SlERF36 OEx lines (Figure 1d;
Figure S2b). A comprehensive analysis of SlERF36 targets using
tagged versions would shed more light on the complexities of
SlERF36 regulation on other aspects besides GA homeostasis.
Regardless of the mechanismsgoverning flowering and senes-
cence inSlERF36 altered lines, the overall effect of faster growth
and earliness in flowering, harvest and senescence in OEx lines is
an attribute of significance for farmers.
As a member of the small EAR-motif sub-group of repressors
within the large AP2/ERF family, SlERF36 is likely to function
through recruitment of co-repressors like SIN3 and TOPLESS, like
other EAR-motif repressors (Deng et al., 2022; Song et al., 2005).
EAR-motif repressors, although accounting for a small subgroup
of the large AP2-ERF domain family, seem to have major roles in
modulating hormone responses during development and stress
adaptation. AtERF7, a homologue of SlERF36, reduces ABA
responses in Arabidopsis in stomatal closure leading to water loss
under drought while its suppression increases ABA sensitivity in
germination and stomatal closure (Song et al., 2005). AtERF4
negatively regulated JA responses to Fusarium oxysporum
(McGrath et al., 2005) and also reduced sensitivity to ethylene
and ABA (Yang et al., 2005) while OsERF3 increased JA responses
upon wounding and ethylene responses in drought (Lu
et al., 2011; Zhang et al., 2013). AtERF12 controls the transition
from dormancy to germination by regulating ethylene responses
(Li et al., 2019b) while SlERF12 in tomato negatively regulates the
transition to fruit ripening through suppression of ethylene
biosynthesis genes ACS2 and ACS4 (Deng et al., 2022). Although
SlERF36 governs GA homeostasis and all the major transitions
across development, a simultaneous effect on other hormones
like ABA (akin to its Arabidopsis homologue AtERF7) cannot be
ruled out considering that ABA and GA often function
antagonistically in developmental processes (Liu and Hou, 2018).
Finally, controlling transitions and shortening plant duration in
the field is important in agriculture as early maturity and early

ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–15

harvest can reduce water and nutrient requirement in fields,
reduce exposure to adverse conditions (harsh temperatures,
excess rains, biotic factors) and promote faster crop rotation.
SlERF36 expression reduces plant duration substantially from 6 to
5 months by accelerating growth without reducing yield (Fig-
ures 1 and 2), making it an important candidate for smart
agriculture. Further, studies in our lab show that it is functionally
conserved in Arabidopsis, potentially extending its usefulness
across families. Manipulation of this gene provides an avenue for
tailoring plants in different families for faster growth and a
shorter life cycle without yield loss.
Experimental procedures
Tomato transformation, growth conditions and
phenotypic analysis of transgenic plants
All analyses were carried out in the background of the tomato
variety Arka Vikas in homozygous progeny (T3-T6 generations) of
transgenic SlERF36 over-expression and suppression lines (three
independent lines each). Plants were grown in the glasshouse and
in the field (nethouse) under natural conditions of light
and temperature from August to March/April in four different
years (2018–2021).
The SlERF36 ORF was cloned in pBI121 (Upadhyay et al., 2013,
2014) in sense and anti-sense orientations under the CaMV35S
promoter at BamHI and introduced in tomato through
Agrobacterium-mediated transformation (Madhulatha et al.,
2007). Ten independent lines were generated of which three
lines over-expressing SlERF36, namely OEx1, OEx3 and OEx4 with
threefold, fourfold and 4.5-fold higher SlERF36 transcript levels,
respectively, and three suppression lines, Sup3, Sup6 and Sup7
with SlERF36 transcript levels down to 30%, 35% and 40% of
control, respectively, were chosen for study (Figure S1c). Homo-
zygous plants of these lines in T3 generation were used for all
subsequent studies on phenotyping, gene expression and
hormone estimation as described below:
Germination: Seeds were grown on ½ MS and germination
recorded as radical emergence after two days of darkness at 25°C
(50 seeds/line). Experiments were replicated at least thrice.
Differences due to seed germination were excluded when
collecting data on subsequent plant growth parameters.
Plant height, leaf area and leaf number
These were measured at 15, 30, 45, 60, 75, 90, 120 and
150 days after seedling emergence. The height was measured
from the base to the top of each plant, and the leaf area (4th leaf
from bottom) was measured using a graph chart. For root length,
1-month-old plants were used.
Biomass
Roots and aerial parts (fresh/dry weight) of 15-day and 30-day-old
plants (10 plants each) were cleaned and separately measured.
The weight of the entire aerial part was taken as the shoot
weight. Dry weights were measured after drying in an oven (48 h,
70°C).
Flowering time
Flowering time (monitored on 20 plants) was measured as the
time taken for the first bud to appear on the plant. The time for
fruit harvest of at least 80% of the total fruits was measured
for comparing earliness in harvest.
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SlERF36 controls GA and growth transitions 11
Whole plant and leaf senescence
The onset of whole-plant senescence was considered when more
than 50% of the leaves on the plant had wilted and turned
yellow/brown after fruiting. The extent of leaf senescence was
studied by chlorophyll fluorescence imaging of leaf discs of
control and SlERF36 transgenic leaves using an Imaging-PAM, M-
series Chlorophyll Fluorometer (Walz Effeltrich, Germany) over 4–
5 days. The onset of senescence, indicated by damage to
photosystem II, is seen as a change in fluorescence and decrease
in Fv/Fm values with a colour change from blue to green-yellow-
red and black (Singh et al., 2013).
GA and paclobutrazol (PAC) treatment
Control and transgenic seeds (30 seeds each) were treated
separately with water, 10 lM GA3 and 10 lM paclobutrazol
(Sigma-Aldrich). After radical emergence, the seedlings were
transferred to soilrite and sprayed separately with either water,
100 lM GA3 or 100 lM PAC every third day. Height and leaf
areas for 10–15 plants from each set (watered and GA/
paclobutrazol-treated sets) were measured for 3 months at
seven-day intervals from the day of radical emergence.
RNA isolation and real-time PCR analysis
Transcriptional analysis of various GA pathway genes was carried
out in germinating tomato seedlings and in 2-month-old leaves.
Seedlings were sampled on the third day post-imbibition (DPI),
excluding the first two days of dark period.
RNA was isolated as described (Asif et al., 2000) and cDNA
prepared. Real-time PCR was performed using SYBR Select
(Applied Biosystems Inc, USA) on an ABI StepOnePlus PCR
machine using gene-specific primers (Table S1). Reactions were
run in technical triplicates for three biological samples, and the
data analysed were the mean of biological triplicates normalized
against the validated reference control gene, SlCAC (Expo sito-
Rodrıguez et al., 2008) using the 2 ΔCT method to obtain the fold
change in expression (Livak and Schmittgen, 2001).
Protoplast isolation and transfection for SlERF36
repressor assay
Preparation of effector and reporter constructs
The effector (GAL4-DBD and GAL4-DBD-SlERF36) and reporter
constructs [GAL4(4X)::GUS] were prepared as described previ-
ously (Singh et al., 2019; Wang et al., 2007) in the pUC19 vector
background under the control of the double CaMV35S promoter
at Nde1 and Sac1 restriction sites with the full-length SlERF36
ORF cloned in-frame with an N-terminal GAL4 DNA binding
domain.
Protoplast Isolation
Arabidopsis protoplasts were isolated largely as described by
Abel and Theologis (1994) but without vacuum infiltration.
Following incubation (22–24°C, 6–8 h in dark with gentle
swirling), the protoplasts were filtered using nylon filter
(70 lm) and centrifuged at 100 9 g for 5 min. The pellet
was washed once with pre-chilled W5 buffer and resuspended
in 20 mL of the same (Abel and Theologis, 1994). Following a
30 min incubation on ice, the protoplasts were centrifuged at
300 9 g for 5 min and resuspended in pre-chilled MaMg
solution (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES, pH
5.7). Transfection was carried out as described (Abel and

12 Rashmi Garg et al.
Theologis, 1994) using 100 lL protoplasts (2 9 104 cells) mixed
with 15 lg plasmid (effector and reporter constructs) followed
by addition of 110 lL of 40% PEG solution (containing 0.2 M
mannitol and 0.1 M CaCl2). After incubation at room
temperature for 30 min, the reaction mixture was diluted
slowly with 0.44 mL of W5 solution, centrifuged at 300 9 g
for 3 min, and the protoplast pellet was resuspended in
1 mL W5 solution and incubated in dark for 16–18 h at room
temperature. GUS activity was assayed as described by
Jefferson et al. (1987) on a microtitre plate.
Arabidopsis protoplasts were isolated largely as described by
Abel and Theologis (1994) but without vacuum infiltration.
Following incubation (22–24°C, 6–8 h in dark with gentle
swirling), the protoplasts were filtered using nylon filter (70 lm)
and centrifuged at 100 9 g for 5 min. The pellet was washed
once with pre-chilled W5 buffer and resuspended in 20 mL of the
same (Abel and Theologis, 1994). Following a 30 min incubation
on ice, the protoplasts were centrifuged at 300 9 g for 5 min
and resuspended in pre-chilled MaMg solution (0.4 M mannitol,
15 mM MgCl2, and 4 mM MES, pH 5.7). Transfection was
carried out as described (Abel and Theologis, 1994) using 100 lL
protoplasts (2 9 104 cells) mixed with 15 lg plasmid (effector
and reporter constructs) followed by addition of 110 lL of 40%
PEG solution (containing 0.2 M mannitol and 0.1 M CaCl2). After
incubation at room temperature for 30 min, the reaction mixture
was diluted slowly with 0.44 mL of W5 solution, centrifuged at
300 9 g for 3 min, and the protoplast pellet was resuspended in
1 mL W5 solution and incubated in dark for 16–18 h at room
temperature. GUS activity was assayed as described by Jefferson
et al. (1987) on a microtitre plate.
Yeast one-hybrid and luciferase assays
The yeast one-hybrid protein-DNA interactions were performed
in Y1H gold cells essentially as described in the manufacturer’s
protocol (Takara, Japan). Three bait sequences representing
promoters of SlGA2ox3 ( 1931 bp), SlGA2ox4 ( 2498 bp) and
SlDELLA ( 1983 bp) were cloned in the pAbAi vector,
integrated in Y1H gold yeast genome and selected on SD/-
uracil agar plates. A minimum inhibitory concentration of
aureobasidin A required to inhibit growth of cells in absence of
prey (100 ng/mL AbA) was established. For the prey construct,
the open reading frame of SlERF36 (666 bp) driven by the
constitutive ADH1 promoter was cloned in pGADT7-AD vector
at BamHI and selected in yeast on SD/ leu plates. Following
mating, the yeast recombinants, where both bait and prey
interacted, were selected by their ability to grow on SD/ leu
and the inhibitory concentration of AbA (100 ng/mL) at 30°C
for 3–5 days on different dilutions.
The above promoters were also used to drive luciferase
expression in tobacco leaves by cloning in the Gateway cloning
vector pGWB435 (Invitrogen). The coding sequence of SlERF36
was separately cloned in pGWB402 under the CaMV35S
promoter to generate effectors. Empty vectors (pGWB435,
pGWB402) and Pro::Luc reporters (with individual promoters)
were used as negative and positive controls, respectively. The
constructs were introduced in A. tumefaciens (strain GV3101),
assays performed by agro-infection in N. tabacum leaves and
visualized by the ability of SlERF36 (co-infiltrated with Pro::Luc
vectors) to suppress fluorescence as described (Zhou et al., 2018).
For each promoter, interactions were performed in a single leaf
divided into three parts. Experiments were repeated at least thrice
in different leaves.
ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–15
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Electrophoretic mobility shift assay (EMSA)
The full-length coding region of SlERF36 was cloned into pET28a
and expressed in Rosetta (DE-3) strain of E. coli. The His-tagged
protein was induced at 37°C for 16 h. with 1 mM IPTG and
purified on Ni-NTA agarose.
For EMSA, the probes covering the GCC box in the promoters
of SlGA2ox3 ( 1343 bp), SlGA2ox4 ( 1366 bp) and SlDELLA
( 1166 bp) were labelled with biotin using the 3՛ end biotinyla-
tion kit (Thermo Fisher) and double-stranded oligonucleotide
prepared. Unlabelled probe was used for competitive binding. In
the mutated version of the probe, ‘GCCGCC’ was changed to
‘AAAAA’ and biotinylated. The probe (15 lg) and purified fusion
protein were incubated with 10x binding buffer containing
10 mM MgCl2, 1 lg/ll poly(dI.dC), 1% NP-40, 50% glycerol for
30 min at room temperature. The reaction product was resolved
on a 6% native polyacrylamide gel, transferred to positively
charged nylon membrane (Bright star-plus, Thermo Fisher
Scientific) and the membrane visualized on a Chemidoc system
(Bio-Rad).
GA estimation
GA estimation was performed with fresh leaf tissue as described
(Hou et al., 2008) with slight modifications. Crushed samples
(1 g) were extracted by ultrasonication in 90% methanol (1 mL)
on ice bath (with 30 s on/off cycles for 3 min). After
centrifugation (10 000 9 g, 15 min at 4°C), the supernatant
was collected and pellet re-extracted with 0.5 mL extraction
solvent thrice. The supernatants were combined, dried under a
nitrogen stream and re-dissolved in 1 mL methanol followed by
centrifugation (10 000 9 g, 5 min at 4°C) and filtration
through a 0.22 lm PTFE filter. Samples (5 lL) were analysed
against standards of GA (Sigma-Aldrich, Bengaluru, India) using
liquid chromatography-mass spectrometry (SCIEX Exion LC 2.0
system – SCIEX Triple-TOF 5600 + system). LC separation was
achieved using the SCIEX Exion LC 2.0 system (Framingham,
MA, USA) and a Phenomenex Kinetex 1.8 lm, EVO C18 100  A
LC column 100 9 2.1 mm (Torrance, California, USA). A 4 min
isocratic flow of water with 0.1% formic acid (5%) and
methanol with 0.1% formic acid (95%) was used, with a flow
rate of 0.5 mL/min. 5 lL of the sample was injected into the LC
system. The SCIEX Triple-TOF 5600 + system (Framingham,
MA, USA) was operated with the Turbo V ion source using an
electrospray ionization (ESI) probe. MRM-HR transitions were
monitored for GA3 and GA4 in negative mode by observing the
product of 345.0 Da and 331.0 Da, respectively. The source
parameter for mass analysis was GS1 (55.0 psi), GS2 (45.0 psi),
CUR (35.0 psi), TEM (500°C), ISVF (4500 V), TOF Mass range
(50–500 Da) and accumulation time (0.100 s). The compound
parameter for GA3 and GA4 was 30 and 40 declustering
potential, 35 was the collision energy with 2.0 collision energy
spread. Data acquisition was performed using Analyst TF 1.8.1
software.
Data processing of mass spectrometry acquired data was
performed using MultiQuant 3.0.3 software in which calibration
curves and peak integration statistics were generated.
Statistical analysis
Statistical analysis was carried out using data from different
biological replicates. Significant differences between control and
transgenic SlERF36 over-expression and suppression lines were
analysed using Student’s t-test with the GraphPad Prism software

where * indicates P < 0.05, ** indicates P < 0.01, and ***
indicates P < 0.001.
Acknowledgements
The authors thank Prof. David Weiss (Hebrew University of
Jerusalem, Rehovot, Israel) for the kind gift of procera mutant
seeds and Dr Shucai Wang (Northeast Normal University,
Changchum China) for the effector and reporter constructs for
the repressor assay. The authors thank Mr. Ram Awadh
for maintaining plants in the glasshouse and nethouse. The
authors are grateful to Mr. Shiv Narayan and Dr. PA Shirke (Dept
of Plant Physiology, CSIR-NBRI) for help with the PAM imaging
system for leaf disc senescence studies. RG, HM and UC were
supported by Senior Research fellowships from CSIR, India and
from SERB, while PD was supported by CSIR funds. Financial
support for the work was provided to APS by SERB (Science and
Engineering Research Board), Department of Science
and Technology, Govt of India, under the project EMR/2016/
007736 (GAP3455).
Author contributions
APS conceived the idea; RG performed the genetic manipulations
and real-time expression analysis; RG, HM performed the
phenotypic analysis and GA/paclobutrazol treatments; RG/UC/
HM performed the yeast-one-hybrid and luciferase assays while
HM performed the repression studies; HM/PD performed the
EMSA studies; RG/UC/HM prepared samples for GA estimation;
RST/NGA performed the GA estimations, VAS analysed the data;
RG, VAS and APS analysed all the results; RG, APS wrote the
paper.
CSIR-NBRI manuscript number: CSIR-NBRI_MS/2023/04/12.
Competing interests
The authors have no competing interests.
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Supporting information
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
Table S1 List of oligonucleotides used in the study.
ª 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 1–15
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SlERF36 controls GA and growth transitions 15
Figure S1 SlERF36 expression in different tissues and in different
OEx and suppression lines.
Figure S2 SlERF36 manipulation regulates various growth
processes.
Figure S3 Plant phenotypes of control and transgenic SlERF36
lines upon exogenous treatment with GA3 and paclobutrazol.
Figure S4 qRT-PCR analysis showing relative changes in transcript
levels of GA2ox genes.