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Stress-Inducible Overexpression of SlDDF2 Gene Improves Tolerance against

Multiple Abiotic Stresses in Tomato Plant

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 horticulturae

Article
Stress-Inducible Overexpression of SlDDF2 Gene Improves
Tolerance against Multiple Abiotic Stresses in Tomato Plant
Taghleb Al-Deeb 1, *, Mohammad Abo Gamar 2 , Najib El-Assi 3 , Hmoud Al-Debei 3 , Rabea Al-Sayaydeh 4
and Ayed M. Al-Abdallat 3, *

1 Department of Biological Sciences, Faculty of Science, Al al-Bayt University, Mafraq 25113, Jordan
2 Department of Biological Sciences, Faculty of Science, Yarmouk University, Irbid 21163, Jordan;
mohammad.abogamar@yu.edu.jo
3 Department of Horticulture and Crop Science, School of Agriculture, The University of Jordan,
Amman 11942, Jordan; najibasi@ju.edu.jo (N.E.-A.); debeih@ju.edu.jo (H.A.-D.)
4 Department of Agriculture Sciences, Shoubak College, Al-Balqa Applied University, Al-Salt 19117, Jordan;
rabea.sayaydeh@bau.edu.jo
* Correspondence: taghleb@aabu.edu.jo (T.A.-D.); a.alabdallat@ju.edu.jo (A.M.A.-A.);
Tel.: +962-2-629-7000 (ext. 3570) (T.A.-D.); +962-6-535-5000 (ext. 22331) (A.M.A.-A.)

Abstract: Dehydration-responsive element-binding protein 1 (DREB1)/C-repeat binding factor (CBF)

family plays a key role in plant tolerance against different abiotic stresses. In this study, an orthologous
gene of the DWARF AND DELAYED FLOWERING (DDF) members in Arabidopsis, SlDDF2, was
identified in tomato plants. The SlDDF2 gene expression was analyzed, and a clear induction



in response to ABA treatment, cold, salinity, and drought stresses was observed. Furthermore,


two transgenic lines (SlDDF2-IOE#6 and SlDDF2-IOE#9) with stress-inducible overexpression of
Citation: Al-Deeb, T.; Abo Gamar, M.;
SlDDF2 under Rd29a promoter were generated. Under stress conditions, the gene expression of
El-Assi, N.; Al-Debei, H.;
SlDDF2 was significantly higher in both transgenic lines. The growth performance, as well as
Al-Sayaydeh, R.; Al-Abdallat, A.M.
physiological parameters, were evaluated in wild-type and transgenic plants. The transgenic lines
Stress-Inducible Overexpression of
showed growth retardation phenotypes and had higher chlorophyll content under stress conditions
SlDDF2 Gene Improves Tolerance
against Multiple Abiotic Stresses in
in plants. However, the relative decrease in growth performance (plant height, leaf number, and leaf
Tomato Plant. Horticulturae 2022, 8, area) in stressed transgenic lines was lower than that in stressed wild-type plants, compared with
230. https://doi.org/10.3390/ nonstressed conditions. The reduction in the relative water content and water loss rate was also lower
horticulturae8030230 in the transgenic lines. Compared with wild-type plants, transgenic lines showed enhanced tolerance
to different abiotic stresses including water deficit, salinity, and cold. In conclusion, stress-inducible
Academic Editors: Pirjo Mäkelä,
Mercè Llugany, Peter A. Roussos and
expression of SlDDF2 can be a useful tool to improve tolerance against multiple abiotic stresses in
Mumtaz Cheema tomato plants.

Received: 15 February 2022 Keywords: abiotic stresses; bioinformatics; DREB1; stress-inducible promoter; tomato; transcription
Accepted: 4 March 2022
factor
Published: 7 March 2022

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil- 1. Introduction
iations. Abiotic stresses, such as cold, drought, high salinity, and extreme heat have adverse
effects on plant growth and development. They are considered major constraints for plant
production in many areas around the globe. Among them, drought is considered a major
limiting factor for the productivity of any given crop. To minimize the negative impact
Copyright: © 2022 by the authors.
of abiotic stresses on plants, it is necessary to develop new plants that utilize water more
Licensee MDPI, Basel, Switzerland.
efficiently and tolerate such stresses [1]. In this perspective, a basic strategy is based on the
This article is an open access article
cloning of key regulatory genes and the introduction of their active forms into plants so
distributed under the terms and
that they can acquire abiotic stress tolerance phenotypes [2].
conditions of the Creative Commons
Attribution (CC BY) license (https://
Under stress, several morphological, physiological, and molecular processes are al-
creativecommons.org/licenses/by/
tered in different organs to improve plant tolerance [3]. Plants have developed different
4.0/). defense mechanisms against abiotic stresses that involve the interaction among a group

Horticulturae 2022, 8, 230. https://doi.org/10.3390/horticulturae8030230 https://www.mdpi.com/journal/horticulturae

Horticulturae 2022, 8, 230 2 of 13

of transcription factors and the activation of key effector genes [4]. Transcription factors
control plant responses to different environmental factors through sequence-specific interac-
tions with cis-regulatory DNA elements, which are found in promoter and enhancer regions
of their target genes [5]. Thus, the levels of expression of different abiotic stress-responsive
genes are influenced by the manipulation of stress-responsive transcription factors [6].
Therefore, the manipulation of these transcription factors can improve the tolerance of
different plants against different biotic and abiotic stress conditions [7]. In this perspec-
tive, members of dehydration-responsive element-binding protein 1 (DREB1)/C-repeat
binding factor (CBF) family, encoding AP2 transcription factors, are known to regulate the
expression of several stress-responsive genes by binding to C-repeat/dehydration-responsive
cis-element in their promotors, thus enhancing cold, high salinity, and drought tolerance
of plants [8]. In many plants, DREB genes work as the connecting points for multiple
plant-response pathways to different stress factors, such as salinity, drought, ABA, and cold
pathways [9–11]. For instance, the DWARF AND DELAYED FLOWERING (DDF) genes
were upregulated in Arabidopsis plant under cold, drought, salinity stress conditions [12],
while the overexpression of CBF4 gene in Arabidopsis plants improved drought tolerance
that was associated with upregulation of several stress-responsive genes and resulted
in [13].
Tomato (Solanum lycopersicon L.) is one of the most grown vegetable crops all over
the world, and it is mainly cultivated under irrigated conditions. Tomato yield is greatly
affected in many growing areas by different abiotic stresses including drought, salinity,
and low temperatures [14]. There is a growing need to improve stress tolerance in tomato
plants to withstand such adverse conditions. In this study, a new member of DREB
transcription factors in tomato plants (named SlDDF2) that is closely related to the DDF
gene in Arabidopsis was isolated and characterized. The SlDDF2 gene was expressed in
response to a variety of abiotic stimuli, implying a potential role in tolerance against abiotic
stresses. Transgenic tomato plants with different levels of inducible overexpression of the
SlDDF2 were generated and analyzed for their growth, physiological, biochemical, and
gene expression responses to multiple abiotic stress factors. Transgenic tomato plants with
inducible overexpression of SlDDF2 showed reduced water loss and improved tolerance
against multiple abiotic stresses.

2. Materials and Methods

2.1. Cloning of SlDDF2 Gene and Bioinformatics Analysis
To clone a DDF orthologous gene in tomato plants, bioinformatics and comparative
genomics analyses were performed based on a previously published full-length sequence
of Arabidopsis DDF1 gene (GenBank accession number: NM_101131 [15]). A TBLASTN
search was performed utilizing DDF1’s amino acid sequence against the annotated ITAG2.3
predicted tomato cDNA sequences database [16]. The full-length coding sequences of
novel and unstudied tomato DNA sequences encoding DDF transcription factors in tomato
were retrieved and further analyzed. The predicted coding sequences of a selected tomato
DDF gene (SlDDF2; Solyc08g007820) were used to design specific primers (SlDDF2fwd: 50 -
ATGAATAACGACTCGAGTTTG-30 and SlDDF2Rev: 50 -TCAAATACTATAACTCCACA-30 )
using the NCBI Primer-BLAST tool to isolate its full-length CDS as described previously by
Al-Abdallat et al. [16].
For SlDDF2 cloning, leaves from two-week-old tomato cv. “Money Maker” plants
were collected and used for total RNA extraction using the SV Total RNA Isolation System
Kit (Promega, Madison, WI, USA), as directed by the manufacturer. Following the manu-
facturer’s instructions, the extracted RNA was used to synthesize the first-strand cDNA
library using the SuperScript® First-Strand Synthesis System (Invitrogen, Carlsbad, CA,
USA) and the oligo T(18) primer. The full-length CDS of SlDDF2 gene was then amplified
from the synthesized cDNA using specific pair of primers (SlDDF2Fwd and SlDDF2Rev) in
a PCR with a total volume of 25 µL containing 5 µL of cDNA as a template, 2.5 µL of dNTPs
(100 µM), 5 µL of 5× PCR buffer, 0.5 µM of each primer and 0.25 µL of 5 U/µL GoTaq
Horticulturae 2022, 8, 230 3 of 13

DNA polymerase (Promega, Madison, WI, USA). The thermal reaction was conducted
using GeneAmp® PCR system 9700 (Applied Biosystems, Carlsbad, CA, USA) under the
following conditions: 94 ◦ C for 5 min, followed by 35 cycles at 94 ◦ C for 1 min, 50 ◦ C for 30 s
and 72 ◦ C for 2 min and a final extension of 72 ◦ C for 10 min. The amplified CDS fragments
were resolved on a horizontal 1% agarose gels stained with ethidium bromide. The PCR
products (estimated size 735 bp) were then eluted from the agarose gel using Wizard®
SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and then cloned into
pGEM® -T Easy Vector System (Promega, Madison, WI, USA). DNA Plasmids containing
PCR products were selected, and the DNA products were then sequenced using the M13
reverse and forward sequencing primers by ABI 3730XL machine by Macrogen (Seoul, Ko-
rea). The sequenced SlDDF2 cDNA and its deduced amino acids sequences were analyzed
by the Vector NTI software (https://www.thermofisher.com/jo/en/home/life-science/
cloning/vector-nti-software.html, accessed on 1 July 2021; Invitrogen™, Carlsbad, CA,
USA) and further confirmed by a BLAST search (https://solgenomics.net/tools/blast/,
accessed on 1 February 2019) for DNA and amino acid sequence homology to further verify
the identity of the cloned cDNA.
The Sol Genomics Network [17] was used to retrieve and analyze DNA and amino acid
sequences, as well as chromosomal position and annotation prediction of several members
of DREB-A1 and DREB-A2. Phylogenetic trees were constructed using MEGA version
10 software [18] and the amino acids sequences of SlDDf2 and selected ERF subfamily
proteins from tomato and Arabidopsis from the DREB-A1 and DREB-A2 groups [19] that
were retrieved from the Phytozome databases [20] were included. The retrieved sequences
were aligned using the ClustalW algorithm and the alignment was used to calculate
distance matrices for neighbor-joining analyses with the Kimura two-parameter model
and Bootstrap analysis with 10,000 replicates was performed to test the robustness of the
internal branches, as described previously by Alhindi and Al-Abdallat [21].

2.2. Plant Material and Stress Treatments

For gene expression analysis, tomato cv. “Moneymaker” (MM) plants were grown
under growth conditions and were subjected to different treatments that included water
deficit, salinity, cold, and ABA. Transgenic tomato lines with inducible overexpression
of the SlDDF2 gene, on the other hand, were utilized to compare abiotic stress tolerance
in MM plants in response to drought, salinity, and cold. The transgenic plants were
generated using the binary plasmid pCABIMA1302 harboring the SlDDF2 gene down-
stream of the stress-inducible Rd29a promoter. For this purpose, the mgfp5 gene was
replaced with SlDDF2 CDS at the NcoI and BstEII sites to generate pCAMBIA1302/SlDDF2.
Thereafter, the Rd29a promoter (GenBank accession number: AY973635.1) was cloned into
pCAMBIA1302/SlDDF2 by replacing the CaMV 35S promoter using EcoRI and NcoI sites to
produce pCAMBIA1302/Rd29a::SlDDF2. The constructs were then introduced into tomato
cv. “MM” using Agrobacterium-mediated transformation, and two positive plants carrying
a single copy of the transgene were identified and selected, as described previously by
Al-Abdallat et al. [22]. Gene expression analysis levels in T2 homozygous selected trans-
genic lines were analyzed using the quantitative RT–PCR approach under water deficit and
control conditions.
For stress treatments, tomato seeds of the selected genotypes were submerged in water
for 24 h at 25 ◦ C, and then, they were washed with sterilized water and sown into small pots
(10 cm diameter × 10 cm depth) filled with acid-washed sand. The pots were incubated
under controlled conditions (at constant temperature (25 ◦ C) and photoperiod of 16 h light–
8 h dark, with 250 µmol·m−2 ·s−1 photon flux density) till full germination. The tomato
seedlings were then irrigated daily with a fixed amount of 1× Hoagland solution (Sigma-
Aldrich, Gillingham, UK). For water deficit treatment, two-week-old tomato seedlings were
exposed to water withholding for 0, 3, 5, and 7 days for gene expression analysis in MM
plants and for 7 days for inducible expression analysis in transgenic plants. Furthermore,
stress tolerance and wilting behavior in response to water withholding for 10 days were
Horticulturae 2022, 8, 230 4 of 13

investigated on transgenic and nontransgenic lines, in which 20 plants from each line were
used per treatment and the survival percentage was calculated at the end of the dehydration
treatment.
To investigate the effect of high salt treatment on SlDDF2 gene expression, the roots
of two-week-old MM seedlings were submerged in saline water (300 mM NaCl) for 0, 2,
4, 8, 12, and 24 h. For salinity stress tolerance in transgenic and nontransgenic lines, two-
week-old seedlings (20 plants from each line were used per treatment) were irrigated every
three days with a fixed volume of 1× Hoagland solution supplemented with 100 mM for
12 days and the wilting behavior was monitored, and survival percentage was calculated
at the end of the high salt treatment. For the effect of cold treatment on SlDDF2 gene
expression, two-week-old MM seedlings were incubated at 4 ◦ C for 0, 2, 4, 8, 12, and 24 h.
For cold stress tolerance in transgenic lines and wild-type plants, two-week-old seedlings
(20 plants from each line were used per treatment) were incubated for 24 h at 4 ◦ C, and the
wilting behavior was monitored, and the survival percentage was calculated. The gene
expression of SlDDF2 was analyzed in response to ABA treatment, two-week-old MM
seedlings were sprayed with 100 µM ABA solution, and leaf tissues were collected after 0,
2, 4, 8, 12, and 24 h.

2.3. Growth and Physiological Measurements

Relative water content (RWC) was determined in well-watered and stressed plants
(subjected to 10 days of water withholding period), as described in Al-Abdallat et al. [16],
using fully expanded leaves, and the RWC was calculated according to Barrs and Weath-
erley [23]. The water loss rate was measured using fully expanded leaves excised from
the well-watered and stressed transgenic lines (subjected to 10 days of water withholding
period) that were placed on filter paper for 2 h, as described in Al-Abdallat et al. [16], and
the water loss rate was measured according to Ristic and Jenks [24]. Stomatal resistance
(s·cm−1 ) was measured using a fully expanded leaf from well-watered and stressed plants
after 10 days of water withholding by using a Porometer device (AP4, Delta-T Devices,
Cambridge, UK). The determination of chlorophyll content (Chla, Chlb, and total Chl)
was carried out using excised leaves from stress-treated transgenic lines and MM plants
following a modified protocol, as described previously by Al-Abdallat et al. [16]. Finally,
growth-related measurements (leaf number, plant height (in cm), and leaf area (cm2 ) were
collected from stress-treated (subjected to 10 days of water withholding period) and well-
watered transgenic lines and MM plant. Five replicates were used for all physiological and
growth parameters, and the standard error of means was used to compare means.

2.4. Gene Expression Analysis

For quantitative real-time PCR (qRT–PCR) analysis, total RNA was extracted from
leaf samples collected from treated plants at indicated time points using the SV Total RNA
Isolation System Kit (Promega, Madison, WI, USA). The extracted RNA was used to syn-
thesize the first-strand cDNA library as described above. Specific primers pairs for SlDDF2
expression analysis (SlDDF2Efwd: 50 -ATGAATAACGACTCGAGTTTG-30 and SlDDF2ERev:
50 -TCAAATACTATAACTCCACA-30 ) were used. The qRT–PCR analysis was performed
using the stress-inducible Le16 gene (Solyc10g075090, a phospholipid transfer protein from
tomato also known as Le16 (Lycopersicon esculentum protein 16) and Solyc03g078400 (en-
coding actin, a house-keeping gene used as an internal reference control for relative gene
expression analysis) as described in Al-Abdallat et al. [16]. All cDNA samples were an-
alyzed in triplicate, and each replicate was derived from two biological replicates. The
relative changes in gene expression were quantified as described in Vandesompele et al. [25].

2.5. Statistical Analysis

The data are presented as mean ± SD of three technical replicates from two biological
replicates (n = 6) for gene expression analysis and five biological replicates for the growth
performance and physiological parameters’ measurements. Student’s t-test was used
 Horticulturae 2022, 8, 230 5 of 13

to determine significant group differences, and means were considered as statistically

significant if p < 0.05.

3. Results and Discussion

3.1. Identification of DDF Orthologous Genes in Tomato
To identify DDF orthologous gene in tomato plants, a TBLASTN search was conducted
against the annotated ITAG2.3 predicted tomato cDNA sequences database using the
full-length amino acids sequence data of the DDF1 gene from Arabidopsis (GenBank
Accession: NM_101131). Using this approach, three tomato orthologous genes were
identified: Solyc12g056430, Solyc08g007820, and Solyc08g007830, and the three genes were
named SlDDF1, SlDDF2, and SlDDF3, respectively. Phylogenetic analysis using ERF
subfamily proteins from tomato and Arabidopsis belonging to groups DREB-A1 and DREB-
A2 revealed the identity of SlDDF proteins, which clustered with DDF1 and DDF2 proteins
from Arabidopsis (Figure 1). The DDFs are members of the DREB-A1 subfamily of the
ERF/AP2 transcription factor family in Arabidopsis, and they are implicated in stress
responses and GA biosynthesis regulation [12], indicating that SlDDF may have a role in
stress tolerance in tomato plants. To investigate this role in tomato plants, the SlDDF2
gene was selected and cloned using specific pair of primers. The SlDDF2 gene was found
Horticulturae 2022, 8, x FOR PEER REVIEW
on the upper arm of chromosome 8, and it was annotated as Solyc08g007820 that encodes 14 o
ethylene-responsive transcription factor 10 with a single ORF (735 bp with a single exon)
intron and a total length of 245 amino acids.

1. Phylogenetic
Figure 1.
Figure Phylogeneticanalysis of Arabidopsis
analysis proteins
of Arabidopsis belonging
proteins to the DREB-A1
belonging and DREB-A2
to the DREB-A1 and DREB
groups and their closest orthologs in tomato plants.
groups and their closest orthologs in tomato plants.

3.2. Expression Analysis of SlDDF2

The expression behavior of SlDDF2 in response to ABA and abiotic stresses was
alyzed in MM plants using quantitative real-time PCR. The SlDDF2 expression lev
Horticulturae 2022, 8, 230 6 of 13

3.2. Expression Analysis of SlDDF2

The expression behavior of SlDDF2 in response to ABA and abiotic stresses was
analyzed in MM plants using quantitative real-time PCR. The SlDDF2 expression levels
were compared with Lycopersicon esculentum protein 16; Solyc10g075090 (Le16), a stress-
inducible gene from tomato encoding a phospholipid transfer protein. The expression of
the SlDDF2 gene in MM plants was highly induced after two hours of ABA treatment before
returning to the basal expression level of the control (zero time) after 4 h (Figure 2A). This
is in general agreement with Li et al. [26], who reported that the ABA-induced expression
of SlDREB in tomato plants started from 1 to 6 h after treatment before it returned to
pre-treatment levels after 12 h. The expression of the Le16 stress-responsive gene, on the
other hand, was induced as expected in response to ABA treatment and peaked after 24 h,
which is consistent with previous studies [27,28]. The expression patterns of SlDDF2 and
Le16 genes in response to cold treatment (4 ◦ C for 0, 2, 4, 8, 12, and 24 h) were also analyzed
in tomato MM plants. The expression of SlDDF2 was induced after 4 h, with a higher level
of induction observed at 12 h of cold treatment, while Le16 gene expression was at the
highest level after 12 h of cold treatment (Figure 2B). These results are similar to the findings
of Zhang et al. [29], who found that the expression of the LeCBF1 gene was upregulated
upon exposure to low temperature, reaching its highest level after 8 h before returning to
its pretreatment levels after 24 h.
The expression of the SlDDF2 gene in MM plants was induced after 2 h in response to
100 mM NaCl treatment, reaching its highest expression at 4 h of incubation (Figure 2D).
Le16 gene expression was induced in response to NaCl with time, which is in agreement
with Al-Abdallat et al. [22], and the highest expression level was observed at 24 h. The
upregulation of SlDDF2 in response to high salt stress suggests a potential role in salinity
tolerance, which was previously reported by Sakuma et al. [30] and Magome et al. [12], who
found that DDF1 gene expression was induced in Arabidopsis roots under high salinity
conditions. These results are also in agreement with Hichri et al. [31], who reported the
inducible expression of SlDREB2 in tomato plants in response to NaCl treatments.
The expression patterns of the SlDDF2 gene and Le16 were analyzed in MM tomato
plants in response to water deficit treatment by water withholding for 3, 5, and 7 days. The
expression of the Le16 gene in stressed MM tomato plants was induced under water deficit
with time, reaching the highest expression level after seven days (Figure 2D), which is in
general agreement with Al-Abdallat et al. [16]. Similarly, the expression of the SlDDF2
gene was induced in response to water deficit after five days of stress, reaching its highest
level after seven days (Figure 2D). Similar results were described by Hichri et al. [31], who
reported drought-induced expression of SlDREB2 in tomato plants.

3.3. Stress-Inducible Overexpression of SlDDF2 in Tomato

To check if SlDDF2 inducible overexpression can enhance tolerance of tomato against
various abiotic stresses, the coding sequence of SlDDF2 was cloned into a binary plasmid
under the control of the rd29A, a stress-inducible promoter from Arabidopsis plant, or
under the control of the CaMV 35S constitutive promoter. Several independent transgenic
tomato lines were generated with transgenic lines carrying the CaMV 35S constitutive
promoter showed severe growth retardation phenotypes and did not produce any seeds and,
therefore, were discarded from further analysis (data not shown). Severe pleotropic effects
of constitutive overexpression of stress-related DREB transcription factors in tomato plants
were reported previously [32]. Similarly, the overexpression of DDF1 in Arabidopsis plants
resulted in dwarfism and late-flowering phenotypes [12]. On the other hand, transgenic
lines carrying the rd29A stress-inducible promoter were generated, and two transgenic lines
carrying a single insertion event as revealed by real-time PCR analysis were selected for
further analysis (SlDDF2-IOE#6 and SlDDF2-IOE#9). To validate the inducible expression
of SlDDF2 in response to stress, transgenic lines and MM plants were subjected to water
withholding for seven days. The expression of the SlDDF2 gene was significantly higher in
both transgenic lines, compared with wild type; however, the expression level was much
 al. [12], who found that DDF1 gene expression was induced in Arabidopsis roots under
high salinity conditions. These results are also in agreement with Hichri et al. [31], who
reported the inducible expression of SlDREB2 in tomato plants in response to NaCl
treatments.
Horticulturae 2022, 8, 230 7 of 13
The expression patterns of the SlDDF2 gene and Le16 were analyzed in MM tomato
plants in response to water deficit treatment by water withholding for 3, 5, and 7 days.
The expression of the Le16 gene in stressed MM tomato plants was induced under water
higherwith
deficit in stressed plants than
time, reaching the that in nonstressed
highest expression plants (Figure
level after 3A).
seven Furthermore,
days (Figure 2D), the
expression levels in SlDDF2-IOE#9 plants were significantly higher than that of SlDDF2-
which is in general agreement with Al-Abdallat et al. [16]. Similarly, the expression of the
IOE#6 plants
SlDDF2 under
gene was both treatments.
induced in responseStress-inducible
to water deficitexpression
after five of DREB
days the rd29A
genes reaching
of stress,
stress-inducible promoter was reported previously in different plants species including
its highest level after seven days (Figure 2D). Similar results were described by Hichri et
tomato
al. [33,34].
[31], who reported drought-induced expression of SlDREB2 in tomato plants.

Figure 2. Relative gene expression analysis of SlDDF2 and Le16 in response to (A) ABA, (B) cold,
Figure
(C) NaCl, Relative
2. and gene expression
(D) water deficit treatments. of SlDDF2
analysisMM and Le16
plants were in response
compared to (A) ABA,
to untreated (B)(con-
plants cold,
(C) NaCl, and (D) water deficit treatments. MM plants were compared to untreated
trol). The stress-responsive Le16 (Solyc10g075090) gene was included as a control. Values are the plants (con-
trol). ±The
means SD.stress-responsive Le16 (Solyc10g075090)
of six replicates. Relative expressions aregene was included
significantly as afrom
different control.
thoseValues
at zeroare the
time
atmeans
different levels:
± SD. * preplicates.
of six < 0.05, *** Relative
p < 0.001.expressions are significantly different from those at zero time
at different levels: * p < 0.05, *** p < 0.001.
 expression of the SlDDF2 gene was significantly higher in both transgenic lines, com-
pared with wild type; however, the expression level was much higher in stressed plants
than that in nonstressed plants (Figure 3A). Furthermore, the expression levels in
SlDDF2-IOE#9 plants were significantly higher than that of SlDDF2-IOE#6 plants under
Horticulturae 2022, 8, 230 both treatments. Stress-inducible expression of DREB genes the rd29A stress-inducible
8 of 13
promoter was reported previously in different plants species including tomato [33,34].

Figure 3.3.(A)
Figure (A)Relative
Relativegene expression
gene analysis
expression of SlDDF2
analysis in MMinand
of SlDDF2 MMSIDDF2-IOE#6 and SIDDF2-
and SIDDF2-IOE#6 and
IOE#9 transgenic
SIDDF2-IOE#9 plants in response
transgenic plants in to water withholding
response for seven days;
to water withholding valuesdays;
for seven are the means
values ± the
are SE
means
of ± SE of six(B)
six replicates; replicates; (B) plant
plant height, (C) height, (C) leaf
leaf number, andnumber, and
(D) leaf (D)ofleaf
area area of two-week-old
two-week-old seedlings
seedlings
of of SIDDF2-IOE#6
SIDDF2-IOE#6 and SIDDF2-IOE#9
and SIDDF2-IOE#9 transgenictransgenic
lines andlines
MMand MM under
plants plants normal
under normal and
and water
water withholding
withholding fordays
for seven seven days (stress).
(stress). Values
Values are are the±means
the means ± SD. Different
SD. Different lower-case
lower-case letters
letters indicate
aindicate a significant
significant differencedifference between transgenic
between transgenic and wild-type
and wild-type plants underplants under nonstressed
nonstressed conditions, con-
and
different capital letters indicate a significant difference between transgenic and wild-type plants
under stress conditions (p < 0.05).

When compared with the wild-type plants, the two selected transgenic lines (SlDDF2-
IOE#6 and SlDDF2-IOE#9) showed growth retardation phenotypes under normal and
water deficit conditions, with clear shorter plants and shorter internodes phenotypes
(Figure 3B). The wild-type and SlDDF2-IOE#9 plants showed a significant reduction in
leaf number mean values under stress conditions when compared with nonstressed plants
(Figure 3C). In addition, the SlDDF2-IOE#9 plants showed a significant reduction in leaf
area means values under stress conditions when compared with well-watered plants and
SlDDF2-IOE#6 plants (Figure 3D). On the contrary to the findings of this study, the use
of the stress-inducible rd29A promoter for the overexpression of AtDREB1A in transgenic
tomato did not show negative effects on plant growth and development [33]. However, the
observed growth retardation phenotypes in the SlDDF2-IOE lines are similar to previous
phenotypes reported in rd29A:DREB1A transgenic tobacco [35,36] and rd29A:AtCBF3 potato
plants, in which growth retardation phenotypes were observed. The differences in growth
retardation phenotypes between the two transgenic lines can be explained by the higher
expression levels in SlDDF2-IOE#9, as reported previously by Pino et al. [36]. Addition-
ally, the observed behaviors were comparable to those found in transgenic tomato plants
with constitutive overexpression of SlDREB, which has been linked to reduced internode
elongation due to lower gibberellin levels [26].
Under water deficit conditions, the transgenic plants were found to have darker green
leaf color, which was associated with increased chlorophyll a, chlorophyll b, and total
chlorophyll pigments concentrations in comparison with the wild-type plants (Figure 4).
 SlDDF2-IOE#9, as reported previously by Pino et al. [36]. Additionally, the observed
behaviors were comparable to those found in transgenic tomato plants with constitutive
overexpression of SlDREB, which has been linked to reduced internode elongation due to
lower gibberellin levels [26].
Horticulturae 2022, 8, 230 Under water deficit conditions, the transgenic plants were found to have darker 9 of 13
green leaf color, which was associated with increased chlorophyll a, chlorophyll b, and
total chlorophyll pigments concentrations in comparison with the wild-type plants (Fig-
ure
The 4). The of
levels levels
Chla,ofChlb,
Chla,andChlb, andchlorophyll
total total chlorophyll were increased
were increased only water
only under under deficit
water
deficit stress conditions compared with the nonstressed groups. These
stress conditions compared with the nonstressed groups. These findings are in general findings are in
general
agreement agreement
with Li with
et al.Li[26]
et al.
and[26] and Al-Abdallat
Al-Abdallat et al.who
et al. [16], [16], observed
who observed increased
increased total
total chlorophyll
chlorophyll pigments
pigments in transgenic
in transgenic tomatooverexpressing
tomato plants plants overexpressing DREB
DREB genes, genes,
which was
which waspreviously
attributed attributed previously
to reduced to GAreduced
levels inGA levels in lines
transgenic transgenic
[37]. lines [37].

Figure 4. (A) Total

Total chlorophyll
chlorophyll(green),
(green),Chlorophyll
Chlorophylla a(blue),
(blue),and
andChlorophyll
Chlorophyll b (yellow)
b (yellow) contents
contents in
in
MM MM and
and SIDDF2-IOE#6
SIDDF2-IOE#6 andand SIDDF2-IOE#9
SIDDF2-IOE#9 transgenic
transgenic plants
plants in response
in response to water
to water withholding
withholding for
for seven
seven days
days orstress
or no no stress conditions;
conditions; (B) detached
(B) detached leavesleaves
from from stressed
stressed and nonstressed
and nonstressed wild-type
wild-type plants
plants and the two transgenic lines plants. Values are the means ± SD. Different lower-case
and the two transgenic lines plants. Values are the means ± SD. Different lower-case letters indicate letters
indicate a significant difference between transgenic and wild-type plants under nonstressed con-
a significant difference between transgenic and wild-type plants under nonstressed conditions, and
ditions, and different capital letters indicate a significant difference between transgenic and
different capital letters indicate a significant difference between transgenic and wild-type plants
wild-type plants under stress conditions (p < 0.05).
under stress conditions (p < 0.05).

The physiological behavior of the transgenic plants under water deficit stress was
compared with MM plants. Initially, the relative water content was measured immediately
after detaching leaves from each plant. As shown in Figure 5, MM plants exposed to stress
conditions showed lower RWC than the transgenic lines, which indicates a better water
status in cells of transgenic lines. To investigate the effect of water deficit on water loss rate
(g·h−1 ·g−1 DW), fully expanded wild-type and transgenic-line leaves from both treatments
were detached and subjected to dehydration for 2 h. The results showed that the water loss
rate from the stressed transgenic lines was lower than that in control and stressed wild-type
plants (Figure 5). These results suggest that the drought resistance of the transgenic plants
overexpressing the SlDDF2 gene was improved, compared with MM plants. It has been
reported that transgenic tomato plants overexpressing DREB genes showed enhanced
drought tolerance by maintaining higher water content and reduced water loss rate [38].
To analyze the impact of water deficit on physiological responses of SlDDF2 transgenic
lines, two-weeks old transgenic and MM (included as control) seedlings were grown
under stress conditions for 10 days by water withholding and observed for their growth
and wilting behaviors at the end of treatment. Under drought stress conditions, the
majority of MM plants were welted (60% survival rate), and an obvious, adverse effect
was observed, while transgenic tomato lines showed enhanced tolerance to water deficit
stress and showed a delayed wilting behavior and higher survival rate when compared
with MM plants (Figure 6B). For salinity stress tolerance, SlDDF2-IOE#9 transgenic plants
displayed improved tolerance to high salt stress (survival rate 50%), followed by SlDDF2-
IOE#6 plants (survival rate 50%), while MM wild-type plants suffered severely from
salinity stress (Figure 6C). For cold stress tolerance, the survival rate of the wild-type
plants was 10%, whereas the SlDDF2-IOE transgenic plants showed enhanced tolerance
to cold stress and higher survival rate when compared with wild-type plants, with 45%
and 75% for SlDDF2-IOE#6 and the SlDDF2-IOE#9, respectively (Figure 6D). These results
 ately after detaching leaves from each plant. As shown in Figure 5, MM plants exposed to
stress conditions showed lower RWC than the transgenic lines, which indicates a better
water status in cells of transgenic lines. To investigate the effect of water deficit on water
loss rate (g·h−1·g−1 DW), fully expanded wild-type and transgenic-line leaves from both
Horticulturae 2022, 8, 230 10 of 13
treatments were detached and subjected to dehydration for 2 h. The results showed that
the water loss rate from the stressed transgenic lines was lower than that in control and
stressed wild-type plants (Figure 5). These results suggest that the drought resistance of
suggested that the overexpression of the SlDDF2 gene improved drought, cold, and salt
the transgenic plants overexpressing the SlDDF2 gene was improved, compared with
stresses tolerance in tomato plants. In line with our results, the overexpression of SlDREB2
MM plants.
enhanced It has been
Arabidopsis reported
and tomato that transgenic
tolerance tomato
to salinity stress plants
(125 mm overexpressing
NaCl) [31], while the DREB
genes showed enhanced drought tolerance by maintaining higher
stress-inducible overexpression of Arabidopsis CBF1 in transgenic tomato plants water content and
improved
reduced
tolerancewater
againstloss
lowrate [38].
temperatures, water-deficit, and high salt treatments [34].

Figure
Figure5.5. Relative watercontent
Relative water contentofof leaves
leaves of MM
of MM andand SIDDF2-IOE#6
SIDDF2-IOE#6 and SIDDF2-IOE#9
and SIDDF2-IOE#9 transgenic
transgenic
lines
linesafter
after 10 days
days ofof(A)
(A)water
water withholding
withholding andand (B) water
(B) water loss as
loss rate, rate, as measured
measured by decrease
by decrease in in
fresh
freshweight aftertwo
weight after twohours
hoursinin detached
detached leaves
leaves fromfrom
MM MM and SIDDF2-IOE#6
and SIDDF2-IOE#6 and SIDDF2-IOE#9
and SIDDF2-IOE#9
transgenic
transgenic plants. Valuesare
plants. Values arethe
the means
means ± SD.
± SD. Different
Different lower-case
lower-case lettersletters indicate
indicate a significant
a significant
difference
difference between transgenicand
between transgenic and wild-type
wild-type plants
plants underunder nonstressed
nonstressed conditions,
conditions, and different
and different
capital
capitalletters indicateaasignificant
letters indicate significant difference
difference between
between transgenic
transgenic and wild-type
and wild-type plants plants under stress
under stress
conditions
conditions (p < 0.05).
(p < 0.05).

To analyze the impact of water deficit on physiological responses of SlDDF2 trans-

genic lines, two-weeks old transgenic and MM (included as control) seedlings were
grown under stress conditions for 10 days by water withholding and observed for their
growth and wilting behaviors at the end of treatment. Under drought stress conditions,
the majority of MM plants were welted (60% survival rate), and an obvious, adverse ef-
fect was observed, while transgenic tomato lines showed enhanced tolerance to water
 wild-type plants, with 45% and 75% for SlDDF2-IOE#6 and the SlDDF2-IOE#9, respec-
tively (Figure 6D). These results suggested that the overexpression of the SlDDF2 gene
improved drought, cold, and salt stresses tolerance in tomato plants. In line with our
results, the overexpression of SlDREB2 enhanced Arabidopsis and tomato tolerance to
Horticulturae 2022, 8, 230
salinity stress (125 mm NaCl) [31], while the stress-inducible overexpression of 11Ara-
of 13
bidopsis CBF1 in transgenic tomato plants improved tolerance against low temperatures,
water-deficit, and high salt treatments [34].

Figure 6. Representative MM and SIDDF2-IOE#6 and SIDDF2-IOE#9 transgenic lines grown under
Figure 6. Representative MM and SIDDF2-IOE#6 and SIDDF2-IOE#9 transgenic lines grown under
(A) normal conditions, (B) water deficit, (C) salinity, and (D) cold stresses (percentages are describing
(A) normal conditions, (B) water deficit, (C) salinity, and (D) cold stresses (percentages are de-
survivalsurvival
scribing rate outrate
of 20out
plants
of 20per treatment).
plants per treatment).
4. Conclusions
4. Conclusions
The SlDDF2 gene was identified in tomato plants, and the phylogenetic analysis
The SlDDF2
clustered gene
it with the was identified
DREB1 in tomato
family, indicating plants, and
a potential rolethe phylogenetic
in abiotic analysis
stress tolerance.
clustered it with
Furthermore, theexpression
gene DREB1 family, indicating
analysis of SlDDF2 a potential role in abiotic
showed inducible stress tolerance.
expression patterns in
Furthermore, gene expression analysis of SlDDF2 showed inducible expression
response to multiple abiotic stresses including cold, salinity, and drought. Stress-induciblepatterns
inoverexpression
response to ofmultiple abiotic stresses including cold, salinity, and
the SlDDF2 gene in tomato plants enhanced tolerance against different drought.
Stress-inducible
abiotic stresses overexpression
when comparedofwith the SlDDF2
MM plants,genewith
in tomato plants enhanced
clear pleotropic effectstolerance
observed
against
on them. The identified stress-related SlDDF2 gene could be a useful tool pleotropic
different abiotic stresses when compared with MM plants, with clear for tomato
effects observedand
improvement on tolerance
them. Theunder
identified stress-related
abiotic SlDDF2 gene could be a useful tool
stress conditions.
for tomato improvement and tolerance under abiotic stress conditions.
Author Contributions: T.A.-D. and A.M.A.-A. conceived and had designed the experiments, ana-
Author Contributions:
lyzed the T.A.-D.
data, and wrote and A.M.A.-A.
the manuscript. conceived
R.A.-S. helped inand had designed
molecular the experiments,
work, RNA ana-
extraction, relative
lyzed
gene the data, andand
expression, wrote theanalysis.
data manuscript. R.A.-S.
M.A.G., helpedand
N.E.-A. in molecular work, RNA
H.A.-D. helped extraction,
in stress rela-
experiments,
tive gene expression,
physiological and dataand
measurements, analysis. M.A.G., N.E.-A.
data analysis. andedited
All authors H.A.-D.
andhelped in stress
provided experiments,
a critical review of
the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded by the Deanship of Scientific research/The University of Jordan
(Grants Number: 1014).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets supporting the results of this article are freely available
upon reasonable request from A.M.A.-A.
Acknowledgments: We sincerely thank Jamal Ayad and Shireen Qasrawi, for their technical assis-
tance. We also gratefully acknowledge the financial support of the Deanship of Scientific research/The
University of Jordan to A.M.A.-A.
Conflicts of Interest: The authors declare no conflict of interest.
Horticulturae 2022, 8, 230 12 of 13

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