Functional characterization of plant specific Indeterminate Domain (IDD) transcription factors in tomato (Solanum lycopersicum L.)

Abstract

Plant-specific transcription factors (TFs) are responsible for regulating the genes involved in the development of plant-specific organs and response systems for adaptation to terrestrial environments. This includes the development of efficient water transport systems, efficient reproductive organs, and the ability to withstand the effects of terrestrial factors, such as UV radiation, temperature fluctuations, and soil-related stress factors, and evolutionary advantages over land predators. In rice and Arabidopsis, INDETERMINATE DOMAIN (IDD) TFs are plant-specific TFs with crucial functions, such as development, reproduction, and stress response. However, in tomatoes, IDD TFs remain uncharacterized. Here, we examined the presence, distribution, structure, characteristics, and expression patterns of SlIDDs. Database searches, multiple alignments, and motif alignments suggested that 24 TFs were related to Arabidopsis IDDs. 18 IDDs had two characteristic C2H2 domains and two C2HC domains in their coding regions. Expression analyses suggest that some IDDs exhibit multi-stress responsive properties and can respond to specific stress conditions, while others can respond to multiple stress conditions in shoots and roots, either in a tissue-specific or universal manner. Moreover, co-expression database analyses suggested potential interaction partners within IDD family and other proteins. This study functionally characterized SlIDDs, which can be studied using molecular and bioinformatics methods for crop improvement.

Full text

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Functional characterization
of plant specic Indeterminate
Domain (IDD) transcription factors
in tomato (Solanum lycopersicum
L.)
Sujeevan Rajendran
1,6, Yu Mi Kang
2,6, In Been Yang
1, Hye Bhin Eo
1, Kyung Lyung Baek
1,
Seonghoe Jang
3, Assaf Eybishitz
4, Ho Cheol Kim
1, Byeong Il Je
2, Soon Ju Park
5 &
Chul Min Kim
1*
Plant-specic transcription factors (TFs) are responsible for regulating the genes involved in
the development of plant-specic organs and response systems for adaptation to terrestrial
environments. This includes the development of ecient water transport systems, ecient
reproductive organs, and the ability to withstand the eects of terrestrial factors, such as UV
radiation, temperature uctuations, and soil-related stress factors, and evolutionary advantages over
land predators. In rice and Arabidopsis, INDETERMINATE DOMAIN (IDD) TFs are plant-specic TFs with
crucial functions, such as development, reproduction, and stress response. However, in tomatoes, IDD
TFs remain uncharacterized. Here, we examined the presence, distribution, structure, characteristics,
and expression patterns of SlIDDs. Database searches, multiple alignments, and motif alignments
suggested that 24 TFs were related to Arabidopsis IDDs. 18 IDDs had two characteristic C2H2 domains
and two C2HC domains in their coding regions. Expression analyses suggest that some IDDs exhibit
multi-stress responsive properties and can respond to specic stress conditions, while others can
respond to multiple stress conditions in shoots and roots, either in a tissue-specic or universal
manner. Moreover, co-expression database analyses suggested potential interaction partners within
IDD family and other proteins. This study functionally characterized SlIDDs, which can be studied
using molecular and bioinformatics methods for crop improvement.
Keywords Tomato, Transcription factor, Indeterminate Domain, Development, Stress response
In the past ve decades, the global population has increased by four billion and is predicted to increase rapidly
from the current eight billion individuals1. e reduction of arable land and the water crisis in agriculture will
be a great challenge in the future2. Climate change projections indicate that intense rains will cause oods and
long droughts, reducing cultivation periods in the future3. Increase in global population, reduction in arable
land, and reduction in cultivation periods will exponentially increase the need for intensive farming methods
and new crop varieties. Currently, widespread plant breeding methods are likely to limit yield limitation in the
near future. erefore, plant breeders are obliged to discover new tools and principles to increase crop yield.
Owing to their sessile nature, plants have evolved to withstand and counteract biotic and abiotic stress4,5. Stress
signals from unfavorable conditions, such as temperature, waterlogging, drought, oxidative stress, proton stress,
heavy metals, salinity, light, viruses, bacteria, fungi, and insects are perceived by receptor complexes, and the
perceived signals are transduced to TFs to activate stress response genes6,7. TFs interact with the cis-regulatory
elements of a target gene and modulate its expression of their target genes8. Changes in cis-regulatory elements
OPEN
1Department of Horticulture Industry, Wonkwang University, Iksan 54538, Republic of Korea. 2Department
of Horticultural and Life Science, Pusan National University, Milyang 50463, Korea. 3World Vegetable Center
Korea Oce (WKO), Wanju-gun, Jeollabuk-do 55365, Republic of Korea. 4World Vegetable Center, P.O. Box 42,
Tainan 74199, Shanhua, Taiwan. 5Division of Applied Life Science (BK21 Four), Plant Molecular Biology and
Biotechnology Research Center (PMBBRC), Gyeongsang National University, Jinju, Korea. 6
These authors
contributed equally: Sujeevan Rajendran and Yu Mi Kang. *email: chulmin21@wku.ac.kr
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result in alterations in target gene expression, which can alter cellular activities9–12. TFs sequences specically
bind to transcription factor binding motifs (TFBMs) to activate or repress downstream genes with a DNA-binding
domain13,14. TFs also contain oligomerization, transcription, and nuclear localization domain15. Changes in the
domain architecture of TFs can be a driving force in plant evolution and changes in the expression can result
in morphological variations16,17. Plant-specic TFs regulate genes related to the development of plant-specic
organs and response systems for adaptation to terrestrial environments18. ese include the development of
ecient water transport systems, ecient reproductive organs, the ability to withstand the eects of terrestrial
factors such as UV radiation, temperature uctuations, soil-related stress factors, and evolutionary advantages
over land predators19–21. INDETERMINATE DOMAIN (IDD) TFs are plant-specic TFs with crucial functions
in rice and Arabidopsis, including development, reproduction and stress response22–28.
Among the vast array of TFs, IDD, a class of C2H2 zinc-nger TFs, is specic to plants22,25,29,30. e N-termi-
nus of the IDD contains two C2H2 DNA-binding domains and 2C2HC protein-binding domains. e C-terminus
also contains protein interaction domain24,25,29. In Arabidopsis, 12 of 18 identied IDD TFs have been charac-
terized for their roles. IDDs in Arabidopsis are involved in various cellular and developmental functions such
as seed germination, tissue patterning, responses to external cues, and abiotic stress. Some IDDs can produce
transcript variants, depending on the conditions (see review)22.
Tomatoes (Solanum lycopersicum) are one of the most cultivated crops in the fresh and processed market.
Owing to its relatively small genome size and chromosomal architecture, the tomato is also an excellent model
plant for studying Solanaceae species31,32. Tomatoes also bear berry fruits, which can be used as models for
studying fruit development and metabolite analysis33–35. Studies on the abiotic and biotic responses in tomatoes
have been widely conducted. To understand the IDD family genes in tomato (SlIDDs), this study was conducted
to identify and explore the basic information of SlIDDs and to understand their expression dynamics under
developmental stages and stress conditions in tomato.
Results
Identication and phylogenetic analysis of SlIDD family genes in tomato
To identify candidate SlIDD family genes, a BLAST search was conducted using Gramene (https:// www. grame
ne. org) and Plaza (https:// bioin forma tics. psb. ugent. be/ plaza) databases. Overall, 25, 24, and 20 genes were iden-
tied by search results in tomato, rice, and Arabidopsis respectively. Arabidopsis and rice have 16 and 15 IDD
genes, respectively. e evolutionary relationships among IDD family genes were determined using phylogenetic
analysis. Phylogenetic analysis suggested that IDD genes may have structural dierences between monocots and
dicots (Fig.1a). Four subgroups of IDD-like genes have been identied in tomato plants. Here, 16 Arabidopsis
and 15 rice IDD genes were clustered with 19 tomato IDD-like genes. Among these clades, rice Ehd2 showed the
lowest homology with other IDD genes. 12 genes clustered with the AtSTOP1 group and seven genes showed
distant homology with IDD genes (Fig.1b).
Structure and distribution of SlIDD genes
Multiple sequence alignments showed conserved C2H2 and C2HC motifs among SlIDD genes (Fig.2a, Fig.S1).
However, Solyc03g098070 does not possess the rst C2H2 motif. Solyc05g054030 possesses a C2HR motif in
the second zinc nger domain with a less reactive arginine stead of Histidine36 (Fig.2a). seven ortholog groups
were found within tomato IDD-like genes. Block 6 and block 7 contained four and three orthologs respec-
tively (Fig.2b and TableS1). Among them Solyc01g005060, Solyc04g080130, Solyc04g008500, Solyc05g054030,
Solyc07g053570, Solyc08g063040 and Solyc09g065670 did not show orthologs. Synteny between Arabidopsis,
rice and tomato revealed that the rice IDD family showed the least synteny when compared with Arabidopsis
and tomato (Fig.S2). Sequences with two complete C2H2 complete C2HC domains were considered as true IDD
TFs. Aer conrming the number of IDD genes in tomatoes, the distribution of IDD genes were determined
(Fig.2c, TableS2). Twelve IDDs showed synteny, indicating duplication events in all chromosomes. Dispersed
duplications were accounted for the majority (80%) of IDD like genes and other genes were duplicated by seg-
mental duplication events (TableS3). Solyc03g098070, which lacks the rst C2H2 motif, exhibited synteny with
Solyc06g072360. 18 conrmed IDD genes were distributed among 11 chromosomes, excluding chromosome 12.
Motif analysis revealed structural variations among IDD-like genes. Among the 25 sequences, 18 IDD TFs had
four prominent motifs corresponding to two C2H2 and two C2HC domains in the C-terminus. Other IDD-like
genes lacked one or more zinc-nger domains (Fig.2d and Fig.S3). Ka/Ks analysis revealed that all IDD genes
evolved under high selection pressure (TableS4). In addition to the primary isoforms of IDD TFs, our analyses
revealed that multiple IDDs have splice variants. IDD1, IDD2, IDD12 and IDD13 showed two isoforms. Sur-
prisingly, IDD4 and IDD11 had three and ve isoforms, respectively, indicating complex post-transcriptional
regulatory mechanisms present in IDD TFs (TableS5).
3D structure of SlIDD TFs
Following the identication of SlIDD TFs, 3D structures were predicted using AlphaFold2.0 (https:// alpha fold.
ebi. ac. uk) to verify the structural similarity of the conrmed TFs using BLAST with UniProt (https:// www.
unipr ot. org) accession numbers (TableS6). e 3D structures showed prominent zinc nger domains in the C
terminus regions of the primary isoforms. However, Solyc03g098070 had only three zinc nger domains, which
conrmed the results from motif analysis and multiple alignments, and Solyc08g063040 showed an incomplete
4th C2HC domain, even though the alignments and motifs were intact (Fig.3).
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Cis-regulatory element analysis of SlIDD promoters
e promoter sequences of 18 SlIDD genes (3000bp) were scanned for cis-regulatory elements using Arabidopsis
DAP motifs with a cut-o p-value of 1 × 10−4. A total of 518 binding elements were present in all 18 promoter
sequences, and VRN1, REM19, and DOF4.7 binding elements were relatively more enriched (14.65%) than other
promoters (Fig.4a, b and TableS7). Most of the enriched elements showed functions related to environmental
signal response and development (Fig.4c).
Interaction networks of SlIDDs
Coexpression network analysis revealed complex interactions between SlIDDs and STOP-like TFs. Unlike tissue
expression patterns, co-expression networks suggested possible dierences in temporal expression patterns and
provided clues to gene regulation networks in dierent tissues (Fig.5). SlIDD4 showed close association with
SlIDD2 similar to the tissue-specic expressions. However, SlIDD12 did not interact with other SlIDDs, including
SlIDD15, SlIDD16, SlIDD17, and SlIDD18. SlIDD3 showed multiple interactions with the other SlIDDs. SlIDD10
interacted with SlIDD2, SlIDD7, and SlIDD11; however, the tissue-specic expression patterns were distant from
those of SlIDD11. SlIDD13 and SlIDD14 interacted with SlIDD2 but showed similar expression patterns in tissues.
Compared to other interactions, there are less data on SlIDDs, therefore, databases have shown that SlIDDs are
co-expressed with TFIIIA and SlkdsA.
Expression of IDD TFs under abiotic stresses
Cis-regulatory analysis suggests that the binding elements in the promoters are highly responsive to environmen-
tal signals. Moreover, SlIDD1, SlIDD8, SlIDD9, and SlIDD16 show increased expression under various abiotic
Figure1. Phylogenetic analyses of IDD family genes in major plant species. (a) Unrooted phylogenetic tree
of IDD family genes in ten major plant species. Red branches indicate monocots and green indicate dicots. (b)
Phylogenetic tree of IDD family genes in rice, Arabidopsis, and maize. Tomato sequences are indicated in bold
letters [branch values (MYA)].
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100|....:.110|....:.120|....:.130|....:.140|....:.150|....:.160|....:.170|....:.180|....:.190|....:.200|....:.210|....:.220|....:.230|....:.240|
FVCEICSKGFQRDQNLQLHRRGHNLPWKLRQRSSN-----EVK-KRVYVCPESSCVHHDPSRALGDLTGIKKHFCRKHGE-KKWKCDKCSKKYAVQS-----DLKAHSKICGTREYKCDCGTLFSRRDSFITHRAFCDALA
FVCEICNKGFQRDQNLQLHRRGHNLPWKLRQRSSN-----EVR-KRVYVCPETTCVHHDPSRALGDLTGIKKHFCRKHGE-KKWKCDKCSKKYAVQS-----DLKAHLKICGTKEYKCDCGTMFSRRDSFITHRAFCDVLA
FVCEICNKGFQRDQNLQLHRRGHNLPWKLRQRTSN-----EVK-KRVYVCPESSCVHHDPSRALGDLTGIKKHFCRKHGE-KKFKCERCTKKYAVHS-----DWKAHMKTCGTREYRCDCGTLFSRRDSFITHRAFCDALA
FICEICNKGFQRDQNLQLHRRGHNLPWKLKQRNKN-----EVIKKKVYICPEKTCIHHDPSRALGDLTGIKKHFSRKHGE-KKWKCEKCSKKYAVQS-----DWKAHTKICGTREYKCDCGTLFSRKDSFITHRAFCDALA
FVCEICNKGFQRDQNLQLHRRGHNLPWKLRQRTTA-----EVK-KRVYICPEPTCVHHNPSRALGDLTGIKKHYSRKHGE-KKWKCDKCSKKYAVQS-----DWKAHQKTCGTREYKCDCGTIFSRRDSFITHRAFCDALA
FLCEICGKGFQRDQNLQLHRRGHNLPWKLKQRTSK-----EVR-KRVYVCPEKVCVHHHPSRALGDLTGIKKHYCRKHGE-KKWKCDKCSKRYAVQS-----DWKAHSKTCGTREYRCDCGTIFSRRDSFVTHRAFCDALA
FVCEICNKGFQRDQNLQLHRRGHNLPWKLKQRTSK-----EIR-KKVYVCPETSCVHHDPARALGDLTGIKKHFCRKHGE-KKWKCEKCSKRYAVQS-----DWKAHSKTCGTKEYRCDCGTLFSRRDSFITHRAFCDALA
FVCEICNKGFQRDQNLQLHRRGHNLPWKLKQRSNK-----EVK-KKVYVCPEASCVHHHPSRALGDLTGIKKHFCRKHGE-KKWKCEKCSKRYAVQS-----DWKAHSKICGTREYRCDCGTLFSRRDSFITHRAFCDALT
FFCEICNKGFQRDQNLQLHRRGHNLPWKLKKRENK-----EVVRKKVYICPESSCVHHDPSRALGDLTGIKKHFSRKHGE-KKWKCEKCSKRYAVQS-----DCKAHFKTCGTREYKCECGTIFSRRDSFITHRAFCETLA
FICEICNKGFQRDQNLQLHRRGHNLPWKLKQRNKQ-----EIVKKKVYICPEKSCVHHDPSRALGDLTGIKKHFSRKHGE-KKWKCEKCSKKYAVQS-----DWKAHTKTCGTREYKCDCGTLFSRKDSFITHRAFCDALA
FVCEICNKGFQRDQNLQLHRRGHNLPWKLKQRTNK-----ENK-KKAYVCPEPTCVHHHPSRALGDLTGIKKHFCRKHGE-KKWKCDKCSKIYAVQS-----DWKAHSKTCGTKEYRCDCGTLFARKDSFVTHRAFCDALA
FICEICNKGFQRDQNLQLHRRGHNLPWKLKQRNKL-----EQVKKKVYICPEKTCIHHDPSRALGDLTGIKKHFSRKHGE-KKWKCEKCSKKYAVQS-----DWKAHSKTCGTREYKCDCGTLFSRKDSFITHRAFCDALA
FICEVCNKGFQREQNLQLHRRGHNLPWKLKQKTSN-----EIK-KRVYICPESSCIHHNPSRALGDLTGIKKHFSRKHGE-KKWKCEKCSKKYAVQS-----DWKAHSKTCGTKEYKCDCGTIFSRRDSFVTHRAFCDALA
FLCEVCNKGFQREQNLQLHRRGHNLPWKLKQKNTK-----EVAKRKVYLCPEPTCVHHEPSRALGDLTGIKKHYFRKHGE-KKFKCEKCSKKYAVQS-----DWKAHTKTCGTREYRCDCGTLFSRRDSFITHRAFCDALV
FVCEICNKGFQRDQNLQLHRRGHNLPWKLKQRNNK-----EVIKKKVYICPEKSCVHHDPSRALGDLTGVKKHYSRKHGE-KKWKCEKCSKKYAVQS-----DWKAHSKICGTREYKCDCGTLFSRKDSFITHRAFCDALA
YICEICNQGFQRDQNLQMHRRRHKVPWKLLKRETP------IVKKRVFVCPEPTCLHHDPCHALGDLVGIKKHFRRKHSNHKQWVCEKCNKGYAVQS-----DYKAHLKTCGTRGHSCDCGRVFSRVESFIEHQDACSFGR
YICDICDQGFQRDQNLQMHRRRHKVPWKLVKREI------E-VKKRVFVCPEPSCLHHDPCHALGDLVGIKKHFRRKHSDNKQWICDKCGKAYAVQS-----DYKAHLKTCGTRGHSCDCGRVFSRVESFIEHQDSCTIRR
YVCEICNLSFQREQNLQMHRRRHKVPWKLKKKEEEKNEMDQVIKKRVYVCPEPSCVHHDPCHALGDLVGIKKHFRRKRSNYKQWICQKCNKGYAVQS-----DYKAHIKTCGTRGHSCDCGRVFSRVETFIEHQDSCKPQS
-----------------MHRRRHKVPWKLLKRETP------IVRKRVFVCPEPTCLHHDPCHALGDLVGIKKHFRRKHSNHKQWVCEKCSKGYAVQS-----DYKAHLKTCGTRGHSCDCGRVFSRVESFIEHQDACSMGR
VPCELIDRIARLKHGGTYKVLKNLLKHKLVHHDSSK----YDGFRLTYLGYDFLAIKTLVNRGMFNGVGRQIGVGKESDIFEVVKEDGTVLAMKLHRLG-RVSFRAVKSKRDYLRHRSSYNWLYLSRLAALKEFAFMKALE
QLQELVHLIVGRRGQDEVQGNDLIVQQQQLITADLT-------SIIVQLISTAGSLLPTMKYTLSSAIPAASQLGQVGGVTVPSTAGTSAG--------------GLTCNDGVAKLEDQSNHIDQLRDCGIEHNHAADEHE
SLQKFLSDSVNSNTLLGQHQMN-------MVSDEIT-------SAIHQIIVNGAALLSSAQLTNPPPPPPPPPR---------------------------------PSSSAELKINLKSNHKRSFPEF--------DRRD
NVGAFSLQTRRPNAHGGLHTRPNSSALQPLSNRTQK---FANHIRASPLEEWEGRIKVGMSNSVTTAIRGSVRDMAIGKTKTTEKADRATVEQAIDPR----TRMVLFKMLNRGVFHDINGCISTGKEANVYHATKADGQE
QVQSLATMFITPDNQTIIHPPPESIS---MIIANMG-------TLIQEIITTSSSLMFSCQKIVLDSTSLSQNSSRYREPSQ-NDVGHGQGQGQVDHLLQDYDWYVDNYNSNCNTHEDNKNHVTSSSTIIASSTISHDNYY
QLRELVRIIVDHRSLAGIQGSDLSIQQQQLITADLT-------SIIIQLISTAGSLLPTVKHQAN---PPTKRLEQFGGASVPSETGTNIG--------------ALTCNGYVPKAKDQLSHVDQMGDC------FVDEHE
Solyc02g085580_
SlIDD1
Solyc02g062940_SlIDD17
Solyc04g080130_SlIDD18
Solyc06g075250_SlIDD7
Solyc09g074780_SlIDD3
Solyc07g053570_SlIDD8
Solyc03g121660_SlIDD11
Solyc08g063040_SlIDD16
Solyc06g062670_SlIDD2
Solyc09g007550_SlIDD13
Solyc09g065670_SlIDD15
Solyc10g084180_SlIDD14
Solyc01g099340_SlIDD10
Solyc04g008500_SlIDD4
Solyc11g069240_SlIDD6
Solyc06g072360_SlIDD9
Solyc01g005060_SlIDD12
Solyc05g054030_SlIDD5
Solyc03g098070
Solyc01g007120
Solyc11g017140
Solyc04g056320
Solyc08g080850
Solyc11g066420
Solyc06g065440
Tree scale: 1
MotifSymbol Motif Consensus
1. VIALSPKTLLATNRFVCEICNKGFQRDQNLQLHRRGHNLPW
2. HGEKKWKCEKCSKKYAVQSDW
3. RCDCGTLFSRRDSFITHRAFCDA
4. QRSKEEVRKRVYVCPEPTCVHHDPSRALGDLTGIKKHFCRK
5. AAPSPHMSATALLQKAAQMGATTSSSS
6. SAPPAKKKRNLPGNPDPDAE
a
d
c
0 Mb
9 Mb
18 Mb
27 Mb
36 Mb
45 Mb
54 Mb
63 Mb
72 Mb
81 Mb
90 Mb
SL4.0Ch01
Solyc01g005060.3.1
Solyc01g099340.3.1
SL4.0Ch02
Solyc02g085580.4.1
Solyc02g062940.3.1
SL4.0Ch03
Solyc03g121660.3.1
Solyc03g098070.3.1
SL4.0Ch04
Solyc04g008500.4.1
Solyc04g080130.3.1
SL4.0Ch05
Solyc05g054030.3.1
SL4.0Ch06
Solyc06g072360.3.1
Solyc06g062670.3.
1
Solyc06g075250.3.
1
SL4.0Ch07
Solyc07g053570.4.1
SL4.0Ch08
Solyc08g063040.4.1
SL4.0Ch09
Solyc09g007550.3.1
Solyc09g065670.3.1 Solyc09g074780.3.1
SL4.0Ch10
Solyc10g084180.2.1
SL4.0Ch11
Solyc11g069240.2.1
SL4.0Ch12
SL4.0ch01
SL4.0ch02
SL4.0ch03
SL4.0ch04
SL4.0ch05
SL4.0ch06
SL4.0ch07
SL4.0ch08
SL4.0ch09
SL4.0ch10
SL4.0ch11
SL4.0ch12
Solyc01g099340.3.1
Solyc02g062940.3.1
Solyc02g085580.4.1
1
.3.070890g30cyloS
Solyc03g121660.3.1
Solyc06g065440.1.1
Solyc06g075250.3.1
Solyc06g072360.3.1
Solyc06g062670.3.1
Solyc09g007550.3.1
Solyc09g074780.3.1
Solyc11g017140.3.1
Solyc11g069240.2.1
Solyc10g084180.2.1
b
Figure2. Comparison, conrmation, and distribution of tomato IDD sequences. (a) Multiple alignment of
IDD-like genes in tomato. (green; cystine motifs, Blue; histidine motifs, Red; histidine–cystine motifs. Red bold
R indicates the arginine in the C2HR motif). (b) e synteny analysis of the SlIDD family in tomato. e genes
linked by red lines represent homologs. (c) Distribution of 18 IDD TFs in tomato chromosomes (Black lines
indicate synteny). (d) Phylogenetic relationship and gene structure of 18 conrmed IDD genes (le) and protein
motifs in corresponding sequences (right).
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Figure3. 3D models of tomato IDD TFs predicted by AlphaFold2.0. Light blue chains show zinc nger
domains. 2D images were taken for visibility of zinc nger domains (see TableS6 for AlphaFold2.0 accession
numbers to access 3D models).
Solyc04g008500
Solyc09g065670
Solyc09g074780
Solyc01g099340
Solyc10g084180
Solyc07g053570
Solyc04g080130
Solyc02g062940
Solyc05g054030
Solyc01g005060
Solyc06g075250
Solyc06g062670
Solyc06g072360
Solyc08g063040
Solyc11g069240
Solyc09g007550
Solyc02g085580
Solyc03g098070
0 300 600 900 1200 1500 1800 2100 2400 2700 3000 3300
5' 3'
FRS9
VRN1
BPC5
BPC1
AT5G60130
AT5G02460
CDF3
AT1G69570
AT2G20110
REM19
TCX2
At4g38000
SOL1
AT5G66940
OBP3
AT2G28810
Adof1
ATHB23
AT1G04880
COG1
1
1
2
2
4
5
16
21
84
86
92
0102030405060708090100
Localization (GO:0051179)
Rhythmic process (GO:0048511)
Reproductive process (GO:0022414)
Reproduction (GO:00 00003)
Signaling (GO:0023052)
Multicellular organi smal process (GO:0032501)
Developmental process (GO:0032502)
Response to stimulus (GO:0050896)
Metabolic process (GO:0008152)
Biological re gulation (GO:0065007)
Cellular process (GO:0009987)
0500 1000 150
02
00
0
At1g76110
AT2G17410
TSO1
AT3G52440
MYB1
ATHB24
AT1G47655
At3g45610
OBP4
At1g64620
AT2G28810
COG1
ATHB23
CDF3
BPC1
Adof1
BPC5
TCX2
AT1G04880
AT2G20110
SOL1
AT5G60130
AT5G02460
FRS9
AT1G69570
AT5G66940
OBP3
At4g38000
REM19
VRN1
Number of binding mo tifs
Predicted binding elements
ab
c
Figure4. Cis-regulatory element analysis of SlIDD family genes. (a) Promoter binding sites of tomato IDD TFs.
(b) Enriched promoter binding elements in tomato IDD TFs (50% of enriched elements). (c) GO term analysis
for enriched promoter elements.
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stress conditions37,38. To conrm whether other SlIDDs were also responsive to abiotic stress, 3-week-old plants
grown under greenhouse nursery conditions were subjected to salt, pH, and ood stress, which represent the
basic stress conditions that can occur under greenhouse conditions (see Materials and Methods). Expression
analysis was conducted to determine the expression level of each SlIDD TFs.
Expression of SlIDD TFs under salt stress
Salt stress can aect plants by restricting water and nutrient uptake, resulting in reduced root biomass and
reduced productivity39. Nutrient imbalance owing to NaCl-induced conductivity stress reduces fruit quality
under greenhouse conditions40. To determine the expression patterns of SlIDD TFs, 3-week-old tomato seedlings
were treated with 200mM NaCl and sampled at 2- and 24h intervals.
Expression analysis revealed that the levels of multiple SlIDDs were upregulated in the roots under salt stress
conditions (Fig.6a). SlIDD12 and SlIDD14 showed over 100- and 30-fold increases in expression, respectively,
whereas SlIDD3, SlIDD4, and SlIDD9 showed only signicant increases in expression. Other SlIDDs such as
SlIDD1 and SlIDD2 showed signicantly reduced expression. SlIDD4 and SlIDD13 were upregulated aer 24h
of treatment, whereas SlIDD6 showed increased expression only aer 2h. However, in the shoots, SlIDD12 and
SlIDD14 did not show a signicant increase in expression, whereas SlIDD15 and SlSlIDD18 showed a dramatic
increase in expression. Signicant increases in expression were observed in SlIDD13 and SlIDD17 in 2h. SlIDD1,
Solyc01 g007120_STOP-like1
Solyc01g099340_SlIDD10
Solyc02g085580_SlIDD1
Solyc03g098070_SlIDD-like1
Solyc03g121660_SlIDD11
Solyc04g008500_SlIDD4
Solyc04g056320_SlSTOP-like6
Solyc05g054030_SlIDD5
Solyc06g062670_SlIDD2
Solyc06 g065440_SlSTOP-like3
Solyc06g072360_SlIDD9
Solyc06g075250_SlIDD7
Solyc07g053570_SlIDD8
Solyc08g080850_STOP-like2
Solyc09 g007550_SlIDD13
Solyc09g074780_SlIDD3
Solyc10g084180_SlIDD14
Solyc11g017140_SlSTOP-like4
Solyc11g066420_SlSTOP-like5
Solyc11g069240_SlIDD6
-1 01
Solyc01g005060_SlIDD12
Solyc09g065670_SlIDD15
Solyc08g063040_SlIDD16
Solyc02g062940_SlIDD17
Solyc04g080130_SlIDD18
TFIIIA
A0A3Q7JN10
A0A3Q7GI92
A0A3Q7HXU8
A0A3Q7J4Y0
A0A3Q7GW43
A0A3Q7F8R2
A0A3Q7I9Y8
A0A3Q7JTU6
A0A3Q7JY69
A0A3Q7IZY4 (SlIDD6)
A0A3Q7H0A7_SlIDD7
TFIIIA
A0A3Q7JN10
A0A3Q7GI92
A0A3Q7HXU8
A0A3Q7J4Y0
A0A3Q7GW43
A0A3Q7F8R2
A0A3Q7I9Y8
A0A3Q7JTU6
A0A3Q7JY69
A0A3Q7I4C6_SlIDD15
TFIIIA
A0A3Q7JN10
A0A3Q7GI92
A0A3Q7HXU8
A0A3Q7J4Y0
A0A3Q7GW43
A0A3Q7F8R2
A0A3Q7I9Y8
A0A3Q7JTU6
A0A3Q7JY69
A0A3Q7HF56_SlIDD8
A0A3Q7JN10
A0A3Q7GI92
A0A3Q7HXU8
A0A3Q7H204
A0A3Q7J4Y0
KdsA
A0A3Q7GW43
A0A3Q7F8R2
A0A3Q7I9Y8
A0A3Q7JTU6
a
b
Figure5. Coexpression networks for SlIDDs. (a) Coexpression network between SlIDDs, SlIDD-like1, and
STOP1-like TFs. (b) Interaction network of SlIDD6, SlIDD7, SlIDD15, and SlIDD8 with other proteins.
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SlIDD7, SlIDD12, SlIDD16, SlIDD2, SlIDD4, and SlIDD-like1 showed signicantly higher levels in 24h. SlIDD3
and SlIDD9 showed a signicant reduction in expression under salt stress conditions (Fig.6b and TableS8).
Expression of SlIDD TFs under pH stress
Low pH is an occasional problem in greenhouse vegetable production, as it can aect the quality and quantity
of produce by aecting soluble ions in the media41,42. SlSTOP1 is an essential TF that is closely related to SlIDD
TFs and crucial for proton stress tolerance43,44. To examine the expression of SlIDD TFs, plants were subjected
to low pH conditions (pH = 4.2) in 3-week-old plants.
e expression of SlIDD12 was up-regulated in the roots by over 50-fold. SlIDD8 also showed a signicant
increase in the roots (Fig.7). In contrast, SlIDDlike-1, SlIDD13, SlIDD15, SlIDD16, and SlIDD17 showed sig-
nicant reductions in expression levels in the roots. However, in the shoots, SlIDD6 showed a 40-fold increase
in expression aer 24h. Notably, SlIDD15 and SlIDD17 showed signicant increases in the shoots, but not in
the roots. SlIDD8 expression was signicantly higher in both tissues at both time points (Fig.7b and TableS9).
Expression of SlIDD TFs under ooding stress
Flooding stress is a major problem for eld-cultivated tomatoes because of the intensive rainfall patterns that
induce climate change45. Waterlogging reduces oxygen availability to the submerged plant parts, which subse-
quently leads to cell death and, eventually severe yield losses46,47. Because IDD TFs are plant-specic, they can
potentially respond to ood stress. To determine the response of IDD TFs to ood stress, 3-week-old tomato
seedlings were submerged in water up to the crown, and RNA was extracted from the roots and shoots at 2h
and 24h intervals.
Unlike salt and pH stress, less severe reaction of SlIDD TFs were observed in the roots (Fig.8a and TableS9).
Among strongly responded seven genes to ood, SlIDD12 showed an 80-fold increase in expression. Moreover,
SlIDD3, SlIDD6, SlIDD9 and SlIDD18 showed signicantly increased expression (Fig.8b). In contrast, SlIDD2
was downregulated in roots and upregulated in shoots at 24h time points. In the shoots, all genes except SlIDD7,
SlIDD13, and SlIDD14 showed increased expression levels. In particular, SlIDD18 showed more than tenfold
and 28-fold increase at the 2h and 24h intervals, respectively. SlIDD11 and SlIDD15 also exhibited dramatic
increases in the shoots under ood stress (TableS10).
Control Root 2h
Control Root 24h
Salt Root 2h
Salt Root 24h
Control Shoot 2h
Control Shoot 24h
Salt Shoot 2h
Salt Shoot 24h
IDD9
IDD3
IDD10
IDD18
IDD17
IDD-like1
IDD15
IDD16
IDD7
IDD5
IDD1
IDD2
IDD11
IDD6
IDD8
IDD12
IDD14
IDD4
IDD13
ab
Shoots Roots
2 hours 24 hours 2 hours 24 hours
0
1
2
ControlTreated
Relatveexpression
SlIDD12
**
0
1
2
3
ControlTre ated
Relatveexpression
SlIDD13
**
0
1
2
ControlTreated
Relatve expressio n
SlIDD14
**
0
10
20
30
40
ControlTreated
Relatveexpression
SlIDD15
**
0
2
4
6
8
10
12
ControlTreated
Relatveexpression
SlIDD18
**
0
1
2
ControlTre ated
Relative expression
SlIDD3
*
0
1
2
ControlTre ated
Relatveexpression
**
0
1
2
3
4
ControlTreated
**
0
50
100
150
ControlTreated
**
0
5
10
15
ControlTre ated
**
0
1
2
3
4
ControlTreated
0
1
2
3
4
5
6
ControlTreated
0
1
2
ControlTreated
0
1
2
ControlTreated
**
0
10
20
30
40
50
ControlTreated
**
0
10
20
30
40
50
60
ControlTreated
**
0
5
10
15
20
ControlTre ated
0
1
2
ControlTreated
0
1
2
ControlTreated
**
0
10
20
30
40
ControlTre ated
0
1
2
ControlTreated
*
0
1
2
ControlTreated
**
0
1
2
ControlTreated
**
0
1
2
3
ControlTreated
*
0
1
2
3
4
ControlTreated
**
0
1
2
ControlTre ated
**
0
1
2
3
4
5
6
ControlTreated
**
0
1
2
ControlTreated
SlIDD9
0.00004 0.47316
Figure6. Expression patterns of screened zinc nger TFs under salt stress. (a) Clustergram for IDD expression
levels under NaCl induced salt stress in 3-week-old tomato seedlings (Scales represented as relative values). (b)
Expression levels of high responsive SlIDDs under salt stress. (**P < 0.01; *P < 0.05).
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Discussion
Functional analysis of SlIDDs
e present study systematically analyzed IDD TFs belonging to tomatoes, as IDDs in Arabidopsis, rice, and maize
have already been examined for their existence and properties22–25,27,48–51. Currently, there are 16, 15, and 22 IDD
TFs identied in Arabidopsis, rice, and maize, respectively. Consistent with our results for 18 IDDs in tomatoes,
the IDD family genes might have played crucial roles in a species-specic manner. IDD TFs are also plant-specic
and can participate in multiple plant-specic functions such as vascular development, photosynthesis, light sign-
aling, owering etc52. Moreover, plant-specic TFs are also involved in shaping the phenotypic and physiological
factors of plants for the adaptation of plants to land-based environments, where the plants need to withstand
biotic and abiotic stress conditions53. Functional characterization can shed light on the IDD TFs role in plants.
Moreover, some plant-specic TFs show dierences in the number of monocot and dicot species54,55. Phylogenetic
analysis of IDD TFs from the model plant Arabidopsis and major model crops such as tomato and rice suggested
that IDD transcription factors other than higher conservation of their functional motifs in monocots and dicots
and structural elements are potentially specialized within each of these two lineages.
Phylogenetic analysis revealed closely related IDD groups in rice, tomato, and Arabidopsis. SlIDD1 showed
close relationships with AtIDD1, AtIDD2, OsIDD1, SlIDD17, and SlIDD18 (Fig.1b). AtIDD1 is involved in gib-
berellin signaling by forming activator and repressor complexes upstream of gibberellin biosynthesis genes56.
Notably, AtIDD1 is a direct target of PHYTOCHROME INTERACTING FACTOR 3-LIKE5 (PIL5), which inhibits
seed germination in dark conditions by regulating abscisic acid (ABA) and Gibberellic acid (GA)57. AtIDD2
(GAF1) also shows light-responsive properties where AtIDD2 acts as a transcriptional activator and repressor of
GA20OX under dierent light conditions and regulates owering and plant size56. OsIDD1 along with OsIDD6
potentially have redundant functions in oral transition58. OsIDD1 also regulates the expression of JA-related
genes related to herbivore resistance59. SlIDD1 expression was signicantly down-regulated in salt-stressed roots
and increased in salt-stressed shoots aer 24h. Under acidic conditions, SlIDD1 expression was reduced within a
short time and recovered aer 24h. Under ooding conditions, the shoots showed signicantly higher expression
levels, suggesting a pivotal role in the transition under stress conditions (Figs.6, 7, 8). SlIDD17 is signicantly
responsive to salt and acidic stress. Previous studies have shown increased expression of SlIDD17 under heat
stress and during fruit development60,61.
Control Root 2h
Control Root 24h
pH Root 2h
pH Root 24h
Control Shoot 2h
Control Shoot 24h
pH Shoot 2h
pH Shoot 24h
IDD16
IDD14
IDD12
IDD4
IDD10
IDD9
IDD6
IDD3
IDD8
IDD7
IDD5
IDD11
IDD-like1
IDD15
IDD17
IDD18
IDD1
IDD2
IDD13
ab
Shoots Roots
2 hours 24 hours 2 hour
s2
4 hours
**
0
2
4
6
8
10
12
ControlTreated
Relatveexpression
**
0
2
4
6
8
10
ControlTre ated
**
0
2
4
6
8
ControlTreated
**
0
1
2
3
4
ControlTreated
SlIDD8
0
1
2
3
4
ControlTre ated
Relatveexpression
**
0
5
10
15
20
ControlTre ated
**
0
10
20
30
40
50
60
ControlTreated
0
1
2
3
4
ControlTreated
SlIDD9
0
1
2
3
ControlTre ated
Relatveexpr ession
*
0
1
2
3
4
ControlTreated
**
0
25
50
75
100
125
150
ControlTreated
**
0
2
4
6
8
10
ControlTre ated
SlIDD12
0
1
2
ControlTre ated
Relatve expression
**
0
5
10
ControlTreated
0
1
2
3
4
ControlTreated
**
0
1
2
ControlTreated
SlIDD13
**
0
2
4
6
8
10
12
ControlTreated
Relatveexpression
**
0
2
4
6
ControlTreated
0
1
2
ControlTreated
**
0
1
2
ControlTreated
SlIDD15
0
1
2
3
ControlTreated
Relatve expression
**
0
1
2
3
4
ControlTreated
**
0
1
2
ControlTreated
**
0
1
2
ControlTreated
SlIDD17
*
0
1
2
3
4
5
6
7
ControlTre ated
Relatveexpression
**
0
10
20
30
40
50
60
ControlTre ated
**
0
5
10
15
20
25
30
ControlTreated
0
1
2
3
ControlTreated
SlIDD6
0.00012 0.4700
Figure7. Expression patterns of screened zinc nger TFs under proton Stress. (a) Clustergram for IDD
expression levels under pH stress in 3-week-old tomato seedlings (Scales represented as relative values). (b)
Expression levels of high responsive SlIDDs under pH stress. (**P < 0.01; *P < 0.05).
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SlIDD2 is closely related to AtIDD7, AtIDD11, OsIDD2, OsIDD11, SlIDD10 and SlIDD11 (Fig.1b). AtIDD7
shows higher activity during phosphorus starvation and owering62,63. In rice, OsIDD2 negatively regulates the
transcription of genes involved in lignin biosynthesis64. OsIDD11 hypothesized to have drought tolerance via
stomatal control65. In the current study, under stress conditions, SlIDD2 transcripts were signicantly down-
regulated in the roots and increased in the leaves. SlIDD2 is down-regulated in the base margin tissue of tf-2 leaf
patterning-decient mutants66. When treated with auxin and ethylene, SlIDD2 showed increased and decreased
activity in fruits, respectively, and reduced expression in LATERAL ORGAN BOUNDARIES(LOB) TF and SlLOB1
RNAi lines, with reduced soening and increased shelf life67,68. In Arabidopsis, AtIDD7 shows dierential expres-
sion under phosphorus starvation69, early ower development63, and low temperature70. However, the precise
function of AtIDD7 is currently unknown22. AtIDD11 shares structural homology with and is potentially involved
in leaf patterning22,71. SlIDD10 showed higher expression levels in maturing fruits and the root exodermis in
previous studies60,61,72. Interestingly, SlIDD11 showed a sudden dramatic increase in expression in shoots under
all stress conditions, suggesting a role similar to OsIDD11. GWAS studies suggest that SlIDD11 is associated with
isothermality and shows allele specicity in exotic land races73,74. Notably, SlIDD11 produced ve isoforms that
may be expressed under specic stress conditions to respond specically (TableS5).
SlIDD3 grouped along with AtIDD12 and SlIDD15. SlIDD3 showed higher expression patterns in the exoder-
mis and the cortex72. SlIDD15 shows a gradual reduction from young to mature fruits60. Under stress, SlIDD15
showed higher levels of expression in roots, especially under salt stress. SlIDD3 also showed varying expression
patterns in the roots and shoots under stress. AtIDD12s function is currently unknown, but it shows higher
activity in seeds75.
SlIDD4, AtIDD5, and AtIDD6 grouped in phylogenetic analysis (Fig.1b). The Arabidopsis homolog
AtIDD5/RAVEN interacts with DELLA28 and promotes anisotropic growth by positively regulating STARCH
SYNTHASE 4 (SS4)50 possibly regulating root tissue patterning through asymmetric cell division76. AtIDD6 is
also involved in the tissue patterning of roots during development77. However, in tomatoes, stress treatments
showed a signicant response to salt and acid stress in roots and to ood stress in shoots, suggesting a role in
Control Root 2h
Control Root 24h
Flood Root 2h
Flood Root 24h
Control Shoot 2h
Control Shoot 24h
Flood Shoot 2h
Flood Shoot 24h
IDD14
IDD12
IDD3
IDD1
IDD15
IDD6
IDD16
IDD9
IDD10
IDD8
IDD4
IDD17
IDD5
IDD11
IDD18
IDD7
IDD-like1
IDD2
IDD13
ab
Shoots Roots
2 hours 24 hours 2 hours 24 hours
**
0
1
2
3
ControlTre ated
Relatve expression
**
0
2
4
6
8
10
ControlTre ated
**
0
20
40
60
80
100
ControlTreated
**
0
5
10
15
ControlTre ated
SlIDD12
0
1
2
ControlTreated
Relatveexpression
**
0
1
2
3
4
ControlTreated
**
0
1
2
3
4
5
ControlTreated
**
0
2
4
6
8
10
12
ControlTreated
SlIDD3
0
1
2
ControlTreated
Relatveexpr ession
**
0
2
4
6
8
ControlTreated
**
0
2
4
6
8
ControlTreated
0
1
2
ControlTreated
SlIDD6
*
0
1
2
3
ControlTre ated
Relatveex pression
**
0
2
4
6
8
10
ControlTreated
**
0
2
4
6
8
ControlTreated
**
0
1
2
3
ControlTreated
SlIDD9
**
0
5
10
15
ControlTreated
Relatveex pression
**
0
10
20
30
40
ControlTre ated
0
1
2
ControlTreated
**
0
1
2
3
ControlTreated
SlIDD18
**
0
1
2
3
4
5
ControlTreated
0
1
2
ControlTreated
*
0
1
2
3
ControlTreated
**
0
5
10
15
20
25
ControlTreated
Relatve expression
SlIDD11
**
0
5
10
15
20
25
ControlTreated
Relatveex pression
**
0
5
10
15
ControlTre ated
0
1
2
ControlTreated
0
1
2
ControlTreated
SlIDD15
0.00012 0.65
Figure8. Expression patterns of screened zinc nger TFs under ood stress. (a) Clustergram for IDD
expression levels under ood stress in 3-week-old tomato seedlings (Scales represented as relative values). (b)
Expression levels of high responsive SlIDDs under ood stress. (**P < 0.01; *P < 0.05).
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root patterning under both developmental stages and stress tolerance in both roots and shoots (Figs.6, 7, 8).
Notably, SlIDD4 produces four isoforms (TableS5).
Even though SlIDD5 contains a C2HR motif instead of a C2H2 motif, evidence suggests that the replace-
ment of Histidine by Arginine might not have any major eect on transcriptional activity (Fig.2). However,
C2HR motifs have been shown to interact with other proteins36. SlIDD5/OBV was highly expressed in the leaves
and vegetative phases of the meristems. Heterobaric leaves contains Bundle sheath extensions in the leaves
which provide mechanical strength. SlIDD5 mutants failed to produce bundle sheath extension cells (homo-
baric leaves)78. Increased chlorophyll content has been observed in obv mutants, such as M82 and CRISPR/
Cas9 mutants of Micro-Tom, where the absence of BSE allowed chloroplast development in leaf veins and
reduced water conductivity79,80. Moreover, OBV also regulates the leaf insertion angle, leaf margin serration, and
fruit shape, and has been shown to work together with auxin signaling80. SlIDD5 binds to the promoter FUL2
which then regulates fruit shape81. Arabidopsis AtIDD14, AtIDD15, and AtIDD16 show structural homology
with SlIDD5 and have similar functions in leaf shape, ower development, plant architecture, and gravitrophic
responses by regulating auxin biosynthesis and transport factors23. Surprisingly, OsIDD14/LPA1 also shows simi-
lar functions in plant architecture and has been extensively studied. LPA1 determines rice tiller angle and shoot
gravitropism by aecting the sedimentation rate of amyloplasts and binds to the promoter region of PIN182,83.
LPA1 also exhibits water conservation properties by reducing the rate of transpiration from rice leaves84. However,
data for OsIDD12 and OsIDD13 were unavailable.
AtIDD9, AtIDD10, AtIDD13, SlIDD6, SlIDD7, SlIDD13, and SlIDD14 grouped together in the phylogenetic
analysis (Fig.1b). Reduced pH also caused a higher accumulation of SlIDD6 in shoots (Fig.6) and showed heat-
induced expression in tomato leaves85. SlIDD7 expression patterns were similar to those of SlIDD10. Under Salt
stress conditions, roots showed reduced expression and shoots showed increased expression. SlIDD7 expression
increases in leaves and stems under heat stress61 and is negatively correlated with CYC-B in developing fruits,
indicating its possible role in the regulation of lycopene accumulation in developing fruits60. Both SlIDD13 and
SlIDD14 regulate stem thickness and leaf shape, and mutants are tolerant to necrotrophic infection51. Under
salt stress, the roots showed higher levels of IDD14 transcripts. IDD13 also showed a signicant increase in the
shoots of the salt-stressed tomato seedlings (Fig.8).
SlIDD8 showed homology with OsIDD8, AtIDD3, and AtIDD8 which showed increased expression under salt
and heat stress (Fig.1b)86. AtIDD3 and AtIDD8 are involved in root development. Moreover, AtIDD8 regulates
oral transition and sugar metabolism22.
SlIDD9 is highly expressed in roots and developing fruits, and shows increased expression under abiotic stress
conditions87. Arabidopsis AtSTOP1 shows close homology with SlIDD9 and is involved in proton toxicity and
aluminum tolerance by activating the malate transporter AtALMT188,89. AtSTOP1 also modulates the response to
drought and salt levels by regulating root growth and guard cell movement90. SlIDD-like1 showed close homology
with OsLPA1. Even though SlIDD-like1 does not contain the rst C2H2 domain, it shares close homology with
SlIDD12 and SlIDD5. SlIDD-like1/Se3.1 controls stigma extortion or insertion with Style3.1 which determines
the rate of self-pollination91,92. Under stressful conditions, SlIDD-like1 showed less severe changes in expression.
SlIDD16/SlZF-31 mutants showed reduced salt and drought tolerance38. SlIDD16 showed reduced expres-
sion in RIN mutants, suggesting its potential role in fruit ripening93,94. e rice orthologs SlIDD16, OsIDD11
are involved in drought tolerance by regulating stomatal movement and starch composition in rice65,95. OsIDD2
regulates the secondary cell wall (SCW) formation by directly binding to SCW biosynthesis genes48. OsIDD2 is
responsible for plant height, leaf strength, and resistance to fungal infection64,96.
In the interaction analysis, more clues and the possible applicability of SlIDDs were revealed. SlIDD6 and
SlIDD7 SlIDD15 showed co-expressed with TFIIIA during viral infection. Arabidopsis thaliana experiments
have hypothesized that TFIIIA acts as a bridge between the viroid template and DNA polymerase II during
viroid-derived RNA replication97,98. SlIDD8 closely interacts with SlkdsA, a Kdo-8-P synthase associated with
cell division99.
Stress experiments revealed that SlIDD10, SlIDD5, SlIDD7, SlIDD13, and SlIDD16 showed less dramatic
changes in expression, suggesting that these TFs are highly involved in development51,61,91. However, SlIDD16
mutants are tolerant to salt and drought stress, suggesting SlIDD16 is stress specic38. In contrast, SlIDD3, SlIDD8,
SlIDD9, and SlIDD12 showed multi-stress responses, suggesting that these TFs should be further studied for
their eects on tomato survival and productivity.
Future perspectives for IDDs for breeding climate-resilient and high-producing crops
e evolution of plants from aquatic to terrestrial habitats is noteworthy. Unlike in aquatic environments, land
plants have to increase their survivability by specializing in organs to compartmentalize functions, such as
developing eective root systems and vascular systems for water transport, increasing photosynthetic ability and
survival adaptations, such as distinguishing benecial organisms from pathogens and predators, and adapting to
dry terrain100. Plant-specic transcription factors drive adaptation through genome and gene duplication events
and specialize in downstream fuctions19,21,101,102.
Climate change threatens crop productivity due to changes in agro-climatic conditions, and current breed-
ing programs are exploring possibilities to develop climate-resilient cultivars for better productivity103–106. A
better alternative for escaping climate catastrophes without breeding for climate resilience is to cultivate crops
in protected environments, such as greenhouses. However, due to the fact that the cultivated crops are highly
adapted to pre-climate change era, breeding programs should focus on the evolution of terrestrial plants to
identify evolutionarily signicant candidate genes for plant breeding.
Evolutionary analyses of SlIDD TFs indicated that these genes were selected under high selection pressure
and all genes were crucial for survival. In particular, IDDs are plant-specic and are involved in functions such
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as herbivore resistance and starvation responses from germination to fruit ripening. Our current data and those
of previous studies show that these SlIDDs are potential candidates for improving the productivity of protected
house cultivation and land cultivation60,61,88,89,107. In the case of land cultivation, IDDs respond to abiotic stresses,
such as drought, salt, ooding, pH changes, and starvation, along with the development of roots. Other IDDs,
such as SlIDD2 showed leaf-patterning roles, and SlIDD5 showed chlorophyll content, which can be used to
increase photosynthetic capacity and productivity. Under changing climates, indoor farming can reduce expo-
sure to harsh climates, which can reduce the energy spent on defense mechanisms108,109. However, it is possible
to reduce the stress response to eliminate pests and stress in well-protected houses, which renders the stress
response elements in plants insignicant110–112. Stress response-related genes can be down-regulated to force
plants to focus on productivity by diverting energy allocations112,113. Finally, the marketability of produce is a
crucial factor in increasing the net returns from tomato cultivation114–116. SlIDDs such as SlIDD5 and SlIDD16
showed functions related to fruit shape and ripening, which can be further studied to improve fruit shape and
shelf life, and increase market value and post-harvest quality. Plants produce isoforms to diversify their roles by
alternative splicing (AS), from a single coding region to multiple protein derivatives for specialized roles117. is
mechanism allows the plants to eliminate the necessity to harbor additional genetic information in the genome
and increase the transcriptome plasticity and proteome complexity118. With this mechanism, plants are able to
respond against a large array of environmental stresses and cellular damages119–123. e isoforms of SlIDDs can
be further dissected based on their specic roles in growth and development, where a single SlIDD responded
to various stress conditions in our study (Figs.6, 7, 8). Studying the role of isoforms can provide insights into
the isolation of stress responses and developmental elements from a single TF.
Conclusions
Amid climate change manifesting in real time, food security must be ensured in every corner of the world. Agro
climatic factors may also change with the increase in average global temperature and humans may have to modify
crops to ensure cultivation in limited resources and possibly indoors under articial conditions124,125. e current
analysis identied 18 IDDs (SlIDDs) in tomatoes. Functionally, only a few SlIDD have been characterized based
on molecular evidence. Current study revealed the multi-role potentials of the the SlIDD TFs in tomato growth,
development and plasticity. Notably, SlIDD1, SlIDD3, SlIDD4, SlIDD6 to 9, SlIDD11, SlIDD16 and SlIDD17
showed potential roles in abiotic stress responses where SlIDDs 4 and 11 showed three and ve isoforms respec-
tively. Which indicates the functionally diverse role of these TFs. Moreover, previous studies showed SlIDD13
and SlIDD14 are involved in abiotic stress response50,97. On the other hand, SlIDDs 2 to 5, SlIDD10, SlIDD15,
SlIDD17 showed potential roles in growth, organ pattering51,61,91. ese results indicate that the SlIDDs are
capable of regulating overall plant development, plasticity and physiology in a well-coordinated manner. Based
on the results presented here, the functions of SlIDDs may be applied well beyond stress tolerance, productivity,
and quality of tomato production, where some mutants of SlIDDs show crucial agroeconomic traits that can aid
in breeding climate-resilient, high-producing tomato cultivars with the aid of the tomato PAN genome. Based
on current expression patterns and ortholog functions, embryo lethality is possible. However, other techniques,
such as promoter engineering8,11,12 or RNAi126–129 can be employed to study the molecular functions of SlIDDs.
Natural disasters and temperature uctuations have increasingly challenged the future of agriculture. TFs that
play a major role in land adaptation can be repurposed to adapt to the current climate crisis, and SlIDDs can be
pivotal for this purpose.
Methods
Database search and BLAST
A BLAST search was conducted using three dierent databases for tomatoes (Solgenomics network; https://
solge nomics. net/, Plaza 5.0; https:// bioin forma tics. psb. ugent. be/ plaza/ and Gramene; https:// www. grame ne.
org. Arabidopsis and Rice sequences were veried using TAIR10 (https:// www. arabi dopsis. org/) and RAP-DB
(https:// rapdb . dna. arc. go. jp/) respectively. Default parameters were used as the conditions for BLAST searches.
Multiple alignment and phylogenetic tree construction
Multiple protein sequence alignments were performed using ClustalW and visualized using the ALIGNMENT-
VIEWER (https:// github. com/ sande rlab/ align mentv iewer) soware. A phylogenetic tree was constructed using
MEGA (version 11.0; Penn State University, PA, USA) and the maximum likelihood tree method (bootstrap 1000
replicates). Sequences for the multiple alignments and phylogenetic tree and accession numbers of OsIDDs and
AtIDDs are available in TableS12–S14. e iTOL web tool was used to construct the evolutionary tree (https://
itol. embl. de).
Chromosomal location, synteny analysis, motif visualization, and 3D structure visualization
e locations of candidate genes were acquired from the Solgenomics network (https:// solge nomics. net/), and
positions were visualized using MG2C v2.1 (htt p:// mg2c. iask. in/ mg2c_ v2.1/). Synteny analysis and Ka/Ks values
were calculated using TBtools130. Gene duplications were assessed by using R package “Doubletrouble” (https://
github. com/ almei dasil va f/ do ub l etr o u ble) 131. e MEME suite was used to identify and visualize conserved motifs
among candidate genes (https:// meme- suite. org/ meme/ tools/ meme). Motifs were searched among the given
sequences, and the remainder were set to default. e 3D structure was identied using a UniProt (https:// www.
unipr ot. org) database search and visualized using Afphafold2.0 (htt ps :// www. unipr ot. org/ da tab ase? q uery= (name:
Alpha FoldDB) & direct).
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Cis-regulatory motif analysis and Coexpression network construction
Promoter sequences of 3Kb of each SlIDD gene were used to scan and identify cis-regulatory elements using
FIMO (https:// meme- suite. org/ meme/ tools/ mo) against Arabidopsis promoter matrices (http:// bar. utoro nto.
ca/ ~nprov art/ Arabi dopsi sDAPv1. meme) based on previous reports11,66,132,133. e cuto values for the p- and
q-values were 1.99E-14 and 1.63E-09 respectively. TB tools were used to visualize the architectural positions of
the major promoter elements130. A coexpression network was constructed using the TomExpress database134.
STRING (https:// string- db. org) was used to identify interaction partners of SlIDDs.
Plant materials and growth conditions
All experiments were conducted using Solanum lycopersicum cv. M82 seeds kindly provided for the experiments
by Prof. Soon Ju Park from Gyeongsang National University, Jinju, Korea. e plants were grown under long-day
conditions and controlled temperatures in a greenhouse at Wonkwang University, Iksan, South Korea. Plants
were grown under natural and supplemental light from a natrium, and halogen lamps were applied in the early
morning and late evening. e light/dark cycle was 16h/8h/day. Plants were supplied with nutrients in the
irrigation water one month aer transplanting, following the manufacturer’s guidelines (S-feed, 1kg/10 a/day;
https:// www . farmh annong. com/ kor/ produ ct/ produ ct_ ct01/ view. do? seq= 4392 (accessed on 08 November 2023).
Abiotic stress treatment
Stress treatments were applied using potting media to ensure regular greenhouse growth. Salt stress was induced
by saturating the potting medium with tap water mixed with 200mM NaCl at an adjusted pH of 6.8. Proton
stress was induced by saturating the plants with tap water at pH 4.2. Flood stress was induced by submerging
plant roots in potting media in water at a pH of 6.8. All stress treatments were performed under greenhouse
conditions. Control plants were saturated with water at pH 6.8. Shoot and root samples were collected at 2 and
24h aer treatment.
RNA extraction and quantitative real time PCR for stress-responsive SlIDDs
To extract RNA from shoots and roots, 3weeks old control and treated plants were harvested at 2pm in a green-
house. Total RNA was extracted using the AccuPrep® Universal RNA extraction kit (Bioneer, Daejeon, Korea)
and treated with RNase-free DNase to remove DNA fragments (Qiagen, Hilden, Germany). One microgram
of total RNA was used to synthesize cDNA with AccuPower® RT PreMix (Bioneer, Daejeon, Korea). qRT-PCR
was performed using a T100TM ermocycler system (Bio-Rad, Hercules, CA, USA). Primer information is
provided in TableS11. Reactions (10µL nal volume) were prepared using 5µL of LaboPass™ SYBR Green Q
master kit (Cosmogenetech, Dajeon, Korea). Next, 0.5pmol of a primer pair, and 0.5µL of cDNA template. Four
biological samples and two technical replicates were used for quantication. Ubiquitin was used as a reference.
gene expression analysis was performed with the 2^ − ΔΔCt method using Bio-Rad CFX Maestro soware v.4.0
(Bio-Rad). e baseline and threshold levels were set according to the manufacturer’s instructions.
Ethics approval and consent to participate
All experiments were conducted in greenhouses situated at Wonkwang University using wild-type plants. Ethical
guidelines provided by the ethics committee were followed when conducting the experiments.
Data availability
All data related to the expression analyses are available in the GEO repository under accession number
GSE248090 (https:// www. ncbi. nlm. nih. gov/ geo/ query/ acc. cgi? acc= GSE24 8090). Sequences related to the bio-
informatics analyses are included in the Supplementary Tables.
Received: 14 November 2023; Accepted: 4 April 2024
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Acknowledgements
We thank all members of the Plant Molecular Breeding Laboratory at Wonkwang University and Plant Develop-
ment and Genetics lab at Gyeongsang National University for their valuable suggestions and assistance.
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Author contributions
Conceptualization, S.R., S.J.P., C.M.K.; Investigation, S.R., C.M.K.; Methodology, S.R., C.M.K.; Resources, A.E.,
S.J., H.C.K., C.M.K.; Data Collection, S.R., Y.M.K., I.B.Y., H.B.E., K.L.B.; Data analysis, S.R., Supervision A.E.,
S.J., H.C.K., B.I.J., C.M.K.; Validation A.E., S.J., H.C.K., B.I.J., C.M.K.; writing—review and editing S.R., S.J., A.E.,
H.C.K., S.J.P., C.M.K. All authors have read and agreed to the published version of the manuscript.
Funding
is work was supported by a grant from the New Breeding Technologies Development Program (Project No.
RS-2024-00322297) of the Rural Development Administration, Republic of Korea. is work was supported in
part by a grant from the World Vegetable Center Korea Oce (WKO #10000379) and by long-term strategic
donors to the World Vegetable Center: Taiwan, UK, aid from the UK government, the United States Agency for
International Development (USAID), the Australian Center for International Agricultural Research (ACIAR),
Germany, ailand, Philippines, Korea, and Japan.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 024- 58903-0.
Correspondence and requests for materials should be addressed to C.M.K.
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