Introduction

In the past five decades, the global population has increased by four billion and is predicted to increase rapidly from the current eight billion individuals1. The 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 floods 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. Therefore, 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 result in alterations in target gene expression, which can alter cellular activities9,10,11,12. TFs sequences specifically 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-specific TFs regulate genes related to the development of plant-specific organs and response systems for adaptation to terrestrial environments18. These include the development of efficient water transport systems, efficient reproductive organs, the ability to withstand the effects of terrestrial factors such as UV radiation, temperature fluctuations, soil-related stress factors, and evolutionary advantages over land predators19,20,21. INDETERMINATE DOMAIN (IDD) TFs are plant-specific TFs with crucial functions in rice and Arabidopsis, including development, reproduction and stress response22,23,24,25,26,27,28.

Among the vast array of TFs, IDD, a class of C2H2 zinc-finger TFs, is specific to plants22,25,29,30. The N-terminus of the IDD contains two C2H2 DNA-binding domains and 2C2HC protein-binding domains. The C-terminus also contains protein interaction domain24,25,29. In Arabidopsis, 12 of 18 identified IDD TFs have been characterized 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 analysis33,34,35. 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

Identification and phylogenetic analysis of SlIDD family genes in tomato

To identify candidate SlIDD family genes, a BLAST search was conducted using Gramene (https://www.gramene.org) and Plaza (https://bioinformatics.psb.ugent.be/plaza) databases. Overall, 25, 24, and 20 genes were identified by search results in tomato, rice, and Arabidopsis respectively. Arabidopsis and rice have 16 and 15 IDD genes, respectively. The evolutionary relationships among IDD family genes were determined using phylogenetic analysis. Phylogenetic analysis suggested that IDD genes may have structural differences between monocots and dicots (Fig. 1a). Four subgroups of IDD-like genes have been identified 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).

Figure 1
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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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 first C2H2 motif. Solyc05g054030 possesses a C2HR motif in the second zinc finger 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 respectively (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. After confirming 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 segmental duplication events (Table S3). Solyc03g098070, which lacks the first C2H2 motif, exhibited synteny with Solyc06g072360. 18 confirmed 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-finger 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. Surprisingly, IDD4 and IDD11 had three and five isoforms, respectively, indicating complex post-transcriptional regulatory mechanisms present in IDD TFs (Table S5).

Figure 2
figure 2

Comparison, confirmation, 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) The synteny analysis of the SlIDD family in tomato. The 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 confirmed IDD genes (left) and protein motifs in corresponding sequences (right).

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3D structure of SlIDD TFs

Following the identification of SlIDD TFs, 3D structures were predicted using AlphaFold2.0 (https://alphafold.ebi.ac.uk) to verify the structural similarity of the confirmed TFs using BLAST with UniProt (https://www.uniprot.org) accession numbers (Table S6). The 3D structures showed prominent zinc finger domains in the C terminus regions of the primary isoforms. However, Solyc03g098070 had only three zinc finger domains, which confirmed 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).

Figure 3
figure 3

3D models of tomato IDD TFs predicted by AlphaFold2.0. Light blue chains show zinc finger domains. 2D images were taken for visibility of zinc finger domains (see Table S6 for AlphaFold2.0 accession numbers to access 3D models).

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Cis-regulatory element analysis of SlIDD promoters

The promoter sequences of 18 SlIDD genes (3000 bp) were scanned for cis-regulatory elements using Arabidopsis DAP motifs with a cut-off 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).

Figure 4
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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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 differences in temporal expression patterns and provided clues to gene regulation networks in different tissues (Fig. 5). SlIDD4 showed close association with SlIDD2 similar to the tissue-specific 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-specific 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.

Figure 5
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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Expression of IDD TFs under abiotic stresses

Cis-regulatory analysis suggests that the binding elements in the promoters are highly responsive to environmental signals. Moreover, SlIDD1, SlIDD8, SlIDD9, and SlIDD16 show increased expression under various abiotic stress conditions37,38. To confirm whether other SlIDDs were also responsive to abiotic stress, 3-week-old plants grown under greenhouse nursery conditions were subjected to salt, pH, and flood 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 affect 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 significant increases in expression. Other SlIDDs such as SlIDD1 and SlIDD2 showed significantly reduced expression. SlIDD4 and SlIDD13 were upregulated after 24 h of treatment, whereas SlIDD6 showed increased expression only after 2 h. However, in the shoots, SlIDD12 and SlIDD14 did not show a significant increase in expression, whereas SlIDD15 and SlSlIDD18 showed a dramatic increase in expression. Significant increases in expression were observed in SlIDD13 and SlIDD17 in 2 h. SlIDD1, SlIDD7, SlIDD12, SlIDD16, SlIDD2, SlIDD4, and SlIDD-like1 showed significantly higher levels in 24 h. SlIDD3 and SlIDD9 showed a significant reduction in expression under salt stress conditions (Fig. 6b and Table S8).

Figure 6
figure 6

Expression patterns of screened zinc finger 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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Expression of SlIDD TFs under pH stress

Low pH is an occasional problem in greenhouse vegetable production, as it can affect the quality and quantity of produce by affecting 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.

The expression of SlIDD12 was up-regulated in the roots by over 50-fold. SlIDD8 also showed a significant increase in the roots (Fig. 7). In contrast, SlIDDlike-1, SlIDD13, SlIDD15, SlIDD16, and SlIDD17 showed significant reductions in expression levels in the roots. However, in the shoots, SlIDD6 showed a 40-fold increase in expression after 24 h. Notably, SlIDD15 and SlIDD17 showed significant increases in the shoots, but not in the roots. SlIDD8 expression was significantly higher in both tissues at both time points (Fig. 7b and Table S9).

Figure 7
figure 7

Expression patterns of screened zinc finger 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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Expression of SlIDD TFs under flooding stress

Flooding stress is a major problem for field-cultivated tomatoes because of the intensive rainfall patterns that induce climate change45. Waterlogging reduces oxygen availability to the submerged plant parts, which subsequently leads to cell death and, eventually severe yield losses46,47. Because IDD TFs are plant-specific, they can potentially respond to flood stress. To determine the response of IDD TFs to flood 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 flood, SlIDD12 showed an 80-fold increase in expression. Moreover, SlIDD3, SlIDD6, SlIDD9 and SlIDD18 showed significantly 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 flood stress (Table S10).

Figure 8
figure 8

Expression patterns of screened zinc finger TFs under flood stress. (a) Clustergram for IDD expression levels under flood stress in 3-week-old tomato seedlings (Scales represented as relative values). (b) Expression levels of high responsive SlIDDs under flood stress. (**P < 0.01; *P < 0.05).

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Discussion

Functional analysis of SlIDDs

The present study systematically analyzed IDD TFs belonging to tomatoes, as IDDs in Arabidopsis, rice, and maize have already been examined for their existence and properties22,23,24,25,27,48,49,50,51. Currently, there are 16, 15, and 22 IDD TFs identified 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-specific manner. IDD TFs are also plant-specific and can participate in multiple plant-specific functions such as vascular development, photosynthesis, light signaling, flowering etc52. Moreover, plant-specific 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-specific TFs show differences 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 gibberellin 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 different light conditions and regulates flowering and plant size56. OsIDD1 along with OsIDD6 potentially have redundant functions in floral transition58. OsIDD1 also regulates the expression of JA-related genes related to herbivore resistance59. SlIDD1 expression was significantly down-regulated in salt-stressed roots and increased in salt-stressed shoots after 24 h. Under acidic conditions, SlIDD1 expression was reduced within a short time and recovered after 24 h. Under flooding conditions, the shoots showed significantly higher expression levels, suggesting a pivotal role in the transition under stress conditions (Figs. 6, 7, 8). SlIDD17 is significantly responsive to salt and acidic stress. Previous studies have shown increased expression of SlIDD17 under heat stress and during fruit development60,61.

SlIDD2 is closely related to AtIDD7, AtIDD11, OsIDD2, OsIDD11, SlIDD10 and SlIDD11 (Fig. 1b). AtIDD7 shows higher activity during phosphorus starvation and flowering62,63. 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 significantly downregulated in the roots and increased in the leaves. SlIDD2 is down-regulated in the base margin tissue of tf-2 leaf patterning-deficient 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 softening and increased shelf life67,68. In Arabidopsis, AtIDD7 shows differential expression under phosphorus starvation69, early flower 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 specificity in exotic land races73,74. Notably, SlIDD11 produced five isoforms that may be expressed under specific stress conditions to respond specifically (Table S5).

SlIDD3 grouped along with AtIDD12 and SlIDD15. SlIDD3 showed higher expression patterns in the exodermis 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 significant response to salt and acid stress in roots and to flood stress in shoots, suggesting a role in 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 replacement of Histidine by Arginine might not have any major effect 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 (homobaric 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, flower development, plant architecture, and gravitrophic responses by regulating auxin biosynthesis and transport factors23. Surprisingly, OsIDD14/LPA1 also shows similar functions in plant architecture and has been extensively studied. LPA1 determines rice tiller angle and shoot gravitropism by affecting 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 significant 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 floral 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 first 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 expression in RIN mutants, suggesting its potential role in fruit ripening93,94. The 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 specific38. In contrast, SlIDD3, SlIDD8, SlIDD9, and SlIDD12 showed multi-stress responses, suggesting that these TFs should be further studied for their effects on tomato survival and productivity.

Future perspectives for IDDs for breeding climate-resilient and high-producing crops

The 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 effective root systems and vascular systems for water transport, increasing photosynthetic ability and survival adaptations, such as distinguishing beneficial organisms from pathogens and predators, and adapting to dry terrain100. Plant-specific 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 breeding programs are exploring possibilities to develop climate-resilient cultivars for better productivity103,104,105,106. 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 significant 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-specific and are involved in functions such 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, flooding, 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 exposure 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 insignificant110,111,112. 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 cultivation114,115,116. 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. This 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 damages119,120,121,122,123. The isoforms of SlIDDs can be further dissected based on their specific 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 artificial conditions124,125. The current analysis identified 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 five isoforms respectively. 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. These 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 RNAi126,127,128,129 can be employed to study the molecular functions of SlIDDs. Natural disasters and temperature fluctuations 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 different databases for tomatoes (Solgenomics network; https://solgenomics.net/, Plaza 5.0; https://bioinformatics.psb.ugent.be/plaza/ and Gramene; https://www.gramene.org. Arabidopsis and Rice sequences were verified using TAIR10 (https://www.arabidopsis.org/) and RAP-DB (https://rapdb.dna.affrc.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 ALIGNMENTVIEWER (https://github.com/sanderlab/alignmentviewer) software. 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. The iTOL web tool was used to construct the evolutionary tree (https://itol.embl.de).

Chromosomal location, synteny analysis, motif visualization, and 3D structure visualization

The locations of candidate genes were acquired from the Solgenomics network (https://solgenomics.net/), and positions were visualized using MG2C v2.1 (http://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/almeidasilvaf/doubletrouble)131. The 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. The 3D structure was identified using a UniProt (https://www.uniprot.org) database search and visualized using Afphafold2.0 (https://www.uniprot.org/database?query=(name:AlphaFoldDB)&direct).

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/fimo) against Arabidopsis promoter matrices (http://bar.utoronto.ca/~nprovart/ArabidopsisDAPv1.meme) based on previous reports11,66,132,133. The cutoff 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. The 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. The light/dark cycle was 16 h/8 h/day. Plants were supplied with nutrients in the irrigation water one month after transplanting, following the manufacturer’s guidelines (S-feed, 1 kg/10 a/day; https://www.farmhannong.com/kor/product/product_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 after 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 greenhouse. 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 Thermocycler system (Bio-Rad, Hercules, CA, USA). Primer information is provided in Table S11. Reactions (10 µL final 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 quantification. Ubiquitin was used as a reference. gene expression analysis was performed with the 2^ − ΔΔCt method using Bio-Rad CFX Maestro software v.4.0 (Bio-Rad). The 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.