We used eggplant (S. melongena) variety BARI Begun-4 which was developed by the Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur, Bangladesh. Specifically, 3-week old seedlings were grown hydroponically in Yoshida nutrient solution (YS; HIMEDIA, Mumbai, India) and subjected to different stresses like heat (45°C), salt (200 mM NaCl), and drought (150 mM) mannit to understand the response of SmsHSP24.1 expression in leaf tissues. Plants irrigated with water served as treatment controls (CT). Eggplant leaves were collected at 0, 1, 2, 4, 6, 12, and 24 h after stress treatment and used for RNA isolation followed by subsequent gene expression analysis.

For physiological analysis of SmsHSP24.1 overexpressing transgenic eggplant lines, three different southern positive lines (OE1, OE3, and OE7) were used under both normal and stress conditions. Specifically, 2-week old seedlings of OE1, OE3, and OE7 lines along with wild-type plants (WT) shifted in soil pots were used for heat and drought stress experiments, and seedlings transferred to Yoshida nutrient solutions were used for salt stress. After 1 week of adaptation, seedlings were exposed to heat shock (45°C) for up to 36 h. Yoshida nutrient solution was supplemented with 200 mM NaCl for salt treatment for 12 h and as drought treatment, water was withheld for 10 consecutive days to study the growth parameters of transgenic lines (OE) compared with the control (WT). Similarly, after combined heat (45°C) and dt stress by withdrawal water for 25 days at ICGEB net house field, leave tissues were collected. These samples were used to study oxidative damaged, biochemical and enzymatic assays as well as expression profiling of the target transcripts, i.e., NADH-ubiquinone oxidoreductase, quinol-cytochrome c- oxidoreductase, aminopeptidase (PepA), auxin-responsive gene (SAUR), superoxide dismutase (SOD1, SOD2), catalase (CAT), and ascorbate peroxidase (APX) transcript by qRT-PCR.

Genomic DNA was extracted from leaves of 21-days-old eggplant seedlings using the PureLink Genomic Plant DNA Purification Kit (Invitrogen¹, United States) according to the instructions of the manufacturer. Total RNA was extracted from eggplant tissues using the PureLink Plant RNA Reagent kit (Invitrogen; see text footnote 1). RNA was treated with RNase-free DNase (NEB², Ipswich, MA, United States) and used for first-strand cDNAs synthesis according to Verso cDNA Synthesis Kit (Thermo Fisher Scientific, United States; see text footnote 1). Real-time qRT-PCR was performed with SYBR Green PCR Master Mix and Applied Biosystems in 7500 Real-Time PCR Systems (Thermo Fisher Scientific, United States; see text footnote 1). Eggplant 18S rRNA-specific primer pair was used as an internal reference gene. All primer pairs used for RT-PCR and qRT-PCR are mentioned in Supplementary Table 2.

The putative eggplant mitochondria localized nuclear SmsHSP24.1 gene was identified using NCBI expressed sequence tags (ESTs) database (Supplementary Figure 1A). The full-length SmsHSP24.1 cDNA was amplified by nested PCR using a high-fidelity DNA polymerase (KOD plus, Toyobo, Japan) with primer pairs of SmsHSP24.1_F1/R1 and F2/R2 (Supplementary Table 2) and cloned into PCR-4-TOPO vector (Invitrogen, United States). Sequence confirmation was done using universal M13 forward and reverse primers through the Macrogen sequencing platform (Seoul, Republic of Korea) (macrogen.com/ko). MODELLER 9 version 11 (Sali et al., 1995) program was used for homology modeling of SmsHSP24.1 against Mito_AtHsp21 (Protein databank ID P31170) (Supplementary Figure 1B). Evaluation tools ProCheck (Laskowski, 1993) and Verify3D (Eisenberg et al., 1997) were applied to assess the predicted three-dimensional model of SmsHSP24.1 protein. The Muscle (Edgar, 2004) program was used for multiple sequence alignment, and a phylogenetic tree was constructed using MEGAX (Kumar et al., 2018) according to the neighbor-joining method with bootstrap value 1,000 (Supplementary Figure 1C).

Bioinformatics-based prediction tools TargetP³ (Denmark) and MitoFates (Fukasawa et al., 2015) were used to check the SmsHSP24.1 protein targeting signal (Supplementary Figure 2). To further in vivo confirmation, SmsHSP24.1 protein-coding sequence without stop codon was cloned into mGFP based pCAMBIA1302 expression vector with hygromycin selection. BglII and SpeI restriction enzymes were used for the cloning of this sequence (Supplementary Figures 3A-C). Primers used for cloning confirmation are enlisted in Supplementary Table 2. Agrobacterium strain EHA105 was used for transient expression of the final expression cassette in tobacco leaf epidermal cells by following the method described by Kokkirala et al. (2010). After 72 h of inoculation, the leaf tissues were observed under a confocal fluorescence scanning microscope (Zeiss LSM510, Germany).

Similarly, stably integrated eggplant cell suspension culture with the same construct was used to further confirmation by co-localization with mitochondria specific dye MitoTracker^TM Red FM (Cat no: M22425; Invitrogen, United States) following a method similar to the one developed in the crop improvement lab of Reddy (data unpublished, ICGEB). Samples were prepared by adding 10 μl of suspended cells on a slide, and photographs were taken by laser scanning confocal microscopy (Zeiss LSM510, Germany). The fluorescence signal was collected using an emission filter of a 500–535 nm bandpass for GFP and 581–644 nm bandpass for MitoTracker^TM Red FM. All fluorescence experiments were independently repeated at least three times.

The SmsHSP24.1 coding region (NdeI + NotI) was first cloned into Gateway compatible entry vector (pL12R34-Ap) under constitutive cauliflower mosaic virus promoter (CaMV 35S) (KpnI + NdeI) and nopaline synthase terminator (Nos) (NotI + SacI) and then transferred into the plant transformation vector pMDC100 (Invitrogen, United States) containing nptII (kanamycin-resistant) gene as a plant selection marker (Supplementary Figures 3D-G). Agrobacterium strain, EHA105 was used for plant transformation, and 21-days-old cotyledonary leaves were used as starting material. Kanamycin (100 mg/l) was used at a 10-day interval to select transformed lines. The putative transgenic lines were screened by PCR using nptII and SmsHSP24.1 gene-specific primers. For Southern blot analysis of putative transgenic lines, approximately 20 μg genomic DNA was digested with the restriction enzyme, NdeI. DNA fragments were separated on a 0.8% agarose gel and blotted onto the Hybond^TM N⁺ nylon membrane (GE Healthcare Limited, United Kingdom). Subsequently transferred N⁺ nylon membrane was hybridized with a 500 bp of SmsHSP24.1 gene-specific DIG-labeled probe (PCR DIG Probe Synthesis Kit, Roche, Germany). The blot was then washed and detected according to the instructions of the manufacturer (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche, Germany).

The protein-coding sequence SmsHSP24.1 was cloned between NdeI and NotI restriction sites of pET28a vector (Supplementary Figures 3H-K; Primers enlisted in Supplementary Table 2) for recombinant protein production in E. coli BL21 (DE3) cells. 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was used for induction at different temperatures (37, 28, and 18°C) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE).

For studying the thermotolerance capacity of SmsHSP24.1 protein, we followed a method similar to the one described by Wang et al. (2017). Escherichia coli harboring pET28a:SmsHSP24.1 and pET28a blank plasmid were used for the assay at 37, 42, 48, 52, 55, and 58°C, respectively. Cell viability was estimated by counting the number of colony-forming units. Parallelly total soluble protein was also analyzed from supernatants and pellets in SDS–PAGE, visualized by Coomassie Blue staining.

For the chaperone-like activity assay, thermal-induced aggregation of alcohol dehydrogenase (ADH) (50°C) and citrate synthase (CS) (45°C) were measured in the presence or absence of SmsHSP24.1 protein at various molar ratios, according to Ihara et al. (1999). Protein aggregation was monitored by measuring light scattering at 360 nm in three replicates for each treatment. The measurements were performed using Helios Gamma Spectrophotometer (Thermo Spectronic, Cambridge, United Kingdom). For further investigation, electron microscopic analysis was performed with purified SmsHSP24.1 protein and the model substrate, citrate synthase (CS) before and after thermal-induced aggregation according to Zhang et al. (2015). Electron micrographs were recorded with a JEOL 1400 transmission electron microscope operated at 100 kV in ICGEB, India facility.

Seed germination assay of WT and OE lines was done under normal and abiotic stress conditions. MS growth medium was supplemented with 200 mM NaCl for salt stress and 150 mM mannitol for drought stress. For heat stress, seeds were first kept in an incubator at 45°C heat for 2 and 4 h then placed in full strength MS growth medium. A growth room condition of 16 h photoperiod at 25 ± 2°C temperature was provided. Germination rates were calculated each day, up to 7-day incubation, and representative seedlings were photographed.

Total chlorophyll content of OE and WT seedlings under untreated and stressed conditions were measured using a spectrophotometric method described by Lichtenthaler and Wellburn (1976) with slight modification. Approximately 150 mg of leaf tissue was used for chlorophyll measurement, with each reaction was performed in three replicates. The absorbance was measured at 441, 646, 652, and 663 nm using a Helios Gamma Spectrophotometer (Thermo Spectronic, Cambridge, United Kingdom). Physiological parameters, namely plant height (cm), shoot length, root length, fresh weight, and dry weight of transgenic and WT lines were measured under normal and abiotic stress conditions.

Transgenic lines and WT plants from untreated and stressed conditions were harvested for assay of antioxidant enzyme activity. Approximately, 200 mg of fresh leaves were finely ground in ice-cold 0.2 M phosphate buffer (pH 8) and centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was kept on ice. The superoxide dismutase (SOD) activity was performed as previously described by Beyer and Fridovich (1987). Absorbance was measured at 560 nm Ascorbate peroxidase (APX) activity was determined by following dihydrogen dioxide (H₂O₂) dependent oxidation of ascorbic acid (ASC) at 290 nm. The assay for catalase (CAT) activity was performed according to the protocol of Beaumont et al. (1990), which monitored the dismutation of H₂O₂ at 240 nm. Electrolyte leakage was measured as previously described (Sairam et al., 2002). Measurement of proline was also performed according to the protocol of Bates (1973).

Leaf discs from both WT and transgenic plants of similar age were treated with 10 μM methyl viologen (MV; paraquat) for oxidative damage, 200 mM NaCl as salt stress, and 150 mM mannitol as drought stress. For MV stress, leaf discs were incubated for 6 h at 28°C dark in a shaker incubator and then exposed to light. Salt and drought-imposed leaf discs were observed after 72 h of incubation under the light. Generation of H₂O₂ was detected with 3, 3-diaminobenzidine (DAB) as previously reported by Thordal-Christensen et al. (1997). In vivo generation of O^–2 in tissues after applying the stresses mentioned was detected by staining with 1% nitro blue tetrazolium (NBT) described by Fryer et al. (2002). Similarly, cell death was also detected in treated samples after staining with lactophenol trypan blue as described by Keogh et al. (1980).

Total RNA was extracted from the leaves of 3-week-old WT and T2 generation transgenic eggplants (OE7) under either normal or heat stress (45°C) conditions using the RNAeasy Plant Mini Kit (Qiagen⁴, Germany) according to the instructions of the manufacturer. The quality and quantity of RNA were confirmed by RNase-free agarose gel electrophoresis and a Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). Library construction and RNA sequencing were carried out by Agri-Genome Labs, Kerala, India. Only samples with RNA integrity of N7.0 were used for RNA-seq analysis. Illumina HiSeq 2500 platform (Illumina Inc., CA, United States) was used for RNA sequencing. All the RNA-seq data were then successfully submitted to the NCBI SRA database (PRJNA750594). The pre-processed and rRNA removed reads were aligned to the tomato reference genome, and the gene model downloaded from Sol Genomics Network⁵. After aligning the reads with the reference genome, the aligned reads were used to estimate genes and the expression of transcripts using the cufflinks program (version 2.2.1, United States). Differential expression analyses of all control and treated samples were performed using the cufflinks program. Levels of gene expression were measured by the fragments per kilobase of transcript per million fragments mapped (FPKM) plot. Log2 fold change cut-off 2 and p-value cut-offs.01 and.05 were used separately as cut-offs for up and down-regulated genes and isoforms. Clustered heat maps of up- and down-regulated genes of sample comparison with a p-value cut-off of.05 were generated. Gene ontology (GO) enrichment analysis was performed using the GOSeq tool (default parameters; United States). GOseq functions in calculating the significance of over-representation of each GO category amongst differentially expressed genes (DEGs). The KEGG pathway analysis was carried out for the available differentially expressed genes using a bio-conductor tool, path view with the tomato reference genome.

Statistical analyses were performed by one-way ANOVA and the differences between means were compared with Tukey HSD.5 values obtained for the particular dataset.

The putative 1032 bp gene was retrieved by NCBI ESTs blast using the tomato Mito-slHSP23 (BAA32547.1) full-length cDNA as a query. In silico sequence analysis using eggplant draft genome database (Hirakawa et al., 2014), we found one copy of mitochondria-targeted small HSP gene at Sme2.5_00899.1_g00005.1 contig with two exons and an open reading frame (ORF) of 636 bp. We amplified the SmsHSP24.1-ORF with nested PCR primers that encode a protein with an apparent molecular weight of 24.1 kDa and an isoelectric point of (pI) 4.84. Blastp homology search and multiple sequence alignment and homology modeling indicated that SmsHSP24.1 contained a conserved α-crystalline domain (ACD) of 81-amino-acid at the C-terminus (positions + 115 to + 196). Phylogenetic analysis with the deduced amino acid sequences from a diverse range of species revealed that this protein was closely related to tomato (SlsHSP23.5) and Arabidopsis (AtsHSP26.4). Therefore, we designated our protein as SmsHSP24.1 and submitted it to the NCBI gene bank (AXS76128.1).

We had investigated SmsHSP24.1 transcript abundance to understand its physiological role under abiotic stress conditions. The level of SmsHSP24.1 transcript in leaves under heat (45°C), salt (200 mM NaCl), and osmotic stress (150 mM mannitol) were compared with untreated seedlings as relative fold-change (Figures 1A-C). The relative fold change of SmsHSP24.1 under heat stress was found to be the highest among all the treatments. An elevated transcript level was observed within 1 h after the heat treatment which reached a maximum of 9-fold change at 2 h time point and then gradually declined (Figure 1A). In the case of salt stress, the expression dynamics of the SmsHSP24.1 transcript were found to be different from the heat stress. An approximately 5-fold increase of SmsHSP24.1 transcript was observed at the 4 h time point of salt stress after which it reduced gradually until 12 h and then a significant upregulation was observed again at the 24 h timepoint (Figure 1B). A quick expression of SmsHSP24.1 was witnessed when treated with mannitol reaching a peak as soon as this osmotic agent was added to the plant. However, after 2 h the level of SmsHSP24.1 transcript started to decline steadily up to 12 h and at 24 h, it was 1.5-fold lower than the level transcript observed at 1 h of drought stress (Figure 1C).

In silico analysis using TargetP (see text footnote 3) and MitoFates (Fukasawa et al., 2015) revealed that the SmsHSP24.1 protein was localized in mitochondria with a significantly high score of 0.792 and 0.997, respectively. Similarly, in vivo, transient expression assay of Agrobacterium harboring SmsHSP24.1 fused with mGFP on tobacco leaf (3-week-old seedlings) also indicated discreet localization other than the nucleus (Figures 1D,E). Furthermore, co-localization analysis with mitochondria specific dye (MitoTracker^TM, United States) FM (Cat no: M22425; Invitrogen) in eggplant cell suspension-culture conclusively confirmed that this novel SmsHSP24.1 was targeted to the mitochondria (Figure 1F).

We performed a series of experiments to analyze whether the novel SmsHSP24.1 protein can function as a chaperone and enhance survivability under severe stress conditions. First, we had expressed the coding sequence of SmsHSP24.1 protein in the BL21(DE3) strain of E. coli using the expression vector pET28a. Western blot analysis with an anti-His-tag antibody demonstrated the presence of the recombinant protein in the E. coli cells (Figures 2A-C). A Thermotolerance assay was conducted with a pET28a vector carrying SmsHSP24.1 and blank pET28a as an experimental control (Figure 2D). No growth difference was observed under normal growth conditions, but 65% of E. coli culture carrying pET28a-SmsHSP24.1 construct found to be effectively tolerated temperature up to 55°C for 1 h, whereas the control cells failed to survive (Figure 2E). Only 10% of the cells carrying the blank pET28a at 50°C survived 1 h compared with 50% of E. coli carrying the pET28a-SmsHSP24.1 construct. SDS–PAGE analysis of the soluble fractions further revealed a significant increase in total soluble proteins in the pET28a-SmsHSP24.1 carrying BL21 cells compared with pET28a-blank at different temperatures (Figure 2F).

To further understand, we conducted a transmission electron microscopic thermal-aggregation assay of two commonly used heat-sensitive model substrate proteins, ADH, and CS, at different molar ratios of SmsHSP24.1. Thermal inactivation and aggregation of CS and ADH were detected by light scattering at 45°C and 50°C, respectively (Figures 2G,H). SmsHSP24.1 delayed aggregation of CS at a molar ratio of 1:1 (SmsHSP24.1:CS), and at a 2:1 molar ratio, it fully protected CS from aggregation. Similarly, the aggregation of ADH was also found to be entirely prevented at a 2:1 (SmsHSP24.1:ADH) molar ratio. Transmission electron microscopic (TEM) analysis further showed CS is in its native state at 4°C, whereas it was completely denatured and aggregated at 45°C without the presence of SmsHSP24.1 (Figure 2I), On the other hand, while incubated with SmsHSP24.1, the formation of polyhedron assembly under TEM was observed which correlates with the thermal protection of CS at 45°C up to 60 min (Figure 2J).

To further evaluate the physiological role of SmsHSP24.1, we developed independent transgenic eggplant lines constitutively overexpressing SmsHSP24.1 protein (Supplementary Figure 4A). PCR screening with two sets of primers (NptII primer sets ∼0.45 kb and SmsHSP24.1 + NosT terminator junction primer set ∼0.9 kb) found 17 out of 20 putative T0 plants to be positive (Supplementary Figure 4B). Homozygous T2 OE lines were selected under 100 mg/L kanamycin and reconfirmed by PCR (Supplementary Figures 4C,D). Same lines were further used for Southern blot analysis to confirm the copy number of transgene integration using the SmsHSP24.1 gene-specific probe. We obtained two single, two double, one triple, and one multi-copy (containing five copies of insertions) transgenic events with morphological parameters similar to WT eggplants (Supplementary Figure 4E). Similarly, the copy number of Mito-sHSP (SmsHSP24.1) in the WT eggplants genome was also confirmed by Southern blot analysis (Supplementary Figure 4F). qRT-PCR analysis showed a higher level of transgene expression compared with the WT (Supplementary Figure 4G). Henceforth, we chose single copy integrated transgenic lines (OE1, OE3, and OE7) with high transgene expression from the T2 generation for subsequent physiological analysis.

Transgenic (OE1, OE3, and OE7) and WT plants were tested under normal and stress conditions. Both grew well and produced new leaves in the untreated condition. However, upon heat stress (45°C) for 36 h, OE lines exhibited marked thermotolerance compared with WT (Figure 3A) and wholly recovered when the stress was withdrawn. During drought stress induced by withholding water for 10 days, WT plants exhibited severe wilting, and some died, while only minor signs of dehydration were observed in OE lines. After 1 week of re-watering, 80% of the OE lines revived compared with 30% WT plants (Supplementary Figure 5). Also, when seedlings were treated with 200 mM NaCl for 3 days, WT plants either exhibited significant growth inhibition or died compared with OE lines. It was also observed that seedlings of OE lines were less prone to chlorosis compared with WT plants (Figure 3B). Furthermore, the OE seedlings exhibited significantly greater fresh weight, dry weight, root length, shoot length, and chlorophyll content under heat, salt, and drought stress compared with the WT plants (Figures 3C-E and Supplementary Figures 5A-C).

A field study of combined heat and drought stress was also conducted at the ICGEB net house facility, New Delhi. In here, two and half-month-old seedlings of both OE lines and WT plants were subjected to 25 days of water withdrawal and an average of 43°C air and 50°C soil temperature at the reproductive stage. Under such conditions, most WT seedlings were found to be severely injured, whereas OE lines remained green and healthy (Figures 4A-C). OE lines (OE1, OE3, and OE7) recovered quickly without any effect on flowering and fruit setting after withdrawal of stress, whereas the WT plants were completely retarded (Figure 4D). Physiological parameters also showed higher fresh and dry weight in all OE lines compared with WT under control and stress conditions (Figure 4E). Besides, semi-quantitative RT-PCR analysis showed increased accumulation of SmsHSP24.1 transcript in all OE lines both in control and combined stressed plots compared with WT (Figure 4F). Enzymatic activity of ROS-scavenging enzymes, such as SOD, APX, and CAT was also monitored in both OE and WT lines from the combined stress plot. OE lines were found to maintain a high level of all the ROS-scavenging enzymes compared with WT plants (Figure 4G). Similarly, proline content showed an increase in overexpressed lines, whereas MDA content had decreased significantly after stress treatment in the OE lines compared with WT plants (Figures 4H,I). This data was found to correlate with RNA-seq, and semi-quantitative RT-PCR analysis carried out on 3-week-old heat stress OE lines.

To evaluate the impact of overexpressed SmsHSP24.1 in mitigating ROS damage, we conducted leaf disc assay separately for heat (45°C heat for 36 h), osmotic stress (150 mM Mannitol), salt (200 mM NaCl), and MV (10 μM) to investigate the H₂O₂, O^2– contents and cell death after stress (Figure 5 and Supplementary Figure 6). Reduced and slow chlorosis was observed for SmsHSP24.1 overexpressing event compared with WT plants after stress treatments. The OE lines overexpressing SmsHSP24.1 showed a significantly lower level of ROS production and cell death compared with WT plants after MV (Supplementary Figure 6A) and heat treatment as were evident by DAB, NBT, and Trypan blue staining (Figure 5A). We also noticed that the H₂O₂ levels in WT plants were approximately 3- to 4-fold higher than OE lines after the heat, salt, mannitol, and MV treatment (Figure 5B). However, it was interesting to note that under normal conditions, the OE lines maintained a slightly higher cellular H₂O₂ level compared with WT plants. However, we have observed that this slight increment of cellular ROS in OE lines did not hamper the growth under normal environmental conditions, while no lethal increment in the level of H₂O₂ was observed in OE lines compared with WT plants upon stress treatment (Figure 5B). This finding was also supported by a comparative analysis of chlorophyll to be retained and ion leakage between OE lines and WT plants. OE lines were found to prevent chlorophyll damaged and reduced ion leakage probably due to the protection from oxidative damage through decreased ROS production under stress conditions (Figures 5C,D). As described in the previous data (Figure 4C), OE lines are able to maintain a higher level of ROS scavenging enzymes compared with WT plants due to which ROS production could not be increased up to lethal level.

We observed a significant rate of early seed germination and seedling vigor, both in vitro and field condition in OE lines (OE1, OE3, and OE7) compared with WT (Supplementary Figure 7A). Furthermore, a 95% germination rate was observed within 3 to 4 days in OE lines (OE1, OE3, and OE7) in the growth medium, while 6 days were required to attain an 80% germination rate in WT (Supplementary Figure 7A). A similar result was observed when seeds were sown in the field under normal conditions. Physiological parameters obtained at 2 weeks after germination revealed significantly higher total fresh weight, dry weight, root length, shoot length, and chlorophyll content in all OE seedlings compared with WT seedlings (Supplementary Figures 7B-D).

We further conducted transcriptome analysis to understand the possible molecular mechanisms underlying the effect of early seed germination and seedling vigor. The comprehensive transcriptome data have been presented separately; however, changes in transcription were noticed in three key regulatory pathways; ETC and GSH, as well as auxin biosynthesis and transport (Figures 6A,B and Supplementary Figure 8). In ETC, NADH-ubiquinone oxidoreductase transcript was found to be significantly upregulated while quinol-cytochrome c- oxidoreductase was slightly down-regulated in OE lines compared with WT lines under both normal and stressed conditions. qPCR cross-validation also revealed similar results (Figure 6C). PepA (aminopeptidase) gene from the GSH pathway was found to be highly upregulated in OE lines. It was approximately 5-fold higher in OE lines compared with WT (Figure 6C). Similarly, significant upregulation was also observed for auxin carrier proteins like auxin-responsive SAUR-like genes. Likewise, RNA-seq values of differentially expressed transcripts of SOD1, SOD2, CAT, and APX in OE lines, further confirmed by qPCR (Figure 6C).

Transcriptome profiling of OE lines compared with WT plants further provide evidence of a significant impact on global transcriptome due to the overexpression of SmsHSP24.1. During this experiment, an average of 56.1 and 83.5 million raw reads (2 × 100 bp) were generated from transgenic (OERT) and WT (WTRT) lines, respectively, under controlled environmental conditions. Similarly, an average of 73.5 and 82.5 million raw reads (2 × 100 bp) were generated under the heat-stressed conditions at 45°C for 2 h from transgenic (OE2hH) and WT (WT2hH) line respectively.

After adaptor trimming and base quality check, it was found that more than 95% of total reads were of high quality (HQ) except for the WT2hH plant, in which after the removal of rRNA, the value was 87%. Mapping of cleaned reads with tomato reference genome and gene model downloaded from Sol Genomics Network (Fernandez-pozo et al., 2015) found 65.6% of OERT, 52.8% of WTRT, 56.88% of OE2hH, and 57.76% of WT2hH reads to be mapped (Supplementary Table 3). Differential gene expression analysis revealed significant transcription alteration in some of the physiologically important pathways (Figures 6A,B and Table 1). Heat map illustration with cluster dendrogram based on FPKM plot and GO classification of DEGs in WT and OE also revealed the up- and down-expression of important genes (Figures 7, 8 and Supplementary Figures 9–11). Among the several DEGs, Photosystem II reaction center protein M (Solyc09g064580), Chloroplastic matK (Solyc09g061390), genes involved in electron transport chain (Solyc09g074540, Solyc11g044636) were significantly upregulated in OE_vs_WT samples at RT (Figure 7 and Table 1), while SOD (Solyc01g067740), ABC transporter that regulates lipid transporter (Solyc03g113070), GLB1(nitrogen regulatory P-II-like protein) regulated fatty acid biosynthesis (Solyc07g008240) (Duan et al., 2017), in OE_vs._WT samples after heat treatment, respectively (Supplementary Figure 9). Similarly, significant down-regulation was also observed in transmembrane transporters like F-box domain-containing protein (Solyc07g044920), iron-nicotianamine transporter (Solyc03g082620), calcium-binding protein CML44 (Solyc04g058170), and LOB1 (Solyc06g064540) under normal conditions, while ADF-H actin-binding protein (Solyc02g063450), cell division cycle protein- CDC27B (Solyc03g061590), cell number control protein- PLAC (Solyc02g079390), and FA_desaturase domain-containing protein (Solyc10g011810) in WT eggplants compared with OE lines upon heat treatment (Supplementary Figure 10 and Supplementary Table 1). GO term-based gene classification for DEGs in WT, and OE samples also revealed a large number of genes affected significantly. Twelve major pathway-related genes were found to have distinctive differential expression in OE treated lines compared with WT (Figure 8 and Table 1). Similarly, a total of 185 genes are down-regulated in the same event (Figure 8 and Supplementary Table 1). Among the genes for transcription factors, BZIP transcription factor (Solyc01g109880.3), ethylene-responsive factor 2; ethylene-binding protein (Solyc09g075420.3), and NAC4 domain protein (Solyc11g017470.2) were found to be down-regulated whereas a total of 18 different transcription factors (Tfs), most of which were auxin-responsive/ethylene-responsive, DREB, GATA, and TCP family transcripts were found to be upregulated in OE lines.

Well-known ROS-scavenging genes such as SOD, catalase, ascorbate-peroxidase were significantly upregulated in OE lines under both normal and treated conditions. An extensive set of genes involved in fatty acid biosynthesis (28 in number, Table 1), chlorophyll biosynthesis, genes involved in the developmental pathway (32 in number), and mitochondrial integral membrane protein have identified upregulated. Among phytohormones, auxin biosynthesis/carrier-related transcripts upregulated in OE lines (Table 1). A total of 14 auxin pathway genes were significantly upregulated in OE lines compared with WT lines. Transcript for auxin-induced SAUR-like protein showed 4.3-fold upregulation. Significant upregulation was also observed for cytokinin oxidoreductase and cytokine-responsive transcripts in OE lines. This considerable alteration in transcription might have significant effects on OE lines, and our RNA-Seq data reveal substantial transcriptional reprogramming in several physiologically essential pathways of the OE lines (Table 1). DEGs involved in other essential pathways were also found to be down-regulated (Supplementary Table 1), such as 8 DEGs involved in hydrolase activity [GO:0016787], 6 DEGs with ubiquitin-protein ligase activity [GO:0061630], 5 DEGs of cell wall organization or biogenesis pathway [GO:0071554] and 2 each of auxin catabolic process [GO:0009852] and chlorophyll catabolic process [GO:0015996].