Heat shock transcription factors regulate thermotolerance gene networks in tomato (Solanum lycopersicum) flower buds

Abstract

Tomato (Solanum lycopersicum) is an important fruit and vegetable crop in worldwide. The fertility of tomato reproductive organs can be dramatically decreased when ambient temperatures rise above 35 °C, reducing tomato fruit yield. It is necessary to identify transcription factors (TFs) and target genes involved in heat stress response (HSR) signaling cascades in tomato flower buds to improve tomato plant thermotolerance. In this study, we profiled genes expressed in three developmental stages of tomato flower buds. Red and turquoise modules for heat stress (HS) were identified through gene co-expression network analysis, and the genes within these modules were enriched in HS-related pathways. By focusing on the TFs in the two modules, we identified several novel HSR-related TFs, including SIWRKY75, SIMYB117, and SINAM. Furthermore, homology analysis illustrated a conserved signaling cascade in tomato. Lastly, we identified and experimentally validated four HSF-regulated genes, namely SIGrpE, SIERDJ3A, SITIL, and SIPOM1, that likely modulate thermotolerance in plants. These results provide a high-resolution atlas of gene expression during tomato flower bud development under HS conditions, which is a valuable resource for uncovering potential regulatory networks associated with the HSR in tomato.

Headnote

Received 14 February 2023; Accepted 25 June 2023; Available online 14 December 2023

ABSTRACT

Tomato (Solanum lycopersicum) is an important fruit and vegetable crop in worldwide. The fertility of tomato reproductive organs can be dramatically decreased when ambient temperatures rise above 35 °C, reducing tomato fruit yield. It is necessary to identify transcription factors (TFs) and target genes involved in heat stress response (HSR) signaling cascades in tomato flower buds to improve tomato plant thermotolerance. In this study, we profiled genes expressed in three developmental stages of tomato flower buds. Red and turquoise modules for heat stress (HS) were identified through gene co-expression network analysis, and the genes within these modules were enriched in HS-related pathways. By focusing on the TFs in the two modules, we identified several novel HSR-related TFs, including SIWRKY75, SIMYB117, and SINAM. Furthermore, homology analysis illustrated a conserved signaling cascade in tomato. Lastly, we identified and experimentally validated four HSF-regulated genes, namely SIGrpE, SIERDJ3A, SITIL, and SIPOM1, that likely modulate thermotolerance in plants. These results provide a high-resolution atlas of gene expression during tomato flower bud development under HS conditions, which is a valuable resource for uncovering potential regulatory networks associated with the HSR in tomato.

Keywords: Tomato; Heat stress; Transcriptome; Transcription factor; Thermotolerance

1. Introduction

Global temperatures will increase by 2-5 °C by the end of the twenty-first century (Gonzalo et al, 2021), bringing frequent extreme weather and potentially causing extensive agricultural production losses (Xie et al., 2022). Tomato (Solanum lycopersicum), the highest-value fruit and vegetable crop in worldwide, is thermophilic but cannot withstand high temperatures (Gonzalo et al., 2021). Ambient temperatures over 35 °C during the day are considered heat stress (HS) for tomato growth and development (Camejo et al., 2005; Golam et al., 2012). HS can inhibit pollination and stimulate flower abscission, dramatically reducing the yield of tomato fruit (Alsamir et al., 2021). Therefore, it is crucial to understand the regulatory mechanisms of thermotolerance in tomato to alleviate HS damage and cultivate heat-tolerant tomato varieties.

Plants activate the HS response (HSR) under high temperatures to mitigate the detrimental effects of HS (Tian et al., 2017; Chaturvedi et al., 2021; Xie et al., 2022). In tomato, the HSR involves a signaling network containing HS transcription factors (HSFs) and other transcription factor (TF) families (Hahn et al, 2011). In previous studies, HSFA1, HSFA2, HSFA7a, and HSFC have been found to play important roles in the HSR of Arabidopsis thaliana (Lin et al., 2018), pepper (Capsicum annuum) (Albert and Chang, 2014), tall fescue (Festuca arundinacea) (Zhuang et al., 2018), and wheat (Triticum aestivum) (Xue et al., 2015). Moreover, HSFA3 and HSFB1 showed high expression in mature tomato pollen under HS, whereas HSFB2b positively responds to HS postmeiosis (Keller et al., 2018). Apart from HSFs in plants, members of other TF families, such as MYB (v-myb avian myeloblastosis viral oncogene homolog), ERF (ethylene responsive factor), NAC (Petunia NAM, Arabidopsis ATAF1/2 and CUC2), and MBF1C (multi protein bridging factor 1c), also play key roles in the HSR (Zhao et al., 2020; Haider et al., 2022). In wheat, TaMYB80 overexpression upregulated the expression of the ERF family TF DREB2A under HS through an abscisic acid-dependent pathway (Zhao et al., 2017). DREB2A directly regulated HSFA3 transcription under HS in Arabidopsis via a coactivator complex consisting of Nuclear factor Y, Subunit A2 (NF-YA2), NF-YB3, and DNA polymerase II Subunit B3-1 (DPB3-10)/NF-YC10 (Sato et al., 2014; Zhao et al., 2020). In rice (Oryza sativa) and wheat, the NAC TFs OsNTL3 and TaNAC2L activate the expression of HSFAs to enhance plant thermotolerance (Guo et al., 2015; Liu et al., 2020). In Arabidopsis, HSFA1s regulated MBF1C, a highly conserved transcriptional coactivator and key regulator of thermotolerance (Suzuki et al., 2011).

Moreover, HSFs rapidly induce the expression of heat shock proteins (HSPs) and other target genes to enhance thermotolerance in plants (Ohama et al., 2017; Wang et al., 2020; Zhao et al. 2020, 2021). HSPs are chaperones synthesized in the cytosol and organelles that maintain protein stability and prevent cellular collapse under HS (Krishna, 2003). In recent years, several other HSF-regulated genes involved in the plant HSR have been described, such as TaGAAP and TaRCA in wheat (Xue et al., 2015), GolS1 in Arabidopsis (Busch et al., 2005), and seven genes, namely Cu/Zn-SOD, GST8, MDAR1, UBP5, UBP18, RPN10a, and ATG10 in tomato (Xie et al., 2022). Nevertheless, the mechanisms regulating tomato thermotolerance remain elusive, especially in reproductive organs.

In this study, we analyzed the transcriptome of tomato flower buds under HS (37 °C) to elucidate HS-mediated thermotolerance mechanisms. We detected two modules of co-expressed genes for HS, and some genes within these modules were enriched in HS-related pathways. From these data, we identified several novel HSR-related TFs, such as SIWRKY75, SIMYB117, and ЯМАМ. Furthermore, we identified and experimentally validated four thermotolerance genes, namely SIGrpE, SIERDJ3A, SITIL, and SIPOM1, regulated by HSFs in the conserved HSR signaling cascade in tomato. Our system-level approach provides critical functional insight relevant to understanding the potential regulatory networks associated with the HSR in tomato flower buds. 2. Materials and methods

2.1. Plant material and experimental conditions

Tomato plants (5. lycopersicum 'Heinz 1706') were cultivated in a growth chamber under a 16 h/8 h light/dark cycle with day and night temperatures of 25 °C and 20 °C, respectively, and 65%-70 % relative humidity. Plants were grown to harvest flower buds of three stages (namely 4-, 6-, and 8-mm) based on the pollen development stages (pollen mother cell stage, tetrad stage, and uninucleate microspore stage). Thirty eight-week-old plants were divided evenly into two groups and transferred to a growth incubator with a 16 h/8 h light/dark cycle and a day temperature of 25 °C (control, CK), or 37 °C for 0.5 h or 1 h (heat stress, HS). Under HS, the flower buds per stage of 0.5 h and 1 h were combined as a biological duplication. The flower buds were harvested, rapidly frozen in liquid nitrogen and then stored at -80 °C for further RNA sequencing (RNA-seq) analysis. All experiments were performed with three biological replicates, and each replicate consisted of five buds of equal size.

2.2. Total RNA extraction and RNA-sequencing library preparation

Total RNA of tomato flower buds under CK and HS was extracted with the Quick RNA isolation Kit following the manufacturer's protocol (Huayueyang, China). The extracted total RNA was stored at -80 °C. The concentration and quality of total RNA samples were analyzed using a NanoDrop 2000 (Thermo, MA, USA). The A>60o/A>g0 ratios of individual samples were all above 2, indicating that RNA is pure. The 285/185 ratio and the RNA integrity number values were determined using an Agilent 2100 system (Agilent, California, USA). RNA libraries were constructed and sequenced using the platform of Beijing Genomics Institute (Beijing, China). In addition, about 1.5 mg of each RNA sample was used for cDNA synthesis (Maxima H Minus cDNA Synthesis Master Mix, with dsDNase, Thermo Scientific).

2.3. Transcriptome bioinformatics analyses

Raw RNA-seq reads were processed to trim terminal low quality bases and adaptor sequences refer to Yan et al. (2013). High-quality RNA-seq reads were aligned to the tomato genome with version 4.0 (The Tomato Genome Consortium, 2012) using Hisat2 (version 2.0.3) (Kim et al., 2019) with default parameters. Transcript expression levels, in fragments per kilobase per million reads mapped (FPKM), were determined for each sample using featureCounts (version 2.2.1) (Liao et al., 2014). Differentially expressed genes (DEGs) were analyzed with DESeq2 (version 1.24.0) (Love et al., 2014). The thresholds for statistically significant DEGs were P < 0.05 and |log,foldChange| > 1 (Zhu et al., 2023). The expression patterns of DEGs between different samples were displayed using pheatmap package. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of genes were calculated with the hypergeometric distribution based on a specific number of genes from one pathway or class in a gene list. Principal component analysis (PCA) was performed using the prcomp function in R (version 3.6.0) on detected genes filtering coefficient of variation more than zero. For detailed insight of gene expression in the transcriptome, the k-means clustering approach was performed with the Mfuzz package (version 2.56.0) (Kumar and Futschik, 2007). All transcription factors (TFs) were identified from the Plant Transcription Factor Database v5.0 (http:// planttfdb.gao-lab.org/) using the full-length coding sequence of all genes in tomato. The genomic region 2 kb upstream of the translation start codon (ATG) of all genes was extracted from the GFF file (Generic feature format version 4; ITAG4.0). Prediction of transcription factor target genes was performed using the FIMO tool with version 5.5.0 (Grant et al., 2011). Co-expression networks were constructed using the WGCNA package (version 1.69) (Langfelder and Horvath, 2008) and the networks were visualized using Cytoscape (version 3.9.1). Protein functional domains were determined by querying protein sequences in the Pfam database and NCBI Conserved Domain Database. Calculation of Pearson's coefficients between TFs and their candidate target genes was tested by Hmisc package (version 4.7.0). Phylogenetic analysis of predicted candidate gene families from tomato and five other species [Arabidopsis (A. thaliana), rice (O. sativa), wheat (T. aestivum), potato (Solanum tuberosum) and Populus trichocarpa] was performed through protein sequence similarity using Blastp (for protein-protein comparisons based on BLAST 2.12.0). Above software was used with default parameters.

2.4. Reverse transcription quantitative PCR analysis

Reverse transcription quantitative PCR (qRT-PCR) was performed using template-specific primer pairs (Table S1) and performed on an ABI 7900 using SYBR Premix (Roche), according to the manufacturer's instructions. PCR reactions were performed in technical triplicates and repeated independently three biological replicates. Expression levels were normalized to SIEXP (Solyc07g025390) to obtain the expression data. The relative quantification (27"4C') method was used to calculate the expression levels of target genes among different treatments (Wang et al., 2015).

2.5. Statistical analysis

Comparisons between two groups were performed using the unpaired two-tailed Student's t-test in Microsoft Excel (2021). Results were represented as mean standard deviation (s.d). P < 0.05 were considered statistically significant, with levels of significance as follows: ·P < 0.05; ··Р < 0.01; ···P < 0.001.

3. Results

3.1. Construction of an expression atlas of tomato flower bud thermotolerance

To construct a global gene expression profile of developing tomato flower buds under CK and HS conditions, we conducted RNA-seq of three developmental stages with three biological replicates and obtained at least 5.84 Gb of data for each sample (Table S2). After aligning the sequenced reads to the tomato reference genome SL4.0 (The Tomato Genome Consortium, 2012), we detected 22 712 genes (sum of FPKM in all samples >20) expressed in all samples (Fig. 1, A) and found a high correlation among the three biological replicates (0.84 < r < 0.99; Fig. S1). Principal component analysis showed that the 18 samples were clustered into two groups based on temperature treatment (Fig. 1, B), and the three developmental stages of tomato flower buds were distinctly distinguished in both CK and HS conditions (Fig. S2), indicating the significant influence of HS on gene expression in tomato flower buds.

To identify the candidate genes expressed during HS, we analyzed the DEGs between HS and CK for each developmental stage. We identified 2 976, 1 643, and 1984 DEGs at the 4-, 6-, and 8-mm stages, respectively (Fig. 1, C). Among these DEGs, approximately 60 % were highly expressed in flower buds under HS, suggesting that the expression of genes likely involved in flower bud thermotolerance is triggered by HS. Furthermore, we found that 38.13 % of DEGs at the 4-mm stage were unique to this stage, which was higher than the percentage of unique DEGs in the 6- (9.12 %) and 8-mm (20.59 %) stages. This finding indicates that tomato flower buds at the 4-mm stage are likely more susceptible to HS than those at the 6- and 8-mm stages.

In addition, we identified 645 (14.31 %) DEGs shared among the three developmental stages, comprising 85 downregulated and 560 upregulated genes (Fig. 1, C). Next, GO analysis in the biological process category of these 645 genes showed significant enrichment (P < 0.01) in "response to heat stress" (5.58 % of DEGs), "response to cadmium ion" (3.10 % of DEGs), "response to oxidative stress" (4.19 % of DEGs), "response to reactive oxygen species" (2.95 % of DEGs), and "response to toxic substance" (3.88 % of DEGs) (Fig. 1, D; Table 53). These data showed that these candidate DEGs may play critical roles in tomato flower buds under HS. We further analyzed the expression levels of the 645 shared DEGs to explore the possible biological functions of the candidate genes and found three clusters representing differential expression patterns at all stages. Of these shared DEGs, only 85 genes (13.18 %) were assigned to cluster 1, showing downregulation under HS. In contrast, 560 genes (86.82 %) in clusters 2 and 3 were dramatically upregulated under HS (Fig. 1, E). Functional analysis showed that all genes enriched in the "response to heat stress" biological process were dispersed in clusters 2 and 3. These findings suggest that these upregulated genes under HS may be involved in HSR to maintain the viability of tomato reproductive organs.

3.2. Gene co-expression network analysis under heat stress

Genes with parallel expression profiles may have similar biological functions. We measured the correlation between gene expression level and developmental stage using weighted gene co-expression network analysis to identify gene sets influencing flower bud thermotolerance under HS (Langfelder and Horvath, 2008). A total of 12 453 genes with high coefficients of variation (greater than 0.3) and high expression levels (sum of FPKM in all samples > 20) were used to construct a co-expression network. As a result, we identified eight stage-specific co-expression modules. Each module contained 33 (tan) to 4 504 (turquoise) genes (Fig. 2, A). One or more co-expression modules were distinctly associated with each stage of tomato flower bud development. These results suggest that genes in different modules likely play different roles in the flower bud developmental stages under HS.

To understand the correlations between each co-expression module and each developmental stage, we performed an indepth analysis of gene expression patterns. The eight coexpression modules were divided into two distinct categories showing a significant correlation (|r| > 0.50) with either HS at a single stage or all stages. Among the eight modules, two modules (blue and greenyellow) were positively correlated with HS at the 4-mm stage, while the turquoise module was negatively correlated with HS at every stage (Fig. 2, A). We also identified three modules (pink, red, and green) that were strongly correlated with HS at the 8-mm stage.

To further clarify the relationship between the co-expression modules and HS, we prioritized five modules (red, green, tan, black, and turquoise) that were significantly associated with HS at all stages. Because many TFs have been identified in plant heat stress response pathways, we focused on 11 TF families whose members play known roles in the HSR and were present in the five modules. Intriguingly, we found most TFs in the blue and turquoise modules, including 51 bHLHs, 45 ERFs, and 37 MYBs (Fig. 2, В). Furthermore, we detected the previously identified genes SIHSFA1 and SIHSFA2 (Ohama et al., 2017; Zhao et al, 2020; Haider et al., 2022) in the turquoise and red module (Fig. 2, C). We further analyzed all HSFs among these five modules and found that their expression patterns could be divided into two categories (Fig. 2, C). Among them, SIHSFA1, SIHSFA2, SIHSFA7a, SIHSFB1, SIHSFB2b, and SIHSFA4c.2, as core genes in the HSR, were strongly up-regulated under HS, whereas other HSFs were down-regulated. Notably, the HSFs up-regulated under HS were in the blue, red, and turquoise modules (Fig. 2, C). In addition, the expression levels of 11 HSPs in the red and turquoise modules dramatically increased under HS (Fig. 2, D). These data indicate that genes from the red and turquoise modules may respond to HS in tomato flower buds.

3.3. Prediction of novel HSR-related TFs in the HS co-expression network

Basal thermotolerance is an intrinsic ability of plants to resist mildly high temperatures. We analyzed 36 genes enriched in the "response to heat" biological process to elucidate the mechanism of basal thermotolerance (Fig. 1, D). Among these genes, we identified HSFs, ERFs, and HSPs that were all positively regulated by HS. Further analysis showed that all 36 genes belonged to the red and turquoise modules (Fig. 3, A). KEGG analysis showed that genes in both these modules were significantly enriched in multiple cellular functional processes, including "protein processing in endoplasmic reticulum," "cell cycle," and "DNA replication" (Fig. 3, B). These results indicate that HS could affect protein folding and gene expression, resulting in plant cell apoptosis, in line with the findings of previous study (Zhao et al, 2020).

Next, we predicted the target genes of TFs involved in the HSR. To this end, we performed motif analysis of the promoters of all genes in the red and turquoise modules. From this analysis, we detected 706 candidate target genes (Fig. 3, C). We found the top 10 hub genes, including SIHSFA1, SIHSFA4c, SIHSFA7a, SIHSFB2a, SIHSFB2b, SIHSFB3, and SIHSFC1, which have known important functions in the plant HSR. And then we identified novel HSRrelated TFs and their target genes. Expression analysis of their target genes among various flower bud developmental stages using our RNA-seq data showed that a large subset of these.

genes was primarily expressed in all developmental stages under HS (Fig. 3, A). Furthermore, we found that more target genes may be regulated by SIHSFA1 than SIHSFA2, suggesting an important role for this TF in the early HSR. However, the 11 HSPs identified in both modules showed low connectivity, indicating that these HSPs may participate in downstream HSR processes (Fig. 3, С). In addition, the expression patterns of 57 TFs changed significantly under HS, including ERFs, MYBs, NACs, and NF-YC (Fig. 3, D). Among these, the expression levels of 35 TFs, such as SIDREBs (Guo and Wang, 2011; Thirumalaikumar et al., 2018), SICBF1, SICIP2b, and SIWRKY75, were positively regulated by HS among three stages. In contrast, eight TFs, such as SIMYB117 and SINAM, were downregulated under HS. These findings indicate that these TFs have potential functions in the HSR of tomato flower buds.

3.4. Identification of thermotolerance genes in the HS signaling cascade

Both HSF and HSP family members play central roles in the HSR and induce thermotolerance through the HSR signaling cascade (Zhao et al., 2020; Haider et al., 2022). In this study, we found several homologs of previously identified HSR-related genes in tomato flower buds, including five early response genes (SIUB1, SIHSFA1s, SIRCF2, SIHSP70, and SIHSP90) and four late response genes (SIHSFA2, SIDREB2A, SIHSFA7a, and SIHSFBs) for acute signaling cascades. For each signal ramification, at least one gene showed strong expression in one flower bud developmental stage under HS, except for SIRCF2 (Fig. 4). These results suggest a conserved HSR pathway in plants.

Interestingly, we identified SIHSFA4c, a TF homologous to AtHSFA4c (AT5G45710), a gene involved in reproductive organ development (Wang et al., 2008). SIHSFA4c was specifically expressed in all stages of flower bud development. Prediction using the 2-kb upstream sequence of SIHSFA4c targets in the red and turquoise co-expression modules showed that the expression of SIHSFA2, SIHSFA7a, SIHSFB1, and SIHSFB2b might be positively regulated by SIHSFA4c. We also found that the expression of SIMBF1c and SIDREB2A might be positively regulated by SIHSFBs, in addition to the mutual regulation of SIHSFA1s and SIHSFBs in the biology of the stress response system. These observations indicate that the HS regulatory pathway is more complex than previously thought.

To identify thermotolerance genes directly regulated by key HSFs in the HSR, we found several strongly expressed genes from 243 HSF-regulated genes in the red and turquoise modules (Table S4). Among these genes, eight were predicted and regulated by HSF family members, namely SIHSFA1, SIHSFA7a, SIHSFB1, and SIHSFB2b. Further analysis showed that these target genes, such as HSPs, were temperature induced. Additionally, we identified SIPOM1, a gene encoding a chitinase-like protein that might be upregulated by SIHSFA1 and SIHSFB1, as well as SlerSHSP, a gene encoding a small HSP that might be positively regulated by SIHSF7a, SIHSFB1, and SIHSFB2b. Furthermore, SIHSFA7a might uniquely regulated SIGolS1, encoding a hexosyltransferase. Two genes, SIHSP101 and SITIL, which play important roles in the response to high temperatures, might be regulated by HSFBs (SIHSFB1 and SIHSFB2b) under HS in tomato flower buds. However, we should be cautious, and these genes need to be further validated in different experiments. Overall, our findings indicate that these HSF-regulated genes likely play key roles in tomato thermotolerance.

3.5. Validation of four thermotolerance genes

We further measured gene pairs that had highly synchronized expression under HS based on the correlation between HSFs and their target thermotolerance genes. In total, we found four HSFs and four target thermotolerance genes that were highly correlated in expression (r > 0.50; Fig. 5, A). Among the four target thermotolerance genes, SIGrpE, a gene encoding a nucleotide-exchange factor of the HSP70 complex, was positively correlated with all four HSFs (r > 0.70). Moreover, the expression of SIERDJ3A, a gene encoding a J domain protein localized in the endoplasmic reticulum lumen, was highly correlated (r > 0.70) with that of SIHSFA1, SIHSFA7a, and SIHSFB2b. We also found that the correlation coefficients between SIHSFB2b and SITIL, SIHSFA1 and SIPOM1, and SIHSFB2b and SIPOM1 were greater than 0.85.

Furthermore, homology analyses showed that four target thermotolerance genes had high protein sequence similarities to Arabidopsis genes (Fig. 5, B). Their homologous genes, AT4G26780 (Hu et al., 2012), AT3G08970 (Yamamoto et al., 2020), AT5G58070 (Chi et al., 2009), and AT1G05850 (Kwon et al., 2007), have been shown to respond to heat shock. Phylogenetic analyses revealed orthologs of SIERDJ3A, SIPOM1, SIGrpE, and SİTİL based on protein sequence similarity (Fig. 5, С and D; Fig. 53). Based on the phylogenetic tree of ERDJ3A and POM1 from tomato and other plants, we found that these proteins were assigned to different clades (Fig. 5, C and D), indicating that ERDJ3A and POM1 proteins are highly conserved in different species. These results indicate that SIGrpE, SIERDJ3A, SITIL, and SIPOM1 may have the capacity to improve tomato heat resistance.

We tested the expression levels of these genes in tomato flower buds under HS to validate the four HSFs and four target thermotolerance genes. As expected, all genes were significantly highly expressed following 0.5 h of HS, whereas their expression dramatically decreased after 1 h of HS (Fig. 5, E and F), demonstrating that these genes may play critical roles in the initial phase of the HS response. Taken together, these data suggest that four thermotolerance genes, namely SIGrpE, SIERDJ3A, SITIL, and SIPOM1, may have the potential to trigger tomato thermotolerance.

4. Discussion

Tomato is an important fruit and vegetable crop worldwide. The tomato HSR is induced to maintain cellular homeostasis and restore plant morphology when environmental temperatures rise above 35 °C (Camejo et al., 2005; Fragkostefanakis et al., 2015a). HSFs are the key TFs of the HSR and play central roles in the response to abiotic stress (Fragkostefanakis et al., 2015b). In this study, we performed RNA-seg on tomato flower buds of different developmental stages subjected to HS and constructed gene coexpression networks of TFs under HS (Fig. 3, C).

From our co-expression networks, we identified target genes encoding TFs from the MBF1C, NAC, ЕВЕ, and MYB families, which may be involved in the regulation of HSR genes. Among these TF genes, HSFA1s target MBF1C, which encodes a multiprotein bridging factor that is a highly conserved transcriptional coactivator and a key regulator of thermotolerance (Suzuki et al, 2011). Herein, we found that Solyc03g026280/SICBF1 and Solyc02g069960/SIUB1 were highly expressed in tomato flower buds under HS, indicating that they may regulate the transcription of HS resistance genes to promote thermotolerance. In rice and wheat, the expression of HSFAs could be activated by the NAC TFs OsNTL3 (Liu et al., 2020) and TaNAC2L (Guo et al., 2015) to enhance plant thermotolerance. In Arabidopsis, the expression of DREB2A under HS could also be regulated by the NAC TF JUNGBRUNNEN1 (JUB1) (Shahnejat-Bushehri et al., 2012). In addition, we found that Solyc10g005760/SIMYB80 was more highly expressed at the 4-mm stage than at the 6- and 8-mm stages in tomato flower buds, indicating that SIMYB80 may have a specific spatiotemporal expression pattern. We also found that SIWRKY75 and SIWRKY81 transcripts accumulated after HS treatment, and our motif-binding prediction analysis revealed that HSFs may regulate these WRKY TFs. These results are consistent with previous studies оп WRKY TFs in Arabidopsis and tomato (Li et al., 2010, 2011; Wang et al., 2024). Taken together, our findings revealed that, except for HSFs, some novel TFs belonging to the MBF1C, NAC, ЕВЕ, and MYB families may play essential roles in the tomato flower bud HSR.

HSFs and HSPs play central roles in the HSR and regulate thermotolerance in plants (Sanmiya et al., 2004; Hahn et al., 2011; Hu et al, 2012; Ohama et al., 2017; Haider et al., 2022). In this study, we found that many HSFs (Solyc03g097120/SIHSFA1, Solyc08g062960/SIHSFA2, Solyc09g065660/SIHSFA7a, and SIHSFBs) and HSPs (Solyc11g020040/SIHSP70, and Solyc06g036290/SIHSP90) were significantly upregulated among the three stages of tomato flower bud development under HS. Of these genes, SIHSFA7a accumulated during the tomato HSR and might regulate a few important genes in the response to HS, which is consistent with previous studies in Arabidopsis (Lin et al., 2018) and pepper (Albert and Chang, 2014). Apart from SIHSP70 and SIHSP90, HS also rapidly triggered Solyc03g115230/SIHSP101, Solyc06g076570/ SIHSP20, and small HSPs (Solyc03g082420/SIHSP21, Solyc11g020330/Sler-sHSP, and Solyc08g078700/SIMT-sHSP) (Fig. 3, C). In Arabidopsis (Queitsch et al., 2000), maize (Zea mays) (NietoSotelo et al., 2002), and tomato (Werghi et al., 2021), HSP101 overexpression enhanced plant thermotolerance. Moreover, the plastidial HSP21 overexpression in tomato increased the tolerance of photosystem II to heat-induced oxidative stress (NetaSharir et al., 2005). Similarly, thermotolerance in tobacco (Nicotiana tabacum) plants was enhanced through heterologous expression of the tomato mitochondrial small heat shock protein (MT-sHSP) (Sanmiya et al., 2004). These data indicate conserved regulatory mechanisms in the plant HSR.

Although the majority of genes responding to HS were HSPs facilitated by HSFs, other genes are required for thermotolerance in plants, such as NCED3, NCED6, and RD29A (Wang et al., 2018), in addition to H3K4me2 and H3K4me3 methylation (Lamke et al, 2016). In this study, we identified four target thermotolerance genes regulated by HSFs: SIGrpE, SIERDJ3A, SITIL, and SIPOMI. Their orthologous genes in Arabidopsis respond to heat or drought stress (Kwon et al., 2007; Chi et al., 2009; Ни et al., 2012; Yamamoto et al., 2020). For example, GrpE acted as a nucleotide-exchange factor in the HSP70 chaperone complex during long-term HS (Hu et al., 2012), and ERDJ3B maintained Arabidopsis anther development under high temperatures (Yamamoto et al., 2020). Moreover, TIL was upregulated in wheat and Arabidopsis during HS (Charron et al., 2005; Chi et al., 2009). Similarly, we observed that HS rapidly induced the TIL ortholog in tomato flower buds. Additionally, HOT2, the SIPOM1 homolog in Arabidopsis, was highly expressed at 42 °C and regulated seedling thermotolerance (Kwonetal., 2007). Therefore, we speculated that these four genes may have the capacity to trigger tomato thermotolerance.

5. Conclusions

Our dataset presents a high-resolution atlas of gene expression in different developmental stages of tomato flower buds under HS. The gene networks provide insight into the regulation of the tomato HSR and illustrate that the associated signaling cascade is conserved in tomato. Furthermore, our analysis identified novel TFs as important candidate genes in the plant HSR signaling cascade. Finally, we detected four target genes regulated by four HSFs as important candidates for regulating tomato thermotolerance. Overall, the TFs and target genes revealed here provide new resources for the scientific research community and may be used to improve tomato plant thermotolerance through future breeding programs.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 32072571), the 111 Project (Grant No. B17043), and the Construction of Beijing Science, and Technology Innovation and Service Capacity in Top Subjects (Grant No. CEFF-PXM2019_014207_000032).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.1016/j.hpj.2023. 06.004.

Sidebar

2468-0141/Copyright © 2023 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS). Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Footnote

* Corresponding authors.

* These authors contributed equally to this work.

E-mail addresses: [email protected]; [email protected]

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)

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