Physiological and Transcriptomic Analyses of the Effects of SlBRI1 Expression Levels on the Drought Tolerance of Tomato Seedlings

Brassinosteroids (BRs) not only influence plant growth and development but also regulate various stress responses in plants. BRASSINOSTEROID INSENSITIVE 1 (BRI1) acts as a BR receptor, sensing BRs and then activating BR signalling. In this study, we researched how SlBRI1 regulates the drought tolerance of tomato at both the physiological and transcriptomic levels. We used SlBRI1-overexpressing and SlBRI1-weak mutant (abs) plants that were of the same background to research the mechanism underlying their drought tolerance. Physiological analyses revealed that, compared with Money Maker (MM) plants, abs plants had a greater net photosynthesis rate (Pn) and less wilting, and abs plants also accumulated lower amounts of peroxide (H2O2) and superoxide (O2−) through increased antioxidant enzyme activities under drought conditions. RNA sequencing (RNA-seq) analysis showed that the expression of 1425 and 840 genes was induced and repressed in response to drought in MM plants, respectively. However, the expression of 768 (53.9%) of drought induced 1425 genes and 418 (49.8%) of drought repressed 840 genes was induced and repressed, respectively, in abs plants under normal conditions. Moreover, the expression of 158 genes whose expression was induced in response to drought and 43 genes whose expression was repressed in response to drought was further upregulated and downregulated, respectively, in abs plants under drought conditions. In-depth analysis of these differentially expressed genes (DEGs) revealed that the expression of genes related to abscisic acid (ABA) metabolism and polyamine biosynthesis as well as oxidoreductase activity was upregulated in abs under both normal and drought conditions. Furthermore, analysis of transcription factor (TF) expression suggested that the drought tolerance of abs differed mainly through the regulation of WRKY, ERF, bHLH and MYB TFs. However, the expression of most of these genes was the same or opposite in the SlBRI1OE plants compared with the MM plants. Our results affirm that SlBRI1 expression is negatively involved in the drought response of tomato. Furthermore, our study provides valuable information for future breeding to appropriately reduce the expression of SlBRI1 and improve drought tolerance without affecting plant growth.


Introduction
Water availability is one of the most important environmental factors limiting plant species distribution and agricultural production (Yordanov et al. 2000). When plants are subjected to moderate water stress, its photosynthesis and respiration rates decrease, and also produced a series of physiological and biochemical responses to adapt to drought stress (Jaleel et al. 2008). More severe water stress can inhibit growth and damage the ultrastructure of cells and organelles, which results in cellular dehydration, the accumulation of toxic substances, the loss of cell membrane permeability, the inactivation of enzymes, and changes in protein structure. All of these factors eventually lead to metabolic disturbances and even plant death (Chaves and Oliveira 2004;Sofo et al. 2005;Morales et al. 2013;Talbi et al. 2015). Furthermore, drought stress can induce enhanced production of reactive oxygen species (ROS), which leads to oxidative stress ).
To resist drought-induced oxidative stress, plants can eliminate excess ROS by upregulating the activity of enzymatic and nonenzymatic antioxidants (Boaretto et al. 2014). Indeed, increased antioxidant enzyme activities play an important role in plant drought tolerance (Wang et al. 2012).
Brassinosteroids (BRs) are steroid hormones that exist in all plant tissues. BRs play important roles in plant growth and development processes, such as the promotion of seed germination, cell elongation and division, pollen fertility, vascular differentiation, fruit set, and seed set (Li et al. 1996(Li et al. , 2020Feng et al. 2021;Rozhon et al. 2019). In addition, BRs can enhance plant photosynthesis, and increase chlorophyll contents, delay ageing, and increase stress resistance (Wenhai et al. 2006). To date, well-developed BR signal transduction models have been established in Arabidopsis. BRs first bind to the plasma membrane-localized receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the coreceptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) (Li and Chory 1997;Wang et al. 2001;Wu et al. 2011;Qiao et al. 2017). BRI1 interacts with BAK1 and activates BR signalling, after which the BR signals are transduced to downstream components. Finally, dephosphorylated bri1-EMS suppressor 1 (BES1) and Brassinazoleresistant 1 (BZR1) accumulate in the nucleus to regulate the expression of thousands of BR-response genes (Wang et al. 2008;Qinsong et al. 2018).
BR signalling intensity not only influences plant growth and development but also regulates various stress responses of plants. Oryza sativa glycogen synthase kinase 3-like gene 1 (OsGSK1) is an orthologue of Arabidopsis brassinosteroid insensitive 2 (BIN2), and knocking out OsGSK1 enhances tolerance to cold, heat, salt, and drought stresses (Koh et al. 2007). The Arabidopsis elongated-D mutant, a BAK1 singlebase gain-of-function mutant, presents increased BR signalling and is more vulnerable to bacterial pathogens and salinity stress (Chung et al. 2012). The Arabidopsis transcription factor (TF) RESPONSIVE TO DESICCATION 26 (RD26) inhibits BR-regulated growth but increases drought tolerance by upregulating the expression of drought-induced genes. RD26 mediates the crosstalk between the drought response and BR signalling . Moreover, WRKY46, WRKY54, and WRKY70 positively regulate BR signalling and plant growth, whilst WRKYs negatively regulate drought tolerance by inhibiting dehydration-induced gene expression (Chen et al. 2017). The AP2/ERF TF TINY negatively regulates plant growth and compromises BR-responsive gene expression; TINY positively regulates drought tolerance by promoting drought-responsive gene expression (Xie et al. 2019). Similarly, wheat brassinazole-resistant 2 (TaBZR2) positively regulates BR signalling and the drought response. TaBZR2 directly activates the expression of the Triticum aestivum glutathione transferase 1 (TaGST1) gene, whose product scavenges drought-induced superoxide (O 2 − ) anions (Cui et al. 2019). The expression level of the BR receptor BRI1 directly influences BR signal intensity and drought tolerance. RNA interference (RNAi) of a BRI1 homologue in Brachypodium distachyon decreases the growth potential of plants whilst increasing their tolerance to drought and drought-responsive gene expression (Feng et al. 2015). The drought tolerance of the Arabidopsis gain-of-function BR mutant bes1-D is negatively regulated by repression of the expression of drought response genes, whilst the BR-weak mutant bri1-5 exhibits increased drought tolerance by upregulating the expression of these genes . Overexpression of the vascular BR receptor BRL3 increases drought tolerance and the accumulation of osmoprotectant metabolites without affecting plant growth (Fàbregas 2018).
In a previous study, we used SlBRI1-overexpressing plants of the tomato cultivar Micro-Tom to increase BR signalling and decrease drought tolerance (Nie et al. 2019). We further found that SlBRI1-weak mutants in the tomato cultivar Money Maker (MM) exhibited altered brassinolide sensitivity (abs) and had a missense mutation in the kinase domain, delayed growth and enhanced drought tolerance (Bajwa et al. 2013;Nie et al. 2019). To further understand the relationship between SlBRI1 expression level and drought tolerance, we obtained SlBRI1-overexpressing and SlBRI1-weak mutant (abs) plants from the same background to research the mechanism underlying drought tolerance.

Plant Materials, Growth Conditions and Plant Transformation
Seeds of the tomato cultivar MM, abs (SlBRI1-weak mutant) and T2-generation transgenic tomato plants were germinated at 28 °C in Petri plates lined with two layers of filter paper moistened with deionized water. Tomato MM and abs seeds were obtained from staff of the Northwest A&F University Xiaofeng Wang laboratory. The germinated seeds were then sown in plastic pots (8 cm × 8 cm × 9 cm) filled with 70 g of a mixture of peat and vermiculite (v/v = 7:3), with one seed per pot. The seedlings were grown in a growth chamber with a temperature of 25 °C, a photosynthetic photon flux density (PPFD) of 500 µmol m -2 s -1 , and a photoperiod of 16 h of light/8 h of darkness.
35S:SlBRI1 overexpression transgenic tomato plants were obtained according to the methods of Nie et al. (Park et al. 2003;Nie et al. 2017). Two independent homozygous SlBRI1 overexpression plants (SlBRI1-OE-6 and SlBRI1-OE-7) were used for drought experiments because of their high expression levels of SlBRI1. abs plants were sown 30 d earlier than other tomato plants were because abs plants grow slowly.

Drought Experiments
abs seeds were sown 30 d earlier than other tomato seeds were because abs plants grow slowly. All the plants were grown to the six-leaf stage. Plants of the same size were selected for subsequent experiments. The selected plants were not watered for 12 d. There were 40 seedlings in each group.

Measurements of Relative Water Content (RWC) and Electrolyte Leakage
Young fully expanded leaves were harvested to measure the RWC and electrolyte leakage. The RWC and electrolyte leakage were measured according to the methods of Liu et al. (2015).

Measurement of Photosynthesis Parameters
The net photosynthesis rate (Pn), stomatal conductance (Gs), intercellular CO 2 concentration (Ci), and transpiration rate (Tr) of the seedlings were measured using a portable photosynthesis system (LI-6400-40, LI-COR, Lincoln, NE, USA). All the measurements were carried out at 400 µmol mol -1 CO 2 , 25 °C, and 500 µmol m -2 s -1 light intensity. For each measurement, eight leaves were measured from different seedlings per treatment (Liu et al. 2020).

Antioxidant Enzyme Activities and the Accumulation of ROS
Antioxidant enzyme activities and the accumulation of ROS were determined in accordance with the methods of Nie et al. (2019).

Western Blot Analysis
Total proteins were extracted from young leaves (0.2 g) of 25-d-old tomato plants. The procedure was performed as previously described (Wang et al. 2016).

RNA Extraction and Quantitative Real-time PCR (qRT-PCR) Analysis
Total RNA was extracted and reverse-transcribed according to the methods of Nie et al. (2017). qRT-PCR was performed following the three-step protocol of BioEasy Master Mix (Bioer Technology) in a CFX96 real-time system (Bio-Rad) as described in the manufacturer's instructions. Each assay included three biological replicates. We used the UBI3 gene as an internal control. The sequences of all the specific primers used are listed in Table S1.

RNA Sequencing (RNA-seq) and Transcriptomic Analysis
Total RNA samples were collected from the meristem and young leaves using an RNAprep Pure Plant Kit (Tiangen Biotech) following the manufacturer's instructions. RNA quality and concentration were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and a Nan-oDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). For the transcriptome analyses, RNA-seq libraries were prepared from cDNA by Majorbio and sequenced on an Illumina HiSeq 4000 instrument. The raw sequences were quality filtered by SeqPrep (https:// github. com/ jstjo hn/ SeqPr ep) and Sickle (https:// github. com/ najos hi/ sickle) and subsequently mapped to the Solanum_lycopersicum SL4.0 reference genome (https:// www. solge nomics. net/ organ ism/ Solan umlyc opers icum/ genome/) using TopHat2 (http:// ccb. jhu. edu/ softw are/ tophat/ index. shtml). DESeq2 was used to analyse the differential expression between the RNA-seq expression profiles. |log2(fold change (FC))|≥ 1 and P adjusted < 0.05 were set as thresholds. The genes that were expressed at significantly different levels were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) functional analysis and GO enrichment analysis. A metabolic pathway heatmap was generated using Multiple Array Viewer.

Statistical Analysis
All the data in this study were analysed using SPSS version 17.0 and the least significant difference (LSD) test. The means and standard errors were calculated, and P < 0.05 was considered statistically significant in comparison with MM plants.

SlBRI1 Transgenic Plants Exhibit Increased SlBRI1 Expression Levels
To investigate the association of drought tolerance with SlBRI1 expression, we generated transgenic tomato plants in the MM background in which SlBRI1, driven by the constitutive cauliflower mosaic virus 35S promoter (35S), was overexpressed. The transcript levels of SlBRI1 in two transgenic lines, SlBRI1-OE-6 and SlBRI1-OE-7, were 5.4 and 16.5 times greater, respectively, than those in the MM plants ( Fig. 1A). Moreover, the SlBRI1 protein levels in the SlBRI1-OE-6 and SlBRI1-OE-7 lines were confirmed by Western blot analysis (Fig. 1B); in addition, the expression levels of the BR biosynthesis-related genes 6-DEOXOCASTAS-TERONE OXIDASE (DWARF) and CONSTITUTIVE PHO-TOMORPHOGENESIS AND DWARF (CPD) were significantly lower than those in the MM plants ( Fig. 1C, D). These results showed that transgenic plants exhibited increased SlBRI1 expression levels and BR signalling intensity.

SlBRI1 Expression Alters Plant Growth, Leaf RWC and Electrolyte Leakage under Drought Stress
To investigate whether SlBRI1 expression is related to the drought tolerance of tomato seedlings, SlBRI1-overexpressing, MM and abs (SlBRI1-weak mutants) tomato seedlings were subjected to drought stress (water withheld) for 12 d.
After 12 d of drought stress, all the plants showed different degrees of leaf wilting. The abs plants exhibited slight wilting, whilst wilting was the most severe for the SlBRI1-OE-6 and SlBRI1-OE-7 plants ( Fig. 2A, B).
Drought stress decreased the leaf RWC, and the leaf RWC of SlBRI1-overexpressing lines was significantly lower than that of the MM plants. However, the RWC of the abs plants was significantly greater than that of the MM plants after 10 d of drought stress and was 24.5%, 30.6% and 35.6% greater than that of the MM, SlBRI1-OE-6 and SlBRI1-OE-7 plants, respectively (Fig. 2C). Drought stress resulted in increased electrolyte leakage levels in all the studied plants. The leaf electrolyte leakage levels of the SlBRI1-overexpressing lines were significantly greater than those of the MM plants, whilst the electrolyte leakage levels of the abs plants were significantly lower than those of the MM plants after 10 d of drought stress and were 19.1%, 33.6% and 40.5% lower than those of the MM, SlBRI1-OE-6 and SlBRI1-OE-7 plants, respectively (Fig. 2D). Taken together, these results indicated that SlBRI1 expression negatively regulated the drought tolerance of tomato seedlings.

SlBRI1 Expression Affects Gas Exchange under Drought Stress
Compared with the control plants, the SlBRI1-overexpressing plants presented a slightly increased Pn and Gs before drought stress. However, drought stress obviously decreased the Pn, Gs and Tr but increased the Ci of all plants ( Fig. 3A-D). After 10 d of drought stress, the Pn and Tr of the abs plants were significantly greater than those of the other plants, and the Ci was obviously lower; however, for the SlBRI1-overexpressing plants, the Pn and Tr were obviously lower than those of the other plants, and the Ci was markedly greater (Fig. 3A-D). For example, the Pn of the abs plants was 27.1%, 49.2% and 56.5% greater than that of the MM, SlBRI1-OE-6 and SlBRI1-OE-7 plants, respectively. However, there were no significant differences in Gs amongst any of the plants (Fig. 3D).

SlBRI1 Expression Affects the Accumulation of H 2 O 2 and O 2 − and Antioxidant Enzyme Activities under Drought Stress
Histochemical observations were used to assess the accumulation of hydrogen peroxide (H 2 O 2 ) and O 2 − in the tomato leaves. Before drought stress, there were no significant differences in H 2 O 2 and O 2 − accumulation amongst any of the plants (Fig. 4A, B). After 10 d of drought stress, the accumulation of H 2 O 2 and O 2 − in the abs leaves was lower than that in the MM leaves, but the leaves of the SlBRI1-overexpressing plants accumulated much greater levels than did the leaves of the MM plants (Fig. 4A, B). Therefore, compared with the MM plants, the abs plants had lower ROS levels under drought stress, and SlBRI1overexpressing plants had greater ROS levels.
Drought stress obviously increased the antioxidant enzyme activities in all plants. The superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities of the abs plants were significantly greater than those of other plants after 10 d of drought stress and were 39.9%, 47.9% and 29.8% greater than those of the SlBRI1-OE-6 plants, respectively. The ascorbate peroxidase (APX) activities of the MM and abs plants were significantly greater than those of the SlBRI1-overexpressing plants after drought stress ( Fig. 4C-F). Taken together, these results showed that BR signalling was negatively related to the antioxidant enzyme activities of tomato seedlings under drought stress.

Sequencing of Plants with Different SlBRI1 Expression Levels via RNA-seq
To further understand how SlBRI1 negatively regulates drought responses, we performed global gene expression studies involving MM, SlBRI1-OE-7 and abs plants via highthroughput RNA-seq. As shown in Table 1, six cDNA libraries (three replicates per library) were constructed. RNA-seq of these libraries generated approximately 47-62 million total reads, with an average of 98.9% clean reads obtained after quality filtering. The clean reads from each library had a match rate of approximately 95% to the tomato genome, indicating that the sequencing data could be used for subsequent transcriptome analysis (Table 1).

SlBRI1 Negatively Regulates Drought-responsive Gene Expression
Differentially expressed genes (DEGs) were analysed between every group pair (MMDS/SlBRI1OEDS, MMDS/absDS, MM/abs, abs/absDS, MM/MMDS, SlBRI1OE/SlBRI1OEDS and MM/SlBRI1OE) based on fragments per kilobase of transcript per million mapped reads (FPKM), with a false discovery rate (FDR) threshold of < 0.05 and a FC > 2. There were 3144 DEGs (2096 and 1048 whose expression was up-and downregulated, respectively) between MMDS and absDS, 6162 DEGs (3873 and 2289 whose expression was up-and downregulated, respectively) between MM and abs, and 2265 DEGs (1425 and 840 whose expression was up-and downregulated, respectively) between MM and MMDS. There were only 85 DEGs between MM and SlBRI1OE, comprising 59 and 26 whose expression was up-and downregulated, respectively (Fig. 5). We selected the above four groups for a comprehensive analysis. The number of DEGs whose expression was upregulated was greater than that of the DEGs whose expression was downregulated across all four comparison groups (Fig. 5). However, we further found that 768 (53.9%) of 1425 drought-induced and 418 (49.8%) of 840 genes whose expression was repressed by drought were regulated in abs in the same direction under normal conditions (Fig. 6A, B). There were 158 and 43 genes whose expression was induced and repressed, respectively, by drought that were further upregulated and downregulated in absDS, respectively (Fig. 6A, B). These results indicated that abs may regulate the expression of a number of drought-related genes, which results in the inhibition of plant growth under normal conditions, which is consistent with the growth phenotype of abs.
To further investigate how drought-related gene expression is affected in SlBRI1-OE and abs, we performed clustering analysis of the genes whose expression was upregulated and downregulated in each treatment. Under normal conditions, the genes whose expression was upregulated and downregulated in MM were repressed and induced, respectively, under drought conditions (Fig. 6C). However, Fig. 3 Effects of different SlBRI1 expression levels on gas exchange parameters in tomato seedlings under drought stress. A Photosynthetic rate (Pn), B Stomatal conductance (Gs), C Intercellular CO 2 concentration (Ci), and D Transpiration rate. Data values are the means ± SD of three independent biological samples. Different letters indicate significant differences according to Tukey's test (P < 0.05) the expression of many drought stress-induced genes and repressed genes was already upregulated and downregulated, respectively, in abs under normal conditions (cluster b). Many drought stress-induced genes were expressed at greater levels in absDS under drought conditions, whilst many drought stress-repressed genes were expressed at lower levels in absDS (cluster a). Overall, our transcriptome analyses support a role of SlBRI1 expression in modulating drought-responsive gene expression, largely in an antagonistic manner.

GO and GO Enrichment Analyses of Different SlBRI1 Levels under Normal Conditions
To explore the effects of SlBRI1 overexpression under normal conditions, we performed GO analyses, focusing on differences between the MM group and the SlBRI1OE group. There were 71, 72 and 74 DEGs enriched in the "biological process", "cellular component" and "molecular function" categories, respectively (Fig. 7, Table S2). The main biological process categories were "metabolic process" and "cellular process", DEGs in the molecular function category were related to "catalytic activity" and "binding", and most DEGs were assigned to the "cell" and "membrane part" cellular component categories (Fig. 7, Table S2). These results highlighted the involvement of cellular processes and metabolic processes in SlBRI1-OE, consistent with the growth regulation of these plants under normal conditions.
To explore how abs functions under normal conditions, we performed GO enrichment analyses, focusing on differences between the MM group and abs group. The top 20 most obviously enriched pathways are shown in Fig. 8. The DEGs were enriched for "photosynthesis, light harvesting in photosystem I", "photosynthesis, light harvesting", "protein phosphorylation", "phosphorylation", "plastoglobule", "cellular protein modification process", "protein modification process", "response to abiotic stimulus", "lipid metabolic process", "phosphorus metabolic process", "phosphate-containing compound metabolic process", "carbohydrate metabolic process" and "intrinsic component of membrane" (Fig. 8, Table S3). In addition, the most enriched category for DEGs in this study was "catalytic activity", with a total of 2445 DEGs. A total of 148 DEGs were annotated as "response to abiotic stimulus" (Fig. 8, Table S3). Taken together, these results indicated that the expression of a number of stress response-related genes is upregulated in abs under normal conditions.

Differences in Stress Response-related Gene Expression Between Different Treatments
To further explore how the drought tolerance of abs increases, we selected stress-related metabolic pathways from GO enrichment analyses. The expression of four abscisic acid (ABA) biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase 2 (Solyc08g016720.1), notabilis 9-cis-epoxycarotenoid dioxygenase (Solyc07g056570.1), beta-carotene (Pfam: PF05834; Solyc06g074240.3), and zeaxanthin epoxidase (Solyc02g090890.4), was upregulated in MM under drought stress (Table S4). Like in MM, the expression of these genes was also induced in abs under normal conditions. However, the expression of these genes did not obviously differ between the SlBRI1OE plants and the MM plants. The expression of Solyc08g016720.1 and Solyc06g074240.3 was upregulated between MMDS and absDS (Table S4). The expression of eleven of 13 genes involved in polyamine biosynthesis was upregulated in MM under drought stress, and the expression of all 13 genes was upregulated in abs compared with MM under normal conditions. However, the expression of these genes did not obviously differ between the SlBRI1OE plants and the MM plants. Moreover, the expression of eight of these 13 genes was upregulated between MMDS and absDS. These results indicated that ABA and polyamine contents in abs may increase via increased ABA-and polyamine biosynthesisrelated gene expression under both normal and drought conditions (Table S5). The expression of even of 11 oxidoreductase activity-related genes was upregulated in MM under drought stress, and unlike in MM, the expression   Table S6). Taken together, these results indicated that abs may reduce the reactive oxygen content by inducing the expression of genes involved in oxidoreductase activity and further enhance antioxidant enzyme activities under both normal and drought conditions. TFs play a critical role in abiotic stress via gene regulatory networks. We selected 25 TFs across 7 different families from the MMDS group, 18 of 7 whose expression was up-and downregulated, respectively. Most of the differentially expressed TFs participate in the drought stress response, and the majority is members of the WRKY, ERF, bHLH and MYB families ( Fig. 9b; Table S7). The expression of 18 drought stress-induced TFs was also upregulated under normal conditions, and the expression of 7 of these drought-downregulated TFs was downregulated in abs. Seventeen TFs did not show exhibit changes in expression between the SlBRI1OE plants and MM plants; 4 TFs, ERF (Solyc06g068360.3), AP2-ERF (Solyc10g084340.2), M Y B 7 5 ( S o lyc 1 0 g 0 8 6 2 5 0 . 2 ) a n d b H L H 0 7 9 (Solyc02g078130.3), had the same expression direction, whilst 4 TFs, WRKY33 (Solyc09g014990.4), WRKY46 (Solyc08g067340.4), MYB76 (Solyc05g008250.2) and bHLH150 (Solyc09g065100.3), had opposite expression changes in SlBRI1OE plants and drought-exposed plants (Fig. 8B, Table S5). Eighteen of 25 TFs exhibited the same expression direction in absDS compared with MMDS, and the expression of 7 of 25 TFs did not obviously differ (Fig. 9B, Table S7). In the abs plants, reduced BR signalling . The x-axis shows the rich factor. Blue represents a high adjusted P value, and red represents a low adjusted P value. The y-axis shows the top 20 KEGG pathways. The bigger size of spot, the more DEGs enriched

Validation of RNA-seq Data by qRT-PCR Analysis
To validate the RNA-seq data, we selected 15 DEGs in the MM vs abs comparison group for qRT-PCR. We then compared the results obtained from qRT-PCR with those generated from the RNA-seq data. The expression trends were consistent for all transcripts in both analyses, with a correlation coefficient of R 2 = 0.883 (Fig. 10). These results confirmed the reliability of the RNA-seq data.

Discussion
Plants frequently encounter various environmental stresses, such as drought, salt and extreme temperature, which severely affect plant growth and yield. In many previous studies, it has been reported that application of BRs can improve the drought tolerance of plants. In recent years, we found that tomato cultivar Micro-Tom overexpressing SlBRI1 present increased BR signalling but decreased drought tolerance. We further found that SlBRI1-weak mutants in the MM background (abs) exhibited delayed growth and enhanced drought tolerance (Nie et al. 2019). To further understand the relationship between SlBRI1 expression and drought tolerance of tomato, we generated SlBRI1overexpressing and SlBRI1-weak mutant plants in the same background to examine their differences in terms of drought tolerance. The expression of SlBRI1 in the transgenic plants was significantly greater than that in the MM plants (Fig. 1A, B). Furthermore, the transgenic plants already expressed SlBRI1 proteins. In addition, the expression of the BR biosynthesis-related genes DWARF and CPD was significantly inhibited in the transgenic plants (Fig. 1C, D). Taken together, these results indicated that the SlBRI1 transgenic plants had obviously increased BR signalling.
Abiotic stress can lead to a reduction in photosynthesis through stoma-dependent and stoma-independent pathways. If Ci and Gs are reduced simultaneously, the reduction in the Pn can be considered to be due primarily to stomatal factors. In contrast, if Gs is reduced but Ci does not change or is increased, the reduction in Pn can be considered to be due to nonstomatal factors (Yin et al. 2005;Jia et al. 2008;Begcy et al. 2012;Zhang et al. 2013). In our study, drought stress decreased the Pn of all the plants. Furthermore, the Pn and Tr of the abs plants were significantly greater than those of other plants, whilst the Ci of the abs plants was significantly lower than that of other plants under drought conditions (Fig. 3A-D). These results were consistent with BRI1 B. distachyon RNAi plants having greater a photosynthesis ability than wild-type plants under drought stress (Feng et al. 2015). In contrast, the Pn and Tr of the SlBRI1overexpressing plants were significantly lower than those of the other plants under drought conditions, whilst the Ci was significantly greater than that of other plants (Fig. 3A,  C, D). Therefore, compared with that of the other plants, the photosynthesis ability of the abs plants was less affected by drought stress. These results indicated that the reduction in Pn in all the plants could be attributed to both stomatal factors and nonstomatal factors. The nonstomatal factors may include a reduced activity and efficiency of key enzymes Rubisco of the Calvin cycle, disruption to the photosynthetic apparatus and other organelles and slowed transport of photosynthesis products (Sapeta et al. 2013).
Normally, the formation and elimination of ROS are balanced and steady, but this balance is disrupted when plants are subjected to drought stress. In this study, drought stress obviously increased the accumulation of H 2 O 2 and O 2 − in all the plants, as determined by nitro blue tetrazolium (NBT) and 3, 3'-diaminobenzidine (DAB) staining (Fig. 4A, B). The accumulation of H 2 O 2 and O 2 − in abs leaves was lower than that in MM leaves, but the leaves of SlBRI1-overexpressing plants accumulated much more H 2 O 2 and O 2 − than did the leaves of MM plants (Fig. 4A, B). Therefore, compared with the MM plants, the abs plants had lower ROS levels under drought stress, and the SlBRI1-overexpressing plants had greater ROS levels. These results were consistent with the observations that the SOD, POD, CAT and APX activities of the abs plants were significantly greater than those of other plants under drought stress (Fig. 4C-F). The activities of these enzymes in the SlBRI1-overexpressing plants were significantly lower than those in the MM and abs plants after drought stress. Furthermore, our RNA-seq data indicated that the expression of 11 oxidoreductase activity genes was induced in the abs plants under normal conditions. The expression of 7 of 11 genes was upregulated in the MM plants under drought stress. However, the expression of most of these genes did not obviously change in the SlBRI1OE plants. In addition, the expression of 9 of these same 11 genes was greater in the absDS group than in the MMDS group (Fig. 9A). SlLC6D RNAi lines increased expression of SlSOD and SlPOD and activities of SOD and POD, which leaded to reduce ROS contents in RNAi lines under chilling stress (Hu et al. 2021). Therefore, these results indicated that abs plants may upregulate the expression of some oxidoreductase genes and further enhance antioxidant enzyme activities, which resulted in lower ROS level than MM and SlBRI1OE plants under drought stress (Choudhury et al. 2017).
The pleiotropic roles of BRs are complex, BRs play roles in developmental processes and multiple types of stress responses in plants. To further understand the relationship between SlBRI1 expression and drought tolerance of tomato, we used RNA-seq to characterize the influence of SlBRI1 expression on gene expression under both normal and drought conditions. We focused on comparing the differences in gene expression between MM and MMDS, MM and abs, MM and SlBRI1OE and MMDS and absDS. We found that the expression of 768 (53.9%) of 1425 genes whose expression was induced and 418 (49.8%) of 840 genes whose expression was repressed by drought was regulated in the same direction in abs under normal conditions (Fig. 6A, B). There were 158 and 43 genes whose expression was induced and repressed, respectively, by drought whose expression was further upregulated and downregulated, respectively, in absDS (Fig. 6A, B). These results were consistent with the observations that RNAi of a BRI1 homologue in B. distachyon decreased the growth potential of plants whilst increasing both their tolerance to drought and their droughtresponsive gene expression (Feng et al. 2015). ABA plays a crucial role in drought tolerance. Drought stress usually induces the expression of ABA biosynthesis-related genes, which further increases the ABA content and eventually enhances the drought tolerance of plants (Nie et al. 2019). Therefore, we compared stress-related metabolic pathways identified via GO enrichment analyses. Our results indicated that drought stress upregulated the expression of 4 ABA biosynthesis-related genes whose expression was already induced in the abs plants under normal conditions. Additionally, the expression of 2 ABA biosynthesis-related genes was greater in the absDS group than in the MMDS group (Table S4). Taken together, these results indicated that ABA contents may increase and that drought tolerance may further improve by the induction of ABA biosynthesis-related gene expression in abs under both normal and drought conditions. Dopamine also plays an important role in the drought tolerance of plants (Liang et al. 2018). A previous study showed that dopamine could regulate photosynthetic oxygen reduction and photophosphorylation in chloroplasts (Elstner et al. 1976). Furthermore, dopamine could increase the drought tolerance of apple seedlings by improving the photosynthesis ability and antioxidant enzyme activity of the plants (Li et al. 2019). Our results indicated that the expression of 13 polyamine biosynthesis-related genes was upregulated in the abs plants under normal conditions. The expression of eleven of these 13 was induced in MM under drought stress. Furthermore, the expression of 8 genes was greater in the absDS group than in the MMDS group. However, most of these genes did not exhibit obvious changes in expression between the SlBRI1OE plants and the MM plants (Table S5). Therefore, these results indicated that polyamine contents may increase and drought tolerance may further improve by the induction of polyamine biosynthesis-related gene expression in abs under both normal and drought conditions.
TFs play an important role in drought stress signalling. Some TFs, including WRKYs, NACs, ERFs, bHLHs, bZIPs and MYBs, are major components of signalling networks. In our study, 25 TF-encoding genes were differentially expressed between the different treatments, mainly TFs from the WRKY, ERF, bHLH and MYB families. Furthermore, the most abundant TF members were members of the WRKY TF family (Fig. 9B, C, Table S7). Previous studies have shown that WRKY38 participates in the drought stress response and that WRKY40 is involved in ABA signalling (Han et al. 2010;Albayrak et al. 2012). Moreover, overexpression of OsWRKY30 obviously improved the drought tolerance of rice (Shen et al. 2012). Compared with the MM group, the MMDS group exhibited upregulated expression of drought-related TFs such as WRKY81, WRKY33, WRKY41, WRKY42 and WRKY46. However, in the abs plants, the expression of these genes was already upregulated under normal conditions. Moreover, the expression of these genes in the absDS group was greater than that in the MMDS group, whilst the expression of these genes was downregulated in the SlBRI1OE plants compared with the MM plants (Fig. 9B, Table S7). The overexpression of AtWRKY46 reduced drought tolerance and regulated the expression of a number of genes involved in cellular osmoprotection and redox homeostasis under dehydration stress (Ding et al. 2014). Overexpression of GhWRKY41 enhanced the drought and salt stress tolerance of transgenic Nicotiana benthamiana by increasing stomatal closure and the expression of antioxidant-related genes (Chu et al. 2015). Therefore, the expression of drought-related WRKY TFs may be upregulated in abs plants, further improving drought tolerance under both normal and drought conditions. We performed a preliminary analysis of the gene structure of SlWRKY42 and verified its function through the use of transgenic plants.
AP2/ERF TFs are involved in the regulation of plant drought responses and plant growth. The overexpression of stress-inducible AP2/ERF TFs inhibited plant growth but improved the drought tolerance of transgenic plants. TINY positively regulates the drought response by inducing the expression of drought-responsive genes and promoting ABA-regulated stomatal closure. BR signalling negatively regulates TINY through BIN2 phosphorylation, and TINY inhibits BR-mediated growth through antagonistic TINY-BES1 interactions (Elstner et al. 1976). The expression of three AP2/ERF TFs each was upregulated and downregulated in the MMDS group compared with the MM group. However, the expression of these same genes was already upregulated and downregulated under normal conditions in the abs plants (Fig. 9B). The expression of most of the same genes did not different between the SlBRI1OE plants and the MM plants. In plants, stress resistance is often related to decreased growth rates and productivity (Ulrike and Benjamin 2018). TINY belongs to the AP2/ERF family of TFs, and overexpression of TINY increases drought tolerance by upregulating drought-responsive gene expression and inhibits plant growth by negatively regulating BR signalling (Sun et al. 2008). These results were consistent with the results for abs. We also observed similar results for several bHLH and MYB family TFs in abs plants and SlBRI1OE plants (Fig. 9B). In Arabidopsis, overexpression of TaMYB30-B increased drought tolerance during the germination and seedling stages . Therefore, in abs plants, the bri1 mutation leads to reduced BR signalling and may upregulate or downregulate the expression of droughtrelated TFs, further regulating drought stress-related gene expression and ultimately improving drought tolerance under both normal and drought conditions. These results were consistent with the growth phenotype of abs. We also preliminary analysed the structure of the SlMYB43 gene and verified the function of this gene in transgenic plants.

Conclusions
In conclusion, physiological analyses revealed that abs plants were more drought tolerant than MM plants were via two mechanisms. First, compared with MM plants, abs plants had a greater Pn and RWC as well as lower wilting and electrolyte leakage; second, abs plants had lower accumulation of H 2 O 2 and O 2 − through greater antioxidant enzyme activities under drought conditions. In contrast, these indicators in SlBRI1OE plants were opposite those in the abs plants: SlBRI1OE overexpression reduced the drought tolerance of transgenic plants. RNA-seq was used to characterize the influence of SlBRI1 expression on the drought tolerance of tomato. In total, the expression of 768 (53.9%) of 1425 genes whose expression was induced in response to drought and 418 (49.8%) of 840 genes whose expression was repressed in response to drought was regulated in abs in the same direction under normal conditions. Moreover, the expression of 158 and 43 genes whose expression was induced and repressed, respectively in response to drought was further upregulated and downregulated in abs plants under drought conditions. However, the major genes whose expression is regulated by SlBRI1OE were found to be associated with cellular processes and metabolic processes, which is consistent with the regulation of growth by SlBRI1OE under normal conditions. Furthermore, the expression of most droughtinduced genes whose expression was repressed by drought in the SlBRI1OE plants changed to the opposite direction (or there were no obvious changes) compared with that in the MM plants under drought conditions. Using GO enrichment analyses, we further found that the expression of genes related to ABA metabolism and polyamine biosynthesis, as well as oxidoreductase activity, is regulated in abs under both normal and drought conditions. Furthermore, the drought tolerance of abs tomato may be affected by regulating the expression of WRKY, ERF, bHLH and MYB TFs. Conversely, the expression of most of the same genes changed in the opposite direction (or no obvious change was detected) in the SlBRI1OE plants compared with the abs plants. Therefore, our study provides valuable information for future breeding to appropriately reduce the expression of SlBRI1 and improve drought tolerance without affecting plant growth.