Whole exome sequencing identifies a novel mutation in Annexin A4 that is associated with recurrent spontaneous abortion.

Background: Recurrent spontaneous abortion (RSA) is a multifactorial disease, the exact causes of which are still unknown. Environmental, maternal, and genetic factors have been shown to contribute to this condition. The aim of this study was to investigate the presence of mutations in the ANXA4 gene in patients with RSA. Methods: Genomic DNA was extracted from 325 patients with RSA and 941 control women with a normal reproductive history for whole-exome sequencing (WES). The detected variants were annotated and filtered, and the pathogenicity of the variants was predicted through the SIFT online tool, functional enrichment analyses, Sanger sequencing validation, prediction of changes in protein structure, and evolutionary conservation analysis. Furthermore, plasmid construction, Western blotting, RT-qPCR, and cell migration, invasion and adhesion assays were used to detect the effects of ANXA4 mutations on protein function. Results: An ANXA4 mutation (p.G8D) in 1 of the 325 samples from patients with RSA (RSA-219) was identified through WES. This mutation was not detected in 941 controls or included in public databases. Evolutionary conservation analysis revealed that the amino acid residue affected by the mutation (p.G8D) was highly conserved among 13 vertebrate species, and the SIFT program and structural modeling analysis predicted that this mutation was harmful. Furthermore, functional assays revealed that this mutation could inhibit cell migration, invasion and adhesion. Conclusion: Our study suggests that an unreported novel ANXA4 mutation (p.G8D) plays an important role in the pathogenesis of RSA and may contribute to the genetic diagnosis of RSA.

SlBTB19 interacts with SlWRKY2 to suppress cold tolerance in tomato via the CBF pathway.

Cold stress restricts the metabolic and physiological activities of plants, thereby affecting their growth and development. Although broad-complex, tramtrack, and bric-à-brac (BTB) proteins are essential for diverse biological processes and stress responses, the mechanisms underlying BTB-mediated cold responses remain not fully understood. Here, we characterize the function of the cold-induced SlBTB19 protein in tomato (Solanum lycopersicum). Overexpression of SlBTB19 resulted in increased plant sensitivity to cold stress, whereas SlBTB19 knockout mutants exhibited a cold-tolerance phenotype. Further analyses, including protein-protein interaction studies and cell-free degradation assays, revealed that SlBTB19 interacts with and destabilizes the transcription factor SlWRKY2. Using virus-induced gene silencing (VIGS) to silence SlWRKY2 in both wild-type and slbtb19 mutants, we provided genetic evidence that SlWRKY2 acts downstream of SlBTB19 in regulating cold tolerance. Importantly, we demonstrated that SlWRKY2 positively regulates cold tolerance in a CRT/DRE binding factor (CBF)-dependent manner. Under cold stress, SlWRKY2 binds to the W-box in the CBF1 and CBF3 promoters, directly activating their expression. In summary, our findings identify a SlBTB19-SlWRKY2 module that negatively regulates the CBF-dependent cold tolerance pathway in tomato.

SlDELLA interacts with SlPIF4 to regulate arbuscular mycorrhizal symbiosis and phosphate uptake in tomato.

Arbuscular mycorrhizal symbiosis (AMS), a complex and delicate process, is precisely regulated by a multitude of transcription factors. PHYTOCHROME-INTERACTING FACTORS (PIFs) are critical in plant growth and stress responses. However, the involvement of PIFs in AMS and the molecular mechanisms underlying their regulator functions have not been well elucidated. Here, we show that SlPIF4 negatively regulates the arbuscular mycorrhizal fungi (AMF) colonization and AMS-induced phosphate uptake in tomato. Protein-protein interaction studies suggest that SlDELLA interacts with SlPIF4, reducing its protein stability and inhibiting its transcriptional activity towards downstream target genes. This interaction promotes the accumulation of strigolactones (SLs), facilitating AMS development and phosphate uptake. As a transcription factor, SlPIF4 directly transcriptionally regulates genes involved in SLs biosynthesis, including SlCCD7, SlCDD8, and SlMAX1, as well as the AMS-specific phosphate transporter genes PT4 and PT5. Collectively, our findings uncover a molecular mechanism by which the SlDELLA-SlPIF4 module regulates AMS and phosphate uptake in tomato. We clarify a molecular basis for how SlPIF4 interacts with SLs to regulate the AMS and propose a potential strategy to improve phosphate utilization efficiency by targeting the AMS-specific phosphate transporter genes PTs.

SlCPK27 cross-links SlHY5 and SlPIF4 in brassinosteroid-dependent photo- and thermo-morphogenesis in tomato.

ELONGATED HYPOCOTOYL5 (HY5) and PHYTOCHROME INTERACTING FACTORs (PIFs) are two types of important light-related regulators of plant growth, however, their interplay remains elusive. Here, we report that the activated tomato (Solanum lycopersicum) HY5 (SlHY5) triggers the transcription of a Calcium-dependent Protein Kinase SlCPK27. SlCPK27 interacts with and phosphorylates SlPIF4 at Ser-252 and Ser-308 phosphosites to promote its degradation. SlPIF4 promotes hypocotyl elongation mainly by activating the transcription of SlDWF, a key gene in brassinosteroid (BR) biosynthesis. Such a SlHY5-SlCPK27-SlPIF4-BR cascade not only plays a crucial role in photomorphogenesis but also regulates thermomorphogenesis. Our results uncover a previously unidentified mechanism that integrates Ca2+ signaling with the light signaling pathways to regulate plant growth by modulating BR biosynthesis in response to changes in ambient light and temperature.

The RALF2-FERONIA-MYB63 module orchestrates growth and defense in tomato roots.

Plant secreted peptides RAPID ALKALINISATION FACTORs (RALFs), which act through the receptor FERONIA (FER), play important roles in plant growth. However, it remains unclear whether and how RALF-FER contributes to the trade-off of plant growth-defense. Here, we used a variety of techniques such as CRISPR/Cas9, protein-protein interaction and transcriptional regulation methods to investigate the role of RALF2 and its receptor FER in regulating lignin deposition, root growth, and defense against Fusarium oxysporum f. sp. lycopersici (Fol) in tomato (Solanum lycopersicum). The ralf2 and fer mutants show reduced primary root length, elevated lignin accumulation, and enhanced resistance against Fol than the wild-type. FER interacts with and phosphorylates MYB63 to promote its degradation. MYB63 serves as an activator of lignin deposition by regulating the transcription of dirigent protein gene DIR19. Mutation of DIR19 suppresses lignin accumulation, and reverses the short root phenotype and Fol resistance in ralf2 or fer mutant. Collectively, our results demonstrate that the RALF2-FER-MYB63 module fine-tunes root growth and resistance against Fol through regulating the deposition of lignin in tomato roots. The study sheds new light on how plants maintain the growth-defense balance via RALF-FER.

Corrigendum: Melatonin mitigates cadmium phytotoxicity through modulation of phytochelatins biosynthesis, vacuolar sequestration, and antioxidant potential in Solanum lycopersicum L.

[This corrects the article DOI: 10.3389/fpls.2015.00601.].

Bioinspired Thermal Conductive Cellulose Nanofibers/Boron Nitride Coating Enabled by Co-Exfoliation and Interfacial Engineering.

Thermal conductive coating materials with combination of mechanical robustness, good adhesion and electrical insulation are in high demand in the electronics industry. However, very few progresses have been achieved in constructing a highly thermal conductive composites coating that can conformably coat on desired subjects for efficient thermal dissipation, due to their lack of materials design and structure control. Herein, we report a bioinspired thermal conductive coating material from cellulose nanofibers (CNFs), boron nitride (BN), and polydopamine (PDA) by mimicking the layered structure of nacre. Owing to the strong interfacial strength, mechanical robustness, and high thermal conductivity of CNFs, they do not only enhance the exfoliation and dispersion of BN nanoplates, but also bridge BN nanoplates to achieve superior thermal and mechanical performance. The resulting composites coating exhibits a high thermal conductivity of 13.8 W/(m·K) that surpasses most of the reported thermal conductive composites coating owing to the formation of an efficient thermal conductive pathway in the layered structure. Additionally, the coating material has good interface adhesion to conformably wrap around various substrates by scalable spray coating, combined with good mechanical robustness, sustainability, electrical insulation, low-cost, and easy processability, which makes our materials attractive for electronic packaging applications.

Far-red light inhibits lateral bud growth mainly through enhancing apical dominance independently of strigolactone synthesis in tomato.

The ratio of red light to far-red light (R:FR) is perceived by light receptors and consequently regulates plant architecture. Regulation of shoot branching by R:FR ratio involves plant hormones. However, the roles of strigolactone (SL), the key shoot branching hormone and the interplay of different hormones in the light regulation of shoot branching in tomato (Solanum lycopersicum) are elusive. Here, we found that defects in SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 in tomato resulted in more lateral bud growth but failed to reverse the FR inhibition of lateral bud growth, which was associated with increased auxin synthesis and decreased synthesis of cytokinin (CK) and brassinosteroid (BR). Treatment of auxin also inhibited shoot branching in ccd mutants. However, CK released the FR inhibition of lateral bud growth in ccd mutants, concomitant with the upregulation of BR synthesis genes. Furthermore, plants that overexpressed BR synthesis gene showed more lateral bud growth and the shoot branching was less sensitive to the low R:FR ratio. The results indicate that SL synthesis is dispensable for light regulation of shoot branching in tomato. Auxin mediates the response to R:FR ratio to regulate shoot branching by suppressing CK and BR synthesis.

Characterization and quality evaluation of QiXueShuFu Decoction based on fingerprint and ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometry.

QiXueShuFu Decoction (QXSFD) modified from the Bazhen Decoction which was originally from the classic Ming Dynasty is a traditional folk formula that boosts the body's immune system. However, its ambiguous chemical components limited its quality control evaluation. In this study, ultra-performance liquid chromatography (UPLC) fingerprint combined with multivariate analysis was used to evaluate the quality of 15 batches of QXSFD, and UPLC quadrupole-orbitrap mass spectrometry was used to further examine the chemical components in QXSFD, after which representative compounds from each disassembled prescription were selected for comparison. Fifteen batches of samples had 33 common peaks in which 11 differential components could be used as a reference for subsequent quality control. One hundred forty-three components were identified from QXSFD. Saponins were mainly derived from the monarch, terpenes from the minister, and polysaccharides and glycosides from the assistant. In addition, quantitative assay revealed that the content of ferulic acid, chlorogenic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside and 3,6'-disinapoyl sucrose in the whole prescription were higher than the contents of each disassembled prescription. This is the first comprehensive quality report on the chemical components of QXSFD, which is important for pharmacodynamic material basis and quality control.

The transcription factor SPL13 mediates strigolactone suppression of shoot branching by inhibiting cytokinin synthesis in Solanum lycopersicum.

Plant architecture imposes a large impact on crop yield. IDEAL PLANT ARCHITECTURE 1 (IPA1), which encodes a SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor, is a target of molecular design for improving grain yield. However, the roles of SPL transcription factors in regulating tomato (Solanum lycopersicum) plant architecture are unclear. Here, we show that the expression of SPL13 is down-regulated in the lateral buds of strigolactone (SL)-deficient ccd mutants and is induced by GR24 (a synthetic analog of SL). Knockout of SPL13 by CRISPR/Cas9 resulted in higher levels of cytokinins (CKs) and transcripts of the CK synthesis gene ISOPENTENYL TRANSFERASES 1 (IPT1) in the stem nodes, and more growth of lateral buds. GR24 suppresses CK synthesis and lateral bud growth in ccd mutants, but is not effective in spl13 mutants. On the other hand, silencing of the IPT1 gene inhibited bud growth of spl13 mutants. Interestingly, SL levels in root extracts and exudates are significantly increased in spl13 mutants. Molecular studies indicated that SPL13 directly represses the transcription of IPT1 and the SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and MORE AXILLARY GROWTH 1 (MAX1). The results demonstrate that SPL13 acts downstream of SL to suppress lateral bud growth by inhibiting CK synthesis in tomato. Tuning the expression of SPL13 is a potential approach for decreasing the number of lateral shoots in tomato.

SlMPK1- and SlMPK2-mediated SlBBX17 phosphorylation positively regulates CBF-dependent cold tolerance in tomato.

B-box (BBX) proteins are an important class of zinc finger transcription factors that play a critical role in plant growth and stress response. However, the mechanisms of how BBX proteins participate in the cold response in tomato remain unclear. Here, using approaches of reverse genetics, biochemical and molecular biology we characterized a BBX transcription factor, SlBBX17, which positively regulates cold tolerance in tomato (Solanum lycopersicum). Overexpressing SlBBX17 enhanced C-repeat binding factor (CBF)-dependent cold tolerance in tomato plants, whereas silencing SlBBX17 increased plant susceptibility to cold stress. Crucially, the positive role of SlBBX17 in CBF-dependent cold tolerance was dependent on ELONGATED HYPOCOTYL5 (HY5). SlBBX17 physically interacted with SlHY5 to directly promote the protein stability of SlHY5 and subsequently increased the transcriptional activity of SlHY5 on SlCBF genes under cold stress. Further experiments showed that cold-activated mitogen-activated protein kinases, SlMPK1 and SlMPK2, also physically interact with and phosphorylate SlBBX17 to enhance the interaction between SlBBX17 and SlHY5, leading to enhanced CBF-dependent cold tolerance. Collectively, the study unveiled a mechanistic framework by which SlMPK1/2-SlBBX17-SlHY5 regulated transcription of SlCBFs to enhance cold tolerance, thereby shedding light on the molecular mechanisms of how plants respond to cold stress via multiple transcription factors.

HY5 functions as a systemic signal by integrating BRC1-dependent hormone signaling in tomato bud outgrowth.

Light plays an important role in determining plant architecture, which greatly influences crop yield. However, the precise mechanisms by which light signaling regulates bud outgrowth remain to be identified. Here, we show that light regulates bud outgrowth via both HY5 and brassinosteroid (BR)-dependent pathways in tomato. Inactivation of the red-light photoreceptor PHYB, or deficiencies in PHYB or the blue-light photoreceptor CRY1a, inhibits bud outgrowth and leads to decreased accumulation of HY5 protein and increased transcript level of BRANCHED1 (BRC1), a central integrator of branching signals. HY5, functioning as a mobile systemic signal from leaves, promotes bud outgrowth by directly suppressing BRC1 transcript and activating the transcript of BR biosynthesis genes within the lateral buds in tomato. Furthermore, BRC1 prevents the accumulation of cytokinin (CK) and gibberellin (GA) by directly inhibiting the transcript of CK synthesis gene LOG4, while increasing the transcript levels of CK and GA degradation genes (CKX7, GA2ox4, and GA2ox5), leading to an arrest of bud outgrowth. Moreover, bud outgrowth occurs predominantly in the day due to the suppression of BRC1 transcript by HY5. These findings demonstrate that light-inducible HY5 acts as a systemic signaling factor in fine-tuning the bud outgrowth of tomato.

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato.

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.

The APETALA2a/DWARF/BRASSINAZOLE-RESISTANT 1 module contributes to carotenoid synthesis in tomato fruits.

Ethylene (ET) signaling plays a critical role in the ripening of climacteric fruits such as tomato. Brassinosteroids (BRs) were found to promote the ripening of both climacteric and non-climacteric fruits. However, the mechanism of interaction between ET and BRs during fruit ripening is unclear. Here, we found that BR synthesis and signaling increased after the onset of fruit ripening. Overexpression of the BR synthesis gene DWARF (DWF) promotedfruit softening, lycopene synthesis and ET production, whereas defect of DWF inhibited them. BRASSINAZOLE RESISTANT 1 (BZR1) as a key component of BR signaling, enhanced fruit lycopene content by directly activating the transcription of PSY1 gene. Interestingly, the increases in BR synthesis and BZR1 protein levels were dependent on ET signaling. Knocking out the ET-induced APETALA2a (AP2a) suppressed the expression of DWF and BR accumulation. Molecular assays demonstrated that AP2a was a positive regulator of DWF expression. Furthermore, 28-homobrassinolide, a bioactive BR, partially compensated the defects of lycopene accumulation and expression of PSY1 in ap2a mutant fruits. The results demonstrated that AP2a mediated ET signaling to regulate BR synthesis and signaling. BRs played critical roles in lycopene synthesis after onset of fruit ripening.

S-Nitrosoglutathione Reductase Contributes to Thermotolerance by Modulating High Temperature-Induced Apoplastic H2O2 in Solanum lycopersicum.

S-nitrosoglutathione reductase (GSNOR) is considered as a critical regulator of plant stress tolerance for its impacts on protein S-nitrosylation through regulation of the S-nitrosothiol (SNO) level. However, the mechanism of GSNOR-mediated stress tolerance is still obscure. Here, we found that GSNOR activity was induced by high temperature in tomato (Solanum lycopersicum) plants, whereas mRNA level of SlGSNOR1 exhibited little response. Suppressing SlGSNOR1 expression by virus-induced gene silencing (VIGS) increased accumulation of SNO and nitrites under high temperature and reduced thermotolerance. The compromised thermotolerance was associated with less accumulation of abscisic acid (ABA) and salicylic acid (SA), attenuated activation of mitogen-activated protein kinase (MAPK) and reduced expression of heat shock protein. Intriguingly, SlGSNOR1 silencing impaired upregulation of RESPIRATORY BURST OXIDASE HOMOLOG1 (SlRBOH1) and apoplastic H2O2 accumulation in response to high temperature, whereas SlRBOH1 silencing abolished activation of GSNOR and led to a similar decline in thermotolerance as in SlGSNOR1-silenced plants. Importantly, H2O2 treatment recovered the thermotolerance and improved antioxidant capacity in SlGSNOR1-silenced plants. Our results suggest that GSNOR plays a role in regulating the SlRBOH1-dependent apoplastic H2O2 production in response to high temperature, while a balanced interaction between SNO and H2O2 is critical for maintaining the cellular redox homeostasis and thermotolerance.

Light-dependent activation of HY5 promotes mycorrhizal symbiosis in tomato by systemically regulating strigolactone biosynthesis.

Light quality affects mutualisms between plant roots and arbuscular mycorrhizal fungi (AMFs), which modify nutrient acquisition in plants. However, the mechanisms by which light systemically modulates root colonization by AMFs and phosphate uptake in roots remain unclear. We used a range of approaches, including grafting techniques, protein immunoblot analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, and dual-luciferase assays, to unveil the molecular basis of light signal transmission from shoot to root that mediates arbuscule development and phosphate uptake in tomato. The results show that shoot phytochrome B (phyB) triggers shoot-derived mobile ELONGATED HYPOCOTYL5 (HY5) protein accumulation in roots, and HY5 further positively regulates transcription of strigolactone (SL) synthetic genes, thus forming a shoot phyB-dependent systemic signaling pathway that regulates the synthesis and accumulation of SLs in roots. Further experiments with carotenoid cleavage dioxygenase 7 mutants and supplementary red light confirm that SLs are indispensable in the red-light-regulated mycorrhizal symbiosis in roots. Our results reveal a phyB-HY5-SLs systemic signaling cascade that facilitates mycorrhizal symbiosis and phosphate utilization in plants. The findings provide new prospects for the potential application of AMFs and light manipulation to effectively improve nutrient utilization and minimize the use of chemical fertilizers and associated pollution.

A Novel Role of Pipecolic Acid Biosynthetic Pathway in Drought Tolerance through the Antioxidant System in Tomato.

With global warming and water shortage, drought stress is provoking an increasing impact on plant growth, development, and crop productivity worldwide. Pipecolic acid (Pip) is an emerging lysine catabolite in plants, acting as a critical element in disease resistance with a related signal pathway of phytohormone salicylic acid (SA). While SA plays a vital role in various abiotic stresses, the role of Pip in plant response to abiotic stresses, especially drought, remains largely unknown. To address this issue, Pip biosynthetic gene Slald1 mutants and hydroxylated modification gene Slfmo1 mutants were generated using CRISPR-Cas9 gene-editing approaches. Drought resistance dramatically increased in Slald1 mutants compared with wild-type, which was associated with increased CO2 assimilation, photosystems activities, antioxidant enzymes activities, ascorbate and glutathione content, and reduced reactive oxygen species accumulation, lipid peroxidation and protein oxidation. On the contrary, Slfmo1 mutants were more sensitive to drought, showing damaged photosystems and impaired antioxidant systems, which were significantly alleviated by exogenous ascorbate. Our results demonstrate that Pip biosynthesis and hydroxylated modification pathways play a critical role in drought tolerance through the antioxidant system in tomato. This knowledge can be helpful to breed improved crop cultivars that are better equipped with drought resistance.

Strigolactones positively regulate abscisic acid-dependent heat and cold tolerance in tomato.

Strigolactones are carotenoid-derived phytohormones that impact plant growth and development in diverse ways. However, the roles of strigolactones in the responses to temperature stresses are largely unknown. Here, we demonstrated that strigolactone biosynthesis is induced in tomato (Solanum lycopersicum) by heat and cold stresses. Compromised strigolactone biosynthesis or signaling negatively affected heat and cold tolerance, while application of the synthetic strigolactone analog GR245DS enhanced heat and cold tolerance. Strigolactone-mediated heat and cold tolerance was associated with the induction of abscisic acid (ABA), heat shock protein 70 (HSP70) accumulation, C-REPEAT BINDING FACTOR 1 (CBF1) transcription, and antioxidant enzyme activity. Importantly, a deficiency in ABA biosynthesis compromised the GR245DS effects on heat and cold stresses and abolished the GR245DS-induced transcription of HSP70, CBF1, and antioxidant-related genes. These results support that strigolactones positively regulate tomato heat and cold tolerance and that they do so at least partially by the induction of CBFs and HSPs and the antioxidant response in an ABA-dependent manner.

The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression.

Iron (Fe) deficiency affects global crop productivity and human health. However, the role of light signaling in plant Fe uptake remains uncharacterized. Here, we find that light-induced Fe uptake in tomato (Solanum lycopersicum L.) is largely dependent on phytochrome B (phyB). Light induces the phyB-dependent accumulation of ELONGATED HYPOCOTYL 5 (HY5) protein both in the leaves and roots. HY5 movement from shoots to roots activates the expression of FER transcription factor, leading to the accumulation of transcripts involved in Fe uptake. Mutation in FER abolishes the light quality-induced changes in Fe uptake. The low Fe uptake observed in phyB, hy5, and fer mutants is accompanied by lower photosynthetic electron transport rates. Exposure to red light at night increases Fe accumulation in wild-type fruit but has little effects on fruit of phyB mutants. Taken together, these results demonstrate that Fe uptake is systemically regulated by light in a phyB-HY5-FER-dependent manner. These findings provide new insights how the manipulation of light quality could be used to improve Fe uptake and hence the nutritional quality of crops.

Crosstalk between Brassinosteroid and Redox Signaling Contributes to the Activation of CBF Expression during Cold Responses in Tomato.

Brassinosteroids (BRs) play a critical role in plant responses to stress. However, the interplay of BRs and reactive oxygen species signaling in cold stress responses remains unclear. Here, we demonstrate that a partial loss of function in the BR biosynthesis gene DWARF resulted in lower whilst overexpression of DWARF led to increased levels of C-REPEAT BINDING FACTOR (CBF) transcripts. Exposure to cold stress increased BR synthesis and led to an accumulation of brassinazole-resistant 1 (BZR1), a central component of BR signaling. Mutation of BZR1 compromised the cold- and BR-dependent increases in CBFs and RESPIRATORY BURST OXIDASE HOMOLOG 1(RBOH1) transcripts, as well as preventing hydrogen peroxide (H2O2) accumulation in the apoplast. Cold- and BR-induced BZR1 bound to the promoters of CBF1, CBF3 and RBOH1 and promoted their expression. Significantly, suppression of RBOH1 expression compromised cold- and BR-induced accumulation of BZR1 and related increases in CBF transcripts. Moreover, RBOH1-dependent H2O2 production regulated BZR1 accumulation and the levels of CBF transcripts by influencing glutathione homeostasis. Taken together, these results demonstrate that crosstalk between BZR1 and reactive oxygen species mediates cold- and BR-activated CBF expression, leading to cold tolerance in tomato (Solanum lycopersicum).