The plant protein phosphatase 2C (PP2C) plays an irreplaceable role in phytohormone signaling, developmental processes, and manifold stresses. However, information about the PP2C gene family in tomato (Solanum lycopersicum) is relatively restricted. In this study, a genome-wide investigation of the SlPP2C gene family was performed. A total of 92 SlPP2C genes were identified, they were distributed on 11 chromosomes, and all the SlPP2C proteins have the type 2C phosphatase domains. Based on phylogenetic analysis of PP2C genes in Arabidopsis, rice, and tomato, SlPP2C genes were divided into eight groups, designated A–H, which is also supported by the analyses of gene structures and protein motifs. Gene duplication analysis revealed that the duplication of whole genome and chromosome segments was the main cause of SLPP2Cs expansion. A total of 26 cis-elements related to stress, hormones, and development were identified in the 3 kb upstream region of these SlPP2C genes. Expression profile analysis revealed that the SlPP2C genes display diverse expression patterns in various tomato tissues. Furthermore, we investigated the expression patterns of SlPP2C genes in response to Ralstonia solanacearum infection. RNA-seq and qRT-PCR data reveal that nine SlPP2Cs are correlated with R. solanacearum. The above evidence hinted that SlPP2C genes play multiple roles in tomato and may contribute to tomato resistance to bacterial wilt. This study obtained here will give an impetus to the understanding of the potential function of SlPP2Cs and lay a solid foundation for tomato breeding and transgenic resistance to plant pathogens.

Metallothionein (MT) is a multifunctional inducible protein in animals, plants, and microorganisms. MT is rich in cysteine residues (10−30%), can combine with metal ions, has a low molecular weight, and plays an essential biological role in various stages of the growth and development of organisms. Due to its strong ability to bind metal ions and scavenge free radicals, metallothionein has been used in medicine, health care, and other areas. Zinc is essential for plant growth, but excessive zinc (Zn) is bound to poison plants, and cadmium (Cd) is a significant environmental pollutant. A high concentration of cadmium can significantly affect the growth and development of plants and even lead to plant death. In this study, the human metallothionein gene HsMT1L under the control of the CaMV 35S constitutive promoter was transformed into tobacco, and the tolerance and accumulation capacity of transgenic tobacco plants to Zn and Cd were explored. The results showed that the high-level expression of HsMT1L in tobacco could significantly enhance the accumulation of Zn²⁺ and Cd²⁺ in both the aboveground parts and the roots compared to wild-type tobacco plants and conferred a greater tolerance to Zn and Cd in transgenic tobacco. Subcellular localization showed that HsMT1L was localized to the nucleus and cytoplasm in the tobacco. Our study suggests that HsMT1L can be used for the phytoremediation of soil for heavy metal removal.

Plant protein phosphatase 2C (PP2C) is recognized as one of the most critical protein family in plants and plays a pivotal role in disease resistance responses. However, the involvement of tomato PP2C family members in resistance to bacterial wilt caused by Ralstonia solanacearum remains poorly understood. In this study, we found that silencing SlPP2C28 increased tomato susceptibility to R. solanacearum; then, we introduced the tomato gene SlPP2C28, which exhibits a strong response to R. solanacearum, into the Nicotiana benthamiana genome via Agrobacterium-mediated transformation, generating high-expression transgenic lines OE-2 and OE-3. Following inoculation with R. solanacearum, the transgenic tobacco plants displayed reduced wilting symptoms, delayed disease onset, lower disease index, reduced stem cross-section damage, decreased internal bacterial colonization, diminished accumulation of reactive oxygen species in leaves, enhanced expression of SlPP2C28, up-regulated expression of defense-related genes NbSOD, NbPOD, and NbPAL along with an increase in the activities of their corresponding enzymes, and elevated expression levels of pathogenesis-related genes NbPR1a, NbPR2, NbPR4, and NbPR10. Collectively, these findings demonstrate that the SlPP2C28 gene has the function of enhancing resistance to bacterial wilt disease.

Tomato bacterial wilt, caused by Ralstonia solanacearum (G⁻), is one of the most devastating plant diseases. Developing effective resistance against this pathogen remains a major challenge in plant disease management. In this study, we constructed a fusion gene BPI-LY by combining the gene encoding the lipophilic functional domains of human bactericidal/permeability-increasing protein (BPI) with the gene of human lysozyme (LY). The recombinant gene BPI-LY was heterologously expressed in yeast and tomato. Preliminary in vitro assays in yeast demonstrated that BPI enhances LY’s antibacterial activity against G⁻ bacteria. Furthermore, overexpression of BPI-LY in tomato delayed onset of the disease in the transgenic lines and lowered the degree of tissue damage and the number of bacteria present in the stems relative to those in the wild-type plant. Additionally, the expression levels of the SlSOD, SlPOD, SlPAL, SlPR5, SlPR10, and SlPR-NP24 genes were indirectly upregulated in the transgenic plants following R. solanacearum inoculation. Collectively, these findings demonstrate that BPI-LY enhances the resistance of transgenic tomato against bacterial wilt caused by R. solanacearum.

The circadian clock genes in tomato are key regulators of cold stress adaptation. However, the low-temperature regulatory mechanism of the circadian clock gene SlPCL1 remains unclear. In this study, we evaluated the role of SlPCL1 in cold tolerance through low-temperature treatment of transgenic plants. Downstream target genes were identified using RNA-seq, RT-qPCR, yeast-one-hybrid (Y1H), dual-luciferase assays, and electrophoretic mobility shift assay (EMSA), while interacting proteins were characterized using yeast-two-hybrid (Y2H), luciferase complementation imaging (LCI), and pull-down assays, thereby elucidating the molecular mechanism underlying SlPCL1-mediated low-temperature regulation. We identified SlPCL1 as a nuclear-localized circadian clock gene with transcriptional repressor activity. Overexpression of SlPCL1 resulted in a cold-sensitive phenotype, whereas virus-induced gene silencing (VIGS)-mediated silencing of SlPCL1 enhanced cold tolerance. SlNPF4.6 functions as an abscisic acid (ABA) transporter involved in ABA transport. RNA-seq and RT-qPCR identified the ABA transporter SlNPF4.6 as a downstream target. Functional assays confirmed that SlPCL1 binds to the MYB element in the SlNPF4.6 promoter to repress its expression. Meanwhile, VIGS-mediated silencing of SlNPF4.6 decreased cold tolerance. Furthermore, the expression levels of the ABA receptor SlPYLs in the silenced lines were significantly reduced, confirming the decrease in intracellular ABA content. SlSUMO1, a ubiquitin-like protein, can influence gene transcription through noncovalent interactions. In addition, SlSUMO1 was found to interact with the SlPCL1 protein, attenuating SlPCL1 transcriptional repression activity. Together, these findings establish an SlSUMO1-mediated fine control mechanism of the SlPCL1-SlNPF4.6 regulatory module. This integration of circadian clock regulation uncovers new molecular mechanisms of cold tolerance and supports the development of cold-resistant breeding materials.