Calmodulin-like (CML) proteins are important Ca²⁺ sensors, which play significant role in mediating plant stress tolerance. In the present study, cold responsive calmodulin-like (ShCML44) gene was isolated from cold tolerant wild tomato (Solanum habrochaites) and functionally characterized. The ShCML44 was differentially expressed in all plant tissues including root, stem, leaf, flower and fruit and was strongly up-regulated under cold, drought and salinity stresses along with plant growth hormones. Under cold stress, progressive increase in the expression of ShCML44 was observed particularly in cold-tolerant S. habrochaites. The ShCML44-overexpressed plants showed greater tolerance to cold, drought and salinity stresses and recorded higher germination and better seedling growth. Transgenic tomato plants demonstrated higher antioxidant enzymes activity, gas exchange and water retention capacity with lower malondialdehyde accumulation and membrane damage under cold and drought stresses compared to wild-type. Moreover, transgenic plants exhibited reduced reactive oxygen species and higher relative water contents under cold and drought stress, respectively. Greater stress tolerance of transgenic plants was further reflected by the up-/down-regulation of stress-related genes including SOD, GST, CAT, POD, LOX, PR and ERD. In crux, these results strengthen the molecular understanding of ShCML44 gene to improve the abiotic stress tolerance in tomato.

Ascorbic acid (AsA) has high antioxidant activities, and its biosynthesis has been well studied by engineering of a single structural gene (SG) in staple crops, such as tomato (Solanum lycopersicum). However, engineering the AsA metabolic pathway by multi-SG for biofortification remains unclear. In this study, pyramiding transgenic lines including GDP-Mannose 3′,5′-epimerase (GME) × GDP-d-mannose pyrophosphorylase (GMP), GDP-l-Gal phosphorylase (GGP) × l-Gal-1-P phosphatase (GPP) and GME × GMP × GGP × GPP, were obtained by hybridization of four key genes to get over-expression transgenic plants (GME, GMP, GGP, and GPP) in tomato. Pyramiding lines exhibited a significant increase in total ascorbate in leaves and red fruits except for GGP × GPP. Expression analysis indicated that increased accumulation of AsA in pyramiding transgenic lines is due to multigene regulation in AsA biosynthesis. Substrate feeding in leaf and fruit suggested that AsA biosynthesis was mainly contributed by the d-Man/l-Gal pathway in leaves, while alternative pathways may contribute to AsA accumulation in tomato fruit. Pyramiding lines showed an enhanced light response, stress tolerance, and AsA transport capacity. Also, fruit shape, fruit size, and soluble solids were slightly affected by pyramiding. This study provides the first comprehensive analysis of gene pyramiding for ascorbate biosynthesis in tomato. SGs pyramiding promotes AsA biosynthesis, which in turn enhances light response and oxidative stress tolerance. Also, the data revealed an alternative ascorbate biosynthesis pathway between leaves and fruit of tomato.

RING finger proteins play an important role in plant adaptation to abiotic stresses. In the present study, a wild tomato (Solanum habrochaites) cold-induced RING-H2 finger gene, ShATL78L, was isolated, which has been identified as an abiotic stress responsive gene in tomato. The results showed that ShATL78L was constitutively expressed in various tissues such as root, lea petiole, stem, flower, and fruit. Cold stress up-regulated ShATL78L in the cold-tolerant S. habrochaites compared to the susceptible cultivated tomato (S. lycopersicum). Furthermore, ShATL78L expression was also regulated under different stresses such as drought, salt, heat, wound, osmotic stress, and exogenous hormones. Functional characterization showed that cultivated tomato overexpressing ShATL78L had improved tolerance to cold, drought and oxidative stresses compared to the wild-type and the knockdown lines. To understand the underlying molecular mechanism of ShATL78L regulating abiotic stress responses, we performed yeast one-hybrid and two-hybrid assays and found that RAV2 could bind to the promoter of ShATL78L and activate its transcription, and CSN5B could interact with ShATL78L to regulate abiotic stress responses. Taken together, these results show that ShATL78L plays an important role in regulating plant adaptation to abiotic stresses through bound by RAV2 and interacting with CSN5B.

This study assessed the effects of auxin analog, para-chlorophenoxyacetic acid (pCPA), on fruit set and development in Micro-Tom tomato (TOMJPF00001) via phenotypic, biochemical, metabolomic, and transcriptomic analyses. pCPA treatment significantly (p < 0.05) reduced floral abscission, increased fruit set and yield, while impacting fruit morphology, ripening, sugar content, organoleptic properties, and phytohormone profiles. Metabolomic profiling revealed 836 differentially accumulated metabolites (DAMs). Among these, several phenylpropanoid-related DAMs (isoferulic acid, 6-methylcoumarin, naringenin, hesperetin 5-O-glucoside, quercetin, and dihydrokawain) were upregulated in immature green, mature green, and breaker fruits, but downregulated in red ripe fruits following pCPA application. Transcriptome analysis yielded 35,501 differentially expressed genes (DEGs), including 740 novel genes, with growth stage-specific expression patterns observed in phenylpropanoid, carotenoid, and flavonoid biosynthetic pathways. Particularly, pCPA treatment downregulated chitinase (Soly04g072000.3) and acidic endochitinase (Soly05g050130.3), potentially enhancing fruit firmness through cell wall stabilization. Reduced accumulation of alpha-L-arabinofuranosidase (Soly10g077080.2) and a UDP-glucose 6-dehydrogenase family protein (Soly06g069550.1) further supported this observation. In red ripe fruits, pCPA decreased organic acids (malic and citric acids), sugars (fructose, glucose and sucrose), soluble solids (TSS/brix), amino acids (aspartic acid, phenylalanine, valine) and nucleotide (uracil, cytosine) levels, correlating with altered sensory attributes. pCPA also influenced lipid biosynthesis in mature green fruits and consistently downregulated sucrose across all developmental stages, suggesting impacts on carbohydrate metabolism. Furthermore, pCPA treatment altered the expression of genes related to carbohydrate metabolism, including beta-amylase (Soly08g007130.3), ADP-glucose pyrophosphorylase (Soly07g056140.3), and beta-glucosidase (Soly11g071640.2). pCPA-induced alterations in fruit development were correspondingly susceptible to alternative splicing patterns. These findings provide insights into the molecular mechanisms underlying pCPA-induced changes in tomato fruit set and development, offering valuable information for optimizing horticultural breeding practices and strategies.