Electroweak Multi-Higgs Production: A Smoking Gun for the Type-I Two-Higgs-Doublet Model.

Extending the Higgs sector of the standard model (SM) by just one additional Higgs doublet field leads to the two-Higgs-doublet model (2HDM). In the type-I Z_{2}-symmetric limit of the 2HDM, all the five new physical Higgs states can be fairly light, O(100) GeV or less, without being in conflict with current data from the direct Higgs boson searches and the B-physics measurements. In this Letter, we establish that the new neutral as well as the charged Higgs bosons in this model can all be simultaneously observable in the multi-b final state. The statistical significance of the signature for each of these Higgs states, resulting from the electroweak (EW) production of their pairs, can exceed 5σ at the 13 TeV high-luminosity Large Hadron collider (HL-LHC). Since the parameter space configurations where this is achievable are precluded in the other, more extensively pursued, 2HDM types, an experimental validation of our findings would be a clear indication that the true underlying Higgs sector in nature is the type-I 2HDM.

SlTCP24 and SlTCP29 synergistically regulate compound leaf development through interacting with SlAS2 and activating transcription of SlCKX2 in tomato.

The complexity of compound leaves results primarily from the leaflet initiation and arrangement during leaf development. However, the molecular mechanism underlying compound leaf development remains a central research question. SlTCP24 and SlTCP29, two plant-specific transcription factors with the conserved TCP motif, are shown here to synergistically regulate compound leaf development in tomato. When both of them were knocked out simultaneously, the number of leaflets significantly increased, and the shape of the leaves became more complex. SlTCP24 and SlTCP29 could form both homodimers and heterodimers, and such dimerization was impeded by the leaf polarity regulator SlAS2, which interacted with SlTCP24 and SlTCP29. SlTCP24 and SlTCP29 could bind to the TCP-binding cis-element of the SlCKX2 promoter and activate its transcription. Transgenic plants with SlTCP24 and SlTCP29 double-gene knockout had a lowered transcript level of SlCKX2 and an elevated level of cytokinin. This work led to the identification of two key regulators of tomato compound leaf development and their targeted genes involved in cytokinin metabolic pathway. A model of regulation of compound leaf development was proposed based on observations of this study.

Genome-wide analysis of Myo-inositol oxygenase gene family in tomato reveals their involvement in ascorbic acid accumulation.

BACKGROUND: Ascorbic acid (Vitamin C, AsA) is an antioxidant metabolite involved in plant development and environmental stimuli. AsA biosynthesis has been well studied in plants, and MIOX is a critical enzyme in plants AsA biosynthesis pathway. However, Myo-inositol oxygenase (MIOX) gene family members and their involvement in AsA biosynthesis and response to abiotic stress remain unclear. RESULTS: In this study, five tomato genes encoding MIOX proteins and possessing MIOX motifs were identified. Structural analysis and distribution mapping showed that 5 MIOX genes contain different intron/exon patterns and unevenly distributed among four chromosomes. Besides, expression analyses indicated the remarkable expression of SlMIOX genes in different plant tissues. Furthermore, transgenic lines were obtained by over-expression of the MIOX4 gene in tomato. The overexpression lines showed a significant increase in total ascorbate in leaves and red fruits compared to control. Expression analysis revealed that increased accumulation of AsA in MIOX4 overexpression lines is possible as a consequence of the multiple genes involved in AsA biosynthesis. Myo inositol (MI) feeding in leaf and fruit implied that the Myo-inositol pathway improved the AsA biosynthesis in leaves and fruits. MIOX4 overexpression lines exhibited a better light response, abiotic stress tolerance, and AsA biosynthesis capacity. CONCLUSIONS: These results showed that MIOX4 transgenic lines contribute to AsA biosynthesis, evident as better light response and improved oxidative stress tolerance. This study provides the first comprehensive analysis of the MIOX gene family and their involvement in ascorbate biosynthesis in tomato.

An allelic variant of GAME9 determines its binding capacity with the GAME17 promoter in the regulation of steroidal glycoalkaloid biosynthesis in tomato.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules found in the family Solanaceae. SGA content varies among different plant species and varieties. However, the genetic mechanisms regulating SGA content remain unclear. Here, we demonstrate that genetic variation in GLYCOALKALOID METABOLISM 9 (GAME9) is responsible for the variation in SGA content in tomato (Solanum lycopersicum). During a sequential analysis we found a 1 bp substitution in the AP2/ERF binding domain of GAME9. The 1 bp substitution in GAME9 was significantly associated with high SGA content and determined the binding capacity of GAME9 with the promoter of GAME17, a core SGA biosynthesis gene. The high-SGA GAME9 allele is mainly present in S. pimpinellifolium and S. lycopersicum var. cerasiforme populations and encodes a protein that can bind the GAME17 promoter. In contrast, the low-SGA GAME9 allele is mainly present in the big-fruited varieties of S. lycopersicum and encodes a protein that shows weak binding to the GAME17 promoter. Our findings provide new insight into the regulation of SGA biosynthesis and the factors that affect the accumulation of SGA in tomato.

Biosynthetic Gene Pyramiding Leads to Ascorbate Accumulation with Enhanced Oxidative 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.

An ATL78-Like RING-H2 Finger Protein Confers Abiotic Stress Tolerance through Interacting with RAV2 and CSN5B in 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, leaf, 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 activates/alters 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. Highlight: RAV2 binds to the promoter of ShATL78L to activates/alters its transcription to adapt the environmental conditions; furthermore, ShATL78L interacts with CSN5B to regulate the stress tolerance.

Overexpression of calmodulin-like (ShCML44) stress-responsive gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses.

Calmodulin-like (CML) proteins are important Ca(2+) 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.

Genome-wide identification, characterization and expression analysis of calmodulin-like (CML) proteins in tomato (Solanum lycopersicum).

Calcium (Ca(2+)) has emerged as a significant secondary messenger that regulates the activities of hormonal and environmental signals that are associated with biotic and abiotic stresses. Ca(2+) binding proteins typically contain a Ca(2+) binding EF-hand (a helix-loop-helix structure) motif. In this study, tomato genes encoding calmodulin-like (CML) proteins that possess EF-hand motifs and no other identifiable functional domains were analyzed. Using genome analysis and BLAST searches in database, 52 CML genes were identified in tomato. Comprehensive analyses, including evolutionary relationships, gene structures, chromosomal locations, functional annotations, and gene duplications, were performed. Distribution mapping exhibited that 52 SlCML proteins containing different intron/exon patterns were unevenly distributed among ten chromosomes. In addition, 24 SlCML proteins were predicted as segmentally duplicated. Conserved motifs, promoter cis-regulatory elements, organ-based expression patterns and expression analyses indicated the potential responsiveness of SlCML proteins to abiotic stresses and phytohormones. These results illustrate the complexity of the CML gene family and indicate a potential vital role for these molecules in tomato growth and development as Ca(2+) signal transducers.