Exploring the influence of a single-nucleotide mutation in EIN4 on tomato fruit firmness diversity through fruit pericarp microstructure.

Tomato (Solanum lycopersicum) stands as one of the most valuable vegetable crops globally, and fruit firmness significantly impacts storage and transportation. To identify genes governing tomato firmness, we scrutinized the firmness of 266 accessions from core collections. Our study pinpointed an ethylene receptor gene, SlEIN4, located on chromosome 4 through a genome-wide association study (GWAS) of fruit firmness in the 266 tomato core accessions. A single-nucleotide polymorphism (SNP) (A → G) of SlEIN4 distinguished lower (AA) and higher (GG) fruit firmness genotypes. Through experiments, we observed that overexpression of SlEIN4AA significantly delayed tomato fruit ripening and dramatically reduced fruit firmness at the red ripe stage compared with the control. Conversely, gene editing of SlEIN4AA with CRISPR/Cas9 notably accelerated fruit ripening and significantly increased fruit firmness at the red ripe stage compared with the control. Further investigations revealed that fruit firmness is associated with alterations in the microstructure of the fruit pericarp. Additionally, SlEIN4AA positively regulates pectinase activity. The transient transformation assay verified that the SNP (A → G) on SlEIN4 caused different genetic effects, as overexpression of SlEIN4GG increased fruit firmness. Moreover, SlEIN4 exerts a negative regulatory role in tomato ripening by impacting ethylene evolution through the abundant expression of ethylene pathway regulatory genes. This study presents the first evidence of the role of ethylene receptor genes in regulating fruit firmness. These significant findings will facilitate the effective utilization of firmness and ripening traits in tomato improvement, offering promising opportunities for enhancing tomato storage and transportation capabilities.

A highly sensitive nanochannel device for the detection of SUMO1 peptides.

SUMOylation is an important and highly dynamic post-translational modification (PTM) process of protein, and its disequilibrium may cause various diseases, such as cancers and neurodegenerative disorders. SUMO proteins must be accurately detected to understand disease states and develop effective drugs. Reliable antibodies against SUMO2/3 are commercially available; however, efficient detectors are yet to be developed for SUMO1, which has only 50% homology with SUMO2 and SUMO3. Here, using phage display technology, we identified two cyclic peptide (CP) sequences that could specifically bind to the terminal dodecapeptide sequence of SUMO1. Then we combined the CPs and polyethylene terephthalate conical nanochannel films to fabricate a nanochannel device highly sensitive towards the SUMO1 terminal peptide and protein; sensitivity was achieved by ensuring marked variations in both transmembrane ionic current and Faraday current. The satisfactory SUMO1-sensing ability of this device makes it a promising tool for the time-point monitoring of the SENP1 enzyme-catalyzed de-SUMOylation reaction and cellular imaging. This study not only solves the challenge of SUMO1 precise recognition that could promote SUMO1 proteomics analysis, but also demonstrates the good potential of the nanochannel device in monitoring of enzymes and discovery of effective drugs.

Comparative transcriptome analysis of R3a and Avr3a-mediated defense responses in transgenic tomato.

Late blight caused by Phytophthora infestans is one of the most devastating diseases in potatoes and tomatoes. At present, several late blight resistance genes have been mapped and cloned. To better understand the transcriptome changes during the incompatible interaction process between R3a and Avr3a, in this study, after spraying DEX, the leaves of MM-R3a-Avr3a and MM-Avr3a transgenic plants at different time points were used for comparative transcriptome analysis. A total of 7,324 repeated DEGs were detected in MM-R3a-Avr3a plants at 2-h and 6-h, and 729 genes were differentially expressed at 6-h compared with 2-h. Only 1,319 repeated DEGs were found in MM-Avr3a at 2-h and 6-h, of which 330 genes have the same expression pattern. Based on GO, KEGG and WCGNA analysis of DEGs, the phenylpropanoid biosynthesis, plant-pathogen interaction, and plant hormone signal transduction pathways were significantly up-regulated. Parts of the down-regulated DEGs were enriched in carbon metabolism and the photosynthesis process. Among these DEGs, most of the transcription factors, such as WRKY, MYB, and NAC, related to disease resistance or endogenous hormones SA and ET pathways, as well as PR, CML, SGT1 gene were also significantly induced. Our results provide transcriptome-wide insights into R3a and Avr3a-mediated incompatibility interaction.

Inducible positive mutant screening system to unveil the signaling pathway of late blight resistance.

Late blight is the most devastating potato disease and it also causes serious yield loss in tomato. Several disease resistance genes (R genes) to late blight have been cloned from potato in the past decade. However, the resistance mechanisms remain elusive. Tomato and potato belong to the botanical family Solanaceace and share remarkably conserved genome structure. Since tomato is a model system in genetic and plant pathology research, we used tomato to develop a powerful mutant screening system that will greatly facilitate the analysis of the signaling pathway of resistance to Phytophthora infestans. First we proved that the R3a transgenic tomatoes developed specific hypersensitive cell death response (HR) to P. infestans strains carrying the corresponding avirulence gene Avr3a, indicating that the signaling pathway from the R3a-Avr3a recognition to HR is conserved between potato and tomato. Second, we generated transgenic tomatoes carrying both R3a and Avr3a genes, with the latter under the control of a glucocorticiod-inducible promoter. Dexamethasone induced expression of Avr3a and resulted in localized HR. This versatile system can be used to construct a mutant library to screen surviving mutants whose resistance signal transduction was interrupted, providing the basis to identify key genes involved in the resistance to late blight in Solanaceae.

The genome of the cucumber, Cucumis sativus L.

Cucumber is an economically important crop as well as a model system for sex determination studies and plant vascular biology. Here we report the draft genome sequence of Cucumis sativus var. sativus L., assembled using a novel combination of traditional Sanger and next-generation Illumina GA sequencing technologies to obtain 72.2-fold genome coverage. The absence of recent whole-genome duplication, along with the presence of few tandem duplications, explains the small number of genes in the cucumber. Our study establishes that five of the cucumber's seven chromosomes arose from fusions of ten ancestral chromosomes after divergence from Cucumis melo. The sequenced cucumber genome affords insight into traits such as its sex expression, disease resistance, biosynthesis of cucurbitacin and 'fresh green' odor. We also identify 686 gene clusters related to phloem function. The cucumber genome provides a valuable resource for developing elite cultivars and for studying the evolution and function of the plant vascular system.

Molecular isolation of the M gene suggests that a conserved-residue conversion induces the formation of bisexual flowers in cucumber plants.

Sex determination in plants involves a variety of mechanisms. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling gene M. M was identified as a previously characterized putative 1-aminocyclopropane-1-carboxylic acid synthase gene, while the m allele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.

Genetic association of ETHYLENE-INSENSITIVE3-like sequence with the sex-determining M locus in cucumber (Cucumis sativus L.).

Cucumber (Cucumis sativus L.) has served as the model system for sex expression in flowering plants and its sex type is predominantly controlled by two genetic loci, F and M. Ethylene is the major plant hormone that regulates sex expression in cucumber. The current model predicts that ethylene serves as both a promoter of femaleness via the F locus and an inhibitor of the male sex via the M locus. In support of this model, genetic, genomic, and transcript analyses indicate that the F gene encodes a key enzyme in ethylene biosynthesis. In this study, we discovered that the M locus co-segregates with an ETHYLENE-INSENSITIVE3 (EIN3)-like genomic sequence in an F2 population of 96 individuals. This genetic association agrees with the prediction that the M locus is involved in ethylene signaling, thus providing another line of evidence in favor of the model. In addition, we generated an amplified fragment length polymorphism (AFLP) map of the M locus, which was delimited into a genetic interval of 2.5 cM. The genetic association and the local map will assist the molecular isolation of the M gene using the combination of positional cloning and candidate gene approach.

Verticillium dahliae toxin induced alterations of cytoskeletons and nucleoli in Arabidopsis thaliana suspension cells.

In plant cells, cytoskeletons play important roles in response to biotic and abiotic stresses. However, little is known about the dynamics of cytoskeletons when cells are attacked by unphysical stress factors such as elicitors and toxins. We report here that the toxin of Verticillium dahliae (VD toxin) induced changes of microfilaments (MFs) and microtubules (MTs) in Arabidopsis thaliana suspension-cultured cells. When cells were treated with a low concentration of VD toxin, MFs were disrupted ordinally from the cortex to the perinuclear region, and then recovered spontaneously; but the MTs persisted. The MFs in the perinuclear region showed more resistance to VD toxin than the cortical ones. In contrast, when cells were treated with a high concentration of VD toxin, MFs and MTs were disrupted sooner and more severely and did not recover spontaneously. Treatments with high concentrations of VD toxin also induced changes of nucleoli. At the early stages of treatment, a nucleus had a single ring-shaped nucleolus. At the later stages, multiple smaller and more brightly fluorescing nucleoli emerged in a single nucleus. Disrupted MFs could be recovered by removing the VD toxin before the ring-shaped nucleoli appeared. All these results showed that MFs and MTs play important roles in the early defense responses against VD toxin in Arabidopsis suspension cells. The cytoskeletons may be used as sensors and effectors monitoring the defense reactions. The changes of nucleoli induced by VD toxin should be important characteristics of cell death.