Galactinol synthase 2 influences the metabolism of chlorophyll, carotenoids, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyses the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR/Cas9 gene-editing technology in tomato (Solanum lycopersicum) to create the gols2 mutant showing uniformly green fruits without dark-green shoulders, and promoting fruit ripening. Analysis indicated that galactinol was undetectable in the ovaries and fruits of the mutant, and the accumulation of chlorophyll and chloroplast development was suppressed in the fruits. RNA-sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development were down-regulated, including PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, GOLDEN 2-LIKE 2, and CHLOROPHYLL A/B-BINDING PROTEINS. In addition, early color transformation and ethylene release was prompted in the gols2 lines by regulation of the expression of genes involved in carotenoid and ethylene metabolism (e.g. PHYTOENE SYNTHASE 1, CAROTENE CIS-TRANS ISOMERASE, and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2/4) and fruit ripening (e.g. RIPENING INHIBITOR, NON-RIPENING, and APETALA2a). Our results provide evidence for the involvement of GolS2 in pigment and ethylene metabolism of tomato fruits.

Tomato short internodes and pedicels encode an LRR receptor-like serine/threonine-protein kinase ERECTA regulating stem elongation through modulating gibberellin metabolism.

Plant height is an important agronomic trait. Dwarf varieties present several advantages, such as lodging resistance, increased yield, and suitability for mechanized harvesting, which are crucial for crop improvement. However, limited research is available on dwarf tomato varieties suitable for production. In this study, we report a novel short internode mutant named "short internode and pedicel (sip)" in tomato, which exhibits marked internode and pedicel shortening due to suppressed cell elongation. This mutant plant has a compact plant structure and compact inflorescence, and has been demonstrated to produce more fruits, resulting in a higher harvest index. Genetic analysis revealed that this phenotype is controlled by a single recessive gene, SlSIP. BSA analysis and KASP genotyping indicated that ERECTA (ER) is the possible candidate gene for SlSIP, which encodes a leucine-rich receptor-like kinase. Additionally, we obtained an ER functional loss mutant using the CRISPR/Cas9 gene-editing technology. The 401st base A of ER is substituted with T in sip, resulting in a change in the 134th amino acid from asparagine (N) to isoleucine (I). Molecular dynamics(MD) simulations showed that this mutation site is located in the extracellular LRR domain and alters nearby ionic bonds, leading to a change in the spatial structure of this site. Transcriptome analysis indicated that the genes that were differentially expressed between sip and wild-type (WT) plants were enriched in the gibberellin metabolic pathway. We found that GA3 and GA4 decreased in the sip mutant, and exogenous GA3 restored the sip to the height of the WT plant. These findings reveal that SlSIP in tomatoes regulates stem elongation by regulating gibberellin metabolism. These results provide new insights into the mechanisms of tomato dwarfing and germplasm resources for breeding dwarfing tomatoes.

Construction of transcription factor mutagenesis population in tomato using a pooled CRISPR/Cas9 plasmid library.

Adequate mutant materials are the prerequisite for conducting gene function research or screening novel functional genes in plants. The strategy of constructing a large-scale mutant population using the pooled CRISPR/Cas9-sgRNA library has been implemented in several crops. However, the effective application of this CRISPR/Cas9 large-scale screening strategy to tomato remains to be attempted. Here, we identified 990 transcription factors in the tomato genome, designed and synthesized a CRISPR/Cas9 plasmid library containing 4379 sgRNAs. Using this pooled library, 487 T0 positive plants were obtained, among which 92 plants harbored a single sgRNA sequence, targeting 65 different transcription factors, with a mutation rate of 23%. In the T0 mutant population, the occurrence of homozygous and biallelic mutations was observed at higher frequencies. Additionally, the utilization of a small-scale CRISPR/Cas9 library targeting 30 transcription factors could enhance the efficacy of single sgRNA recognition in positive plants, increasing it from 19% to 42%. Phenotypic characterization of several mutants identified from the mutant population demonstrated the utility of our CRISPR/Cas9 mutant library. Taken together, our study offers insights into the implementation and optimization of CRISPR/Cas9-mediated large-scale knockout library in tomato.