Stimulation of Tomato Drought Tolerance by PHYTOCHROME A and B1B2 Mutations.

Drought stress is a severe environmental issue that threatens agriculture at a large scale. PHYTOCHROMES (PHYs) are important photoreceptors in plants that control plant growth and development and are involved in plant stress response. The aim of this study was to identify the role of PHYs in the tomato cv. 'Moneymaker' under drought conditions. The tomato genome contains five PHYs, among which mutant lines in tomato PHYA and PHYB (B1 and B2) were used. Compared to the WT, phyA and phyB1B2 mutants exhibited drought tolerance and showed inhibition of electrolyte leakage and malondialdehyde accumulation, indicating decreased membrane damage in the leaves. Both phy mutants also inhibited oxidative damage by enhancing the expression of reactive oxygen species (ROS) scavenger genes, inhibiting hydrogen peroxide (H2O2) accumulation, and enhancing the percentage of antioxidant activities via DPPH test. Moreover, expression levels of several aquaporins were significantly higher in phyA and phyB1B2, and the relative water content (RWC) in leaves was higher than the RWC in the WT under drought stress, suggesting the enhancement of hydration status in the phy mutants. Therefore, inhibition of oxidative damage in phyA and phyB1B2 mutants may mitigate the harmful effects of drought by preventing membrane damage and conserving the plant hydrostatus.

Functional Characterization of Tomato Phytochrome A and B1B2 Mutants in Response to Heat Stress.

Heat stress (HS) is a prevalent negative factor affecting plant growth and development, as it is predominant worldwide and threatens agriculture on a large scale. PHYTOCHROMES (PHYs) are photoreceptors that control plant growth and development, and the stress signaling response partially interferes with their activity. PHYA, B1, and B2 are the most well-known PHY types in tomatoes. Our study aimed to identify the role of tomato 'Money Maker' phyA and phyB1B2 mutants in stable and fluctuating high temperatures at different growth stages. In the seed germination and vegetative growth stages, the phy mutants were HS tolerant, while during the flowering stage the phy mutants revealed two opposing roles depending on the HS exposure period. The response of the phy mutants to HS during the fruiting stage showed similarity to WT. The most obvious stage that demonstrated phy mutants' tolerance was the vegetative growth stage, in which a high degree of membrane stability and enhanced water preservation were achieved by the regulation of stomatal closure. In addition, both mutants upregulated the expression of heat-responsive genes related to heat tolerance. In addition to lower malondialdehyde accumulation, the phyA mutant enhanced proline levels. These results clarified the response of tomato phyA and phyB1B2 mutants to HS.

Efficient base editing in tomato using a highly expressed transient system.

KEY MESSAGE: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.

Genome editing in PDS genes of tomatoes by non-selection method and of Nicotiana benthamiana by one single guide RNA to edit two orthologs.

The CRISPR/Cas9 system is widely used for targeted mutagenesis in many organisms including plants. For application of this system, tissue culture methods need to be established. In this study, detailed methods for introduction of mutations in tomato and Nicotiana benthamiana plants using the CRISPR/Cas9 system are described. The methods include tissue culture protocols for tomato and N. benthamiana. We also demonstrate the methodology to generate Cas9-free genome edited tomato plants and use of one single guide RNA (sgRNA) to edit two orthologs in N. benthamiana. The examples of editing the PHYTOENE DESATURASE (PDS) genes in these plants are also provided. The Cas9-free tomato line was obtained when tomato plants were cultured on a non-selective medium after transformation with the CRISPR/Cas9 system. Two orthologs of PDS in N. benthamiana were mutated using a sgRNA, because these orthologs contain the same nucleotide sequences with PAM motif. These mutations were inherited to the next generation. The mutations in the PDS genes resulted in an albino phenotype in tomato and N. benthamiana plants. These results demonstrate that the non-selective method is one of the ways to obtain Cas9-free genome editing in tomato plants and that the two orthologs can be edited by one sgRNA in N. benthamiana.