Letter to the editor

Efficient generation of pink-fruited tomatoes using CRISPR/Cas9 system

Retrograde signaling between plastids and the nucleus is vital for chloroplast biogenesis and environmental responses. GENOMES UNCOUPLED1 (GUN1) was proposed to be a central integrator of multiple retrograde signaling pathways in the model plant Arabidopsis thaliana (Arabidopsis). However, the function of GUN1 orthologs in other plant species has not been well studied. Here, we found that many GUN1 orthologs from the Solanaceae family have a short N-terminus before the first pentatricopeptide repeat (PPR) motif which is predicted as intrinsically disordered regions (IDRs). Functional analyses of tomato (Solanum lycopersicum L.) GUN1 (SlGUN1), which does not contain N-terminal IDRs, show that it can complement the GUN phenotype of the Arabidopsis gun1 mutant (Atgun1). However, in contrast to the AtGUN1 protein, which does contain the N-terminal IDRs, the SlGUN1 protein is highly accumulated even after chloroplast biogenesis is completed, suggesting that the N-terminal IDRs may determine the stability of the GUN1 protein. Furthermore, we generated tomato Slgun1 genome-edited mutants via the CRISPR–Cas9 system. The Slgun1 mutants exhibited a typical GUN phenotype under lincomycin (Lin) or norflurazon (NF) treatment. Moreover, Slgun1 mutants are hypersensitive to low concentrations of Lin or NF. Taken together, our results suggest that, although lacking the N-terminal IDRs, SlGUN1 plays conserved roles in plastid retrograde signaling in tomato plants.

High temperature stress is one of the major environmental factors that affect the growth and development of plants. Although WRKY transcription factors play a critical role in stress responses, there are few studies on the regulation of heat stress by WRKY transcription factors, especially in tomato. Here, we identified a group I WRKY transcription factor, SlWRKY3, involved in thermotolerance in tomato. First, SlWRKY3 was induced and upregulated under heat stress. Accordingly, overexpression of SlWRKY3 led to an increase, whereas knock-out of SlWRKY3 resulted in decreased tolerance to heat stress. Overexpression of SlWRKY3 accumulated less reactive oxygen species (ROS), whereas knock-out of SlWRKY3 accumulated more ROS under heat stress. This indicated that SlWRKY3 positively regulates heat stress in tomato. In addition, SlWRKY3 activated the expression of a range of abiotic stress-responsive genes involved in ROS scavenging, such as a SlGRXS1 gene cluster. Further analysis showed that SlWRKY3 can bind to the promoters of the SlGRXS1 gene cluster and activate their expression. Collectively, these results imply that SlWRKY3 is a positive regulator of thermotolerance through direct binding to the promoters of the SlGRXS1 gene cluster and activating their expression and ROS scavenging.

Plant species have evolved diverse metabolic pathways to effectively respond to internal and external signals throughout their life cycle, allowing adaptation to their sessile and phototropic nature. These pathways selectively activate specific metabolic processes, producing plant secondary metabolites (PSMs) governed by genetic and environmental factors. Humans have utilized PSM-enriched plant sources for millennia in medicine and nutraceuticals. Recent technological advances have significantly contributed to discovering metabolic pathways and related genes involved in the biosynthesis of specific PSM in different food crops and medicinal plants. Consequently, there is a growing demand for plant materials rich in nutrients and bioactive compounds, marketed as “superfoods”. To meet the industrial demand for superfoods and therapeutic PSMs, modern methods such as system biology, omics, synthetic biology, and genome editing (GE) play a crucial role in identifying the molecular players, limiting steps, and regulatory circuitry involved in PSM production. Among these methods, clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR/Cas) is the most widely used system for plant GE due to its simple design, flexibility, precision, and multiplexing capabilities. Utilizing the CRISPR-based toolbox for metabolic engineering (ME) offers an ideal solution for developing plants with tailored preventive (nutraceuticals) and curative (therapeutic) metabolic profiles in an ecofriendly way. This review discusses recent advances in understanding the multifactorial regulation of metabolic pathways, the application of CRISPR-based tools for plant ME, and the potential research areas for enhancing plant metabolic profiles.

Agricultural plants have a significant impact on the availability of food and energy for people, but it is deteriorated by numerous abiotic stresses like salinity, and drought due to climate change. These abiotic stresses threaten the food security by reducing crop plants' yields significantly. Breeding abiotic stress-tolerant crop varieties are the most environment friendly and sustainable solution of the problems. These have been developed using conventional plant breeding techniques such as mass selection, pure line selection, bulk method, single seed descent method, plant introduction, development of hybrid varsities, pedigree method, backcross method. However, these techniques are typically laborious and time-consuming. Alternatively, clustered regularly interspaced short palindromic repeats associated with Cas endonuclease (CRISPR/Cas) genome editing has become a novel method for precise and effective genetic alteration with plant genomes. Nowadays, many stress tolerant mutants have been developed by using the CRISPR tool. The effect of salinity and drought on plants is highlighted in this review, along with the CRISPR tool's operation and its use in plants to mitigate the effect of aforesaid stresses. We also discuss recently developed techniques and limitations of the CRISPR genome editing tool that can be used to confer salinity and drought.