Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System

Abstract

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography–tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit. Differential gene expression analysis revealed 728 DEGs between WT and sgr#1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, with fold-change >2 and adjusted p-value < 0.05. Most of the DEGs have functions related to photosynthesis, chloroplasts, and carotenoid biosynthesis. The strong changes in pigment and carotenoid content resulted in the accumulation of key primary metabolites, such as sucrose and its derivatives (fructose, galactinol, and raffinose), glycolytic intermediates (glucose, glucose-6-phosphate, and fructose-6-phosphate), and tricarboxylic acid cycle intermediates (malate and fumarate) in the leaves and fruit of the SGR-KO null lines. Overall, the SGR1-KO null lines developed here provide new evidence for the mechanisms underlying the roles of SGR1 as well as the molecular pathways involved in photosynthesis, chloroplasts, and carotenoid biosynthesis.

Receptor for Activated C Kinase1B (RACK1B) Delays Salinity-Induced Senescence in Rice Leaves by Regulating Chlorophyll Degradation

“…Mendel’s green cotyledon gene stay-green (SGR), that encodes Mg-dechelatase, has recently been recognized as a central regulator of natural and stress-induced chlorophyll breakdown [ 35 , 38 , 54 , 55 , 56 , 57 ]. SGR interacts with all known CCEs and forms a multiprotein complex with CCEs and LHCII proteins for chlorophyll degradation, the proteolytic cleavage of chloroplast, and the channeling of phototoxic intermediate materials into vacuoles [ 36 , 37 , 58 , 59 ].…”

“…Remarkably, chloroplast ribosome-associated proteins are found to support translation during stress conditions [ 79 ]. Over the last decade, several functional studies in Arabidopsis , rice, and other plants have implicated SGR as one of the master regulators of Chl degradation, and sgr mutant plants displayed delayed the Chl-degradation phenotype during natural and stress-induced senescence [ 55 , 56 , 69 , 80 , 81 , 82 , 83 ]. Rice has two SGR homologs, SGR and SGR-like (SGRL), with overlapping biochemical functions [ 59 ].…”

Physicochemical Properties and Antioxidant Activity of CRISPR/Cas9-Edited Tomato SGR1 Knockout (KO) Line

“…The sgr1 null lines "sgr1 #1-6 and sgr1 #2-4", previously reported by [9], were selected and grown in the GMO greenhouse in the Horticulture Department of Horticultural Biotechnology in Hankyoung National University (Korea) in spring 2023. The fertilizer solution was used after adjustment to pH 5.5-5.8 and EC 2.0-2.2 dS m-1 at each growth stage.…”

“…As the tomato fruit ripens, chloroplasts develop into colored bodies, so chlorophyll is lost, and carotenoids accumulate. Previous studies have reported that point mutations in the gene encoding the tomato STAY-GREEN (SGR) protein inhibit chlorophyll degradation during fruit ripening, resulting in an increased chlorophyll and carotenoid content, resulting in brown fruits [9]. In addition, a transformant in which the SGR1 gene was silenced by RNA interference in tomato had a similar phenotype to that of the green flesh mutant [10].…”

Strategies and Methods for Improving the Efficiency of CRISPR/Cas9 Gene Editing in Plant Molecular Breeding

“…They found that only the TaeIF4E triple mutant was completely resistant to WYMV and set fruit normally; the single or double mutant remained susceptible and showed dwarf plants and severely reduced fruit set after susceptibility [ 66 ]. Not only has multi-gene editing been used on a large scale in major crops, but new superior traits have been created through multi-gene editing in a variety of crops such as tomatoes, apples, bananas, and potatoes ( Solanum tuberosum L.) [ 67 , 68 , 69 , 70 , 71 , 72 ].…”