The RNA Editing Factor SlORRM4 Is Required for Normal Fruit Ripening in Tomato

Abstract

RNA editing plays a key posttranscriptional role in gene expression. Existing studies on cytidine-to-uridine RNA editing in plants have focused on maize (), rice (), and Arabidopsis (). However, the importance and regulation of RNA editing in several critical agronomic processes are not well understood, a notable example of which is fruit ripening. Here, we analyzed the expression profile of 33 RNA editing factors and identified 11 putative tomato () fruit ripening-related factors. A rapid virus-induced gene silencing assay indicated that the organelle RNA recognition motif-containing protein SlORRM4 affected tomato fruit ripening. Knocking out SlORRM4 expression using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome editing strategy delayed tomato fruit ripening by lowering respiratory rate and ethylene production. Additionally, the expression of numerous genes associated with fruit ripening and mitochondrial functions changed significantly when was knocked out. Moreover, the loss of function significantly reduced RNA editing of many mitochondrial transcripts, leading to low-level expression of some core subunits that are critical for mitochondrial complex assembly (i.e. Nad3, Cytc1, and COX II). Taken together, these results indicate that SlORRM4 is involved in RNA editing of transcripts in ripening fruit that influence mitochondrial function and key aspects of fruit ripening.

Lycopene Is Enriched in Tomato Fruit by CRISPR/Cas9-Mediated Multiplex Genome Editing

“… Ito et al (2015) reported the CRISPR/Cas9 system can efficiently induce mutations in the tomato RIN gene and affected the accumulation or structure of the RIN protein. Yang et al (2017) showed that maturation was significantly inhibited in mutants after RNA editing factor SlORRM4 was knocked out by CRISPR/Cas9 in tomato. However, CRISPR/Cas9 genome editing also has certain limitations, the genotypes cannot be artificially controlled, and production of large numbers of homozygotes often requires propagation over several generations.…”

Application and future perspective of CRISPR/Cas9 genome editing in fruit crops

“…However, tomato has been a model for studying fleshy fruit development and fruit ripening for decades (Vrebalov et al ; Giovannoni ). CRISPR/Cas9 genome editing was used to investigate gene function as shown in ARGONAUTE7 ( SlAGO7 ) (Brooks et al ), SHORT ROOT (SHR) (Ron et al ), RIPENING INHIBITOR (RIN) (Ito et al ; ), RNA Editing Factor SlORRM4 (Yang et al ), SlMAPK3 in drought tolerance (Wang et al ), auxin signaling SlIAA9 in parthenocarpic fruit (Ueta et al ), and PROCERA , a DELLA protein, in fruit development (Tomlinson et al ). High mutation frequency (83.56%) was observed at SlPDS (phytoene desaturase) and SlPIF4 (Phytochrome interacting factor) in the T 0 lines, and mutations were stably transmitted to the T 1 and T 2 generations (Pan et al ).…”

Genome Editing as a Tool for Fruit Ripening Manipulation

“…In flowering plants, RNA editing by cytidine-to-uridine (C-to-U) conversion is a widespread process that occurs only in plastids and mitochondrial transcripts and plays an important role in developmental processes such as organelle biogenesis, adaptation to environmental changes and signal transduction ( Ichinose and Sugita, 2017 ). In a recent report, Yang et al (2017) aimed to identify RNA editing factors that might play an essential role in the regulation of tomato fruit ripening. A virus-induced gene silencing (VIGS) assay was performed, targeting 11 RNA editing factor genes putatively related to ripening, positively identifying SlORRM4 , which is located in mitochondria ( Yang et al, 2017 ).…”

“…In a recent report, Yang et al (2017) aimed to identify RNA editing factors that might play an essential role in the regulation of tomato fruit ripening. A virus-induced gene silencing (VIGS) assay was performed, targeting 11 RNA editing factor genes putatively related to ripening, positively identifying SlORRM4 , which is located in mitochondria ( Yang et al, 2017 ). Consistently, CRISPR/Cas9-mediated stable slorrm4 mutants resulted in a delay of ripening, and in a diminution of the respiratory rates and ethylene production compared with the wild-type.…”