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Key Message: Combining phenotype and gene expression analysis of the CRISPR/Cas9-induced SlAN2 mutants, we revealed that SlAN2 specifically regulated anthocyanin accumulation in vegetative tissues in purple tomato cultivar 'Indigo Rose.' Anthocyanins play an important role in plant development and also exhibit human health benefits. The tomato genome contains four highly homologous anthocyanin-related R2R3-MYB transcription factors: SlAN2, SlANT1, SlANT1-like, and SlAN2-like/Aft. SlAN2-like/Aft regulates anthocyanin accumulation in the fruit; however, the genetic function of the other three factors remains unclear. To better understand the function of R2R3-MYB transcription factors, we conducted targeted mutagenesis of SlAN2 in the purple tomato cultivar 'Indigo Rose' using clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9). The SlAN2 mutants had a fruit color and anthocyanin content similar to cv. 'Indigo Rose,' while the anthocyanin content and the relative expression levels of several anthocyanin-related genes in vegetative tissues were significantly lower in the SlAN2 mutant relative to cv. Indigo Rose. Furthermore, we found that anthocyanin biosynthesis is controlled by different regulators between tomato hypocotyls and cotyledons. In addition, SlAN2 mutants were shorter, with smaller and lighter fruits than cv. 'Indigo Rose.' Our findings further our understanding of anthocyanin production in tomato and other plant species.

Anthocyanin fruit (Aft) and atroviolacea (atv) were characterized in wild tomato and can enhance anthocyanin content in tomato fruit. However, the gene underlying the Aft locus and the mechanism by which Aft and atv act remain largely unknown. In this study, the Aft locus was fine-mapped to an approximately 145-kb interval on chromosome 10, excluding SlAN2 (Solyc10g086250), SlANT1 (Solyc10g086260) and SlANT1-like (Solyc10g086270), which have previously been suggested as candidates. Thus, the R2R3-MYB transcription factor SlAN2-like (Solyc10g086290) was considered the best candidate gene for Aft. The CRISPR/Cas9-mediated SlAN2-like mutants show a much lower accumulation of anthocyanins associated with the downregulation of multiple anthocyanin-related genes compared to the wild-type tomato, indicating that SlAN2-like is responsible for the Aft phenotype. The repressive function of SlMYBATV also was confirmed through the CRISPR/Cas9 approach. A yeast-two-hybrid assay revealed that SlMYBATV interacts with the bHLH protein SlJAF13. Furthermore, yeast-one-hybrid and dual-luciferase transient expression assays showed that Aft directly binds to the SlMYBATV promoter and activates its expression. The results herein provide candidate genes to enhance anthocyanin content in tomato fruit. This research also provides insight into a mechanism involving the Aft-SlMYBATV pathway that fine-tunes anthocyanin accumulation in tomato fruit., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

High quantities of anthocyanins in plants confer potential protective benefits against biotic and abiotic stressors. Studies have shown that the bZIP transcription factor HY5 plays a key role in controlling anthocyanin accumulation in response to light. However, in hy5 mutants, residual anthocyanins have been detected, indicating that other regulators exist to regulate anthocyanin biosynthesis in an HY5-independent manner. Here, we employed the CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9) system specifically to induce targeted mutagenesis of SlHY5 in the purple tomato cultivar 'Indigo Rose'. The T2 generation of tomato plants homozygous for the null allele of the SlHY5 frameshift mutated by a 1 bp insertion contained a lower anthocyanin content. Transcriptional analysis showed that most of the anthocyanin biosynthesis structural genes and several regulatory genes were down-regulated in the hy5 mutant lines. With transcriptome analyses of the various tissues from hy5 mutant lines, eight candidate transcription factors were identified that may regulate anthocyanin biosynthesis in an HY5-independent manner. These findings deepen our understanding of how light controls anthocyanin accumulation and facilitate the identification of the regulators of anthocyanin biosynthesis in an HY5-independent manner., (� The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Fruit ripening is usually accompanied by anthocyanin accumulation. Ethylene is key in ripening-induced anthocyanin production in many fruits. However, the effects of fruit ripening and ethylene on anthocyanin biosynthesis in purple tomato fruits are unclear. This study shows that bagged fruits of the purple tomato cultivar 'Indigo Rose' failed to produce anthocyanins at the red ripening stage after bag removal. In contrast, the bagged immature fruits accumulated a significant amount of anthocyanins after removing the bags. The transcriptomic analyses between immature and red ripening fruit before and after bag removal revealed that anthocyanin-related genes, including the key positive R2R3-MYB regulator SlAN2-like, were repressed in the red ripening fruit. The 86 identified transcription factors, including 13 AP2/ERF, 7 bZIP, 8 bHLH and 6 MYB, showed significantly different expressions between immature and red ripening fruits. Moreover, subjecting bagged immature fruits to exogenous ethylene treatment significantly inhibited anthocyanin accumulation and the expression of anthocyanin-related genes, including the anthocyanin structure genes and SlAN2-like. Thus, ethylene inhibits anthocyanin biosynthesis by repressing the transcription of SlAN2-like and other anthocyanin-related genes. These findings provide new insights into anthocyanin regulation in purple tomato fruit. [ABSTRACT FROM AUTHOR]