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Free, publicly-accessible full text available January 1, 2025
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Abstract Use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated 9)-mediated genome editing has proliferated for use in numerous plant species to modify gene function and expression, usually in the context of either transient or stably inherited genetic alternations. While extremely useful in many applications, modification of some loci yields outcomes detrimental to further experimental evaluation or viability of the target organism. Expression of Cas9 under a promoter conferring gene knockouts in a tissue-specific subset of genomes has been demonstrated in insect and animal models, and recently in
Arabidopsis . We developed an in planta GFP (green fluorescent protein) assay system to demonstrate fruit-specific gene editing in tomato using aphosphoenolpyruvate carboxylase 2 gene promoter. We then targeted a SET-domain containing polycomb protein, SlEZ2, previously shown to yield pleiotropic phenotypes when targeted via35S-driven RNA interference and we were able to characterize fruit phenotypes absent additional developmental perturbations. Tissue-specific gene editing will have applications in assessing function of essential genes otherwise difficult to study via germline modifications and will provide routes to edited genomes in tissues that could not otherwise be recovered when their germline modification perturbs their normal development. -
Summary Many
Actinidia cultivars are characterized by anthocyanin accumulation, specifically in the inner pericarp, but the underlying regulatory mechanism remains elusive. Here we report two interacting transcription factors, AcMYB 123 and AcbHLH 42, that regulate tissue‐specific anthocyanin biosynthesis in the inner pericarp ofActinidia chinensis cv. Hongyang. Through transcriptome profiling analysis we identified fiveMYB and threebHLH transcription factors that were upregulated in the inner pericarp. We show that the combinatorial action of two of them, AcMYB 123 and AcbHLH 42, is required for activating promoters ofAcANS andAcF3GT1 that encode the dedicated enzymes for anthocyanin biosynthesis. The presence of anthocyanin in the inner pericarp appears to be tightly associated with elevated expression ofAcMYB123 andAcbHLH42 .RNA interference repression ofAcMYB123 ,AcbHLH42 ,AcF3GT1 andAcANS in ‘Hongyang’ fruits resulted in significantly reduced anthocyanin biosynthesis. Using both transient assays inNicotiana tabacum leaves orActinidia arguta fruits and stable transformation in Arabidopsis, we demonstrate that co‐expression ofAcMYB123 andAcbHLH42 is a prerequisite for anthocyanin production by activating transcription ofAcF3GT1 andAcANS or the homologous genes. Phylogenetic analysis suggests that AcMYB 123 or AcbHLH 42 are closely related toTT 2 orTT 8, respectively, which determines proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than regulators in dicots. All these experimental results suggest that AcMYB 123 and AcbHLH 42 are the components involved in spatiotemporal regulation of anthocyanin biosynthesis specifically in the inner pericarp of kiwifruit.