Effective genome editing and identification of a regiospecific gallic acid 4-O-glycosyltransferase in pomegranate (Punica granatum L.)

Lijing Chang,Li Tian,Sheng Wu

doi:10.1038/s41438-019-0206-7

Abstract

Pomegranate (Punica granatum L.) trees are woody perennials that bear colorful and nutritious fruits rich in phenolic metabolites, e.g., hydrolyzable tannins (HTs) and flavonoids. We here report genome editing and gene discovery in pomegranate hairy roots using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), coupled with transcriptome and biochemical analyses. Single guide RNAs (sgRNAs) were designed to target two UDP-dependent glycosyltransferases (UGTs), PgUGT84A23 and PgUGT84A24, which possess overlapping activities in β-glucogallin (a galloylglucose ester; biosynthetic precursor of HTs) biosynthesis. A unique accumulation of gallic acid 3-O- and 4-O-glucosides (galloylglucose ethers) was observed in the PgUGT84A23 and PgUGT84A24 dual CRISPR/Cas9-edited lines (i.e., ugt84a23 ugt84a24) but not the control (empty vector) or PgUGT84A23/PgUGT84A24 single edited lines (ugt84a23 or ugt84a24). Transcriptome and real-time qPCR analyses identified 11 UGTs with increased expression in the ugt84a23 ugt84a24 hairy roots compared to the controls. Of the 11 candidate UGTs, only PgUGT72BD1 used gallic acid as substrate and produced a regiospecific product gallic acid 4-O-glucoside. This work demonstrates that the CRISPR/Cas9 method can facilitate functional genomics studies in pomegranate and shows promise for capitalizing on the metabolic potential of pomegranate for germplasm improvement.

Highlights

The woody plant pomegranate (Punica granatum L.)produces colorful flowers and fruits with ornamental and culinary values
To knockout the activity of PgUGT84A23 or PgUGT84A24, one Single guide RNAs (sgRNAs) for PgUGT84A23 and two sgRNAs for PgUGT84A24 were designed, which are specific for each target gene and away from the Plant Secondary Product Glycosyltransferase (PSPG) motif conserved among plant UDP-dependent glycosyltransferases (UGTs) for binding sugar donors (Fig. 1b)
To eliminate both PgUGT84A23 and PgUGT84A24 activities, sgRNA for PgUGT84A23 (sgRNA23) and sgRNA24-1/sgRNA24-2 were placed into the same expression plasmid (Fig. 1c)

Summary

Introduction

The woody plant pomegranate (Punica granatum L.)produces colorful flowers and fruits with ornamental and culinary values. Pomegranate fruits and juice have been pursued by consumers for their favorable nutritional quality, contributed by the abundant phenolic compounds, e.g., hydrolyzable tannins (HTs) and flavonoids, in these tissues and products. Complementary to the classic breeding approach, new molecular techniques, such as genome editing, can enable targeted modification of key metabolic genes for improved nutritional and commercial quality of pomegranate fruits and products. Among the various genome-editing technologies, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) has gained increasing popularity for its efficiency and ease of use. In this method, a single guide RNA (sgRNA) directs the Cas nuclease to the target gene sequence upstream of a protospacer adjacent motif. Cas creates a break in the double-strand DNA, which is ligated by homology-directed repair or non-homologous

Methods

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Discussion

Conclusion