Targeted insertion of regulatory elements enables translational enhancement in rice

In-locus editing of agronomically-important genes to optimize their spatiotemporal expression is becoming an important breeding approach. Compared to intensive studies on mRNA transcription, manipulating protein translation by genome editing has not been well exploited. Here, we found that precise knock-in of a regulating element into the 5’UTR of a target gene could efficiently increase its protein abundance in rice. We firstly screened a translational enhancer (AMVE) from alfalfa mosaic virus using protoplast-based luciferase assays with an 8.5-folds enhancement. Then the chemically modified donor of AMVE was synthesized and targeted inserted into the 5’UTRs of two genes (WRKY71 and SKC1) using CRISPR/Cas9. Following the in-locus AMVE knock-in, we observed up to a 2.8-fold increase in the amount of WRKY71 protein. Notably, editing of SKC1, a sodium transporter, significantly increased salt tolerance in T2 seedlings, indicating the expected regulation of AMVE knock-in. These data demonstrated the feasibility of such in-locus editing to enhance protein expression, providing a new approach to manipulating protein translation for crop breeding

Application of γ-aminobutyric acid (GABA) and nitrogen regulates aroma biochemistry in fragrant rice

The 2-acetyl-1-pyrroline (2AP) is a key aroma compound in fragrant rice. The present study assessed the γ-aminobutyric acid (GABA) and nitrogen (N) application induced regulations in the biochemical basis of rice aroma formation. Four N levels, that is, 0, 0.87, 1.75, and 2.61 g/pot, and two GABA treatments, that is, 0 mg/L (GABA0) and 250 mg/L (GABA250), were applied to three fragrant rice cultivars, that is, Yuxiangyouzhan, Yungengyou 14, and Basmati-385. Results showed that GABA250 increased 2AP, Na, Mn, Zn, and Fe contents by 8.44%, 10.95%, 25.70%, 11.14%, and 43.30%, respectively, under N treatments across cultivars. The GABA250 further enhanced the activities of proline dehydrogenase (PDH), ornithine aminotransferase (OAT) (both at 15 days after heading (d AH), and diamine oxidase (DAO) (at maturity) by 20.36%, 11.24%, and 17.71%, respectively. Significant interaction between GABA and N for Mn, Zn, and Fe contents in grains, proline content in leaves, GABA content in leaves at 15 d AH and maturity stage (MS), Δ1-pyrroline-5-carboxylic acid (P5C) contents in leaves at 15 d AH, and Δ1-pyrroline-5-carboxylate synthase (P5CS), PDH, and OAT activities in leaves at MS was noted. Moreover, the 2AP contents in grains at MS showed a significant and positive correlation with the proline contents in the leaves at 15d AH. In conclusion, GABA250 enhanced the 2AP, Na, Mn, Zn, and Fe contents, as well as the enzyme activities involved in 2AP biosynthesis. Exogenous GABA and N application improved the 2AP contents and nutrient uptake in fragrant rice