Prolongation of seed viability and grain quality in rice by editing OsLOX1 using CRISPR/Cas9

Changling Mou, Yaping Chen, Ping Zhang, Qikai Tong, Ziyan Zhu, Tengfei Ma, Ping Wang, Kai Fu, Cheng Chen, Yunshuai Huang, Fulin Zhang, Qixian Hao, Min Zhang, Shijia Liu, Ling Jiang & Jianmin Wan

Molecular Breeding; 12 October 2024; vol.137; article 72

Abstract

Deterioration of rice (Oryza sativa L.) affects grain quality and seed viability during storage. Lipoxygenase (LOX), a key enzyme in lipid metabolism, directly affects the rate of ageing. Here, we found that knock-out of lipoxygenase gene OsLOX1 by CRISPR/Cas9 delayed loss of seed viability and quality. Transcriptome analysis showed that during storage, OsLOX1 affected transcription of multiple genes, including genes related to lipid metabolism and antioxidant pathways such as phosphatase and acetaldehyde dehydrogenase, which may regulate the seed storability. The genes significantly down- and up-regulated only in Ningjing 4 after NA for 13 months and 3 days of AA suggesting that OsLOX1 likely promoted seed viability in rice by balancing ageing and storage related genes, and regulated the seed storability through the amino acid synthesis and metabolic pathways. Moreover, knock-out of OsLOX1 without CRISPR/Cas9 not only improved the seed viability, but also had little impact on agronomic traits. More importantly, the OsLOX1 knock-out lines were approved in 2019 (Agricultural Foundation of China Report No. 770). Collectively, our study showed that knock-out of OsLOX1 is beneficial for prolongation of seed viability and can be directly applied to agricultural production.

See https://link.springer.com/article/10.1007/s11032-024-01506-4

Figure 1:

The germination and the physicochemical properties of wild type and OsLOX1 knock-out lines. a-b Germination of Ningjing 4 and cr-lox1 lines after AA of 22 days (a) and NA of 13 months (b), respectively. c Germination rates of Ningjing 4 and cr-lox1 lines with dormancy breaking followed by AA and NA. CK, fresh, untreated seeds; ‘AA-22-d’, seeds treated by AA; ‘NA-13-m’, seeds treated by NA; n = 5. Samples with different letters were significantly different (P < 0.05, One-way ANOVA with Tukey’s post-test). d Relative expression of OsLOX1 in Ningjing 4 and OsLOX1 knock-out lines after breaking dormancy and before aging. n = 3; the data were normalized with the level for Ningjing 4 as 1. The ubiquitin gene (Os03g0234200) was used as the internal control. e Comparison of lipoxygenase activities among Ningjing 4 and OsLOX1 knock-out lines after breaking dormancy and before aging. n = 5. c-e Samples with different letters were significantly different (P < 0.05, One-way ANOVA with Tukey’s post-test). Proportions of linoleic acid (f) and contents of MDA (g) among Ningjing 4, and cr-lox1-I, cr-lox1-II, and cr-lox1-III lines before and after AA for 22 days, n = 5. starch (h), amylose (i) and gel consistency (j) among Ningjing 4, and cr-lox1-I, cr-lox1-II, and cr-lox1-III lines before and after NA for 13 months, n = 5. f-j Samples with different letters were significantly different (P < 0.05, One-way ANOVA with Dunnett’s test). FS, AA-22-d and NA-13-m represent fresh seed, seeds treated by AA for 22 days and seeds treated by NA for 13 months, respectively