Aging and deterioration of rice (Oryza sativa L.) during storage affect grain quality causing in-stage losses and reduced seed viability. Lipid degradation is primary cause of odor and decreasing seed viability associated with grain aging. Lipoxygenase (LOX) is a key enzyme in lipid metabolism, its activity directly affects the rate of aging in rice (Zhang et al., 2021). In our study, lipoxygenase genes OsLOX1, OsLOX2, OsLOX3 were edited by CRISPR/Cas9 in Ningjing 4 (approved by National Varietal Appraisal Committee in 2009, with No. 200940 and high-yielding, high-quality). LOX2 and LOX3 gene deletion significantly improved seed storage tolerance of rice (Supplementary fig 1). However, due to the poor agronomic traits and phenotypes of the LOX2 and LOX3 gene knockout lines(Supplementary fig 2), the research was mainly carried out on the LOX1 knockout lines. Three independent mutants (named cr-lox1-I, cr-lox1-II, and cr-lox1-III) at the target site (Figure 1 a) caused early termination of translation of OsLOX1. Knockout lines with homozygous gene edited target sites without the Cas9 locus were selected for further study by genetic segregation.
By artificial aging (T=40°C, RH=80%) (AA) for 22 days and natural aging (stored at room temperature) (NA) for 13 months we found that germination of knock-out lines after aging was significantly higher than that of Ningjing 4 (Figure 1 b, c, d). After AA treatment, OsLOX1 expression in seeds of knock-out lines had no significant change compared with Ningjing 4, and LOX activity of knock-out lines were significantly decreased (Figure 1 e, f). From these results, we concluded that OsLOX1 knockout abolished lipoxygenase activity and reduced the deterioration rate of seed viability during storage.
Linoleic acid is the main fatty acid and substrate in LOX pathway in rice; malonaldehyde (MDA) is final metabolite in LOX pathway, and its content is an important indicator to measure lipid peroxidation of rice during storage (Ebone et al., 2019). With aging linoleic acid contents of seeds of Ningjing 4 and knockout lines decreased significantly, and MDA began to accumulate. However, the rate of linoleic acid oxidation and MDA accumulation in the knock-out lines was less than in Ningjing 4 (Figure1 g, h). Therefore, OsLOX1 knockout reduced the rate of lipid peroxidation of seeds during storage.
To further understand transcriptional differences between OsLOX1 knockout line and Ningjing 4, seeds of Ningjing 4 and cr-lox1-I knockout line harvested in November 2019 were subjected to transcriptome sequencing before and after AA for 3 days on the basis of NA for 13 months. The difference in seed germination rate was the largest at 3 days and then declined to zero at 9 days (Supplementary fig 3 a). Differentially expressed genes (DEGs) in Ningjing 4 and cr-lox1-I were selected as candidate genes related to storage tolerance. Some candidate genes were selected for qRT-PCR to verify the reliability of RNA-seq (Supplementary fig 3 f). In Ningjing 4, 438 genes were up-regulated and 401 genes were down-regulated before and after AA. Only 16 genes were up-regulated and 23 genes were down-regulated in cr-lox1-I, indicating that RNA stability in dry seeds plays an important, positive role in seed longevity (Supplementary fig 3 b, c).
After KEGG enrichment, we found that the genes with lower expression levels in the cr-lox1-I than in Ningjing 4 after NA included multiple genes encoding phospholipases: OsPLDζ1, OsPLDζ2, OsPLDβ1. Phospholipase D (PLD) is involved in lipid signal transduction, metabolism and degradation by converting hydrated phospholipid to non-hydrated phospholipid during seed storage and processing (Deepika and Singh, 2022). This suggests that during seed storage, phospholipase gene is induced to express, catalyzing the hydrolysis of phospholipids on cell membranes to fatty acids that are then catalyzed by OsLOX1 to harmful peroxides, resulting in seed inactivation. The genes with higher expression levels in cr-lox1-I than in Ningjing 4 contained multiple acetaldehyde dehydrogenases: OsALDH2B1 and OsALDH6B2. Aldehyde dehydrogenase (ALDH) detoxifies cytotoxic compounds in plants, can oxidize aldehydes to corresponding carboxylic acids, which is crucial in maintaining seed longevity (Niranjan et al., 2021). After OsLOX1 knockout, the toxic substances produced by seeds were much lower whereas detoxification ability remained at a high level effectively maintaining seed viability.
Then what changes will happen to these differentially expressed genes in seeds after 3 days of AA? The 272 genes significantly down-regulated only in Ningjing 4 after AA were mainly enriched in endocytosis pathway, linolenic acid metabolism, lipid metabolism and other pathways suggesting that Ningjing 4 seeds were on the verge of inviability at this time, and enzymes that decomposed lipid and protein storage substances in seeds were also significantly down-regulated with depletion of storage substances (Figure 1 i). The 266 genes up-regulated only in Ningjing 4 were mainly enriched in the biosynthesis and metabolic pathways of amino acids such as phenylalanine, tyrosine, tryptophan, cysteine and methionine, illustrating the role of amino acid synthesis and metabolic pathways in regulating seed storability (Figure 1 j). The up-regulated gene only in the cr-lox1-I was Hsp16.9A that produced a small heat shock protein (sHsp), suggesting a protective effect of sHsp in seed subjected to high temperature and high humidity stress.
Starch, as the main seed component determining rice quality, undergoes significant changes during storage. Starch includes amylose and amylopectin, among which amylose has the greatest impact on quality. Gel consistency reflects the colloidal characteristics of rice flour and is an important indicator for evaluating edible quality and storage quality. There were no significant changes in total starch content, amylose content and gel consistency in fresh seeds of cr-lox1 compared with Ningjing 4. After 13 months of NA, the total starch content in seeds was lower, the amylose content was higher, and gel consistency was lower. However, the range of variation in cr-lox1 was lower than for Ningjing 4 (Figure 1 k). OsLOX1 knockout slows down the rate of quality change in Ningjing 4 seeds during storage. Finally, investigation of plant agronomic traits in the field showed that OsLOX1 knockout had little effect on plant agronomic traits (Supplementary fig 3 e).
In summary, LOX gene OsLOX1 in Ningjing 4 was edited by CRISPR/Cas9. LOX inactivation was achieved without affecting nutritional value or agronomic features. After multiple generations of screening and identification, stable genetic storage-tolerant lines were obtained. At the same time, the mutant LOX gene quality traits improvement in rice was successfully applied (Agricultural Foundation of China Report No. 770 (2019)). Transcriptome analysis suggesting the OsLOX1 affected the transcription of multiple genes during storage, and was involved in seed storage tolerance by regulating lipid metabolism and antioxidant pathways such as phosphatase and acetaldehyde dehydrogenase.