Our study examined the mutation efficiency of the CRISPR/Cas9 method for tryptophan aminotransferase BnaTAA1 genes involved in the auxin biosynthesis pathway. We made nine CRISPR/Cas9 constructs with various promoters driving the expression of a Cas9 from Staphylococcus aureus (SaCas9) or a plant-codon-optimized Streptococcus pyogenes Cas9 (pcoCas9). We developed a fast and efficient system for evaluating the variety and frequency of mutations caused by each construct using Brassica napus hairy roots. We showed that pcoCas9 is more efficient in mutating the targeted loci than SaCas9 and the presence of the NLS signal enhanced the chance of mutagenesis by 25%. The mutations were studied further in regenerated lines, and we determined the BnaTAA1 gene expression and heritability of the gene modifications in transgenic plants. Hairy root transformation combined with CRISPR/Cas9-mediated gene editing represents a fast and straightforward system for studying target gene function in the important oilseed crop B. napus.
Brassica napus (rapeseed) is the second most important oilseed crop worldwide. Global rise in average ambient temperature and extreme weather severely impact rapeseed seed yield. However, fewer research explained the phenotype changes caused by moderate-to-high temperatures in rapeseed. To investigate these events, we determined the long-term response of three spring cultivars to different temperature regimes (21/18°C, 28/18°C, and 34/18°C) mimicking natural temperature variations. The analysis focused on the plant appearance, seed yield, quality and viability, and embryo development. Our microscopic observations suggest that embryonic development is accelerated and defective in high temperatures. Reduced viable seed yield at warm ambient temperature is due to a reduced fertilization rate, increased abortion rate, defective embryonic development, and pre-harvest sprouting. Reduced auxin levels in young seeds and low ABA and auxin levels in mature seeds may cause embryo pattern defects and reduced seed dormancy, respectively. Glucosinolates and oil composition measurements suggest reduced seed quality. These identified cues help understand seed thermomorphogenesis and pave the way to developing thermoresilient rapeseed.
Background Plant sexual reproduction is highly sensitive to elevated ambient temperatures, impacting seed development and production. We previously phenotyped this effect on three rapeseed cultivars (DH12075, Topas DH4079, and Westar). This work describes the transcriptional response associated with the phenotypic changes induced by heat stress during early seed development in Brassica napus. Results We compared the differential transcriptional response in unfertilized ovules and seeds bearing embryos at 8-cell and globular developmental stages of the three cultivars exposed to high temperatures. We identified that all tissues and cultivars shared a common transcriptional response with the upregulation of genes linked to heat stress, protein folding and binding to heat shock proteins, and the downregulation of cell metabolism. The comparative analysis identified an enrichment for a response to reactive oxygen species (ROS) in the heat-tolerant cultivar Topas, correlating with the phenotypic changes. The highest heat-induced transcriptional response in Topas seeds was detected for genes encoding various peroxidases, temperature-induced lipocalin (TIL1), or protein SAG21/LEA5. On the contrary, the transcriptional response in the two heat-sensitive cultivars, DH12075 and Westar, was characterized by heat-induced cellular damages with the upregulation of genes involved in the photosynthesis and plant hormone signaling pathways. Particularly, the TIFY/JAZ genes involved in jasmonate signaling were induced by stress, specifically in ovules of heat-sensitive cultivars. Using a weighted gene co-expression network analysis (WGCNA), we identified key modules and hub genes involved in the heat stress response in studied tissues of either heat-tolerant or sensitive cultivars. Conclusions Our transcriptional analysis complements a previous phenotyping analysis by characterizing the growth response to elevated temperatures during early seed development and reveals the molecular mechanisms underlying the phenotypic response. The results demonstrated that response to ROS, seed photosynthesis, and hormonal regulation might be the critical factors for stress tolerance in oilseed rape.
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