Efficient Genetic Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable Overexpression Studies: A Case Study on Rice Lipase 1 and Galactinol Synthase Encoding Genes

Rice is a staple food crop for almost half of the world’s population, especially in the developing countries of Asia and Africa. It is widely grown in different climatic conditions, depending on the quality of the water, soil, and genetic makeup of the rice cultivar. Many (a)biotic stresses severely curtail rice growth and development, with an eventual reduction in crop yield. However, for molecular functional analysis, the availability of an efficient genetic transformation protocol is essential. To ensure food security and safety for the continuously increasing global population, the development of climate-resilient crops is crucial. Here, in this study, the rice transformation protocol has been effectively optimized for the efficient and rapid generation of rice transgenic plants. We also highlighted the critical steps and precautionary measures to be taken while performing the rice transformation. We further assess the efficacy of this protocol by transforming rice with two different transformation constructs for generating galactinol synthase (GolS) overexpression lines and CRISPR/Cas9-mediated edited lines of lipase (Lip) encoding the OsLip1 gene. The putative transformants were subjected to molecular analysis to confirm gene integration/editing, respectively. Collectively, the easy, efficient, and rapid rice transformation protocol used in this present study can be applied as a potential tool for gene(s) function studies in rice and eventually to the rice crop improvement.

A Sugarcane G-Protein-Coupled Receptor, ShGPCR1, Confers Tolerance to Multiple Abiotic Stresses

Sugarcane (Saccharum spp.) is a prominent source of sugar and serves as bioenergy/biomass feedstock globally. Multiple biotic and abiotic stresses, including drought, salinity, and cold, adversely affect sugarcane yield. G-protein-coupled receptors (GPCRs) are components of G-protein-mediated signaling affecting plant growth, development, and stress responses. Here, we identified a GPCR-like protein (ShGPCR1) from sugarcane and energy cane (Saccharum spp. hybrids) and characterized its function in conferring tolerance to multiple abiotic stresses. ShGPCR1 protein sequence contained nine predicted transmembrane (TM) domains connected by four extracellular and four intracellular loops, which could interact with various ligands and heterotrimeric G proteins in the cells. ShGPCR1 sequence displayed other signature features of a GPCR, such as a putative guanidine triphosphate (GTP)-binding domain, as well as multiple myristoylation and protein phosphorylation sites, presumably important for its biochemical function. Expression of ShGPCR1 was upregulated by drought, salinity, and cold stresses. Subcellular imaging and calcium (Ca2+) measurements revealed that ShGPCR1 predominantly localized to the plasma membrane and enhanced intracellular Ca2+ levels in response to GTP, respectively. Furthermore, constitutive overexpression of ShGPCR1 in sugarcane conferred tolerance to the three stressors. The stress-tolerance phenotype of the transgenic lines corresponded with activation of multiple drought-, salinity-, and cold-stress marker genes, such as Saccharum spp. LATE EMBRYOGENESIS ABUNDANT, DEHYDRIN, DROUGHT RESPONSIVE 4, GALACTINOL SYNTHASE, ETHYLENE RESPONSIVE FACTOR 3, SALT OVERLY SENSITIVE 1, VACUOLAR Na+/H+ ANTIPORTER 1, NAM/ATAF1/2/CUC2, COLD RESPONSIVE FACTOR 2, and ALCOHOL DEHYDROGENASE 3. We suggest that ShGPCR1 plays a key role in conferring tolerance to multiple abiotic stresses, and the engineered lines may be useful to enhance sugarcane production in marginal environments with fewer resources.

Overexpression of Galactinol Synthase 1 From Solanum Commersonii (ScGolS1) Confers Freezing Tolerance In Transgenic Potato

Potato (Solanum tuberosum L.) is the fourth largest food crop in the world. Low temperature causes serious damage to potato plants every year, and freezing tolerance has become a hot spot in potato research. Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs), and plays an important role in the response of plants to abiotic stress. In this study, the ScGolS1 gene from S. commersonii was cloned and introduced into the S. tuberosum cultivars ‘Atlantic’ and ‘Desiree’ via Agrobacterium-mediated transformation. Phenotyping assay showed that overexpression of the ScGolS1 could significantly improve freezing tolerance in transgenic potato plants. Further physiological and biochemical results showed that the relative conductivity, malondialdehyde (MDA) content, and 3,3'-Diaminobenzidine (DAB) staining of the transgenic lines decreased, and the plant survival rate increased compared with wild type (WT). Moreover, CBF1, CBF2, CBF3, CBF downstream cold responsive genes COR413, COR47 and ERF transcription factor genes ERF3, ERF4, ERF6 in the ethylene signaling pathway were all induced by freezing treatment, while higher levels were observed in ScGolS1 overexpression lines compared with WT. In addition, other genes such as MIPS, STS and RS genes from RFO metabolic pathway and some sugars content were altered in response to freezing treatment. This indicates that overexpression of the ScGolS1 gene induced both the regulation of the ethylene signaling pathway and the metabolism of raffinose series oligosaccharides, regulating the balance of sugar composition and improved anti-peroxidation capacity, and thereby improved freezing tolerance in potato. These results provide theoretical support and genetic resources for freezing tolerance breeding in potato.

Differentiation of the High Night Temperature Response in Leaf Segments of Rice Cultivars with Contrasting Tolerance

High night temperatures (HNT) affect rice yield in the field and induce chlorosis symptoms in leaves in controlled chamber experiments. However, little is known about molecular changes in leaf segments under these conditions. Transcript and metabolite profiling were performed for leaf segments of six rice cultivars with different HNT sensitivity. The metabolite profile of the sheath revealed a lower metabolite abundance compared to segments of the leaf blade. Furthermore, pre-adaptation to stress under control conditions was detected in the sheath, whereas this segment was only slightly affected by HNT. No unique significant transcriptomic changes were observed in the leaf base, including the basal growth zone at HNT conditions. Instead, selected metabolites showed correlations with HNT sensitivity in the base. The middle part and the tip were most highly affected by HNT in sensitive cultivars on the transcriptomic level with higher expression of jasmonic acid signaling related genes, genes encoding enzymes involved in flavonoid metabolism and a gene encoding galactinol synthase. In addition, gene expression of expansins known to improve stress tolerance increased in tolerant and sensitive cultivars. The investigation of the different leaf segments indicated highly segment specific responses to HNT. Molecular key players for HNT sensitivity were identified.

Comparative Transcriptome Analysis of Two Contrasting Chinese Cabbage (Brassica rapa L.) Genotypes Reveals That Ion Homeostasis Is a Crucial Biological Pathway Involved in the Rapid Adaptive Response to Salt Stress

Salt is the most important limiting factor in plant yield and quality. Different Chinese cabbage cultivars appeared different salt tolerances, but there are few studies attempting to elucidate the mechanism underlying this phenomenon. In this study, 100 mmol L–1 NaCl was found to be the most suitable treatment concentration according to a sprouting bag test of 39 Chinese cabbage cultivars, and through comprehensive comparison and analysis, the relative values of fresh weight and electrolyte leakage in leaves proved to be convenient indicators for the identification of salt tolerance in Chinese cabbage. We analyzed the physiological responses of Qinghua45 (salt-tolerant) and Biyuchunhua (salt-sensitive) in terms of the growth indexes, ion homeostasis and Photosynthesis, the results indicated that Qinghua45 could ensure osmotic regulation, ion homeostasis and photosynthesis under salt stress. Next, we compared the transcriptome dynamics of the two cultivars. Overall, 2,859 differentially expressed genes (DEGs) were identified, and the number of DEGs in Qinghua45 was significantly less than that in Biyuchunhua. VDAC promoted the release of Ca2+, which indirectly promoted the transport of Na+ to vacuoles through the SOS2 pathway. Cation/H (+) antiporter 17 and V-H + -ATPase improve the exchange of Na+ and H+ and maintain Na+ in the vacuoles, thereby reducing the injury affected by salt stress. Increases in galactinol synthase and soluble protein synthesis helped relieve osmotic stress caused by salt, together, they regulated the Na+ content and chlorophyll biosynthesis of the plant and enabled the plant to adapt to salt stress over time.

CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.