Unravelling the raffinose family oligosaccharides metabolic pathway in common bean: From gene discovery to gene editing for improved nutrition

Common bean (Phaseolus vulgaris L.) is a vital source of human nutrition, especially in developing countries, being a rich source of proteins, carbohydrates, vitamins, minerals, and dietary fibres. Despite its nutritional value, consumption is hindered due to the presence of anti-nutritional factors, such as the raffinose family oligosaccharides (RFOs), which cause digestive disturbances in humans and monogastric animals. Lowering the quantity of RFOs in the seeds could improve these beans' nutritional quality and solve the discomforts associated with their consumption. However, a profound understanding of the galactinol and RFO biosynthetis gene family and the expression patterns of the individual genes is a prerequisite for the sustainable reduction of the RFO content in the seeds without compromising plant development and funstioning under normal and abiotic stress conditions.
This research gives an overvies of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes for two important legume crops, soybean and common bean. In common bean, three galactinol synthase genes, two raffinose synthase genes and one stachyose synthase gene were identified for the first time. To discover the expression patterns of the genes in different tissues, two expression atlases have been created through a re-analysis of publicly available RNA-seq data. De novo expression analysis through an RNA-seq study during seed development gave more insight into the expression patterns of these genes during seed development. The results of the expression analysis suggest that different classes of galactinol- and RFO synthase genes have tissue-specific expression patterns. With the obtained knowledge, important galactinol- and RFO synthase genes that specifically play a key role in the accumulation of RFOs in the seeds were identified.
Furthermore, we tried to identify the importance of galactinol and RFOs during drought and salt stress in common bean. Initially, the physiological characteristics of common bean under agronomical relevant abiotic stress conditions were investigated by measuring the growth rate, transpiration rate, chlorophyll concentration and membrane stability to find scientifically sound sample points. Subsequently, the differential gene expression of the galactinol- and RFO synthase genes and the amount of galactinol and RFO molecules were measured in the leaves and roots at these sample points using RT-qPCR and HPAEC-PAD, respectively. The results suggest that both galactinol and raffinose play a role in the protection of the plant against these abiotic stresses and especially isoform galactinol synthase 3 seems to have a specific role during drought stress.
To reduce the RFO content in common bean seeds, the CRISPR/Cas9 gene-editing technique was used. As the stable transformation of common bean is a long and complex process, we initially developed a hairy root transformation system to evaluate in a fast way the on-target activity of sgRNAs and the impact that different promoters have on the gene-editing efficiency. Several highly efficient sgRNAs that induced frameshift mutations of up to 70% were identified. These can be used in future transformation experiments to generate knockout lines of the RFO biosynthetic genes. Additionally, we evaluated the reliability of in silico tools, CRISPOR, CRIPSR RGEN, and inDelphi, to predict the sgRNA efficiencies and resulting insertions and deletions. Although most of the computational models used to predict the sgRNA efficiency did not match the in planta resultys, the Lindel model proved to be the most reliable for common bean, accurately predicting the sgRNA efficiency and the type of induced mutation in most hairy roots. We also found that the inDelphi algorithm could correctly predict deletions and single nucleotide insertions resulting from DNA double-strand breaks in common bean. These results offer promising implications for enhancing precise editing in plants as they can predict repair outcomes. However, we observed that not all sgRNAs with high in silico mutation scores showed similar performance in planta, emphasizing the relevance of conducting an initial sgRNA efficiency check using hairy root transformation.
Common bean has remained recalcitrant to transformation and regeneration, restricting its genetic improvement. We aimed to optimize these processes with a specific focus on generating knock-out lines for the RFO biosynthetic genes using CRISPR/Cas9. We optimised the indirect organogenesis protocol, improving callus induction and increasing callus transformation efficiencies by up to 67.55%. Additionally, a gradual increase in selection pressure led to higher survival rates post-transformation. However, most transformed calli failed to generate viable shoots, underscoring the persistent challenge in common bean regeneration. With these findings, researchers are one step closer towards developing reliable and efficient transformation protocols for common bean and hold significant promise for the succesful formation of knockout lines in the future. Resulting plants with a reduced RFO content in the seeds could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption.