CRISPR/CAS9 mediated gene-editing of HD Zip Class I transcription factors enhances salt tolerance in rice

TRIONFINI, VALENTINA; WELCHEN, ELINA; CHAN, RAQUEL L.; ATTALLAH, CAROLINA V.

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2022

Society for Biochemistry and Molecular Biology Research (SAIB)

Rice (Oryza sativa L.) is one of the most important crops worldwide. However, both biotic and abiotic stress conditions cause enormous economic losses. According to the Food and Agriculture Organization of the United Nations (FAO), the demand for agricultural products will be around 70% by 2050, so the annual production of cereals must be increased accordingly to feed the world population. We study members of the HD-Zip type I family of transcription factors in plants. The overexpression of these proteins in Arabidopsis increased sensitivity to Abscisic acid (ABA), decreased water deficit tolerance, and increased soil salinity. Conversely, insertional mutants with a significant decrease in the expression of these proteins exhibited less sensitivity to ABA treatment and increased tolerance to water and salt stress. To obtain plants with adaptive advantages and of agronomic interest, we generated rice varieties with altered expression of two members of the family of HD-Zip I rice proteins, OsHDZIPI.2 and OsHDZIPI.4, using CRISPR/Cas9 edition technology. We obtained ten edited lines in OsHDZIPI.2 and fifteen edited lines in OsHDZIPI.4. As a selection criterion for the best-edited plants, we performed a germination test using different concentrations of NaCl ranging from 0 to 300 mM. We selected lines showing more than 50% germination in the highest NaCl concentration. We analyzed these edited lines by sequencing, obtaining two homozygous lines for OsHDZIPI.2 and 2 for OsHDZIPI.4. We determined that proteins expressed by the edited plants are aberrant and lost the DNA-binding and dimerization domains characteristic of HD-Zip transcription factors. Seven-day-old seedlings of edited lines for OsHDZIPI.2 and OsHDZIPI.4 showed higher tolerance to 100 mM and 200 mM NaCl relative to Kitaake (WT) plants. Likewise, edited plants exhibited increased tolerance to 50 mM NaCl than WT plants 30 days after sowing. In a field trial under normal growth conditions, the edited plants did not exhibit yield penalty, being the productivity parameters comparable to WT plants. This result must be confirmed and reproduced to incorporate grown under saline soil conditions. We also performed a drought assay following the rehydration and observed that edited lines recovered the healthy phenotype while WT plants died after the stress. In conclusion, both gene-edited rice plants in HD-Zip I transcription factors showed better tolerance to salt conditions than the WT. Gene expression and ABA-related metabolic pathways are being analyzed to characterize the molecular mechanism in this stress-tolerant phenotype observed on the edited plants.