14-3-3 proteins GRF6 and GRF8 positively regulate drought tolerance in Arabidopsis thaliana

14-3-3 proteins constitute a class of highly conserved regulatory proteins present in all eukaryotic cells. In plants, they are involved in diverse biological processes, including responses to abiotic stresses. Here, we investigated the roles of two 14-3-3 proteins, GRF6 and GRF8, in the drought tolerance of Arabidopsis thaliana through comparative transcriptome profiling of wild-type (WT) and grf6 grf8 mutant plants. Overall design: 12-day-old wild-type (WT) and grf6 grf8 mutant plants were transferred from 1/2 Murashige and Skoog (MS) plates to soil and grown for an additional 12 days. Rosette leaves of these 24-day-old plants were harvested after 0, 2, and 4 hours of dehydration, then immediately frozen in liquid nitrogen. Total RNA was extracted using the TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified by RNase-free agarose gel electrophoresis. Following total RNA extraction, eukaryotic mRNA was enriched with Oligo (dT) beads. The enriched mRNA was fragmented into short segments using fragmentation buffer and reverse-transcribed into cDNA with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB#7530, New England Biolabs, Ipswich, MA, USA). Purified double-stranded cDNA fragments underwent end repair, A-tailing, and ligation to Illumina sequencing adapters. The ligation product was purified with AMPure XP Beads (1.0×) and amplified by polymerase chain reaction (PCR). The resulting cDNA library was sequenced on an Illumina NovaSeq 6000 platform.