GSE201015
Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation
The stability of mRNA is an important determinant of its abundance and, consequently, protein production. There has been extensive research on the pathways governing mRNA stability and translation, however, it is unclear the extent to which these processes are modulated by environmental conditions. We previously modelled rapid recovery gene down-regulation (RRGD) following light stress in Arabidopsis thaliana (Arabidopsis) using mathematical calculations to account for transcription in order to predict half-lives and led to the hypothesis of recovery-specific transcript destabilisation. Here, we test this hypothesis by quantifying changes in transcription, mRNA stability, and translation in leaves of mature Arabidopsis undergoing light stress and recovery and investigate processes regulating transcript abundance and fate. Compared to juvenile plants from prior work, here we find that stability is altered for a range of transcripts that encode proteins involved in post-transcriptional processes in mature leaves. We also observe transcript destabilisation during light stress, followed by re-stabilisation upon recovery. Alongside this, we observe fast transcriptional shut-off in recovery that, when paired with transcript destabilisation, promotes rapid down-regulation of stress-induced genes. Translation was dynamic over the course of light stress and recovery, with substantial transcript-specific increases in polysome loading observed during late stress independently of total mRNA abundance. Taken together, we provide evidence for the combinatorial regulation of transcription, mRNA stability, and translation that occurs during light stress and recovery. Overall design: PolyA-enriched RNA-sequencing (mRNA-seq) was performed on Arabidopsis leaves infiltrated with cordycepin or mock [buffer only: 1 mM PIPES (pH 6.25), 1 mM sodium citrate, 1 mM KCl, 15 mM sucrose] harvested at 10, 20, 30, and 40 minutes post-infiltration under unstressed (US, 100 uE), high-light (HL, 1000 uE), and recovery conditions (Rec, 1000 ? 100 uE). The transcriptional inhibitor, cordycepin (3'-deoxyadenosine, Sigma-Aldrich), was syringe-infiltrated on the abaxial side of fully-expanded leaves (true leaves 4-6) of 21-day old plants. Individual leaves were infiltrated with 0.1 mL of incubation buffer [1 mM PIPES (pH 6.25), 1 mM sodium citrate, 1 mM KCl, 15 mM sucrose (Seeley et al. 1992)] with 0.6 mM cordycepin, or without (mock), using a 1 mL needleless syringe (Terumo). Excess liquid was removed from the leaf surface and plants were incubated for a minimum of 10 minutes before further treatment and harvesting. Light-stress was induced by increasing the light intensity to 10× growth irradiance (i.e. 1000 ?mol photons m-2 s -1), resulting in a ?hot high-light? treatment that effectively induces oxidative stress (Jung et al. 2013). For recovery, plants were returned to pre-stress light conditions. Each biological replicate was derived from leaves syringe-infiltrated, with either mock or cordycepin treatment, from the same plant at each time point per condition. Infiltrated leaves were excised, at the appropriate time point under each condition, from the base of the petiole and immediately flash-frozen, in a 2 mL safe-lock microcentrifuge tube (Eppendorf), using liquid nitrogen. Changes in polysome- and monosome-bound, and total-mRNAs were also profiled during the course of light stress and recovery. Plants were sampled at 0, 30, and 60 mins of high-light followed by 7.5, 15, and 30 mins of recovery (67.5, 75, 90 mins). Polysome profiling and mRNA-seq was performed in biological triplicate, consisting of multiple whole rosettes (250 mg) from independntly treated plants. Harvested tissue was snap frozen in liquid nitrogen and stored at -80 °C until ready for processing. [Dec-16-2022] The 'time' information was corrected for GSM6048558-GSM6048627.