Cordycepin 3 deoxyadenosine

The half-life of mRNA was determined in rice plants as previously described (Lidder et al. 2005 (link); Park et al. 2012 (link)). Briefly, two-week-old rice seedlings were transferred soil to tap water for 2 days. After adaptation, the rice seedlings were treated with 1 mM of cordycepin (3′-deoxyadenosine) (Sigma, USA) through roots uptake for 30 min. After the treatment, the leaf tissues were harvested at indicated time points. RNA extraction, first-strand synthesis, and qRT-PCR were performed as described in “RNA Isolation and Quantitative Real-Time PCR Analysis.” Half-lives were calculated using Sigma plot software (Sigma Plot v10.0; http://www.systat.com).

The half-life (t_1/2) of transcripts was determined as described previously (Fedak et al., 2016 (link)). In brief, Col-0 (wild-type) seedlings were grown vertically on plates containing ½ MS medium for 10 days and then transferred to ½ MS liquid medium and maintained at 22°C or shifted to 4°C for 4 h under the same light conditions. Seedlings from both temperatures were transferred into 12-well plates and incubated in buffer A (1 mM PIPES at pH 6.25, 1 mM trisodium citrate, 1 mM KCl, and 15 mM sucrose) at 22°C or 4°C, respectively. After 30 min of incubation, 150 mg/L cordycepin (3′-deoxyadenosine, Sigma Aldrich) was added and vacuum-infiltrated twice for 5 min. Samples were collected after 0, 15, 30, 60, 120, and 300 min in triplicate (15 seedlings per replicate), followed by total RNA extraction and RT-qPCR analysis using gene-specific primers (Supplemental Table 1). EIF4A1 and EXPL1 were used as assay controls (Perea-Resa et al., 2012 (link); Fedak et al., 2016 (link)). C_t values were normalized with the C_t value at 0 min [Ct(n) = (ln(C_t/C_t(0)) × (−10)] and the slope to determine the half-life of the transcripts was calculated as follows [t_1/2 = (ln₂)/slope] of transcript.

RNAs were isolated from leaf and stem samples using Qiagen RNeasy Plant mini kits (Qiagen) with on-column DNase treatment. Plant RNA purification reagent (Invitrogen) was also used for total RNA extraction followed with DNase treatment [27 (link)]. RNA concentration was measured by Nanodrop (Thermo, USA). M-MLV reverse transcriptase (Promega, USA) was used for reverse transcription reactions. Real-time PCR was performed with Power SYBR Green PCR Master (Applied Biosystems, USA) and run in ABI7900HT. All samples were run in triplicates and data was analyzed with RQ manager at a pre-set Ct value (Applied Biosystems, USA). PCR primers were listed in S1 Table. Cordycepin treatments and mRNA stability analysis were performed as described before [7 (link)]. Twelve-day-old seedlings were incubated in MS medium with cordycepin (3′-deoxyadenosine; Sigma-Aldrich) and DMSO treatment was used as a mock control. Data derived from the mock control was used for normalization for mRNA stability assays. Total RNA was extracted from samples harvested at various time points using Plant RNA extraction reagent (Invitrogen). Actin2 was used as an internal mRNA control.