Plant material and growth conditions
Arabidopsis thaliana wild type Columbia-0 (Col-0) and the T-DNA insertion lines, SALK_005448 (named here rpl23aa) and SAIL_597_B08 (named here as rpl23ab), were obtained from the Arabidopsis Biological Resource Center (ABRC). Seeds were first treated for 2 minutes in 75% ethanol, then treated for 6 minutes in commercial bleach and rinsed at least 3 times with sterile distilled water. Solid medium consisted of 2.2 g/L Murashige and Skoog basal salt mixture (Phyto Tech Labs), 10 g/L sucrose, and 8 g/L agar. pH was adjusted to 5.6 with KOH before autoclaving. When required, BASTA (GOLDBIO) was added at a final concentration of 125 μg/L. Seeds were sown in a water suspension, using a 1.5mL pipette tip, in 150 mm Petri dishes filled with 120 ml of solid culture medium, at a density of 150 regularly spaced seeds per plate. Once inoculated, the Petri dishes were sealed with Micropore Scotch 3M surgical tape, which prevented contamination but allowed gaseous exchange, and placed in 4℃ for 24h. Growth was allowed to proceed at 22℃ in Percival tissue culture chambers under long day conditions (16 hours light and 8 hours dark). 10-day seedlings were then transplanted to pots containing a 1:2:2 mixture of perlite, vermiculite and soil at 22℃ under long day conditions from a combination of incandescent and fluorescent lamps (10000 lux). Plants were watered twice a week with nutrient solution.
RNA isolation and RT-PCR
50 mg seedlings from 14-day-old Col-0, rpl23aa, and rpl23ab were harvested and immediately frozen in liquid nitrogen. RNA was extracted using RNAiso Plus(TAKARA BIO INC). In the elution step, RNA was resuspended in DEPC-treated water. cDNA was obtained by reverse transcription of 1μg of RNA with the PrimeScriptTMRT reagent Kit with gDNA Eraser(TAKARA BIO INC).
Plasmid construction and generation of transgenic plants
In order to construct the pRPL23aA::RPL23aA and pRPL23aB::RPL23aB plasmids, a 3 001 bp DNA fragment (including the promoter region) of RPL23aA (AT2G39460) and a 2 016 bp DNA fragment (including the promoter region) of RPL23aB (AT3G55280) were amplified from Col-0 genomic DNA using Phusion polymerase (Thermo Scientific). The primers used are shown in Table S1 (Additional file 12). The amplified DNA sequences were cloned in pEG301 [24] to result in pRPL23aA::RPL23aA and pRPL23aB::RPL23aB. The plasmids were used to transform rpl23aa. For pRPL23aA::RPL23aB construction, the promoter region (about 1.5 kb) of RPL23aA plus the coding region of RPL23aB were synthesized by a commercial company (GENEWIZ SuZhou), then the synthesized DNA fragment was sequenced and was cloned in pEG301. The promoter regions of RPL23aA (about 1.5 kb) and RPL23aB (about 1.5 kb) were cloned into pMDC162 [24] to generate the plasmids pRPL23aA::GUS and pRPL23aB::GUS, which were then used to transform Col-0 plants. Floral dip transformation was performed as described by Clough and Bent [25]. T1 transgenic plants were screened on solid 1/2 Murashige & Skoog (MS) medium with 25 mg/L Hygromycin B or 0.002% BASTA and verified by PCR. GUS staining was carried out with plants in the T2 generation.
GUS staining assay
8-days-old seedlings and 36-days-old inflorescences, immature and mature flowers, immature and mature siliques of Col-0, pRPL23aA:GUS and pRPL23aB:GUS were subjected to histochemical GUS staining according to the standard protocol [26].
Transcripts profiling
RNA-seq data was obtained from a public website (http://travadb.org/browse/DeSeq/), and the average value of normalized absolute read counts from two biological replicates was extracted. We also downloaded the original RNA-seq data of A. thaliana different organs and developmental stages from NCBI Sequence Read Archive (project ID PRJNA314076 for samples except meristem and project ID PRJNA268115 for the meristem samples). The RPKM (Reads Per Kilobase per Million mapped reads) value of RPL23aA (AT2G39460), RPL23aB (AT2G39460), and ACT2 (AT3G18780) were calculated. Our calculated RPKM value is consistent with the value of normalized absolute read counts obtained from the public website (http://travadb.org/browse/DeSeq/).
Polysome profiling
Polysome profiling was performed as described by Mustroph et al. [25] (Mustroph et al. 2009). Briefly, 2 g of 14-day-old seedlings were collected and ground to a fine powder using sufficient liquid nitrogen, and the powder was resuspended in 8 mL of ice-cold polysome extraction buffer by gentle shaking. The lysate was incubated on ice for 10 min and centrifuged at 4 ℃, 16, 000 x g for 15 min. The supernatant was filtered through Miracloth and centrifuged at 4 ℃, 16, 000 x g for another 15 min. The supernatant was gently transferred to the top of a sucrose cushion and then centrifuged at 4 ℃, 50,000 r.p.m. for 3 h to obtain the polysome pellet. The pellet was resuspended in ice-cold resuspension buffer and loaded onto a 4.5 mL sucrose gradient (20-60% w/v) for fractionation of polysomes by ultracentrifugation, after which the sucrose gradient was pumped through a UV detector and absorbance at 254 nm was recorded.