Fig. 4: The A/GC-type Kozak motif relies on HOT3 to achieve high translation efficiency.
a Diagrams of the Dual-LUC system and Kozak motif of LHCB5 tested. The 5ʹ UTR and the first two codons were inserted into the Dual-LUC system. The WT version is A/GC, while the mutated versions are C/TG and C/AT. b, c The Dual-LUC assay for LHCB5 with different Kozak motifs was performed in WT protoplasts. The signals of firefly luciferase (FLUC) and renilla luciferase (RLUC) were recorded, and their ratios were used to represent the relative translational activity of FLUC (b). FLUC and RLUC transcripts were quantified using qRT-PCR, and their ratios were used to represent the relative RNA level of FLUC (c). d, e Dual-LUC assays performed for the A/GC-type Kozak motifs of LHCA1, PsaD2, and LHCB5 in WT and hot3-2 protoplasts. Relative protein levels (d) and RNA levels (e) are shown. For (b-e), each point represents an independent biological replicate, with n = 3 for (b, c) and n = 4 for (d, e). Statistical analysis was done using Prism version 8.4.0. Statistical significance was determined with Ordinary one-way ANOVA using Dunnett’s multiple comparisons test with adjusted p values shown for (b, c), and the Mann–Whitney test with two-tailed p values shown for (d, e). f Dual-LUC assays for LHCB5A/GC-LUC and LHCB5C/AT-LUC performed with WT and hot3-2 protoplasts. Relative protein and RNA levels in two biological replicates (br) are shown. The average FLUC/RLUC value in hot3-2 were divided by that in WT to generate the relative FLUC/RLUC (hot3-2 vs. WT) for A/GC- or C/AT- type Kozak motifs. g Genome browser views of Ribo-seq and RNA-seq for LHCB5 in inflorescences and seedlings. The translation initiation sites (TISs) are indicated by gray triangles and red texts. The values on the Y axes represent normalized coverage for Ribo-seq and RNA-seq. The mean values of three biological replicates were marked in each panel for rPS, TE, Ribo-seq density (Ribo, FPKM value of the CDS downstream of the ATG start codon), and total RNA levels (RNA, FPKM value of the entire transcript). h The distribution of total RNAs from inflorescences and seedlings of WT and hot3-2 in sucrose gradient fractions. Methylene-blue staining was used to indicate patterns of 25S rRNA in the 60S ribosomal subunit and 18S rRNA in the 40S ribosomal subunit, with RNA sizes shown on the right. Twelve fractions from 1 to 12 were examined, with fractions of 6 to 8 and 10 to 12 designated as light polysomes (LP) and heavy polysomes (HP), respectively. i The distribution patterns of LHCB5 RNA in sucrose gradient fractions from inflorescences and seedlings tissues. j A bar plot to show the relative LP/HP ratios for LHCB5, LHCA1, PsaD2, AT1G25580, and AT1G21160 in hot3-2 compared with those in WT (hot3-2 vs. WT) in inflorescences (orange color) and seedlings (blue color). For (i) and (j), two biological replicates (n = 2) were performed to generate the mean value shown for each fraction. k Immunoblotting for LHCA1, PsaD, and RPN6 were performed in seedlings of WT and hot3-2, with RPN6 serving as the internal control. Quantification was carried out using ImageJ 1.52a version. Rep1, 2, and 3 are three independent replicates. Kilodalton (kDa), molecular weight.
