Translational profile of coding and non-coding RNAs revealed by genome wide profiling of ribosome footprints in grapevine

Abstract

Translation is a crucial process during plant growth and morphogenesis. In grapevine (Vitis vinifera L.), many transcripts can be detected by RNA sequencing; however, their translational regulation is still largely unknown, and a great number of translation products have not yet been identified. Here, ribosome footprint sequencing was carried out to reveal the translational profile of RNAs in grapevine. A total of 8291 detected transcripts were divided into four parts, including the coding, untranslated regions (UTR), intron, and intergenic regions, and the 26 nt ribosome-protected fragments (RPFs) showed a 3 nt periodic distribution. Furthermore, the predicted proteins were identified and classified by GO analysis. More importantly, 7 heat shock-binding proteins were found to be involved in molecular chaperone DNA J families participating in abiotic stress responses. These 7 proteins have different expression patterns in grape tissues; one of them was significantly upregulated by heat stress according to bioinformatics research and was identified as DNA JA6. The subcellular localization results showed that VvDNA JA6 and VvHSP70 were both localized on the cell membrane. Therefore, we speculate that DNA JA6 may interact with HSP70. In addition, overexpression of VvDNA JA6 and VvHSP70, reduced the malondialdehyde (MDA) content, improved the antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), increased the content of proline, an osmolyte substance, and affected the expression of the high-temperature marker genes VvHsfB1, VvHsfB2A, VvHsfC and VvHSP100. In summary, our study proved that VvDNA JA6 and the heat shock protein VvHSP70 play a positive role in the response to heat stress. This study lays a foundation for further exploring the balance between gene expression and protein translation in grapevine under heat stress.

Keywords: DNA JA6; Hsp70; Ribo-seq; grape; heat stress.

Figure 1

Translation landscape in grapes. (A) Overview of the ribosome sequencing approach, the mRNA-tethered ribosomes were extracted and enriched, which performed library preparation and deep sequencing. (B) mRNA protected by ribosome fragment enriched at 23-31nt. (C) Protein translation regions are distributed in coding, UTR, Intron and Intergenic. (D) Ribosomes move in units of 3 nt nucleotides in the initial codon. (E) Ribosomes move in units of 3 nt nucleotides in a stop codon.

Figure 2

GO secondary function analysis 8291 transcripts are translated in grapes. The blue bar chart represents a cellular component, the orange bar chart represents a molecular function, red bar chart represents the molecular function.

Figure 3

Comparation and visualization of translation efficiency in heat shock binding proteins. (A) RPKM in Different heat shock binding proteins. Reads coverage in VIT_00s0324g00040 (B), VIT_06s0080g01230 (C), VIT_08s0056g01490 (D), VIT_09s0002g07210 (E), VIT_13s0073g00560 (F), VIT_15s0021g02090 (G), VIT_18s0001g14090 (H).

Figure 4

Different expression patterns of tissue and reaction by abiotic stresses in grapevine. (A) Tissue expression in root, stem and leaf. After heat stress (B), cold stress (C), drought stress (D) and salt stress (E) for 0 h, 2 h, 6 h and 8 h, the different expression profiles in grapevine. The results perform the mean ± SE of three independent replicates (P < 0.05).

Figure 5

Bioinformatics Analysis with DNA JA6 protein. (A) Evolutionary tree analysis. (B) Conservative domain analysis. All protein names are uniformly named with NCBI accession ID. VIT_00s0324g00040 counterparted as XP 002274349 and marked with a red box.

Figure 6

DNA JA6 may interact with HSP70 in Grapevine. (A) Relative expression of HSP70 in root, stem and leaf. (B) Different expression patterns of HSP70 in heat stress, cold stress, drought stress and salt stress. (C) Subcellular localization of DNA JA6 and HSP70. The results perform the mean ± SE of three independent replicates (P < 0.05).

Figure 7

DNA JA6 and HSP70 play a positive role in response to heat stress. (A) The phenotype of WT, DNA JA6 and HSP70 transient over expression in tobacco after heat stress. The observation of NBT staining (B) and the test of O₂ ^•-content (C). The observation of DAB staining (D) and the test of H₂O₂ content (E). Analysis of physiological indexes SOD activity (F), POD activity (G), CAT activity (H) and proline content (I) in WT, GFP, DNA JA6 and HSP70 transient over expression tobacco after heat stress. Relative expression of heat stress marker gene VvHsfB1 (J), VvHsfB2A (K), VvHsfc (L) and VvHSP100 (M) in transient over expression tobacco after heat stress. The results perform the mean ± SE of three independent replicates (P < 0.05). “#” represent the object being compared. “*” represent difference. "**" represent significant difference. “***” represent extremely significant difference.