Gene expression quantification through genomics methods is crucial for understanding diverse biological contexts. Among these methods, ribosome profiling (Ribo-seq) stands out as a valuable tool for uncovering post-transcriptional gene expression regulation by providing a comprehensive view of the translatome. While current protocols are time-intensive with limited variations, we introduced the use of the Benzonase enzyme to generate ribosome footprints from a polysome-enriched fraction, streamlining the workflow and reducing time, cost and potential sources of bias. Comparing translatome from Benzonase- and RNAse I-derived footprints reveals minimal differences, with both separating from RNA-seq-based transcriptome quantification. Benzonase acts more gently on ribosomes, yielding larger footprints and a weaker triplet periodicity compared to RNAse I, while enabling its application to low-input samples. We further demonstrate Ribo-seq application in primary neuronal cultures, using Benzonase to digest the post-mitochondrial supernatant, thereby bypassing the labor-intensive ribosome/polysome purification step. The introduction of such protocol variations for Ribo-seq, especially for challenging low-input samples, offers a significant advancement of this resource for the community.

Gene expression quantification through genomics methods is crucial for understanding diverse biological contexts. Among these methods, ribosome profiling (Ribo-seq) stands out as a valuable tool for uncovering post-transcriptional gene expression regulation by providing a comprehensive view of the translatome. While current protocols are time-intensive with limited variations, we introduced the use of the Benzonase enzyme to generate ribosome footprints from a polysome-enriched fraction that exhibit expected characteristics in size, transcriptome mapping, and periodicity. Comparing translatome from Benzonase- and RNAse I-derived footprints reveals minimal differences underscoring Benzonase’s potential to streamline the protocol, reducing time, cost and bias. We further demonstrate Ribo-seq application in primary neuronal cultures, using Benzonase to digest the post-mitochondrial supernatant, thereby bypassing the labor-intensive ribosome/polysome purification step. The introduction of such protocol variations for Ribo-seq, especially for challenging low-input samples, offers a significant advancement of this resource for the community. GRAPHICAL

The TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are a plant-specific family proposed to function as peripheral membrane proteins that contribute to abiotic stress tolerance in Arabidopsis, likely by maintaining cell wall integrity through brassinosteroid signaling. Previously, we identified a TTL gene that was differentially regulated at the translational level in nitrogen-fixing soybean plants under water deficit (WD) conditions. This finding prompted the characterization of the soybean TTL gene family. Using the Glycine max v4.0 proteome, we identified ten TTL homologs (GmTTL1 to GmTTL10), which are unevenly distributed across five chromosomes. Phylogenetic and structural analyses grouped these genes into three clades and revealed a highly conserved exon-intron organization. Likewise, GmTTL proteins display a conserved number and arrangement of TPR and TRXL motifs. To gain insights into their potential biological functions, we integrated co-expression and differential expression analyses. This approach identified a co-expression module enriched for translationally downregulated genes related to the Gene Ontology terms: cellular anatomical entity, membrane, cell periphery, cell wall modification, nitrate assimilation, and cell wall organization or biogenesis. Protein-protein interaction network analysis of this specific subset of genes uncovered a novel GmTTL connection with two nitrate reductase enzymes in nitrogen-fixing plants subjected to WD, potentially linking the TTL gene family to new functions or roles. This study provides a framework for future functional studies of GmTTL proteins and their contribution to abiotic stress adaptation in soybean.

The TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are a plant-specific family proposed to function as peripheral membrane proteins that contribute to abiotic stress tolerance in Arabidopsis, likely by maintaining cell wall integrity through brassinosteroid signaling. In previous work of our group, we identified a GmTTL gene that showed differential abundance in the polysome-associated RNA (PAR) fraction in nitrogen-fixing soybean plants under water restriction (WR). This finding, together with the lack of knowledge about this gene family in soybean, prompted us to perform a genome-wide characterization of this family. In this work, using the Glycine max v4.0 proteome, we identified ten homologs (GmTTL1–GmTTL10) distributed across five chromosomes. Phylogenetic and structural analyses grouped these genes into three clades and revealed a highly conserved exon–intron organization, as well as conserved numbers and arrangements of TPR and TRXL motifs. Moreover, to investigate their potential biological functions, we reanalyzed by STRING the co-expression module enriched with 367 downregulated genes at the PAR level to which GmTTL7 belong. Interestingly, the protein–protein interaction (PPI) network analysis showed the over-representation of Gene Ontology (GO) terms associated with “cellular anatomical entity”, “membrane”, “cell periphery”, “cell wall modification”, “cell wall organization or biogenesis”, and “nitrate assimilation”. Notably, the PPI network revealed a novel predicted functional association between GmTTL and two nitrate reductase enzymes in nitrogen-fixing plants subjected to WR. This study provides a framework for future functional studies of GmTTL proteins and their contribution to abiotic stress adaptation in soybean.