The effector molecule is a short RNA duplex, composed of two strands of approximately 21 nucleotides, with two overhanging bases at the 3’ end (^{Elbashir et al., 2001}). However, several types of precursors can trigger gene silencing, such as long double-stranded RNAs (dsRNAs) from 200 to 800 base pairs (bp) (^{Guo et al., 2018}), hairpin RNAs (hpRNAs) (^{Hussain et al., 2019}), small interfering RNAs (siRNAs) (^{Guo et al., 2018}), short hairpin RNAs (shRNAs) (^{Wang et al., 2018}), among others. Notoriously, an ingenious and inexpensive way to produce shRNAs in large quantities was developed, via in vitro transcription of a circularized DNA template, generating multimeric shRNAs (mshRNAs) (^{Seyhan et al., 2006}; ^{Abe et al., 2012}). In the last decade, such multimeric shRNAs generated by rolling circle transcription have been used by other groups and have been shown to be an interesting option for RNAi (^{Wang et al., 2015}; ^{Shopsowitz et al., 2016}; ^{Wu et al., 2016}; ^{Kim et al., 2018}).^{7 8}

Background The progress of climate change demands the adoption of crop cultivars more tolerant to water-deficient conditions. Sugarcane (Saccharum spp.) is a major crop used for the production of sucrose, biomass, and alternative biofuels. Breeding sugarcane cultivars is a complex process, limited by the genetic make-up of the crop. The way around these difficulties depends on the development of biotechnological tools, including genetically modified products. Transcriptome studies provide a reliable source for selecting candidate genes for target modification. However, the choice needs to be effective due to the intrinsic biological challenges of sugarcane. Here, we developed a strategy based on systems biology to demonstrate the reutilization of transcriptomic data in search of central genes and mechanisms involved in response to water deficit, exploring systems biology, co-expression networks studies, and prediction models. Results Sixteen RNA-seq BioProjects of sugarcane grown under contrasting water conditions were retrieved from the SRA-NCBI repository. The 352 libraries were analyzed for differentially expressed genes (DEGs), which were mapped against available sugarcane genomes and assembled transcriptomes. Canonical ontology enrichment and annotation analyses of the combined DEGs allowed the identification of candidate genes, some highly represented and frequently associated with water deficit response, such as inositol 3-alpha-galactosyltransferase. Next, we categorized the 1,415,808 DEGs into 342 functional groups, proposed based on terms commonly related to plant responses to water deficit, and supported by a biological network. All functional groups contain DEGs and their connections; therefore, the member frequencies and number of occurrences among projects allowed us to determine those deviating from normal distribution. We identified 109 functional groups associated with specific pathways that are more active in response to water deficit. The network links enabled the identification of the most central components controlling the mechanisms and pathways. Conclusions The approach proposed here is efficient in providing information on relevant functional groups and the key genes, pathways, and mechanisms associated with those groups. The identification of candidates within the most frequent group effectively demonstrates the relevance of our proposed approach. The strategy offers a robust method for filtering candidates compared to canonical transcriptomic analyses and allows the reuse of data in meta-analyses.

Anhydrobiosis refers to a state of suspended animation in which some organisms enter when exposed to extreme desiccation, ensuring them an outstanding tolerance to several physical stresses due to molecular and cellular adaptations. Metallothioneins (MTs) are short cysteine-rich metal-chelating proteins that work as a cellular protection element in metal ion-rich conditions. Here we aimed to investigate possible molecular signatures in primary and tertiary structures in anhydrobiotic cyanobacterial MTs. Anhydrobiotic and non-anhydrobiotic cyanobacterial MT amino acid sequences were retrieved from NCBI database and aligned in Clustal Omega server. Additionally, the amino acid compositions of these sequences were determined by GeneRunner. Further, we carried out homology-modeling via SWISS-MODEL, structural superposition in UCSF Chimera 1.4 Matchmaker tool and ligand-binding site prediction via COFACTOR. In silico analyses revealed specific divergences in amino acid positions between MT groups, evidencing positive and negative selections, however without affecting final protein structures. Some of these changes on polypeptide sequence potentially enhance protein stabilization during desiccation, whereas others possibly act as additional metal-ion coordinating residues. Analyses on the molecular adaptations on anhydrobiotic cyanobacterial MTs help shed light on their molecular functions and biological roles, as well as may have applications on the development of desiccation- and metal-tolerant organisms.

Sugarcane and energy cane (Saccharum spp. hybrids) yields are significantly threatened by smut disease, caused by the fungus Sporisorium scitamineum. Gene expression studies are essential for understanding molecular responses in this pathosystem; however, reference gene stability may vary among genotypes, plant organs, and across infection stages, highlighting the need for systematic validation under the specific conditions of each experiment. In this study, eight candidate genes were assessed in the buds of sugarcane IACSP02-1064 (intermediate resistance) and energy cane Vertix 10 (susceptible) during the course of the infection (12, 24, 48, and 120 hpi). Expression stability was assessed using geNorm, NormFinder, and BestKeeper algorithms. For IACSP02-1064, ACAD (Acyl-CoA dehydrogenase family member 10) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were the most stable genes, whereas GAPDH/ACAD and ACAD/UBQ2 (Ubiquitin 2) were identified as optimal pairs. For Vertix 10, GAPDH, UBQ2, and ACAD showed the highest stability, while GAPDH/UBQ2, ACAD/GAPDH, and ACAD/SARMp1 were recommended for normalization. The selected reference genes were further evaluated by analyzing ScSPL19 (SQUAMOSA promoter-binding protein-like 19 from Saccharum spp.) expression, a member of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor family, whose members have previously been reported to respond to smut infection, using the most and least stable reference gene pairs. This analysis revealed differential temporal regulation of ScSPL19 when normalized with the most stable reference gene pair (GAPDH/ACAD). These results support the suitability of this reference gene combination for accurate relative gene expression analysis under the evaluated conditions.