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Defensins are a prominent family of antimicrobial peptides. They play sophisticated roles in the defense against pathogens in all living organisms, but few works address their expression under different conditions and plant tissues. The present work prospected defensins of Manihot esculenta Crantz, popularly known as cassava. Five defensin candidates (MeDefs) were retrieved from the genome sequences and characterized. Considering chromosome distribution, only MeDef1 and 2 occupy adjacent positions in the same chromosome arm. All 3D structures had antiparallel ß-sheets, an α-helix, and amphipathic residues distributed throughout the peptides with a predominance of cationic surface charge. MeDefs expression was validated by RT-qPCR, including two stress types (biotic: fungus Macrophomina pseudophaseolina, and abiotic: mechanical injury) and a combination of both stresses (fungus+injury) in three different tissues (root, stem, and leaf). For this purpose, ten reference genes (RGs) were tested, and three were chosen to characterize MeDef expression. MeDef3 was up-regulated at roots in all stress situations tested. MeDef1 and MeDef5 were induced in leaves under biotic and abiotic stresses, but not in both stress types simultaneously. Only MeDef2 was down-regulated in the stem tissue also with biotic/abiotic combined stresses. These results indicate that although defensins are known to be responsive to pathogen infection, they may act as preformed defense or, still, have tissue or stress specificities. Aspects of their structure, stability and evolution are also discussed.

Pluripotency-associated transcription factors (PATFs) are involved in several cellular processes, such as transcription initiation, cellular metabolism, and cell fate determination in preimplantation embryos. Despite growing understanding of PATFs in pluripotent human and mouse cells, their roles in other species or cell types remain limited. The work aimed to determine the transcript availability of OCT4 and four non-canonical PATFs (NAC1, NR0B1, RONIN, and ZFP281) in goat ovaries, eggs, and cumulus cells. Goat ovaries were collected at slaughterhouses and cumulus-oocyte complexes (COC) were retrieved from 3 to 6 mm follicles. The COCs were subject to in vitro maturation in TCM-199 medium containing 2 mM l-glutamine, 5.0 μg mL−1 FSH, 10% FBS, and antibiotics at 38.5 °C with saturated humidity and 5% CO2 during 24 h. Gene activity was detected by both conventional reverse transcription quantitative polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). In silico analysis were carried out for protein physical properties, phenetic analysis, and protein domains within goat, mouse, and human PATF orthologs. Products of all five PATFs analyzed (NAC1, NR0B1, OCT4, RONIN, and ZFP281) were detected in goat eggs, but NR0B1 was not detected in the ovary. Surprisingly, NR0B1, RONIN, and ZFP281 were also expressed in cumulus cells. All PATFs and their binding partners (i.e., OCT4-SOX2, RONIN HCF1, and NANOG-NR0B1-ZFP281-NAC1) showed similar protein size, molecular weight, and isoelectric point among species. The phenetic analysis clustered more closely goat and human proteins for OCT4, SOX2, NANOG, and NR0B1. Finally, both DNA-binding and transactivation domains were also highly conserved among species, particularly between goats and humans, suggesting that these PATFs may have conserved functions and similar target genes. In conclusion, non-canonical PATFs have distinctive transcript abundance in goat eggs and cumulus cells, while in silico approaches suggest that these genes may play similar roles to their mouse and human orthologs.

SummaryPluripotency-associated transcription factors (PATFs) modulate gene expression during early mammalian embryogenesis. Despite a strong understanding of PATFs during mouse embryogenesis, limited progress has been made in ruminants. This work aimed to describe the temporal expression of eight PATFs during both sheep and cattle preimplantation development. Transcript availability of PATFs was evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in eggs, cleavage-stage embryos, morulae, and blastocysts. Transcripts of five genes were detected in all developmental stages of both species (KLF5, OCT4, RONIN, ZFP281, and ZFX). Furthermore, CMYC was detected in all cattle samples but was found from cleavage-stage onwards in sheep. In contrast, NR0B1 was detected in all sheep samples but was not detected in cattle morulae. GLIS1 displayed the most significant variation in temporal expression between species, as this PATF was only detected in cattle eggs and sheep cleavage-stage embryos and blastocysts.In silicoanalysis suggested that cattle and sheep PATFs share similar size, isometric point and molecular weight. A phenetic analysis showed two patterns of PATF clustering between cattle and sheep, among several mammalian species. In conclusion, the temporal expression of pluripotency-associated transcription factors differs between sheep and cattle, suggesting species-specific regulation during preimplantation development.