The mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) participates in protein synthesis initiation, translational repression of specific mRNAs, and nucleocytoplasmic shuttling. Multiple isoforms of eIF4E are expressed in a variety of organisms, but their specific roles are poorly understood. We investigated one Caenorhabditis elegans isoform, IFE-4, which has homologues in plants and mammals. IFE-4::green fluorescent protein (GFP) was expressed in pharyngeal and tail neurons, body wall muscle, spermatheca, and vulva. Knockout of ife-4 by RNA interference (RNAi) or a null mutation produced a pleiotropic phenotype that included egg-laying defects. Sedimentation analysis demonstrated that IFE-4, but not IFE-1, was present in 48S initiation complexes, indicating that it participates in protein synthesis initiation. mRNAs affected by ife-4 knockout were determined by DNA microarray analysis of polysomal distribution. Polysome shifts, in the absence of total mRNA changes, were observed for only 33 of the 18,967 C. elegans mRNAs tested, of which a disproportionate number were related to egg laying and were expressed in neurons and/or muscle. Translational regulation was confirmed by reduced levels of DAF-12, EGL-15, and KIN-29. The functions of these proteins can explain some phenotypes observed in ife-4 knockout mutants. These results indicate that translation of a limited subset of mRNAs is dependent on a specific isoform of eIF4E.

Determination of reducing sugars is carried out routinely in the food industry, in biological research, or pharmaceutical and biomedical quality control to estimate metabolically assimilable sugars. Widespread detection methods are complex, expensive, or highly polluting. Here, we propose the use of spectrophotometric quantification for reducing sugars (Benedict_q) based on the qualitative method of Benedict. The protocol was validated, to verify its reproducibility and precision. With the proposed method (Benedict_q), the reducing sugar glucose can be determined in a range of 0.167–10 mg mL^–1, with an R² of 0.997 and accuracy (expressed as % of recovery) greater than 97%. Other reducing sugars, such as maltose, fructose, and lactose, showed similar values. The method robustness was verified for pH values greater than or equal to 4. In the case of protein presence, a correction is proposed in the range of 0–1.67 mg mL^–1. Modifications implemented in the protocol reduce cost, working time, and reaction volumes with respect to the original assay without detriments in accuracy and precision. In addition, waste reduction represents an important contribution of the method.

Maize somatic embryogenesis is induced from the immature zygotic embryo in darkness and under the appropriate hormones’ levels. Small RNA expression is reprogrammed and certain miRNAs become particularly enriched during induction while others, characteristic to the zygotic embryo, decrease. To explore the impact of different environmental cues on miRNA regulation in maize somatic embryogenesis, we tested specific miRNA abundance and their target gene expression in response to photoperiod and hormone depletion for two different maize cultivars (VS-535 and H-565). The expression levels of miR156, miR159, miR164, miR168, miR397, miR398, miR408, miR528 and some predicted targets (SBP23, GA-MYB, CUC2, AGO1c, LAC2, SOD9, GR1, SOD1A, PLC) were examined upon staged hormone depletion in the presence of light photoperiod or darkness. Almost all examined miRNA, except miR159, increased upon hormone depletion, regardless photoperiod absence/presence. miR528, miR408 and miR398 changed the most. On the other hand, expression of miRNA target genes was strongly regulated by the photoperiod exposure. Stress-related miRNA targets showed greater differences between cultivars than development-related targets. miRNA/target inverse relationship was more frequently observed in darkness than light. Interestingly, miR528, but not miR159, miR168 or miR398, was located on polyribosome fractions suggesting a role for this miRNA at the level of translation. Overall our results demonstrate that hormone depletion exerts a great influence on specific miRNA expression during plant regeneration independently of light. However, their targets are additionally influenced by the presence of photoperiod. The reproducibility or differences observed for particular miRNA-target regulation between two different highly embryogenic genotypes provide clues for conserved miRNA roles within the SE process.

Summary: Maize embryonic axes contain stored mRNAs, some of which are able to undergo cap‐independent translation initiation during germination. The Hsp101 mRNA, encoding a heat shock protein, is essential for thermo‐tolerance induction and is present among the stored transcripts. This research aimed to investigate whether the Hsp101 transcript is IRES‐driven regulated upon heat stress. Hsp101 transcribed either in vitro or in vivo was efficiently translated via a cap‐independent mechanism. This was observed either in an animal in vitro translation system containing proteolytically cleaved eukaryotic initiation factor eIF4G or in a plant system lacking both eIF4E and eIFiso4E initiation factors. Deletion of the 5′ untranslated region (UTR) from the Hsp101 mRNA abolished its cap‐independent translation indicating that this nucleotide sequence is required to confer cap‐independent initiation. Bicistronic constructs containing the Hsp101 mRNA 5′UTR in sense and anti‐sense directions between two reporter genes were translated in both cap‐independent systems. A similar bicistronic construct containing a viral internal ribosome entry site (IRES) element between the reporter genes was used as control. Internal translation of the second reporter gene was observed when the Hsp101 5′UTR was in the sense but not in the anti‐sense orientation in the bicistronic construct. Taken together, these data suggest that the 5′UTR of maize Hsp101, a plant cellular mRNA, functions as an IRES‐like element accounting for its cap‐independent translation during heat stress.