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.The most highly regulated phase of protein synthesis is initiation (8, 39). A different class of initiation factors catalyzes each of the individual steps (12). A ternary complex of eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNA i binds to the 40S ribosomal subunit to form the 43S initiation complex. The next step, recruitment of mRNA to the 43S initiation complex to form the 48S initiation complex, is rate limiting for initiation and requires the recognition of the 5Ј-terminal m 7 G-containing cap by eIF4E and the 3Ј-terminal poly(A) tract by the poly(A)-binding protein. The complex of eIF4E, eIF4G, and eIF4A unwinds mRNA secondary structure at the expense of ATP. Global regulation of initiation involves modulation of the canonical initiation factor activities, whereas mRNA-specific regulation is often mediated through proteins that bind cis-regulatory sequences in the 5Ј-or 3Ј-untranslated regions (UTRs) (23

Antagonism between unrelated plant viruses has not been thoroughly described. Our studies show that two unrelated viruses, papaya ringspot virus (PRSV) and papaya mosaic virus (PapMV) produce different symptomatic outcomes during mixed infection depending on the inoculation order. Synergism occurs in plants infected first with PRSV or in plants infected simultaneously with PRSV and PapMV, and antagonism occurs in plants infected first with PapMV and later inoculated with PRSV. During antagonism, elevated pathogenesis-related (PR-1) gene expression and increased reactive oxygen species production indicated the establishment of a host defense resulting in the reduction in PRSV titers. Polyribosomal fractioning showed that PRSV affects translation of cellular eEF1α, PR-1, β-tubulin, and PapMV RNAs in planta, suggesting that its infection could be related to an imbalance in the translation machinery. Our data suggest that primary PapMV infection activates a defense response against PRSV and establishes a protective relationship with the papaya host.

SummaryMaize 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 thermotolerance 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 capindependent translation during heat stress.

Maize somatic embryogenesis (SE) 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 SE, 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.

Insulin and ‘insulin‐like’ growth factors (IGFs) are known to regulate cell growth in eukaryotes by stimulating a signal transduction pathway that exerts translational control. Intermediate kinases of this pathway, target of rapamycin (TOR) and ribosomal protein S6 kinase (S6K), have been reported in Arabidospsis thaliana and Zea mays. However, upstream signal inducers and downstream targets of the pathway are not well known in plants. The objective of this work is to inquire whether plant growth is regulated by a signal transduction pathway similar to the insulin/IGF‐stimulated pathway in other metazoans. Insulin as well as Zea mays insulin‐related peptide (ZmIGF), which is a maize, 20‐kDa peptide fraction recognized by insulin antibody, were used as effectors to stimulate maize axes growth from germinating seeds. ZmIGF expression was identified in axes from germinating maize seeds and immunolocalized in the meristems of these tissues. Significant enhancement of specific de novo protein synthesis of the translational apparatus components was found in the stimulated axes. Reverse‐transcription‐polymerase chain reaction analysis of total and polysomal RNA pools in ZmIGF‐ or insulin‐stimulated axes confirmed these data by revealing specific mRNA recruitment into polysomes. In addition, the same stimuli induced activation of S6 ribosomal protein kinase (ZmS6K) in germinating maize axes. All the above effects were inhibited by rapamycin, indicating that they depend on TOR activity. We conclude that a TOR–S6K signal transduction pathway is functional in maize germination, as that found for non‐photosynthetic eukaryotes. The evolutionary implications of these findings are discussed.