Raúl Aguilar

Expression of maize eukaryotic initiation factor (eIF) iso4E is regulated at the translational level.

Mechanisms for regulation of gene expression at the translational level have been reported at specific developmental stages in eukaryotes. Control of eukaryotic initiation factor (eIF) 4E availability by insulin/growth factors constitutes a main point of translational regulation. The aim of the present research was to understand the regulatory mechanism(s) behind the differential expression of two main 4E factors present in maize embryonic axes during germination. De novo synthesis of eIFiso4E initiates earlier and is faster than that of eIF4E in maize axes. Insulin addition to maize axes stimulated de novo synthesis of the eIFiso4E protein, but not that of eIF4E. Specific recruitment of the eIFiso4E transcript into polysomes was observed in these axes after insulin stimulation. Inhibitors of the insulin signal-transduction pathway, wortmannin and rapamycin, reversed the insulin effect. In vitro translation of maize poly(A)(+) RNAs by S6 ribosomal protein (rp)-phosphorylated ribosomes demonstrated a strong increase in eIFiso4E synthesis, as compared with its translation by S6 rp-non-phosphorylated ribosomes. Other mRNAs from the poly(A)(+) RNA set, including the eIF4E mRNA, did not show differential translation with regard to the S6-phosphorylated status of the ribosomes. The overall results indicate that eIFiso4E, but not eIF4E, cell content is regulated by de novo synthesis in maize axes during germination, most probably by specific mRNA recruitment into polysomes via a signal-transduction pathway involving S6 rp phosphorylation.

Tight translational control by the initiation factors eIF4E and eIF(iso)4E is required for maize seed germination

A characteristic mechanism of gene expression regulation during seed germination is the selective translation of mRNAs. Previous findings indicate that the two cap-binding complexes eIF4F (with eIF4E and eIF4G subunits) and eIF(iso)4F [with eIF(iso)4E and eIF(iso)4G subunits] are differentially expressed during maize seed germination. In addition, several studies in vitro have suggested that these factors may participate in selective mRNA translation. The translational activities of eIF4E and eIF(iso)4E were tested in vitro using transcripts from two different sets: dry (0h) and 24-h-imbibed maize embryonic axes. In vitro translation of these mRNA pools in the presence of the recombinant eIF4E or eIF(iso)4E, and the native cap-binding complexes from dry- or 24-h-imbibed axes, produced different profiles of proteins which were visualized by two-dimensional protein gels and autoradiography. The data indicated that eIF(iso)4E was particularly required for translation of the stored mRNAs from dry seeds, and that eIF4E was unable to fully replace the eIF(iso)4E activity. In addition, the dry seed mRNA pool was translated by the cap-binding complex isolated from dry seeds better than by the complex isolated from 24-h-imbibed seeds, whereas the translational efficiency of the mRNA pool from 24-h-imbibed seeds was similar between the capbinding complexes from these two stages. Interestingly, eIF(iso)4E was more abundant than eIF4E in dry seeds, while both cap-binding proteins were present at similar levels in 24-h-imbibed seeds. These results suggest that the ratio of eIF(iso)4E to eIF4E in the corresponding eIF4F complex is critical for the mechanisms of translational control during maize germination.

Biochemical Characterization of a New Maize (Zea mays L.) Peptide Growth Factor

Coordination of cell growth and cell division is very important for living organisms in order for these to develop harmonically. The present research is concerned with the purification and characterization of a new peptide hormone, namely ZmIGF (Zea mays insulin-like growth factor), which regulates growth and cell division in maize tissues. ZmIGF is a peptide of 5.7 kDa, as determined by mass spectroscopy. It was isolated either from maize embryonic axes of 48-h germinated seeds or from embryogenic callus and purified through several chromatographic procedures to obtain a single peak as shown by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). This peptide exhibits a well defined α-helix structure by circular dichroism analysis, similar to that reported for Insulin or for Insulin-like growth factor (IGF-1). Further, ZmIGF seems to perform, in maize, a similar function to that reported for insulin or peptides from the IGF family in animals. Indeed, maize tissues stimulated either by ZmIGF or insulin showed to induce selective synthesis of ribosomal proteins as well as of DNA. Taken together, the previously mentioned data strongly suggest that plants contain a peptide hormone of the IGF family, highly conserved through evolution that regulates growth and development.

Distinctive characteristics of protein synthesis in maize embryos during the early stages of germination.

Abstract Quiescent maize embryos were found to contain significant amounts of poly-A-rich pre-formed RNA. 14C-amino acid incorporation into trichloroacetic acid precipitable material was detected at slow rate at the begining of imbibition and fastly increased near 18 to 24 h. Polysomal formation was measured during this period. Addition of - amanitin to the incubation system at two 6h-pulse periods showed significant inhibition of the 14C-amino acid incorporation for the 18–24 h-period, but not for the 0–6 h-period.

Coordination of cell growth and cell division in maize (Zea mays L.) relevance of the conserved TOR signal transduction pathway

Mechanisms that bring about coordination of cell growth and cell division in different organisms are biological events not yet clearly revealed. In maize, insulin effector of the phosphatidylinositol 3-kinase (PI3K)–target of rapamycin (TOR) signal transduction pathway in metazoan or an intrinsic maize growth factor similar to insulin has shown to regulate cell growth. This research has been undertaken to analyze the role of PI3K–TOR signal transduction pathway in maintaining coordinated regulation of cell growth and cell division in maize tissues. Results indicate that DNA synthesis as well as mitotic index increased in maize callus in vitro cultures after insulin or maize factor stimulation. Biomass and ribosomal protein synthesis also showed significant increment after this stimulation, and the cell morphology composition of the cultures drastically changed. Two proteins related to cell cycle, D-type cyclins and proliferating cell nuclear antigen, were selectively synthesized under these conditions. Reverse transcription-polymerase chain reaction analysis of total and polysomal RNAs revealed that this effect was mainly due to specific mobilization of the correspondent mRNAs into polysomes rather than to transcriptional activation. All these events were sensitive to rapamycin inhibition, indicating that the stimulatory effect was mediated by TOR kinase activation. It is concluded that the evolutionary conserved PI3K–TOR pathway might coordinately regulate cell growth and cell division in maize.

Amino acid pools and protein synthesis in germinating maize embryos.

Amino acid pools were analyzed in maize (Zea mays L.) axes germinated for 0, 6 and 24 h. Proteins synthesized at early (0–6 h) and late (18–24 h) stages were characterized by gel electrophoresis and fluorography after either 14C-leucine or 14C-lysine pulses. An increase of amino acid incorporation after 18–24 h was observed, as well as changes in the protein patterns of the corresponding fluorographs. Analysis of the endogenous amino acid pools showed major changes in contents of proline, alanine, isoleucine, valine, leucine and lysine. A selective increase of lysine incorporation into proteins during the late stage was detected.

Contribution of the Zea mays insulin-like growth factor (ZmIGF) to the embryogenic competence of maize tissue cultures

Establishment of somatic embryogenic cultures is highly influenced by the plant genotype and the explant type. In maize, immature embryos generate embryogenic callus (E), whereas quiescent embryos produce non-embryogenic callus (NE). E callus shows active growth and high capacity of plant regeneration, while NE callus shows slow growth and no regeneration capacity. Active growth is needed for the establishment of embryogenic cultures; therefore, lack of this characteristic pose a handicap for plant regeneration from NE callus. To correct the slow growth on NE callus, the Zea mays insulin-like growth factor (ZmIGF), a peptide that promotes growth by activating the target of rapamycin (TOR) pathway, was used as media supplement. Additionally, since the TOR pathway is connected to the auxin pathway, ZmIGF participation in cell differentiation was considered. To this end, this research explores ZmIGF effect, beyond growth and proliferation on a Mexican maize landrace, which has shown high somatic embryogenic capacity. Expression levels of reported genes involved in the embryogenesis and differentiation processes were evaluated in maize E, NE, and NE-ZmIGF (NE-Zm) growth-activated calluses. A tendency to upregulate messenger RNA (mRNA) expression was observed for genes encoding transcription factors and auxin transport. Some genes related to epigenetic control showed downregulation. Global DNA methylation and chromatin modifications results suggest an epigenetic activation state on E callus and a repression status on NE callus. ZmIGF induced modifications at DNA methylation and chromatin over NE callus, which changed its original repression state to an active one. Overall, results suggest that the expression of genes related to auxin signaling, mainly transport and efflux carriers, are essential for accomplishing plant regeneration through somatic embryogenesis (SE).

REGULATORY MECHANISMS OF REM SLEEP IN THE CAT

Today it is universally accepted that mammals and primates present at least two basic stages of sleep. The state of sleep is first characterized electroencephalographically by the appearance of 14–18 Hz cortical spindles, which are subsequently replaced by 2–4Hz slow waves. At the same time, high-voltage (500–800μV) sharp waves are recorded from the hippocampus, while the electromyogram (EMG) decreases slightly. Usually after some 30–40 minutes the electrophysiological signs of slow wave sleep (SWS) are replaced by low-voltage fast cortical EEG activity, regular hippocampal theta rhythm (5–6 Hz), isoelectric EMG, burst of rapid eye movements (REM), and pontogeniculooccipital (PGO) waves. PGO waves appear in SWS approximately one minute before all REM sleep periods. These PGO spikes exhibit a fairly constant daily rate of about 14,000 in the cat (18), may exist in man (30) and are made up of simple Type I and complex Type II spikes (24).