E. Sánchez de Jiménez

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.

Regulation of Rubisco activity during grain-fill in maize: possible role of Rubisco activase

Levels of ribulose bisphosphate carboxylase (Rubisco) and Rubisco activase were compared in leaves above the ear in two genetically related populations (Z 0 and Z 20 ) of maize ( Zea mays L.). Z 20 was obtained from Z 0 after twenty agronomic selection cycles for grain yield improvement ( c . 90% above Z 0 ). Plants were cultivated in the highlands of Mexico and leaves were sampled weekly during the grain-filling period. Chlorophyll, soluble protein and Rubisco activity were measured. Chlorophyll and soluble protein content slowly decreased during this period, the former faster than the latter, with no significant differences between populations. During the first 40 days after anthesis, Rubisco activity was significantly greater in the high-yielding population (Z 20 ), although Western blot analysis of Rubisco showed similar values for both populations within this period. However, the same analysis for Rubisco activase indicated a greater amount of this protein in the higher-yielding population (Z 20 ) than the original one (Z 0 ) during the early and middle part of the grain-filling period. The addition of Rubisco activase and an ATP-generating system to Z 0 leaf extracts resulted in increased Rubisco activity. It was concluded that during grain-fill in maize, the level of Rubisco activase has a regulatory effect on Rubisco activity expression.

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.

Regulation of ribosome biogenesis in maize embryonic axes during germination.

Ribosome biogenesis is a pre-requisite for cell growth and proliferation; it is however, a highly regulated process that consumes a great quantity of energy. It requires the coordinated production of rRNA, ribosomal proteins and non-ribosomal factors which participate in the processing and mobilization of the new ribosomes. Ribosome biogenesis has been studied in yeast and animals; however, there is little information about this process in plants. The objective of the present work was to study ribosome biogenesis in maize seeds during germination, a stage characterized for its fast growth, and the effect of insulin in this process. Insulin has been reported to accelerate germination and to induce seedling growth. It was observed that among the first events reactivated just after 3 h of imbibition are the rDNA transcription and the pre-rRNA processing and that insulin stimulates both of them (40-230%). The transcript of nucleolin, a protein which regulates rDNA transcription and pre-rRNA processing, is among the messages stored in quiescent dry seeds and it is mobilized into the polysomal fraction during the first hours of imbibition (6 h). In contrast, de novo ribosomal protein synthesis was low during the first hours of imbibition (3 and 6 h) increasing by 60 times in later stages (24 h). Insulin increased this synthesis (75%) at 24 h of imbibition; however, not all ribosomal proteins were similarly regulated. In this regard, an increase in RPS6 and RPL7 protein levels was observed, whereas RPL3 protein levels did not change even though its transcription was induced. Results show that ribosome biogenesis in the first stages of imbibition is carried out with newly synthesized rRNA and ribosomal proteins translated from stored mRNA.

Influence of carbon source and CO2-enrichment on biochemical parameters associated with photomixotrophia in maize callus cultures

Summary To learn about the biochemical processes underlying the induction of photomixotrophia in maize cell culture, maize calli were cultured in medium containing either glucose or starch as the carbon source. The effect of a CO 2 -enriched atmosphere on different parameters was tested. Levels of chlorophyll and CO 2 -fixing enzymes were measured to assess the greening process concomitant to histological observations of chloroplast development. Both starch and glucose promoted higher chlorophyll accumulation in callus cultured under light than sucrose. Histological analysis of green callus grown on glucose-containing medium revealed the formation of poorly developed chloroplasts containing starch grains, whereas in starch medium a large number of elongated chloroplasts containing thylakoids were observed. Exposure of these calli to a CO 2 -enriched atmosphere enhanced the plastid differentiation process up to mature chloroplasts with grana and intergranal thylakoids. Western-blot analysis demonstrated the presence of CO 2 -fixing enzymes, Rubisco (EC 4.1.1.39) and PEP carboxylase (EC 4.1.1.31), as well as Rubisco activase in greening callus. Rubisco and PEP carboxylase activities showed large values when starch was the carbon source in the medium. Results of histological analysis and a/b chlorophyll ratios indicated that the chloroplasts formed were of the C 3 -type. PEP carboxylase kinetic properties were also consistent with a C 3 -type enzyme involved in anaplerotic functions. It is concluded that under the experimental conditions tested, starch plus CO 2 -enriched atmosphere are the best carbon source for inducing and supporting photomixotrophia in maize cultures, as indicated by several biochemical parameters.

Translation of Ribosomal Protein mRNAs in Maize Axes

The objective of this research is to study the mechanism(s) regulating ribosomal protein (rp) mRNA translation during maize germination. Northern analysis of RNA from non-germinated maize embryonic axes revealed rp-transcripts within the stored mRNA pool. S4 and S6 rp transcripts were stored as mature mRNAs while others seemed to be as pre-mRNAs. Preferential translation of rp-mRNAs was observed early in germination, as indicated by in vivo pulse labelling experiments with 35S-methionine. Mobilization of S6-transcript from ribonucleoprotein particles to polysomes was followed during germination. The effects of different hormones and environmental stresses were tested on this process. Recruitment of S6 rp-transcript was found to be affected by insulin and stress. Inhibitors of either protein-phosphatases or kinases altered this process suggesting that it could be regulated by phosphorylation/dephosphorylation of protein(s) related to the translational apparatus. Insulin caused an increase in phosphorylation of the ribosome integrated S6 protein. These data suggest that S6 phosphorylation might be involved in the mechanism responsible for rp-mRNA recruitment into polysomes. Consequently, in maize axes translation of rp-mRNA might be regulated by a signal transduction mechanism similar to other eukaryotic cells.