Estela Sánchez de Jiménez

SYNTHESIS OF RIBOSOMAL PROTEINS FROM STORED MRNAS EARLY IN SEED GERMINATION

In several eukaryotic organisms the mRNA expression for ribosomal proteins (RPs) is highly regulated at the translational level. The present work addresses the synthesis of RPs early in germination and particularly the origin of their transcripts. Excised maize axes were labeled with [35S]methionine for 6 h, and both cytoplasmic and ribosomal proteins were analyzed by electrophoresis and fluorography. It was found that some RPs are newly synthesized and already incorporated into ribosomes during this period. Synthesis of RPs was also observed under conditions of transcription inhibition, suggesting the presence of stored-RP transcripts in the embryonic axes. In vitro translation of polysomes isolated from α-amanitin-treated axes early in germination (3 and 9 h) was performed. Immunoprecipitation of the products revealed the synthesis of rps. Four heterologous probes two each of the small subunit (S4 and S6) and large (L3, L16) subunit rps were used to detect the correspondent transcripts within the stored mRNA stock of the embryonic axes. Both, by slot blot and northern analysis, a positive cross-reaction occurred for all the tested samples. Although only two of them (S4 and S6) seem to be stored as mature mRNA.

DNA synthesis and cell division in embryonic maize tissues during germination

Summary The relationship between the beginning of DNA replication and the onset of cell division in embryonic maize tissues during germination was examined. Biochemical and cytological evidence indicates that DNA replication starts after around 14 h of imbibition of maize embryos. Between 15 to 20 h typical replicative DNA synthesis can be detected, reaching its highest value at 40 h. The first mitotic figures appear in mesocotyl 28 h after imbibition followed by seminal (32 h) and primary root (36 h) meristems. The kinetic pattern observed for the dividing cells throughout the germination period suggests that me- ristematic tissues consist of distinct cell populations, which might re-enter the cycle at different times during seed germination.

Distinctive expression and functional regulation of the maize (Zea mays L.) TOR kinase ortholog

TOR (Target of rapamycin) kinase is a central component of a signal transduction pathway that regulates cellular growth in response to nutrients, mitogens and growth factors in eukaryotes. Knowledge of the TOR pathway in plants is scarce, and reports in agronomical relevant plants are lacking. Previous studies indicate that Arabidopsis thaliana TOR (AtTOR) activity is resistant to rapamycin whereas maize TOR (ZmTOR) is not, suggesting that plants might have different regulation mechanisms for this signal transduction pathway. In the present work maize ZmTOR cDNA was identified and its expression regulation was analyzed during germination on different tissues at various stages of differentiation and by the main ZmTOR regulators. Our results show that ZmTOR contains all functional domains characteristic of metazoan TOR kinase. ZmTOR expression is highly regulated during germination, a critical plant development period, but not on other tissues of contrasting physiological characteristics. Bioinformatic analyses indicated that maize FKBP12 and rapamycin form a functional structure capable of targeting the ZmTOR protein, similar to other non-plant eukaryotes, further supporting its regulation by rapamycin (in contrast with the rapamycin insensitivity of Arabidopsis thaliana) and the conservation of rapamycin regulation through plant evolution.

Regulation of Glutamine Synthetase/Glutamate Synthase Cycle in Maize Tissues, Effect of the Nitrogen Source

Summary Glutamine synthetase (EC. 6.3.1.2) and NADH- and ferredoxin-dependent glutamate synthase (EC. 1.4.1.14) activities were measured in leaves, roots and callus tissues of maize exposed to different nitrogen sources. NADH-dependent glutamate synthase activity was detected in roots where the enzyme level was increased by ammonium citrate, NH 4 Cl plus nitrate, glutamate and urea, and in callus tissue where the level was increased by nitrate. No NADH-dependent glutamate synthase could be detected in leaves, which showed a high ferre-doxin-dependent glutamate synthase activity. The level of this latter enzyme was reduced by all nitrogen sources used except for ammonium citrate and glutamate. The results suggest that NADH-dependent glutamate synthase is regulated in different ways in the tissues tested. Glutamine synthetase was strongly regulated in the leaf but not in the root. Glutamine synthetase activity was reduced by NH4CI and increased by nitrate, ammonium citrate, glutamate, glutamine and urea. The results suggest that in the root the GS/GOGAT cycle is used as such, but that GS also synthesized glutamine, which the tissue exports for the synthesis of glutamate.

Physiological traits associated with mass selection for improved yield in a maize population

Abstract In an attempt to identify genetic traits related to increased productivity, physiological and biochemical parameters were compared between two cycles of selection of a maize (Zea mays L.) population in a field study during the summer seasons of 1993 and 1994. Stratified mass selection for yield (based on ear phenotype) over 22 cycles has altered phenotype and increased the productivity of the original population (C0). Plants of the selected population (C22) were 0.5 m taller, had almost twice the amount of leaf area at anthesis (2600 versus 1330 cm2/pl), accumulated more than twice the amount of aboveground dry matter (294 versus 133 g/pl), had a 30% larger harvest index, and yielded 2.7× as much as C0 per unit area and 2.9× as much on a per plant basis. At anthesis, the diurnal pattern of carbon assimilation from single leaves revealed greater photosynthetic rates in C22, whereas dark respiration rates were 20% greater in C0 at this stage. Maximum transpiration rates were similar in both cycles, although a rapid, early decline during the day to lower levels occurred in C22. Rubisco and sucrose phosphate synthase activities were greater in C22 beginning early in development. The data indicate that selection for enhanced yield has altered the expression of all parameters studied.

Comparative DNA-RNA hybridization in differentiated cells☆

Abstract Some hydrodynamic and spectral properties, as well as the capability to form RNA-DNA hybrids, have been measured for avian DNA. The DNA sources were red cells from adult chickens and from embryos at particular stages of differentiation, and selected adult chicken tissues. Sedimentation coefficients corresponded to an average molecular weight of 15 × 10 6 for red cell DNA, and to 11.4 × 10 6 for DNA from adult tissues. Determinations of T m for these DNA samples showed values ranging from 85.0 to 85.6 °C with an average of 85.3 ± 0.3 °C, corresponding to a G + C content of 39.0%. The hybridization conditions used permitted specific interaction between chicken DNA and reticulocyte 10 s RNA. Escherichia coli , Bacillus cereus and crab DNA tested in this sytem produced less than 15% hybridization on a scale placing chicken DNA at 100%. Competition experiments with total yeast RNA or chicken ribosomal RNA, added at concentrations as high as ten times that of the 10 s RNA, did not diminish the percentage of 10 s RNA hybridized. On the other hand, cold 10 s RNA showed a competitive effect under the same experimental conditions. In experiments designed to test whether a highly differentiated organism keeps the same genomal information in various tissues, no differences in capacity to hybridize with “Hb”-messenger RNA were detected. The same level of hybridization of reticulocyte 10 s RNA was detected with DNA from red cells at different stages of differentiation and with DNA from tissues of markedly different embryonic origin. It was not possible to reach saturation concentrations of 10 s RNA for the chicken DNA. When 30 times more RNA than DNA was present, approximately 2.5% hybridization was obtained. The meaning of this high percentage of hybridization is discussed.

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.

Effect of Nitrate, Ammonium and Glutamine on Nitrogen Assimilation Enzymes during Callus Growth of Maize

Summary The activities of the enzymes glutamine synthetase, glutamate synthase and glutamate dehydrogenase were measured during the cell cycle in maize tissue cultures grown in different nitrogen sources: 20 mM nitrate, 2 mM ammonium, 5 mM glutamine and a mixture of 40 mM nitrate plus 20 mM ammonium.Similar growth curves were obtained with all the nitrogen sources employed, except for ammonium.The callus grown on nitrate presented two GS activity peaks.While with the mixture of nitrate and ammonium only one peak was present and in the presence of glutamine no definitive peaks of activity were observed.All nitrogen sources showed three GOGA T activity peaks, with the exception of nitrate where only two were observed.GDH-NADH displayed three peaks in cultures grown on nitrate, ammonium and glutamine.In the presence of the mixture only one peak was observed.

Biochemical parameters to assess cell differentiation of Bouvardia ternifolia Schlecht callus.

Calli derived from leaves and radicles of B. ternifolia were grown on Murashige and Skoog (MS) basal medium, and the effects of different nitrogen sources on the rate of callus growth and on the enzymes related to nitrogen assimilation were studied. Ammonium alone did not support callus growth unless a Krebs-cycle intermediate was added to the medium. The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (EC 1.4.1.2) were measured in homogenates of callus grown on media supplied with different nitrogen sources. The results indicate that leaf and root calli have similar levels of these enzymes when grown on MS medium (Murashige and Skoog 1962. Physiol. Plant. 15, 473–497). However, when the calli were supplied with glutamine as the sole nitrogen source, the activity of glutamate synthase increased in leaf callus but was almost completely inhibited in root callus. The results indicate that calli originated from different B. ternifolia tissues do not have the same biochemical dedifferentiated state.