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.

In maize, two distinct ribulose 1,5-bisphosphate carboxylase/oxygenase activase transcripts have different day/night patterns of expression.

Several cDNAs encoding ribulose-1,5-bisphosphate carboxylase/oxygenase activase (Rubisco activase, RCA) were isolated from a maize (Zea mays L.) leaf cDNA library. Although all the cDNAs encoded the same polypeptide, the RCA beta isoform, they showed two different downstream-like elements (DST-like) at their 3' untranslated regions (UTRs). The Zmrca1 cDNAs had the subdomain I, and II and the Zmrca2 cDNAs, besides these subdomains, showed two repeats of the subdomain III. The presence of at least two different rca genes in the maize genome was demonstrated by Southern, and by PCR analysis using primers specific for the two cDNAs. Northern analysis with probes specific for each gene showed that the Zmrca2 was expressed as a 1.8 kb transcript, the Zmrca1 corresponded to a 1.4 kb transcript, and a 1 kb band was a stable degradation product of one or both transcripts. Although both mRNAs showed cyclic variations during a day/night period, with their highest levels before dawn, the Zmrca2 transcript showed stronger changes than the Zmrca1 transcript, presenting a twofold larger highest to lowest RNA accumulation ratio than the Zmrca1 transcript, implying that they have different turnover rates. Our results suggest that post-transcriptional mechanisms, mediated by the DST-like element might be involved in the circadian expression of the maize rca transcripts.

A maize insulin-like growth factor signals to a transduction pathway that regulates protein synthesis in maize.

Insulin and insulin-like growth factors (IGFs) are well-characterized regulators in higher eukaryotic cells that control biological processes such as cell growth and survival, and selective translation of mRNAs. This research presents the purification of a 20 kDa protein, isolated from maize tissue, with IGF activity. The protein was purified from 48 h-germinated maize embryonic axes by G-50 Sephadex fractionation followed by affinity chromatography through a bovine insulin antibody-Sepharose column. This protein proved to significantly speed up maize germination and seedling growth. At the molecular level, Zea mays IGF (ZmIGF) enhanced phosphorylation of S6 ribosomal protein (rp) on the 40 S ribosomal subunit, in a similar way as observed when bovine insulin is applied to maize axes during germination. Rapamycin, a specific inhibitor of the insulin-stimulated signal transduction pathway, prevented S6 rp phosphorylation in maize axes. Moreover, ZmIGF stimulated [(35)S]methionine incorporation into rps, above the level of overall cytoplasmic proteins. Either incubation with anti-insulin antibody, heat treatment (60 degrees C) or trypsin digestion abolished this ZmIGF effect. It is proposed that ZmIGF is an endogenous maize growth factor that regulates the synthesis of specific proteins through a pathway similar to that of insulin or IGFs in animal tissues.

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.

Ribonucleoprotein particles of quiescent maize embryonic axes.

Certain RNA molecules are known to be sequestered and stored as ribonucleoprotein particles (RNPs) in many different tissues, particularly at some stages of metabolic quiescence. In this research RNPs from embryonic axes of mature maize seeds were isolated by sucrose and CsCl gradient centrifugation and characterized based on their RNA and protein contents. Two types of RNP particles of non-ribosomal nature were identified by northern blot analysis with specific probes: the 7S RNP and the signal recognition particle (SRP) particles which contain 5S rRNA and 7S RNA respectively. The proteins associated to these RNA molecules were the transcription factor TFIIIA-homologous protein associated to 7S RNP, and the p72, p68 and p54-GTPase proteins associated to SRP.

S6 ribosomal protein phosphorylation and translation of stored mRNA in maize.

This article focuses on the effect that S6 ribosomal protein phosphorylation might have in regulating mRNA translation. Maize axes of either 4 or 14 h of germination were pulse-labelled for 1 h with [32P]-orthophosphate. Analysis of their ribosomal proteins by gel electrophoresis and autoradiography showed distinctive levels of S6 ribosomal protein phosphorylation for both ribosomal sets. Axes at these two stages of germination were treated with alpha-amanitin to ensure transcription inhibition and pulse-labelling with [35S]-methionine. The [35S]-proteins, resulting from stored mRNA translation, when analysed by 2-D-gel electrophoresis and fluorography revealed distinctive [35S]-protein patterns. In vitro translation of stored mRNA on ribosomes from either 4 or 14 h germinated-maize axes produced different [35S]-protein patterns. Further, addition of 7methyl-GTP-Sepharose to the translation system showed differential cap-dependent protein synthesis inhibition depending on the set of ribosomes tested. It is concluded that translation of stored mRNA in germinating maize axes is at least partially regulated by a mechanism that involves S6 ribosomal protein phosphorylation.

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 alpha-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.

Rubisco activase, a possible new member of the molecular chaperone family.

The present research addresses the question of whether Rubisco activase (R-A), the enzyme reported to activate Rubisco, is actually a molecular chaperone rather than a conventional enzyme. Several biochemical properties known to be characteristics of molecular chaperones were tested for R-A with positive results. The experiments were performed either in vitro with purified spinach Rubisco and Rubisco activase or in vivo in maize seedling leaves. Our results confirmed that activation of Rubisco by R-A is an ATP hydrolysis-dependent process and further demonstrated that (a) R-A binds preferably to non-native Rubisco protein, than to the native form, and dissociates from this complex after addition of ATP, (b) R-A increases during heat shock treatment in maize seedling leaves, and (c) a large recovery of Rubisco activity is achieved from heat-inactivated Rubisco by addition of R-A and an energy source. We conclude that R-A characteristics strongly suggest that this protein belongs to the molecular chaperone group. The possible role of R-A on maintaining Rubisco activity in vivo is discussed.

Regulation of ribulose-1,5-bisphosphate carboxylase expression in second leaves of maize seedlings from low and high yield populations.

Ribulose-1,5-bisphosphate carboxylase oxygenase (EC 4.1.1.39) (Rubisco) activity, Rubisco-protein, and Rubisco large and small subunit gene (rbcL and rbcS) transcripts were measured at seven stages of development in the second leaf of maize (Zea mays L.) seedlings belonging to low and high yield populations. During the three early stages of development, when the leaf has not yet expanded, it was determined that increments in Rubisco-activity were caused by increases in Rubisco-protein and its mRNAs. Afterward, the rbcS level decreased sharply down to nondetectable levels at the seventh stage, when the leaf was at the beginning of senescence. As a contrast, rbcL transcript decreased slowly and Rubisco-protein accumulated up to the fifth stage, when the leaf reached its maximum expansion. A slight decrease in Rubisco-protein was then observed. These results suggest that at early stages of development Rubisco-activity and Rubisco-protein are regulated mainly at the transcriptional level. At the later phase the regulation seems to be at other biochemical levels. Neither Rubisco activity nor Rubisco-protein showed correlation with yield for both maize populations at this stage of development. Slightly higher levels of both transcripts were observed in the high yield population.

Polyamine distribution among maize embryonic tissues and its relation to seed germination.

Polyamines are known to participate in the control of cell division. In the present report, the levels and distribution of polyamines in the embryonic tissues of maize seeds were studied to relate them to the reinitiation of cell division during seed germination. Results indicate higher contents of polyamines in scutellum than in axis tissues of dry seeds. After germination, larger increments of the three polyamines were obtained in axes, rather than in scutella. Seeds of low viability did not show putrescine increments after germination. It is suggested that spermidine and spermine are directly related to the germination process.

Glutamate synthase in greening callus of Bouvardia ternifolia Schlecht.

The distribution of the two glutamate-synthase (GOGAT) activities known to exist in higher plants (NADH dependent, EC 2.6.1.53; and ferredoxin dependent, EC 1.4.7.1) was studied in non-chlorophyllous and chlorophyllous cultured tissue as well as in young leaves of Bouvardia ternifolia. The NADH-GOGAT was present in all three tissues. Using a sucrose gradient we found it in both the soluble and the plastid fraction of non-chlorophyllous and chlorophyllous tissue, but exclusively in the chloroplast fraction of the leaves. Ferredoxin-GOGAT was found only in green tissues and was confined to the chloroplasts. Ferredoxin-GOGAT activity increased in parallel with the chlorophyll content of the callus during the greening process in Murashige-Skoog medium (nitrate and ammonium as the nitrogen sources), while NADH-GOGAT was not affected by the greening process in this medium. Furthermore, both activities were differentially affected by either nitrate or ammonium as the sole nitrogen source in the medium during this process. It is suggested that each GOGAT activity is a different entity or is differently regulated.

Alternative Pathways for Ammonium Assimilation in Bouvardia ternifolia Cell Suspension Cultures.

Cell suspension cultures of young leaves (L) and roots (R) of Bouvardia temifolia were cloned and their growth was followed, measuring cell volume and fresh weight. Both cultures were depleted of nitrogen and subcultured in different nitrogen sources. The enzymes for ammonium assimilation and the ammonium pools were measured during the growth cycle. Results indicate that the two ammonium assimilation pathways, the GS/GOGAT, and the GDH-GS, are functional in both cultures. The pathway used is dependent upon the tissue and the nitrogen source in the medium. GDH seems to have an anabolic role on root cultures and is regulated by the internal concentration of ammonium.