[show abstract][hide abstract] ABSTRACT: 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 (0 h) 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 cap-binding 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The reactivity and selectivity of the the captodative olefins 1-acylvinyl benzoates 1a–1f and 3a as heterodienes in hetero-Diels–Alder reactions in the presence of electron-rich dienophiles is described. Heterodienes 1 undergo regioselective cycloaddition with the alkyl vinyl etherdienophiles 6a,b and 9 to give the corresponding dihydro-2H-pyrans 7, 8, and 10 under thermal conditions. The reactivity of these cycloadditions depends, to a large extent, on the electronic demand of the substituent in the aroyloxy group of the heterodiene. Frontier-molecular-orbital (FMO; ab initio) and density-functional-theory (DFT) calculations of the ground and transition states account for the reactivity and regioselectivity observed in these processes.
[show abstract][hide abstract] ABSTRACT: Ribosomal protein S6 (S6rp) is phosphorylated by the p70S6K enzyme in mammals, under mitogen/IGF regulation. This event has been correlated with an increase in 5'TOP mRNA translation. In this research, a maize S6 kinase (ZmS6K) was isolated from maize (Zea mays L.) embryonic axes by human p70S6K antibody immunoprecipitation. This enzyme, a 62 kDa peptide, proved to be specific for S6rp phosphorylation, as revealed by in vivo and in vitro kinase activity using either the 40S ribosomal subunit or the RSK synthetic peptide as the substrates. ZmS6K activation was achieved by phosphorylation on serine/threonine residues. Specific phospho-Threo recognition by the p70S6K antibody directed to target phospho-Threo residue 389 correlated with ZmS6K activation. The ZmS6K protein content remained almost steady during maize seed germination, whereas the ZmS6K activity increased during this process, consistent with Zm6SK phosphorylation. Addition of insulin to germinating maize axes proved to increase ZmS6K activity and the extent of S6rp phosphorylation. These events were blocked by rapamycin, an inhibitor of the insulin signal transduction pathway in mammals, at the TOR (target of rapamycin) enzyme level. We conclude that ZmS6K is a kinase, structurally and functionally ortholog of the mammalian p70S6K, responsible for in vivo S6rp phosphorylation in maize. Its activation is induced by insulin in a TOR-dependent manner by phosphorylation on conserved serine/threonine residues.
[show abstract][hide abstract] ABSTRACT: Electrophilic aromatic substitution of activated benzenes with the captodative olefin 1‐acetylvinyl‐1‐p‐nitrobenzoate (9), and with the electron‐deficient alkenes methyl acrylate (8a), methylvinylketone (8b), and acrolein (8c) were evaluated under Lewis acid catalysis. Olefin 9 proved to be much more reactive than alkenes 8a–8c. We also describe a one‐step synthesis of the antifungal and larvicidal natural product methyl 3‐(2,4,5‐trimethoxyphenyl)propionate (6), by reaction of 1,2,4‐trimethoxybenzene with 8a under microwave irradiation.
[show abstract][hide abstract] ABSTRACT: Acidic ribosomal proteins (ARPs) are highly conserved phosphoproteins in eukaryotic organisms. They participate in translation regulation by interacting with eEF-2 elongation factors in the peptide elongation process. During maize germination, protein synthesis is tightly regulated by different mechanisms that are not yet clearly understood. The objective of this research is to characterize the expression patterns of the two maize ARPs (P1 and P2) and their phosphorylated status in germinating maize embryonic axes. Expression of P1 and P2 mRNA transcripts was analyzed by Northern blots with specific cDNA probes. Results indicated that both transcripts are among the mRNA stored pool of the quiescent axes and each displays a distinctive expression pattern during germination. P1 and P2 synthesis initiates very early in germination, as demonstrated by [(35)S]methionine pulse-labeling experiments. This synthesis was not insulin/IGF-stimulated as the synthesis of the bulk of ribosomal proteins that was responsive to this stimulus. P1 and P2 proteins were purified from ribosomes of maize embryonic axes and their physicochemical characteristics determined. A cytoplasmic pool of dephosphorylated P1 and P2 proteins was found in axes of quiescent and germinated stages that freely assembled into the ribosomes. IEF analysis of ARPs revealed one P1 (P1-1) and two P2 (P2-1 and P2-2) forms in the ribosomes of 24 h germinated axes. Kinetic studies of ARP phosphorylation during germination revealed a specific order of phospho-ARP appearance, suggesting that this process is under regulation within this period. It is concluded that P1 and P2 phosphorylation rather than ARP expression or assembly into ribosomes is the main step that regulates ARP function in axes during maize germination.
[show abstract][hide abstract] ABSTRACT: Auxin is known to stimulate protein synthesis in many plant tissues, but the mechanisms involved in this process are unknown. The present research inquires whether auxin might regulate selective translation of mRNAs by inducing S6 ribosomal protein phosphorylation on the 40S ribosomal subunit in maize (Zea mays L.). Maize embryonic axes auxin-stimulated by natural (IAA) or synthetic (Dicamba or 1-NAA) auxins, selectively increased ribosomal protein synthesis. This effect was not reproduced by auxin inactive analogue 2-NAA. Enhanced S6 ribosomal protein phosphorylation on the 40S ribosomal subunit was also observed after auxin stimulation, as measured by [32P] incorporation into this protein. This increment did not occur when stimulation was performed with the inactive auxin analogue. Further, increased recruitment into polysomes of two 5'TOP-like mRNAs, encoding for the initiation translation factor eIF-iso4E and the S6 ribosomal protein, was also found after auxin stimulation of maize axes. A positive correlation was established between the levels of S6 ribosomal protein phosphorylation and the S6 ribosomal protein transcript recruitment into polysomes by means of okadaic acid or heat shock application to maize axes. These data indicate that auxin stimulates S6 ribosomal protein phosphorylation on maize ribosomes, concomitant to the recruitment of specific mRNAs (5'TOP-like mRNAs) into polysomes for translation. It is proposed that by this mechanism auxin regulate the synthesis of specific proteins in maize tissues.
Physiologia Plantarum 07/2002; 115(2):291-297. · 3.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: A short and regioselective synthesis of γ-hydroxycyclohexenones is described, using 3-p-nitrobenzoyloxy-3-buten-2-one (2a) as a ketene equivalent in Diels-Alder reactions with substituted dienes. Oxidation with MCPBA of the α-acetylcyclohexenol derivative, obtained by hydrolysis of the cycloadducts, led to the corresponding γ-hydroxycyclohexenones in moderate overall yields. Evidence of the mechanism is provided.
[show abstract][hide abstract] ABSTRACT: The objective of this research was to determine the role of acidic ribosomal protein (ARP) phosphorylation in translation. Ribosomes (Rbs) from germinated maize (Zea mays L.) axes had four ARP bands within 4.2 to 4.5 isoelectric points when analyzed by isoelectric focusing. Two of these bands disappeared after alkaline phosphatase hydrolysis. During germination a progressive change from nonphosphorylated (0 h) to phosphorylated ARP (24 h) forms was observed in the Rbs; a free cytoplasmic pool of nonphosphorylated ARPs was also identified by immunoblot and isoelectric focusing experiments. De novo ARP synthesis initiated very slowly early in germination, whereas ARP phosphorylation occurred rapidly within this period. ARP-phosphorylated versus ARP-nonphosphorylated Rbs were tested in an in vitro reticulocyte lysate translation system. Greater in vitro mRNA translation rates were demonstrated for the ARP-phosphorylated Rbs than for the non-ARP-phosphorylated ones. Rapamycin application to maize axes strongly inhibited S6 ribosomal protein phosphorylation, but did not interfere with the ARP phosphorylation reaction. We conclude that ARP phosphorylation does not depend on ARP synthesis or on ARP assembly into Rbs. Rather, this process seems to be part of a translational regulation mechanism.
[show abstract][hide abstract] ABSTRACT: The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [(32)P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of (32)P incorporation and the electrophoretic patterns were dependent on (32)P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K(m) values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.