Polyamine anabolic/catabolic regulation along the woody grapevine plant axis

The distribution of the endogenous PA fractions throughout the entire perennial woody grapevine (Vitis vinifera L.) plant was studied, along with the expression profiles of the PA anabolic and catabolic genes and their substrates and secondary metabolites. Putrescine fractions increased with increasing leaf age, although the expression of its biosynthetic enzymes Arg and Orn decarboxylases decreased. Orn transport from young organs dramatically enhanced putrescine biosynthesis in older tissues, via the Orn decarboxylase pathway. S-adenosylmethionine decarboxylase and spermidine synthase genes were down-regulated during development in a tissue/organ-specific manner, as were spermidine and spermine levels. In contrast, amine oxidases, peroxidases and phenolics increased from the youngest to the fully developed vascular tissues; they also increased from the peripheral regions of leaves to the petioles. Hydrogen peroxide generated by amine oxidases accumulated for the covalent linkage of proteins via peroxidases during lignification. These results could be valuable for addressing further questions on the role of PAs in plant development.     

Genes, enzymes and regulation of arginine biosynthesis in plants.

Arabidopsis genes encoding enzymes for each of the eight steps in L-arginine (Arg) synthesis were identified, based upon sequence homologies with orthologs from other organisms. Except for N-acetylglutamate synthase (NAGS; EC 2.3.1.1), which is encoded by two genes, all remaining enzymes are encoded by single genes. Targeting predictions for these enzymes, based upon their deduced sequences, and subcellular fractionation studies, suggest that most enzymes of Arg synthesis reside within the plastid. Synthesis of the L-ornthine (Orn) intermediate in this pathway from L-glutamate occurs as a series of acetylated intermediates, as in most other organisms. An N-acetylornithine:glutamate acetyltransferase (NAOGAcT; EC 2.3.1.35) facilitates recycling of the acetyl moiety during Orn formation (cyclic pathway). A putative N-acetylornithine deacetylase (NAOD; EC 3.5.1.16), which participates in the "linear" pathway for Orn synthesis in some organisms, was also identified. Previous biochemical studies have indicated that allosteric regulation of the first and, especially, the second steps in Orn synthesis (NAGS; N-acetylglutamate kinase (NAGK), EC 2.7.2.8) by the Arg end-product are the major sites of metabolic control of the pathway in organisms using the cyclic pathway. Gene expression profiling for pathway enzymes further suggests that NAGS, NAGK, NAOGAcT and NAOD are coordinately regulated in response to changes in Arg demand during plant growth and development. Synthesis of Arg from Orn is further coordinated with pyrimidine nucleotide synthesis, at the level of allocation of the common carbamoyl-P intermediate.     

Effects of salt and osmotic stresses on free polyamine content and expression of polyamine biosynthetic genes in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Grape (Vitis vinifera L.) seedlings grown in vitro were treated with either 200 mM NaCl or 350 mM mannitol for 7 d. Both salinity and osmotic stress caused significant increase in electrolyte leakage. From the three commonly occurring free polyamines (PA), only conspicuous accumulation of putrescine was found in the NaCl-treated seedlings. Four PA biosynthetic genes encoding arginine decarboxylase (pVvADC), S-adenosylmethionine decarboxylase (pVvSAMDC), spermidine synthase (pVvSPDS) and spermine synthase (pVvSPMS) were successfully isolated. While induction of pVvADC was observed from the 1^st day of salt treatment, pVvSAMDC and pVvSPMS were induced only at late stage of stress. As for expression levels of genes in the mannitol-treated seedling, either temporary (pVvADC at day 1) or late (pVvSPMS at days 5 and 7) induction was observed.     

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [¹⁴C]Arg, [¹⁴C]Orn, and [¹⁴C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [¹⁴C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.     

Influence of arginine,ornithine, DFMO and polyamines on division of the generative nucleus in cultured pollen tubes of <Emphasis Type="Italic">Aechmea fasciata</Emphasis> (Bromeliaceae)

During in vitro pollen tube growth of Aechmea fasciata the second pollen mitosis (PM II) that produces two sperm cells was influenced by exogenous amino acids. Arginine (Arg) as single amino acid was the limiting factor for the second mitosis of the generative nucleus and thus the formation of sperm cells in cultured pollen tubes of A. fasciata. The involvement of Arg was probably related to protein synthesis. The need for Arg was not related to polyamine (PA) biosynthesis, since PA added to the germination medium were unfavourable for sperm cell production. Both ornithine (Orn) and difluoromethylornithine (DFMO) inhibited the second mitosis in cultured pollen tubes of A. fasciata. The addition of Arg during the first 2 h of pollen germination was necessary to establish the division of the generative nucleus 6 h later.     

Molecular Phylogenies and Evolution of crt Genes in Algae

ABSTRACT

The carotenoids constitute the most widespread class of pigments in nature. Most previous work has concentrated on the identification and characterization of their chemical physical properties and bioavailability. In recent years, significant amounts of research have been conducted in an attempt to analyze the genes and the molecular regulation of the genes involved in the biosynthesis of carotenoids. However, it is important not to lose sight of the early evolution of carotenoid biosynthesis. One of the major obstacles in understanding the evolution of the respective enzymes and their patterns of selection is a lack of a well-supported phylogenic analysis. In the present research, a major long-term objective was to provide a clearer picture of the evolutionary history of genes, together with an evaluation of the patterns of selection in algae. These phylogenies will be important in studies characterizing the evolution of algae. The gene sequences of the enzymes involved in the major steps of the carotenoid biosynthetic pathway in algae (cyanobacteria, rhofophyta, chlorophyta) have been analyzed. Phylogenetic relationships among protein-coding DNA sequences were reconstructed by neighbor-joining (NJ) analysis for the respective carotenoid biosynthetic pathway genes (crt) in algae. The analysis also contains an estimation of the rate of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N), synonymous nucleotide substitution per synonymous site (d_S), and the ratio of nonsynonmous (d_N/d_S) for the test of selection patterns. The phylogenetic trees show that the taxa of some genera have a closer evolutionary relationship with other genera in some gene sequences, which suggests a common ancient origin and that lateral gene transfer has occurred among unrelated genera. The d_N values of crt genes in the early pathway are relatively low, while those of the following steps are slightly higher, while the d_N values of crt genes in chlorophyta are higher than those in cyanobacteria. Most of the d_N/d_S values exceed 1. The phylogenetic analysis revealed that lateral gene transfer may have taken place across algal genomes and the d_N values suggest that most of the early crt genes are well conserved compared to the later crt genes. Furthermore, d_N values also revealed that the crt genes of chlorophyta are more evolutionary than cyanobacteria. The amino acids' changes are mostly adaptive evolution under the influence of positive diversity selection.