Translational and post-translational regulation of polyamine metabolic enzymes in plants.

Polyamines are small organic and basic polycations that perform essential regulatory functions in all living organisms. Fluctuations in polyamine content have been observed to occur during growth, development and under stress conditions, implying that polyamines play pivotal roles in diverse cellular and physiological processes. To achieve polyamine homeostasis, the entire metabolic pathway is subjected to a fine-tuned regulation of its biosynthetic and catabolic genes and enzymes. In this review, we describe and discuss the most important mechanisms implicated in the translational and post-translational regulation of polyamine metabolic enzymes in plants. At the translational level, we emphasize the role of polyamines in the modulation of upstream open reading frame (uORF) activities that control the translation of polyamine biosynthetic and catabolic mRNAs. At the post-translational level, different aspects of the regulation of polyamine metabolic proteins are depicted, such as the proteolytic activation of enzyme precursors, the importance of dimerization in protein stability as well as in protein intracellular localization.

Retention of a new-defined intron changes pharmacology and kinetics of the full-length P2X2 receptor found in myenteric neurons of the guinea pig.

P2X2 plays an important role in ATP signaling in guinea pig myenteric plexus. Here, we cloned and characterized three P2X2 isoforms expressed in myenteric neurons. RT/PCR was used to amplify the cDNA of P2X2 variants. These were expressed in Xenopus oocytes, and nucleotide-induced membrane currents were recorded with the two-electrode voltage clamp technique. Three P2X2 cDNAs were identified in myenteric single neurons, named P2X2-1, P2X2-2 and P2X2-4. Based on the analysis of the structural organization of these variants we predicted that P2X2-2 is the fully processed variant, which lead us to propose a new exon-intron arrangement of P2X2 receptor gene with 12 exons and 11 introns. In agreement with this new model, the intron 11 is retained in P2X2-1 and P2X2-4 variants by alternative splicing. Expression of P2X2-1, P2X2-2 and P2X2-4 were found in 92, 42 and 37%, respectively, out of 40 analyzed single neurons. P2X2-4 does not form functional channels, and homomeric channels formed by P2X2-1 and P2X2-2 have different pharmacological profile. Thus, the former receptor is more sensitive to ATP, BzATP, and PPADS, whereas, suramin inhibited both receptors in a biphasic- and monophasic-manner, respectively. α,ß-meATP has very low efficacy on either channel. Furthermore, ionic currents mediated by P2X2-1 have slower desensitization than P2X2-2. These results indicate that P2X2-1 was the most common P2X2 transcript in myenteric neurons and displays significant phenotypical changes implicating that retention of the intron 11 plays a major role in ATP signaling in the intestinal myenteric plexus.

Functional characterization of an acidic SK(3) dehydrin isolated from an Opuntia streptacantha cDNA library.

Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.

Differential expression of Phaseolus vulgaris genes induced during the interaction with Rhizoctonia solani.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption; however, bean production is affected by several diseases such as Rhizoctonia root rot. Few bean cultivars have been identified that effectively resist the attack of this fungus. Herein, we used the P. vulgaris Pv-2094 landrace, which is less susceptible to Rhizoctonia root rot, for the construction of a suppressive subtractive hybridization cDNA library in order to isolate plant defense-related genes. Total RNAs obtained after 8 and 16 h from inoculated and non-inoculated roots with R. solani Kühn, were used as the source of the "tester" and the "driver" samples, respectively. A total of 136 unigenes were obtained and classified into 12 functional categories. Six unigenes were selected to analyze for differential expression by qRT-PCR, including a receptor-like kinase (PvRK20-1), an acid phosphatase associated to defense (PA), a pathogenesis related protein (PR1), an ethylene responsive factor (ERF), a polygalacturonase inhibitor protein (PGIP), and an alpha-dioxygenase (α-DOX). These genes were found to be differentially expressed in a time-dependent manner in bean roots during the interaction with R. solani. Data generated from this study will contribute to the understanding of the molecular mechanisms associated with plant defense against root rot in common bean.

Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae).

Seeds of Opuntia spp. have physiological dormancy; they need a period of after-ripening to break dormancy, and the embryos have low growth potential. We evaluated the combined effects of seed age and presence of fungi on the testa on germination of Opuntia streptacantha, an abundant species in the Chihuahuan Desert (Mexico), assuming that older seeds have broken seed dormancy and fungi can reduce mechanical resistance to germination. In a preliminary experiment, we found no germination of 9-year-old (1998) and freshly collected (2007) seeds. However, we obtained 67% and 27% germination from 9-year-old and fresh non-sterilized seeds, respectively, and found fungi growing on the testa of all germinated seeds. Two fungal strains were isolated and identified using ribosomal internal transcribed spacer (ITS) sequence analysis: Penicillium chrysogenum and Phoma sp. In a second experiment, we inoculated seeds with strains of P. chrysogenum and Phoma sp., as well as Trichoderma koningii and binucleate Rhizoctonia (Gto17S2), to evaluate their ability to break seed dormancy. Seeds inoculated with P. chrysogenum, Phoma sp. and T. koningii had higher germination than controls for both seed ages, but germination was higher in older seeds. Scanning electron microscopy showed that these fungi eroded the funiculus, reducing its resistance. Binucleate Rhizoctonia did not lead to germination and controls had almost no germination. Our results strongly indicate that fungi are involved in breaking seed dormancy of O. streptacantha, and that the effect of fungi on seeds is species-specific.

Disruption of gene YlODC reveals absolute requirement of polyamines for mycelial development in Yarrowia lipolytica.

Polyamines are required for cellular growth and differentiation. In mammals and fungi they are synthesized via a pathway involving ornithine decarboxylase (ODC), which transforms ornithine into putrescine. We have cloned and disrupted the gene coding for ODC in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus. Substrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence. A single transcript 1.7 kb in size was identified by Northern hybridization, and confirmed by rapid amplification of cDNA ends (RACE). Null mutants lacked ODC activity and behaved as polyamine auxotrophs. When low levels of polyamines were supplied to the null mutant, only yeast-like, but not mycelial growth was sustained. This phenomenon was confirmed by introduction of the YlODC gene under the control of an inducible promoter into the null mutant.