Juan Francisco Jiménez-Bremont

Proline accumulation in two bean cultivars under salt stress and the effect of polyamines and ornithine

Proline accumulation in two different bean (Phaseolus vulgaris L.) cultivars, one drought-sensitive (Canario 60) and one drought-resistant (Pinto Villa) was investigated. Both tolerated salt concentrations up to 150 mM NaCl, but the sensitive Canario 60 did not survive at 400 mM NaCl. In response to salt stress, both cvs. accumulated proline in all the analyzed tissues, the lowest contents were detected in roots. Pinto Villa accumulated higher proline concentrations than Canario 60 only at 400 mM NaCl. The addition of polyamines or ornithine increased proline content in plant tissues without stress, while they decreased it under salt stress.

Effect of salt stress on the regulation of maize (Zea mays L.) genes involved in polyamine biosynthesis

A cDNA for spermidine synthase (SPDS), which converts putrescine to the higher polyamine spermidine using decarboxylated S-adenosylmethionine as a cofactor, was isolated from Zea mays leaves (Zmspds2A). Comparison of the deduced amino acid sequence revealed a high homology (81.9%) with Oryza sativa SPDS2. RT-PCR analyses showed that Zmspds2A was equally expressed in leaves, stem and roots. In contrast, transcripts of other genes related to polyamine biosynthesis (Zmodc, adc and samdc) showed tissue-specific regulation. The effect of salt stress on the expression of all these genes in maize leaves exposed to NaCl solutions of different concentrations was analysed. Our results showed that only Zmodc and Zmspds2A were up-regulated by salt stress; whereas the other two genes were barely affected by this treatment. In addition to Zmspds2A, a second transcript encoding a maize spermidine synthase (Zmspds2B) that also became up-regulated by salt stress, was identified. Comparison of partial cDNA sequences of transcripts Zmspds2A and Zmspds2B with the corresponding genomic DNA region revealed the existence of alternative splicing mechanism, opening a new aspect in plant polyamine biosynthesis modulation under abiotic stress.

Polyamine metabolism in maize tumors induced by Ustilago maydis

Alterations occurring in polyamine metabolism of maize in tumors formed during the interaction with the biotrophic pathogenic fungus Ustilago maydis were analyzed. During the process, a striking increase in maize polyamine biosynthesis, mainly free and conjugated putrescine occurred in the tumors induced by the fungus, and in the neighbor plant tissues. This increase correlated with an activation mainly of Adc, Samdc1, Zmsamdc2 and Zmsamdc3, but not of Zmodc, Zmspds1 and Zmspds2 genes, and an elevation in arginine decarboxylase activity, confirming a predominant role of this enzyme in the process. Evidences for a possible contribution of spermidine and spermine degradation by polyamine oxidase activity, probably related to cell wall stiffening or lignification during tumor growth, were also obtained. It is suggested that polyamines, mainly putrescine, might play an active role in the pathosystem maize-U. maydis.

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.

Periplasmic expression and recovery of human interferon gamma in Escherichia coli

A synthetic human interferon gamma (hIFN-gamma) gene was fused to SP1 and SP3, two Sec-dependent artificial signal peptides to transport the hIFN-gamma to the periplasm of Escherichia coli BL21-SI. The processing efficiency of both SP1-hIFN-gamma and SP3-hIFN-gamma was dependent on the culture medium as well as the post-induction temperature. Both precursors were processed completely when cells were cultured using minimal medium and a post-induction temperature of 32.5 degrees C, and only the processed hIFN-gamma was detected. The SP3 signal peptide was more efficient than SP1 for the secretion of hIFN-gamma. Sixty percent of the total hIFN-gamma was secreted to the periplasm using the SP3 signal peptide and a post-induction temperature of 20 degrees C. Using Tris-sucrose-dithiothreitol (TSD) hypertonic buffer, the periplasmic soluble hINF-gamma was recovered with a purity of 85%.

The Epl1 and Sm1 proteins from Trichoderma atroviride and Trichoderma virens differentially modulate systemic disease resistance against different life style pathogens in Solanum lycopersicum.

Fungi belonging to the genus Trichoderma, commonly found in soil or colonizing plant roots, exert beneficial effects on plants, including the promotion of growth and the induction of resistance to disease. T. virens and T. atroviride secrete the proteins Sm1 and Epl1, respectively, which elicit local and systemic disease resistance in plants. In this work, we show that these fungi promote growth in tomato (Solanum lycopersicum) plants. T. virens was more effective than T. atroviride in promoting biomass gain, and both fungi were capable of inducing systemic protection in tomato against Alternaria solani, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst DC3000). Deletion (KO) of epl1 in T. atroviride resulted in diminished systemic protection against A. solani and B. cinerea, whereas the T. virens sm1 KO strain was less effective in protecting tomato against Pst DC3000 and B. cinerea. Importantly, overexpression (OE) of epl1 and sm1 led to an increase in disease resistance against all tested pathogens. Although the Trichoderma WT strains induced both systemic acquired resistance (SAR)- and induced systemic resistance (ISR)-related genes in tomato, inoculation of plants with OE and KO strains revealed that Epl1 and Sm1 play a minor role in the induction of these genes. However, we found that Epl1 and Sm1 induce the expression of a peroxidase and an α-dioxygenase encoding genes, respectively, which could be important for tomato protection by Trichoderma spp. Altogether, these observations indicate that colonization by beneficial and/or infection by pathogenic microorganisms dictates many of the outcomes in plants, which are more complex than previously thought.

Further evidence from the effect of fungi on breaking Opuntia seed dormancy.

Recently, we found that fungi are involved in breaking seed dormancy of Opuntia streptacantha, and that the effect of fungi on seeds is species-specific. However, the effect of fungi on seed germination from other Opuntia spp has not been evaluated. Thus, we evaluated the effect of four fungal species (Penicillium chrysogenum, Phoma sp., Trichoderma harzianum, Trichoderma koningii) on the germination of Opuntia leucotricha, an abundant species in the Chihuahuan Desert, Mexico. We found that seeds inoculated with the four fungal species had higher germination than control seeds. Trichoderma spp. were the most effective. Our results strongly indicate that fungi are involved in breaking seed dormancy of O. leucotricha. Thus, we suggest that these fungi could promote seed germination from other Opuntia species.

Genomic organization of plant aminopropyl transferases

Aminopropyl transferases like spermidine synthase (SPDS; EC 2.5.1.16), spermine synthase and thermospermine synthase (SPMS, tSPMS; EC 2.5.1.22) belong to a class of widely distributed enzymes that use decarboxylated S-adenosylmethionine as an aminopropyl donor and putrescine or spermidine as an amino acceptor to form in that order spermidine, spermine or thermospermine. We describe the analysis of plant genomic sequences encoding SPDS, SPMS, tSPMS and PMT (putrescine N-methyltransferase; EC 2.1.1.53). Genome organization (including exon size, gain and loss, as well as intron number, size, loss, retention, placement and phase, and the presence of transposons) of plant aminopropyl transferase genes were compared between the genomic sequences of SPDS, SPMS and tSPMS from Zea mays, Oryza sativa, Malus x domestica, Populus trichocarpa, Arabidopsis thaliana and Physcomitrella patens. In addition, the genomic organization of plant PMT genes, proposed to be derived from SPDS during the evolution of alkaloid metabolism, is illustrated. Herein, a particular conservation and arrangement of exon and intron sequences between plant SPDS, SPMS and PMT genes that clearly differs with that of ACL5 genes, is shown. The possible acquisition of the plant SPMS exon II and, in particular exon XI in the monocot SPMS genes, is a remarkable feature that allows their differentiation from SPDS genes. In accordance with our in silico analysis, functional complementation experiments of the maize ZmSPMS1 enzyme (previously considered to be SPDS) in yeast demonstrated its spermine synthase activity. Another significant aspect is the conservation of intron sequences among SPDS and PMT paralogs. In addition the existence of microsynteny among some SPDS paralogs, especially in P. trichocarpa and A. thaliana, supports duplication events of plant SPDS genes. Based in our analysis, we hypothesize that SPMS genes appeared with the divergence of vascular plants by a processes of gene duplication and the acquisition of unique exons of as-yet unknown origin.

uORF, a regulatory mechanism of the Arabidopsis polyamine oxidase 2

The translational efficiency of an mRNA can be modulated by elements located in the 5′-untranslated region. The flavin-containing polyamine oxidases catabolize oxidative deamination of spermidine and spermine, thus contributing to polyamine homeostasis as well as diverse biological processes through their reaction products. In this study, we characterized the uORF of AtPAO2 gene using the GUS reporter gene. Transgenic lines harboring the native AtPAO2 promoter or the constitutive CaMV 35S promoter show that the uORF negatively affects GUS expression. Exogenous applications of PAs positively modulate GUS expression, thus alleviating the negative effect of AtPAO2 uORF, while treatments with MGBG inhibitor show an opposite effect. Our data suggest that AtPAO2 uORF regulatory mechanism is modulated by polyamines. In addition, we present a comparative in silico study of the uORFs identified in several plant transcripts encoding polyamine oxidases in both mono- and dicotyledonous plants as well as in the Bryophyte Physcomitrella patens. The polyamine oxidase uORF-encoded peptides are conserved among families and share conserved features such as their position, length, and amino acid sequence. Our findings provide new insights into the regulatory mechanism of polyamine oxidase genes and encourage further exploration to assess the biological significance of uORFs in the polyamine catabolic pathway.

A dehydrin-dehydrin interaction: the case of SK3 from Opuntia streptacantha

Dehydrins belongs to a large group of highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins. It is well known that dehydrins are intrinsically disordered plant proteins that accumulate during the late stages of embryogenesis and in response to abiotic stresses; however, the molecular mechanisms by which their functions are carried out are still unclear. We have previously reported that transgenic Arabidopsis plants overexpressing an Opuntia streptacantha SK3 dehydrin (OpsDHN1) show enhanced tolerance to freezing stress. Herein, we show using a split-ubiquitin yeast two-hybrid system that OpsDHN1 dimerizes. We found that the deletion of regions containing K-segments and the histidine-rich region in the OpsDHN1 protein affects dimer formation. Not surprisingly, in silico protein sequence analysis suggests that OpsDHN1 is an intrinsically disordered protein, an observation that was confirmed by circular dichroism and gel filtration of the recombinantly expressed protein. The addition of zinc triggered the association of recombinantly expressed OpsDHN1 protein, likely through its histidine-rich motif. These data brings new insights about the molecular mechanism of the OpsDHN1 SK3-dehydrin.