Evidence for in vivo interactions between dehydrins and the aquaporin AtPIP2B.

Plants have developed mechanisms that allow them to tolerate different abiotic stresses. Among these mechanisms, the accumulation of specific proteins such as dehydrins (DHNs) and aquaporins (AQPs) can protect other proteins from damage during dehydration and may allow the control of water loss, respectively. Although both types of proteins are involved in plant protection against dehydration stress, a direct interaction between them has not been explored. A previous screen to identify potential OpsDHN1 protein interactions revealed an aquaporin as a possible candidate. Here, we used the Bimolecular Fluorescence Complementation (BiFC) approach to investigate the direct interaction of the cactus OpsDHN1 protein with the Arabidopsis plasma membrane PIP family aquaporin AtPIP2B (At2G37170). Since AtPIP2B is a membrane protein and OpsDHN1 is a cytosolic protein that may be peripherally associated with membranes, we propose that OpsDHN1/AtPIP2B interaction takes place at cellular membranes. Furthermore, we also demonstrate the interaction of AtPIP2B with the three Arabidopsis dehydrins COR47 (AT1G20440), ERD10 (At1g20450), and RAB18 (At5g66400).

Hetero- and homodimerization of Arabidopsis thaliana arginine decarboxylase AtADC1 and AtADC2.

The arginine decarboxylase enzyme (ADC) carries out the production of agmatine from arginine, which is the precursor of the first polyamine (PA) known as putrescine; subsequently, putrescine is turned into the higher PAs, spermidine and spermine. In Arabidopsis thaliana PA production occurs only from arginine and this step is initiated by two ADC paralogues, AtADC1 and AtADC2. PA production is essential for A. thaliana life cycle. Here, we analyzed the sub-cellular localization of AtADC1 and AtADC2 enzymes through GFP translational fusions. Our data revealed that the A. thaliana arginine decarboxylase enzymes exhibit a dual sub-cellular localization both in the cytosol and chloroplast. Moreover, we examined the protein dimer assembly using a Bimolecular Fluorescence Complementation (BiFC) approach, which showed that AtADC1 and AtADC2 proteins were able to form homodimers in the cytosol and chloroplast. Interestingly, we found the formation of AtADC1/AtADC2 heterodimers with similar sub-cellular localization than homodimers. This study reveals that both ADC proteins are located in the same cell compartments, and they are able to form protein interaction complexes with each other.

Heteromeric channels with different phenotypes are generated when coexpressing two P2X2 receptor isoforms.

To investigate if channels with different stoichiometry are formed from P2X2 receptor isoforms during their heterologous co-expression. The two-electrode voltage-clamp technique was used to measured ATP induced currents in Xenopus laevis oocytes. We used a mutant (P2X2-2bm) because its ATP sensitivity is lower than P2X2-2b receptors, which highlights the differences with its splice variant P2X2-1a.Currents through homomeric channels had significantly different Hill coefficients. P2XR are trimeric proteins with three agonist binding sites; therefore, only two homomeric and two heteromeric stoichiometries are possible when both P2X2 isoforms are coexpressed, the heteromeric channels might be formed by: i) 2(P2X2-1a)+1(P2X2-2bm); or ii) 1(P2X2-1a)+2(P2X2-2bm). Because P2X2 channels open when two binding sites are occupied, these stoichiometries are expected to have different ATP sensitivities. Thus, co-expressing both P2X2 isoforms, two oocyte populations were distinguished based on their sensitivity to ATP and Hill coefficients. For the first population (P2X2-1a like), the ATP EC50 and the Hill coefficient were not different than those of homomeric P2X2-1a channels similarly, for the second population (P2X2-2bm like), these variables were also not different than for those of homomeric P2X2-2bm channels. Various findings indicate that homomeric channel expression is not responsible for such differences. Our observations indicate that two heteromeric channels can be assembled from two P2X2 receptor isoforms. Our data support a current model, according to which, ATP activation of two subunits can open P2X2 channel. However, PPADS appears to bind to all three subunits in order to inhibit ATP effects on P2X2 receptors.

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.

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%.

Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear.

Proline (Pro) is one of the most accumulated osmolytes in salinity and water deficit conditions in plants. In the present study, we measured the Pro content, the activity and the expression level of delta 1-pyrroline-5-carboxylate synthetase (P5CS: gamma-glutamyl kinase, EC 2.7.2.11 and glutamate-5-semialdehyde dehydrogenase, EC 1.2.1.41), a key regulatory enzyme involved in the biosynthesis of Pro, in cactus pear (Opuntia streptacantha) subjected to 6, 9 and 11 days of salt stress. Treatment with NaCl of O. streptacantha young plants resulted in a decrease in the cladode thickness and root length, and in a significant and gradual accumulation of Pro in young cladodes, in a time- and concentration-dependent manner. P5CS activity, studied as gamma-glutamyl kinase, was reduced at all times as a consequence of salt treatment, except at the sixth day at 75 and 150mM of NaCl, where a slight increase was observed. We isolated an open reading frame (ORF) fragment of p5cs gene. The deduced amino acid sequence of the P5CS protein exhibited 90.4% of identity with the P5CS protein from Mesembryanthemum crystallinum. RT-PCR analysis revealed that the Osp5cs gene of O. streptacantha was induced by salt stress at 9 and 11 days of treatment. Furthermore, ABA-induced Osp5cs gene expression was observed in cladodes of cactus pear young plants. We observed an evident correlation between the transcript up-regulation and the Pro accumulation under salt stress; however, these results do not parallel with the changes in P5CS enzymatic activity. This Pro accumulation might function as an osmolyte for the intracellular osmotic adjustment and might be playing a critical role in protecting photosynthetic activity in O. streptacantha plants under salt stress.

Modulation of spermidine and spermine levels in maize seedlings subjected to long-term salt stress.

Salinity is one of the major abiotic stresses affecting plant agriculture worldwide. Polyamines, a group of aliphatic amines, are known to accumulate under salt stress conditions in different plant systems, resulting in presumed protective effects, acting as free radical scavengers, stabilizing cellular membranes and maintaining cellular ionic balance under these conditions. In the present study, we measured the polyamine content in maize leaves of semi-hydroponically grown seedlings subjected to 1 and 7 days of salt stress. We observed that the maize plants tend to maintain or accumulate the levels of spermidine and spermine, while putrescine levels fluctuate depending on the NaCl concentration. The effect of salt stress on the expression of the main genes involved in polyamine biosynthesis was also assessed. Our data show a time and NaCl dependent regulation of the Zmspds2 and Zmspds1 genes, suggesting that the former might be hyperosmotic responsive while the later NaCl responsive. Interestingly, the maize adc, Zmspds1 and Zmspds2 genes are regulated at the transcriptional level by the plant growth regulator abscisic acid. A connection between polyamine metabolism, abiotic stress and abscisic acid is discussed.

In vivo evidence for homo- and heterodimeric interactions of Arabidopsis thaliana dehydrins AtCOR47, AtERD10, and AtRAB18.

Dehydrins (DHNs) are intrinsically disordered proteins that play central roles in plant abiotic stress responses; however, how they work remains unclear. Herein, we report the in planta subcellular localization of Arabidopsis thaliana DHNs AtCOR47, AtERD10, and AtRAB18 through GFP translational fusions. To explore the dimerization ability of the Arabidopsis acidic DHNs AtCOR47 and AtERD10, we conducted an in planta DHN binding assay using the Bimolecular Fluorescence Complementation (BiFC) technique. Our analyses revealed homodimeric interactions for AtCOR47 and AtERD10; interestingly, heterodimeric associations also occurred with these DHNs, and these interactions were observed in the cytosol of tobacco cells. Furthermore, we evaluated whether Arabidopsis basic DHNs, such as AtRAB18, could also interact with itself and/or with AtCOR47 and AtERD10 in the BiFC system. Our data revealed homodimeric RAB18 complexes in the nucleus and cytosol, while heterodimeric associations between AtRAB18 and acidic DHNs occurred only in the cytosol. Finally, we demonstrated the presence of heterodimeric complexes among Arabidopsis AtCOR47, AtERD10, and AtRAB18 DHNs with their acidic ortholog the OpsDHN1 from Opuntia streptacantha; these heterodimeric interactions showed different subcellular distributions. Our results guide DHN research toward a new scenario where DHN/DHN oligomerization could be explored as a part of their molecular mechanism.

Environmental Growth Conditions of Trichoderma spp. Affects Indole Acetic Acid Derivatives, Volatile Organic Compounds, and Plant Growth Promotion.

Trichoderma species are soil-borne filamentous fungi widely utilized for their many plant health benefits, such as conferring improved growth, disease resistance and abiotic stress tolerance to their hosts. Many Trichoderma species are able to produce the auxin phytohormone indole-3-acetic acid (IAA), and its production has been suggested to promote root growth. Here we show that the production of IAA is strain dependent and diverse external stimuli are associated with its production. In in vitro assays, Arabidopsis primary root length was negatively affected by the interaction with some Trichoderma strains. In soil experiments, a continuum effect on plant growth was shown and this was also strain dependent. In plate assays, some strains of Trichoderma spp. inhibited the expression of the auxin reporter gene DR5 in Arabidopsis primary roots but not secondary roots. When Trichoderma spp. and A. thaliana were physically separated, enhancement of both shoot and root biomass, increased root production and chlorophyll content were observed, which strongly suggested that volatile production by the fungus influenced the parameters analyzed. Trichoderma strains T. virens Gv29.8, T. atroviride IMI206040, T. sp. "atroviride B" LU132, and T. asperellum LU1370 were demonstrated to promote plant growth through volatile production. However, contrasting differences were observed with LU1370 which had a negative effect on plant growth in soil but a positive effect in plate assays. Altogether our results suggest that the mechanisms and molecules involved in plant growth promotion by Trichoderma spp. are multivariable and are affected by the environmental conditions.

Simultaneous Silencing of Two Arginine Decarboxylase Genes Alters Development in Arabidopsis.

Polyamines (PAs) are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2) catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC). The generated transgenic lines (amiR:ADC-L1 and -L2) showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes.

Arabidopsis AtDjA3 Null Mutant Shows Increased Sensitivity to Abscisic Acid, Salt, and Osmotic Stress in Germination and Post-germination Stages.

DnaJ proteins are essential co-chaperones involved in abiotic and biotic stress responses. Arabidopsis AtDjA3 gene encodes a molecular co-chaperone of 420 amino acids, which belongs to the J-protein family. In this study, we report the functional characterization of the AtDjA3 gene using the Arabidopsis knockout line designated j3 and the 35S::AtDjA3 overexpression lines. Loss of AtDjA3 function was associated with small seed production. In fact, j3 mutant seeds showed a reduction of 24% in seed weight compared to Col-0 seeds. Expression analysis showed that the AtDjA3 gene was modulated in response to NaCl, glucose, and abscisic acid (ABA). The j3 line had increased sensitivity to NaCl and glucose treatments in the germination and cotyledon development in comparison to parental Col-0. Furthermore, the j3 mutant line exhibited higher ABA sensitivity in comparison to parental Col-0 and 35S::AtDjA3 overexpression lines. In addition, we examined the expression of ABI3 gene, which is a central regulator in ABA signaling, in j3 mutant and 35S::AtDjA3 overexpression lines. Under 5 µM ABA treatment at 24 h, j3 mutant seedlings displayed higher ABI3 expression, whereas in 35S::AtDjA3 overexpression lines, ABI3 gene expression was repressed. Taken together, these results demonstrate that the AtDjA3 gene is involved in seed development and abiotic stress tolerance.

Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif.

The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

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.

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.

Overexpression of AtGRDP2, a novel glycine-rich domain protein, accelerates plant growth and improves stress tolerance.

Proteins with glycine-rich signatures have been reported in a wide variety of organisms including plants, mammalians, fungi, and bacteria. Plant glycine-rich protein genes exhibit developmental and tissue-specific expression patterns. Herein, we present the characterization of the AtGRDP2 gene using Arabidopsis null and knockdown mutants and, Arabidopsis and lettuce over-expression lines. AtGRDP2 encodes a short glycine-rich domain protein, containing a DUF1399 domain and a putative RNA recognition motif (RRM). AtGRDP2 transcript is mainly expressed in Arabidopsis floral organs, and its deregulation in Arabidopsis Atgrdp2 mutants and 35S::AtGRDP2 over-expression lines produces alterations in development. The 35S::AtGRDP2 over-expression lines grow faster than the WT, while the Atgrdp2 mutants have a delay in growth and development. The over-expression lines accumulate higher levels of indole-3-acetic acid and, have alterations in the expression pattern of ARF6, ARF8, and miR167 regulators of floral development and auxin signaling. Under salt stress conditions, 35S::AtGRDP2 over-expression lines displayed higher tolerance and increased expression of stress marker genes. Likewise, transgenic lettuce plants over-expressing the AtGRDP2 gene manifest increased growth rate and early flowering time. Our data reveal an important role for AtGRDP2 in Arabidopsis development and stress response, and suggest a connection between AtGRDP2 and auxin signaling.

Physiological and molecular implications of plant polyamine metabolism during biotic interactions.

During ontogeny, plants interact with a wide variety of microorganisms. The association with mutualistic microbes results in benefits for the plant. By contrast, pathogens may cause a remarkable impairment of plant growth and development. Both types of plant-microbe interactions provoke notable changes in the polyamine (PA) metabolism of the host and/or the microbe, being each interaction a complex and dynamic process. It has been well documented that the levels of free and conjugated PAs undergo profound changes in plant tissues during the interaction with microorganisms. In general, this is correlated with a precise and coordinated regulation of PA biosynthetic and catabolic enzymes. Interestingly, some evidence suggests that the relative importance of these metabolic pathways may depend on the nature of the microorganism, a concept that stems from the fact that these amines mediate the activation of plant defense mechanisms. This effect is mediated mostly through PA oxidation, even though part of the response is activated by non-oxidized PAs. In the last years, a great deal of effort has been devoted to profile plant gene expression following microorganism recognition. In addition, the phenotypes of transgenic and mutant plants in PA metabolism genes have been assessed. In this review, we integrate the current knowledge on this field and analyze the possible roles of these amines during the interaction of plants with microbes.

Isolation of UmRrm75, a gene involved in dimorphism and virulence of Ustilago maydis.

Ustilago maydis displays dimorphic growth, alternating between a saprophytic haploid yeast form and a filamentous dikaryon, generated by mating of haploid cells and which is an obligate parasite. Induction of the dimorphic transition of haploid strains in vitro by change in ambient pH has been used to understand the mechanisms governing this differentiation process. In this study we used suppression subtractive hybridization to generate a cDNA library of U. maydis genes up-regulated in the filamentous form induced in vitro at acid pH. Expression analysis using quantitative RT-PCR showed that the induction of two unigenes identified in this library coincided with the establishment of filamentous growth in the acid pH medium. This expression pattern suggested that they were specifically associated to hyphal development rather than merely acid pH-induced genes. One of these genes, UmRrm75, encodes a protein containing three RNA recognition motifs and glycine-rich repeats and was selected for further study. The UmRrm75 gene contains 4 introns, and produces a splicing variant by a 3'-alternative splicing site within the third exon. Mutants deleted for UmRrm75 showed a slower growth rate than wild type strains in liquid and solid media, and their colonies showed a donut-like morphology on solid medium. Interestingly, although ΔUmRrm75 strains were not affected in filamentous growth induced by acid pH and oleic acid, they exhibited reduced mating, post-mating filamentous growth and virulence. Our data suggest that UmRrm75 is probably involved in cell growth, morphogenesis, and pathogenicity in U. maydis.

Galactose induces the expression of penicillin acylase under control of the lac promoter in recombinant Escherichia coli.

The expression of penicillin acylase (PA), cloned in the pPA102 plasmid under control of the wild-type lac promoter and using galactose as inducer in Escherichia coli JM101, JM103 and JM105 transformant cells, was analyzed. The E. coli JM101/pPA102 cultures attained the highest specific activity of PA. For large scale PA production based on E. coli JM101/pPA102 a culture media with galactose instead of isopropyl-thio-galactopyranoside as inducer would be as successful and less expensive.