Verónica Lira-Ruan

Synthesis of hemoglobins in rice (Oryza sativa var. Jackson) plants growing in normal and stress conditions

In rice (Oryza sativa var. Jackson) at least three copies of hemoglobin (hb) gene exist. Rice hb1 and hb2 genes are differentially expressed in roots and leaves from mature plants. We used polyclonal antibodies raised to recombinant rice Hb1 and Western blotting to analyze the synthesis of Hbs in rice plants growing under normal or stress conditions. Results showed that rice Hbs are synthesized in coleoptiles, seminal roots and embryos from seeds germinated for 6 days, and also in leaves and roots from plants 2-14 weeks after germination. Analysis of Hb synthesis in stressed rice showed that: (i) level of Hbs was higher in etiolated than control plants, (ii) level of Hbs increased in roots from flooded rice, and (iii) level of Hbs did not change under oxidative (H(2)O(2)), nitrosative (SNP) and hormonal (2,4-D) stresses. These results suggest that the effect of light withdrawal in etiolated leaves and O(2)-limiting conditions in flooded roots, but not oxidative, nitrosative and hormonal stresses, modulate the synthesis of rice Hbs.

In silico analysis of a flavohemoglobin from Sinorhizobium meliloti strain 1021

Hemoglobins (Hbs) have been characterized from a wide variety of eubacteria, but not from nitrogen-fixing rhizobia. Our search for Hb-like sequences in the Sinorhizobium meliloti genome revealed that a gene coding for a flavohemoglobin (fHb) exists in S. meliloti (SmfHb). Computer analysis showed that SmfHb and Alcaligenes eutrophus fHb are highly similar and could fold into the same tertiary structure. A FNR-like box was detected upstream of the smfhb gene and mapping analysis revealed that the smfhb gene is flanked by nos and fix genes. These observations suggest that smjhb is regulated by the concentration of O2 and that SmfHb functions in some aspects of nitrogen metabolism.

In silico characterization of a nitrate reductase gene family and analysis of the predicted proteins from the moss Physcomitrella patens.

Assimilatory nitrate reductase (NR; EC 1.7.1.1-3) catalyzes the reduction of nitrate to nitrite. This enzyme has a conserved structure common to fungi, algae and plants. However, some differences in the amino acid sequence between plant and algal NR suggest that the activity regulation mechanisms have changed during plant evolution. Since only NRs from angiosperms have been studied, the search and analysis of NR genes and proteins from the moss Physcomitrella patens, a basal land plant, was performed to widen the knowledge of land plant NR structure. A family of three nr genes, named ppnia1;1, ppnia1;2 and ppnia2, was localized in the P. patens genome. The predicted proteins are canonical NRs with the conserved domains Molybdene-Cytochorme b –Cytochrome b reductase and possess 20 amino acid residues important for the enzymatic function conserved in plant and algal NRs. Interestingly, moss NRs lack a consensus sequence, common to angiosperm NRs, that is a target for posttranslational regulation. A phylogenetic tree with embryophyte and green algae NR sequences was constructed and P. patens NRs localized at the base of embryophyte NR evolution. The data presented here suggest that bryophytes and vascular plants have different systems to regulate NR activity.

The nitric oxide production in the moss Physcomitrella patens is mediated by nitrate reductase.

During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.

Expression of non-symbiotic hemoglobin 1 and 2 genes in rice (Oryza sativa) embryonic organs

Rice (Oryza sativa) contains five copies of the non-symbiotic hemoglobin (hb) gene, namely hb1 to hb5. Previous analysis by RT-PCR revealed that rice hb1 expresses in roots and leaves and hb2 expresses in leaves. However, it is not known whether or not hb1 and hb2 express in rice embryonic organs. Here, we report the expression of hb1 and hb2 genes in rice embryonic organs using RT-PCR and specific oligos for Hb1 and Hb2. Our results indicate that hb1 and hb2 genes express in embryonic organs in rice growing under normal conditions. Specifically, hb1 expresses in rice embryos and seminal roots, and hb2 expresses in embryos, coleoptiles and seminal roots. These observations suggest that Hb1 and Hb2 coexist and function in rice embryonic organs.

Identification of a novel carbohydrate esterase from Bjerkandera adusta: structural and function predictions through bioinformatics analysis and molecular modeling.

A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three‐dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p‐nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg−1 protein on 2‐naphthyl acetate. No activity was detected on p‐nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N‐acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin. Proteins 2015; 83:533–546.

Heuristic aspect of the lateral root initiation index: A case study of the role of nitric oxide in root branching.

Premise of the study: Lateral root (LR) initiation (LRI) is a central process in root branching. Based on LR and/or LR primordium densities, it has been shown that nitric oxide (NO) promotes LRI. However, because NO inhibits primary root growth, we hypothesized that NO may have an opposite effect if the analysis is performed on a cellular basis. Using a previously proposed parameter, the LRI index (which measures how many LRI events take place along a root portion equivalent to the length of a single file of 100 cortical cells of average length), we addressed this hypothesis and illustrate here that the LRI index provides a researcher with a tool to uncover hidden but important information about root initiation. Methods and Results: Arabidopsis thaliana roots were treated with an NO donor (sodium nitroprusside [SNP]) and/or an NO scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide [cPTIO]). LRI was analyzed separately in the root portions formed before and during the treatment. In the latter, SNP caused root growth inhibition and an increase in the LR density accompanied by a decrease in LRI index, indicating overall inhibitory outcome of the NO donor on branching. The inhibitory effect of SNP was reversed by cPTIO, showing the NO-specific action of SNP on LRI. Conclusions: Analysis of the LRI index permits the discovery of otherwise unknown modes of action of a substance on the root system formation. NO has a dual action on root branching, slightly promoting it in the root portion formed before the treatment and strongly inhibiting it in the root portion formed during the treatment.

Isolation and characterization of endophytes from nodules of Mimosa pudica with biotechnological potential

Legumes establish symbiotic relationships with different microorganisms, which could function as plant growth promotion microorganisms (PGPM). The finding of new PGPM strains is important to increase plant production avoiding or diminishing the use of industrial fertilizers. Thus, in this work we evaluated the plant growth promotion traits of ten strains isolated from Mimosa pudica root nodules. According to the 16S rDNA sequence, the microorganisms were identified as Enterobacter sp. and Serratia sp. To the best of our knowledge this is the first report describing and endophytic interaction between Mimosa pudica and Enterobacter sp. These strains have some plant growth promoting traits such as phosphate solubilization, auxin production and cellulase and chitinase activity. Strains identified as Serratia sp. inhibited the growth of the phytopathogenic fungi Fusarium sp., and Alternaria solani and the oomycete Phytophthora capsici. According to their biochemical characteristics, three strains were selected to test their plant growth promoting activity in a medium with an insoluble phosphate source. These bacteria show low specificity for their hosts as endophytes, since they were able to colonize two very different legumes: Phaseolus vulgaris and M. pudica. Seedlings of P. vulgaris were inoculated and grown for fifteen days. Enterobacter sp. NOD1 and NOD10, promoted growth as reflected by an increase in shoot height as well as an increase in the size and emergence of the first two trifolia. We could localize NOD5 as an endophyte in roots in P. vulgaris by transforming the strain with a Green Fluorescent Protein carrying plasmid. Experiments of co-inoculation with different Rhizobium etli strains allowed us to discard that NOD5 can fix nitrogen in the nodules formed by a R. etli Fix- strain. The isolates described in this work show biotechnological potential for plant growth promoting activity and production of indoleacetic acid and siderophores.