Phylogenomic and Microsynteny Analysis Provides Evidence of Genome Arrangements of High-Affinity Nitrate Transporter Gene Families of Plants.

Nitrate transporter 2 (NRT2) and NRT3 or nitrate-assimilation-related 2 (NAR2) proteins families form a two-component, high-affinity nitrate transport system, which is essential for the acquisition of nitrate from soils with low N availability. An extensive phylogenomic analysis across land plants for these families has not been performed. In this study, we performed a microsynteny and orthology analysis on the NRT2 and NRT3 genes families across 132 plants (Sensu lato) to decipher their evolutionary history. We identified significant differences in the number of sequences per taxonomic group and different genomic contexts within the NRT2 family that might have contributed to N acquisition by the plants. We hypothesized that the greater losses of NRT2 sequences correlate with specialized ecological adaptations, such as aquatic, epiphytic, and carnivory lifestyles. We also detected expansion on the NRT2 family in specific lineages that could be a source of key innovations for colonizing contrasting niches in N availability. Microsyntenic analysis on NRT3 family showed a deep conservation on land plants, suggesting a high evolutionary constraint to preserve their function. Our study provides novel information that could be used as guide for functional characterization of these gene families across plant lineages.

Fibrillarin evolution through the Tree of Life: Comparative genomics and microsynteny network analyses provide new insights into the evolutionary history of Fibrillarin.

Fibrillarin (FIB), a methyltransferase essential for life in the vast majority of eukaryotes, is involved in methylation of rRNA required for proper ribosome assembly, as well as methylation of histone H2A of promoter regions of rRNA genes. RNA viral progression that affects both plants and animals requires FIB proteins. Despite the importance and high conservation of fibrillarins, there little is known about the evolutionary dynamics of this small gene family. We applied a phylogenomic microsynteny-network approach to elucidate the evolutionary history of FIB proteins across the Tree of Life. We identified 1063 non-redundant FIB sequences across 1049 completely sequenced genomes from Viruses, Bacteria, Archaea, and Eukarya. FIB is a highly conserved single-copy gene through Archaea and Eukarya lineages, except for plants, which have a gene family expansion due to paleopolyploidy and tandem duplications. We found a high conservation of the FIB genomic context during plant evolution. Surprisingly, FIB in mammals duplicated after the Eutheria split (e.g., ruminants, felines, primates) from therian mammals (e.g., marsupials) to form two main groups of sequences, the FIB and FIB-like groups. The FIB-like group transposed to another genomic context and remained syntenic in all the eutherian mammals. This transposition correlates with differences in the expression patterns of FIB-like proteins and with elevated Ks values potentially due to reduced evolutionary constraints of the duplicated copy. Our results point to a unique evolutionary event in mammals, between FIB and FIB-like genes, that led to non-redundant roles of the vital processes in which this protein is involved.

The SoNAP gene from sugarcane (Saccharum officinarum) encodes a senescence-associated NAC transcription factor involved in response to osmotic and salt stress.

Climate change has caused serious problems related to the productivity of agricultural crops directly affecting human well-being. Plants have evolved to produce molecular mechanisms in response to environmental stresses, such as transcription factors (TFs), to cope with abiotic stress. The NAC proteins constitute a plant-specific family of TFs involved in plant development processes and tolerance to biotic and abiotic stress. Sugarcane is a perennial grass that accumulates a large amount of sucrose and is a crucial agro-industry crop in tropical regions. Our previous transcriptome analyses on sugarcane that were exposed to drought conditions revealed significant increases in the expression of several NAC TFs through all of the time-point stress conditions. In this work, we characterize all previously detected sugarcane NAC genes, utilizing phylogenetics and expression analyses. Furthermore, we characterized a sugarcane NAC gene orthologous to the senescence-associated genes AtNAP and OsNAP via transient expression in tobacco calluses, from Arabidopsis and rice respectively, thus we named it the SoNAP gene. Transient localization assays on onion epidermal cells confirmed the nuclear localization of the SoNAP. Expression analysis showed that the SoNAP gene was induced by high salinity, drought, and abscisic acid treatments. The overexpression of the SoNAP gene in tobacco calluses caused a senescence associated phenotype. Overall, our results indicated that the SoNAP gene from sugarcane is transcriptionally induced under abiotic stress conditions and conserved the predicted senescence-associated functions when it was overexpressed in a heterologous plant model. This work provides key insights about the senescence mechanisms related to abiotic stress and it provides a benchmark for future work on the improvement of this economically important crop.

New insights into the phylogeny of the TMBIM superfamily across the tree of life: Comparative genomics and synteny networks reveal independent evolution of the BI and LFG families in plants.

The Transmembrane BAX Inhibitor Motif containing (TMBIM) superfamily, divided into BAX Inhibitor (BI) and Lifeguard (LFG) families, comprises a group of cytoprotective cell death regulators conserved in prokaryotes and eukaryotes. However, no research has focused on the evolution of this superfamily in plants. We identified 685 TMBIM proteins in 171 organisms from Archaea, Bacteria, and Eukarya, and provided a phylogenetic overview of the whole TMBIM superfamily. Then, we used orthology and synteny network analyses to further investigate the evolution and expansion of the BI and LFG families in 48 plants from diverse taxa. Plant BI family forms a single monophyletic group; however, monocot BI sequences transposed to another genomic context during evolution. Plant LFG family, which expanded trough whole genome and tandem duplications, is subdivided in LFG I, LFG IIA, and LFG IIB major phylogenetic groups, and retains synteny in angiosperms. Moreover, two orthologous groups (OGs) are shared between bryophytes and seed plants. Other several lineage-specific OGs are present in plants. This work clarifies the phylogenetic classification of the TMBIM superfamily across the three domains of life. Furthermore, it sheds new light on the evolution of the BI and LFG families in plants providing a benchmark for future research.

Novel Ribonuclease Activity Differs between Fibrillarins from Arabidopsis thaliana.

Fibrillarin is one of the most important nucleolar proteins that have been shown as essential for life. Fibrillarin localizes primarily at the periphery between fibrillar center and dense fibrillar component as well as in Cajal bodies. In most plants there are at least two different genes for fibrillarin. In Arabidopsis thaliana both genes show high level of expression in transcriptionally active cells. Here, we focus on two important differences between A. thaliana fibrillarins. First and most relevant is the enzymatic activity by AtFib2. The AtFib2 shows a novel ribonuclease activity that is not seen with AtFib1. Second is a difference in the ability to interact with phosphoinositides and phosphatidic acid between both proteins. We also show that the novel ribonuclease activity as well as the phospholipid binding region of fibrillarin is confine to the GAR domain. The ribonuclease activity of fibrillarin reveals in this study represents a new role for this protein in rRNA processing.

Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea.

Fibrillarin is a well conserved methyltransferase involved in several if not all of the more than 100 methylations sites in rRNA which are essential for proper ribosome function. It is mainly localized in the nucleoli and Cajal bodies inside the cell nucleus where it exerts most of its functions. In plants, fibrillarin binds directly the guide RNA together with Nop56, Nop58, and 15.5ka proteins to form a snoRNP complex that selects the sites to be methylated in pre-processing of ribosomal RNA. Recently, the yeast counterpart NOP1 was found to methylate histone H2A in the nucleolar regions. Here we show that plant fibrillarin can also methylate histone H2A. In Brassica floral meristem cells the methylated histone H2A is mainly localized in the nucleolus but unlike yeast or human cells it also localize in the periphery of the nucleus. In specialized transport cells the pattern is altered and it exhibits a more diffuse staining in the nucleus for methylated histone H2A as well as for fibrillarin. Here we also show that plant fibrillarin is capable of interacting with H2A and carry out its methylation in the rDNA promoter.

Antibacterial activity of defensin PaDef from avocado fruit (Persea americana var. drymifolia) expressed in endothelial cells against Escherichia coli and Staphylococcus aureus.

Antimicrobial therapy is a useful tool to control infectious diseases in general and rising antibiotic resistant microorganisms in particular. Alternative strategies are desirable, and antimicrobial peptides (AMP) represent attractive control agents. Mexican avocado (Persea americana var. drymifolia) is used in traditional medicine; however, the AMP production has not been reported in this plant. We obtained a cDNA library from avocado fruit and clone PaDef was identified, which has a cDNA (249 bp) encoding a protein (78 aa) homologous with plant defensins (>80%). We expressed the defensin PaDef cDNA (pBME3) in the bovine endothelial cell line BVE-E6E7. Polyclonal and clonal populations were obtained and their activity was evaluated against Escherichia coli, Staphylococcus aureus, and Candida albicans. E. coli viability was inhibited with 100 µg/mL of total protein from clones (>55%). Also, S. aureus viability was inhibited from 50 µg/mL total protein (27-38%) but was more evident at 100 µg/mL (52-65%). This inhibition was higher than the effect showed by polyclonal population (~23%). Finally, we did not detect activity against C. albicans. These results are the first report that shows antimicrobial activity of a defensin produced by avocado and suggest that this AMP could be used in the control of pathogens.

Analysis of expressed sequence tags (ESTs) from avocado seed (Persea americana var. drymifolia) reveals abundant expression of the gene encoding the antimicrobial peptide snakin.

Avocado is one of the most important fruits in the world. Avocado "native mexicano" (Persea americana var. drymifolia) seeds are widely used in the propagation of this plant and are the primary source of rootstocks globally for a variety of avocado cultivars, such as the Hass avocado. Here, we report the isolation of 5005 ESTs from the 5' ends of P. americana var. drymifolia seed cDNA clones representing 1584 possible unigenes. These avocado seed ESTs were compared with the avocado flower EST library, and we detected several genes that are expressed either in both tissues or only in the seed. The snakin gene, which encodes an element of the innate immune response in plants, was one of those most frequently found among the seed ESTs, and this suggests that it is abundantly expressed in the avocado seed. We expressed the snakin gene in a heterologous system, namely the bovine endothelial cell line BVE-E6E7. Conditioned media from transfected BVE-E6E7 cells showed antimicrobial activity against strains of Escherichia coli and Staphylococcus aureus. This is the first study of the function of the snakin gene in plant seed tissue, and our observations suggest that this gene might play a protective role in the avocado seed.

MAP kinase-like activity in transformed Catharanthus roseus hairy roots varies with culture conditions such as temperature and hypo-osmotic shock.

Mitogen activated protein (MAP) kinase-like activity was determined in extracts obtained from transformed Catharanthus roseus hairy roots by the ability to phosphorylate myelin basic protein (MBP). Both in solution and in gel kinase assays showed variation in activity, depending on root developmental stage. In gel kinase assays, using the extract soluble fraction, revealed a 56 kDa polypeptide with phosphorylation activity on MBP. In addition, another 75 kDa polypeptide was observed in the particulate fraction. Immunodetection with monoclonal antibodies against ERK-1, a mammalian MAP kinase, and with anti-phosphotyrosine antibodies cross-reacted with the 56 kDa polypeptide, named SMK56, from the soluble fraction, suggesting that this polypeptide could be related with members of the MAP kinase family. Antibodies against the dually phosphorylated threonine-tyrosine motif, characteristic of active forms of MAP kinases, also cross-reacted with this 56 kDa polypeptide. Changes in the levels of SMK56 were detected within the first 30 min of root exposure to low temperatures or hypo-osmotic shock, suggesting that this protein may be involved in the perception of environmental changes.