Molecular and Functional Characterization of Novel Fructosyltransferases and Invertases from Agave tequilana

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Exoidrolases de frutanos e sua importância no metabolismo de frutanos em Agave tequilana Weber var. azul

Fructans are carbohydrates of diverse chemical structure that are distributed in different taxa. In plants, in addition to be an important source of carbon, they have been associated with tolerance to different types of stress. The biosynthesis of these compounds is carried out by the action of enzymes called fructosyltransferases, while their degradation is mediated by fructan exohydrolases, both types are part of the 32 family of glycosidic hydrolase enzymes. Fructan exohydrolases are exo-enzymes that sequentially release terminal fructose residues from fructans to make them available as carbon sources for different cellular processes or as signaling molecules. In this review, a description is made of fructans, y of fructosyl exohydrolases in some plants important for human consumption or for industrial use, such as the Agave genus, specifically in A. tequilana. We conclude that the study of fructan exohydrolases in agaves could be useful in various biotechnological applications, for example, in the hydrolysis of undigested fructan structures during the agave cooking stage in the tequila production process. However, the first step is to determinate the enzymatic activity in which they are involved, for its posterior inclusion in biotechnology processes.Los fructanos son carbohidratos de estructura química diversa distribuidos en diferentes taxa. En las plantas, además de constituir una importante fuente de carbono, han sido asociados con la tolerancia a diferentes tipos de estrés. La biosíntesis de estos compuestos se lleva a cabo por la acción de las enzimas fructosiltransferasas, mientras que su degradación es mediada por las exohidrolasas fructosílicas, ambos tipos forman parte de la familia 32 de las enzimas hidrolasas glicosídicas. Las exohidrolasas fructosílicas son exo-enzimas que liberan secuencialmente residuos de fructosa terminales de los fructanos para hacerlos disponibles como fuente de carbono en diferentes procesos celulares o bien moléculas de señalización. En esta revisión se hace una descripción de los fructanos y las exohidrolasas fructosílicas en algunas plantas importantes para el consumo humano y para el aprovechamiento industrial, con un enfoque particular en el género Agave, específicamente en A. tequilana. Concluimos, que el estudio de las exohidrolasas fructosílicas en agaves podría ser útil en varias aplicaciones biotecnológicas como en la hidrólisis de estructuras de fructanos no digeridos durante la etapa de cocción del agave en el proceso de producción de tequila.  Sin embargo, el primer paso es conocer la funcionalidad de estas enzimas, lo que podría facilitar su incorporación en diferentes procesos biotecnológicos.Os frutanos são um conjunto de carboidratos de estrutura química diversificada que são distribuídos em diferentes filos da vida. Nas plantas, ocorrem em dicotiledôneas e monocotiledôneas, nas quais, além de ser uma importante fonte de carbono, têm sido associadas à tolerância a diferentes tipos de estresse. A biossíntese desses compostos é realizada pela ação de enzimas chamadas fructosiltransferases, enquanto sua degradação é mediada por frutossil exoidrolases, ambos os tipos fazem parte da família das 32 enzimas glicosídeos hidrolases. As frutossil exohidolases são exoenzimas que liberam seqüencialmente resíduos terminais de frutose dos frutanos para torná-los disponíveis para diferentes processos celulares. Nesta revisão, é feita uma descrição dos frutanos presentes nas plantas, principalmente das monocotiledôneas, e das frutossil exo-hidrolases como elemento indispensável para seu uso. Da mesma forma, a presença dessas enzimas no Agave tequilana Weber var. azul, uma espécie de significado ecológico, cultural e econômico no México

Analysis and optimization of bulk DNA sampling with binary scoring for germplasm characterization.

The strategy of bulk DNA sampling has been a valuable method for studying large numbers of individuals through genetic markers. The application of this strategy for discrimination among germplasm sources was analyzed through information theory, considering the case of polymorphic alleles scored binarily for their presence or absence in DNA pools. We defined the informativeness of a set of marker loci in bulks as the mutual information between genotype and population identity, composed by two terms: diversity and noise. The first term is the entropy of bulk genotypes, whereas the noise term is measured through the conditional entropy of bulk genotypes given germplasm sources. Thus, optimizing marker information implies increasing diversity and reducing noise. Simple formulas were devised to estimate marker information per allele from a set of estimated allele frequencies across populations. As an example, they allowed optimization of bulk size for SSR genotyping in maize, from allele frequencies estimated in a sample of 56 maize populations. It was found that a sample of 30 plants from a random mating population is adequate for maize germplasm SSR characterization. We analyzed the use of divided bulks to overcome the allele dilution problem in DNA pools, and concluded that samples of 30 plants divided into three bulks of 10 plants are efficient to characterize maize germplasm sources through SSR with a good control of the dilution problem. We estimated the informativeness of 30 SSR loci from the estimated allele frequencies in maize populations, and found a wide variation of marker informativeness, which positively correlated with the number of alleles per locus

qRT-PCR expression profiles of <i>A. tequilana</i> fructosyltransferase and invertase genes in different plant tissues and developmental stages.

<p>A. <i>Atq1-SST-2,</i> B. <i>AtqVinv-1</i>, C. <i>Atq6G-FFT-1</i>, D. <i>AtqCwinv-1.</i> S-stem, BL-Base of leaf and ML-Mid-leaf of 1 and 3, year old plants (vegetative stage) and 5 and 7 year old plants (Post-reproductive stage). Photographic examples of tissues sampled are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035878#pone.0035878.s001" target="_blank">Figure S1</a>.</p

Schematic representation of the genomic structures of <i>A. tequilana</i> Fructosyltransferases and Invertases.

<p>Introns are represented by lines and exons with solid boxes. Exons are distinguished by Roman numerals from left to right. Only <i>AtqCwinv-1</i> shows a distinct pattern of exon/intron number and organization. Drawings are to scale and scale bar represents 250 bp.</p

Alignment of deduced amino acid sequences of fructosyltransferases and Invertases of <i>A. tequilana</i>.

<p>Asterisks, colons and periods indicate identical residues, conserved substitutions, and semi-conserved substitutions, respectively. Putative initiation points of the large and small subunits are arrowed. Potential glycosylation sites are underlined. The β-fructosidase motif, RDP motif and the cysteine catalytic site are boxed. A sucrose-donor substrate motif is shown as a stippled box. Predicted leader sequence cleavage points are shaded in grey. Differences between <i>Atq1-SST-1</i> and SSTAg are shown in italics.</p

Activity of recombinant 6G-FFT-1 protein.

<p>HPAEC chromatograms of the products obtained by the reactions supplied with: A. 100 mM sucrose. B. 50 mM 1-kestose C. both 100 mM sucrose and 50 mM 1-kestose D-amplification of the chromatogram in C. Abbreviations G-glucose, F-fructose, S-sucrose, 1-K-1-kestose, Neo-Neokestose, Nys-nystose, 4c-1^F,6^G-Di-β-D-fructofuranosylsucrose. Unidentified products are indicated by asterisks.</p

Unrooted tree of selected fructosyltransferases, fructan exohydrolases and invertases from monocotyledonous and dicotyledonous plants.

<p>A: branch containing cell wall invertases and FEHs, B: branch containing vacuolar invertases and fructosyltransferases, b1/b2-monocotyledons, b3-dicotyledons. <i>A. tequilana</i> fructosyltransferases and invertases are indicated with a diamond. Accession numbers are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035878#pone.0035878.s005" target="_blank">Table S1</a>.</p