Retroelements and DNA Methylation Could Contribute to Diversity of 5S rDNA in Agave L.

Agave L. is a genus of economic importance, and many of the 166 species in the American plant genus Agave L. inhabit high-stress environments, which makes the genus promising for facing global climate change. However, sustainable use of economically important species without interfering with their ecology and evolution requires generating knowledge about the factors responsible for their genetic variation and diversity and, on this basis, their adaptation and speciation. Few genetic studies exploring the evolutionary relationships, speciation processes, genetic variability and diversity within species of Agave are currently available. Analyses of rDNA loci have been performed with the purpose of determining the genetic variability and diversity of the genus Agave, and these loci have been used as genetic markers of ploidy. However, the factors involved in the diversity of 5S rDNA regions in Agave have not yet been studied in depth. Our study explored the possible mechanisms of genetic (retroelements) and epigenetic (DNA methylation) diversity in 5S rDNA regions in Agave. We characterized the 5S rDNA gene tandem in species of the genus with different ploidy numbers and determined the levels of methylation in 13 haplotypes of 5S rDNA and in four non-transcribed spacers (NTS). Our results showed highly dynamic methylation with a high percentage in haplotypes and NTS of 5S rDNA regions in Agave. The characterization of the 5S rDNA tandem array in Agave revealed vestigial remains of the Cassandra terminal-repeat retrotransposon in miniature (TRIM). Our analysis supported previous results suggesting that in species of Agave L., regulation and diversity of 5S rDNA regions are controlled by coordinated genetic and epigenetic events, which will vary according to the species and the level of ploidy. The artificial pressure to which some agave crops are subjected may affect the mechanisms of evolution of gene 5S rDNA.

Interspecific evolutionary relationships of alpha-glucuronidase in the genus Aspergillus.

The increased availability and production of lignocellulosic agroindustrial wastes has originated proposals for their use as raw material to obtain biofuels (ethanol and biodiesel) or derived products. However, for biomass generated from lignocellulosic residues to be successfully degraded, in most cases it requires a physical (thermal), chemical, or enzymatic pretreatment before the application of microbial or enzymatic fermentation technologies (biocatalysis). In the context of enzymatic technologies, fungi have demonstrated to produce enzymes capable of degrading polysaccharides like cellulose, hemicelluloses and pectin. Because of this ability for degrading lignocellulosic material, researchers are making efforts to isolate and identify fungal enzymes that could have a better activity for the degradation of plant cell walls and agroindustrial biomass. We performed an in silico analysis of alpha-glucoronidase in 82 accessions of the genus Aspergillus. The constructed dendrograms of amino acid sequences defined the formation of 6 groups (I, II, III, IV, V, and VI), which demonstrates the high diversity of the enzyme. Despite this ample divergence between enzyme groups, our 3D structure modeling showed both conservation and differences in amino acid residues participating in enzyme-substrate binding, which indicates the possibility that some enzymes are functionally specialized for the specific degradation of a substrate depending on the genetics of each species in the genus and the condition of the habitat where they evolved. The identification of alpha-glucuronidase isoenzymes would allow future use of genetic engineering and biocatalysis technologies aimed at specific production of the enzyme for its use in biotransformation.

Review and in silico analysis of fermentation, bioenergy, fiber, and biopolymer genes of biotechnological interest in Agave L. for genetic improvement and biocatalysis.

Several of the over 200 known species of Agave L. are currently used for production of distilled beverages and biopolymers. The plants live in a wide range of stressful environments as a result of their resistance to abiotic stress (drought, salinity, and extreme temperature) and pathogens, which gives the genus potential for germplasm conservation and biotechnological applications that may minimize economic losses as a result of the global climate change. However, the limited knowledge in the genus of genome structure and organization hampers development of potential improved biotechnological applications by means of genetic manipulation and biocatalysis. We reviewed Agave and plant sequences in the GenBank NCBI database for identifying genes with biotechnological potential for fermentation, bioenergy, fiber improvement, and in vivo plant biopolymer production. Three-dimensional modeling of enzyme structures in plant accessions revealed structural differences in sucrose 1-fructosyltransferase, fructan 1-fructosyltransferase, fructan exohydrolase (1-FEH), cellulose synthase (CES), and glucanases (EGases) with possible effects in fructan, sugar, and biopolymer production. Although the coding genes of FEH and enzymes involved in biopolymer production (CES, sucrose synthase, and EGases) remain unidentified in Agave L., our results could aid isolation of such genes in Agave. By comparing nucleotide and amino acid sequences in accessions of Agave and other plants, knowledge may be gained about transcriptional regulation and enzymatic activity factors. Future study is needed of biotechnological application of Agave genes for crop breeding aided by genetic engineering and biocatalysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1314-1334, 2018.

Morphological features of different polyploids for adaptation and molecular characterization of CC-NBS-LRR and LEA gene families in Agave L.

Polyploidy has been widely described in many Agave L. species, but its influence on environmental response to stress is still unknown. With the objective of knowing the morphological adaptations and regulation responses of genes related to biotic (LEA) and abiotic (NBS-LRR) stress in species of Agave with different levels of ploidy, and how these factors contribute to major response of Agave against environmental stresses, we analyzed 16 morphological trials on five accessions of three species (Agave tequilana Weber, Agave angustifolia Haw. and Agave fourcroydes Lem.) with different ploidy levels (2n=2x=60 2n=3x=90, 2n=5x=150, 2n=6x=180) and evaluated the expression of NBS-LRR and LEA genes regulated by biotic and abiotic stress. It was possible to associate some morphological traits (spines, nuclei, and stomata) to ploidy level. The genetic characterization of stress-related genes NBS-LRR induced by pathogenic infection and LEA by heat or saline stresses indicated that amino acid sequence analysis in these genes showed more substitutions in higher ploidy level accessions of A. fourcroydes Lem. 'Sac Ki' (2n=5x=150) and A. angustifolia Haw. 'Chelem Ki' (2n=6x=180), and a higher LEA and NBS-LRR representativeness when compared to their diploid and triploid counterparts. In all studied Agave accessions expression of LEA and NBS-LRR genes was induced by saline or heat stresses or by infection with Erwinia carotovora, respectively. The transcriptional activation was also higher in A. angustifolia Haw. 'Chelem Ki' (2n=6x=180) and A. fourcroydes 'Sac Ki' (2n=5x=150) than in their diploid and triploid counterparts, which suggests higher adaptation to stress. Finally, the diploid accession A. tequilana Weber 'Azul' showed a differentiated genetic profile relative to other Agave accessions. The differences include similar or higher genetic representativeness and transcript accumulation of LEA and NBS-LRR genes than in polyploid (2n=5x=150 and 2n=6x=180) Agave accessions, thus suggesting a differentiated selection pressure for overcoming the lower ploidy level of the diploid A. tequilana Weber 'Azul'.