Agave Species Research Articles

Undervalued potential of crassulacean acid metabolism for current and future agricultural production.

The potential for crassulacean acid metabolism (CAM) to support resilient crops that meet demands for food, fiber, fuel, and pharmaceutical products far exceeds current production levels. This review provides background on five families of plants that express CAM, including examples of many species within these families that have potential agricultural uses. We summarize traditional uses, current developments, management practices, environmental tolerance ranges, and economic values of CAM species with potential commercial applications. The primary benefit of CAM in agriculture is high water use efficiency that allows for reliable crop yields even in drought conditions. Agave species, for example, grow in arid conditions and have been exploited for agricultural products in North and South America for centuries. Yet, there has been very little investment in agricultural improvement for most useful Agave varieties. Other CAM species that are already traded globally include Ananas comosus (pineapple), Aloe spp., Vanilla spp., and Opuntia spp., but there are far more with agronomic uses that are less well known and not yet developed commercially. Recent advances in technology and genomic resources provide tools to understand and realize the tremendous potential for using CAM crops to produce climate-resilient agricultural commodities in the future.

Insects associated with the genus Agave spp. (Asparagaceae) in Mexico.

We present a synthesis of the insects reported associated with plants of the genus Agave L. (Asparagaceae) in Mexico, through a detailed review of published records. This work includes two checklists: the first include the species of insects collected in Agave, name of their hosts, their geographical distribution, notes on their biology and, references. Natural enemies of the insects associated with agaves are included in the second checklist with their geographical distribution, insect species host and, references. We found 273 species of insects collected in different Agave species. These species belong to 63 families and 7 orders. The orders of insects with the highest number of species are Coleoptera with 118 species and, Hemiptera with 65. The natural enemies of insects collected in agaves were 98 species from 18 families and 6 orders. The genera Aphytis and Encarsia (Hymenoptera: Aphelinidae) grouped the largest number of natural enemies of insects that were recorded in Agave with 15 and 10 species, respectively. Insects were collected from 42 species of Agave, of which 25 are endemic to Mexico. This study provides biological information on Mexican insects captured in Agave and, their natural enemies. The need for the conservation of these plants and associated insects is highlighted.

Anti-neuroinflammatory effect of agaves and cantalasaponin-1 in a model of LPS-induced damage

Chronic neuroinflammation is a key component of many neurodegenerative disorders. Chronic activation of this process produces pro-inflammatory cytokines, prostaglandins and reactive oxygen species that induce brain injury and neuronal dysfunction. Agave species contain saponins, compounds with anti-inflammatory activity. Extracts from A. tequilana (At), A. angustifolia (Aan), A. Americana (Aam) (125 mg/kg) and cantalasaponin-1 (5 and 10 mg/kg, isolated from Aam) were administered to male ICR mice with lipopolysaccharide (LPS)-induced neuroinflammation, after which inflammatory cytokines were measured in brain homogenates by using an enzyme-linked immunoassay (ELISA) test. All agave extracts and cantalasaponin-1, reduced brain concentration of LPS-induced pro-inflammatory cytokines IL-6 and TNF-α. Moreover, Cantalasaponin-1 increased the brain concentration of the anti-inflammatory cytokine IL-10. Agave extracts and derived compounds show promising results in the development of novel drugs for neuroinflammatory disease therapy.

Caracterización morfológica de Agave angustifolia y su conservación en Guerrero, México

El Agave angustifolia presenta una amplia variación morfológica, que con frecuencia dificulta su taxonomía. La diferenciación entre las especies de Agave, se hace a partir de características morfológicas homólogas e independientes entre sí, cuya variación puede ser ontogenética y genética. El objetivo de esta investigación fue caracterizar la morfología del A. angustifolia denominado ‘sacatoro’ del estado de Guerrero. La investigación se realizó de 2016 a 2017, en seis poblaciones de A. angustifolia y dos poblaciones como control, A. angustifolia Haw y A. cupreata. Se midieron 32 variables del tallo y hojas de plantas madre e hijuelos a cada ejemplar, de acuerdo con la guía técnica para la descripción varietal de Agave spp. Los datos se analizaron mediante componentes principales (ACP) y agrupamiento aglomerativo jerárquico (AJA). Las variables que permitieron discernir las poblaciones fueron: forma, borde, color y textura de la hoja, estrías en espinas laterales y longitud de la espina terminal, todas con una correlación positiva. Los resultados obtenidos del ACP y el AJA permitieron observar la relación entre variables y especímenes en función de los atributos morfológicos que comparten las especies estudiadas. A pesar de la variación morfológica entre el maguey sacatoro y el A. angustifolia Haw., se muestra una estrecha relación positiva entre ambos. Por lo que se infiere que las características fenotípicas que sub agrupan al maguey sacatoro, pudieran mostrar como resultado una posible variante de A. angustifolia Haw que permitiría ubicar taxonómicamente a este grupo para contribuir a la ordenación del género Agave y su conservación.

Transcriptome Dataset of Leaf Tissue in Agave H11648

Sisal is widely cultivated in tropical areas for fiber production. The main sisal cultivar, Agave H11648 ((A. amaniensis × A. angustifolia) × A. amaniensis) has a relatively scarce molecular basis and no genomic information. Next-generation sequencing technology has offered a great opportunity for functional gene mining in Agave species. Several published Agave transcriptomes have already been reused for gene cloning and selection pressure analysis. There are also other potential uses of the published transcriptomes, such as meta-analysis, molecular marker detection, alternative splicing analysis, multi-omics analysis, genome assembly, weighted gene co-expression network analysis, expression quantitative trait loci analysis, miRNA target site prediction, etc. In order to make the best of our published transcriptome of A. H11648 leaf, we here represent a data descriptor, with the aim to expand Agave bio information and benefit Agave genetic researches.

Scyphophorus acupunctatus (Coleoptera: Dryophthoridae): A Weevil Threatening the Production of Agave in Mexico

Scyphophorus acupunctatus Gyllenhal (Coleoptera: Dryophthoridae), which also is known as agave weevil, is distributed across 5 continents. In Mexico, their presence has been reported in 12 states. Many species of Agave (Asparagaceae) are suitable hosts, though not all are equally suitable. Plant infestation can start with either female or male weevils, and their presence can be detected by gummy secretions emanating from feeding sites. The punctures are observed principally in the lower part of the trunk and external roots of the infested plants. Eggs are deposited singly or in clusters of up to 4 eggs at the feeding punctures after tissue decay has begun. The eggs hatch after about 5 d, and young larvae begin to create tunnels in the plant tissue. The larvae display 11 instars, and require about 50 to 90 d for development. The pupae normally require 11 to 14 d. The life cycle lasts between 105 to 137 d depending on the agave species with which the weevil is associated. The active insect can attack during any mo of the yr, although it is more frequent in the rainy season. Scyphophorus acupunctatus dispersion is determined by the attraction of volatile agave compounds. This insect apparently introduces different microorganisms that have been associated with the agave maladies. Plant extracts and seed powders have been assessed as potential botanical insecticides on larvae and pupae, with plant extracts causing mortality of 43 to 53% in larvae and 3 to 15% in pupae, and with seed powders causing 90 to 100% larval mortality. Synthetic insecticides have not been successful because the larvae and adults live inside the plant tissues. Alternative effective control tactics are needed to avoid increased damage and destruction of this important crop.

Morphological comparison among populations of Agave salmiana Otto ex Salm-Dyck (Asparagaceae), a species used for mezcal production in Mexico

Agave salmiana represents a high economic and cultural relevance in Mexican society. Cultivated in arid zones of Mexico, it is the agave species primarily used for the production of mezcal, a traditional Mexican alcoholic beverage. In this study, we performed a morphological comparison among 145 accessions of Agave salmiana ssp. crassispina and ssp. salmiana from 14 municipalities in northern Guanajuato. A total of 12 morphological variables of leaf size, spines and teeth were measured. In order to perform this evaluation, agave plants were considered either as cultivated or wild-type. Statistical analyzes were conducted and a dendrogram was constructed with the nearest neighbor method, analysis of variance (ANOVA) and Fisher’s Least Significant Difference (LSD) multiple comparisons. Several groups were established within the dendrogram and the distance among the groups was found to be small. The ANOVA shows significant differences among the subspecies and the populations, whereas the multiple comparisons detected several groups. The statistical analyses showed a morphological variation among the studied plants. However, the whole population under study falls within a range of morphological homogeneity.

Fermentative capabilities of native yeast strains grown on juices from different Agave species used for tequila and mezcal production.

The Asparagaceae family is endemic from America, being the Agave genus the most important. The Agave species possess economic relevance and are use as raw material to produce several distilled alcoholic beverages, as bacanora, tequila, and mezcal. The fermentation process has been carry out either spontaneously or by adding a selected yeast strain. The latter is generally responsible for the production of ethanol and volatile compounds. This study comprised five Agave species (A. angustifolia, A. cupreata, A. durangensis, A. salmiana, and A. tequilana) and eight endogenous yeast strains: five of them were non-Saccharomyces (Torulaspora delbrueckii, Zygosaccharomyces bisporus, Candida ethanolica, and two Kluyveromyces marxianus) and three Saccharomyces cerevisiae strains. The results showed that the S. cerevisiae strains were not able to grow on A. durangensis and A. salmiana juices. The Kluyveromyces marxianus strains grew and fermented all the agave juices and displayed high ethanol production (48-52gL-1) and volatile compounds. The ethanol production was higher on A. angustifolia juice (1.1-2.8-fold), whereas the volatile compound was dependent on both yeast strain and the Agave species. The use of endogenous non-Saccharomyces yeast strains is feasible, as they may outperform S. cerevisiae regarding the production of fermented beverages from agave plants with a high content of ethanol and aromatic compounds. Graphical abstract.

De Novo Transcriptome Assembly of Agave H11648 by Illumina Sequencing and Identification of Cellulose Synthase Genes in Agave Species.

Agave plants are important crassulacean acid metabolism (CAM) plants with multiple agricultural uses, such as being used in tequila and fiber production. Agave hybrid H11648 ((A. amaniensis Trel. and Nowell × A. angustifolia Haw.) × A. amaniensis) is the main cultivated Agave species for fiber production in large tropical areas around the world. In this study, we conducted a transcriptome analysis of A. H11648. About 49.25 million clean reads were obtained by Illumina paired-end sequencing. De novo assembly produced 148,046 unigenes with more than 40% annotated in public databases, or matched homologs in model plants. More homologous gene pairs were found in Asparagus genome than in Arabidopsis or rice, which indicated a close evolutionary relationship between Asparagus and A. H11648. CAM-related gene families were also characterized as previously reported in A. americana. We further identified 12 cellulose synthase genes (CesA) in Asparagus genome and 38 CesA sequences from A. H11648, A. americana, A. deserti and A. tequilana. The full-length CesA genes were used as references for the cloning and assembly of their homologs in other Agave species. As a result, we obtained CesA1/3/4/5/7 genes with full-length coding region in the four Agave species. Phylogenetic and expression analysis revealed a conserved evolutionary pattern, which could not explain the distinct fiber traits in different Agave species. We inferred that transcriptional regulation might be responsible for Agave fiber development. This study represents the transcriptome of A. H11648, which would expand the number of Agave genes and benefit relevant studies of Agave fiber development.

Cuatro especies nuevas de Agave (Asparagaceae, Agavoideae) del sur de México

Antecedentes y Objetivos: El género Agave está conformado por 159 especies, es el octavo más diverso de la flora mexicana. En este trabajo se describen cuatro especies nuevas para la ciencia, resultado de la exploración de áreas poco conocidas desde un punto de vista botánico en el sur de México.Métodos: Como resultado de trabajo de campo y de herbario en los estados de Guerrero, Oaxaca y Puebla, se encontraron poblaciones de agaves cuyas características morfológicas no corresponden con especies descritas con anterioridad, por lo que después de un análisis morfológico y una revisión bibliográfica se describen formalmente. El estado de conservación se evaluó con base en los criterios de la NOM-059-SEMARNAT-2010 y el método propuesto por la Unión Internacional para la Conservación de la Naturaleza.Resultados clave: Se describen e ilustran Agave lyobaa, del subgénero Agave grupo Ditepalae, y tres especies del subgénero Littaea, A. gypsicola del grupo Choritepalae y A. quiotepecensis y A. megalodonta del grupo Marginatae. Para cada una se proporciona información sobre su distribución, hábitat, fenología, posibles relaciones morfológicas con otras especies del género y usos. Según los criterios de la NOM-059-SEMARNAT-2010, las cuatro especies se proponen en la categoría amenazada (A). De acuerdo con el método propuesto por la IUCN, las dos primeras especies se encuentran en la categoría En Peligro (EN) y las dos últimas como Casi Amenazada (NT).Conclusiones: Se describen cuatro especies endémicas de México, distribuidas en los estados de Guerrero, Oaxaca y Puebla; todas ellas con poblaciones vulnerables que requieren acciones prioritarias de conservación.

De novo assembly of Agave sisalana transcriptome in response to drought stress provides insight into the tolerance mechanisms

Agave, monocotyledonous succulent plants, is endemic to arid regions of North America, exhibiting exceptional tolerance to their xeric environments. They employ various strategies to overcome environmental constraints, such as crassulacean acid metabolism, wax depositions, and protective leaf morphology. Genomic resources of Agave species have received little attention irrespective of their cultural, economic and ecological importance, which so far prevented the understanding of the molecular bases underlying their adaptations to the arid environment. In this study, we aimed to elucidate molecular mechanism(s) using transcriptome sequencing of A. sisalana. A de novo approach was applied to assemble paired-end reads. The expression study unveiled 3,095 differentially expressed unigenes between well-irrigated and drought-stressed leaf samples. Gene ontology and KEGG analysis specified a significant number of abiotic stress responsive genes and pathways involved in processes like hormonal responses, antioxidant activity, response to stress stimuli, wax biosynthesis, and ROS metabolism. We also identified transcripts belonging to several families harboring important drought-responsive genes. Our study provides the first insight into the genomic structure of A. sisalana underlying adaptations to drought stress, thus providing diverse genetic resources for drought tolerance breeding research.

Tempo and mode in coevolution of Agave sensu lato (Agavoideae, Asparagaceae) and its bat pollinators, Glossophaginae (Phyllostomidae)

The genus Agave sensu lato contains ca. 211 described species, many of which are considered keystone species because of their ecological dominance and the quantity of resources they provide with their massive, nectar-rich inflorescences. The large diversity of Agave species has been hypothesized as being related to their reproductive strategy (predominantly monocarpic) and diverse pollinators (e.g., bats, hummingbirds, hawkmoths). In particular, Agave species provide resources that a few genera of nectar feeding bats from the subfamily Glosophaginae are dependent upon. To explore a possible coevolutionary relationship between Agave and the bat species that pollinate them, we calibrated molecular phylogenies of both groups and looked for a correlation in their dates of divergence. One coding and two non-coding regions of the chloroplast genome were sequenced from 49 species of the Agavoideae (Asparagaceae), and the mitochondrial gene Cyt-b and nuclear coding gene RAG2 were either sequenced or obtained from gene bank for 120 Phyllostomid bats. Results from the analyses indicate that Agave sensu lato is a young genus (estimated crown age 2.7–8.5/stem age 4.6–12.3 Ma), with an increasing diversification rate, and the highest speciation rate among Agavoideae’s clades. The origin of the Glossophaginae bats (stem age 20.3–23.5 Ma) occurred prior to the stem age of Agave sensu lato, while the origin of the current pollinators of Agave species, members of the genera Glossophaga, Leptonycteris, Anoura, Choeronyscus, Musonycteris and Choeronycteris, was estimated to be around 6.3–16.2 Ma, overlapping with the stem age of Agave sensu lato, supporting the hypothesis of diffuse coevolution.

Metagenomic Microbial Diversity in Aguamiel from Two Agave Species During 4-Year Seasons

ABSTRACTAguamiel is the sap of “maguey pulquero” and can be consumed as a fresh beverage or after a cooking process. Further, it is also fermented for the production of “pulque,” a traditional Mexican alcoholic beverage. Use of aguamiel as a functional beverage has been suggested due to prebiotic activity of its fructooligosaccharides and probiotic activity of lactic acid bacteria that it contains. There are some reports about the culturable microbial diversity in aguamiel; however, the total microbial diversity has not been characterized. The objective of this study was to identify metagenomic populations in aguamiel using denaturing gradient gel electrophoresis and sequencing of the 16S rRNA gene and 26SrRNA gene regions. Microbial diversity in aguamiel from two maguey species was evaluated: Agave salmiana and Agave atrovirens, during the four seasons of the year. The highest diversity was found during summer season in aguamiel samples from both Agave species, detecting presence of Leuconostoc sp., Leuconostoc gelidum, Lactococcus lactis, Enterococcus casseliflavus, Pediococcus sp., uncultured Trichococcus sp., uncultured Leuconostoc, uncultured Lactococcus, Kazachstania zonata (not previously reported in aguamiel), Kluyveromyces marxianus, and Saccharomyces cerevisiae. Knowledge about the microbial populations of this beverage throughout the different seasons will provide advantages through understanding of aguamiel for application areas such as syrup elaboration and pulque making. In addition, this beverage could be industrialized as a functional beverage with a consistent quality.

Transcriptome Comparison Reveals Distinct Selection Patterns in Domesticated and Wild Agave Species, the Important CAM Plants.

Agave species are an important family of crassulacean acid metabolism (CAM) plants with remarkable tolerance to heat and drought stresses (Agave deserti) in arid regions and multiple agricultural applications, such as spirit (Agave tequilana) and fiber (Agave sisalana) production. The agave genomes are commonly too large to sequence, which has significantly restricted our understanding to the molecular basis of stress tolerance and economic traits in agaves. In this study, we collected three transcriptome databases for comparison to reveal the phylogenic relationships and evolution patterns of the three agave species. The results indicated the close but distinctly domesticated relations between A. tequilana and A. sisalana. Natural abiotic and biotic selections are very important factors that have contributed to distinct economic traits in agave domestication together with artificial selection. Besides, a series of candidate unigenes regulating fructan, fiber, and stress response-related traits were identified in A. tequilana, A. sisalana, and A. deserti, respectively. This study represents the first transcriptome comparison within domesticated and wild agaves, which would serve as a guidance for further studies on agave evolution, environmental adaptation, and improvement of economically important traits.

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.

Genetic variation and relationships among agaves related to the production of Tequila and Mezcal in Jalisco

The study of evolutionary history allows us to examine diversification, selection and domestication processes. Mexico belongs to Mesoamerica, one of the world’s most important centers of origin and diversification of plants. One of the plants that has sustained its peoples for over 10,000 years is the agave (Agave sp.). Mexico is the center of diversity of the genus, with 75% of the species. Two agave products, tequila and mezcal, are of great economic and biocultural importance for Mexico. The description of genetic diversity and the identification of the wild relatives of the agave species used to produce these emblematic beverages is fundamental information for their production and conservation. Previous studies have proposed wild populations of A. angustifolia in Jalisco as possible wild relatives of blue agave or tequila (Agave tequilana). We use microsatellite (eight loci) to study the genetic diversity and the relationships between wild populations of A. angustifolia and traditional cultivars of the Agave species utilized in the production of tequila and mezcal in Jalisco. The studied taxa present intermediate genetic variation, with the exception of A. tequilana “Azul” which had the same genotype. A Structure analysis indicates that the “Azul” is closely related to A. angustifolia mainly to wild populations from southern Jalisco. Agave rhodacantha and the cultivars of A. tequilana (“Sigüin” and “Chato”) form a group separate from Agave angustifolia y A. tequilana “Azul”.

Identification and characterization of a new satellite-like DNA family in three Agave species

Repeated DNA analysis was conducted in three Agave species (A. angustifolia Haworth, A. tequilana Weber and A. fourcroydes Lemaire). Self-priming PCR was initially used to generate repeated PCR products and their sequence information was then used to build different primers sets. The ATsat102-113 primer set was able to amplify repeated PCR products, including probably multimers without becoming a typical ladder profile, in all Agave accessions analyzed. Sequence analysis of these amplifications identified two subfamilies of repeated DNA which share six conserved short motifs but exhibit a high genetic distance and different sequence traits between them. Consensus monomers obtained have a length higher than 100 bp and are rich in Adenine/Thymine percentage, as many satellites of plants. A representative probe of these consensus monomers was able to hybridize in some PCR amplified products and the more frequent short motif 1 (CTYRAGTTSG) also exhibits probably multimers without becoming a typical ladder profile. These Agave satellite-like subfamilies have different homogenization rates and the subfamily 2 is more conserved. These satellite-like monomers are transcribed and exhibited a strong relation with transposable element signatures of the LTR/Gypsy families and with A. tequilana TSA sequences, some which exhibits transposon-related domains in different locations to the satellite-like sequences identified. FISH analysis of these satellites-like subfamilies only may be reliable determined in the diploid (A. angustifolia Haworth var. Marginata and A. tequilana Weber ‘Azul’) accessions. In these generally showed a subtelomeric hybridization with less interstitial signals.

Diel rewiring and positive selection of ancient plant proteins enabled evolution of CAM photosynthesis in Agave

BackgroundCrassulacean acid metabolism (CAM) enhances plant water-use efficiency through an inverse day/night pattern of stomatal closure/opening that facilitates nocturnal CO2 uptake. CAM has evolved independently in over 35 plant lineages, accounting for ~ 6% of all higher plants. Agave species are highly heat- and drought-tolerant, and have been domesticated as model CAM crops for beverage, fiber, and biofuel production in semi-arid and arid regions. However, the genomic basis of evolutionary innovation of CAM in genus Agave is largely unknown.ResultsUsing an approach that integrated genomics, gene co-expression networks, comparative genomics and protein structure analyses, we investigated the molecular evolution of CAM as exemplified in Agave. Comparative genomics analyses among C3, C4 and CAM species revealed that core metabolic components required for CAM have ancient genomic origins traceable to non-vascular plants while regulatory proteins required for diel re-programming of metabolism have a more recent origin shared among C3, C4 and CAM species. We showed that accelerated evolution of key functional domains in proteins responsible for primary metabolism and signaling, together with a diel re-programming of the transcription of genes involved in carbon fixation, carbohydrate processing, redox homeostasis, and circadian control is required for the evolution of CAM in Agave. Furthermore, we highlighted the potential candidates contributing to the adaptation of CAM functional modules.ConclusionsThis work provides evidence of adaptive evolution of CAM related pathways. We showed that the core metabolic components required for CAM are shared by non-vascular plants, but regulatory proteins involved in re-reprogramming of carbon fixation and metabolite transportation appeared more recently. We propose that the accelerated evolution of key proteins together with a diel re-programming of gene expression were required for CAM evolution from C3 ancestors in Agave.

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.

Recalcitrance Assessment of the Agro-industrial Residues from Five Agave Species: Ionic Liquid Pretreatment, Saccharification and Structural Characterization

Agave has recently shown its potential as a bioenergy feedstock with promising features such as higher biomass productivity than leading bioenergy feedstock while at the same time being drought-resistant with low water requirements and high sugar to ethanol conversion using ionic liquid (IL) pretreatment. IL pretreatment was studied to develop the first direct side-by-side comparative recalcitrance assessment of the agro-industrial residues from five Agave species [Agave americana (AME), A. angustifolia (ANG), A. fourcroydes (FOU), A. salmiana (SAL), and A. tequilana (TEQ)] using compositional analysis, X-ray diffraction, and the lignin syringyl/guaiacyl subunit ratio (S/G) by pyrolysis molecular beam mass spectrometry (PyMBMS). Prominent calcium oxalate peaks were found only in unpretreated AME, SAL, and TEQ. The S/G ratios of all five unpretreated Agave species were between 1.27 and 1.57 while the IL-pretreated samples were from 1.39 to 1.72. The highest overall sugar production was obtained with IL-pretreated FOU with 492 mg glucose/g biomass and 157 mg xylose/g biomass at 120 °C and 3 h using 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]). An estimated theoretical ethanol yield from the studied agro-industrial residues from the five Agave species was in the range of 1060 to 5800 L ethanol/ha/year. These comparison results demonstrate the potential of the Agave spp. as a suitable biofuel feedstock which can be employed within a biorefinery scheme.