Pre-contact Agave domesticates - living legacy plants in Arizona's landscape.

BACKGROUND AND SCOPE: Agaves played a central role as multi-use plants providing food, fibre and beverage to pre-contact and historical Mesoamerican cultures. However, their importance to Indigenous Peoples in the Southwest USA and northern Mexico, where they occur because of adaptations such as CAM photosynthesis, is less well known. Archaeological research indicates the Hohokam and other pre-contact Southwestern agrarian people increased agricultural potential in this region by engineering riverine terraces and bajadas for agave dry farming. Agricultural features such as terraces and rock piles were especially characteristic of post-1000 CE with the increase of dense, aggregated populations. We present an overview of six pre-contact agave domesticates (PCADs) the Hohokam and other cultures cultivated, and their ecological and cultural attributes. These PCADs are Agave murpheyi, A. delamateri, A. phillipsiana, A. sanpedroensis, A. verdensis and A. yavapaiensis. CONCLUSION: Pre-contact agriculturists cultivated at least six once cryptic domesticated agave species in the modern Arizona landscape associated with pre-contact agricultural features, such as rock structures. Because of the longevity and primarily asexual reproduction of these agaves, relict clones have persisted to the present day, providing an opportunity to study pre-contact nutrition, trade, migration and agricultural practices. Taxonomic data imply that pre-contact farmers selected desirable attributes, initiating domestication processes that resulted in discrete lineages. These agaves are morphologically and genetically distinct from Southwest US and northern Mexico wild agaves and Mesoamerican wild and domesticated species. Additionally, the remnant clones present a rare opportunity to examine domesticates virtually unchanged since they were last cultivated prehistorically. These discoveries underline the need to view landscapes and some plant species from a cultural, rather than 'natural', perspective and discern potential cryptic species veiled by traditional taxonomic treatments. Protecting and understanding the distribution, and ecological and cultural roles of these plants require interdisciplinary collaboration between botanists, archaeologists, federal agencies and Indigenous Peoples.

Management, morphological and genetic diversity of domesticated agaves in Michoacán, México.

BACKGROUND: Pulque is a fermented beverage prepared with sap of Agave species in Mexico. Management of agaves for this purpose has motivated domestication of some species and high phenotypic variation that commonly causes uncertainty about the taxonomic identity of varieties traditionally managed by people. This study assumed that varieties of crop species continually arise from mutations, sexual reproduction and hybridization, among other processes, and some of them are favoured and maintained by humans. Identifying these varieties may be difficult and a challenging issue for botanists and evolutionary biologists studying processes of domestication. Through a case study, we analysed the traditional varieties of agaves used to produce pulque in Michoacán, Mexico. We aimed at identifying the varieties, analysing the relatedness among them and developing a methodological approach that could help solve taxonomic problems and study variation under domestication of this and other plant groups. We documented (1) the traditional varieties of agave used and their identity, (2) how these varieties are perceived, used and managed by the local people and (3) how management influences phenotypic and genetic variation among varieties. METHODS: We interviewed pulque producers in two localities of the state of Michoacán, Mexico, where we recorded management practices of agaves, the traditional varieties used, the attributes characterizing those varieties, the varieties preferred by people, and features and mechanisms of selection. We conducted multivariate analyses of morphological features of the agave varieties, as well as genetic diversity and genetic distance studies among agave varieties through 11 nuclear microsatellites. RESULTS: Seven traditional varieties of Agave were recorded in the study area. Multivariate analyses of morphology identified varieties belonging to the species A. salmiana, A. mapisaga and, presumably, A. americana. The preferred varieties have morphological features selected to make easier their management and produce higher sap yields. Genetic diversities (HE = 0. 470 to 0.594) were high compared with other Agave species with similar life history traits and use. Genetic distance analyses grouped the varieties "Verde" and "Negro" (identified as A. salmiana), whereas the varieties "Tarímbaro" and "Listoncillo" (identified as A. mapisaga) formed another group. The varieties "Blanco" and "Carrizaleño" (most probably being A. americana) clustered with varieties of A. salmiana, whereas the variety "Cenizo" appeared as a distinct group. Bayesian analysis indicated that most individuals of varieties of A. salmiana form a group and those of the varieties of A. mapisaga form another, whereas individuals of the varieties putatively belonging to A. americana clustered in similar proportions with both groups. CONCLUSIONS: The traditional pulque production in the study area is an ongoing practice. It is still an important source of products for direct consumption by households and generation of economic incomes and as part of the cultural identity of local people. The most used traditional variety exhibited a marked gigantism, and although these agaves are mainly asexually propagated, populations have high genetic diversity. The local producers promote the maintenance of different traditional varieties. Our study shows the value of an integral research approach including ethnobiological, morphological and genetic information to clarify the state of variation influenced by humans on agaves, but it would be helpful to study other organisms under domestication. In addition, such approach would help to document human and non-human mechanisms generating crop varieties managed by local people.

Transcriptome analysis of bolting in A. tequilana reveals roles for florigen, MADS, fructans and gibberellins.

BACKGROUND: Reliable indicators for the onset of flowering are not available for most perennial monocarpic species, representing a drawback for crops such as bamboo, agave and banana. The ability to predict and control the transition to the reproductive stage in A. tequilana would represent an advantage for field management of agaves for tequila production and for the development of a laboratory model for agave species. RESULTS: Consistent morphological features could not be determined for the vegetative to reproductive transition in A. tequilana. However, changes in carbohydrate metabolism where sucrose decreased and fructans of higher degree of polymerization increased in leaves before and after the vegetative to reproductive transition were observed. At the molecular level, transcriptome analysis from leaf and shoot apical meristem tissue of A. tequilana plants from different developmental stages identified OASES as the most effective assembly program and revealed evidence for incomplete transcript processing in the highly redundant assembly obtained. Gene ontology analysis uncovered enrichment for terms associated with carbohydrate and hormone metabolism and detailed analysis of expression patterns for individual genes revealed roles for specific Flowering locus T (florigen), MADS box proteins, gibberellins and fructans in the transition to flowering. CONCLUSIONS: Based on the data obtained, a preliminary model was developed to describe the regulatory mechanisms underlying the initiation of flowering in A. tequilana. Identification of specific promoter and repressor Flowering Locus T and MADS box genes facilitates functional analysis and the development of strategies to modulate the vegetative to reproductive transition in A. tequilana.

Steroidal Saponin and Flavonol Content and Antioxidant Activity during Sporophyte Development of Maguey (Agave salmiana).

Agave salmiana Otto ex Salm-Dyck has traditionally been used for the production of fermented beverage known as "pulque" that has recently gained acceptance as a functional food. However, the plant requires up to 10 years to be used as raw material. The objective of this work was to evaluate the antioxidant and bioactive principles of Agave salmiana during different stages of development. Wild grown plants from Coahuila, Mexico, were identified based on leaf and spine traits to obtain a representative sample from six different stages of development (I-VI) from 1 to 7 years old. Total phenolic content (TPC), antioxidant activity (AOX), as well as composition and content of flavonols and saponins by HPLC-MS-TOF and HPLC-ELSD-PDA were evaluated. Concentrations of TPC were found to be between 5 to 13 mg gallic acid equivalents/g, reaching a maximum at stage II. The AOX presented a negative tendency from stage I to stage VI (from 148 to 50 µmol Trolox equivalents/g respectively). Kaempferol, quercetin and five saponins were identified. Similar to AOX, flavonols presented a negative concentration tendency with a reduction of 65% between the stage I and VI. Plants of stage III and IV presented the highest content of saponins, particularly chlorogenin glycoside, containing 3.19 and 2.90 mg protodioscin equivalents/g, respectively. These data suggest that plants from stages I to IV may be used as a source of antioxidant and bioactive principles, and that the content of these metabolites could be used as a marker to determine the developmental stage of the plant.

Morphological and genetic divergence between Agave inaequidens, A. cupreata and the domesticated A. hookeri. Analysis of their evolutionary relationships.

Agave inaequidens and A. cupreata are wild species with some populations under incipient management, while A. hookeri is exclusively cultivated, used for producing the fermented beverage pulque. These species are closely related and sympatric members of the Crenatae group, but taxonomists have previously hypothesized that A. inaequidens is the most probable ancestor of A. hookeri. Our study aims at evaluating patterns of morphological and genetic divergence among populations of the three species, in order to analyze their ecological and possible evolutionary relationships. We studied 24 agave populations, 16 of them of Agave inaequidens, four of A. cupreata and four of A. hookeri. Population morphometric and genetics studies were performed using 39 morphological characters and 10 nuclear microsatellites, respectively. We estimated levels of morphological and genetic diversity and dissimilarity, as well as genetic structure and gene flow among populations and species. The three species were clearly differentiated by general plant size, lateral teeth, terminal spines, flowers and fruit size. The largest plants were those of A. hookeri followed by A. inaequidens and the smallest were A. cupreata. Multivariate analyses indicated greater morphological similarity between A. hookeri and cultivated A. inaequidens, while A. cupreata consistently appeared as a separate group. We identified similar levels of morphological diversity index (MDI) in the three species, but higher genetic diversity in A. inaequidens (MDI = 0.401-0.435; HE = 0.704-0.733), than in A. cupreata (MDI = 0.455-0.523; HE = 0.480-0.510) and the predominantly vegetative propagated crop A. hookeri (MDI = 0.335-0.688; HE = 0.450-0.567), a pattern consistent with our expectations. The morphological and genetic similarities between cultivated A. inaequidens and A. hookeri support the hypothetical evolutionary relationships among these species, but studies with cpDNA and SNPs, and including other member of the Crenatae group are necessary to further resolve these relationships.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process.

KEY MESSAGE: Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

Functionally different PIN proteins control auxin flux during bulbil development in Agave tequilana.

In Agave tequilana, reproductive failure or inadequate flower development stimulates the formation of vegetative bulbils at the bracteoles, ensuring survival in a hostile environment. Little is known about the signals that trigger this probably unique phenomenon in agave species. Here we report that auxin plays a central role in bulbil development and show that the localization of PIN1-related proteins is consistent with altered auxin transport during this process. Analysis of agave transcriptome data led to the identification of the A. tequilana orthologue of PIN1 (denoted AtqPIN1) and a second closely related gene from a distinct clade reported as 'Sister of PIN1' (denoted AtqSoPIN1). Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis showed different patterns of expression for each gene during bulbil formation, and heterologous expression of the A. tequilana PIN1 and SoPIN1 genes in Arabidopsis thaliana confirmed functional differences between these genes. Although no free auxin was detected in induced pedicel samples, changes in the levels of auxin precursors were observed. Taken as a whole, the data support the model that AtqPIN1 and AtqSoPIN1 have co-ordinated but distinct functions in relation to auxin transport during the initial stages of bulbil formation.

Morphological variation, management and domestication of 'maguey alto' (Agave inaequidens) and 'maguey manso' (A. hookeri) in Michoacán, México.

BACKGROUND: Agave inaequidens and A. hookeri are anciently used species for producing the fermented beverage 'pulque', food and fiber in central Mexico. A. inaequidens is wild and cultivated and A. hookeri only cultivated, A. inaequidens being its putative wild relative. We analysed purposes and mechanisms of artificial selection and phenotypic divergences between wild and managed populations of A. inaequidens and between them and A. hookeri, hypothesizing phenotypic divergence between wild and domesticated populations of A. inaequidens in characters associated to domestication, and that A. hookeri would be phenotypically similar to cultivated A. inaequidens. METHODS: We studied five wild and five cultivated populations of A. inaequidens, and three cultivated populations of A. hookeri. We interviewed agave managers documenting mechanisms of artificial selection, and measured 25 morphological characters. Morphological similarity and differentiation among plants and populations were analysed through multivariate methods and ANOVAs. RESULTS: People recognized 2-8 variants of A. inaequidens; for cultivation they select young plants collected in wild areas recognized as producing the best quality mescal agaves. Also, they collect seeds of the largest and most vigorous plants, sowing seeds in plant beds and then transplanting the most vigorous plantlets into plantations. Multivariate methods classified separately the wild and cultivated populations of A. inaequidens and these from A. hookeri, mainly because of characters related with plant and teeth size. The cultivated plants of A. inaequidens are significantly bigger with larger teeth than wild plants. A. hookeri are also significatly bigger plants with larger leaves but lower teeth density and size than A. inaequidens. Some cultivated plants of A. inaequidens were classified as A. hookeri, and nearly 10% of A. hookeri as cultivated A. inaequidens. Wild and cultivated populations of A. inaequidens differed in 13 characters, whereas A. hookeri differed in 23 characters with wild populations and only in 6 characters with cultivated populations of A. inaequidens. CONCLUSIONS: Divergence between wild and cultivated populations of A. inaequidens reflect artificial selection. A. hookeri is similar to the cultivated A. inaequidens, which supports the hypothesis that A. hookeri could be the extreme of a domestication gradient of a species complex.

Effect of acid concentration and pulp properties on hydrolysis reactions of mercerized sisal.

The influence of sulfuric acid concentration (H2SO4 5-25%, 100°C), crystallinity and fibers size on the hydrolysis reaction of sisal pulps were investigated, with the goal of evaluating both the liquor composition, as an important step in the production of bioethanol, and the residual non-hydrolyzed pulp, to determine its potential application as materials. Aliquots were withdrawn from the reaction media, and the liquor composition was analyzed by HPLC. The residual non-hydrolyzed pulps were characterized by SEM, their average molar mass and crystallinity index, and their size distribution was determined using a fiber analyzer. Sulfuric acid 25% led to the highest glucose content (approximately 10gL(-1)), and this acid concentration was chosen to evaluate the influence of both the fiber size and crystallinity of the starting pulp on hydrolysis. The results showed that fibers with higher length and lower crystallinity favored glucose production in approximately 12%, with respect to the highly crystalline shorter fibers.

Class I KNOX genes are associated with organogenesis during bulbil formation in Agave tequilana.

Bulbil formation in Agave tequilana was analysed with the objective of understanding this phenomenon at the molecular and cellular levels. Bulbils formed 14-45 d after induction and were associated with rearrangements in tissue structure and accelerated cell multiplication. Changes at the cellular level during bulbil development were documented by histological analysis. In addition, several cDNA libraries produced from different stages of bulbil development were generated and partially sequenced. Sequence analysis led to the identification of candidate genes potentially involved in the initiation and development of bulbils in Agave, including two putative class I KNOX genes. Real-time reverse transcription-PCR and in situ hybridization revealed that expression of the putative Agave KNOXI genes occurs at bulbil initiation and specifically in tissue where meristems will develop. Functional analysis of Agave KNOXI genes in Arabidopsis thaliana showed the characteristic lobed phenotype of KNOXI ectopic expression in leaves, although a slightly different phenotype was observed for each of the two Agave genes. An Arabidopsis KNOXI (knat1) mutant line (CS30) was successfully complemented with one of the Agave KNOX genes and partially complemented by the other. Analysis of the expression of the endogenous Arabidopsis genes KNAT1, KNAT6, and AS1 in the transformed lines ectopically expressing or complemented by the Agave KNOX genes again showed different regulatory patterns for each Agave gene. These results show that Agave KNOX genes are functionally similar to class I KNOX genes and suggest that spatial and temporal control of their expression is essential during bulbil formation in A. tequilana.

Isolation and immunomodulatory effect of homoisoflavones and flavones from Agave sisalana Perrine ex Engelm.

Three known flavones and seven known homoisoflavonoids were isolated from the methanolic extract of the leaves of Agave sisalanaPerrine ex Engelm. Their structures were elucidated on the basis of spectroscopic analysis. The isolated compounds were also evaluated for immunopharmacological activity. PBMC were used as target cells, and cell proliferation was determined by (3)H-thymidine uptake. (+/-)-3,9-Dihydroeucomin (4), dihydrobonducellin (5), and 5,7-dihydroxy-3-(4'-hydroxybenzyl)-4-chromanone (7) showed inhibitory effects on PBMC proliferation activated by PHA with IC(50) values 19.4, 73.8, and 58.8 microM, respectively. All three compounds significantly inhibited the production of IL-2 and IFN-gamma in activated PBMC in a concentration-dependent manner.

Surface modification of natural fibers using bacteria: depositing bacterial cellulose onto natural fibers to create hierarchical fiber reinforced nanocomposites.

Triggered biodegradable composites made entirely from renewable resources are urgently sought after to improve material recyclability or be able to divert materials from waste streams. Many biobased polymers and natural fibers usually display poor interfacial adhesion when combined in a composite material. Here we propose a way to modify the surfaces of natural fibers by utilizing bacteria ( Acetobacter xylinum) to deposit nanosized bacterial cellulose around natural fibers, which enhances their adhesion to renewable polymers. This paper describes the process of modifying large quantities of natural fibers with bacterial cellulose through their use as substrates for bacteria during fermentation. The modified fibers were characterized by scanning electron microscopy, single fiber tensile tests, X-ray photoelectron spectroscopy, and inverse gas chromatography to determine their surface and mechanical properties. The practical adhesion between the modified fibers and the renewable polymers cellulose acetate butyrate and poly(L-lactic acid) was quantified using the single fiber pullout test.