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While most plants die below a threshold of water content, desiccation-tolerant species display specific responses that allow them to survive extreme dehydration. Some of these responses are activated at critical stages during water loss and could represent the difference between desiccation tolerance (DT) and death. Here, we report the development of a simple and reproducible system to determine DT in Selaginella species. The system is based on exposure of excised tissue to a dehydration agent inside small containers, and subsequent evaluation for tissue viability. We evaluated several methodologies to determine viability upon desiccation including: triphenyltetrazolium chloride (TTC) staining, the quantum efficiency of PSII, antioxidant potential, and relative electrolyte leakage. Our results show that the TTC test is a simple and accurate assay to identify novel desiccation-tolerant Selaginella species, and can also indicate viability in other desiccation-tolerant models (i.e. ferns and mosses). The system we developed is particularly useful to identify critical points during the dehydration process. We found that a desiccation-sensitive Selaginella species shows a change in viability when dehydrated to 40% relative water content, indicating the onset of a critical condition at this water content. Comparative studies at critical stages could provide a better understanding of DT mechanisms and unravel insights into the key responses to survive desiccation.

The

Resilience of growing in arid and semiarid regions and a high capacity of accumulating sugar-rich biomass with low lignin percentages have placed Agave species as an emerging bioenergy crop. Although transcriptome sequencing of fiber-producing agave species has been explored, molecular bases that control wall cell biogenesis and metabolism in agave species are still poorly understood. Here, through RNAseq data mining, we reconstructed the cellulose biosynthesis pathway and the phenylpropanoid route producing lignin monomers in

The advent of next generation sequencing has opened up the possibility to carry out detailed molecular genetic analysis on non-model species. Here we present strategies to carry out RNAseq analysis in Agave tequilana which for the first time have permitted the identification and characterization of genes involved in fructan metabolism and flowering time in Agave species. Based on in silico data, expression patterns for individual genes could be determined and later confirmed by qRT-PCR and cDNA´s of genes of interest were cloned and functionally characterized in heterologous systems such as Pichia pastoris and Arabidopsis thaliana. We also present data obtained by RNAseq analysis of the C. lindemuthianum–P. vulgaris pathosystem. Using strains of C. lindemuthianum expressing the green fluorescent protein, the infection process can easily be followed. RNAseq was carried out at different time points during the initial stages of infection by C. lindemuthianum on P. vulgaris cultivar BAT93 using virulent or non-virulent races of C. lindemuthianum (Race 1088 and Race 256 respectively). Differentially expressed genes specific to compatible and non-compatible reactions could therefore be identified.

Generations of farmer selection have produced a unique collection of traditional maize varieties adapted to the environmental challenges of the central Mexican highlands. In addition to agronomic and cultural value, Mexican highland maize represents a good system for the study of local adaptation and acquisition of adaptive phenotypes under cultivation. In this study, we characterized a recombinant inbred line population derived from the cross of the B73 reference line and the Mexican highland maize variety Palomero Toluqueño. Evaluation over multiple years in lowland and highland field sites in Mexico identified genomic regions linked to yield components and putatively adaptive morphological traits. A region on chromosome 7 associated with ear weight showed antagonistic allelic effects in lowland and highland fields, suggesting a trade-off consistent with local adaptation. We identified several alleles of highland origin associated with characteristic highland traits, including reduced tassel branching, increased stem pigmentation and the presence of stem macrohairs. The oligogenic architecture of characteristic morphological traits supports their role in adaptation, suggesting they have arisen from consistent directional selection acting at distinct points across the genome. We discuss these results in the context of the origin of phenotypic novelty during selection, commenting on the role ofde novomutation and the acquisition of adaptive variation by gene flow from endemic wild relatives.

Plants can tolerate some degree of dehydration but below a threshold of water content most plants die. However, some species display specific physiological, biochemical, and molecular responses that allow survival to desiccation. Some of these responses are activated at critical stages during water loss and could represent the difference between desiccation tolerance (DT) and death. Here, we report the development of a simple and reproducible system to determine DT in Selaginella species. This system is based on the use of excised tissue (explants), exposed to a dehydration agent inside small containers, rather than whole plants making it faster, better controlled, and potential use under field conditions. We also report that the triphenyltetrazolium chloride test is a simple and accurate assay to determine tissue viability. The explant system is particularly useful to identify critical points during the dehydration process and was applied to identify novel desiccation-tolerant Selaginella species. Our data suggest that desiccation-sensitive Selaginella species have a change in viability when dehydrated to 40% RWC, indicating the onset of a critical condition at this water content. Comparative studies at these critical stages could provide a better understanding of DT mechanisms and unravel insights into the key responses to survive desiccation.HighlightIn this article, we developed a simple and efficient system to determine desiccation tolerance and critical stages during the dehydration process in Selaginella that can be applied to other plant species.

Plant Glycoside Hydrolase Family 32 (PGHF32) contains the fructosyltransferases and fructan exohydrolase enzymes responsible for fructan metabolism, in addition to closely related vacuolar and cell wall acid invertases. Agave species produce complex and dynamic fructan molecules (agavins) requiring 4 different fructosyltransferase activities (1-SST, 1-FFT, 6G-FFT and 6-SFT) for their synthesis. Combined analysis of RNAseq and genome data for A. tequilana led to the characterization of the genes encoding 3 fructosyltransferases for this species and support the hypothesis that no separate 6-SFT type enzyme exists in A. tequilana, suggesting that at least one of the fructosyltransferases identified may have multiple enzymatic activities. Structures for PGHF32 genes varied for A. tequilana and between other plant species but were conserved for different enzyme types within a species. The observed patterns are consistent with the formation of distinct gene structures by intron loss. Promoter analysis of the PGHF32 genes identified abundant putative regulatory motifs for light regulation and tissue-specific expression, and these regulatory mechanisms were confirmed experimentally for leaf tissue. Motifs for phytohormone response, carbohydrate metabolism and dehydration responses were also uncovered. Based on the regulatory motifs, full-length cDNAs for MYB, GATA, DOF and GBF transcription factors were identified and their phylogenetic distribution determined by comparison with other plant species. In silico expression analysis for the selected transcription factors revealed both tissue-specific and developmental patterns of expression, allowing candidates to be identified for detailed analysis of the regulation of fructan metabolism in A. tequilana at the molecular level.

The Agave genus is composed of approximately 210 species distributed from south United States to Colombia and Venezuela. Numerous Agave species have been used for the preparation of alcoholic beverages and have attracted interest in the pharmaceutical and food industry. Despite their economic importance, there are few initiatives for the improvement and selection of characteristics of interest. This is mainly due to its morphology, long lifecycles, and monocarpic nature. Micropropagation is a feasible alternative to the improvement of Agave species. It has been used for multiple purposes, including massive propagation, induction of somaclonal variation to enhance agronomic characteristics of interest, maintenance of specific genotypes, and genetic transformation using molecular techniques. In this report, we summarize the most outstanding findings regarding the micropropagation of Agave species mediated by multiple regeneration responses. We also describe the media and growth regulators for each of the previously described methods. In addition, we discuss how micropropagation has allowed the development of transformation protocols. Exploitation of this technology may be a feasible strategy to introduce genes and improve certain traits. Genetic transformation also offers an opportunity for studying molecular mechanisms. This represents advantages for optimizing production in the field and for implementing breeding programs.

Methodology combining mass spectrometry imaging (MSI) with ion mobility separation (IMS) has emerged as a biological imaging technique due to its versatility, sensitivity and label-free approach. This technique has been shown to separate isomeric compounds such as lipids, amino acids, carboxylic acids and carbohydrates. This report describes mass spectrometry imaging in combination with traveling-wave ion mobility separation and matrix-assisted laser desorption/ionization (MALDI). Positive ionization mode was used to locate fructans on tissue printed sections of Agave rhizome and stem tissue and distinguished fructan isoforms. Here we show the location of fructans ranging from DP3 to DP17 to be differentially abundant across the stem tissue and for the first time, experimental collision cross sections of endogenous fructan structures have been collected, revealing at least two isoforms for fructans of DP4, DP5, DP6, DP7, DP8, DP10, and DP11. This demonstrates that complex fructans such as agavins can be located and their isoforms resolved using a combination of MALDI, IMS, and MSI, without the need for extraction or derivatization. Use of this methodology uncovered patterns of fructan localization consistent with functional differences where higher DP fructans are found toward the central section of the stem supporting a role in long term carbohydrate storage whereas lower DP fructans are concentrated in the highly vascularized central core of rhizomes supporting a role in mobilization of carbohydrates from the mother plant to developing offsets. Tissue specific patterns of expression of genes encoding enzymes involved in fructan metabolism are consistent with fructan structures and localization.

Together with their undeniable role in the ecology of arid and semiarid ecosystems, Agave species are emerging as a model to dissect the relationships between crassulacean acid metabolism and high efficiency of light and water use, and as an energy crop for bioethanol production. Transcriptome resources from economically valuable Agaves species, such as Agave tequilana and A. salmiana, as well as hybrids for fibers, are now available, and multiple gene expression landscape analyses have been reported. Key components in molecular mechanisms underlying drought tolerance could be uncovered by analyzing gene expression patterns of roots. This study describes an efficient protocol for high-quality total RNA isolation from phenolic compounds-rich Agave roots. Our methodology involves suitable root handling and collecting in the field and using saving-time commercial kits available. RNA isolated from roots free of lignified out-layers and clean cortex showed high values of quality and integrity according to electrophoresis and microfluidics-based platform. Synthesis of long full-length cDNAs and PCR amplification tested the suitability for downstream applications of extracted RNA. The protocol was applied successfully to A. tequilana roots but can be used for other Agave species that also develop lignified epidermis/exodermis in roots.

Background Desiccation tolerant Selaginella species evolved to survive extreme environmental conditions. Studies to determine the mechanisms involved in the acquisition of desiccation tolerance (DT) have focused on only a few Selaginella species. Due to the large diversity in morphology and the wide range of responses to desiccation within the genus, the understanding of the molecular basis of DT in Selaginella species is still limited. Results Here we present a reference transcriptome for the desiccation tolerant species S. sellowii and the desiccation sensitive species S. denticulata. The analysis also included transcriptome data for the well-studied S. lepidophylla (desiccation tolerant), in order to identify DT mechanisms that are independent of morphological adaptations. We used a comparative approach to discriminate between DT responses and the common water loss response in Selaginella species. Predicted proteomes show strong homology, but most of the desiccation responsive genes differ between species. Despite such differences, functional analysis revealed that tolerant species with different morphologies employ similar mechanisms to survive desiccation. Significant functions involved in DT and shared by both tolerant species included induction of antioxidant systems, amino acid and secondary metabolism, whereas species-specific responses included cell wall modification and carbohydrate metabolism. Conclusions Reference transcriptomes generated in this work represent a valuable resource to study Selaginella biology and plant evolution in relation to DT. Our results provide evidence of convergent evolution of S. sellowii and S. lepidophylla due to the different gene sets that underwent selection to acquire DT

Methodology combining mass spectrometry imaging (MSI) with ion mobility separation (IMS) has emerged as a biological imaging technique due to its versatility, sensitivity and label-free approach. This technique has been shown to separate isomeric compounds such as lipids, amino acids, carboxylic acids and carbohydrates. This report describes mass spectrometry imaging in combination with traveling-wave ion mobility separation and matrix-assisted laser desorption/ionization (MALDI). Positive ionization mode was used to locate fructans on tissue printed sections of Agave rhizome and stem tissue and distinguished fructan isoforms. Here we show the location of fructans ranging from DP3 to DP17 to be differentially abundant across the stem tissue and for the first time, experimental collision cross sections of endogenous fructan structures have been collected, revealing at least two isoforms for fructans of DP4, DP5, DP6, DP7, DP8, DP10, and DP11. This demonstrates that complex fructans such as agavins can be located and their isoforms resolved using a combination of MALDI, IMS, and MSI, without the need for extraction or derivatization. Use of this methodology uncovered patterns of fructan localization consistent with functional differences where higher DP fructans are found toward the central section of the stem supporting a role in long term carbohydrate storage whereas lower DP fructans are concentrated in the highly vascularized central core of rhizomes supporting a role in mobilization of carbohydrates from the mother plant to developing offsets. Tissue specific patterns of expression of genes encoding enzymes involved in fructan metabolism are consistent with fructan structures and localization.

The Agave angustifolia complex, distributed from Mexico to Costa Rica, comprises four species and five varieties, including three species used for mescal production. The complex is represented in the Mexican state of Oaxaca by two wild taxa, A. angustifolia var. angustifolia and A. angustifolia var. rubescens, the cultivated form A. angustifolia “Espadin” and the partially cultivated species A. rodacantha. The aims of this study were to investigate the morphological and genetic variation of the A. angustifolia complex in the state of Oaxaca and to identify traits useful for taxonomic delimitation. Four wild and three cultivated populations of A. angustifolia from Oaxaca, one population of A. tequilana from Guanajuato and one population of A. angustifolia from Sonora were sampled for morphological, genetic and cytometric analyses. We showed that cultivated populations of A. angustifolia “Espadin,” A. rhodacantha and A. tequilana could be clearly differentiated from wild populations. Furthermore, the domesticated populations of A. angustifolia, known locally as “Espadin,” had a higher ploidy level and lower genetic variation than their related wild populations. The population of A. angustifolia from Sonora could be recognized as a different entity. Populations of A. rhodacantha need to be studied throughout their entire distribution area to further evaluate their taxonomic delimitation.

Together with their undeniable role in the ecology of arid and semiarid ecosystems, Agave species are emerging as a model to dissect the relationships between crassulacean acid metabolism and high efficiency of light and water use, and as an energy crop for bioethanol production. Transcriptome resources from economically valuable Agaves species, such as Agave tequilana and A. salmiana, as well as hybrids for fibers, are now available, and multiple gene expression landscape analyses have been reported. Key components in molecular mechanisms underlying drought tolerance could be uncovered by analyzing gene expression patterns of roots. This study describes an efficient protocol for high-quality total RNA isolation from phenolic compounds-rich Agave roots. Our methodology involves suitable root handling and collecting in the field and using saving-time commercial kits available. RNA isolated from roots free of lignified out-layers and clean cortex showed high values of quality and integrity according to electrophoresis and microfluidics-based platform. Synthesis of long full-length cDNAs and PCR amplification tested the suitability for downstream applications of extracted RNA. The protocol was applied successfully to A. tequilana roots but can be used for other Agave species that also develop lignified epidermis/exodermis in roots.

While terahertz imaging has been used before for the determination of water content in vegetative tissue, most studies have either presented measurements of the temporal evolution of water content at a single-point of the plant or have presented two-dimensional images of leaves, demonstrating the potential of the technique, but relatively little of such information has been used to support biologically relevant conclusions. In this article we introduce terahertz time-domain spectroscopic imaging as a technique for the determination of the three-dimensional distribution of water in succulent plant tissues. We present the first three-dimensional water mapping of an agave leaf, which demonstrates an unprecedented capability to study the water retention mechanisms within succulent plants. We found that agave leaves are composed of a low-hydration outer tissue layer, defined by the outermost layer of vascular tissue that surrounds a high-hydration tissue, the carbohydrate rich hydrenchyma. The findings are supported by histological images and the correlation between the water content and carbohydrate presence is consistent with recently published findings of a remarkably large hydration shell associated with agave fructans.

Over 70% of Agave species, (159 of 206) are found in Mexico and are well adapted to survive under hot, arid conditions, often in marginal terrain, due to a unique combination of morphological and physiological attributes. In the pre-Columbian era agaves were also key to human adaptation to desert terrain. In contrast to other species such as cacti or resurrection plants, Agaves store carbohydrates in the form of fructan polymers rather than starch or sucrose, however, properties specific to fructans such as a strong hydration shell, the ability to be transported through phloem, variable composition throughout the Agave life-cycle and accumulation in succulent tissues and flowers suggest a potential for multiple functional roles. This mini-review summarizes current knowledge of molecular and biochemical aspects of fructan metabolism in Agave species.

Bixa orellana L. is a plant from the Bixaceae family commercialized for its high bixin contents. Recently, demand for this pigment has led to the development of strategies for its genetic improvement. Through molecular markers obtained by sequence-related amplified polymorphism (SRAP), one β-cyclase marker and the two-way “pseudo-testcross” strategy an integrated genetic linkage map of Bixa orellana L. has been developed. Genetic maps were constructed for each parent and an integrated map using an F1 population with 157 individuals was produced. A total of 167 markers were used in linkage analysis. The maternal map covers a length of 306 cM, consisting of 52 markers with an average distance between them of 13.21 cM, distributed over seven linkage groups. The paternal map covers a length of 509 cM, consisting of 70 markers with an average distance between them of 12.28 cM, distributed over 12 linkage groups. The integrated genetic map covers a length of 879.054 cM, comprising 103 markers (102 SRAP and the β-cyclase), of which, 68% segregated in a 1:1 ratio and 32% in a 3:1 ratio, with an average distance of 14.6 cM between them, and distributed over 20 linkage groups. The size of the B. orellana genome was estimated between 1514 and 1728 cM. This study provides a basis for future construction of a high-density map and for genomic studies of B. orellana, in relation to bixin production and fruit dehiscence. It will also be useful in marker-assisted selection (MAS) within breeding programs of B. orellana and other species.

Phaseolus vulgaris line A193 has been shown to be widely resistant to Colletotrichum lindemuthianum, including race 1472, one of the most virulent races of C. lindemuthianum characterized. Resistance to C. lindemuthianum race 1472 in P. vulgaris line A193 was investigated in segregating F2 and F2.3 populations from a cross between A193 and Flor de Mayo Bajio (a commercial cultivar highly susceptible to C. lindemuthianum). Resistance to 1472 in A193 was determined to be conditioned by a single dominant gene. Inoculation of crosses between A193 and cultivars Michigan Dark Red Kidney and Perry Marrow with race 1472 suggest that resistance in A193 is conditioned by the Co-1 gene. Inoculation of the cross A193 × Perry Marrow with C. lindemuthianum race 2, demonstrated that resistance to race 2 in Perry Marrow is also conditioned by a single dominant gene and is distinct to resistance in A193 or Michigan Dark Red Kidney. Three AFLP markers (ECAG/MACC-1, EACA/MAGA-2, EAGG/MAAC-8) linked in repulsion to the Co-1 locus were identified by screening the A193 × Flor de Mayo F2 population with 314 random amplified polymorphic DNA, amplified fragment length polymorphism, and restriction fragment length polymorphism markers. The two most closely linked markers should be useful in marker-assisted selection for Co-1.

As maize was domesticated in Mexico around 9,000 years ago, local farmers have selected and maintained seed stocks with particular traits and adapted to local conditions. In the present day, many of these landraces are still cultivated; however, increased urbanization and migration from rural areas implies a risk that this invaluable maize germplasm may be lost. In order to implement an efficient mechanism of conservation in situ, the diversity of these landrace populations must be estimated. Development of a method to select the minimum number of samples that would include the maximum number of alleles and identify germplasm harboring rare combinations of particular alleles will also safeguard the efficient ex‐situ conservation of this germplasm. To reach this goal, a strategy based on SSR analysis and a novel algorithm to define a minimum collection and rare genotypes using landrace populations from Puebla State, Mexico, was developed as a “proof of concept” for methodology that could be extended to all maize landrace populations in Mexico and eventually to other native crops. The SSR‐based strategy using bulked DNA samples allows rapid processing of large numbers of samples and can be set up in most laboratories equipped for basic molecular biology. Therefore, continuous monitoring of landrace populations locally could easily be carried out. This methodology can now be applied to support incentives for small farmers for the in situ conservation of these traditional cultivars.

Transcriptome analysis of different tissues and developmental stages of A. tequilana plants led to the identification of full length cDNAs and the corresponding amino acid sequences for enzymes involved in starch metabolism in this species. Comparison with sequences from other species confirmed the identities of putative A. tequilana starch metabolism genes and uncovered differences in the evolutionary patterns of these genes between gramineous and non-gramineous monocotyledons. In silico expression patterns showed high levels of expression of starch metabolism genes in shoot apical meristem tissue and histological studies showed the presence of starch in leaf primordia surrounding the shoot apical meristem and in the primary thickening meristem of the stem. Starch was also found to accumulate significantly in developing floral organs and immature embryos. Low levels of starch were observed overall in leaf tissue with the exception of stomatal guard cells where starch was abundant. In root tissue, starch was only observed in statoliths at the root tip. A. tequilana starch grains were found to be small in comparison to other species and have an almost spherical form. The data for gene expression and histological localization are consistent with a role for starch as a transient carbohydrate store for actively growing tissues in A. tequilana.

Observation of a differential expression pattern, including strong expression in meristematic tissue of an Agave tequilana GlsA/ZRF ortholog suggested an important role for this gene during bulbil formation and developmental changes in this species. In order to better understand this role, the two GlsA/ZFR orthologs present in the genome of Arabidopsis thaliana were functionally characterized by analyzing expression patterns, double mutant phenotypes, promoter-GUS fusions and expression of hormone related or meristem marker genes. Patterns of expression for A. thaliana show that GlsA/ZFR genes are strongly expressed in SAMs and RAMs in mature plants and developing embryos and double mutants showed multiple changes in morphology related to both SAM and RAM tissues. Typical double mutants showed stunted growth of aerial and root tissue, formation of multiple ectopic meristems and effects on cotyledons, leaves and flowers. The KNOX genes STM and BP were overexpressed in double mutants whereas CLV3, WUSCHEL and AS1 were repressed and lack of AtGlsA expression was also associated with changes in localization of auxin and cytokinin. These results suggest that GlsA/ZFR is an essential component of the machinery that maintains the integrity of SAM and RAM tissue and underline the potential to identify new genes or gene functions based on observations in non-model plants.

Puya raimondii, the giant Peruvian and Bolivian terrestrial bromeliad, is an emblematic endemic Andean species well represented in Huascarán National Park in Peru. This park is the largest reserve of puna (high altitude plateau) vegetation. The objective of this study is to report on genetic variation in populations of P. raimondii from Huascarán and neighboring areas. AFLP profiles with four selective primer combinations were retrieved for 60 individuals from different zones. Genetic variability was estimated and a total of 172 bands were detected, of which 79.1% were polymorphic loci. The results showed genetic differentiation among populations, and gene flow. A cluster analysis showed that individuals of P. raimondii populations located in different mountain systems could be grouped together, suggesting long distance dispersal. Thus, conservation strategies for P. raimondii have to take into account exchange between populations located far apart in distance in order to preserve the genetic diversity of this showy species. Puya raimondii, uma bromélia terrestre gigante do Peru e da Bolívia, é uma espécie Andina endêmica e emblemática bem representada no Parque Nacional Huascarán, no Peru. O objetivo deste estudo é caracterizar a variação genética entre e dentro de populações de P. raimondii de Huascarán e áreas vizinhas. Perfis de AFLP com quatro combinações de oligonucleotidios foram recuperados de 60 indivíduos provenientes de zonas diferentes. A variabilidade genética foi estimada, e um total de 172 bandas foram detectadas, das quais o 79,1% foram locos polimórficos. Os resultados mostraram a diferenciação genética entre as populações e o fluxo gênico. A análise de cluster mostrou que os indivíduos de populações de P. raimondii localizados em diferentes sistemas montanhosos agruparam-se, sugerindo dispersão a longa distância. Assim, estratégias de conservação para P. raimondii deve considerar o intercâmbio de indivíduos entre as populações localizadas a longa distância, a fim de preservar a diversidade genética desta espécie.

Genetic variation in three forms of asexually propagated Agave tequilana Weber var. 'Azul' plants namely offsets, bulbils and in vitro cultured individuals was studied by AFLP analysis. Low levels of variation were observed between mother plants and offsets and a higher level between mother plant and bulbils. Families obtained from commercial plantations showed lower levels of variation in comparison to families grown as ornamentals. No variation was observed between the original explant and four generations of in vitro cultured plants. Epigenetic variation was also studied by analyzing changes in methylation patterns between mother plants and offspring in each form of asexual reproduction. Offsets and bulbils showed an overall decrease in methylation whereas in vitro cultured plants showed patterns specific to each generation: Generations 1 and 4 showed overall demethylation whereas Generations 2 and 3 showed increased methylation. Analysis of ESTs associated with transposable elements revealed higher proportions of ESTs from Ty1-copia-like, Gypsy and CACTA transposable elements in cDNA libraries obtained from pluripotent tissue suggesting a possible correlation between methylation patterns, expression of transposable element associated genes and somaclonal variation.

Agave tequilana is a monocarpic perennial species that flowers after 5-8 years of vegetative growth signaling the end of the plant's life cycle. When fertilization is unsuccessful, vegetative bulbils are induced on the umbels of the inflorescence near the bracteoles from newly formed meristems. Although the regulation of inflorescence and flower development has been described in detail for monocarpic annuals and polycarpic species, little is known at the molecular level for these processes in monocarpic perennials, and few studies have been carried out on bulbils. Histological samples revealed the early induction of umbel meristems soon after the initiation of the vegetative to inflorescence transition in A. tequilana. To identify candidate genes involved in the regulation of floral induction, a search for MADS-box transcription factor ESTs was conducted using an A. tequilana transcriptome database. Seven different MIKC MADS genes classified into 6 different types were identified based on previously characterized A. thaliana and O. sativa MADS genes and sequences from non-grass monocotyledons. Quantitative real-time PCR analysis of the seven candidate MADS genes in vegetative, inflorescence, bulbil and floral tissues uncovered novel patterns of expression for some of the genes in comparison with orthologous genes characterized in other species. In situ hybridization studies using two different genes showed expression in specific tissues of vegetative meristems and floral buds. Distinct MADS gene regulatory patterns in A. tequilana may be related to the specific reproductive strategies employed by this species.

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterial pathogen causing bacterial wilt and canker of tomato (Solanum lycopersicum), producing economic losses worldwide. In this study, gene expression analysis was conducted using several resistant tomato-related wild species, including Solanum peruvianum LA2157, S. peruvianum LA2172, and Solanum habrochaites LA2128, and a tomato susceptible species, to identify genes involved in disease response. Using cDNA-amplified fragment length polymorphism (AFLP), 403 differentially expressed transcripts were identified. Among those, several genes showed contrasting expression patterns among resistant and susceptible species, including genes involved in the ubiquitin-mediated protein degradation pathway and secretory peroxidase. These genes were up-regulated in resistant species, but down-regulated in susceptible species, suggesting their likely involvement in early plant defense responses following C. michiganensis subsp. michiganensis infection. These identified genes would serve as new candidate bacterial wilt disease resistance genes and should be subjected to further functional analyses to determine the molecular basis of incompatibility between wild species of tomato and C. michiganensis subsp. michiganensis. This would then contribute to the development of more effective and sustainable C. michiganensis subsp. michiganensis control methods.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption; however, bean production is affected by several diseases such as Rhizoctonia root rot. Few bean cultivars have been identified that effectively resist the attack of this fungus. Herein, we used the P. vulgaris Pv-2094 landrace, which is less susceptible to Rhizoctonia root rot, for the construction of a suppressive subtractive hybridization cDNA library in order to isolate plant defense-related genes. Total RNAs obtained after 8 and 16 h from inoculated and non-inoculated roots with R. solani Kühn, were used as the source of the "tester" and the "driver" samples, respectively. A total of 136 unigenes were obtained and classified into 12 functional categories. Six unigenes were selected to analyze for differential expression by qRT-PCR, including a receptor-like kinase (PvRK20-1), an acid phosphatase associated to defense (PA), a pathogenesis related protein (PR1), an ethylene responsive factor (ERF), a polygalacturonase inhibitor protein (PGIP), and an alpha-dioxygenase (α-DOX). These genes were found to be differentially expressed in a time-dependent manner in bean roots during the interaction with R. solani. Data generated from this study will contribute to the understanding of the molecular mechanisms associated with plant defense against root rot in common bean.

Different species of Agave are grown commercially in Mexico for the production of alcoholic beverages and fibers. These plants are well adapted to dry, arid conditions and can be cultivated on land which is unsuitable for staple crops such as corn or beans. A substantial amount of waste material in the form of discarded leaves or stem tissue is produced from commercial applications and an attractive alternative proposition is to employ this waste for bioenergy production. To date little basic research at the moleculargenetic level in agave has been carried out and more detailed and directed work in this area is necessary in order to fully develop agave species as bioenergy crops. The current genomic resources available for agave and the potential for transcriptome mining in relation to bioenergy applications are discussed.

El género Agave pertenece a la familia Agavaceae y cuenta con alrededor de 200 especies, con diversos usos. Agave tequilana es una de las especies más importantes a nivel industrial en México, ya que los azúcares que se acumulan en sus tallos son usados para producir la bebida alcohólica llamada tequila. La producción de agave es soportada por una intensiva propagación clonal y supresión de la reproducción sexual, lo cual ha conducido a reducción en la variabilidad genética y mayor susceptibilidad a plagas y enfermedades en plantaciones comerciales. No obstante, este estudio con plantas de un campo de cultivo del Estado de Guanajuato, México, llevado a cabo con marcadores AFLP, muestra que existen diferencias a nivel molecular entre hijuelos y bulbillos producidos asexualmente provenientes de la misma planta madre (75.08 % y 86.06 % de loci polimórficos, respectivamente). Aunque se observa un nivel significativo de polimorfismo entre hijuelos de rizoma, los niveles son aún más altos entre bulbillos con valores comparables a los encontrados entre plantas producidas por semilla (90.1 %). Por ello se propone un estudio más detallado de los factores que están causando la variabilidad genética entre individuos de Agave tequilana propagados asexualmente.

Roots are vital for the uptake of water and nutrients, and for anchorage in the soil. They are highly plastic, able to adapt developmentally and physiologically to changing environmental conditions. Understanding the molecular mechanisms behind this growth and development requires knowledge of root transcriptomics, proteomics, and metabolomics. Genomics approaches, including the recent publication of a root expression map, root proteome, and environment-specific root expression studies, are uncovering complex transcriptional and post-transcriptional networks underlying root development. The challenge is in further capitalizing on the information in these datasets to understand the fundamental principles of root growth and development. In this review, we highlight progress researchers have made toward this goal.

Agave tequilana Weber var. azul is the raw material used in the production of tequila. This species has a life cycle of approximately 6–8 years; however, owing to the practice of removing the inflorescence to conserve accumulated sugar reserves, the main form of reproduction is asexual. Little attention has, therefore, been paid to the process of flowering and the factors leading to low levels of germination and seedling viability have not been investigated in detail. The objective of this study was to document gametophyte development, seed production, and germination in A. tequilana under different pollination treatments and in an interspecies cross with Agave americana L. Seed production and germination efficiency was low for both A. tequilana and A. americana under the different pollination treatments, although interspecies crosses did produce some viable seeds. Development of the male gametophyte in both species is of the successive type, producing pollen grains with dicolpate morphology. Female gametophyte development is of the Polygonum monosporic type. The results obtained suggest that genetic incompatibility, inbreeding effects, factors affecting pollen development and germination, or errors in female gametophyte development may contribute to the low fertility observed for A. tequilana and A. americana.

A genetic linkage map of the fungal plant pathogen Mycosphaerella fijiensis, causal agent of black leaf streak disease of banana was developed. A cross between the isolates CIRAD86 (from Cameroon) and CIRAD139A (from Colombia) was analyzed using molecular markers and the MAT locus. The genetic linkage map consists of 298 AFLP and 16 SSR markers with 23 linkage groups, containing five or more markers, covering 1,879 cM. Markers are separated on average by around 5.9 cM. The MAT locus was shown to segregate in a 1:1 ratio but could not be successfully mapped. An estimate of the relation between physical size and genetic distance was approximately 39.0 kb/cM. The estimated total haploid genome size was calculated using the genetic mapping data at 4,298.2 cM. This is the first genetic linkage map reported for this important foliar pathogen of banana. The great utility of the map will be for anchoring contigs in the genome sequence, evolutionary studies in comparison with other fungi, to identify quantitative trait loci (QTLs) associated with aggressiveness or oxidative stress resistance and with the recently available genome sequence, for positional cloning.

In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

Pathogenic and genetic characterizations of 96 isolates of Macrophomina phaseolina from Mexico and other countries were carried out in order to define the pathogenic and genetic patterns of diversity and to gain insights into the pathogenic and genetic specialization in this fungus. Isolates were collected from different hosts in Mexico (23 isolates), Italy, Australia, Japan, Argentina, USA, Colombia and Brazil (73 isolates). Pathogenicity was evaluated in seeds of two Phaseolus vulgaris L. cultivars under in vitro conditions, while genotype was determined on the basis of amplified fragment-length polymorphisms (AFLPs). The most frequent pathotypes were 4095 (15 isolates) and 0 (nine isolates), while 59 isolates had a unique pathotype (61%). Cluster analysis showed two contrasting groups of isolates on the basis of pathogenicity, where one group mainly included the most aggressive isolates from Mexico and Colombia. The AFLP analysis produced 418 amplified products and 92.8% were polymorphic. Cluster analysis showed no clear association between AFLP genotype and geographical origin. When subgroups of isolates were re-analysed, we found a clear differentiation between Mexican and non-Mexican isolates. Results confirmed the significant pathogenic and genetic diversity of Macrophomina and showed a clear differentiation between Mexican isolates from all others.

Several varieties of Agave tequilana Weber have been described, but only A. tequilana Weber var. “azul” is accepted for the production of tequila. Discrimination between the different varieties is difficult and the relationships between them at the genotype level are unclear. In addition genetic diversity within and between populations of A. tequilana Weber var. “azul” has been shown to be low. AFLP analysis of nine A. tequilana varieties indicated that certain varieties were genotypically close to A. tequilana Weber var. “azul”. A significant level of diversity was also observed among A. tequilana Weber var. “azul” samples obtained from the same plantation and from different plantations in Guanajuato State, Mexico.

Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the initial step in the biosynthesis of polyamines, the conversion of ornithine to putrescine. Based on the most conserved regions of fungal ODCs, we designed and synthesized oligonucleotides to amplify homologous fragments of three important plant pathogenic Pyrenomycete fungi (Ascomycota), Magnaporthe grisea, Colletotrichum lindemuthianum and Fusarium solani, and one insect pathogenic fungus Metarhizium anisopliae. Cloning and sequencing of the amplified fragments revealed homologies of between 37 to 88% with other fungal ODCs. The predicted peptide sequences were compared by Clustal analysis and conserved sequences corresponding to the substrate and cofactor binding sites were identified. Comparative analyses of the ODC fragments isolated in this study, revealed high homology between them (68.3–81.1%) and also with other Pyrenomycetes such as Neurospora crassa (order Sordariales; 68.6–72.9%) and Fusarium graminearum (order Hypocreales; 70.8–88.1%). D...

Although several reports have described the occurrence of the teleomorphic state of Glomerella lindemuthiana (anamorph, Colletotrichum lindemuthianum), there has been a lack of continuity in this research. To identify G. lindemuthiana isolates capable of developing the teleomorphic state, 19 Mexican isolates were analyzed. Three types of response were observed: (i) negative, where only mycelial growth with or without acervuli was observed; (ii) potential, where in addition to the above, spherical perithecia-like structures were observed; (iii) positive, where perithecia containing asci and ascospores were observed. All strains were self-sterile and only one combination of strains produced fertile perithecia. From this fertile combination 168 individual ascospore cultures were isolated, including five from a single ascus. Forty-four monoascospore cultures were characterized with AFLP, confirming that these individuals were progeny from a sexual cross between the original two G. lindemuthiana isolates and that sexual reproduction in G. lindemuthiana is heterothallic in nature. Analysis of the parental strains with degenerate PCR primers indicated that sequences homologous to the HMG box of the MAT1-2 idiomorph are present in both parental isolates. This supports previous observations in other Glomerella species where the standard ascomycete configuration of distinct idiomorphs at the MAT locus does not hold true. The significance of these results is discussed.

The Mexican pineapple germplasm collection represents the genetic diversity of cultivated pineapple [Ananas comosus (L.) Merr.] in that country and includes important genotypes from Hawaii, Ivory Coast, and Latin America. The collection has been partially characterized at the morphological level, but a molecular characterization has been lacking. With this aim, 39 genotypes of A. comosus var. comosus Coppens & Leal, two of A. comosus var. bracteatus (Lindl) Coppens & Leal, two of A. comosus var. ananassoides (Baker) Coppens & Leal, and three from the related genus Bromelia L. were analyzed with a total of 169 amplified fragment length polymorphism (AFLP) markers. A dendrogram representing the genetic relationships between these samples based on the AFLP results, showed a low level of diversity in the Mexican pineapple collection. In general, the molecular classification of the materials agreed well with the morphological classification. Several groups of genotypes showed distances of

Three transgenic sugarcane populations produced by Agrobacterium tumefaciens infection were analysed for the presence of genomic variability. Plants of the original cultivar, plants regenerated without transformation, as well as transformed and untransformed calli were used as control treatments. Amplified fragment length polymorphism (AFLP) of DNA extracted from leaves or calli assessed genomic profiling. The average DNA polymorphism within each population was determined by calculating the polymorphism index, while the extent of genomic dissimilarity among individual plants within transgenic populations was verified in unweighted pair group method using arithmetic averages dendrograms. The results showed that the production of transgenic sugarcane plants by A. tumefaciens infection is accompanied by limited but detectable genomic changes and that, on average, these occur at the same rate in plant populations carrying different transgenes. Main factors contributing to somaclonal variation in transgenic sugarcane plants have been verified by pre-existing DNA polymorphism into the donor genotype and in vitro culture steps during the transformation procedure. The relevant practical conclusion from this finding is that the AFLP analysis may be effectively used to identify individual transgenic plants with the least genomic deviation from the parental ones. The selected genotype would be conserved as cultivated sugarcane is asexually propagated.

The pathotypes of 17 isolates of Colletotrichum lindemuthianum from the central region of Mexico were characterized to determine the genetic relationship among isolates from this region and other regions of Mexico, and to evaluate the resistance present in the elite germ plasm collection of Phaseolus vulgaris at INIFAP. Eight pathotypes were identified, including pathotype 292, which is reported for the first time in Mexico. The lack of isolates infecting cultivar TU carrying the Co-5 resistance gene suggests that this cultivar is a useful source of resistance. Six pathotypes produced susceptible reactions on only differential cultivars of Middle American origin, one pathotype on a single cultivar of Andean origin, and one pathotype on cultivars of both Middle American and Andean origin. Comparison of amplified fragment length polymorphism (AFLP) genotypes of 21 isolates confirmed suggestions that populations of C. lindemuthianum are comprised of asexually reproducing clonal lineages. Analysis of five different pathotypes of C. lindemuthianum on 21 elite genotypes of P. vulgaris identified four genotypes from different races of P. vulgaris resistant to all five pathotypes. This information will allow breeders and farmers to select the resistant genotypes most suited to their needs.

Sucrose phosphate synthase (SPS) is a key enzyme in the translocation of photoassimilates from sink-to-source tissues and the remobilization of reserves from storage tissues during seed germination. The tissue specific distribution of SPS is mainly determined by transcriptional mechanisms. To gain further insight into the mechanisms that regulate the transcriptional activity of SPS genes, we carried out a deletion analysis of the rice sps1 promoter using transient expression assays and transgenic rice plants. The sps1 promoter is atypical since it lacks recognizable TATA and initiator sequences near the transcription start site. It was found that the minimal active sps1 promoter consists of a 146 bp 5′ flanking sequence that is capable of directing activity in photosynthetic tissues in rice. No positive or negative regulatory elements upstream to position −146 influencing expression in photosynthetic tissues were found. It was also observed that the sps1 promoter fragment containing 5′ sequences upstream to position −1830 conferred expression only in the scutellum of germinating seeds, whereas shorter promoter versions directed expression in the scutellum but also in the aleurone layer, suggesting the presence of a negative element upstream of position −1188 that suppresses expression in the aleurone. Gain of function experiments suggest that the negative element present upstream of position −1188 is necessary but not sufficient to suppress expression in the aleurone.

Patterns of variation were determined in anastomosis, pathotype and genotype of Colletotrichum lindemuthianum samples collected from individual plants of common bean cultivars from four locations in the Mexican highlands. In Chihuahua, 22 polymorphic AFLP bands in isolates taken from a single plant identified five distinct genotypes. In Michoacan, nine genotypes were identified based on a total of only six polymorphic bands. Combined cluster analysis of all isolates from individual plants grouped them geographically. In Chihuahua, in isolates from 16 individual plants, only nine genotypes were identified and all samples were found to belong to the same anastomosis group. However, analysis of selected isolates revealed two new pathotypes not reported previously from Chihuahua (races 449 and 467). In contrast, in Michoacan all 13 isolates from individual plants were found to have distinct genotypes, and in Mexico State only two pairs of isolates among 20 samples had identical genotypes. Although no pathotype variation was determined, five anastomosis groups were identified in Michoacan and three in Mexico State. These results suggest that patterns of variation in genotype and anastomosis groups are complex in the different locations sampled, and that no strong relationship exists between genotype, pathotype or anastomosis group.

With the development of methodologies to introduce and express foreign genes in plant cells, in less than two decades it has been possible to produce transgenic plants with improved insect and disease resistance, seeds and fruits with enhanced nutritional qualities, and plants that are better adapted to adverse environmental conditions. Transgenic plants producing vaccines for important human diseases and other important products have also been developed. Many more agronomic and quality traits are currently been engineered in both academic and industrial laboratories and the application of new strategies to improve plants by transgenic technology is only limited by our poor knowledge of plant gene function. The new functional genomic strategies for the identification and characterization of genes promise to provide a wealth of information with an enormous potential to genetically engineer plants for specific purposes.

The postembryonic developmental program of the plant root system is plastic and allows changes in root architecture to adapt to environmental conditions such as water and nutrient availability. Among essential nutrients, phosphorus (P) often limits plant productivity because of its low mobility in soil. Therefore, the architecture of the root system may determine the capacity of the plant to acquire this nutrient. We studied the effect of P availability on the development of the root system in Arabidopsis. We found that at P-limiting conditions (

Alterations in water relations, growth and histopathology caused by Macrophomina phaseolina , causal agent of charcoal rot, and drought stress were characterized in Phaseolus vulgaris L. under controlled conditions. P. vulgaris cultivars BAT 477 and TLP 19 (resistant) and Pinto UI-114 and Rio Tibagi (susceptible) were cultivated under irrigation and drought stress conditions in infested or uninfested pots with a highly virulent isolate of M. phaseolina . Drought stress showed higher negative effects than M. phaseolina on water relations, vegetative growth and histopathology in P. vulgaris . Drought stress decreased transpiration rate, water potential, osmotic potential, turgor potential, relative water content, leaf area and dry weight of all vegetative structures of P. vulgaris . Drought stress increased charcoal rot development and stomatal resistance, and increased the association among physiological and growth characteristics and charcoal rot development. M. phaseolina invaded between epidermal cells of BAT 477 and Pinto UI-114 hypocotyls. The fungus infected cortex tissues, vascular cylinder, and pith cells of Pinto UI-114, but only epidermal and parenchyma cells of BAT 477. Typical symptoms caused by M. phaseolina were found to be associated with damage caused by the fungus on host tissues, and they were related to drought stress.

Macrophomina phaseolina infects a wide range of plant species. In the case of Phaseolus vulgaris (the common bean), the fungus causes charcoal rot which can lead to severe losses especially under arid conditions. In order to combat the disease, it is necessary to have information on the population structure of the pathogen in terms of pathogenesis on different species which may be grown simultaneously or successively within a region. Previous reports have demonstrated the heterogeneity of the pathogen both in terms of morphological characteristics and molecular genotype; however no systematic attempt has been made to compare pathogenesis of different isolates in relation to genotype, species of origin or geographical location. With this aim 84 isolates of M. phaseolina from different plant species in different geographical regions of Mexico were analysed by inoculation on a set of P. vulgaris cultivars differing in reaction to the fungus and by determination of their amplified fragment length polymorphism (AFLP) genotype. Forty three distinct pathotypes were identified, only two of which were found in more than one species. All 84 isolates had distinct AFLP genotypes and cluster analysis showed a tendency of the isolates to form groups related to geographical origin or species of origin.

We previously isolated and sequenced the major trypsin inhibitor from Amaranthus hypochondriacus seeds. This amaranth trypsin inhibitor (AmTI) is a 69 amino acid protein with high homology to members of the potato-1 inhibitor family. This paper describes the cloning and expression of a cDNA encoding this trypsin inhibitor in various vegetative tissues of the amaranth plant during seed development and imbibition, and investigates the possible induction of AmTI expression by wounding.