Publications by authors named "June Simpson"

33 Publications

Localization and Composition of Fructans in Stem and Rhizome of Weber var. azul.

Front Plant Sci 2020 20;11:608850. Epub 2021 Jan 20.

Metabolomics and Mass Spectrometry Laboratory, National Laboratory of Genomics for Biodiversity, Unidad de Genómica Avanzada (CINVESTAV), Irapuato, Mexico.

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 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.
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http://dx.doi.org/10.3389/fpls.2020.608850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855178PMC
January 2021

Improved method for isolation of high-quality total RNA from Weber roots.

3 Biotech 2021 Feb 15;11(2):75. Epub 2021 Jan 15.

CONACYT Research Fellow, Colegio de Postgraduados Campus Campeche, Carretera Haltunchen-Edzna km 17.5, Sihochac, 24450 Campeche, Mexico.

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 and , 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 roots but can be used for other Agave species that also develop lignified epidermis/exodermis in roots.
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http://dx.doi.org/10.1007/s13205-020-02620-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810796PMC
February 2021

Comparative transcriptome analysis suggests convergent evolution of desiccation tolerance in Selaginella species.

BMC Plant Biol 2020 Oct 12;20(1):468. Epub 2020 Oct 12.

National Laboratory of Genomics for Biodiversity (Langebio), Unit of Advanced Genomics, CINVESTAV, 36824, Irapuato, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.1186/s12870-020-02638-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549206PMC
October 2020

Corrigendum: The Sweet Taste of Adapting to the Desert: Fructan Metabolism in Species.

Front Plant Sci 2020 28;11:659. Epub 2020 May 28.

Department of Genetic Engineering, Cinvestav Unidad Irapuato, Guanajuato, Mexico.

[This corrects the article DOI: 10.3389/fpls.2020.00324.].
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http://dx.doi.org/10.3389/fpls.2020.00659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326099PMC
May 2020

The Sweet Taste of Adapting to the Desert: Fructan Metabolism in Species.

Front Plant Sci 2020 24;11:324. Epub 2020 Mar 24.

Department of Genetic Engineering, Cinvestav Unidad Irapuato, Guanajuato, Mexico.

Over 70% of 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, 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 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 species.
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http://dx.doi.org/10.3389/fpls.2020.00324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105686PMC
March 2020

Three-dimensional water mapping of succulent Agave victoriae-reginae leaves by terahertz imaging.

Sci Rep 2020 Jan 29;10(1):1404. Epub 2020 Jan 29.

Centro de Investigaciones en Optica A.C., Loma del Bosque 115, Lomas del Campestre, Leon, Guanajuato, 37150, Mexico.

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.
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http://dx.doi.org/10.1038/s41598-020-58277-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989691PMC
January 2020

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

BMC Genomics 2019 Jun 10;20(1):473. Epub 2019 Jun 10.

Department of Genetic Engineering, Cinvestav Unidad Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-León, Apdo. Postal 629, 36821, Irapuato, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.1186/s12864-019-5808-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558708PMC
June 2019

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

J Exp Bot 2019 11;70(22):6521-6537

Instituto de Ecología, Universidad Nacional Autónoma de México, Unidad Hermosillo, Sonora, México.

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.
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http://dx.doi.org/10.1093/jxb/erz223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883259PMC
November 2019

Starch accumulation is associated with active growth in A. tequilana.

Plant Physiol Biochem 2018 Sep 11;130:623-632. Epub 2018 Aug 11.

Department of Genetic Engineering, Cinvestav Unidad Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-León, Apdo. Postal 629, 36821, Irapuato, Guanajuato, Mexico. Electronic address:

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.
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http://dx.doi.org/10.1016/j.plaphy.2018.08.011DOI Listing
September 2018

Allele specific expression analysis identifies regulatory variation associated with stress-related genes in the Mexican highland maize landrace Palomero Toluqueño.

PeerJ 2017 23;5:e3737. Epub 2017 Aug 23.

Unidad de Genómica Avanzada (LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato, Mexico.

Background: Gene regulatory variation has been proposed to play an important role in the adaptation of plants to environmental stress. In the central highlands of Mexico, farmer selection has generated a unique group of maize landraces adapted to the challenges of the highland niche. In this study, gene expression in Mexican highland maize and a reference maize breeding line were compared to identify evidence of regulatory variation in stress-related genes. It was hypothesised that local adaptation in Mexican highland maize would be associated with a transcriptional signature observable even under benign conditions.

Methods: Allele specific expression analysis was performed using the seedling-leaf transcriptome of an F individual generated from the cross between the highland adapted Mexican landrace Palomero Toluqueño and the reference line B73, grown under benign conditions. Results were compared with a published dataset describing the transcriptional response of B73 seedlings to cold, heat, salt and UV treatments.

Results: A total of 2,386 genes were identified to show allele specific expression. Of these, 277 showed an expression difference between Palomero Toluqueño and B73 alleles under benign conditions that anticipated the response of B73 cold, heat, salt and/or UV treatments, and, as such, were considered to display a prior stress response. Prior stress response candidates included genes associated with plant hormone signaling and a number of transcription factors. Construction of a gene co-expression network revealed further signaling and stress-related genes to be among the potential targets of the transcription factors candidates.

Discussion: Prior activation of responses may represent the best strategy when stresses are severe but predictable. Expression differences observed here between Palomero Toluqueño and B73 alleles indicate the presence of -acting regulatory variation linked to stress-related genes in Palomero Toluqueño. Considered alongside gene annotation and population data, allele specific expression analysis of plants grown under benign conditions provides an attractive strategy to identify functional variation potentially linked to local adaptation.
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http://dx.doi.org/10.7717/peerj.3737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572453PMC
August 2017

An SSR-based approach incorporating a novel algorithm for identification of rare maize genotypes facilitates criteria for landrace conservation in Mexico.

Ecol Evol 2017 03 10;7(6):1680-1690. Epub 2017 Feb 10.

Department of Plant Genetic Engineering CINVESTAV -Irapuato Irapuato Guanajuato Mexico.

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.
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http://dx.doi.org/10.1002/ece3.2754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5355182PMC
March 2017

Arabidopsis thaliana gonidialess A/Zuotin related factors (GlsA/ZRF) are essential for maintenance of meristem integrity.

Plant Mol Biol 2016 May 29;91(1-2):37-51. Epub 2016 Jan 29.

Department of Plant Genetic Engineering, CINVESTAV Unidad Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-León, Apdo. Postal 629, 36821, Irapuato, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.1007/s11103-016-0439-xDOI Listing
May 2016

New insights into plant glycoside hydrolase family 32 in Agave species.

Front Plant Sci 2015 5;6:594. Epub 2015 Aug 5.

Department of Genetic Engineering, Centro de Investigación y Estudios Avanzados Irapuato, Mexico.

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.
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http://dx.doi.org/10.3389/fpls.2015.00594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524927PMC
August 2015

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

J Exp Bot 2015 Jul 23;66(13):3893-905. Epub 2015 Apr 23.

Department of Plant Genetic Engineering, Cinvestav Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-León, Apdo. Postal 629, 36821 Irapuato, Guanajuato, Mexico

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.
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http://dx.doi.org/10.1093/jxb/erv191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473989PMC
July 2015

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

PLoS One 2013 19;8(11):e79936. Epub 2013 Nov 19.

Department of Plant Breeding, Universidad Autónoma Agraria Antonio Narro, Saltillo, Coahuila, Mexico.

The strategy of bulk DNA sampling has been a valuable method for studying large numbers of individuals through genetic markers. The application of this strategy for discrimination among germplasm sources was analyzed through information theory, considering the case of polymorphic alleles scored binarily for their presence or absence in DNA pools. We defined the informativeness of a set of marker loci in bulks as the mutual information between genotype and population identity, composed by two terms: diversity and noise. The first term is the entropy of bulk genotypes, whereas the noise term is measured through the conditional entropy of bulk genotypes given germplasm sources. Thus, optimizing marker information implies increasing diversity and reducing noise. Simple formulas were devised to estimate marker information per allele from a set of estimated allele frequencies across populations. As an example, they allowed optimization of bulk size for SSR genotyping in maize, from allele frequencies estimated in a sample of 56 maize populations. It was found that a sample of 30 plants from a random mating population is adequate for maize germplasm SSR characterization. We analyzed the use of divided bulks to overcome the allele dilution problem in DNA pools, and concluded that samples of 30 plants divided into three bulks of 10 plants are efficient to characterize maize germplasm sources through SSR with a good control of the dilution problem. We estimated the informativeness of 30 SSR loci from the estimated allele frequencies in maize populations, and found a wide variation of marker informativeness, which positively correlated with the number of alleles per locus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079936PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833943PMC
July 2014

De novo transcriptome assembly of drought tolerant CAM plants, Agave deserti and Agave tequilana.

BMC Genomics 2013 Aug 19;14:563. Epub 2013 Aug 19.

DOE Joint Genome Institute, Walnut Creek, CA, USA.

Background: Agaves are succulent monocotyledonous plants native to xeric environments of North America. Because of their adaptations to their environment, including crassulacean acid metabolism (CAM, a water-efficient form of photosynthesis), and existing technologies for ethanol production, agaves have gained attention both as potential lignocellulosic bioenergy feedstocks and models for exploring plant responses to abiotic stress. However, the lack of comprehensive Agave sequence datasets limits the scope of investigations into the molecular-genetic basis of Agave traits.

Results: Here, we present comprehensive, high quality de novo transcriptome assemblies of two Agave species, A. tequilana and A. deserti, built from short-read RNA-seq data. Our analyses support completeness and accuracy of the de novo transcriptome assemblies, with each species having a minimum of approximately 35,000 protein-coding genes. Comparison of agave proteomes to those of additional plant species identifies biological functions of gene families displaying sequence divergence in agave species. Additionally, a focus on the transcriptomics of the A. deserti juvenile leaf confirms evolutionary conservation of monocotyledonous leaf physiology and development along the proximal-distal axis.

Conclusions: Our work presents a comprehensive transcriptome resource for two Agave species and provides insight into their biology and physiology. These resources are a foundation for further investigation of agave biology and their improvement for bioenergy development.
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http://dx.doi.org/10.1186/1471-2164-14-563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765226PMC
August 2013

Architecture and evolution of a minute plant genome.

Nature 2013 Jun 12;498(7452):94-8. Epub 2013 May 12.

Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, 36821 Irapuato, Guanajuato, México.

It has been argued that the evolution of plant genome size is principally unidirectional and increasing owing to the varied action of whole-genome duplications (WGDs) and mobile element proliferation. However, extreme genome size reductions have been reported in the angiosperm family tree. Here we report the sequence of the 82-megabase genome of the carnivorous bladderwort plant Utricularia gibba. Despite its tiny size, the U. gibba genome accommodates a typical number of genes for a plant, with the main difference from other plant genomes arising from a drastic reduction in non-genic DNA. Unexpectedly, we identified at least three rounds of WGD in U. gibba since common ancestry with tomato (Solanum) and grape (Vitis). The compressed architecture of the U. gibba genome indicates that a small fraction of intergenic DNA, with few or no active retrotransposons, is sufficient to regulate and integrate all the processes required for the development and reproduction of a complex organism.
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http://dx.doi.org/10.1038/nature12132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972453PMC
June 2013

High genetic and epigenetic stability in Coffea arabica plants derived from embryogenic suspensions and secondary embryogenesis as revealed by AFLP, MSAP and the phenotypic variation rate.

PLoS One 2013 13;8(2):e56372. Epub 2013 Feb 13.

Unité Mixte de Recherche Résistance des Plantes aux Bioagresseurs, Centre de Coopération Internationale en Recherche Agronomique pour le Développement, Montpellier, France.

Embryogenic suspensions that involve extensive cell division are risky in respect to genome and epigenome instability. Elevated frequencies of somaclonal variation in embryogenic suspension-derived plants were reported in many species, including coffee. This problem could be overcome by using culture conditions that allow moderate cell proliferation. In view of true-to-type large-scale propagation of C. arabica hybrids, suspension protocols based on low 2,4-D concentrations and short proliferation periods were developed. As mechanisms leading to somaclonal variation are often complex, the phenotypic, genetic and epigenetic changes were jointly assessed so as to accurately evaluate the conformity of suspension-derived plants. The effects of embryogenic suspensions and secondary embryogenesis, used as proliferation systems, on the genetic conformity of somatic embryogenesis-derived plants (emblings) were assessed in two hybrids. When applied over a 6 month period, both systems ensured very low somaclonal variation rates, as observed through massive phenotypic observations in field plots (0.74% from 200,000 plant). Molecular AFLP and MSAP analyses performed on 145 three year-old emblings showed that polymorphism between mother plants and emblings was extremely low, i.e. ranges of 0-0.003% and 0.07-0.18% respectively, with no significant difference between the proliferation systems for the two hybrids. No embling was found to cumulate more than three methylation polymorphisms. No relation was established between the variant phenotype (27 variants studied) and a particular MSAP pattern. Chromosome counting showed that 7 of the 11 variant emblings analyzed were characterized by the loss of 1-3 chromosomes. This work showed that both embryogenic suspensions and secondary embryogenesis are reliable for true-to-type propagation of elite material. Molecular analyses revealed that genetic and epigenetic alterations are particularly limited during coffee somatic embryogenesis. The main change in most of the rare phenotypic variants was aneuploidy, indicating that mitotic aberrations play a major role in somaclonal variation in coffee.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056372PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572038PMC
August 2013

Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana.

PLoS One 2012 30;7(4):e35878. Epub 2012 Apr 30.

Department of Plant Genetic Engineering, Cinvestav-Irapuato, Irapuato, Guanajuato, Mexico.

Fructans are the main storage polysaccharides found in Agave species. The synthesis of these complex carbohydrates relies on the activities of specific fructosyltransferase enzymes closely related to the hydrolytic invertases. Analysis of Agave tequilana transcriptome data led to the identification of ESTs encoding putative fructosyltransferases and invertases. Based on sequence alignments and structure/function relationships, two different genes were predicted to encode 1-SST and 6G-FFT type fructosyltransferases, in addition, 4 genes encoding putative cell wall invertases and 4 genes encoding putative vacuolar invertases were also identified. Probable functions for each gene, were assigned based on conserved amino acid sequences and confirmed for 2 fructosyltransferases and one invertase by analyzing the enzymatic activity of recombinant Agave protein s expressed and purified from Pichia pastoris. The genome organization of the fructosyltransferase/invertase genes, for which the corresponding cDNA contained the complete open reading frame, was found to be well conserved since all genes were shown to carry a 9 bp mini-exon and all showed a similar structure of 8 exons/7 introns with the exception of a cell wall invertase gene which has 7 exons and 6 introns. Fructosyltransferase genes were strongly expressed in the storage organs of the plants, especially in vegetative stages of development and to lower levels in photosynthetic tissues, in contrast to the invertase genes where higher levels of expression were observed in leaf tissues and in mature plants.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035878PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340406PMC
September 2012

Polymorphism and methylation patterns in Agave tequilana Weber var. 'Azul' plants propagated asexually by three different methods.

Plant Sci 2012 Apr 13;185-186:321-30. Epub 2012 Jan 13.

Department of Plant Genetic Engineering, CINVESTAV-IPN, Campus Irapuato, Apdo. Postal 629, Km. 9.6, Libramiento Norte Carretera Irapuato-León, Zip Code 36821, Irapuato, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.1016/j.plantsci.2012.01.004DOI Listing
April 2012

Agave tequilana MADS genes show novel expression patterns in meristems, developing bulbils and floral organs.

Sex Plant Reprod 2012 Mar 20;25(1):11-26. Epub 2011 Oct 20.

Department of Genetic Engineering, CINVESTAV Unidad Irapuato, Km 9.6 Libramiento Norte, Carr. Irapuato-León, Apdo. Postal 629, CP 36821, Irapuato, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.1007/s00497-011-0176-xDOI Listing
March 2012

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

J Exp Bot 2010 Sep 13;61(14):4055-67. Epub 2010 Jul 13.

Department of Plant Genetic Engineering, Cinvestav Irapuato, 36821 Irapuato, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.1093/jxb/erq215DOI Listing
September 2010

Analysis of gene expression and physiological responses in three Mexican maize landraces under drought stress and recovery irrigation.

PLoS One 2009 Oct 30;4(10):e7531. Epub 2009 Oct 30.

Centro de Investigación y Estudios Avanzados, Departamento de Ingeniería Genética de Plantas, Libramiento Norte Carretera Irapuato-León, Irapuato, Guanajuato, Mexico.

Background: Drought is one of the major constraints for plant productivity worldwide. Different mechanisms of drought-tolerance have been reported for several plant species including maize. However, the differences in global gene expression between drought-tolerant and susceptible genotypes and their relationship to physiological adaptations to drought are largely unknown. The study of the differences in global gene expression between tolerant and susceptible genotypes could provide important information to design more efficient breeding programs to produce maize varieties better adapted to water limiting conditions.

Methodology/principal Findings: Changes in physiological responses and gene expression patterns were studied under drought stress and recovery in three Mexican maize landraces which included two drought tolerant (Cajete criollo and Michoacán 21) and one susceptible (85-2) genotypes. Photosynthesis, stomatal conductance, soil and leaf water potentials were monitored throughout the experiment and microarray analysis was carried out on transcripts obtained at 10 and 17 days following application of stress and after recovery irrigation. The two tolerant genotypes show more drastic changes in global gene expression which correlate with different physiological mechanisms of adaptation to drought. Differences in the kinetics and number of up- and down-regulated genes were observed between the tolerant and susceptible maize genotypes, as well as differences between the two tolerant genotypes. Interestingly, the most dramatic differences between the tolerant and susceptible genotypes were observed during recovery irrigation, suggesting that the tolerant genotypes activate mechanisms that allow more efficient recovery after a severe drought.

Conclusions/significance: A correlation between levels of photosynthesis and transcription under stress was observed and differences in the number, type and expression levels of transcription factor families were also identified under drought and recovery between the three maize landraces. Gene expression analysis suggests that the drought tolerant landraces have a greater capacity to rapidly modulate more genes under drought and recovery in comparison to the susceptible landrace. Modulation of a greater number of differentially expressed genes of different TF gene families is an important characteristic of the tolerant genotypes. Finally, important differences were also noted between the tolerant landraces that underlie different mechanisms of achieving tolerance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007531PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2766256PMC
October 2009

Functional genomics of root growth and development in Arabidopsis.

Curr Opin Plant Biol 2009 Apr 29;12(2):165-71. Epub 2008 Dec 29.

Duke University, Department of Biology and Center for Systems Biology, United States.

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.
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http://dx.doi.org/10.1016/j.pbi.2008.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683257PMC
April 2009

Construction of a genetic linkage map of the fungal pathogen of banana Mycosphaerella fijiensis, causal agent of black leaf streak disease.

Curr Genet 2008 May 26;53(5):299-311. Epub 2008 Mar 26.

Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, Calle 43 No. 130. Colonia Chuburná de Hidalgo, 97200 Mérida, Yucatán, Mexico.

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.
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http://dx.doi.org/10.1007/s00294-008-0186-xDOI Listing
May 2008

Development of a molecular genetic linkage map for Colletotrichum lindemuthianum and segregation analysis of two avirulence genes.

Curr Genet 2007 Feb 7;51(2):109-21. Epub 2006 Dec 7.

Department of Genetic Engineering, CINVESTAV, Unidad Irapuato, Apdo. Postal 629, Irapuato, Guanajuato, México.

A framework genetic map was developed for the fungal pathogen Colletotrichum lindemuthianum, the causal agent of anthracnose of common bean (Phaseolus vulgaris L.). This is the first genetic map for any species within the family Melanconiaceae and the genus Colletotrichum and provides the first estimate of genome length for C. lindemuthianum. The map was generated using 106 haploid F1 progeny derived from crossing two Mexican C. lindemuthianum isolates differing in two avirulence genes (AvrclMex and AvrclTO). The map comprises 165 AFLP markers covering 1,897 cM with an average spacing of 11.49 cM. The markers are distributed over 19 major linkage groups containing between 5 and 25 markers each and the genome length was estimated to be approximately 3,241 cM. The avirulence genes AvrclMex and AvrclTO segregate in a 1:1 ratio supporting the gene for gene hypothesis for the incompatible reaction between C. lindemuthianum and P. vulgaris, but could not be incorporated into the genetic map. This initial outline map forms the basis for the development of a more detailed C. lindemuthianum linkage map, which would include other types of molecular markers and allow the location of genes previously isolated and characterized in this species.
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http://dx.doi.org/10.1007/s00294-006-0111-0DOI Listing
February 2007

Heterothallic mating observed between Mexican isolates of Glomerella lindemuthiana.

Mycologia 2005 Jul-Aug;97(4):793-803

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. (INIFAP), Campo Experimental del Bajío, Celaya, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.3852/mycologia.97.4.793DOI Listing
March 2006

Genotyping of Ochrobactrum spp. by AFLP analysis.

J Bacteriol 2005 Apr;187(7):2537-9

Secretaría de Salud de Guanajuato, Tamazuca 4, Colonia Centro, 36000 Guanajuato, Gto., México.

AFLP was used to analyze the genetic diversity among Ochrobactrum strains. AFLP patterns showed a great genomic variability that separated the samples into three distinct clusters. Ochrobactrum intermedium was found to be closely related to Brucella abortus S99.
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http://dx.doi.org/10.1128/JB.187.7.2537-2539.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1065246PMC
April 2005

Transgenic plants: an historical perspective.

Methods Mol Biol 2005 ;286:3-32

Departamento de Ingeniería Genética, de Plantas, Centro de Investigación y de Estudios Avanzados, Guanajuato, Mexico.

The development of technologies that allow the introduction and functional expression of foreign genes in plant cells has extended in less than two decades to the production of 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. Vaccines against serious human diseases and other important products have also been developed using transgenic plants. Many more agronomic and quality traits are currently being engineered in both academic and industrial laboratories, which are limited only by our poor knowledge of plant gene function. The emergence of new functional genomic strategies for the identification and characterization of genes promises to provide a wealth of information with an enormous potential to enhance traditional plant breeding and to genetically engineer plants for specific purposes. This chapter describes some of the highlights in the development of these technologies and some of the major achievements in production and commercialization of transgenic crops. We also discuss some of the biosafety issues related to release of this novel class of plants into the environment.
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http://dx.doi.org/10.1385/1-59259-827-7:003DOI Listing
December 2004

Analysis of Pathotypes of Colletotrichum lindemuthianum Found in the Central Region of Mexico and Resistance in Elite Germ Plasm of Phaseolus vulgaris.

Plant Dis 2004 Feb;88(2):152-156

Department of Genetic Engineering, CINVESTAV, Unidad Irapuato, Apdo. Postal 629, Irapuato, Guanajuato, Mexico.

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.
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http://dx.doi.org/10.1094/PDIS.2004.88.2.152DOI Listing
February 2004