SUSTAINABLE AND INTEGRAL EXPLOITATION OF AGAVE

Transcription

1 SUSTAINABLE AND INTEGRAL EXPLOITATION OF AGAVE Editor Antonia Gutiérrez-Mora Compilers Benjamín Rodríguez-Garay Silvia Maribel Contreras-Ramos Manuel Reinhart Kirchmayr Marisela González-Ávila Index 1. Scientific trends on Agave 2. Science and technology of Agave beverages and other derivatives 3. Biological effects of Agave fructans and other by-products 4. Industrial processing of Agave wastes and subproducts Citation example: Gutiérrez-Aguilar, P.R., Gil-Vega, K.C. and Simpson, J Development of an Agrobacterium tumefasciens mediated transformation protocol for two Agave species by organogenesis. In: A. Gutiérrez-Mora (Ed.), B. Rodríguez-Garay, S. M. Contreras-Ramos, M. R. Kirchmayr, M. González-Ávila (Comps.), Sustainable and Integral Exploitation of Agave. Retrieved from: ISBN: Published online: December 22, 2014

2 Sustainable and Integral Exploitation of Agave 2014 Analysis of differentially expressed genes during the transition from the vegetative to reproductive stage in Agave tequilana José Natzul Santoyo Villa & June Simpson* Centro de Investigación y de Estudios Avanzados del IPN, Km 9.6 Irapuato-León, CP 36821, Irapuato, Gto, México. Agave tequilana is a very important crop for the economy in Mexico, since it is the only agave species from which we can obtain tequila. Once agave plants pass from the vegetative to reproductive state, an inflorescence meristem develops indicating the optimum state for harvesting. The developing inflorescence depletes stored carbohydrates and in turn decreasing the efficiency of production of tequila and for this reason the inflorescence is removed manually. Since most agave species including A. tequilana are perennial, monocarpic plants, it is unknown as to how the bolting process is triggered or what changes occur at the genetic and molecular level during the transition from the vegetative to reproductive stage. In order to study this process at the genetic level, analysis based on deep RNA sequencing (RNA-seq) was used to analyze genes that are expressed differentially during 4 phases of the transition from vegetative to reproductive phase. The phases are defined as: vegetative adult (Mv), initiation of bolting (Mch), inflorescence 10cm (In10cm) and inflorescence 30cm (In30cm). Adjacent stages "Mv vs Mch", "Mch vs In10cm" and "In10cm vs In30cm" were compared in terms of differential expression patterns differentially expressed transcripts were found during the transition, where the genes reported as inducers of flowering such as FT and SOC1, showed low levels of expression in the vegetative and 10cm stages and high levels in initiation of bolting and 30cm stages. Some genes reported as repressors of flowering showed inverse patterns of expression as expected. We found other genes that have similar expression patterns to SOC1 and FT that might be acting as inducers or facilitating the transition to the reproductive stage, some are related to carbohydrate metabolism or flow of auxin whereas others were not annotated. This work gives us an idea of what happens at the genetic and molecular level in the transition from vegetative to reproductive stage in Agave tequilana, laying the groundwork for future research to predict and control the floral transition and to determine optimal harvesting for increased production of tequila. Key words: A. tequilana, bolting, transcriptome, expression analysis, RNAseq. *Corresponding author: jsimpson@ira.cinvestav.mx

3 INTRODUCTION Agave tequilana is a semi arid, monocarpic plant species which produces a single inflorescence at the end of its life cycle then dies. A. tequilana plants take 6 to 12 years to mature depending on the environment, soil and agronomic treatments. A. tequilana Weber, var. azul is a domesticated crop, exploited commercially for the production of tequila. Initiation of bolting signals that plants are ready to harvest and this process must be controlled in order to reduce loss of stored carbohydrates. Although of economical and cultural importance, there is not much information or research at the molecular and genetic level on the process of bolting in A. tequilana. Due to its long life cycles and undesirable loss of stored carbohydrates by the developing inflorescence, we do not know how this process is triggered and what is happening at the genetic level inside the plant. Recently, Delgado Sandoval et al. (2012) described morphologically the stage close to the transition between the vegetative and reproductive stages. In the vegetative stage, the leaves of A tequilana grow as a triangle where the center leaves seem to grow faster than the others forming a parabolic shape, when the plant is ready to switch to the reproductive stage the leaves in the center grow more slowly than the others, producing what seems to be a sinking morphology at the center of the plant as shown in Figure 1. The improved technology and increasingly low cost of transcriptome analysis based on deep RNA sequencing has become a very powerful and reliable tool to perform the identification and quantification of almost all transcripts present in a cell or tissue, either in a specific developmental stage or physiological condition. These advances allowed us to perform a transcriptomic analysis based on RNA-seq technology in order to have a better understanding about what is happening at the genetic and physiological level during the transition from the vegetative to reproductive stage in A. tequilana. Our aim is to generate and characterize transcriptome data, based on RNA-seq at four distinct stages during the transition from the vegetative to the reproductive stage of Agave tequilana. Here we present preliminary data of this transcriptome analysis. METHODOLOGY Plant Material and sampling We took 12 meristems from Agave tequilana plants located in Real de Penjamo, Penjamo Guanajuato, Mexico. The plants correspond to 4 different stages of the transition: vegetative adult stage, initiation of bolting, inflorescence of 10cm and inflorescence of 30cm (Fig 1), with 3 biological replicates per stage. Figure 1. a) vegetative meristem. b) initiation of bolting. c) inflorescence of 10 cm. d) inflorescence of 30 cm. (adapted from Delgado Sandoval et al. 2012) 20

4 Plants where dissected to obtain just the apical meristem as shown in Figure 2 which was then frozen in liquid nitrogen. Figure 1. Agave tequilana apical meristem. RNA extraction RNA was obtained from the shoot apical meristem at 4 stages during the transition from the vegetative to reproductive stage as described by Delgado Sandoval et al. (2012). Sequencing The total RNA extracted was sent to the Advanced Genomics Unit (UGA) at CINVESTAV Irapuato to be sequenced using the Illumina MiSeq 2X250 platform. Assembling and quality control A global De novo assembling was performed as described in the Trinity protocol (Hass B J et al. 2013) and quality control was performed with Trimmomatic (Bolger et al. 2014). Mapping and quantification Each read per library was mapping to the transcriptome assembled as a reference, the aligned and quantification was performed RSEM (Li et al. 2011). Differential expression and annotation Differential expression analysis was performed with R thru the library edger (Reinhardt et al. 2010) taking as a cut of a false discovery rate (FDR) from The annotation step was performed with Blast2go with an e-value from 0.1 e-5 against Refseq for plants. RESULTS AND DISCUSSION The de novo assembled transcriptome data reflected the high quality of the sequencing data obtained. 221,686 isoforms were reconstructed representing 0.250Gb assembled with a N50 statistic of 1669 bases, minimum length of 201 bases and a maximum of 15,804 bases hits were obtained from different proteins in Refseq for plants which is around 14% of the reconstituted reads. Differential expression was carried with the isoforms. In Table 1, we show how many transcripts are up and down regulated during the transition from the vegetative stage to the reproductive stage. As expected, we found some genes that are flowering inducers such as FT and SOC1 that have low counts in the vegetative stage and when bolting initiates increase. We also identified some novel genes in agave that have interesting expression patterns during bolting (Figure 3) and seem closely linked to the transition stage. Some are related to carbohydrate metabolism, auxin efflux, transcription factors and also include nonannotated sequences, whereas others are down regulated and potentially act as repressors. 21

5 Table 1. Number of differentially expressed genes. Comparation Upregulateregulated Down- Total Mv vs Mch Mch vs In10cm In10cm vs In30cm CONCLUSION Figure 3. Heatmap of differential expression. Our analysis provides detailed data on the changes in gene expression during bolting in A. tequilana and reveals genes involved in specific metabolic pathways important for the vegetative-reproductive transition. This will allow us to understand, predict and eventually control the floral transition in A. tequilana. ACKNOWLEDGEMENTS Financial support from CONACyT for project CB and scholarship to JNSV. All my lab mates for their support and advice. BIBLIOGRAPHY Bolger A. M. et al Trimmomatic: A Flexible Trimmer For Illumina Sequence Data. Bioinformatics, Btu170.fsaf Delgado Sandoval S. del C. et al. (2012). Sex Plant Reprod 25(1): Hass B J et al. (2013). Full-Length Transcriptome Assembly From Rna-Seq Data Without A Reference Genome. Nat Biotechnol. Nature Protocols 8: Li, B. and Dewey, C. N. (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 12:323. Reinhardt D. et al. (2010). edger: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26, pp. -1.Saitou N, Nei M