Genomic Resources and Transcriptome Mining in Agave Tequilana

GCB Bioenergy (2011) 3, 25–36, doi: 10.1111/j.1757-1707.2010.01079.x

OPINION Genomic resources and transcriptome mining in Agave tequilana

JUNE SIMPSON*,AI´ DA MARTI´ NEZ HERNA´ NDEZw ,MARI´ AJAZMI´ NABRAHAM JUA´ REZ*,SILVIADELGADOSANDOVAL*,ALFREDOSA´ NCHEZ VILLARREALw and CELSO CORTE´ SROMERO* *Department of Plant Genetic Engineering, Cinvestav Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-Leoń, Apdo, Postal 629, Zip Code 36821. Irapuato, Guanajuato, Me´xico, wColegio de Postgraduados Campus Campeche, Km 17.5 Carretera federal Haltuncheń-Edzna´, Zip code 24750. Sihochac, Champotoń, Campeche, Mexico

Abstract 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 molecular- genetic 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. Keywords: A. tequilana, bioenergy, data mining, genomic resources, transcriptome

Received 21 July 2010; revised version received 16 October 2010 and accepted 2 November 2010

annually with consequently less economic impact (Con- Introduction sejo Mexicano Regulador de la Calidad del Mezcal, Agave plants have been exploited in Mexico since 2009, http://www.comercam.org). For growers how- around 10 000–8 000 years BC (Garcıá-Mendoza, 1992) ever, investment in agave plantations for tequila pro- and pre-Columbian cultures used them for fiber, food, duction can be risky, depending on both environmental construction and religious ceremonies. Agave plants conditions and demand. These problems are exacer- played such an important role in these societies that bated by the long life-cycle of the plant (5–8 years) they were awarded a specific deity, the goddess ‘Maya- and the restrictions which only allow Agave tequilana huel’ who is represented in different forms in several Weber cultivar ‘azul’ grown in specified regions of surviving codices (Lima, 1986). Today agave plants are Mexico to be harvested for tequila production. In recent still strong Mexican cultural icons, appearing in films, years some growers have been unable to find a market works of art and many forms of publicity. for materials from plantations initiated several years The best known modern agave products are of course before and many of these plantations can be found tequila and mezcal. Around 249 million liters of tequila untended and abandoned. are produced annually of which more than half are Until the latter part of the 20th century Mexico was exported, accounting for around $900 million dollars in also a major producer of agave fibers and textiles, income. Around 100 thousand hectares of agave are however this industry has declined from a total produc- grown for tequila production and the industry directly tion of 210 000 tons in 1916 to 32 000 tons in 2005 employs around 60 000 people (SAGARPA, 2009, (Eastmond & Robert, 2000; Caceres-Farfan et al., 2008), http://www.sagarpa.gob.mx; CRT:Consejo Regulador substantially reducing the potential sources of income del Tequila, 2009, http://www.crt.org.mx ). Production in the Yucatań peninsula, the traditional centre of fiber of mezcal is much lower, around 680 thousand liters production in Mexico. These examples underline the need to seek alterna- Correspondence: June Simpson, e-mail: tives for the exploitation of agaves in Mexico and [email protected] several areas such as the use of agave sugars as dietary r 2010 Blackwell Publishing Ltd 25 26 J. SIMPSON et al. supplements and substitutes for sugar and fats (Lopez et al., 2003; Ortiz-Basurto et al., 2008; Urias-Silvas et al., 2008; Gomez et al., 2009; Ravenscroft et al., 2009; Leach & Sobolik, 2010) or for the production of paper (Idarraga et al., 1999) have been explored. Recently however, attention has focused on the potential of agave species as bioenergy crops (Borland et al., 2009; Somerville et al., 2010). Indeed, agaves offer many advantages for this goal: they are succulent, CAM plants which grow naturally in arid or semiarid conditions making them good candidates for the exploitation of marginal or uncultivated land and their exploitation for bioenergy production would not divert resources from staple food crop production as in the case of maize when used for bioenergy production, an extremely important consid- Fig. 1 Schematic representation of the taxonomy of the Agava- eration in Mexico. Among the advantages these plants ceae family. offer are: management in a similar manner to forest species with harvesting over several years, production of large amounts of biomass, methodology for ethanol mezcal (Agave angustifolia) and tequila (Agave tequilana) production from sugars well developed and low-main- are found within the Rigidae group and are underlined. tenance in terms of water and agrochemicals. An added More recently, molecular-genetic techniques have advantage is the possibility to also use the bagasse been employed to study the taxonomy, diversity and produced from the plants harvested for the production evolution of the Agavaceae (Colunga-GarciaMarin et al., of alcoholic beverages and fibers for bioethanol produc- 1999; Eguiarte et al., 1999; Martinez-Palacios et al., 1999; tion, effectively exploiting what would otherwise be a Piven et al., 2001; Navarro-Quezada et al., 2003; Gil-Vega waste product. A recent report (Hernandez-Salas et al., et al., 2006; Good-Avila et al., 2006; Bousios et al., 2007; 2009) describes the feasibility of exploiting not only the Gil-Vega 2007; Abraham-Juarez et al., 2009; Parker et al., sugar component of agave bagasse but also the cellu- 2010). Concerns have been raised over the very narrow losic component. germplasm pool exploited for Agave plants used in Although the current and potential economic impor- commercial applications, since plants are asexually tance of agaves is irrefutable, perhaps surprisingly, propagated and for tequila production by law only a relatively little basic research has been carried out on single genotype (A. tequilana Weber var. azul) can be these species, especially at the genetic and molecular grown (http://www.crt.org.mx). This leads to vulner- level. This is in contrast to comparable species such as ability in the crop under adverse environmental condi- pineapple where specific genes have been characterized tions or attack by pests and pathogens. Indeed the (Antony et al., 2008), transcriptome data generated combination of increased disease incidence and the (Moyle et al., 2005a) and a dedicated website developed international ‘boom’ in tequila sales led to a severe (Moyle et al., 2005b). shortage A. tequilana plants towards the end of the 1990s (Bowen & Valenzuela-Zapata, 2008; Bowen & Valenzuela-Zapata, 2009). However several molecular Available resources for genetic studies in Agave studies have shown that a greater diversity exists with- Although many details of agave taxonomy remain to be in A. tequilana than was first thought (Gil-Vega et al., defined, several excellent works are available, including 2006; Bousios et al., 2007; Abraham-Juarez et al., 2009) the classical morphological descriptions of Gentry and this diversity could be exploited if the terms of the (Gentry, 1982), Dahlgren (Dahlgren et al., 1985) and controlled designation of origin (Bowen & Valenzuela- Garcıá-Mendoza (Garcıá-Mendoza, 1992; Garcıá-Mendoza, Zapata, 2008) were widened. This situation is less 2000; Garcia-Mendoza & Chiang 2003; Garcia-Mendoza, critical for mezcal and fiber production since many 2010). A simplified overview of the classification of the different cultivars and even different species may be Agavaceae family taken from these descriptions is used. shown in Fig. 1. Within the family the genus Agave is No formal reports are available of genetic or breeding economically the most important and for this genus studies although intra and interspecific crosses have alone, 200 species and 247 taxa have been been successful albeit at low efficiencies (Escobar- described (Garcıá-Mendoza 2000). The main species Guzmań et al., 2008) and hybridization has been exploited economically for fiber (Agave fourcroydes), observed in the field (Valenzuela, 1997).

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Table 1 Ploidy levels for Agave species of the subgenus (a) Littaea and (b) Agave

Ploidy level Species

(a) DIPLOID, 2 , 60 chromosomes A. celsii, A. ﬁlifera, A. nizandensis, A. pendula, A. schotti, A. striata, A. stricta, A. toumeyana, A. univittata, A. victoriae-reginae, A. xalapensis, A. xylocantha TRIPLOID, 3 , 90 chromosomes A. warelliana, A. ornithobroma TETRAPLOID, 4 , 120 chromosomes A. kerchovei, A. lechugilla, A. victoriae-reginae HEXAPLOID, 6 , 180 chromosomes A. gilbeyi, A. giesbrechtii ANEUPLOID, No. chromosomes in parenthesis A. lechugilla (55 110), A. stricta (50), A. warelliana (100)

(b) DIPLOID, 2 , 60 chromosomes A. amaniensis, A .americana, A. angustifolia, A. asperrima, A. avellanidens, A. brevispina, A. colorata, A. deserti, A. fourcroydes, A. gigantensis, A. palmeri, A.parryi, A. potatorum, A. rodocantha, A. shawii, A. tequilana TRIPLOID, 3 , 90 chromosomes A. angustifolia, A. cantala, A.lurida, A. vexans TETRAPLOID, 4 , 120 chromosomes A. americana, A. angustifolia, A. ferox, A. goldmaniana, A. lurida, A. parryi, A. salmiana A. tequilana, A. utahuensis PENTAPLOID, 5 , 150 chromosomes A. fourcroydes, A. mapisaga, A. sisalana, A. tequilana HEXAPLOID, 6 , 180 chromosomes A. americana, A. asperrima, A. angustifolia, A. decipiens, A. atrovirens, A. vivipara, A. wightii OCTAPLOID, 8 , 240 chromosomes A. americana ANEUPLOID, No. chromosomes in parenthesis A. americana (20, 44,96,104,110,115,118,119,125,134,226), A. asperrima (148- 186), A. deserti (59,118), A. expansa (119), A. fourcroydes (140,138), A. lurida (100, 108, 114), A. rigida (124-136), A. sisalana (138, 140-149), A. tequilana (16,17), A. vexans (87,174), A. vivipara (58), A. wightii (52).

Species with more than one ploidy level are shown in bold.

Data on ploidy levels and karyotypes is available for In vitro regeneration of most Agave species tested is 81 taxa of the Agave genus including 56 species, 5 relatively easy and can be achieved either by indirect subspecies and 20 varieties (Palomino et al., 2003; organogenesis or through in vitro suspension culture Moreno-Salazar et al., 2007; Palomino et al., 2007; Robert (Robert et al., 1987; Nikam et al., 2003; Robert et al., 2006; et al., 2008). Ploidy levels have been shown to range Valenzuela-Sanchez et al., 2006; Ramirez-Malagon et al., from diploid to octaploid with a basic chromosome 2008). Indeed in vitro propagated A. tequilana plants number of 30 and the karyotype is arranged in a now account for an increasingly large proportion of bimodal pattern, conserved over the range of different the materials in commercial cultivation (Dr. Ignacio del ploidies with five large and 25 small chromosome pairs Real, Tequila Sauza, personal communication). (Castorena-Sańchez et al., 1991; Cavallini et al., 1996). Modern genetic analysis in any organism normally For some species such as Agave americana and A. angu- requires an efficient transformation system and stifolia several ploidy levels have been reported. A although many reports describe regeneration of agave, detailed analysis of ploidy levels for different agave only one report on transformation protocols for Agave species is reported in Palomino et al. (2007) and Table 1 salmiana based on in vitro suspension cultures and both summarizes these results. Agrobacterium mediated and biobalistic methods has Several authors have also estimated the genome size of been published (Flores-Benitez et al., 2007). different Agave species. In general diploid genomes are Agave plants have many unique and interesting estimated to contain between 6.0 pg (Banerjee & Sharma, biological characteristics and in addition to extensive 1987) and 9.6 pg (Bennett & Smith, 1991) cited by Palo- studies by taxonomists, have also attracted the interest mino et al. (2007). Using the definition 1 pg 5 980 Mbp of plant physiologists notably in the field of CAM (Bennett et al., 2000) the basic, haploid (1Cx) genome size (Nobel, 1976; Nobel & Hartsock, 1978; Nobel & Hart- for Agave can be estimated at between 2940 and 4704 Mbp sock, 1979; Nobel, 1985; Nobel & Valenzuela, 1987; of DNA and most recent publications suggest a size of Wang & Nobel, 1998) and their adaptation to arid approximately 4100 Mbp (Palomino et al., 2003; Moreno- climates (Lujan et al., 2009). Ecologists have studied Salazar et al., 2007; Palomino et al., 2007) although Robert the interaction between agave species and other organ- et al. (Robert et al., 2008) report a 1Cx value of 7.61 pg isms such as birds, insects and bats (Delrio & Eguiarte, which would give a genome size of 7458 Mbp. 1987; Colunga-GarciaMarin et al., 1999; Rocha et al., r 2010 Blackwell Publishing Ltd, GCB Bioenergy, 3, 25–36 28 J. SIMPSON et al.

2005; Silva-Montellano & Eguiarte, 2003) and driven by direction by the Sanger method (Sanger et al., 1977). interest from commercial sectors, carbohydrate struc- Sequenced fragments ranged from 800 to 1400 bp and in ture and metabolism has also been addressed (Mancilla- total around 36 500 high quality sequences were ob- Margalli & Lopez 2002; Lopez et al., 2003; Mancilla- tained. In addition pooled RNA samples from all tissues Margalli & Lopez, 2006; De Leon-Rodriguez et al., 2006; and conditions were sequenced using the first-genera- Michel-Cuello et al., 2008; Garcia-Aguirre et al., 2009). tion 454 pyrosequencing (Rothberg & Leamon, 2008) Despite these research interests however, the agave method to obtain around 300 000 short 50–150 bp se- research community as a whole is small and since these quences. The combined sequencing data covers over species are only of commercial interest in a few regions 4 800 000 bases which could be arranged into 15 516 outside of Mexico, very little investment has been contigs. Almost 73% of the sequences could be func- obtained for ongoing projects. Molecular biologists tionally annotated using BLAST and GENE ONTOLOGY and and geneticists have also been wary to accept the around 2.6% of the sequences potentially represent challenge of working with plants characterized by: a previously uncharacterized genes. lack of basic genetic knowledge, large genome size, long The basic transcriptome is therefore a potential source life cycle, an inefficient or nonexistent transformation for a wealth of genetic information from agave, how- system and few funding opportunities, making them ever the lack of molecular tools for any agave species, the direct opposite morphologically and in terms of necessitated the development of an experimental strat- research to Arabidopsis thaliana. However, if a convin- egy which would allow efficient identification and cing argument can be made for agave species as viable characterization of genes of interest. For this purpose bioenergy crops this scenario will hopefully change. the class I KNOX homeodomain genes were chosen as a A search of GenBank reveals that only around 290 model. These genes have been previously well charac- sequences have been deposited for the whole Agave terized in different plant species including maize and genus, 40 of which are from A. tequilana. The deposited A. thaliana and mutant lines for several of these genes sequences are mainly ribosomal genes, Ty1-copia trans- are available. Class I KNOX genes have been shown to poson like sequences and chaperones. To date no large be involved in development and maintenance of mer- scale genomic or transcriptomic sequencing data is istem tissue (Hay et al., 2006; Hay & Tsiantis, 2010) and available. regulation of this process is essential when plants In an attempt to remedy this situation, researchers at switch from vegetative to floral development and vice Cinvestav, Unidad Irapuato and the Colegio de Post- versa. One of the distinguishing characteristics of agave graduados, Campus Campeche, Mexico have been col- plants is their capacity for both sexual and asexual laborating in order to produce general transcriptome reproduction. In particular the formation of bulbils on data for A. tequilana. The details of this initiative will be inflorescences when sexual reproduction has been published elsewhere (A. Martıńez-Hernańdez et al., in unsuccessful is a rare and typical trait associated with preparation) and on publication the data will be publicly these species (Fig. 2a). Bulbil formation involves a available, however researchers interested in searching the change in developmental programming from sexual agave transcriptome database are encouraged to directly reproduction to a vegetative growth pattern with the contact June Simpson ([email protected]) or formation of new meristem structures (Abraham-Juarez Aı´da Martıńez-Hernańdez ([email protected]). A brief et al., 2010). KNOX genes have been shown to be description of the results obtained and examples of necessary for the formation and maintenance of the transcriptome data mining for genes of potential interest meristem structure (Hake & Ori, 2002; Hake et al., for bioenergy applications will be presented below. 2004) and to be expressed specifically in the meriste- matic dome but not in the surrounding leaf primordia (Jasinski et al., 2007). Consequently changes in the The current A. tequilana transcriptome expression patterns of Agave KNOX genes during the Two sampling strategies were employed to develop an initial stages of bulbil formation would be expected. initial A. tequilana transcriptome: (1) General samples Searches within the A. tequilana transcriptome identified were obtained from the most important organs and several sequences encoding two distinct Class 1 KNOX tissues (leaves, roots, anthers, stem etc.) under different genes. Based on sequence information specific oligonu- growth conditions (light, dark) and from plants of cleotide primers could be designed which allowed different ages. (2) Multiple samples were obtained from endogenous expression analysis of the agave KNOX specific tissues (developing bulbils and inflorescence genes to be carried out by in situ hybridization and meristems). From these samples individual cDNA RT-PCR analysis in A. tequilana plants and the effects of libraries were constructed and around 2000 random ectopic expression in wild type and mutant A. thaliana clones from each library were sequenced in a single lines to be observed (Abraham-Juarez et al., 2010).

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Fig. 2 Analysis of agave KNOX genes. (a) Bulbils developing on inﬂorescence tissue. (b) In situ hybridization showing the expression pattern of the AtqKNOX2 gene during the initial stages of bulbil development. (c) Ectopic expression of agave KNOX genes in Arabidopsis thaliana. Leaves from A. thaliana ecotype Columbia transformed with AtqKNOX1 or AtqKNOX2 are shown ﬂanking a wild- type leaf.

Fig. 2b shows an example of an in situ hybridization experiment where AtqKNOX2 gene expression is observed specifically in cells where a new meristem will form very close to the bracteoles on an agave inflorescence. For heterologous expression in A. thaliana,40indepen- dent, phosphinothricin resistant T0 plants were selected for each construction. Lines segregating 3 : 1 (6 for Atq- KNOX1, 5 for AtqKNOX2) in the T1 generation were grown to select T2 homozygotes. Three homozygous lines for each Agave KNOX gene construct were then analyzed by quantitative real time PCR. KNOX gene expression is normally repressed in leaf tissue and fig. 2c shows the severely lobed leaf phenotype produced by ectopic expression of the AtqKNOX1 gene (left hand leaf) and the AtqKNOX2 gene, (right hand leaf) in leaves of transformed A. thaliana plants. Fig. 3 Examples of harvested stems from different Agave species. Species from left to right are A. mapisaga, A. atrovirens, A. This phenotype is very similar to the overexpression of asperrima and A. americana. The inset shows a dissected Agave endogenous A. thaliana KNOX genes in this tissue tequilana stem. S, stem, LB, leaf base. (Douglas et al., 2002; Venglat et al., 2002). In the center a wild type leaf A. thaliana leaf is shown for comparison. Genes involved in carbohydrate metabolism The results obtained from the analysis of the agave KNOX genes demonstrate the feasibility of exploiting In agave, fructans accumulate throughout the life cycle the A. tequilana transcriptome to identify EST’s for genes of the plant and are stored in the stem (also known as of interest and to characterize their expression and the ‘pinã’ in Spanish due to the resemblance of the function. harvested stems to pineapples see Fig. 3) which can grow extensively. A comparison of these tissues taken from different agave species and with the leaves re- Mining of the A. tequilana transcriptome for genes moved is shown in Fig. 3. The inset shows an example with potential bioenergy applications of a dissected, harvested stem of A. tequilana (S) where In agave there are two main areas where genetic manip- the basal parts of the leaves are still attached. From left ulation could have an impact on bioenergy production: to right the samples on the ground correspond to: Agave (1) optimization of normal fructan production and mapisaga, measuring 310 cm in diameter and weighing storage, (2) exploitation of the cellulosic content of leaf 471.85 kg, Agave atrovirens, 215 cm, 280.4 kg, Agave as- tissue. The first option is not only related to effects on perrima, 225 cm, 222.5 kg and A. americana, 172 cm, general carbohydrate metabolism but also photosynth- 76.2 kg. All samples were taken close to maturity when esis and biomass production (this will be discussed the plants begin to flower and all were obtained from in companion articles in this volume and is therefore different regions of Guanajuato state, Mexico. Statisti- not dealt with in detail here). The second option could cally valid data on sucrose and fructan accumulation in be explored by modification of cellulose and lignin different Agave species are difficult to obtain since only synthesis. commercially exploited species such as A. tequilana and r 2010 Blackwell Publishing Ltd, GCB Bioenergy, 3, 25–36 30 J. SIMPSON et al.

A. angustifolia can easily be obtained in representative Lopez, 2006) which may vary depending on the geno- numbers. However for the samples in Fig. 3, obrix and type and the environment. Agave fructans are formed percentage of total reducing sugars (TRS) were esti- initially from a basic sucrose molecule by b(2-1) and mated based on 500 g of crude stem samples and found b(2-6) linkages between fructose residues to form to be as follows: A. tequilana 30obrix/25% TRS, 1-kestose by sucrose:sucrose 1 fructosyl transferase A. mapisaga,11obrix, 8% TRS, A. atrovirens,20obrix, (1-SST) and 6-kestose by sucrose:fructan 6-fructosyl- 17% TRS, A. asperrima,21obrix, 13%TRS and A. amer- transferase (6-SFT) (Fig. 4). Neokestose is formed icana,7obrix, 4% TRS (S. Delgado and J. Simpson, from1-kestose by addition of fructose at position 6 on unpublished results). the glucose residue by fructan:fructan 6G fructosyl- In other studies different species have also been transferase (6G-FFT) and bifurcose form 1-kestose by shown to accumulate fructans to different levels, ran- addition of fructose in a b(2-6) linkage by 6-SFT (Fig. 4). ging from around 350 mg g1 of dry matter reported for At least 1 other enzyme, fructan:fructan 1-fructosyl- A. angustifolia to 4700 mg g1 dry matter reported for transferase (1-FFT) is thought necessary in order to A. tequilana (Mancilla-Margalli & Lopez, 2006). In that complete the synthesis of the long and complex fructan study samples were obtained from contrasting geogra- structures (agavins and graminans) described for agave. phical and climatic regions and this factor also affects To date however, the only enzyme from agave which fructan accumulation since A. tequilana plants cultivated has been functionally characterized is 1-SST from in either Jalisco or Guanajuato states showed levels of A. tequilana (Avila-Fernandez et al., 2007). Other en- fructan accumulation of around 730 and 490 mg g1, zymes such as invertases and fructanexohydrolases respectively although the overall percentage of water are needed to break down these complex molecules soluble carbohydrates was found to be around 80% for when sugars reserves are accessed by the plant. plants sampled in both locations. Similar results were In biological terms fructan accumulation probably reported for A. angustifolia grown in Oaxaca (around aids the agave plants to withstand the hot semiarid 530 mg g1) or in Sonora states (around 360 mg g1) conditions which are their natural habitat and serve as a These differences may be related to climatic and or carbohydrate source for the rapid growth of the huge soil conditions in specific regions (Mancilla-Margalli & inflorescence (6–8 m or more in many cases) at the end Lopez, 2006). Genetic differences also play a role how- of the life cycle. ever exemplified by the differences in fructan accumu- Genes encoding enzymes involved in fructan and lation between Agave potatorum (around 500 mg g1) general carbohydrate metabolism are excellent candi- and Agave cantala (around 360 mg g1) obtained from dates for genetic manipulation in order to optimize the same location in Oaxaca state (Mancilla-Margalli & fructan accumulation, with important applications for Lopez, 2006). both tequila and bioenergy production. A search of the As the most efficient fructan producer A. tequilana agave transcriptome database revealed 33 sequences was traditionally favored by tequila manufacturers and encoding fructosyltransferases or invertases. When legally tequila production is now restricted to A. tequi- compared with sequences for the same gene families lana weber cultivar ‘azul’ and protected by a ‘controlled from other plant species the agave sequences were designation of origin’ (CRT:Consejo Regulador del Te- found to group with the monocotyledons in the family quila, 2009, http://www.crt.org.mx). Plants for tequila Asparagales as would be expected. Figure 5 shows the production take around 6–8 years to mature and a normalized distribution of these sequences in the dif- recent study (Arrizon et al., 2010) has shown different ferent cDNA libraries comprising the transcriptome. To levels of fructan accumulation in stem tissue in plants of compare the relative frequency of a particular gene different ages. Whereas 2-year-old plants have around between the different libraries, normalization of the 69% fructans 4- and 6.5-year-old plants contain around data was carried out by taking the number of high 97%. Fructans also accumulate in leaf tissue, although to quality sequences (short readso50 bp and highly am- much lower levels with an increasing gradient from the biguous reads removed) from a library designated as leaf tip to the leaf base estimated as ranging from 3.3% ‘reference’ and dividing by the number of high quality total soluble sugars at the tip to 13.1% at the leaf base sequences in the library to be normalized to produce a attached to the stem (Arrizon et al., 2010). Leaves ‘normalization factor’. The number of cDNA’s identi- are normally discarded in the field but could potentially fied for each gene was then multiplied by this factor to be exploited for other applications such as biofuel give the normalized frequency. These numbers were production. then represented graphically. Agave fructans have been shown to have complex Six different genes could be unambiguously molecular structures of the graminan and neofructan identified. Four which encode two different enzymes (agavin) types (Lopez et al., 2003; Mancilla-Margalli & (sucrose:sucrose 1-fructosyltransferase or 1-SST and

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Fig. 4 Simpliﬁed outline of enzymes involved in fructan metabolism in Agave and their activities. Black boxes-glucose residues, Open boxes-fructose residues, 1-SST, sucrose:sucrose 1-fructosyltransferase; 6-SFT, sucrose:fructan 6-fructosyl transferase; 6G-FFT, fructan:- fructan 6G-fructosyltransferase; 1-FFT, fructan:fructan 1-fructosyltransferase.

cDNA’s for all enzyme types were found in the ovary specific library whereas only invertase cDNA’s were found in the root library. Transcripts for individual genes also varied between tissues. cDNA’s for 1-SST2 were only found in the ovary specific library whereas 1- SST1 cDNA’s were found in ovary, inflorescence, meristem, bulbil and leaf libraries. 6G-FFT1 cDNA’s were found in ovaries, bulbils and anthers whereas 6G-FFT2 was found in anther and stem tissue in this analysis. In addition, only 6G-FFT cDNA’s were found in anther and stem tissue. When w2 analysis was carried out on the data a significant difference in transcript numbers was found only for the Cwinv cDNA’s in root tissue.

Cellulose and lignin metabolism During production of tequila and mezcal, the leaves of Fig. 5 Distribution of fructosyltransferase/invertase sequences the agave plants are normally discarded as waste, based in cDNA libraries obtained from different agave tissues. 1-SST- roughly on the data from (Iniguez-Covarrubias et al., sucrose:sucrose 1,fructosyltransferase; 6G-FFT, fructan:fructan 2001) about 3000 tons of waste in the form of agave 6G-fructosyltransferase; Inv-invertase; Cwinv-cell wall inver- leaves are produced each year during the production of tase. tequila with a proportionately smaller amount produced from mezcal production. Additionally bagasse in the form of heads or stems is produced during the fructan:fructan 6G-fructosyltransferase or 6G-FFT) in- ﬁber manufacturing process (Caceres-Farfan et al., volved in fructan biosynthesis and two different inver- 2008). Reports have shown that residual fructans can tases. Perhaps surprisingly, cDNA’s for 1-FFT and 6-SFT be rescued from these tissues and used for bioethanol were not identiﬁed in the transcriptome sequences. production (Caceres-Farfan et al., 2008; Hernandez- r 2010 Blackwell Publishing Ltd, GCB Bioenergy, 3, 25–36 32 J. SIMPSON et al.

Salas et al., 2009). As in the case of other bioenergy crops but this is a valuable characteristic which could be the ‘holy grail’ is to access the cellulosic content of the exploited for bioenergy production. bagasse which would make the exploitation of agave for Several reports have shown that manipulation of bioenergy a much more attractive proposition. An genes encoding enzymes involved in the lignin bio- understanding of the cellulose and lignin metabolism synthesis pathway by transgenic strategies can greatly in agave and the genes involved could eventually lead affect the overall levels of lignin and also the underlying to the development of strategies for the exploitation of molecular structures of lignin polymers (Boerjan et al., both the fructan and cellulosic components for bioe- 2003; Chen & Dixon 2007; Nakashima et al., 2008). Ten nergy production. genes have been described to encode enzymes involved As with fructan synthesis the fiber quality and con- in the lignin biosynthesis pathway (PAL-L-phenylanine tent of agave leaves varies between species and de- ammonia-lyase, C4H-Cinnamate 4-hydroxylase, 4CL-4 pends on the ligno-cellulose structures formed. A. coumarate:CoA ligase, CCR-Cinnamoyl CoA reductase, fourcroydes was chosen for commercial fiber production CAD-Cinnamoyl alcohol dehydrogenase, HCT-hydro- since it produces fibers of adequate length and strength. xycinnamoyl CoA:shikimate/quinate hydroxycinna- Although no detailed reports are available on the ge- moly transferase, C3H-Coumaroyl shikimate 3- netics of fiber quality and strength, certain species such hydroxylase, CCoAOMT-caffeoyl CoA 3-O-methyl- as A. americana or A. mapisaga have typically weak or esterase, F5H-ferulate 5-hydroxylase and COMT-Caf- reflexed leaves, whereas others such as A. tequilana have feic acid 3-O-methyltransferase) (Boerjan et al., 2003; straight, rigid leaves (Gentry, 1982) and these pheno- Chen & Dixon, 2007), making them excellent candidates types probably relate to the underlying fiber structure for genetic manipulation and warranting more detailed of the leaves. It is therefore possible that different study in agave. species harbor different alleles for either genes control- The tissues and developmental stages sampled to ling lignin or cellulose metabolism or those encoding produce the transcriptome data were not specifically enzymes involved in this process. An example of the aimed at genes involved in lignin and cellulose meta- different leaf phenotypes is shown in Figs. 6a and b. bolism however a search for cell wall related cellulases Few reports are available of detailed analysis of lignin identified 57 sequences. The normalized (see descrip- levels and structure in Agave species however the tion above) distribution of the sequences within the degree of acylation of lignin fibers was shown to be different cDNA libraries is shown in Fig. 7. Putative related to levels of b-b0resinol linkages (Martinez et al., genes encoding the three different classes of cellulases 2008). It is thought that fewer b-b0resinol linkages lead were found. Whereas only three sequences for exoglu- to lignin structures which are more amenable to chemi- canases were identified, 22 and 32 sequences were cal and/or biological degradation and it was observed found for endoglucanases and b-endoglucanases re- that A. sislana has much lower levels (4%) of these spectively. b-Glucanase cDNA’s were found in libraries linkages in relation to C. sativa (22%). Further work must be carried out on other Agave species to determine how variable this trait is within the Agavaceae family

Fig. 7 Distribution of cellulase cDNA’s in libraries obtained Fig. 6 Comparison of leaf morphology in two different Agave from different agave tissues. EXO, exoglucanses; GUN, endoglu- species. (a) Agave tequilana. (b) A. americana marginata. canases; BGLU, b-glucanases.

r 2010 Blackwell Publishing Ltd, GCB Bioenergy, 3, 25–36 GENOME AND TRANSCRIPTOME MINING IN A. TEQUILANA 33

and PAL which also showed significantly different numbers of transcripts in root tissue based on w2 analysis. Root tissue proved an excellent source for lignin biosynthesis cDNA&pos;s since 8 of the 10 enzymes analyzed were found in this tissue and only CCR and F5H were absent. Taking into account all tissues at least two different cDNA’s were identified for each enzyme. These examples demonstrate that the A. tequilana transcriptome is an extremely efficient resource for the identification of different general classes of sequences as shown by the analysis of cellulases and invertases or for the identification of specific enzymes within a biochemical pathway as shown by the analysis of the lignin biosynthesis genes. Based on the sequence data, detailed quantitative gene expression analysis for the different genes identified can be carried out and the complete coding sequences obtained in order to study Fig. 8 Distribution of lignin biosynthesis associated cDNA’s in in detail their characteristics and modes of regulation. libraries obtained from different agave tissues. PAL-L-phenylanine ammonia-lyase, C4H-Cinnamate 4-hydroxylase, 4CL-4 cou- Conclusions and perspectives marate:CoA ligase, CCR-Cinnamoyl CoA reductase, CAD- Cinnamoyl alcohol dehydrogenase, HCT-hydroxycinnamoyl The cultivation of agave for bioenergy production in CoA:shikimate/quinate hydroxycinnamoly transferase, C3H- Mexico is an attractive alternative use for these species, Coumaroyl shikimate 3-hydroxylase, CCoAOMT-caffeoyl CoA especially in the wake of the decline in the production 3-O-methylesterase, F5H-ferulate 5-hydroxylase,COMT-Caffeic of henequen fiber and the unpredictable and highly acid 3-O-methyltransferase. controlled market for production of tequila and mezcal. The potential to exploit the bagasse produced by these from all tissue types and endoglucanases in all but stem traditional industries is also an added advantage. tissue. However exoglucanase cDNA’s were only found In order to fully exploit their potential however, many in anther and meristem libraries. Based on w2 analysis questions remain to be answered. These range from significant differences were observed in numbers of basic biological questions such as which species should cDNA’s identified for exoglucanases in inflorescence be cultivated and in which regions of the country, (lower than expected) and anther tissue and for b-gluca- through economic considerations such as accessibility nases in inflorescence, root (greater than expected) and and transport for processing and the construction of anther tissues (lower than expected). No significant dif- new installations for the process or the adaptation of ferences were detected in any tissue for endoglucanases. existing factories, to the potential for optimization In the case of lignin metabolism, a search was carried of bioenergy production and in particular the exploita- out for genes encoding the specific enzymes known to tion of the cellulosic components of the plants. To be involved in lignin biosynthesis based on previous resolve these problems, collaboration between research- reports (Chen & Dixon 2007; Nakashima et al., 2008). ers in different disciplines, industry and the govern- Sequences for all 10 enzyme encoding genes were found ment will be necessary. and the normalized (see description above) distribution Our interest is in the development of molecular- among the cDNA libraries is shown in Fig. 8. genetic tools which can be accessed and exploited by As can be noted from the figure, although sequences the community of agave researchers to advance their were found for each enzyme of the lignin biosynthesis own particular research goals. The agave transcriptome pathway, the number and distribution of the sequences database and stored cDNA libraries have proven ex- within the different cDNA libraries differed widely. tremely efficient for identification of cDNA’s of interest, Sequences for C3H were only found in root tissue and including some of those with potential applications for this observation was shown by w2 analysis to be sig- bioenergy production as demonstrated in the examples nificant, strongly suggesting a higher expression level described above. The existing cDNA libraries have not for C3H genes in roots. In contrast CAD sequences were yet been sequenced to saturation and the availability of found in all tissues sampled. All other genes encoding stored, in many cases full-length transcripts, facilitates lignin biosynthesis genes showed no significant differ- the analysis of genes of interest as does the relative ease ences in transcript levels with the exception of COMT of in situ expression analysis and heterologous analysis r 2010 Blackwell Publishing Ltd, GCB Bioenergy, 3, 25–36 34 J. SIMPSON et al. in A. thaliana. Although the construction of genomic Bowen S, Valenzuela-Zapata A (2008) Designations of origin and libraries and a full-scale agave genome sequencing socioeconomic and ecological sustainability: the case of tequila endeavor would be of enormous advantage, currently in Mexico. Cahiers Agricultures, 17, 552–560. the most important stumbling block is the lack of an Bowen S, Valenzuela-Zapata A (2009) Geographical indications, efficient agave transformation protocol. terroir, and socioeconomic and ecological sustainability: the case of tequila. Journal of Rural Studies, 25, 108–119. 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