Luis F. Maceda-López

Resilience of growing in arid and semiarid regions and a high capacity of accumulating sugar-rich biomass with low lignin percentages have placed Agave species as an emerging bioenergy crop. Although transcriptome sequencing of fiber-producing agave species has been explored, molecular bases that control wall cell biogenesis and metabolism in agave species are still poorly understood. Here, through RNAseq data mining, we reconstructed the cellulose biosynthesis pathway and the phenylpropanoid route producing lignin monomers in A. tequilana, and evaluated their expression patterns in silico and experimentally. Most of the orthologs retrieved showed differential expression levels when they were analyzed in different tissues with contrasting cellulose and lignin accumulation. Phylogenetic and structural motif analyses of putative CESA and CAD proteins allowed to identify those potentially involved with secondary cell wall formation. RT-qPCR assays revealed enhanced expression levels of AtqCAD5 and AtqCESA7 in parenchyma cells associated with extraxylary fibers, suggesting a mechanism of formation of sclerenchyma fibers in Agave similar to that reported for xylem cells in model eudicots. Overall, our results provide a framework for understanding molecular bases underlying cell wall biogenesis in Agave species studying mechanisms involving in leaf fiber development in monocots.

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

Spines are key plant modifications developed to deal against herbivores; however, its physical structure and chemical composition have been little explored in plant species. Here, we took advantage of high-throughput chromatography to characterize chemical composition of Agave fourcroydes Lem. spines, a species traditionally used for fiber extraction. Analyses of structural carbohydrate showed that spines have lower cellulose content than leaf fibers (52 and 72%, respectively) but contain more than 2-fold the hemicellulose and 1.5-fold pectin. Xylose and galacturonic acid were enriched in spines compared to fibers. The total lignin content in spines was 1.5-fold higher than those found in fibers, with elevated levels of syringyl (S) and guaiacyl (G) subunits but similar S/G ratios within tissues. Metabolomic profiling based on accurate mass spectrometry revealed the presence of phenolic compounds including quercetin, kaempferol, (+)-catechin, and (−)-epicatechin in A. fourcroydes spines, which were also detected in situ in spines tissues and could be implicated in the color of these plants’ structures. Abundance of (+)-catechins could also explain proanthocyanidins found in spines. Agave spines may become a plant model to obtain more insights about cellulose and lignin interactions and condensed tannin deposition, which is valuable knowledge for the bioenergy industry and development of naturally dyed fibers, respectively.