How to awaken a sleeping giant: antagonistic expression of Flowering locus T homologs and elements of the age-related pathway are associated with the flowering transition in Agave tequilana.

KEY MESSAGE: Antagonistic expression of Flowering locus T proteins and the ageing pathway via miRNAs and sugar metabolism regulate the initiation of flowering in A. tequilana. Flowering in commercial plantations of Agave tequilana signals that plants are ready to harvest for tequila production. However, time of flowering is often unpredictable and a detailed understanding of the process would be beneficial in the field, for breeding and for the development of future research. This report describes the functional analysis of A. tequilana FLOWERING LOCUS T (FT) genes by heterologous expression in A. thaliana and in situ hybridization in agave plants. The gene structures of the Agave tequilana FT family are also described and putative regulatory promoter elements were identified. Most Agave species have monocarpic, perennial life cycles that can last over 25 years during which plants do not respond to the normal environmental signals which induce flowering, suggesting that the ageing pathway as described in Arabidopsis may play an important role in determining flowering time in these species. Elements of this pathway were analyzed and in silico data is presented that supports the regulation of SQUAMOSA PROMOTER BINDING LIKE proteins (SPL), APETALA2 (AP2) proteins and members of Plant Glycoside Hydrolase Family 32 (PGHF32) by interactions with miRNAs 156, 172 and 164 during the initiation of flowering in A. tequilana.

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

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.

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

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.

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

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.

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

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.