a Exploiting the potential of Agave for bioenergy in marginal lands

Abstract

Drylands cover approximately 40% of the global land area, with minimum rainfall levels, high temperatures in the summer months, and they are prone to degradation and desertification. Drought is one of the prime abiotic stresses limiting crop production. Agave plants are known to be well adapted to dry, arid conditions, producing comparable amounts of biomass to the most water-use efficient C3 and C4 crops but only require 20% of water for cultivation, making them good candidates for bioenergy production from marginal lands. Agave plants have high sugar contents, along with high biomass yield. More importantly, Agave is an extremely water-use efficient (WUE) plant due to its use of Crassulacean acid metabolism. Most of the research conducted on Agave has centered on A. tequilana due to its economic importance in the tequila production industry. However, there are other species of Agave that display higher biomass yields compared to A. tequilana. These include A. mapisaga and A. salmiana and A. fourcroydes Lem has been reported to possess high fructan content making it a promising plant for biofuel feedstock. Also, fructans act as osmo-protectants by stabilizing membranes during drought and other abiotic stress. This project set out to examine several hypotheses. In the first experimental chapter (Chapter 2), the central aim was to start identifying traits for the improvement of Agave species for biomass production on arid lands by first examining if the capacity of CAM, and fructan accumulation are linked traits. To address this question 3 species of Agave varying in succulence were compared under different water regimes. Measurements were made of leaf, gas exchange and titratable acidities as markers of CAM and of soluble sugar and fructan content using high performance liquid chromatography (HPLC). High leaf succulence is associated with increased magnitude of CAM, manifested as higher H+ and nocturnal CO2 uptake and fructan accumulation also increased with leaf succulence in Agave. Sucrose provided most, if not all of the substrate required for dark CO2 uptake. At the leaf level, highest CAM activity was found in the tip region whilst most fructan accumulation occurred in the base of the leaf. These results indicate that CAM and fructan accumulation are subject to contrasting anatomical and physiological control processes. v In Chapter 3, the aim was to test 4 hypotheses relating to succulence and biochemical capacity for C3 and C4 carboxylation in Agave. The first hypothesis tested the abundance of PEPC and its variation between species in relation to leaf succulence and age and will vary along the leaf, in line with differences in CAM activity. The second hypothesis looked into the abundance of Rubisco and Rubisco activase and its variation between species in relation to leaf succulence and age and will vary along the leaf, in line with differences in CAM activity. The third hypothesis the more succulent Agave species, drought will have less impact on the abundance of PEPC, Rubisco and Rubisco activase compared to the less succulent species. And the abundance of Rubisco activase will vary over the diel cycle, particularly in leaves of more succulent species of Agave. Results showed that leaf succulence influenced the abundance of PEPC. Thus, the optimal anatomy for nocturnal malic acid accumulation is accompanied by high PEPC abundance in leaves with higher vacuolar storage capacity. In contrast, the abundances of Rubisco and Rubisco activase showed an inverse relationship to succulence and CAM activity. The aim of Chapter 4, was to identify other species of Agave that could be exploited as sources of biofuel from semi-arid marginal lands. Some 14 different species of Agave that showed varying levels of succulence were compared, evaluating the capacity for CAM, fructan content, carbohydrate composition, osmotic pressure and the relationship with succulence. Results demonstrated that Inter-specific variations in the magnitude of expression of CAM in Agave are dependent on leaf succulence. Also, Agave displays flexibility in the use of carbohydrate source pools to sustain dark CO2 uptake. Some species appear to use fructans and others sucrose as substrate for dark CO2 uptake. The final experimental Chapter’s aim was to develop a method to identify vacuolar sugar transporters in Agave related to sucrose turnover and fructan accumulation. First, identifying the tonoplast by testing activity of ATPase and PPiase of leaf vesicles of Agave Americana marginata, and its sensitivity to inhibition by known ATPase inhibitors. Second, was to use a proteomics approach, analysing of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS, to identify vacuolar sugar transporter proteins which are hypothesized to play a key regulatory role in determining sucrose turnover for CAM and fructan accumulation and as such, vi could represent future targets for genetic engineering of increased sugar content for plants grown for bioenergy. The capacity of the vacuole as a sink for carbohydrate maybe an important determinant of CAM expression and has important implications for plant growth and productivity. Combining tonoplast proteomics with the interrogation of diel transcriptome data is a potentially powerful approach to identify candidate vacuolar sugar transporters in Agave.