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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.

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Nature exhibits huge diversity in organ shape, and yet all organs start as small bud-like peripheral outgrowths. Combinations of different spatial and temporal developmental switches in shape determine final organ shape. In plants shape arises through growth, which is defined by axiality and growth rates. Here I tested three hypotheses for how developmental switches in shape could arise: (1) growth rates alone are altered, (2) axiality alone is altered (3) both growth rates and axiality are altered. Using a multidisciplinary approach I explored which of the hypotheses was true for developmental switches in shape during organ development in two monocot models: early grass leaf development and the Hooded barley mutant. Developmental switches in shape were first volumetrically described using 3D imaging. Using this framework, computational models were generated to formulate hypotheses which could account for the switches in shape. Model predictions were then tested using whole-mount immunolocalisation of SISTER OF PINFORMED 1 (SoPIN1), gene expression, and cell division and shape analyses. Synthetic biology was also used to generate a set of transgenic tools for future testing of the models. I found that a developmental switch in shape during early grass leaf development may arise through alterations in growth rates alone (hypothesis 1). In contrast, ectopic flower and wing formation in Hooded may arise through modulation of growth rates and axiality combined (hypothesis 2). In this case a single gene, BKn3, triggers the growth change, possibly through directly influencing tissue cell polarity (if axiality is defined by a polarity based axiality system), with differential effects on shape depending on where it is expressed. This suggests that novel developmental switches in shape could evolve due to single gene mutations, and that during evolution, modulation of growth may have been redeployed in different spatial and temporal patterns to trigger novel changes in shape, ultimately changing final form.

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The correlation between the structure and function of cells may be studied in a number of ways. Some of these methods have been used in this thesis in an attempt to study the elongation zone of geotropically stimulated maize root tips and wheat internodes. Using a cytochemical technique, adenyl cyclase activity has been investigated in the cells of maize root tips and other tissues using a specific substrate developed originally for mammalian tissues. Heavy deposits were found in the plasma membrane, endoplasmic reticulum and nuclear membrane, and the findings are discussed in relation to the reported localization of adenyl cyclase in animal and plant cells. As with adenyl cyclase, no distinct changes were found in the localization of ATPase and peroxidase activities in relation to the geotropic response. The effect of aldehyde fixation of excised roots and of subcellular fractions of maize were studied in relation to the preservation of ATP-ase and peroxidase activities. Total peroxidase was little affected by either formaldehyde or glutaraldehyde whereas ATP-ase showed a considerable loss of activity, particularly with glutaraldehyde. The activity remaining after fixation was dependent on both the concentration of fixative and the pH of fixation.