Investigating low temperature resistance in Brachypodium distachyon, a cold stress and crop model
Low temperature poses one of the most biologically taxing and economically significant stresses for a variety of agricultural crops. To combat cold stress, resilient plants can undergo cold acclimation, a process involving intricately programmed genetic, metabolic, and physiological changes. Ice-binding proteins (IBPs), if present, are upregulated under cold stress and present great biotechnological potential for their ability to protect crops against pathogen-associated freezing damage responsible for widespread economic devastation. Until recently, functional investigation of IBPs via knockdown had not been attempted in any organism, but it was accomplished for the first time in the model crop, Brachypodium distachyon, which has emerged as an ideal experimental system for low temperature tolerance in plants. B. distachyon itself has no economical value of more than an invasive weed; however, the use as a model crop refers to the ease of use for research in place of typical crop species. These first IBP knockdowns proved problematic likely due to the pleiotropic effects of constitutive miRNA expression. Here I present a new strategy to generate transgenic B. distachyon containing a temporal miRNA-facilitated IBP knockdown wholly generated without using tissue culture or traditional genotyping techniques. Plants with temporal knockdown of IBPs confirmed the functional importance of IBPs under cold stress, without other obvious pleiotropic effects. Wildtype plants that had not been cold acclimated produced low, basal levels of IBP expression, suggesting that IBPs may be constitutively expressed for developmental needs and only upregulated under cold stress for protective measures. Although it could not be included as part of this thesis, preliminary assessment of the plasma membrane proteome showed that IBPs do not localize to the plasma membrane, with some potential new protein candidates for future studies on cold stress highlighted. Perhaps most importantly, the methods employed here for transgenic plant generation present an unprecedented level of processivity and low cost so that studies on these many newly identified proteins are now feasible. Thus, an additional legacy of this thesis could be an increasing accessibility and attractiveness of transgenic monocot research in the crucial years to come for humanity.