Living organisms continuously monitor, interpret, and respond to their environments. They do so through an assortment of mechanisms, but the focus of this doctoral dissertation is on two strikingly prevalent mechanisms: calcium signaling and protein phosphoregulation. These two processes are linked through the activities of calcium-regulated protein kinases and phosphatases. This dissertation focuses on calcium signaling processes in plants and the role that calcium-decoding protein kinases, the elusive upstream calcium signal- coding complexes, and the downstream target proteins play in plant environmental responses. The described research builds upon recent advances in the field of plant genomics, merging bioinformatic methods with experimental approaches. Notably, much of the research utilizes genomic and transcriptomic data from an emerging model plant species, the moss Physcomitrella patens. Because all land plants are evolutionarily related, insights from mosses – an early-diverging plant lineage – are useful to classify proteins and assess functional conservation or divergence. In addition to synthesizing relevant published research, this dissertation describes the phylogenomic classification of a family of plant-specific calcium sensors known as calcineurin B-like proteins (CBLs) and their associated protein kinases (CIPKs). Plants feature several families of protein kinases that share a similar overall architecture: an N-terminal serine/threonine kinase domain coupled to a C-terminal autoinhibitory region. Some of these protein kinase families are either directly or indirectly regulated by calcium, the latter exemplified by CIPKs. Calcium-dependent protein kinases (CDPKs) are directly regulated by calcium through calcium-binding EF-hand domains in the autoinhibitory region. Both CIPKs and CDPKs constitute multi- gene families in all sequenced land plant genomes. In contrast, calcium/calmodulin-dependent protein kinases (CCaMKs) are found as single-copy genes in most sequenced plant genomes and have been evolutionarily lost in certain plant lineages, including the preeminent model plant Arabidopsis. The accepted rationale for the inferred independent losses of CCaMK loci has been that CCaMKs fulfill obligate functions in plant-microbe symbioses that are incompatible with some plant lineages due to relatively recent evolutionary events. During the course of characterizing CIPKs, the unexpected observation that Physcomitrella – which is incompetent for canonical plant-microbe symbioses – contains two loci encoding CCaMKs prompted an investigation into CCaMK function in mosses. Through implementation and refinement of a gain- of-function approach in Physcomitrella, this doctoral research demonstrated that activation of a Physcomitrella CCaMK (CCaMK1) was associated with formation of brood cells, a type of stress-induced asexual propagule formed by mosses. In legumes, CCaMK regulates CYCLOPS, a novel type of transcription factor, by phosphorylating it at two sites in its autoinhibitory region. Physcomitrella contains a single CYCLOPS homolog that interacts with CCaMK1 and shows strong sequence conservation at the two key phosphosites identified by prior work in legumes. Expression of a modified form of Physcomitrella CYCLOPS containing two putative phosphomimetic substitutions at these sites also likewise elicited brood cell formation. These observations suggest that Physcomitrella CCaMK and CYCLOPS homologs operate in a similar manner to the legume CCaMK-CYCLOPS module; yet in Physcomitrella, the module tends to a disparate function. This dissertation further describes efforts to identify ion channels that function in calcium signal-coding processes. These efforts contributed to the identification of a novel family of putative cation channels through a combination of heterologous expression, electrophysiological techniques, and bioinformatic approaches. This doctoral dissertation encompasses a broad range of interconnected processes involved in calcium signal generation and interpretation in biology, and the field stands to address many unanswered questions detailed here. To that end, prospective ideas for further investigation are provided in the concluding remarks.

With the threat of climate change becoming more apparent in recent years, it is clear that the increased severity and regularity of extreme weather events will inevitably impact our ability to maintain agricultural productivity in the future. One relatively unexplored avenue towards increasing plant health and resiliency has also recently emerged, however, as the microbes which live in and around plant roots have been shown to influence biomass and yield, as well as resistance to both biotic and abiotic stresses. Therefore, an ability to manipulate and engineer these microbial communities, referred to as the plant microbiome, has the potential to help ensure food security amidst environmental turmoil. Unfortunately, this dream of an engineered, beneficial plant microbiome has yet to be realized. This is in large part due simply to the nascency of the field of research as a whole, with large gaps in our basic understanding still needing to be addressed. Work must be done to generate a more complete understanding of the microbes themselves and the processes by which they are regulated by plant hosts, but also towards developing more robust and efficient plant engineering capabilities in order to realize the potential real-world benefits of an engineered microbiome. The research presented in this dissertation was undertaken in an effort to address these current shortcomings, and hopefully contribute to future efforts aimed at harnessing and utilizing the plant microbiome for societal gain. First, this work investigates factors influencing the fungal microbiome assembly in a forest tree, the coast redwood Sequoia sempervirens. Because most research has been performed in model or crop species in agricultural or fully artificial conditions, investigating patterns of microbiome structure in a gymnosperm growing in a natural forest setting will allow us to assess the universality of previously reported phenomena related to the plant microbiome. We found that, as expected, soil chemistry and pH in particular is a strong determinant of root-associated fungal communities. We also demonstrated that forest continuity and heterogeneity influence the soil microbiome, which shows that principles of landscape ecology can indeed be useful in deepening our understanding of microbial ecology. Finally, we discovered that redwoods have specialized organs which function to morphologically compartmentalize symbiotic arbuscular mycorrhizal fungi, which serves to highlight the need for holistic assessment of factors influencing the plant root microbiome rather than a narrow focus on, for instance, singular -omic techniques. To build upon the theme of holistic rather than narrow microbiome investigations, this research goes on to summarize an ambitious holo-omic characterization of how sorghum and its microbiome interact with drought stress. Termed the EPICON Project, this undertaking serves as an exemplary case-study of using multiple -omic techniques, termed holo-omics, in order to better understand complex interactions governing microbiome assembly and development. Specifically, this work highlighted the correlation between a key metabolite, glycerol-3-phosphate (G3P), as well as transcriptional response to reactive oxygen stress (ROS) mitigation, with reproducible shifts in the sorghum root microbiome under drought. These findings serve as testaments to the power of holo-omic studies in progressing the field of plant microbiome research towards the ultimate goal of being able to engineer desired phenotypes relevant to world agriculture as it faces climate change-induced challenges. Finally, we used the lessons learned through holo-omic investigation of the sorghum root microbiome in conjunction with novel plant transformation techniques and the CRISPR-Cas9 gene editing system in order to provide a framework for future efforts aimed at microbiome engineering. Although significant progress was made in improving overall transformation efficiency in sorghum through the use of the morphogenic genes Babyboom (BBM) and Wuschel (WUS), the successful editing of our target genes remained elusive. Nonetheless, we hope that this work has helped lay the groundwork for future efforts towards realizing an engineered plant root microbiome.

Main conclusion