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Calcium Signaling and Protein Phosphoregulation in Plant Environmental Perception and Responses

  • Author(s): Kleist, Thomas James
  • Advisor(s): Luan, Sheng
  • Lemaux, Peggy G
  • et al.
No data is associated with this publication.
Abstract

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.

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This item is under embargo until May 14, 2020.