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Genetic Analyses Of Calcium Circuits In Arabidopsis Vegetative And Reproductive Development
AuthorRahmati Ishka, Maryam
AdvisorHarper, Jeffrey F.
Biochemistry and Molecular Biology
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Calcium homeostasis is maintained by the action of different calcium transporters including channels, antiporters, and calcium pumps. Calcium channels provide an influx pathway whereas calcium antiporters and pumps provide an efflux system. Together, influx and efflux systems create a calcium circuit. One of the calcium circuits operating in plant cells is associated with the endoplasmic reticulum (ER). In chapter II, results are presented on the functional characterization of three calcium pumps in Arabidopsis with ER and ER-like localization. In chapter III and IV, Arabidopsis pollen transcriptome data are presented, which compare heat-stress induced changes in the pollen transcriptomes of wild type and a mutant harboring a knockout of a cyclic nucleotide gated channel (cngc16).Calcium pumps ACA1, 2, and 7 belong to the type IIB subfamily of Ca2+-ATPases. In a comparison of protein sequences for all ten ACA Ca2+-pumps in Arabidopsis, ACA1, 2, and 7 cluster as a distinct subgroup. Members of this subgroup all appeared to localize to the ER or ER-like structures when expressed in stably transformed plants with a YFP tag and imaged with confocal microscopy. A loss-of-function for all three ACAs, ACA1, 2, and 7, resulted in plants showing a lesion-like phenotype in the leaves. This phenotype was suppressed by the expression of a NahG transgene, which encodes an enzyme that degrades salicylic acid, indicating that the lesion phenotype is dependent on salicylic acid. In addition, a triple knockout of aca1/2/7 resulted in a decrease in pollen transmission efficiency when assayed in competition with wild type pollen. This pollen transmission deficiency was exacerbated when crosses were done under temperature stress conditions. Both phenotypes could be rescued by the expression of any of the threeACA1, 2 or 7 genes, suggesting that all three genes encode Ca2+-pumps that are functionally interchangeable. Together, these results establish ACA1, 2, and 7 as a subgroup of ER-localized autoinhibited Ca2+-ATPases that normally function to suppress an SA-dependent programmed cell death pathway in leaves, and are also important for pollen fitness under normal and temperature stress conditions.Under temperature stress conditions, pollen harboring a cngc16 knockout are nearly sterile, and show a greater than 10-fold decrease in pollen transmission efficiency compared to wild-type pollen. To gain further insight into mechanisms underlying this hypersensitivity, an RNA-Seq experiment was performed to compare the stress-dependent changes in the pollen transcriptomes from wild-type and a cngc16 knockout parent. In response to a heat stress, the wild-type pollen transcriptome showed 2,612 changes (≥2-fold changes and adjusted p<0.01). Of these, 641 changes (~ 25%) failed to occur in the cngc16 knockout, identifying a subset of failed responses that might contribute to the poor stress tolerance displayed by cngc16 pollen. At the same time, the mutant displayed 2,733 changes that were not seen in wild-type pollen. Of these differences between wild type and cngc16, 148 were associated with transcription factors, suggesting that a primary defect in the cngc16 mutant lies in its inability to properly reprogram the transcriptome in response to a heat stress. Overall, the RNA-Seq experiment here provides a great resource for research community to explore genes function of potential importance to pollen thermotolerance.