Browsing by Subject "Arabidopsis"

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    Functional dissection of ERD14 phosphorylation-dependent calcium binding activity
    (2014-12-11) Chacha, Allen R.; Randall, Stephen Karl, 1953-; Watson, John; Kusmierczyk, Andrew
    Drought and cold conditions are among the major factors affecting plant growth and crop production globally. Dehydrins are group II late embryogenesis abundant (LEA) proteins characterized by a conserved K-region (EKKGIMDKIKEKLPG consensus sequence) that accumulate in many plants during drought, low temperature, and high salinity to confer stress tolerance. While it has been demonstrated that overexpression of dehydrins improves cold tolerance in various crop plants, the mechanism leading to cold tolerance is still unclear. Previous studies reported phosphorylation of AtERD14 dehydrin by casein kinase II (CKII) led to an increase in calcium binding activity. Mass spectroscopy analysis determined that the phosphorylation was localized to a poly-serine (S) region. To further characterize the S-region, GST fused ERD14 mutants were created via site-directed mutagenesis and deletion of either the amino or carboxyl ends of ERD14 via the QuickChange® Multi Site-Directed Mutagenesis Kit. Phosphorylation of purified mutant proteins by CKII was analyzed via gel shift and direct phosphorylation assays. The effect of phosphorylation on calcium binding activity was also analyzed. Results showed the serine (S) residue at position 83 was crucial to phosphorylation-dependent molecular mass shift and Ca2+-binding activities followed by the serine residue at position 85 in importance. Mutation of serines at positions 83, 84, and 85 completely eliminated the phosphorylation-dependent gel shift and calcium binding. Examination of truncation mutants determined the N-terminal was an important region for protein structure modification and phosphorylation ability leading to Ca2+ activation. Calcium binding activity of the truncated mutants indicated the calcium binding site was localized in the region between the S-region and the K-region near the C-terminal end. To characterize the acidic dehydrins contribution to cold tolerance in vivo, three single (erd10, erd14, cor47) knockouts (KOs) were characterized. Single KOs produced no cold sensitive phenotype indicating the need for multiple dehydrin KOs in Arabidopsis in order to potentially produce a cold sensitive phenotype.
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    A Lateral Root Defect in the wag1-1/wag2-1 Double Mutant of Arabidopsis
    (2012-08-07) Rowland, Steven D.; Watson, John C., 1953-; Randall, Stephen Karl, 1953-; Chernoff, Ellen
    The root system architecture of higher plants plays an essential role in the uptake of water and nutrients as well as the production of hormones. These root systems are highly branched with the formation of post-embryonic organs such as lateral roots. The initiation and development of lateral roots has been well defined. WAG1 and WAG2 are protein-serine/threonine kinases from Arabidopsis that are closely related to PINOID and suppress root waving. The wag1;wag2 double mutants exhibit a strong root waving phenotype on vertical hard agar plates only seen in wild-type roots when the seedlings are grown on inclined plates. Here an additional root phenotype in the wag1;wag2 mutant is reported. The wag1;wag2 double mutant displays both an increased total number and density of emerged lateral roots (approximately 1.5-fold). An increased LRP density of 1.5-fold over wild-type is observed. To ascertain the role of WAG1 and WAG2 in lateral root development we examined promoter activity in the WAG1::GUS and WAG2::GUS lines. The WAG1 promoter showed no detectable activity at any stage of development. The WAG2 promoter was active in stage IV onward, however there was no detectable activity in the cell types associated with initiation events. The lateral root density and spatial patterning in wild-type, when grown on inclined hard agar plates, was similar to wag1;wag2 on vertical plates. Seedlings of both genotypes were treated with hormones such as auxin and MeJA, and inhibitors. Auxin response in wag1;wag2 was normal with a similar number of LR as the wild-type after treatment. Treatment with MeJA resulted in a similar induction of LRP in both genotypes, however the percent lateral root emergence in wag1;wag2 was reduced while Col-0 was increased compared to controls. Treatment with the calcium blocker tetracaine resulted in wag1;wag2 displaying a wild-type level of LR but had no significant effect on wild-type. Genetic analysis of the wag1;wag2 LR pathway revealed that WAG1 and WAG2 are acting in the same pathway as AUX1, AXR1and PGM1. pgm1-1 was not previously reported to have a LR defect but showed decreased LR formation here, while pgm1;wag1;wag2 had a similar LR density to wag1;wag2. TIR7 and ARG1 were both deduced to operate in separate pathways from WAG1 and WAG2. The data presented here shows that the wag1;wag2 double mutant has an increased number of LR compared to Col-0. This defect appears to be caused by increased pre-initiation events and seems to be tied to the root waving phenotype. However, the treatment with MeJA revealed a possible role for WAG1 or WAG2 in LRP development, potentially under stress conditions. Calcium also seems to play a significant role in the wag1;wag2 LR phenotype, possibly independent of the root waving phenotype.
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    SH3 Domain-Containing Protein 2 Plays a Crucial Role at the Step of Membrane Tubulation during Cell Plate Formation
    (American Society of Plant Biologists, 2017-06) Ahn, Gyeongik; Kim, Hyeran; Kim, Dae Heon; Hanh, Hong; Yoon, Youngdae; Singaram, Indira; Wijesinghe, Kaveesha J.; Johnson, Kristen A.; Zhuang, Xiaohong; Liang, Zizhen; Stahelin, Robert V.; Jiang, Liwen; Cho, Wonhwa; Kang, Byung-Ho; Hwang, Inhwan; Biochemistry and Molecular Biology, School of Medicine
    During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.