183 research outputs found
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CHARACTERIZING THE R2R3 S21 AND S23 MYB TRANSCRIPTION FACTORS IN ARABIDOPSIS THALIANA
No full textCHARACTERIZING THE R2R3 S21 AND S23 MYB TRANSCRIPTION FACTORS INARABIDOPSIS THALIANAAbstractBy Chase Montgomery Beathard, Ph.D. Washington State UniversityJune 2019Chair: Hanjo A. HellmannThe ubiquitin (UBQ) proteasome pathway is a highly conserved mechanism by which plants respond to their environments. The pathway proceeds by an E1-E2-E3 enzymatic cascade that targets proteins with UBQs to signal for their degradation via the 26S proteasome. The E3 ligase component provides specificity to the pathway and physically assembles with a substrate protein. The interaction between the E3 ligase and the substrate is facilitated by a substrate adaptor protein. One type of substrate adaptor that is utilized by CULLIN3-based (CRL3) E3 ligases is BRIC-A-BRAC, TRAM-TRACK AND BROAD COMPLEX (BTB)/POX VIRUS AND ZINC FINGER (POZ)/ MEPRIN AND TUMOR NECROSIS FACTOR RECEPTOR ASSOCIATED FACTOR HOMOLOGY (MATH), or BPM protein.BPM proteins target transcription factors from families including MYELOBLASTOSIS (MYB), HOMEOBOX LEUCINE ZIPPER (HD-ZIP), and ETHYLENE RESPONSE FACTOR/APETALA2 (ERF/AP2) proteins. Two regions have been identified that facilitate a substrate’s interaction with BPMs, a SPOP binding consensus (SBC) domain and a PEST motif that is also associated with protein instability.This work demonstrates that a clade of R2R3 S21 MYB transcription factors, MYB52, MYB54, MYB56, and MYB69, interact with a BPM substrate adaptor and are instable to varying degrees. Moreover, if the MYB56 protein loses its PEST motif, it becomes more stable.We also find that the R2R3 S23 MYB transcription factors, MYB1, MYB25, and MYB109 are instable targets of the UBQ proteasome pathway. The overexpression of MYB25 and MYB109 confers hypersensitivity to salt and an associated stress hormone, abscisic acid (ABA). Transcriptomic data further support the role of MYB25 and MYB109 in salt and ABA response and also show involvement of MYB25 and MYB109 in other cellular processes including defense, light signaling, and general stress response.Collectively, these results broaden the knowledge of R2R3 MYBs that are targeted by CRL3BPM. We provide a first characterization of MYB1, MYB25, MYB52, MYB54, and MYB109 regarding their instability and interaction with BPMs, and further support the PEST motif’s role in protein stability and interaction with CRL3BPM. We show a first phenotypic characterization of MYB25 and MYB109, and provide insight into the broader transcriptomic roles that MYB25 and MYB109 play in plants
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Characterization of PDX1.2 and a novel PDX1.2 interactor
No full textVitamin B6 is a crucial compound in all living organisms based on its capacity to function as a cofactor in more than 140 biochemical processes. In addition the vitamin has been demonstrated to be a powerful antioxidant that prevents cell damage. In plants, the de novo pathway depends on the concerted activities of PDX1 (Pyridoxine Biosynthesis Protein 1) and PDX2 that are able to catalyze the formation of a PLP synthase. In Arabidopsis thaliana, PDX1 encodes for three homologs PDX1.1, PDX1.2, and PDX1.3, but only PDX1.1 and PDX1.3 are able to interact with PDX2. While the functions of PDX1.1 and PDX1.3 have been extensively studied, the role of PDX1.2 has only recently started to become resolved. PDX1.2 has been identified in previous studies to be up-regulated under certain abiotic stressors such as heat, oxidative, and UV-B stress. It is also been observed to stabilize the other PDX1 proteins under a heat stress. It was previously shown via size-exclusion chromatography that AtPDX1.2 assembles into a complex of around 750 kDa, which is around the size of a PLP synthase. Since PDX1.2 does not interact with PDX2, it appeared to be likely that PDX1.2 assembles with another, yet unknown protein. A yeast-2-hybrid screen found an unknown and uncharacterized protein that exclusively interacts with PDX1.2. The work overall provides a better understanding of the role PDX1.2, and its involvement in the biosynthesis of vitamin B6 and stress tolerance. It looks at a novel PDX1.2 interactor called NPI1 and its impact on development, stress tolerance, and a potential role in the biosynthesis of vitamin B6
The Hellmann–Feynman theorem, the comparison theorem, and the envelope theory
No full textAbstractThe envelope theory is a convenient method to compute approximate solutions for bound state equations in quantum mechanics. It is shown that these approximate solutions obey a kind of Hellmann–Feynman theorem, and that the comparison theorem can be applied to these approximate solutions for two ordered Hamiltonians
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THE IMPACT OF PHOSPHOMIMICS ON THE ALLOSTERIC REGULATION OF PHOSPHOENOLPYRUVATE CARBOCYATION (PEPC) FROM SETARIA VIRIDIS
No full textThe carbon concentrating mechanism of C4 photosynthesis has been extensively studied, revealing a complex suite of anatomical and biochemical adaptations required for its high photosynthetic efficiency. While there is growing interest in optimizing this pathway to enhance crop resilience under abiotic stress, many questions remain regarding the regulation and evolution of its primary rate-limiting enzymes, particularly phosphoenolpyruvate carboxylase (PEPC). To support bioengineering efforts aimed at improving carbon assimilation, it is essential to better understand how specific factors influence PEPC kinetics and regulation. This thesis investigates how PEPC kinetic properties and allosteric regulation is fine-tuned through post-translational modifications in the C4 model species Setaria viridis. Focusing on phosphorylation at the conserved N-terminal Ser-11 residue, I explore how phosphomimic substitutions (SvPEPC_S11D and SvPEPC_S11E) at this serine residue impact PEPC kinetic parameters, including substrate affinity (Kₚₑₚ) and sensitivity to key allosteric regulators such as malate and glucose 6-phosphate (G6-P). These findings demonstrate that phosphorylation at Ser-11 plays a key role in modulating the trade-off between PEPC activity and feedback inhibition in C4 photosynthesis. Phosphomimic variants at Ser-11 were generated by substituting this residue with the negatively charged aspartic acid (SvPEPC_S11D) or glutamic acid (SvPEPC_S11E). These PEPC variants had significantly different affinity for PEP (higher KPEP) compared to the wild-type enzyme (SvPEPC_WT), indicating the phosphomimics altered the PEP binding affinity. Additionally, both phosphomimics showed a higher AC50 for G6-P, suggesting enhanced sensitivity to this allosteric activator, while exhibiting elevated IC50 values for malate, indicating reduced susceptibility to feedback inhibition. Caution should be taken when interpreting the AC50 and IC50 data as much higher concentrations of the SvPEPC_WT enzyme were used in these assays compared to SvPEPC_S11D and SvPEPC_S11E enzymes. Together, these shifts in kinetic and allosteric parameters suggest that the phosphomimics do modify PEPC similarly to phosphorylation at Ser-11. However, there were differences between the response of SvPEPC_S11D and SvPEPC_S11E variants to G6-P and malate. The allosteric regulation of the SvPEPC_WT variant from the E. coli expression system also differed from the WT PEPC extracted from leaf tissue. This work provides direct evidence that the phosphomimic in the E. coli expression system have some but limited use in understanding the role of phosphorylation at Ser-11. Additionally, this work demonstrates that PEPC extracted from leaf tissue differs significantly from the enzyme generated in E. coli suggesting the potential importance of other post-translational modifications in PEPC responses to allosteric regulation
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Allosteric Regulation and Kinetic Parameters of a Key Enzyme Driving the Initial Carboxylation of C4 Photosynthesis
No full textThe carbon concentrating mechanism of C4 photosynthesis has been extensively studied over the last 35 years and there has been many discoveries pertaining to the complex set of anatomical and biochemical adaptions required for its efficient operation. Although there is growing interest in optimizing this pathway to alleviate abiotic stress on food crops, there are many questions remaining about the evolution and allosteric regulation of primary rate-limiting enzymes of this mechanism, such as phosphoenolpyruvate carboxylase. Therefore, detailed analysis of amino acid modifications that control kinetic trade-offs and their relationship to carbon capture of atmospheric gasses is necessary to advance such breeding efforts. This dissertation describes some of the genetic and biochemical factors that control CO2 flux into the C4 pathway. First, two grass species, Oropetium thomaeum and Paspalum vaginatum, were used to characterize single amnio acid and region substitutions as they relate to variation in kinetic trade-offs. Although many factors may contribute to variation in kinetic trade-offs, we hypothesized that region II and residue 353 can be manipulated in phosphoenolpyruvate carboxylase and likely drive variation in allosteric regulation and substrate affinity, supporting previous suggestions that the evolution of specific amino acids can be used to bioengineer a more optimal food crops. Second, these diverse isoforms of grass phosphoenolpyruvate carboxylase were used to identify cooperation of multiple amino acids that may drive variation in kinetic trade-offs. No cooperation was observed, but the 780 residue was found to drive a substrate trade-off. These results highlighted the importance of future work on kinetic trade-offs and demonstrate that a larger more diverse collection of phosphoenolpyruvate carboxylase enzymes could unlock a better general understanding of kinetic trade-offs. Finally, kinetic trade-offs were explored across a larger selection of C4 grass species. We observed individual kinetic trade-off responses, but these responses were not consistent across a larger panel of C4 species. Furthermore, we connected variation in allosteric regulation of one substrate to overall fitness of phosphoenolpyruvate carboxylase. Overall, this dissertation advances the understanding of evolutionary substitutions to amino acid composition that drives kinetic trade-offs that impact overall fitness of a rate-limiting enzyme driving C4 photosynthesis
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NOVEL BIOTECHNOLOGICAL APPROACH TO INCREASE ABIOTIC STRESS TOLERANCE AND SEED DEVELOPMENT IN BRASSICA NAPUS
No full textIn plants, E3 ubiquitin ligases play crucial roles in facilitating rapid reactions to environmental stresses by triggering the degradation of proteins. The E3 ligase CRL3BPM regulates the stability of specific transcription factors involved in complex biological processes, including growth, development, and stress response. CRL3BPM can recognize and bind to two specific motifs, called PEST and SBC, on targeted transcription factors. CRL3BPM-substrate interaction leads to the buildup of a chain of ubiquitin molecules that serve as a signal for degradation by the 26S proteasome. From signal perception to substrate degradation, this process may only minutes and because of this, E3 ligases are regarded as regulators of quick responses to environmental changes. However, they also represent interesting targets for affecting substrate stability. By modulating their activities to either delay or promote substrate degradation, they can be a suitable tool to improve critical plant traits such as biomass, seed yield, or abiotic stress tolerance.In this master's thesis, Brassica napus plants were transformed with two expression constructs containing either the PEST or the SBC motif. Both constructs were expressed under the control of a WRINKLED1.2 promoter that confers gene expression in young seedlings and developing seeds. The central hypothesis of this study was that expression of the PEST or the SBC motif at specific developmental stages would result in the transient blocking of CRL3BPM activity, leading to extended half-lives of CRL3BPM substrates. Since substrates of CRL3BPM play roles in abiotic stress responses and seed fatty acid biosynthesis, we tested our hypothesis by exposing germinating seeds to abiotic stress and investigated the transgenic plants for altered seed development. We observed that transgenic B. napus expressing the PEST motif under the control of the WRI1.2 promoter significantly increased tolerance under osmotic and salt stress in germinating seedlings and produced heavier seeds. These data support our hypothesis and validate our approach of using an E3 ligase as a novel tool for generating crop plants with improved traits
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PDX PROTEINS FROM ARABIDOPSIS THALIANA AS NOVEL SUBSTRATES OF CATHEPSIN B: IMPLICATIONS FOR VITAMIN B6 BIOSYNTHESIS REGULATION
No full textVitamin B6 (vitB6) is an important molecule that is critical for metabolism and development in plants. It is a cofactor for a plethora of biochemical reactions that regulate basic cellular function and physiology. Plants can synthesize vitB6 de novo with only two proteins: Pyridoxine Biosynthesis Protein 1(PDX1) and PDX2. VitB6 biosynthesis in plants is well understood, but there are still many open questions in terms of regulatory mechanisms. Because of the central role of the vitamin in cellular metabolism, it is likely that the biosynthesis of vitB6 is highly regulated. One such regulatory mechanism that may impact the rate of vitB6 production is protein proteolysis. There are several ways proteins can be degraded, including the Ubiquitin Proteasome Pathway (UPP) and the autophagy pathway. This work demonstrates that PDX proteins are unstable in a UPP-independent manner. In addition, it shows that cathepsin B, a cysteine protease connected to autophagy, is a novel PDX interactor that facilitates PDX degradation, two novel findings that connect cathepsin activities with vitB6 biosynthesis. This work will discuss the implications of these findings, as well as what is known about vitB6 and PDX proteins in general, including broader applications
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UNDERSTANDING THE GENETIC AND PHYSIOLOGICAL MECHANISMS OF DAY AND NIGHT-TIME HEAT STRESS TOLERANCE RELATED TO PHOTOSYNTHESIS AND KEY AGRONOMIC TRAITS IN WHEAT (Triticum aestivum L.)
No full textWheat (Triticum aestivum L.) is the second most widely cultivated food crop in the world. Abiotic stress factors particularly high temperature stress causes significant yield reduction in wheat. Every 1°C rise in temperature above the optimum has been shown to result in 5-6% yield loss. Heat stress poses threat to the food productions today which will become even a bigger threat in the future with the changing climate. The increase in global nighttime temperature has been predicted to be 1.4 times higher than that for the daytime temperature, but our understanding of its genetic and physiological basis remains very limited. Thus, the goal of this study was to understand the genetic and physiological underpinnings of the daytime and nighttime heat stress tolerance in wheat. There were two aspects to achieve this goal. First, since the information is so limited, a study was conducted to identify the quantitative trait loci (QTLs) and their corresponding candidate genes controlling high night-time (HNT) stress tolerance. Second, to understand the role of Rubisco activase (Rca) in controlling the photosynthetic potential of wheat under daytime heat stress tolerance in comparison to that under control conditions.With the objective to identify genes controlling the HNT trait, first a doubled-haploid (DH) production method using maize pollination system was optimized and used to develop DH mapping populations (Chapter 4). A DH population developed from a cross between a selection out of cultivar ‘Giza 168’ that is HNT tolerant and PBW 343, a mega variety in SE Asia that turned out to be highly susceptible to HNT. The population along with the parents were evaluated under 30°C night-time conditions while keeping the day temperature to normal growing conditions (22-24°C). The same daytime temperature and 16°C night-time temperature was used as a control. These growing conditions negatively impacted all seven agronomic traits (Days to Heading, Plant Height, Spikelet Number, Tiller Number, Total Spike Weight, Biomass, and Grain Yield). Reduction in the trait values ranged between 0.5 to 35% for the HNT tolerant parent compared to 8 to 75% for the HNT susceptible parent. Trait value reduction under HNT among the DH population for the seven agronomic traits ranged between 8 to 50%. QTL mapping using an average of 1 million sequence reads per DH line enriched for the genic fraction identified 32 QTLs, out of which 19 QTLs were detected under HNT stress treatment and the remaining 13 were for traits under normal growing conditions. The 32 QTLs mapped to 25 chromosomal intervals on 13 of the 21 wheat chromosomes, explaining between 9 to 28% of the cumulative phenotypic variance under HNT stress. The size of QTL intervals ranged between 0.021 to 97.48 Mb, with the number of genes in each interval ranging between 2 and 867. A candidate gene analysis for the smallest QTL intervals revealed eight putative candidate genes for four traits in the six QTL intervals for HNT stress tolerance.With the objective to evaluate variation for the effect of daytime heat stress on photosynthesis, various parameters measuring photosynthetic potential were measured under two heat (flash and acclimation) treatments in comparison to control. Using five distinct controlled condition screening protocols, a systematic screening of ~1,200 wheat lines identified 24 lines with varying heat stress tolerance parameters at different stages of wheat growth. The lines were chosen with care to have complementing heat-tolerance mechanisms. Photosynthetic capacity was measured on the selected 24 lines using CO2 gas exchange, chlorophyll fluorescence, Rubisco carboxylation and was compared to that of Rubisco activase (Rca) expression. On an average, heat stress (40°C) decreased net photosynthesis rate (An) by ~20%. The flash treatment had a pronounced effect on photosynthesis metrices. In five wheat genotypes, An increased with heat treatment (e.g. ~12% in KSG1195). Stomatal conductance, internal CO2 concentration, ratio of intercellular to ambient CO2, transpiration rate, water use efficiency, light adapted PSII maximum efficiency, and observed PSII operating efficiency showed differential response to heat stress with an overall reduction in efficiency under heat stress. Heat stress affects various components of photosynthetic machinery of which Rubisco activation inhibition due to heat sensitive Rca is the most prominent. Detailed comparison of Rca coding genes identified a tandem duplication in the grass lineage. Three isoforms of Rca (TaRca1β, TaRca2α, and TaRca2β) are encoded in wheat and gene expression analysis under control (22°C) and heat (40°C) revealed that these isoforms differ in thermostability. Relative expression of Rca correlated with An and the individual contribution of each isoform was established. Expression of TaRca2β isoform had a strong positive correlation with An under control conditions. Expression of TaRca1β was heat inducible with a higher correlation with An under heat treatment as compared to that under control. Detailed analysis of the promoter region of the two Rca gene copies among various plant species showed insertion of several transposable elements harboring heat responsive elements in the heat inducible copy of the gene. Wheat accessions retaining a high photosynthetic potential and Rca expression at elevated temperatures were identified
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CULL-ING CURIOSITY: EXPLORING REGULATION AND NOVEL TARGETS OF CULLIN-BASED E3 LIGASES
No full textRapid turnover of proteins is an essential molecular process for plant development and stress response. The ubiquitin proteasome pathway (UPP) is a conserved mechanism used to regulate protein activities. The pathway uses a three-step enzymatic cascade that marks substrate proteins for degradation via the 26S proteasome. E3 ligases serve as the key step in facilitating substrate recognition and its subsequent ubiquitination. While the basic steps of ubiquitination are well understood, mechanisms that regulate recognition and degradation of target proteins are only poorly resolved. Additionally, one can expect that most substrates of the 26S proteasome have not been identified yet.RELATED TO APETALA2.4 (RAP2.4) is a transcription factor and known substrate of a CULLIN3-based E3 ligase utilizing BTB/POZ/MATH (BPM) proteins (CRL3BPM). To understand how RAP2.4 is regulated by the UPP, we investigated recognized of RAP2.4 by the CRLBPM complex and what signals degradation of RAP2.4. Speckled type POZ Protein (SPOP) is a human homolog of BPMs and RAP2.4 contains an SPOP binding consensus (SBC), a short sequence critical for interaction between SPOP and its targets. Additionally, RAP2.4 has a predicted PEST motif, a region enriched in four amino acids and associated with protein degradation. Loss of the PEST motif significantly impacted the rate of RAP2.4 degradation while loss of the SBC motif only had mild impacts. Change in degradation correlated with reduced interaction with BPMs.RAP2.4 is phosphorylated by ARABIDOPSIS KINASE10 (AKIN10). We hypothesized that phosphorylation contributes to protein stability. Mutagenesis of four identified sites neither influenced the rate of degradation nor interaction of RAP2.4 with BPMs. 14-3-3 proteins in conjunction with phosphorylated proteins participate in many cellular processes and can affect protein stability. For proof of principle, 14-3-3κ was selected and show interaction with RAP2.4 required an intact PEST motif but not phosphorylation. A mild stabilization of RAP2.4 is observed in the presence of 14-3-3κ providing insight of RAP2.4 regulation.Finally, the Salt Tolerance Zinc (STZ) family of six closely related zinc finger proteins were identified as novel targets of a 26S proteasome dependent degradation. While further work is needed, implications of these newly identified targets in plants are discussed
The DDB1a interacting proteins ATCSA-1 and DDB2 are critical factors for UV-B tolerance and genomic integrity in Arabidopsis thaliana
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