R-3 Repository :: Browsing by Author "Braam, Janet"

Browsing by Author "Braam, Janet"

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A maverick in the pectin methylesterase family: PME31 acts in lipid droplet utilization
(2024-08-06) Hamade, Sarah; Bartel, Bonnie; Braam, Janet
In plants, the primary form of energy stored form in seed lipid droplets, triacylglycerol (TAG), is catabolized during germination to support pre-photosynthetic growth. While this process is essential for seedling development, it is incompletely understood. In a screen for Arabidopsis thaliana mutants with delayed lipid droplet coat protein degradation, five independent mutations in PECTIN METHYLESTERASE31 (PME31) were recovered. In addition to delayed coat protein degradation, pme31 mutant seedlings exhibited sustained lipid droplets and elevated levels of several TAG and diacylglycerol species. Although structural prediction classified PME31 as a pectinesterase, it also resembled the putative E.coli lipase, YbhC. A fluorescent PME31 reporter was cytosolic and associated with peroxisomes, the site of fatty acid catabolism, during lipid mobilization. These findings suggest that, in contrast to most PMEs, which modify cell wall pectin, PME31 functions in lipid mobilization at the peroxisome.
Anticipation of Nitric Oxide Stress in the Human Commensal Fungus Candida albicans
(2013-07-24) Lynn, Jed; Bennett, George N.; Braam, Janet; Segatori, Laura; Stewart, Charles R.
Candida albicans is the most common human commensal fungus, able to colonize host niches such as skin, mouth and gastrointestinal tract. Colonization of diverse microenvironments requires the ability to evade or overcome innate host protection and adapt to rapid transitions between environments with different stresses and nutrient availability. Colonization of the gastrointestinal tract requires passage through the stomach containing toxic levels of nitric oxide, generated from acidification of nitrite in the low pH of the stomach. Although resistance of C. albicans to nitric oxide is mediated by the flavohemoglobin Yhb1, little is known about the physiologically relevant ligands that regulate YHB1 expression. Here I propose the hypothesis that nontoxic saliva chemicals induce YHB1 expression and promote resistance to nitric oxide generated in the stomach. Supporting this hypothesis is the observation that two ions actively concentrated in the saliva – nitrate and thiocyanate – induce YHB1 expression. Indeed, whole-genome transcriptional analysis of C. albicans treated with nitrate or thiocyanate produce gene expression profiles nearly identical to cells treated with nitrite or nitric oxide. Pretreatment of C. albicans with either of these two nontoxic compounds increases resistance of the yeast to nitric oxide. I propose that this is an evolved response in which C. albicans anticipates nitric oxide stress generated in the stomach. C. albicans thus upregulates nitric oxide stress response genes in response to saliva signals that precede nitric oxide formation further on in the gut. Only a few examples of anticipatory signaling have so far been identified and it is not known how common this type of regulation is among microbes. Expression of the YHB1 gene in response to nitric oxide is regulated by the transcription factor Cta4. I show that Cta4 binds to the YHB1 promoter in vivo as a homodimer and is necessary, but not sufficient, for nitric oxide, nitrate and thiocyanate induced expression of YHB1. Based on these data I propose a model in which Cta4 transcriptional activation is inhibited under non-inducing conditions by a negative regulator. Understanding the mechanism by which C. albicans senses and responds to nitric oxide, nitrate and thiocyanate remains a question for future research.
Arabidopsis CAMs and CMLs: Regulation and functions of genes encoding potential calcium sensors
(2005) McCormack, Elizabeth; Braam, Janet
The 57-member family of calmodulin (CaM) and CaM-Like (CML) proteins of Arabidopsis, defined and characterized in this thesis, have a strong potential to serve as sensors and interpreters of calcium signals, which are used in plants to convey information about and elicit specific responses to biotic and abiotic stimuli. Despite this opportunity to affect diverse aspects of plant biology, gene expression patterns or physiological function have been only marginally described for less than 30% of this family. The subset of genes characterized here serve as a primer to a comprehensive analysis of CAM and CML gene expression and function. To examine gene expression, mRNA levels were directly measured for 13 genes using northern analysis or quantitative-PCR (Q-PCR). The CAMs and CMLs tested show distinct, but sometimes overlapping, expression patterns both in induction of expression by stimuli and in localization throughout the plant. Indirect gene expression detection using GUS reporter transgenics allows more precise localization of expression for 15 CAMs and CMLs. Additionally, subcellular fractionation shows that CML12/TCH3 protein may be associated with multiple membranes. Physiological functions of the CAMs and CMLs can be elucidated by analysis of mutants expected to lack CaM or CML proteins. From this work, 50 cam or cml mutants were identified. Mutants chosen for in-depth study, cml11 and cml12/tch3, are among the first described mutations in an Arabidopsis CAM or CML. Root border cells accumulate in cml11, and this difference may contribute to root penetration of media. Although CaM and CML proteins share between 16 and 100 percent identity, it is likely that they have independent functions. CAMs and CMLs show non-redundant spatiotemporal patterns of gene expression, indicating that their proteins may function at different times or locations. Understanding gene expression regulation and physiological function for each CAM and CML will help us understand one aspect of how plants decode calcium signals necessary to complete specific developmental processes or survive changing environmental conditions.
Characterization of Proteins Involved in Membrane Fusion- Atlastin and Munc18c
(2013-09-16) Verma, Avani; McNew, James A.; Braam, Janet; Raphael, Robert M.; Shamoo, Yousif; Wagner, Daniel S.
Membranes provide a barrier to cells and organelles, and allow the selective transport of molecules between compartments. Membrane fusion is essential for organelle biogenesis as well as trafficking of molecules between cellular compartments. Membrane fusion is also required for the formation of the branched network of tubules that make up the Endoplasmic Reticulum (ER). One protein implicated in ER fusion is Atlastin, a dynamin like GTPase. Mutations in Atlastin-1, among others, cause Hereditary Spastic Paraplegias (HSP), a group of neurological disorders that cause progressive weakness of lower extremities. We have shown that the C-terminal tail of atlastin is necessary for membrane fusion. The requirement of the C-terminal tail can be partially abrogated in an unstable lipid environment. This implies that the C-terminal tail of Atlastin plays a role in perturbing the lipid bilayer to allow membrane fusion. Understanding the molecular details of how Atlastin drives membrane fusion may help elucidate the pathogenesis of HSP. Intracellular fusion at the plasma membrane is SNARE mediated and regulated by Sec1p/Munc18 (SM) proteins. Increased rate of glucose transport into fat and muscles cells by translocation of glucose transporter GLUT4 in response to insulin is a SNARE regulated fusion process. Recent reports have linked Munc18c and Syntaxin4 with obesity and Type 2 diabetes. We characterized the function of Munc18c, an SM protein, in regulating GLUT-4 containing vesicle fusion with the plasma membrane. We have shown that Munc18c directly inhibits membrane fusion by interacting with its cognate SNARE complexes. Characterization of membrane fusion in a minimal system as the in vitro liposome fusion assay offers a powerful tool with which to finely dissect the mechanistic basis of SM protein function.
Characterization of Structure and Function Relationship between Domains of the ER Membrane Protein Atlastin
(2014-02-06) Desai, Tanvi; McNew, James A.; Huang, Huey W.; Braam, Janet; Stern, Michael; Lwigale, Peter Yunju
The endoplasmic Reticulum (ER) is an important site for lipid synthesis, protein synthesis and transport. ER fusion is an essential process for its maintenance and biogenesis. Mutations in genes involved in this process cause Hereditary Spastic Paraplegia (HSP). These mutations are shown to affect intracellular trafficking and localization of membrane compartment. One of the important proteins causing early onset of HSP is Atlastin. Previous work in McNew lab at Rice University (Moss et al., 2011b) has shown that atlastin is involved in the homotypic fusion of the ER and the C-terminal cytoplasmic region of atlastin is essential for atlastin mediated fusion. During my studies presented in this thesis, I was able to demonstrate that the C-terminal cytoplasmic region of atlastin destabilizes lipid bilayers to facilitate fusion. The requirement of C-terminal cytoplasmic region is minimal when fusing two fluid (or unstable) lipid bilayers. The C-terminal cytoplasmic region of atlastin forms an amphipathic helix and mutations on the hydrophobic phase of the helix reduce fusion. These mutations are not dominant, as presence of full length atlastin on even one of the fusing lipid bilayers can significantly improve fusion during a heterotypic fusion reaction. Additionally, domain swaps between human atlastin-1 and drosophila atlastin show that the role of C-terminal cytoplasmic region is highly conserved. Also, during my research presented here in, I found that when the transmembrane region and C-terminal cytoplasmic region of human atlastin-1 were swapped with drosophila atlastin, it showed functional similarity. These results show that although atlastins in organisms play an important role in the ER fusion, there are likely species specific differences in how this is achieved. An understanding of atlastin mediated fusion should help in unraveling mechanisms of HSP pathogenesis and other disorders arising from dysfunctional ER.
CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production
(Oxford University Press, 2019) Lei, Jiaxin; Jayaprakasha, Guddadarangavvanahally K.; Singh, Jashbir; Uckoo, Rammohan; Borrego, Eli J.; Finlayson, Scott; Kolomiets, Mike; Patil, Bhimanagouda S.; Braam, Janet; Zhu-Salzman, Keyan
CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni. However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.
Circadian control of jasmonates and salicylates: The clock role in plant defense
(Landes Bioscience, 2013) Goodspeed, Danielle; Chehab, E.Wassim; Covington, Michael F.; Braam, Janet
Plants have evolved robust mechanisms to perceive and respond to diverse environmental stimuli.ﾠ The plant phytohormones jasmonates and salicylates play key roles in activating biotic stress response pathways. Recent findings demonstrate that basal levels of both jasmonates and salicylates in Arabidopsis are under the control of the circadian clock and that clock-controlled jasmonate accumulation may underlie clock- and jasmonate-dependent enhanced resistance of Arabidopsis to Trichoplusia ni (cabbage looper), a generalist herbivore. Here we summarize these findings and provide further evidence that a functional plant circadian clock is required for optimal herbivore defense in Arabidopsis.ﾠ When given a choice to feed on wild-type plants or arrhythmic transgenics, T. ni prefer plants lacking robust circadian rhythms. Altogether these data provide strong evidence for circadian clock enabling anticipation of herbivore attack and thus contributing to overall plant fitness.
Elucidating the Roles of PEX19 and Prenylation in Arabidopsis Peroxisomes
(2012-09-05) Stoddard, Jerrad; Bartel, Bonnie; Shamoo, Yousif; Farach-Carson, Cindy; Braam, Janet; Rudgers, Jennifer A.
Peroxisomes are organelles originating from the endoplasmic reticulum. Peroxisome biogenesis requires multiple peroxins, including PEX19, a prenylated protein that helps deliver peroxisomal membrane proteins in yeast and mammals. Arabidopsis thaliana PEX19 is encoded by two isogenes, PEX19A and PEX19B. I demonstrate that pex19A and pex19B insertional mutants lack obvious abberant physiological phenotypes. I provide evidence that pex19A pex19B double mutants are inviable, that PEX19B is more abundant than PEX19A in young seedlings, that Arabidopsis PEX19 is farnesylated in vivo, and that YFP-PEX19 predominantly associates with what appears to be a subcellular membrane regardless of its prenylation state. I show that farnesyltransferase mutants apparently contain only non-prenylated PEX19 and lack phenotypes that would indicate inefficient peroxisome activity. My analysis of PEX19 suggests that PEX19 prenylation is dispensable for peroxisome biogenesis, and has generated tools for future studies of the earliest steps in peroxisome biogenesis in plants.
Engineered nanomaterials and plant interactions: uptake, translocation, transformation and physiological effects
(2014-11-12) Wang, Jing; Alvarez, Pedro J.J.; Schnoor, Jerald L.; Braam, Janet; Li, Qilin
The increasing likelihood of engineered nanomaterial (ENM) releases to the environment and their potential applications in agriculture highlight the importance of understanding ENM interactions with plants, which are cornerstone of most ecosystems. This study investigated how silver nanoparticles (Ag NPs) of different sizes affect plant growth over a wide range of concentrations and how coating charge affects quantum dots (QDs) uptake, translocation and transformation within woody plants. Even though both Ag NPs (5, 10, and 25 nm) and silver ion (Ag+) were phytotoxic to poplars and Arabidopsis above a specific concentration, a stimulatory effect was observed on root elongation, fresh weight and evapotranspiration of both plants at a narrow range of sub-lethal concentrations. Plants were most susceptible to the toxic effects of Ag+, but Ag NPs also showed some toxicity at higher concentrations and this susceptibility increased with decreasing Ag NP size. Both poplars and Arabidopsis accumulated silver, but silver distribution in shoot organs varied between plant species. Arabidopsis accumulated silver primarily in leaves (at ten-fold higher concentrations than in the stem or flower tissues), whereas poplars accumulated silver at similar concentrations in leaves and stems. Uptake of cationic QDs by poplar was faster than anionic QDs, possibly due to electrostatic attraction of cationic QDs to the negatively charged root cell wall. QDs aggregated upon root uptake, and their translocation to poplar shoots was likely limited by the endodermis. After 2-day exposure, both cationic and anionic coatings were likely degraded from the internalized QDs inside the plant, leading to the aggregation of the metallic cores and a “red-shift” of fluorescence. The fluorescence of cationic QD aggregates inside roots was stable through the 11-day exposure period, while that of the anionic QD aggregates was quenched probably due to destabilization of the coating inside the plant, even though these QDs were more stable in the hydroponic solution. Overall, the phyto-stimulatory effect observed in this study precludes the generalization of the phytotoxicity of Ag NPs. The QDs study highlights the importance of coating properties in the rate and extent to which NPs are assimilated by plants and potentially introduced into food webs.
Functions of CML24: A potential calcium sensor of Arabidopsis
(2010) Tsai, Yu-Chang; Braam, Janet
Plants sense environmental conditions and respond by changing development and physiology. Calcium (Ca2+) is a second messenger thought to play a critical role in plant responses to developmental and environmental stimuli. Calmodulin (CaM) is the prototypical Ca2+ sensor that is highly conserved among eukaryotes. The Arabidopsis genome encodes 50 CaM-like (CML) proteins in addition to CaM. CML functions remain largely unknown. CML24 expression is strongly upregulated by diverse stimuli. The encoded protein shares characteristics with CaM, including primary sequence similarity, predicted tertiary structure, and Ca2+-induced conformational change. Plants with epigenetically silenced CML24 are delayed in the transition to flowering and have altered ion sensitivity. To further understand CML24 function, I employed genetic, biochemical, cellular, and physiological approaches. Mass spectroscopy analyses suggest that CML24 cysteines may form disulfides; therefore, CML24 may have the capability of transducing oxidative, in addition to Ca2+, signals. Apparent loss- and gain-of-function cml24 point mutants were identified and determined to have alterations in the timing of flowering induction. Despite the physiological consequences of the cml24 mutant amino acid substitutions, the mutant proteins retain the ability to undergo Ca2+-dependent conformational changes. cml24 mutants are defective in expression of the regulatory genes, CONSTANS and FLOWERING LOCUS C (FLC) and have aberrant nitric oxide (NO) accumulation. Altered NO levels underlie FLC enhanced expression in the late-flowering cml24 mutants and contribute in part to delayed flowering. NO-associated 1 (NOA1), not arginine-dependent NO synthase or nitrate reductase, is implicated, through an indirect mechanism, in NO accumulation in cml24 mutants. CML24 directly binds and may increase the enzymatic activity of Autophagy 4b (ATG4b), a cysteine protease that regulates autophagy, the cellular "self eating" process by which eukaryotic cells remove damaged and nonessential cytoplasmic components. Fluorescence imaging and western analysis of autophagy markers and seedling growth assays in response to nutrient-limitation indicate that the cml24 mutants may have defects in autophagy regulation. Together, this work implicates CML24 as a potential Ca2+ and redox sensor in the regulation of NO accumulation and, through direct interaction with ATG4b, in the regulation of autophagy progression.
Functions of the calmodulin-related TCH3 and TCH4-xyloglucan endotransglycosylase in Arabidopsis plants
(1998) Purugganan, Mary M.; Braam, Janet
Plants sense their environment and respond through changes in their development and physiology. Mechanical perturbation, darkness, temperature shocks, and exogenous hormones upregulate the expression of the TCH genes in Arabidopsis. The objective of this work was to determine the functions of the TCH3 and TCH4 proteins. TCH3, a calmodulin-related protein with six potential Ca$\sp{2+}$ binding sites, binds Ca$\sp{2+}$, as evidenced by its mobility shift on an SDS-polyacrylamide gel in the presence of Ca$\sp{2+}.$ TCH4, a protein related in sequence to the enzyme xyloglucan endotransglycosylase (XET), has XET activity. The TCH4 XET prefers nonfucosylated oligosaccharide acceptor substrates, has a pH optimum of 6 to 6.5, and has unusually high activity at low temperatures, with an optimum of 12 to 18$\sp\circ$C. To address the role of these proteins in Arabidopsis development and responses to the environment, plants were generated with altered expression of TCH3 or TCH4. These plants were downregulated through antisense RNA, a T-DNA insertion in the gene, or cosuppression. Plants were upregulated by a strong, constitutive promoter driving an exogenous copy of the gene. The plants developed normally and did not show any detectable changes in cellular organization, cell wall integrity, or mechanical strength. Several assays were developed to test the responses of the transgenics to environmental stimuli and hormones. The transgenics responded normally to wind, heat, darkness/low light, gravity, mechanical obstruction, osmotic stress, auxin, and brassinosteroids. However, preliminary evidence suggests that the TCH4 transgenics may respond differentially to cold. Plants with reduced TCH4 levels were more sensitive to low temperatures, and plants overexpressing TCH4 were more hardy at low temperatures. The lack of a pronounced mutant phenotype in the transgenic plants suggests genetic redundancy. Nevertheless, under specific conditions, the TCH3 and TCH4 proteins may have essential functions in Arabidopsis.
Genome-wide identification and functional analyses of calmodulin genes inﾠ Solanaceous ﾠspecies
(BioMed Central, 2013) Zhao, Yuan; Liu, Wei; Xu, You-Ping; Cao, Jia-Yi; Braam, Janet; Cai, Xin-Zhong
Background: Calmodulin (CaM) is a major calcium sensor in all eukaryotes. It binds calcium and modulates the activity of a wide range of downstream proteins in response to calcium signals. However, little is known about the CaM gene family in Solanaceous species, including the economically important species, tomato (Solanum lycopersicum), and the gene silencing model plant, Nicotiana benthamiana. Moreover, the potential function of CaM in plant disease resistance remains largely unclear. Results: We performed genome-wide identification of CaM gene families in Solanaceous species. Employing bioinformatics approaches, multiple full-length CaM genes were identified from tomato, N. benthamiana and potato (S. tuberosum) genomes, with tomato having 6 CaM genes, N. benthamiana having 7 CaM genes, and potato having 4 CaM genes. Sequence comparison analyses showed that three tomato genes, SlCaM3/4/5, two potato genes StCaM2/3, and two sets of N. benthamiana genes, NbCaM1/2/3/4 and NbCaM5/6, encode identical CaM proteins, yet the genes contain different intron/exon organization and are located on different chromosomes. Further sequence comparisons and gene structural and phylogenetic analyses reveal that Solanaceous species gained a new group of CaM genes during evolution. These new CaM genes are unusual in that they contain three introns in contrast to only a single intron typical of known CaM genes in plants. The tomato CaM (SlCaM) genes were found to be expressed in all organs. Prediction of cis-acting elements in 5' upstream sequences and expression analyses demonstrated that SlCaM genes have potential to be highly responsive to a variety of biotic and abiotic stimuli. Additionally, silencing of SlCaM2 and SlCaM6 altered expression of a set of signaling and defense-related genes and resulted in significantly lower resistance to Tobacco rattle virus and the oomycete pathogen, Pythium aphanidermatum. Conclusions: The CaM gene families in the Solanaceous species tomato, N. benthamiana and potato were identified through a genome-wide analysis. All three plant species harbor a small set of genes that encode identical CaM proteins, which may manifest a strategy of plants to retain redundancy or enhanced quantitative gene function. In addition, Solanaceous species have evolved one new group of CaM genes during evolution. CaM genes play important roles in plant disease resistance to a variety of pathogens.
High-temperature electrothermal remediation of multi-pollutants in soil
(Springer Nature, 2023) Deng, Bing; Carter, Robert A.; Cheng, Yi; Liu, Yuan; Eddy, Lucas; Wyss, Kevin M.; Ucak-Astarlioglu, Mine G.; Luong, Duy Xuan; Gao, Xiaodong; JeBailey, Khalil; Kittrell, Carter; Xu, Shichen; Jana, Debadrita; Torres, Mark Albert; Braam, Janet; Tour, James M.; NanoCarbon Center and the Rice Advanced Materials Institute; Smalley-Curl Institute
Soil contamination is an environmental issue due to increasing anthropogenic activities. Existing processes for soil remediation suffer from long treatment time and lack generality because of different sources, occurrences, and properties of pollutants. Here, we report a high-temperature electrothermal process for rapid, water-free remediation of multiple pollutants in soil. The temperature of contaminated soil with carbon additives ramps up to 1000 to 3000 °C as needed within seconds via pulsed direct current input, enabling the vaporization of heavy metals like Cd, Hg, Pb, Co, Ni, and Cu, and graphitization of persistent organic pollutants like polycyclic aromatic hydrocarbons. The rapid treatment retains soil mineral constituents while increases infiltration rate and exchangeable nutrient supply, leading to soil fertilization and improved germination rates. We propose strategies for upscaling and field applications. Techno-economic analysis indicates the process holds the potential for being more energy-efficient and cost-effective compared to soil washing or thermal desorption.
Identification and characterization of the xyloglucan endotransglycosylases of Arabidopsis thaliana
(1998) Campbell, Paul Harwell; Braam, Janet
The plant cell wall is a fundamental determinant of cell shape, and therefore plant form. Although the molecular basis of plant morphology is unknown, it most likely involves enzymes capable of modifying different components of the cell wall. One of the major components of the plant cell wall, the polysaccharide xyloglucan, is capable of hydrogen bonding to cellulose microfibrils, potentially acting as a molecular tether between adjacent microfibrils. Therefore, enzymes capable of modifying xyloglucan may play a role in the development of cell shape and plant form. Xyloglucan endotransglycosylases (XETs) are enzymes capable of cleaving xyloglucan polymers endolytically and transferring one of the newly-generated free ends of the polymer to the free end of another xyloglucan chain. The genome of Arabidopsis thaliana encodes at least sixteen XET and XET- related (XTR) genes. I have sequenced 6 novel XTR genes. Several members of this gene family are differentially regulated by environmental stimuli (such as touch, darkness, and temperature extremes) and the growth-promoting hormones auxin and brassinosteroids. The putative XTR proteins share from 34 to 89% identity at the amino acid level. Like XETs from other plant species, the XTRs are predicted to encode signal peptides, N-linked glycosylation motifs, several cysteines with the potential to form disulfide bonds, and a conserved motif related to the proposed active site of the Bacillus beta-glucanases. Four of the Arabidopsis XTRs, TCH4, Meri-5, EXGT, and XTR9, were produced using the baculovirus/insect cell expression system. All four recombinant proteins catalyzed the transglycosylation of xyloglucan in vitro, demonstrating that the proteins are XETs. The isozymes were characterized biochemically for differences in Kms, pH optima, temperature optima, glycosylation, glucanase activity, and the ability to utilize different xyloglucan substrates. In addition, site-directed mutagenesis of TCH4 revealed that the conserved motif shared with Bacillus beta-glucanases encodes at least one amino acid, glutamate 97, essential for XET activity in TCH4.
Insights into peroxisome matrix protein import and IBA metabolism through analysis of IBA-response mutants
(2010) Martinez, Naxhiely; Braam, Janet; Bartel, Bonnie
Peroxisomes are single membrane-bound organelles that function to compartmentalize certain metabolic reactions critical to plant and animal development. I have studied peroxisomal processes in the model plant Arabidopsis thaliana , with a focus on the import of matrix proteins from the cytoplasm into the organelle matrix and the metabolism of the plant hormone indole-3-butryic acid (IBA). In this thesis, I describe my characterization of Arabidopsis thaliana peroxisome defective mutants isolated through forward and reverse genetic screens in physiological and biochemical assays. Peroxisome import depends on more than a dozen pero xin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing a peroxisome t argeting sequence (PTS) into the organelle. PEX5 is the PTS1 receptor, PEX7 is the PTS2 receptor, and in both plants and mammals, PEX7 depends upon PEX5 binding to deliver PTS2 cargo into the peroxisome. I found a pex7 missense mutation, pex7-2, that disrupts PEX7-cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 accumulation in plants. I examined localization of peroxisomally-targeted GFP derivatives in light-grown pex7 mutants and, surprisingly, observed defects not only in PTS2 import, but also in PTS1 protein import. These PTS1 import defects were accompanied by a decrease in PEX5 accumulation in light-grown pex7 mutants. Together, these data suggest that PEX5 and PTS1 import depend on the PEX7 PTS2 receptor in Arabidopsis and reveal a role for the environment in modulating peroxin function. Genetic evidence suggests that indole-3-butyric acid (IBA) is converted to the auxin indole-3-acetic acid (IAA) in Arabidopsis peroxisomes. The IBR1, IBR3, and IBR10 proteins contain peroxisomal targeting signals and are candidates for catalyzing the various steps of IBA beta-oxidation. My analysis of the ibr mutants has provided evidence for the importance of the IBR enzymes and insight into the roles of IAA that derives from IBA beta-oxidation. In humans, deficiencies in peroxins underlie the peroxisomal biogenesis disorders, which are frequently lethal in early infancy. Advancing our understanding of peroxisome biogenesis and metabolism in a genetically distinct model system will allow the continued refinement of our understanding of these essential organelles.
Investigation of pexophagy and autophagy occurrence in Arabidopsis
(2023-04-12) Smith, Kathryn Ann; Bartel, Bonnie; Braam, Janet
Peroxisomes are critical organelles, housing various essential processes conserved across multicellular organisms. Most notable reactions include fatty-acid -oxidation and the detoxification of reactive oxygen species generated as byproducts from diverse oxidative reactions. Peroxisomes also house specialized processes depending on the organism, tissue, and developmental age, such as phytohormone production in plants. Peroxins, or PEX proteins orchestrate the biogenesis and maintenance of these organelles. Despite the evidence for tunability and dynamic changes in peroxisome numbers and function during development and environmental challenges, our understanding of how this organelle is regulated remains limited. Autophagy is a cellular system that targets components of the cell for degradation in the vacuole (of plants or yeast) or the lysosome (of animals). Autophagy can be selective, and the degradation of peroxisomes through this process is called pexophagy. By comparing protein accumulation in various peroxin mutants to accumulation in both wild type and seedlings incapable of autophagy (atg mutants), I found that pex2, pex4, pex10, and pex14 mutants stabilized several peroxisomal proteins similarly to atg mutants in darkness conditions. I also examined autophagy and pexophagy induction using immunoblotting to detect cleavage of autophagy and pexophagy reporters in a variety of conditions. I found that pexophagy occurs during development but is not robustly induced in many autophagy-inducing conditions. Finally, I explored the connection between darkness-induced autophagy and carbon starvation and found that darkness induces autophagy even when fixed carbon is exogenously supplied. My investigations implicate several peroxins in pexophagy modulation and demonstrate that the environmental conditions regulating pexophagy are complex and distinct from the triggers of general autophagy. As future work characterizes subpopulations of peroxisomes within Arabidopsis, the assays described herein provide a foundation for the further elucidation of when, where, and how pexophagy occurs in plants.
Keeping the rhythm: light/dark cycles during postharvest storage preserve the tissue integrity and nutritional content of leafy plants
(BioMed Central, 2015) Liu, John D.; Goodspeed, Danielle; Sheng, Zhengji; Li, Baohua; Yang, Yiran; Kliebenstein, Daniel J.; Braam, Janet
Background: The modular body structure of plants enables detached plant organs, such as postharvest fruits and vegetables, to maintain active responsiveness to environmental stimuli, including daily cycles of light and darkness. Twenty-four hour light/darkness cycles entrain plant circadian clock rhythms, which provide advantage to plants. Here, we tested whether green leafy vegetables gain longevity advantage by being stored under light/dark cycles designed to maintain biological rhythms. Results: Light/dark cycles during postharvest storage improved several aspects of plant tissue performance comparable to that provided by refrigeration. Tissue integrity, green coloration, and chlorophyll content were generally enhanced by cycling of light and darkness compared to constant light or darkness during storage. In addition, the levels of the phytonutrient glucosinolates in kale and cabbage remained at higher levels over time when the leaf tissue was stored under light/dark cycles. Conclusions: Maintenance of the daily cycling of light and dark periods during postharvest storage may slow the decline of plant tissues, such as green leafy vegetables, improving not only appearance but also the health value of the crops through the maintenance of chlorophyll and phytochemical content after harvest.
NOA1: A Tool for Understanding Nitric Oxide Accumulation and Fosmidomycin Resistance in Arabidopsis
(2012) Van Ree, Kalie; Braam, Janet
Chloroplasts generate important cellular signals and synthesize diverse products. The chloroplast-localized protein, Nitric Oxide Associated-1 (NOA1), is implicated in nitric oxide (NO) accumulation and linked to the methylerythritol phosphate (MEP) pathway, but its role is undefined. I report that NOA1 is not essential for NO accumulation because the noa1 mutant accumulates NO when provided sucrose-supplemented media. Therefore, chloroplast function and fixed carbon, but not NOA1 are likely critical for plant NO accumulation. noa1 is also resistant to fosmidomycin, an inhibitor of the MEP pathway. This phenotype led to uncovering a potential link between the MEP and tetrapyrrole pathways. I report that fosmidomycin toxicity is light dependent and reduced by phytol supplementation. Downregulation of the tetrapyrrole pathway enhances fosmidomycin resistance, suggesting that reduced tetrapyrrole biosynthesis alleviates fosmidomycin toxicity. These findings reveal new insight into how impairment of the MEP pathway affects plants and the importance of metabolic balance for chloroplast function.
Plant Interactions with Other Organisms
(2019-08-08) Dai, Yanwan; Braam, Janet; Bonnie, Bartel
With pressures on food availability, improved plant crop productivity and nutritional value are important. To increase crop yield, research into optimizing plant defenses against pests and pathogens can inform new strategies. In addition, efforts to optimize nutrition and health promoting content of plant crops may further contribute to the benefits of plant-rich diets. Arabidopsis thaliana is a model host to investigate defense against fungi. Leaves have often been used to evaluate plant defenses. However, I have found that, unlike rosette leaves, the rosette core, where the leaf petioles join the stem, can prevent fungal infiltration and therefore can display robust fungal resistance. Rosette core fungal resistance is age- and jasmonate-dependent, and may involve formation of an abscission-like zone. Broccoli and cabbage also display rosette core resistance to Botrytis cinerea. Therefore, spatial and developmental aspects of the plant host can play critical roles in fungal resistance and are important to our understanding of plant defense mechanisms. The jasmonate phytohormone is critical to plant biotic resistance. Jasmonate intermediate product (9S,13S)-12-oxo-phytodienoic acid (OPDA) has been implicated as a signaling molecule in fungal defense. To verify this role for OPDA, mutants that block jasmonate biosynthesis at OPDA would be valuable tools. Mutations in OPDA REDUCTASE (OPR3), encoding the only known OPDA reductase, were generated using CRISPR-Cas9. New opr3 mutants were generated, however, these mutants remained capable of jasmonate synthesis. This unexpected finding is consistent with a recent report that Arabidopsis has an OPR3-independent jasmonate biosynthesis pathway. Mutations that block all jasmonate synthesis pathways will be required to verify direct functional roles of OPDA. The plant circadian clock impacts biotic defense. To test whether the time-dependent differential accumulation of plant metabolites is sufficient to confer significant biological effects in feeding organisms, I compared biological effects of broccoli harvested at different times of a day using both the insect herbivore model Trichoplusia ni and a mammalian model rat. Insect choice experiments suggest that time-of-day harvesting may impact feeding behaviors; whereas the rat experiments remain inconclusive. Additional experimentation is required to elucidate how the plant circadian clock may impact host organisms.
Plant responses to environmental stimuli: A screen for mutants aberrant in TCH4 expression behavior, and a full-genome assessment of touch and darkness inducibility
(2005) Lee, Dennis; Braam, Janet
Plants must be capable of detecting and responding to a wide variety of environmental stimuli, yet much remains to be determined about the mechanisms by which these processes occur. I describe herein a proof-of-concept mutant screen for identifying mutants aberrant in their responses to a variety of stimuli including heat, cold, touch, and darkness. Such mutants will undoubtedly serve as tools to investigate the molecular pathways by which plants detect and respond to their environment. In addition, I also describe a large-scale gene expression experiment utilizing Affymetrix ATH1 microarrays which focuses on the short-term responses of plants to touch and darkness. These chips enable an expression level survey of over 22,000 genes simultaneously. We identify 589 genes that have increased expression in plants 30 minutes after touch stimulation and 171 genes that have decreased expression. We also identify 461 genes that have increased expression in plants after 30 minutes of darkness treatment and 72 genes that have decreased expression. The identities of the regulated genes suggest that calcium and kinase signaling, cell wall modification, disease resistance and secondary transcriptional responses may be altered in plants subjected to mechanostimulation or darkness.

Browsing by Author "Braam, Janet"

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