Browsing by Subject "Auxin"

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    Characterization of CRISPR-Cas9 Induced SAUR19 Family Mutants in Arabidopsis.
    (2017-04) Hovland, Austin, S.
    The plant hormone auxin (IAA) regulates many aspects of plant growth and development. Small auxin up RNA (SAUR) genes are highly transcribed in response to auxin, whose differential transport and accumulation creates gradients that regulate various aspects of plant development such as: tropisms, root initiation, and cellular division and differentiation. Previous studies using overexpression of GFP-tagged SAUR proteins have shown that SAURs may function as positive regulators of cellular expansion. To provide additional evidence for this hypothesis, I investigated the effect of creating knockouts of multiple members of the SAUR19 subfamily using CRISPR/Cas9. Additionally, SAUR-promoter-GUS staining was performed to identify the location of SAUR 13, 22, 27, 28, and 29 expression. These studies revealed strong SAUR expression in adult leaf vasculature, expanding stamen filaments, hypocotyls, and petioles. Through analysis of heat-induced growth, we show that knockout of SAURs 19, 20, 21, 22, 24, and 29 conferred a modest decrease in both hypocotyl and petiole length. While not severe, this difference provides supportive evidence that SAURs function as positive regulators of cell expansion, and it also reinforces the hypothesis that SAURs have extensive genetic redundancy. Higher numbers of SAUR knockouts should produce stronger phenotypes and provide definitive evidence of SAUR function.
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    The eta7/csn3-3 auxin response mutant of Arabidopsis defines a novel function for the CSN3 subunit of the COP9 signalosome.
    (2012-07) Huang, He
    The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates plant auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCFTIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit resulted in the pleiotropic cop/det/fus phenotype and caused seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In comparison with eta6/csn1-10, which was identified in the same enhancer screen (Zhang et al., 2008), both csn3-3 and csn1-10 enhanced the auxin response defects of tir1-1. Similar to csn1-10, csn3-3 also confers several phenotypes associated with impaired auxin signaling, including auxin resistant root growth and diminished auxin responsive gene expression. Surprisingly however, unlike csn1-10 as well as other previously characterized csn mutants, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCFTIR1/AFB mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, I observed dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex was unaffected in csn3-3 plants. However, I detected a small CSN3-containing protein complex (sCSN3c) that was altered in csn3-3 plants. I hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a smaller protein complex that is required for proper auxin signaling. Analyses on the purification of sCSN3c suggested that it is not likely a dimer of CSN3, or a CSN subcomplex. My data resulting from sCSN3c purification using various chromatographic steps provide useful information necessary for identifying the components of the complex.
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    Identification of pyruvate decarboxylase/indole pyruvate decarboxylase gene family members from Arabidopsis thaliana.
    (2009-09) Ye, Songqing
    Several biologically important and diverse reactions are regulated by thiamine pyrophosphate (TPP) cofactor-dependent metabolic enzymes, including pyruvate decarboxylase (PDC), indole pyruvate decarboxylase (IPDC), and acetohydroxy acid synthase. PDC is a critical enzyme in plant metabolism that regulates energy production especially during periods of anaerobic stress. IPDC has long been proposed as a key enzyme in the biosynthesis of the plant hormone indole-3-acetic acid (IAA) from tryptophan. Six putative Arabidopsis thaliana PDC gene family members have been individually cloned and expressed in E. coli, and recombinant PDC proteins were purified and biochemically characterized. AtPDC2 was identified as a unique functional PDC based on its measured biochemical activity. The pH and temperature optima for the recombinant protein were 6.2 and 55°C, respectively, and the Km was 3.5 mM. Also, addition of 0.5 mM TPP and 5 mM Mg2+ resulted in the highest activity. However, AtPDC2 lacked any measurable IPDC activity as determined by gas chromatography-mass spectrometry (GC-MS)-based methods. Thus, this mono-functional PDC was different from the more thoroughly studied microbial PDCs, which all have bi-functional activity toward both indole-3-pyruvate and pyruvate substrates. These findings suggest a potential regulatory role for the catalytically inactive PDC proteins in modulation of PDC activity, similar to a mechanism proposed for yeast. None of the pdc mutants showed a change in resistance to chlorsulfuron or imazamox herbicides, and this result was also consistent with the hypothesis that the inactive AtPDC genes may play a role in PDC activity regulation in Arabidopsis. Studies presented here show that the genes most likely to encode proteins with PDC activity or IPDC activity, the PDC gene family, all lack IPDC activity and all except one lack PDC activity. Furthermore, all Arabidopis PDC T-DNA insertion mutants were found to share the same shade avoidance phenotype to as did wild-type plants. These findings bring into question the physiological significance of the IPA pathway for auxin biosynthesis as has been previously proposed. Very low levels of IPDC activity are difficult to measure using procedures developed for the enzyme activity of proteins from bacteria, which produce substantial levels of indole acetaldehyde (IAAld) from indole-3-pyruvate (IPA). To determine the potential activity of plant enzymes, either expressed in E. coli or extracted from Arabidopsis plants, GC-MS assay methods were developed with high sensitivity and specificity. For expressed proteins, IAAld produced from IPA was measured directly using indole carboxaldehyde as an internal standard. This procedure failed, however, to detect IPA in the presence of plant protein extracts; thus, a coupled in vitro reaction with aldehyde dehydrogenase that produced IAA from IPA was developed, and the IAA was quantified using [13C6]IAA as an internal standard, methylation with diazomethane, and GC-MS detection. Together, these methods provide important sensitive and precise methods for the search for IPDC activity in the plant kingdom.
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    MNC1 Negatively Regulates Nectar Production through Auxin and Jasmonic Acid Response Pathways in Arabidopsis thaliana
    (2014-11) Jia, Mengyuan
    Many flowering plants offer a reward for pollinators in the form of nectar. Despite the central role of pollination in reproduction of plants and the considerable amount of energy a plant devotes to produce nectar, little is known of the molecular mechanism of nectar production and its regulation. Previous reports have suggested a significant role for the plant hormone auxin in regulating nectar production. Recent transcriptome studies have made it possible to focus research on several nectary-specific candidate genes with putative roles in the auxin response. In Arabidopsis thaliana this includes a gene termed MEDIAN NECTARY CUPIN 1 (MNC1; At1g74820), which is highly expressed in median nectaries. MNC1 silenced mutants (mnc1) showed more nectar production and increased auxin response activity while MNC1 overexpresser mutant (MNC1 T6) showed significantly less nectar production and less auxin response activity in nectaries. A comparative sequence analysis of proteins with known function shows that MNC1 is a germin-like protein belonging to the RmlC-like cupins superfamily. MNC1 also has a conserved zinc binding domain with known Auxin Binding Protein1. Thus, we hypothesize that MNC1 negatively regulates nectar production, likely through an auxin dependent pathway. PIN6 (At1g77110), an auxin transporter family protein, has been reported to be an auxin transporter localized to the ER modulating cytoplasmic free auxin concentration in nectaries. Arabidopsis thaliana mutant lines with different combinations of crossed target genes were used to understand the feedback mechanism of auxin, nectar production, PIN6, and MNC1 protein behind nectar regulation and production. Transformed Escherichia coli and Pichia pastoris, a methylotrophic yeast species, expressing MNC1 protein were employed for studying its biochemical nature, including auxin binding activity. Jasmonic acid (JA) was also suggested to be required for nectar production, with possible crosstalk to auxin in regulation of nectar production. COI1-independent JA response pathway was found to regulate nectar production by altering the expression of nectary-specific genes such as SWEET9, a sucrose transporter required for nectar production, CWINV4, a cell wall invertase required for nectar production, and MNC1. A potential auxin-JA crosstalk mechanism was constructed based on results in this study and previous studies.
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    New analytical methodologies in the study of auxin biochemistry
    (2014-08) Yu, Peng
    Auxin is the essential plant hormone that regulates many aspects of plant growth and development. Plants typically possess highly complex biochemical networks to regulate the homeostasis of the active hormone, through the regulation of biosynthesis, degradation, transport and conjugation. Biosynthesis, among other processes, has been of particular importance and warranted extensive studies over the decades of auxin research. A number of pathways were proposed and some enzymes potentially involved have been characterized. Stable isotope labeling and turnover studies have proven very useful in these investigative efforts. With the advancement of analytical and computational technologies, it is now feasible to concurrently analyze the turnover patterns of all the precursors in the entire auxin biosynthesis network. I devoted chapter two of the dissertation to establish LC-MS methods to concurrently quantify most of the auxin precursors and deployed different isotopic labeling strategies for turnover studies in Arabidopsis thaliana. Preliminary results showed that indole-3-pyruvate (IPyA) and indole-3-acetaldehyde (IAAld) were among the fastest to be labeled and a key regulatory step existed between IPyA and indole-3-acetic acid (IAA). Chapter three reported the discrepancy and the study of the amount of the total IAA determined by alkaline hydrolysis and that of the summation of all known forms of IAA conjugates in Arabidopsis. My results indicated that chemical artifacts induced by harsh chemical treatments were responsible for a significant portion of the unknown putative IAA conjugates. In chapter four, I described a facile way to directly survey a plant extract for its indole profile, notably IAA conjugates, based on high resolution and accurate mass (HR/AM) liquid chromatography-mass spectrometry (LC-MS). The method was successfully applied to Glycine max, Solanum lycopersicum, Cocos nucifera, and Ginkgo biloba. Together, these investigations and developments have led to an improved understanding of auxin metabolism and now provide useful tools for subsequent studies.