Functional analysis of conserved amino acid residues in the C-terminus of ACC synthase

Abstract only availableEthylene is an important plant hormone that regulates growth, development, and stress response. Synthesis of Ethylene from its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is catalyzed by ACC oxidase. ACC is produced from S-Adenosy1-L-Methionine (SAM) in a reaction catalyzed by ACC synthase (ACS). ACS is the rate limiting enzyme of ethylene biosynthesis. Selected isoforms of ACS are substrates of MPK6 and MPK3, the two Arabidopsis stress-responsive mitogen-activated protein kinases (MAPKs). Phosphorylation of ACS6 by MPK6 stabilizes the ACS protein, thus, elevating the levels of cellular ACS activity and ethylene production. Expression of ACS6DDD, a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, shows constitutive ethylene production and ethylene-induced phenotypes. Analysis of Arabidopsis ACS6 and its orthologs from other species in the database revealed conserved charged amino acids (AAs) in addition to the MAPK phosphorylation sites in their C-termini. We hypothesized that these conserved residues may be involved in the regulation of ACS stability. We used site-directed mutagenesis to mutate the conserved residues to Ala, Ile, or Leu in the ACS6WT or ACS6DDD background using the polymerase chain reaction (PCR). Mutation was confirmed by DNA sequencing. ACS6 mutant gene was transformed into Arabidopsis plants. The stability of ACS6 protein was tested in vivo to determine if the mutation enhances or diminishes its stability. Ethylene production was used as an output reading and the levels of ACS6 protein were determined by immunoblot analysis. Mutation of positively charged AAs makes the ACS6 protein more stable, whereas the mutation of the negatively charged AAs which are close to the phosphorylation sites destabilizes it. Interestingly, deletion of the C-terminus stabilizes the ACS6 protein, suggesting that C-terminus is required for ACS6 degradation. We observed ethylene-regulated morphologies like short hairy main roots and epinastic leaves in ethylene-overproducing seedlings.MU Monsanto Undergraduate Research Fellowshi

Functional analysis of MPK3 and MPK6, two mitogen-activated protine kinases in Arabidopsis thaliana

Abstract only availableMitogen-activated protein kinase (MAPK) cascades are major pathways involved in the transduction of extracellular signals into intracellular responses. A MAPK cascade consists of three kinases; MAPK, MAPK kinase (MAPKK or MEK) and MAPKK kinase (MAPKKK or MEKK). MAPKKK is at the top of this three-tier cascade. Upon its activation by a receptor/sensor, MAPKKK phosphorylates MAPKK, which in turn phosphorylates MAPK and activates it. The activated MAPK can then phosphorylate other protein kinases or be translocated to the nucleus where it can phosphorylate transcription factors and activate gene expression. About 20 MAPKs were identified in the fully sequenced Arabidopsis genome. To study the function of MPK3 and MPK6, the two most closely related MAPKs in Arabidopsis, we isolated the corresponding T-DNA mutants from mutant libraries generated at Wisconsin Arabidopsis Knockout Facility and Salk Institute Genomic Analysis Laboratory. No morphological or developmental phenotypes were observed in the MPK3-/- and MPK6-/- single mutants. In order to determine if MPK3 and MPK6 have overlapping functions, we crossed the two single mutants (MPK3-/- and MPK6-/-) to generate double mutants. Among the 172 F2 plants that we genotyped, no double homozygous (MPK3-/-/MPK6-/-) mutant plants was identified, indicating that this genotype is lethal. We further observed that plants with the MPK3+/-/MPK6-/- genotype are a little smaller and sterile. Reciprocal back cross to wild type plants demonstrated that MPK3+/-/MPK6-/- plants are female sterile. The resilience of the pollens from such plants is still under investigation. In contrast to MPK3+/-/MPK6-/- plants, MPK3-/-/MPK6+/- plants are fertile and apparently normal. Together with the normal phenotype of MPK3-/- and MPK6-/- single mutants, we conclude that MPK3 and MPK6 perform overlapping but not identical roles in the reproduction and development of Arabidopsis thaliana.EXPRESS Progra

Functional analysis of MAP kinases in Arabidopsis thaliana: Fully rescuing the mpk3/mpk6 mutant phenotypes

Abstract only availableMitogen Activated Protein Kinase (MAPK) cascades are three-stage modules involved in signal transduction. MAPKs function at the lower tier of these cascades and they phosphorylate transcription factors and other protein kinases upon activation, ultimately leading to cellular responses. Twenty genes coding for MAPKs were identified in the fully sequenced Arabidopsis genome. MPK3 and MPK6 are the most closely related. Analysis of T-DNA insertional lines revealed no phenotype in the mpk3 and mpk6 single mutants; however, female sterility is observed in MPK3+/-/MPK6-/- plants and embryo lethality results from knocking out both genes. This indicates overlapping function of MPK3 and MPK6. To better understand the function of these two kinases, an attempt was made to rescue these phenotypes by introducing a Dexamethasone (DEX) inducible: MPK6 transgene. This construct led to only partial rescue of the lethal double mutants, and no signs of fertility were evident in MPK3+/-/MPK6-/- plants. In an attempt to attain complete rescue of these phenotypes, new MPK3 and MPK6 constructs were engineered with the following features: • Transgenes regulated by endogenous promoters were used in order to maintain normal cell/tissue specific expression of the protein, which may be essential for normal plant function. • The transgene products were tagged with Yellow Florescent Protein and Green Florescent Protein in order to ascertain their expression patterns. • Genomic DNA, as opposed to complementary DNA, was used as the coding regions in order to ensure the presence of introns, which may be significant for gene function. Currently, T1 generation transgenic plants have been isolated and transgenic lines with good expression of the transgene proteins, in vivo, will be identified by Western Blot analysis. Indication of a full rescue will be verified in the T2 generation. Failure to observe completely rescued lines may indicate protein tag interference, and untagged constructs will then be attempted.MU Monsanto Undergraduate Research Fellowshi

Investigating the developmental defects of rescued mpk3-/ mpk6- Arabidopsis plants [abstract]

Abstract only availableMitogen Activated Protein Kinases (MAPKs) are signaling molecules involved in transducing extracellular signals into intracellular responses. Twenty MAPK genes have been identified in the fully sequenced Arabidopsis genome. To deduce the function of MPK3 and MPK6 (the two most closely related Arabidopsis MAPKs), we obtained the corresponding T-DNA single knockout mutants. Homozygous single mutants had no obvious phenotype. However, we failed to generate double mutants (mpk3-/-mpk6-/- ) and found these plants to be lethal in the early embryo stages. Normal embryogenesis starts with elongation of the zygote followed by cell division to form two cells of different sizes and different fates. However this asymmetric cell division is defective in the double mutants. We generated conditionally rescued double mutants by introducing a steroid (DEX) inducible MPK6 transgene. The resulting (MPK3-/-/MPK6-/-/DEX:MPK6+/+ ) seedlings, however, were abnormal and unable to develop. Their growth was extremely stunted, they had irregular leaf surfaces (stomata clustering) and had no real stem or roots. To understand the molecular mechanisms underlying this embryogenesis defect in the double mutant, we are using reporter genes and Affymetrix GeneChip analysis to investigate the involvement of plant hormones.McNair Scholars Progra

MAP kinases in ovule development: Functional analysis of MPK3 & 6 promoters [abstract]

Abstract only availableMitogen-activated protein kinase (MAPK) cascades are major pathways involved in signal transduction. A MAPK cascade consists of three kinases; MAPK, MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK). Upon activation by a receptor/sensor, MAPKKK phosphorylates MAPKK, which in turn phosphorylates MAPK and activates it (Chang and Karin, 2001; Widmann et al., 1999). Of the nearly 20 MAPKs identified in the fully sequenced Arabidopsis genome, MPK3 and MPK6 are the most similar (Ichimura et al., 2002; Jonak et al., 2002; Tena et al., 2001; Zhang and Klessig, 2001). MPK3 and MPK6 have been found to exhibit overlapping functions (Wang 2006). While MPK3/MPK6 single mutants and MPK3-/-/MPK6+/- plants exhibited normal phenotype, MPK3+/-/MPK6-/- plants were female sterile and double mutants were embryo lethal. In order to rescue the MPK3+/-/MPK6-/- phenotype, we used a technique of "promoter swapping," with the promoter of MPK3 paired with MPK3 or MPK6 cDNA and vice versa. We were able to fully or partially rescue MPK3+/-/MPK6-/- plants with each combination. Now that we know the promoter-swapped transgene can rescue the plants, a green fluorescent protein (GFP) marker can be added to the gene in order to tell where and when in development the MPK3 or MPK6 promoter is activated