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Joshua J Blakeslee

Publications and source records attributed to Joshua J Blakeslee.

12 recordsLinked to original sources
PubMed2006

Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to benzoic acid and enhancement in auxin transport.

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a beta-oxidative or non-beta-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between L-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.

Acyltransferases↗
PubMed2006

Immunophilin-like TWISTED DWARF1 modulates auxin efflux activities of Arabidopsis P-glycoproteins.

The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

PIN proteins perform a rate-limiting function in cellular auxin efflux.

Intercellular flow of the phytohormone auxin underpins multiple developmental processes in plants. Plant-specific pin-formed (PIN) proteins and several phosphoglycoprotein (PGP) transporters are crucial factors in auxin transport-related development, yet the molecular function of PINs remains unknown. Here, we show that PINs mediate auxin efflux from mammalian and yeast cells without needing additional plant-specific factors. Conditional gain-of-function alleles and quantitative measurements of auxin accumulation in Arabidopsis and tobacco cultured cells revealed that the action of PINs in auxin efflux is distinct from PGP, rate-limiting, specific to auxins, and sensitive to auxin transport inhibitors. This suggests a direct involvement of PINs in catalyzing cellular auxin efflux.

ATP-Binding Cassette Transporters↗
PubMed2005

PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots.

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid-reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2005

A possible role for NDPK2 in the regulation of auxin-mediated responses for plant growth and development.

Auxin plays many crucial roles in the course of plant growth and development, such as hook opening, leaf expansion and inhibition of mesocotyl elongation. Although its mechanism of action has not been clarified at the molecular level, recent studies have indicated that auxin triggers the induction of a number of genes known as primary auxin-responsive genes. Hence, the identification of the regulatory components in auxin-mediated cellular responses would help to elucidate the mechanism of the action of this hormone in plant growth and development. NDPK2 encodes a nucleoside diphosphate kinase 2 (NDPK2) in Arabidopsis. We aim to elucidate the possible role of NDPK2 in auxin-related cellular processes, in view of the finding that a ndpk2 mutant displays developmental defects associated with auxin. Interestingly, the ndpk2 mutant exhibits defects in cotyledon development and increased sensitivity to an inhibitor of polar auxin transport (naphthylphthalamic acid; NPA). Consistent with this phenotype, the transcript levels of specific auxin-responsive genes were reduced in the ndpk2 mutant plants treated with auxin. The amount of auxin transported from the shoot apex to the shoot/root transition zone of ndpk2 mutant plants was increased, compared with that in the wild-type plants. These results collectively suggest that NDPK2 appears to participate in auxin-regulated processes, partly through the modulation of auxin transport.

Gene Expression↗
PubMed2005

Auxin transport.

Polar transport of auxin is essential for normal plant growth and development. On a cellular level, directional auxin transport is primarily controlled by an efflux carrier complex that is characterized by the PIN-FORMED (PIN) family of proteins. Detailed developmental studies of PIN distribution and subcellular localization have been combined with the analysis of changes in localized auxin levels to map PIN-mediated auxin movement throughout Arabidopsis tissues. Plant orthologs of mammalian multidrug-resistance/P-glycoproteins (MDR/PGPs) also function in auxin efflux. MDR/PGPs appear to stabilize efflux complexes on the plasma membrane and to function as ATP-dependent auxin transporters, with the specificity and directionality of transport being provided by interacting PIN proteins.

Biological Transport↗
PubMed2005

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1.

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport.

ATP-Binding Cassette Transporters↗
PubMed2004

Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana.

Aglycone flavonols are thought to modulate auxin transport in Arabidopsis thaliana via an as yet undefined mechanism. Biochemical studies suggest that flavonoids interact with regulatory proteins rather than directly with the PIN auxin efflux facilitator proteins. Auxin transport is enhanced in the absence of flavonoids (transparent testa4 [tt4]) and reduced in the presence of excess flavonols (tt7 and tt3). Steady state PIN mRNA levels in roots inversely correlate with auxin movement in tt mutants. PIN gene transcription and protein localization in flavonoid-deficient mutants appear to be modulated by developmental cues and are auxin responsive. Modulation of PIN gene expression and protein distribution by localized auxin accumulations occurs in the wild type as well. Flavonoids inhibit auxin transport primarily at the shoot apex and root tip and appear to modulate vesicular cycling of PIN1 at the root tip. In some auxin-accumulating tissues, flavonoid increases and changes in flavonoid speciation are subsequent to auxin accumulation.

Arabidopsis↗
PubMed2003

The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways.

The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

Arabidopsis↗
PubMed2003

Loss of an MDR transporter in compact stalks of maize br2 and sorghum dw3 mutants.

Agriculturally advantageous reduction in plant height is usually achieved by blocking the action or production of gibberellins. Here, we describe a different dwarfing mechanism found in maize brachytic2 (br2) mutants characterized by compact lower stalk internodes. The height reduction in these plants results from the loss of a P-glycoprotein that modulates polar auxin transport in the maize stalk. The sorghum ortholog of br2 is dwarf3 (dw3), an unstable mutant of long-standing commercial interest and concern. A direct duplication within the dw3 gene is responsible for its mutant nature and also for its instability, because it facilitates unequal crossing-over at the locus.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19.

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrug-resistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.

ATP-Binding Cassette Transporters↗