Auxin, or indole acetic acid (IAA), is an essential, multifunctional plant hormone that influences virtually every aspect of plant growth and development. Although auxin-dependent growth is evident in all plant tissues, it is synthesized primarily in apical regions of the shoot and is then transported in a polar fashion to other sites. When auxin reaches the root apex, it is redistributed away from the root tip through cortical and epidermal tissues. In tropic growth, auxin is diverted laterally to one side of the plant stem or root. As a result, the cells in that portion of the stem or root below the point of redistribution elongate. The result is bending toward light, gravitational pull, or a potential point of attachment.
Auxin is taken up into cells by diffusion augmented by a proton co-transporter, but can only exit from cells via basally-localized efflux carriers. Mutants deficient in auxin transport generally display aberrant morphology. Auxin is thus thought to maintain cellular polarity and, as a result, its own asymmetric transport mechanism.
The directionality of efflux is established primarily by efflux complexes characterized by the PIN family of facilitator proteins. Biochemical evidence suggests that the PIN proteins may mediate basal levels of auxin efflux, but also modulate the acitivity of ATP-dependent p-glycoprotein auxin transport proteins.
Chemiosmotic Model of Polar Auxin Transport
John Raven (1975) and Mary Helen Goldsmith (1976) proposed the currently accepted chemiosmotic model of auxin transport. IAA is protonated (IAAH) in the acidic extracellular space & enters the cell via diffusion or an H+ sympoter (AUX1). In the neutral pH of the cytosol, IAA is anionic & can only exit the cell via basally localized auxin efflux complex (characterized by PIN1), creating & maintaining an auxin gradient.
Testing the Chemiosmotic Model
avp1 null plants have fewer H+ ATPases on the plasma membrane (PM). The extracellular pH is less acidic, therefore IAAH amounts are less, & IAA transport is reduced. AVP1 overexpressors have more H+ ATPases on the PM, more acidic pH, more IAAH & enhanced IAA transport. (Li et al., 2005)
Diffusion effects in small cells vs. larger cells
In meristematic tissue, cells are small & re-diffusion of exported IAA into these cells is greater than in large cells. In small cells, IAA transporters have non-polar localization, & IAA export is greater than the rate of diffusion into the cell. In larger cells (e.g. vascular tissue), IAA transporters have polar localization where IAA re-diffusion is less of a factor. (Blakeslee, Peer & Murphy, 2005a,b)
PGPs are PM anion transporters characterized by 12 membrane-spanning helices, 2 ATP-binding sites, a phosphorylation site, and a C-terminal protein-protein interaction domain. The 21 expressed PGPs in Arabidopsis each have tissue-specific expression & localization. PGPs were initially associated with sauxin transport when they were purified by affinity chromatography with NPA, an auxin efflux inhibitor.
pgp mutants examined thus far have reduced shoot basipetal IAA transport and are dwarfs. pgp1 mutants have slightly altered hypocotyl and root gravitropic bending. pgp19 mutants have hypertropic bending. pgp4 mutants exhibit altered root bending in response to gravity. (Murphy and Taiz, 1999a,b; Murphy et al., 2000; Noh et al., 2001; Murphy et al., 2002; Geisler et al., 2003; Geisler & Blakeslee et al., 2005; Teresaka et al., 2005)
PGPs in Monocots
PGPs and Human Health
APM, an M1 metallopeptidase, is a bifunctional protein with trafficking & catalytic domains. It has one transmembrane domain and occurs in processed & unprocessed forms. We are investigating potential activities of the processed form and whether APM is autocatalytic. APM, a single-copy gene, was identified by NPA-affinity chromatography. APM is most similar to human insulin-responsive aminopeptidase/oxytocinase (IRAP) and APN (CD13). APM is localized in microsomal membranes and on the plasma membrane and is associated with sterol-rich membranes, as is IRAP. Other components of IRAP/GLUT4 system have homologs in Arabidopsis implicated in auxin transport and/or cellular targeting. (Murphy and Taiz, 1999a,b; Murphy et al., 2000, 2002; Muday & Murphy, 2002; Muday, Murphy & Peer, 2003).
APM is strongly expressed in developing seeds and 3-5d seedlings and at regions of differentiation: root & shoot apices, root-shoot junction. Analysis with inducible promoter and RNAi constructs as well as embryonic marker lines are underway to determine the critical spatio-temporal expression necessary for proper development. (Murphy et al., 2002; Bandyopadhyay & Murphy, Hosein & Murphy)
The adaptor complexes and endocytosis are not well-characterized in plants, and this critical component of cellular trafficking is being investigated. (Park & Murphy)
The role of aminopeptidases, particularly aminopeptidase P (APP), in wounding responses are also under investigation. (Makam & Murphy)
APM and Human Health
Loss of traditional diets rich in flavonoids and other nutrients among Americans has contributed to the rise of type II diabetes and obesity. APM/IRAP is involved in intracellular trafficking of proteins related to type II diabetes, and they are also involved in sterol uptake into intestinal cells. Flavonoids have been shown to inhibit both the activity of the M1 proteinases and to modulate intracellular trafficking. Therefore, flavonoid-based therapies and a return to traditional diets can help reduce the incidence of type II diabetes and obesity. The use of herbicides on our food has been linked to cancer. APM activity in food crops reduces the toxicity of the herbicide to the plants, but can increase the carcinogenicity of the herbicides to humans.
Amide herbicide metabolism
A byproduct of the auxin transport research in our lab has been the dissection of amidase activities in plant tissues that hydrolyze amide herbicides like Alanap. We are exploring the metabolism of amide herbicides in planta to determine 1) the extent to which their carcinogenic breakdown products are retained in horticultural crops and 2) whether these compounds enhance susceptibility to plant pathogens. Additionally, we are exploring use of plant and microbe combinations to remediate soils contaminated with either amide herbicides or their polycyclic aromatic hydrocarbon breakdown products.
Flavonoid Signaling
Flavonoids are poylyphenolic compounds that are important flavor and color c onstituents of plant-based foods. Flavonoids are signaling molecules within the plant, between the plant and other organisms (nod gene induction in rhizobacteria), and within other organisms. For example, flavonoids are phytoestrogens and act as mild estrogens in humans.
Flavonoid accumulation in the plant is tissue-specific. Aglycone flavonols are associated with the PM and endomembranes. They act as autocrine effectors within the cells they are synthesized, but may also act as paracrine effectors in adjacent cells, as flavonols appear to be at plasmadesmata. (Murphy et al., 2000; Peer & Murphy, 2005)
Targets
Flavonoids are antioxidants antioxidants and scavenge reactive oxygen species (ROS) thereby potentially regulating the pathways induced by ROS. Flavonoids are also kinase and phosphatase inhibitors. As such, they can modulate signal transduction within the cell. Likely targets are PTEN, PID, RCN1 (PP2a), and PGPs. A major target of ROS is PTEN, a tumor suppressor implicated in breast cancer. Flavonoids (like xanthohumol from hop) can reduce stimulate PTEN and reduce tumor proliferation.
Flavonoids and IAA
IAA treatment induces ROS in Arabidopsis roots. In the absence of flavonoids (tt4), more ROS fluorescence is observed but decreased ROS fluorescence if excess flavonols are present (tt3), due to flavonoid anti-oxidant activity. Flavonol accumulation also occurs after IAA treatment; IAA catabolism induces ROS. A modest increase in flavonols is observed after a modest increase in IAA, but after NPA treatment, which increases the amount of IAA in cells, flavonol accumulation is significantly increased. Flavonols also modulate auxin transport (Peer et al., 2004; Peer & Murphy, 2005)