Undergraduates achieve learning gains in plant genetics through peer teaching of secondary students.

This study tests the hypothesis that undergraduates who peer teach genetics will have greater understanding of genetic and molecular biology concepts as a result of their teaching experiences. Undergraduates enrolled in a non-majors biology course participated in a service-learning program in which they led middle school (MS) or high school (HS) students through a case study curriculum to discover the cause of a green tomato variant. The curriculum explored plant reproduction and genetic principles, highlighting variation in heirloom tomato fruits to reinforce the concept of the genetic basis of phenotypic variation. HS students were taught additional activities related to mole-cular biology techniques not included in the MS curriculum. We measured undergraduates' learning outcomes using pre/postteaching content assessments and the course final exam. Undergraduates showed significant gains in understanding of topics related to the curriculum they taught, compared with other course content, on both types of assessments. Undergraduates who taught HS students scored higher on questions specific to the HS curriculum compared with undergraduates who taught MS students, despite identical lecture content, on both types of assessments. These results indicate the positive effect of service-learning peer-teaching experiences on undergraduates' content knowledge, even for non-science major students.

Polar auxin transport: controlling where and how much.

Auxin is transported through plant tissues, moving from cell to cell in a unique polar manner. Polar auxin transport controls important growth and developmental processes in higher plants. Recent studies have identified several proteins that mediate polar auxin transport and have shown that some of these proteins are asymmetrically localized, paving the way for studies of the mechanisms that regulate auxin transport. New data indicate that reversible protein phosphorylation can control the amount of auxin transport, whereas protein secretion through Golgi-derived vesicles and interactions with the actin cytoskeleton might regulate the localization of auxin efflux complexes.

Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth.

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis.

Polar transport of the plant hormone auxin controls many aspects of plant growth and development. A number of synthetic compounds have been shown to block the process of auxin transport by inhibition of the auxin efflux carrier complex. These synthetic auxin transport inhibitors may act by mimicking endogenous molecules. Flavonoids, a class of secondary plant metabolic compounds, have been suggested to be auxin transport inhibitors based on their in vitro activity. The hypothesis that flavonoids regulate auxin transport in vivo was tested in Arabidopsis by comparing wild-type (WT) and transparent testa (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis, chalcone synthase. In a comparison between tt4 and WT plants, phenotypic differences were observed, including three times as many secondary inflorescence stems, reduced plant height, decreased stem diameter, and increased secondary root development. Growth of WT Arabidopsis plants on naringenin, a biosynthetic precursor to those flavonoids with auxin transport inhibitor activity in vitro, leads to a reduction in root growth and gravitropism, similar to the effects of synthetic auxin transport inhibitors. Analyses of auxin transport in the inflorescence and hypocotyl of independent tt4 alleles indicate that auxin transport is elevated in plants with a tt4 mutation. In hypocotyls of tt4, this elevated transport is reversed when flavonoids are synthesized by growth of plants on the flavonoid precursor, naringenin. These results are consistent with a role for flavonoids as endogenous regulators of auxin transport.

Flavonoid accumulation patterns of transparent testa mutants of arabidopsis.

Flavonoids have been implicated in the regulation of auxin movements in Arabidopsis. To understand when and where flavonoids may be acting to control auxin movement, the flavonoid accumulation pattern was examined in young seedlings and mature tissues of wild-type Arabidopsis. Using a variety of biochemical and visualization techniques, flavonoid accumulation in mature plants was localized in cauline leaves, pollen, stigmata, and floral primordia, and in the stems of young, actively growing inflorescences. In young Landsberg erecta seedlings, aglycone flavonols accumulated developmentally in three regions, the cotyledonary node, the hypocotyl-root transition zone, and the root tip. Aglycone flavonols accumulated at the hypocotyl-root transition zone in a developmental and tissue-specific manner with kaempferol in the epidermis and quercetin in the cortex. Quercetin localized subcellularly in the nuclear region, plasma membrane, and endomembrane system, whereas kaempferol localized in the nuclear region and plasma membrane. The flavonoid accumulation pattern was also examined in transparent testa mutants blocked at different steps in the flavonoid biosynthesis pathway. The transparent testa mutants were shown to have precursor accumulation patterns similar to those of end product flavonoids in wild-type Landsberg erecta, suggesting that synthesis and end product accumulation occur in the same cells.

Auxins and tropisms.

Differential growth of plants in response to the changes in the light and gravity vectors requires a complex signal transduction cascade. Although many of the details of the mechanisms by which these differential growth responses are induced are as yet unknown, auxin has been implicated in both gravitropism and phototropism. Specifically, the redistribution of auxin across gravity or light-stimulated tissues has been detected and shown to be required for this process. The approaches by which auxin has been implicated in tropisms include isolation of mutants altered in auxin transport or response with altered gravitropic or phototropic response, identification of auxin gradients with radiolabeled auxin and auxin-inducible gene reporter systems, and by use of inhibitors of auxin transport that block gravitropism and phototropism. Proteins that transport auxin have been identified and the mechanisms which determine auxin transport polarity have been explored. In addition, recent evidence that reversible protein phosphorylation controls this process is summarized. Finally, the data in support of several hypotheses for mechanisms by which auxin transport could be differentially regulated during gravitropism are examined. Although many details of the mechanisms by which plants respond to gravity and light are not yet clear, numerous recent studies demonstrate the role of auxin in these processes.

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

Basipetal auxin transport is required for gravitropism in roots of Arabidopsis.

Auxin transport has been reported to occur in two distinct polarities, acropetally and basipetally, in two different root tissues. The goals of this study were to determine whether both polarities of indole-3-acetic acid (IAA) transport occur in roots of Arabidopsis and to determine which polarity controls the gravity response. Global application of the auxin transport inhibitor naphthylphthalamic acid (NPA) to roots blocked the gravity response, root waving, and root elongation. Immediately after the application of NPA, the root gravity response was completely blocked, as measured by an automated video digitizer. Basipetal [(3)H]IAA transport in Arabidopsis roots was inhibited by NPA, whereas the movement of [(14)C]benzoic acid was not affected. Inhibition of basipetal IAA transport by local application of NPA blocked the gravity response. Inhibition of acropetal IAA transport by application of NPA at the root-shoot junction only partially reduced the gravity response at high NPA concentrations. Excised root tips, which do not receive auxin from the shoot, exhibited a normal response to gravity. The Arabidopsis mutant eir1, which has agravitropic roots, exhibited reduced basipetal IAA transport but wild-type levels of acropetal IAA transport. These results support the hypothesis that basipetally transported IAA controls root gravitropism in Arabidopsis.

The actin cytoskeleton may control the polar distribution of an auxin transport protein.

The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton.

In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

Inhibition of auxin movement from the shoot into the root inhibits lateral root development in Arabidopsis.

In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [3H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.

In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls.

The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

Cytoplasmic Orientation of the Naphthylphthalamic Acid-Binding Protein in Zucchini Plasma Membrane Vesicles.

Polar transport of the plant hormone auxin is blocked by substances such as N-1-naphthylphthalamic acid (NPA), which inhibit auxin efflux and block polar auxin transport. To understand how auxin transport is regulated in vivo, it is necessary to discern whether auxin transport inhibitors act at the intra- or extracellular side of the plasma membrane. Populations of predominantly in-side-in plasma membrane vesicles were subjected to treatments that reverse the orientation. These treatments, which included osmotic shock, cycles of freezing and thawing, and incubation with 0.05% Brij-58, all increased NPA-binding activity and the accessibility of the binding protein to protease digestion. Marker activities for inside-out vesicles also increased, indicating that these treatments act by altering the membrane orientation. Finally, binding data were analyzed by multiple analyses and indicated that neither the affinity nor abundance of binding sites changed. Kinetic analyses indicated that the change in NPA-binding activity by Brij-58 treatment was due to an increase in the initial rates of both association and dissociation of this ligand. These experiments indicated that the NPA-binding site is on the cytoplasmic face of the plasma membrane in zucchini (Cucurbita pepo L. cv Burpee Fordhook).

Characterization of the growth and auxin physiology of roots of the tomato mutant, diageotropica.

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant (diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([3H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [3H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.

NPA binding activity is peripheral to the plasma membrane and is associated with the cytoskeleton.

N-1-Naphthylphthalamic acid (NPA) binding activity is released into the supernatant when plasma membranes are subjected to high-salt treatment, indicating that this activity is peripherally associated with the membrane. Extraction of plasma membrane vesicles with Triton X-100 resulted in retention of NPA binding activity in the detergent-insoluble cytoskeletal pellet. Treatment of this pellet with KI released NPA binding activity, actin, and alpha-tubulin. Dialysis to remove KI led to the repolymerization of cytoskeletal elements and movement of NPA binding activity into an insoluble cytoskeletal pellet. NPA binding activity partitioned into the detergent-insoluble cytoskeletal pellet obtained from both zucchini and maize membranes and was released from these pellets by KI treatment. Treatment of a cytoskeletal pellet with cytochalasin B doubled NPA binding activity in the resulting supernatant. Together, these experiments indicate that NPA binding activity is peripherally associated with the plasma membrane and interacts with the cytoskeleton in vitro.

Tomato root growth, gravitropism, and lateral development: correlation with auxin transport.

Tomato (Lycopersicon esculentum, Mill.) roots were analyzed during growth on agar plates. Growth of these roots was inhibited by the auxin transport inhibitors naphthylphthalamic acid (NPA) and semicarbazone derivative I (SCB-1). The effect of auxin transport inhibitors on root gravitropism was analyzed by measurement of the angle of gravitropic curvature after the roots were reoriented 90 degrees from the vertical. NPA and SCB-1 abolished both the response of these roots to gravity and the formation of lateral roots, with SCB-1 being the more effective at inhibition. Auxins also inhibited root growth. Both auxins tested has a slight effect on the gravity response, but this effect is probably indirect, since auxins reduced the growth rate. Auxins also stimulated lateral root growth at concentration where primary root growth was inhibited. When roots were treated with both IAA and NPA simultaneously, a cumulative inhibition of root growth was found. When both compounds were applied together, analysis of gravitropism and lateral root formation indicated that the dominant effect was exerted by auxin transport inhibitors. Together, these data suggest a model for the role of auxin transport in controlling both primary and lateral root growth.

Evidence for a Single Naphthylphthalamic Acid Binding Site on the Zucchini Plasma Membrane.

The binding of [2,3,4,5,(n)-3H]N-1-napthylphthalamicacid ([3H]-NPA) to zucchini (Cucurbita pepo L.) plasma membranes was examined in detail using two different filtration assays and the results were rigorously analyzed by saturation curves, double-reciprocal plots, Scatchard plots, Hill plots, and the computer program Ligand (P.J. Munson, D. Rodbard [1980] Anal Biochem 107: 220-239). To facilitate these analyses, a new assay that allows rapid and quantitative analysis of [3H]NPA binding with high reproducibility and ease of manipulation has been developed. These detailed kinetic analyses indicate that only one binding site for [3H]NPA (Kd = 16 nM) was associated with the zucchini plasma membrane. Analysis of [3H]NPA dissociation by several auxin transport inhibitors revealed similar dissociation constants with both plasma and microsomal membrane. Collectively, these data indicate the presence of only one binding site for NPA associated with the zucchini plasma membrane.

Wounding Induces One of Two Isoenzymes of 3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthase in Solanum tuberosum L.

Potato (Solanum tuberosum L.) tubers contain two isoenzymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15), the enzyme that catalyzes the first step of aromatic amino acid biosynthesis. One of the isoenzymes is specifically activated by Mn(2+), and the other requires Co(2+), Mg(2+), or another divalent cation for activity. Monospecific polyclonal antibodies against the Mn(2+)-activated isoenzyme do not cross-react with the other isoenzyme. Wounding of potato tubers induces the Mn(2+)-activated form but not the other. We conclude that two different genes encode two different isoenzymes that catalyze the first step in the shikimate pathway.

Auxin transport and the interaction of phytotropins: probing the properties of a phytotropin binding protein.

We have described the inhibition of polar auxin transport by several phytotropins including 1-N-naphthylphthalamic acid (NPA) and quercetin. Semicarbazones (substituted phenylsemicarbazones of 2-acetylarylcarboxylic acids) are inhibitors consistent with previously predicted general structural requirements for auxin transport inhibitors. The best semicarbazone derivative tested to date, hereafter called SCB-I, binds to the NPA binding protein with high affinity, K(b) = 4 nanomolar. Quantification of the binding of various phytotropins allows us to make some general statements concerning the structure/properties of the NPA binding protein. The data suggest that the ligand binding region of this protein is multifaceted, a conclusion supported by the chemical predictions of Katekar and Geissler ([1977] Plant Physiol 60: 826-829). Although the data do not allow us to make specific conclusions on the structure of the binding site, they do show that both NPA and SCB-I could each occupy two regions of the protein. At least one of these binding regions appears to be common for both inhibitors of auxin transport. We suggest that the diversity of the binding site structure reflects the possible existence of more than one type of natural ligand controlling the process of auxin transport.

The tyrosine repressor negatively regulates aroH expression in Escherichia coli.

The levels of the tryptophan-sensitive isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli, encoded by the aroH gene, were elevated in tyrR and/or trpR mutants. The effect of tyrR and trpR lesions on aroH expression was confirmed by using a lacZ reporter system. The mutational elimination of either repressor led to a threefold increase in beta-galactosidase.