Interactions of PIN and PGP auxin transport mechanisms.

Polarized transport of the plant hormone auxin influences multiple growth processes in plants and is regulated by plasma-membrane-localized efflux and uptake carriers. The PGP (P-glycoprotein) ABC transporters (ATP-binding-cassette transporters), PIN (pin-formed) subfamily of major facilitator proteins and members of AUX/LAX families have been shown to independently transport auxin both in planta and in heterologous systems. However, PIN- and PGP-mediated transport in heterologous systems exhibits decreased substrate specificity and inhibitor-sensitivity compared with what is seen in plants and plant cells. To determine whether PIN-PGP interactions enhance transport specificity, we analysed interactions of the representative auxin-transporting PGPs with PIN1 and AUX1 in planta and in heterologous systems. Here, we provide evidence that PINs and PGPs interact and function both independently and co-ordinately to control polar auxin transport and impart transport specificity and directionality. These interactions take place in protein complexes stabilized by PGPs in detergent-resistant microdomains.

Shade-avoidance responses in two common coastal redwood forest species, Sequoia sempervirens (Taxodiaceae) and Satureja douglasii (Lamiaceae), occurring in various light quality environments.

Shade-avoidance responses were examined for two species common to the coastal redwood forest, Sequoia sempervirens and Satureja douglasii. Sequoia seedlings demonstrated a shade-avoidance response when given end-of-day far-red light by increased hypocotyl, epicotyl, and first-node extension, and greater total number of needles and reduced anthocyanin concentration. Thus, Sequoia seedlings respond as sun-adapted plants. Satureja has several leaf monoterpene chemotypes that occur in different light environments including the redwood forest, and the types responded differently to the light treatments. The pulegone type responded to end-of-day far-red light as a sun-adapted plant with significant extension growth, increased leaf area and chlorophyll, and reduced anthocyanin. The isomenthone type responded as a shade-tolerant plant and did not exhibit extension growth nor a change in other parameters with end-of-day far-red light. However, the carvone and bicyclic types had variable responses depending on the parameter studied, which indicated genetic variation for these traits.

Regulation of auxin transport by aminopeptidases and endogenous flavonoids.

The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, amino acyl-beta-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [3H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [14C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux.

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.

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.