Advances in Understanding the Mechanism of Action of the Auxin Permease AUX1

In over 40 years of research on the cellular uptake of auxin it is somewhat chastening that we have elaborated so little on the original kinetic descriptions of auxin uptake by plant cells made by Rubery and Sheldrake in 1974. Every aspect of that seminal work has been investigated in detail, and the uptake activity they measured is now known to be attributed to the AUX1/LAX family of permeases. Recent pharmacological studies have defined the substrate specificity of AUX1, biochemical studies have evaluated its permeability to auxin in plant cell membranes, and rigourous kinetic studies have confirmed the affinity of AUX1 for IAA and synthetic auxins. Advances in genome sequencing have provided a rich resource for informatic analysis of the ancestry of AUX1 and the LAX proteins and, along with models of topology, suggest mechanistic links to families of eukaryotic proton co-transporters for which crystal structures have been presented. The insights gained from all the accumulated research reflect the brilliance of Rubery and Sheldrake’s early work, but recent biochemical analyses are starting to advance further our understanding of this vitally important family of auxin transport proteins.

THE INFLUENCE OF pH ON AUXIN-INDUCED ADVENTITIOUS ROOT INITIATION IN MALUS DOMESTICA

Many physiological responses in plants are influenced by pH. The present chemiosmotic hypothesis suggests that auxin uptake into plant cells is governed by pH. Since auxin is used widely to enhance rooting, the influence of pH on 1H-indole-3-butyric acid (IBA) induced adventitious root formation was examined. Roots were initiated aseptically in 5 node apical shoot cuttings of micropropagated Malus domestica 'Gala'. Initiation was induced using a four day pulse in IBA and 15 g/L sucrose at pH 5.6 and 30C in the dark. Observations showed pH rose to 7.0 or greater within 1 to 2 days from microcutting placement in unbuffered initiation medium. Root numbers from shoots in media containing 1.5 μM IBA buffered with 10 mM 2[N-morpholino] ethanesulfonic acid (MES) to pH 5.5, 6.0, 6.5 or 7.0 with KOH resulted in average root numbers of 14.2, 10.9, 8.7, and 7.1, respectively, while unbuffered medium yielded 7,6 roots per shoot. Comparison of MES buffered medium at pH 5.5, 6.25 or 7.0 in factorial combination with IBA at 0, 0.15, 1.5, 15.0, and 150.0 μM resulted in a significant pH by IBA interaction for root number. At 0, 0.15 and 1.5 μM IBA root numbers were greatest at pH 5.5. At 15.0 μM IBA, pH 6.25 was optimal and at 150.0 μM IBA all three pH levels produced equivalent root numbers. A calorimetric assay to measure IBA removal from the initiation medium by microcuttings of `Gala' and `Triple Red Delicious' showed more IBA removal at pH 5.5 than at pH 7.0. Possible reasons for the effect of pH on adventitious root formation will be discussed.

Initiation of roots on hypocotyl cuttings of Pinussylvestris, with emphasis on direct rooting, root elongation, and auxin uptake

Direct rooting of hypocotyl cuttings of Pinussylvestris L. is described and compared with rooting via wound tissue. The optimal 3-indolylbutyric acid treatment for direct rooting of Pinussylvestris hypocotyl cuttings under in vitro conditions was 1.23 mM for 24 h. This treatment resulted in 45% rooting within 3 weeks after cutting. During this period, only direct rooting was observed. Three to 4 weeks after cutting, roots developed either directly on the hypocotyl or via wound tissue. After the first month, rooting took place predominantly via wound tissue. The optimal 3-indolylbutyric acid treatment found for in vitro culture could successfully be used for hydroponic culture (under nonsterile conditions). In vitro culture inhibited root elongation. However, after transfer of rooted cuttings from in vitro to hydroponic culture, root elongation increased markedly. Uptake studies with 3-[5(n)-3H]indolylacetic acid mixed with the nutrient solution showed that auxin uptake by the cutting probably occurred mainly through uptake of the nutrient solution. After 24 h treatment with 1.43 mM 3-indolylacetic acid, the concentration taken up by the cuttings was 0.91 nmol/mg fresh weight. Differences between individual cuttings in auxin uptake could not explain the poor direct rooting of Pinussylvestris hypocotyl cuttings cultured in vitro.