A. C. Leopold

Versuche zur Analyse des Auxintransports in der Koleoptile vonZea mays L.

A. Einleitung Das Ph&nomen der Wuchsstoffverlagerung wurde zuerst yon W ~ T (1928) und vA~ I)E~ WEIJ (1932, 1934) an Avenakoleoptilen genauer untersucht. Dabei zeigte sich zuni~chst, da~ der Auxintransport erstaunlich schnell verl&uft; die beobachtete hohe Geschwindigkeit l~i~t sich durch einen einfachen Dfffusionsvorgang kaum erkl&ren. Ferner ergab sich, dal~ die Wuchsstoffwanderung gegen einen Konzentrationsgradienten geschehen kann und yon Lebensprozessen vermittelt wird, dal~ es sich also um einen aktiven Transport handeln muG. Auch wurde gezeigt, dal~ der Auxinstrom polar ausgerichtet ist: nur vom Gipfel des Organs zur morphologischen Basis bin. Viele sp~tere Arbeiten best&tigten das Auftreten dieser Polaritgt und die Tatsache, dal~ es sich um einen metabolisch-aktiven Stofft ransport handelt; die Frage aber, wie strikt die Transportpolariti~t in verschiedenen Organen festgelegt ist, blieb umstri t ten (LEoPoLI) 1961). In neueren Untersuchungen (GOLDSMIT~ 1959; GOLDSMIT~ und T ~ I ~ A ~ 1962) wurde radioaktive Indolylessigsgure benutzt, was alle Transportversuche abkiirzt und erleichtert. Mit dieser Technik sollte nun der Auxintransport in der Maiskoleoptfle untersucht werden. Das Ziel der vorliegenden Arbeit war es, den aktiven Transportsehrit t n~her zu bestimmen, die Polariti~t dazu in Beziehung zu setzen und schliel~lich herauszufinden, wieweit der Transport durch AuBenfaktoren, vor allem dutch die Schwerkraft, beeinflu6t werden kann.

The Specificity of the Auxin Transport System

SummaryIn an effort to examine the specificity of the auxin transport system, the movement of a variety of growth substances and of auxin analogues through corn coleoptile sections was measured in both the basipetal and acropetal directions. In contrast to the basipetal, polar transport of the auxins indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid, no such movement was found for benzoic acid or for gibberellin A1. A comparison of the α- and β-isomers of naphthaleneacetic acid showed that the growth-active α-form is transported, but not the inactive β-analogue. Both the dextro (+) and leavo (-) isomer of 3-indole-2-methylacetic acid showed the basipetal movement characteristic of IAA, the dextro isomer being more readily transported than the (-)-form. In this instance, too, the transport was roughtly proportional to the growth promoting activity. The antiauxin p-chlorophenoxyisobutyric acid inhibited auxin transport as it inhibited auxin-induced growth. These results agree with the hypothesis that processes involved in auxin transport are closely linked to or even identical with the primary auxin action.

Geotropism and the lateral transport of auxin in the corn mutant amylomaize

SummaryIn coleoptiles of the amylomaize corn mutant (AM), the amyloplasts are much reduced in size in comparison with the wild type corn (WT), permitting a comparison of geotropic responsiveness as related to lateral displacement of amyloplasts and lateral transport of auxin. The amyloplasts of AM showed 30–40% lesser lateral redistribution in response to horizontal exposure in comparison with WT. With geotropic stimulation, the lateral transport of auxin in the direction of growth was 40–80% less, and the geotropic curvature by the coleoptiles was also significantly less in the mutant as compared with WT. These correlations support the hypothesis that the starch plastids serve as gravity sensors in the geotropic responses of coleoptiles.

In-vitro Binding of Morphactins and 1-N-Naphthylphthalamic Acid in Corn Coleoptiles and Their Effects on Auxin Transport* **

SummaryUtilizing the specific binding of radioactive 1-N-naphthylphthalamic acid (NPA) to a particulate fraction of corn coleoptiles, a series of NPA and morphactin analogues were tested for competition with the NPA binding site. Competition for this binding site was found for NPA itself, chloro-1-N-naphthylphthalamic acid and dichloro-1-N-naphthylphthalamic acid, to about the same degree. 1-N-phenylphthalamic acid and 4-chloro-1-N-phenylphthalamic acid were less active, and 1-N-naphthylphthalimide did not compete for the NPA binding site Morphactins were found to compete for the NPA site as well; the free fluorenol acids, 2-chloro-9-hydroxylflorene-9-carboxylate and 9-hydroxyfluorene-9-carboxylate, compete better for the NPA binding site than n-butyl-9-hydroxyfluorene-9-carboxylate (Bu-HFC), methyl-2-chloro-9-hydroxyfluorene-9-carboxylate (Me-Cl-HFC) and methyl-2,7-dichloro-9-hydroxyfluorene-9-carboxylate (Me-diCl-HFC). The inhibition of NPA binding has the characteristics of a competitive inhibition when examined in double-reciprocal plots.The transport of auxin (indolyl-3-acetic acid, IAA) is inhibited by both NPA and fluorenol derivatives but whilst the free acids have superior binding activity, transport is more effectively inhibited by the fluorenol esters including Bu-HFC, Me-Cl-HFC and Me-diCl-HFC than by the free fluorenol acids. It is probable that the free fluorenol acids are the active form for inhibition of IAA transport and that the fluorenol esters undergo hydrolysis before acting on IAA transport.

Correlative aging and transport of P32 in corn leaves under the influence of kinetin

Summary1.The course of senescence in leaves of corn seedlings, as measured by the loss of chlorophyll, is shown to be controlled by two correlative influences: a) In the isolated leaf, the base tends to enhance yellowing of the tip region. b) In the leaf attached to the intact plant, correlative aging is delayed by interactions with other parts of the plant.2.Transport of P32 in the isolated leaf is directed from tip to base. It is promoted under conditions enhancing senescence of the tip (application of kinetin to the base). Conditions delaying senescence (leaf attached to intact plant) defer the basipetal P32-transport. This suggests that the pattern of correlative aging in detached leaves is caused by a shift of materials from tip to base (“directed transport” or “mobilization”).3.Kinetin-induced directed transport of P32 was used as a test system to study mobilization. Localized application of kinetin to a leaf causes a) an accumulation of translocated P32 in the treated area, b) an acceleration of P32-transport directed towards the kinetin center.4.The rate of kinetin-induced P32-transport is shown to be mainly determined by the activity of the mobilizing center; but the dissipation of phosphates out of the source region, being a separate variable, may also influence the rate of P32-transport.5.The attracting activity of mobilizing centers is shown to be enhanced by increasing amounts of materials already accumulated. Thus, mobilization is a self-amplifying process. This is also reflected in the sigmoid shape of the time curves of P32-mobilization.Zusammenfassung1.Der Verlauf des Alterns in Maisblättern, gemessen am Verlust des Chlorophylls, wird durch zwei Korrelationen geregelt: a) Im isolierten Blatt hat die Basis die Tendenz, den Verfall in der Spitzenzone zu beschleunigen und sich dadurch die eigene Vitalität zu erhalten. b) Bleibt das Blatt in Verbindung mit der Pflanze, so wird dieses korrelative Altern verzögert.2.32P wird im isolierten Blatt von der Spitze zur Basis transportiert. Wird das Altern der Spitzenzone durch Behandlung der Basis mit Kinetin beschleunigt, so zeigt sich auch der 32P-Transport zur Blattbasis hin gefördert. Er wird aufgehalten, wenn der Verfall des Blattes durch den Anschluß an die Pflanze verhindert wird. Dies spricht dafür, daß eine Verlagerung von Material von der Blattspitze zur Basis hin (“gerichteter Transport”, “Mobilisation”) den Verlauf des korrelativen Alterns bestimmt.3.Am Modellsystem des kinetin-induzierten gerichteten 32P-Transports lassen sich unterscheiden: a) eine Akkumulierung transportierter radioaktiver Phosphate am Kinetinort, b) eine Beschleunigung des dorthin gerichteten Transports um ca. 50%.4.Der gerichtete Transport wird vom mobilisierenden Zentrum induziert und aufrechterhalten. Die Mobilisation kann aber auch vom Lieferbereich beeinflußt werden, besonders in späteren Stadien des Alterns.5.Es wurde gezeigt, daß die Aktivität eines Mobilisationszentrums um so mehr ansteigt, je mehr Material dort bereits akkumuliert ist. Im Verlauf der Stoffverlagerungen verstärkt sich also die Mobilisation selbst. Der autokatalytische Charakter dieses Vorgangs zeigt sich auch in der Zeitkurve des gerichteten Transports von 32P.

The mechanism of kinetin-induced transport in corn leaves

Summary1.Kinetin-induced transport of P32 in detached corn leaves is shown to be limited to the axial direction of the leaves, i.e. along the axis of the vascular bundles. It is not apparently dependent on the water flow in the xylem, and it can be blocked by steam-killed zones or by metabolic inhibitors. It is concluded that kinetin-induced redistribution of phosphates in isolated corn leaves takes place in the phloem.2.The movement of P32 is preferentially toward the base of the excised leaf, indicating a natural mobilization center at the leaf base.3.Kinetin treated parts of leaves attract and accumulate P32. They do not accumulate the radioisotopes Na22, Rb86, Cl36 and I131; but the transport of Na22 directed towards kinetin centers is enhanced.4.Two “mobilizing centers” established by kinetin application along the leaf axis do not attract P32 from intermediate tissue at the same time in opposite directions. They compete with each other for the transport system: both the direction and the velocity of movement are determined by the difference of the “mobilizing forces”. Thus, mobilization is either to one or to the other mobilizing center.5.Under conditions of enhanced transport (i.e. in presence of kinetin centers), the distribution curves of P32 and Na22 are flattened. This change would be expected for the mass-flow type of transport.6.It is concluded that “mobilizing centers” in isolated corn leaves stimulate a mass-flow transport in the phloem. Evidence is presented that they act as suction pumps. Some general aspects of phloem transport are discussed.Zusammenfassung1.Der kinetininduzierte Transport von 32P ist in isolierten Maisblättern an die Längsrichtung, d.h. an die Richtung der parallelen Leitbündel gebunden. Er ist nicht von Wasserbewegungen im Xylem abhängig und wird an abgetöteten oder hemmstoffbehandelten Stellen des Blattes blockiert. Dies deutet darauf hin, daß die kinetininduzierte Verlagerung von Phosphaten im Phloem stattfindet.2.Kinetinbehandelte Blattbereiche induzieren einen auf sie gerichteten Transport von 32P; die transportierten radioaktiven Phosphate akkumulieren sie. Im Gegensatz zu 32P werden die Isotope 22Na, 86Rb, 36Cl und 131J am Kinetinort nicht angereichert. Der Transport von 22Na wird jedoch auf Kinetinzentren zu gefördert.3.Stehen sich zwei “mobilisierende Zentren” in einem Blatt gegenüber, dann wird dazwischen aufgetragenes 32P nicht gleichzeitig in entgegengesetzten Richtungen transportiert. Die beiden Zentren konkurrieren miteinander um das Transportsystem: Richtung und Geschwindigkeit der Stoffbewegung hängen von der Differenz der “mobilisierenden Kräfte” ab.4.Wird der Transport von 32P und 22Na durch Kinetinzentren beschleunigt, so beobachtet man an den Verteilungskurven der Radioaktivität eine Abnahme der Steigung. Diese Veränderung ist zu erwarten, wenn die Stoffe im Phloem mit einer Massenströmung wandern.5.Aus den Ergebnissen wird geschlossen, daß “mobilisierende Zentren” in isolierten Maisblättern einen Massenstromtransport im Phloem auf sich lenken. Der Pumpmechanismus, der die Strömung unterhält, ist wahrscheinlich kein Druckstromsystem, sondern ein einfacher Sog. Einige allgemeine Aspekte des Phloemtransports werden diskutiert.

Two Components of Auxin Transport

The transport of indoleacetic acid-1-(14)C out of sunflower stem sections has been analyzed by a compartmental analysis procedure in which the radioactivity moving out of the tissue (log per cent) is plotted against time. The analysis indicates that indoleacetic acid is transported via a fast transport system (t((1/2)) of about 30 minutes) and a slow transport system (t((1/2)) about 10 hours). While we do not know the sources of these two pools, by analogy with ion transport studies, the fast efflux is characteristic of transport from the cytoplasm across the plasmalemma and the slow efflux is characteristic of transport across the tonoplast and thus out of the vacuole. Both components of transport are inhibited by 2,3,5-triiodobenzoic acid.