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. 2006 May;1(3):122-33.
doi: 10.4161/psb.1.3.2759.

Auxin immunolocalization implicates vesicular neurotransmitter-like mode of polar auxin transport in root apices

Markus Schlicht  1 , Miroslav Strnad, Michael J Scanlon, Stefano Mancuso, Frank Hochholdinger, Klaus Palme, Dieter Volkmann, Diedrik Menzel, Frantisek Baluska
Affiliations

Auxin immunolocalization implicates vesicular neurotransmitter-like mode of polar auxin transport in root apices

Markus Schlicht et al. Plant Signal Behav. 2006 May.

Abstract

Immunolocalization of auxin using a new specific antibody revealed, besides the expected diffuse cytoplasmic signal, enrichments of auxin at end-poles (cross-walls), within endosomes and within nuclei of those root apex cells which accumulate abundant F-actin at their end-poles. In Brefeldin A (BFA) treated roots, a strong auxin signal was scored within BFA-induced compartments of cells having abundant actin and auxin at their end-poles, as well as within adjacent endosomes, but not in other root cells. Importantly, several types of polar auxin transport (PAT) inhibitors exert similar inhibitory effects on endocytosis, vesicle recycling, and on the enrichments of F-actin at the end-poles. These findings indicate that auxin is transported across F-actin-enriched end-poles (synapses) via neurotransmitter-like secretion. This new concept finds genetic support from the semaphore1, rum1 and rum1/lrt1 mutants of maize which are impaired in PAT, endocytosis and vesicle recycling, as well as in recruitment of F-actin and auxin to the auxin transporting end-poles. Although PIN1 localizes abundantly to the end-poles, and they also fail to support the formation of in these mutants affected in PAT, auxin and F-actin are depleted from their end-poles which also fail to support formation of the large BFA-induced compartments.

Keywords: actin; auxin; maize; neurotransmitter; secretion; vesicles.

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Figures

Figure 1
Figure 1
IAA labelings in maize root apices: antibody specificity. (A) Labeling with the IAA antibody immunodepleted with an excess of IAA for 24 h. (B) Labeling with the IAA antibody incubated with an excess of 2,4D for 24 h. (C) Labeling with the IAA antibody incubated with an excess of NAA for 24 h. (D) Labeling with the IAA antibody incubated with an excess of IBA for 24 h. White arrows indicate auxin-enriched end-poles. (E) Comparison of the fluorescence intensities of transition zone cells after treatments with different IAA concentrations. The medium intensities of transition zone cells of 5 roots per treatment are put into graphs. For the comparison, root sections labeled with the same antibody concentration were used. The pictures were recorded with the same exposure time. (CW, cell wall; N, ucleus). Bars: (B) 18 µM, (C) 12 µM, (D) 10 µM.
Figure 2
Figure 2
IAA labelings in maize root apices: subcellular and cellular distributions. (A, B) In the untreated root, IAA enriched cross-walls (end-poles) are prominent in stele cells of the transition zone (A) and the whole quiescent centre (QC) (B). (C, F and G) In BFA-treated root tips (2 h), IAA labeling of end-poles vanishes in the stele while the nuclear labelling gets more prominent. BFA treatment shifts IAA signal into BFA-induced compartments. (D) In cells of the cortex, intensity of the synapse labelling gets weaker while labelling of nuclei increases. White arrowheads point on auxin-enriched BFA-induced compartments. Red Line in (B) and (C) marks the border between meristem and root cap. (S, stele; e, endodermis; C, cortex) Bars: 10 µM.
Figure 3
Figure 3
PIN1 and IAA colocalization in control and BFA-treated root apices. (A) In untreated roots, colocalization of PIN1 with IAA in distinct patches at the end-poles is obvious. (B and C) After 2 h of BFA exposure, PIN1, IAA, and JIM5-positive cell wall pectins colocalize in patch-like structures within endocytic BFA-induced compartments. Bars: 10 µM.
Figure 4
Figure 4
Comparison of IAA labelings at end-poles and within nuclei. (A) Control. (B) TIBA-treatment. (C) IAA-treatment. (D) Wild-type after the BFA treatment. (E) TIBA/BFA treatment. (F) IAA/BFA treatment. Bars: (A–C) and (F) 10 µM; (D and E) 8 µM.
Figure 5
Figure 5
PIN1 labelings in cells of the transition zone of wild-type roots. (A) Control. (B) TIBA treatment. (C) TIBA/BFA treatment the combined treatments consisted of 2 hours of PAT inhibitor followed by two hours of BFA (D) BFA-treatment for ten minutes. (E) BFA-treatment for 2 hours. Bars: 10 µM.
Figure 6
Figure 6
BFA shifts PIN1 from the plasma membrane to endosomes. (A) Aqueous Two-Phase System reveals that BFA induces shift of PIN1 from the plasma mebrane-enriched upper phase (U) into the endomembrane-enriched lower phase (L). Pretreatment with IAA inhibits this BFA-induced shift. (B) Sucrose density gradient analysis of PIN1 localization reveals that BFA induces shift of PIN1 from the plasma membrane into the endosomal fractions, but not to the Golgi apparatus fractions. Pretreatment with TIBA and IAA inhibits this BFA-induced shift. Comparison of treatments with fractions of matchable sucrose density. Sucrose density of fractions increases from left to right.
Figure 7
Figure 7
F-Actin arrangements in cells of the transition zone. (A) Control. (B) BFA treatment. (C) TIBA treatment. (D) NPA treatment. (E) Flurenol treatment. (F) Chlorflurenol treatment. (G) Chlorflurenolmethyl treatment. (H) Latrunculin B treatment. Note the depletion of F-actin from end-poles and disintegration of F-actin cables while nuclei are shifted from their original central position towards the basal cell pole. Bar: 8 µM.
Figure 8
Figure 8
Labeling of the recycling pectin RGII in cells of the transition zone. (A) Control wild-type roots. (B) Latrunculin B-treated roots. (C) TIBA-treated roots. (D) NPA-treated roots. (E) BFA-treated roots. (F) Latrunculin B/BFA-treated roots. (G) TIBA/BFA-treated roots. (H) NPA/BFA-treated roots. (I) Flurenol-treated roots. (J) Chlorflurenol-treated roots. (K) Chlorflurenolmethyl-treated roots. (L) Flurenol/BFA-treated roots. (M) Chlorflurenol/BFA-treated roots. (N) Chlorflurenolmethyl/BFA-treated roots. Note that BFA-induced compartments are smaller in cells of roots pretreated with PAT inhibitors. All the treatments were for two hours, the combined treatments consisted of two hours of PAT inhibitors followed by two hours of BFA. Bar: 8 µM.
Figure 9
Figure 9
Actin, IAA, PIN1, and RGII labelings in root apices of wild-type and maize mutants. Actin (A, F, K, P, U), IAA (B, G, L, Q, V), PIN1 (C, H, M, R, W), and RGII (D, I, N, S, X) and RGII after two hours of BFA-treatment (E, J, O, T, Y) labelings in stele periphery cells of the transition zone the wild-type (A–E), semaphore1 (F–J), lrt1 (K–O), rum1 (P–T), and lrt1/rum1 (U–Y) mutants. Note the depletion of F-actin and IAA from the cellular end-poles, which is correlated with small size of BFA-induced compartments (E, J, O, T, Y) but PIN1 shows still a signal on the end-poles. The only exeption is the lrt1 mutant which is, in contrast to all other mutants, also not affected in PAT. Bars: A,C,F,K, N, P, S, U and X, 8 µM; B, D, E, G, H, I, J, L, M, O, Q, R, T, V,W and Y, 10 µM.
Figure 10
Figure 10
Real-time recordings of auxin uptake into wild-type and, mutant roots. Auxin uptake (transport) shows peak in the distal part of the transition zone (1.0–1.5 mm form the root apex junction). All mutants show a highly reduced auxin influx in this particular root zone, but not in cells of the elongation region. For more information on this technique, see reference .
Figure 11
Figure 11
Schematical comparison of the classical chemiosmotic and the updated endosomal models for the PAT. The most important difference between the models is the involvement of endosomes in both accumulation and regulated secretion of IAA out of exporting cells. Note that the endosomal interior is topologically part of the cell's exterior. This is also consistent with respect to low pH values and endosomal enrichments with cell wall pectins.,,,
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