Nicotiana tabacum actin-depolymerizing factor 2 is involved in actin-driven,auxin-dependent patterning

Polar transport of auxin has been identified as a central element of pattern formation. To address the underlying cellular mechanisms, we use the tobacco cell line (Nicotiana tabacum L. cv. Bright Yellow 2; BY-2) as model. We showed previously that cell divisions within a cell file are synchronized by polar auxin flow, linked to the organization of actin filaments (AF) which, in turn, is modified via actin-binding proteins (ABPs). From a preparatory study for disturbed division synchrony in cell lines overexpressing different ABPs, we identified the actin depolymerizing factor 2 (ADF2). A cell line overexpressing GFP-NtADF2 was specifically affected in division synchrony. The cell division pattern could be rescued by addition of Phosphatidylinositol 4,5-bisphosphate (PIP₂) or by phalloidin. These observations allow to draw first conclusions on the pathway linking auxin signalling via actin reorganization to synchronized cell division placing the regulation of cortical actin turnover by ADF2 into the focus.     

Effect of auxin transport inhibitors and ethylene on the wood anatomy of poplar

The influence of the auxin transport inhibitors naphthylphthalamic acid (NPA) and methyl-2-chloro-9-hydroxyflurene-9-carboxylate (CF), as well as the gaseous hormone ethylene on cambial differentiation of poplar was determined. NPA treatment induced clustering of vessels and increased vessel length. CF caused a synchronized differentiation of cambial cells into either vessel elements or fibres. The vessels in CF-treated wood were significantly smaller and fibre area was increased compared with controls. Under the influence of ethylene, the cambium produced more parenchyma, shorter fibres and shorter vessels than in controls. Since poplar is the model tree for molecular biology of wood formation, the modulation of the cambial differentiation of poplar towards specific cell types opens an avenue to study genes important for the development of vessels or fibres.     

Shoot multiplication in cultures of mature <Emphasis Type="Italic">Alnus glutinosa</Emphasis>

Shoot cultures were initiated from mature trees of Alnus glutinosa. On medium containing 1–5 μM 6-benzylamino purine (BAP), the shoots elongated without branching, formed heavy callus at the base of the stems and readily formed roots. The possibility that these characteristics could be attributed to the strong influence of endogenous auxin was tested on media that contained two auxin transport inhibitors, 1-N- naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), at concentrations of 0.1–3 μM, in combination with 2 μM BAP. On these media, shoots produced numerous branches, less callus and no roots. After 30 weeks (five subcultures) on this medium, leaves were smaller and showed signs of vitrification. These problems were resolved without detriment to shoot proliferation, by reverting to medium without NPA or TIBA. Shoots rooted readily after transfer to medium without growth regulators and were successfully acclimatised after transfer to soil.     

The regulation of sweet cherry fruit abscission by polar auxin transport

Inconsistency of cropping is an important problem for UK sweet cherry production. Premature fruit abscission in Prunus can reduce yields severely, however, the environmental cues and hormonal signals that trigger abscission have not been identified. Auxin (IAA) is known to delay abscission by reducing the sensitivity of cells in the abscission zone to ethylene, a promoter of abscission. Therefore, the capacity for polar auxin transport (PAT) through sweet cherry pedicels was examined in relation to fruit abscission. Cherry ‘spurs’ (short shoots) with similar leaf areas and different fruit numbers were phloem-girdled to restrict assimilate movement. Abscission from spurs with many fruit (eight or more) occurred within 14 days of girdling, whereas abscission from spurs with few (two) fruit was minimal. The pedicels’ capacity for PAT in spurs with different fruit numbers was determined 1, 3 and 9 days after girdling (DAG). Fruit were analysed for endogenous IAA concentration 3, 5, 7 and 9 DAG. PAT inhibitors 2,3,5-triiodobenzoic acid or 1-N-naphthylphtalamic acid were applied to pedicels of fruit not expected to abscise, i.e. on spurs with few fruit. The effect of these inhibitors on fruit abscission was determined 14 DAG. The proportion of the transported [³H]-IAA was lower from the outset in pedicels from spurs with many fruit. By 9 DAG, symptoms of fruit abscission were apparent and 40% less [³H] -IAA was transported through pedicels on spurs with many fruit. Fruit endogenous IAA concentrations were similar in the two groups of spurs. Application of PAT inhibitors shortly after girdling increased fruit abscission by 30%. The results suggest that although a decline in PAT is not the only cause of fruit abscission, the maintenance of PAT contributes to fruit retention.     

MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development

Previous data have suggested an involvement of MDR/PGP-like ABC transporters in transport of the plant hormone auxin and, recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin. Here we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development.     

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca²⁺- and Mg²⁺-ATPase activities of CF1 deficient in its inhibitory ε subunit (CF1-ε) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca²⁺-ATPase activity by CTAB is irreversible. The Ca²⁺-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg²⁺-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-ε or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-ε from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the α/β heterohexamer.     

Stereospecific microbiological 10-O-demethylation of R-(-)-10,11-dimethoxyaporphines

The microbiological O-dealkylation of (+) and (-) 10,11-dimethoxyaporphine and the corresponding N-n-propyl analog 10,11-dimethoxy-N-n-propylnoraporphine utilizing the fungus Cunninghamella elegans (ATCC 9245) was found to proceed with regioselectivity for the 10-position, and with a high degree of substrate stereospecificity for the 6a R(-)enantiomer. Only the (R) 10-demethylated products were isolated i.e. (R) iosapocodeine and (R) N-n-propylnor-isoapocodeine. The products were confirmed by comparison with their GC/MS spectra.     

Promotion of elongation and acid invertase activity in Phaseolus vulgaris L. internode segments by phenylacetic acid

Phenylacetic acid (PAA) significantly stimulated the elongation of isolated Phaseolus vulgaris internodal segments and prevented the decline in acid invertase specific activity observed in segments incubated in the absence of growth substances. Unlike IAA, which stimulated both elongation and invertase activity over a very wide range of concentrations (<10^-4 - 1 mol.m^-3; optimum 10^-2 mol.m^-3), the response to PAA was restricted to a much narrower range of concentrations (3 × 10^-2 - 1 mol.m^-3; optimum ca. 1–2 × 10^-1mol.m^-3). At the optimum concentration of PAA, the stimulation of both responses was about 63–75% of that induced by the optimum concentration of IAA. The differences in the concentration range and magnitude of the responses to IAA and PAA were not due to differences in uptake of the two compounds. The stimulation of elongation by both compounds was prevented by 3.6 × 10^-2mol.m^-3 cycloheximide (CH), and acid invertase activites were greatly reduced compared with samples treated with growth substances alone. A saturating concentration of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA) slightly promoted the growth of control segments, probably by reducing the loss of residual endogenous auxin to the incubation medium. The elongation induced by PAA at its optimum concentration was considerably greater than the elongation induced by NPA, indicating that PAA did not cause growth by preventing the loss of endogenous auxin from the segments. Elongation responses to combinations of IAA and PAA suggested that the compounds were acting additively and that they were affecting growth by the same mechanism.     

Regulatory role of auxin in adventitious root formation in two species of Rumex,differing in their sensitivity to waterlogging

Adventitious rooting in Rumex plants, in which the root systems were in hypoxic conditions, differed considerably between two species. R. palustris, a species from frequently flooded river forelands, developed a large number of adventitious roots during hypoxia, whereas adventitious root formation was poor in R. thyrsiflorus, a species from seldom flooded dykes and river dunes. Adventitious rooting could also be evoked in aerated plants of both species by application of auxin (1-naphthaleneacetic acid or indoleacetic acid) to the leaves. The response to auxin was dose-dependent, but even high auxin doses could not stimulate R. thyrsiflorus to produce as many adventitious roots as R. palustris. Consequently, the difference between the species in the amount of adventitious root formation was probably genetically determined, and not a result of a different response to auxin. A prerequisite for hypoxia-induced adventitious root formation is the basipetal transport of auxin within the shoot, as specific inhibition of this transport by N-1-naphthylphthalamic acid severely decreased the number of roots in hypoxia-treated plants. It is suggested that hypoxia of the root system causes stagnation of auxin transport in the root system. This can lead to an accumulation of auxin at the base of the shoot rosette, resulting in adventitious root formation.     

Polar auxin transport together with AINTEGUMENTA and REVOLUTA coordinate early Arabidopsis gynoecium development

In flowering plants the gynoecium is the female reproductive structure and the site of oogenesis, fertilization, and maturation of the embryo and the seed. Proper development of the gynoecium requires that the early gynoecial primordium be partitioned into distinct spatial domains with divergent fates. Regulated transport of the phytohormone auxin previously has been shown to play a role in the patterning of spatial domains along the apical-basal axis of the gynoecium. Here we establish a role for auxin transport in patterning along the medio-lateral axis of the gynoecial ovary. We demonstrate that auxin transport is required for the development of the medial ovary domain that contains the carpel margin meristem, a vital female reproductive structure. Disruptions in auxin transport enhance the medial domain defects observed in aintegumenta and revoluta mutant genotypes. AINTEGUMENTA and REVOLUTA are likely to function in parallel and partially overlapping pathways required for medial domain development. Our data indicate that different ovary domains are differentially sensitive to the reduction of polar auxin transport and the loss of AINTEGUMENTA and REVOLUTA activity. We suggest that an auxin-mediated positional cue is important for the differential specification of the medial and lateral ovary domains.