Klaus Palme

Ion channels in roots allow the plant to gain access to nutrients. The composition of the individual ion channels and the functional contribution of different α-subunits is largely unknown. Focusing on K ⁺ -selective ion channels, we have characterized AtKC1, a new α-subunit from the Arabidopsis shaker -like ion channel family. Promoter-β-glucuronidase (GUS) studies identified AtKC1 expression predominantly in root hairs and root endodermis. Specific antibodies recognized AtKC1 at the plasma membrane. To analyze further the abundance and the functional contribution of the different K ⁺ channels α-subunits in root cells, we performed real-time reverse transcription–PCR and patch-clamp experiments on isolated root hair protoplasts. Studying all shaker -like ion channel α-subunits, we only found the K ⁺ inward rectifier AtKC1 and AKT1 and the K ⁺ outward rectifier GORK to be expressed in this cell type. Akt1 knockout plants essentially lacked inward rectifying K ⁺ currents. In contrast, inward rectifying K ⁺ currents were present in AtKC1 knockout plants, but fundamentally altered with respect to gating and cation sensitivity. This indicates that the AtKC1 α-subunit represents an integral component of functional root hair K ⁺ uptake channels.

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The Arabidopsis double pore K⁺ channel KCO1 was fused to green fluorescent protein and expressed in tobacco protoplasts. Microscopic analysis revealed a bright green fluorescence at the vacuolar membrane. RT-PCR experiments showed that KCO1 is expressed in the mesophyll. Vacuoles from Arabidopsis wild-type and kco1 knockout plants were isolated for patch-clamp analyses. Currents mediated by slow-activating vacuolar (SV) channels of mesophyll cell vacuoles were significantly smaller in kco1 plants compared to the wild-type. This shows that KCO1 is involved in the formation of SV channels.

The major auxin‐binding protein (ZmERabp1) from maize (Zea mays L.) has been structurally characterized. We determined the position of a disulfide bridge in ZmERabp1 by mass‐spectrometric analysis. We show that Cys2 and Cys61 are covalently linked and that residue Cys155 bears the free sulfhydryl group. By making use of electrospray mass spectrometry, the molecular mass of ZmERabp1 was determined to be 20 243 Da comprising a sugar moiety of 1865 Da, corresponding to a high mannose‐type glycan structure. Due to the high homology among all characterized ABPs, the information on the disulfide bonds will be important for functional analysis of recombinantly expressed ABP1.

Plants contain a novel unique subfamily of Rho GTPases, vital components of cellular signalling networks. Here we report a general role for some members of this family in polarized plant growth processes. We show that Arabidopsis AtRop4 and AtRop6 encode functional GTPases with similar intrinsic GTP hydrolysis rates. We localized AtRop proteins in root meristem cells to the cross‐wall and cell plate membranes. Polar localization of AtRops in trichoblasts specifies the growth sites for emerging root hairs. These sites were visible before budding and elongation of the Arabidopsis root hair when AtRops accumulated at their tips. Expression of constitutively active AtRop4 and AtRop6 mutant proteins in root hairs of transgenic Arabidopsis plants abolished polarized growth and delocalized the tip‐focused Ca2+ gradient. Polar localization of AtRops was inhibited by brefeldin A, but not by other drugs such as latrunculin B, cytochalasin D or caffeine. Our results demonstrate a general function of AtRop GTPases in tip growth and in polar diffuse growth.

A new mutant of Arabidopsis designated bus1-1 (for bushy), which exhibited a bushy phenotype with crinkled leaves and retarded vascularization, was characterized. The phenotype was caused by an En-1 insertion in the gene CYP79F1. The deduced protein belongs to the cytochrome P450 superfamily. Because members of the CYP79 subfamily are believed to catalyze the oxidation of amino acids to aldoximes, the initial step in glucosinolate biosynthesis, we analyzed the level of glucosinolates in a CYP79F1 null mutant (bus1-1f) and in an overexpressing plant. Short-chain glucosinolates derived from methionine were completely lacking in the null mutant and showed increased levels in the overexpressing plant, indicating that CYP79F1 uses short-chain methionine derivatives as substrates. In addition, the concentrations of indole-3-ylmethyl-glucosinolate and the content of the auxin indole-3-acetic acid and its precursor indole-3-acetonitrile were increased in the bus1-1f mutant. Our results demonstrate for the first time that the formation of glucosinolates derived from methionine is mediated by CYP79F1 and that knocking out this cytochrome P450 has profound effects on plant growth and development.

GTP-binding proteins are found in all organisms. They are important switches that cycle between an active and an inactive state, ensuring vectorial flow of information on the expense of guanosine triphosphate (GTP). In this review, we discuss current progress in the molecular characterization and functional analysis of plant genes encoding heterotrimeric and small GTPases. An up-to-date list including all cloned plant GTPase genes is given and a systematic classification is proposed.