Hartwig Lüthen

Oxidoreductases in plant plasma membranes.

Electron transporting oxidoreductases at biological membranes mediate several physiological processes. While such activities are well known and widely accepted as physiologically significant for other biological membranes, oxidoreductase activities found at the plasma membrane of plants are still being neglected. The ubiquity of the oxidoreductases in the plasma membrane suggests that the activity observed is of major importance in fact up to now no plant without redox activity at the plasmalemma is known. Involvement in proton pumping, membrane energization, ion channel regulation, iron reduction, nutrient uptake, signal transduction, and growth regulation has been proposed. However, positive proof for one of the numerous theories about the physiological function of the system is still missing. Evidence for an involvement in signalling and regulation of growth and transport activities at the plasma membrane is strong, but the high activity of the system displayed in some experiments also suggests function in defense against pathogens.

Rapid Auxin-Induced Cell Expansion and Gene Expression: A Four-Decade-Old Question Revisited

The classical effect of the plant hormone auxin is very rapid stimulation of cell expansion followed by sustained growth over a longer time period. However, auxins are also important in other responses, such as cell division and differentiation. Recently, the TRANSPORT INHIBITOR RESPONSE1/AUXIN-

Auxin-induced changes in cell wall extensibility of maize roots

Abstract. The rheological properties of corn (Zea mays L. cv. Garant) root elongation zones were investigated by means of a computer-controlled extensiometer. Creep closely followed a logarithmic time function, which was used to quantify creep activity. Pretreatment with auxin, which inhibits extension growth in roots, lowered the creep activity and the apparent plastic extensibility. While the time course of the inhibition of apparent plastic extensibility lagged behind the cessation of elongation growth, the drop in creep activity matched the growth inhibition more closely. Creep activity and apparent plastic extensibility were not significantly affected by pH. These data support the view that the auxin-induced cell wall stiffening (e.g. by cross-linking processes), while causal for the growth inhibition, is not brought about by a cell wall alkalinization.

Reinvestigation of auxin and fusicoccin stimulation of the plasma-membrane H(+)-ATPase activity

In vivo treatment of maize (Zea mays L.) coleoptile segments with auxin (indole-3-acetic acid; IAA) and fusicoccin (FC) followed by plasma-membrane isolation was used to characterize the effects of these treatments on the plasma-membrane H+-ATPase. Both IAA and FC increased H+ extrusion and elongation rate of the coleoptile segments, FC more strongly than IAA. Plasma membranes isolated after in-vivo treatment with FC showed a twofold stimulation of ATP hydrolysis and a several-fold stimulation of H+ pumping, whereas no effect was observed after IAA treatment, irrespective of whether the plasma membranes were prepared by two-phase partitioning or sucrose-gradient centrifugation. A more detailed investigation of the kinetic properties and pH dependence of the enzyme showed that FC treatment led to a twofold increase in Vmax, a decrease in Km for ATP from 1.5 mM to 0.24 mM, and a change in pH dependence resulting in increased activity at physiological pH levels. Again, IAA treatment showed no effects. Quantitation of the H+-ATPase by immunostaining using four different antibodies revealed no difference between IAA-and FC-treated material, and controls. From these data we conclude that (i) neither IAA nor FC gives rise to an increase in the amount of H+ -ATPase molecules in the plasma membrane that can be detected after membrane isolation, and (ii) if the H+-ATPase is activated by IAA, this activation is, in contrast to FC activation, not detectable after membrane isolation.

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt(diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H+ secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired indgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.

The temporal correlation of changes in apoplast pH and growth rate in maize coleoptile segments

Auxin induces extracellular acidification in growing shoot tissue. The causal relationship between this process and auxin-mediated growth is debated, partly because of contradicting previous reports on the temporal correlation of auxin-induced apoplast pH-drops and growth bursts. We have simultaneously measured both parameters on the background of spontaneously occurring endogenous changes in growth rate and apoplast pH in maize coleoptile segments. Our data demonstrate good temporal correlation, during both the ‘Spontaneous Growth Response’ and the response to exogenous auxin, which is transient under the conditions chosen due to rapid auxin metabolism. We suggest that cell wall pH and growth rate are co-regulated in this organ, and that contradictions in the literature might be due to technical difficulties.

Differential Transcript Expression of Wall-loosening Candidates in Leaves of Maize Cultivars Differing in Salt Resistance

Salt-sensitive crop plants such as maize (Zea mays L.) exhibit a strong and rapid growth reduction in response to NaCl stress. The unique salt-resistant maize hybrid SR03 and the salt-sensitive maize hybrid Lector provide good tools to characterize various genotypic responses to salinity in terms of shoot growth, shoot extensibility, and the expression pattern of wall-loosening candidates. The mRNA transcript levels of wall-loosening candidates such as xyloglucan endotransglucosylase (XET), endo-1,4-β-D-endoglucanase (EGase), α-expansins (EXPA), and the plasma membrane proton pump (PM-H+-ATPase) are correlated with cell-wall extensibility and with shoot growth under NaCl stress. We have found for the salt-sensitive maize that a decrease in the relative transcript abundance of ZmXET1, ZmEXPA1, and the composite PM-H+-ATPase mRNAs correlates with a decrease in wall extensibility and shoot growth. We suggest that this downregulation of wall-loosening candidates contributes to a reduction in extensibility and consequently in growth. In contrast, the decrease in wall extensibility is less strong in the salt-sensitive hybrid SR03. In the salt-resistant maize genotype, an upregulation of ZmXET1, ZmEXPA1 and PM-H+-ATPase transcripts possibly mitigates the salinity-induced decrease in wall extensibility and thus in shoot growth.

Growth: Progress in Auxin Research

Almost exactly 70 years ago, Frits Went (1928) characterized the first plant growth substance, auxin. Soon the natural auxin indole-3-acetic acid (IAA) was identified. Seven decades of attempts to clarify its mode of action followed. Although a bulk of data has been accumulated, most fundamental questions are still under intense debate: what protein is the growth-relevant auxin receptor, and where is it localized? What is the architecture of signalling chain leading to growth stimulation? There seems to be general agreement that auxin induces growth via cell wall loosening, but which molecular mechanism accounts for this? The state of auxin research after such a long time may be disappointing, especially if one considers that many plant physiologists - some of whom were very successful in other fields - have worked on the topic. It appears that auxin physiology is a “never-ending story”, or at least that the difficulties in solving the problem may have been generally underestimated.

Identification of auxins by a chemical genomics approach

Thirteen auxenic compounds were discovered in a screen of 10 000 compounds for auxin-like activity in Arabidopsis roots. One of the most potent substances was 2-(4-chloro-2-methylphenoxy)-N-(4-H-1,2,4-triazol-3-yl)acetamide (WH7) which shares similar structure to the known auxenic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). A selected set of 20 analogues of WH7 was used to provide detailed information about the structure–activity relationship based on their efficacy at inhibiting and stimulating root and shoot growth, respectively, and at induction of gene expression. It was shown that WH7 acts in a genetically defined auxin pathway. These small molecules will extend the arsenal of substances that can be used to define auxin perception site(s) and to dissect subsequent signalling events.

The auxin-induced K+ channel gene Zmk1 in maize functions in coleoptile growth and is required for embryo development

The transcript level and in turn protein density of the K+-uptake channel ZMK1 in maize (Zea mays) coleoptiles is controlled by the phytohormone auxin. ZMK1 is involved in auxin-regulated coleoptile elongation as well as gravi- and phototropism. To provide unequivocal evidence for the role of ZMK1 in these elementary processes we screened for maize plants containing a Mutator-tagged Zmk1 gene. In a site-selected approach, we were able to identify three independent alleles of Mutator-transposon insertions in Zmk1. zmk1-m1::Mu1 plants were characterised by a Mu1 transposon inside intron 1 of ZMK1. When we analysed the Zmk1-transcript abundance in growing coleoptiles of these homozygous mutants, however, we found the K+-channel allele overexpressed. In consequence, elevated levels of K+-channel transcripts resulted in a growth phenotype as expected from more efficient K+-uptake, representing a central factor for turgor formation. Following Zmk1 expression during maize embryogenesis, we found this K+-channel gene constitutively expressed throughout embryo development and upregulated in late stages. In line with a vital role in embryogenesis, the mutations of exon 2 and intron 2 of Zmk1-zmk1-m2::Mu8 and zmk1-m3::MuA2-caused a lethal, defective-kernel phenotype. Thus, these results demonstrate the central role of the auxin-regulated K+-channel gene Zmk1 in coleoptile growth and embryo development.