Does aluminium affect root growth of maize through interaction with the cell wall – plasma membrane – cytoskeleton continuum?

The mechanism of aluminium-induced inhibition of root elongation is still not well understood. It is a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic. The present paper summarises experimental evidence which offers new avenues in the understanding of Al toxicity and resistance in maize. Application of Al for 1 h to individual 1 mm sections of the root apex only inhibited root elongation if applied to the first 3 apical mm. The most Al-sensitive apical root zone appeared to be the 1–2 mm segment. Aluminium-induced prominent alterations in both the microtubular (disintegration) and the actin cytoskeleton (altered polymerisation patterns) were found especially in the apical 1–2 mm zone using monoclonal antibodies. Since accumulation of Al in the root apoplast is dependent on the properties of the pectic matrix, we investigated whether Al uptake and toxicity could be modulated by changing the pectin content of the cell walls through pre-treatment of intact maize plants with 150 mM NaCl for 5 days. NaCl-adapted plants with higher pectin content accumulated more Al in their root apices and they were more Al-sensitive as indicated by more severe inhibition of root elongation and enhanced callose induction by Al. This special role of the pectic matrix of the cell walls in the modulation of Al toxicity is also indicated by a close positive correlation between pectin, Al, and Al-induced callose contents of 1 mm root segments along the 5 mm root apex. On the basis of the presented data we suggest that the rapid disorganisation of the cytoskeleton leading to root growth inhibition may be mediated by interaction of Al with the apoplastic side of the cell wall – plasma membrane – cytoskeleton continuum. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of callose formation is a sensitive marker for genotypic aluminium sensitivity in maize

The screening of 37 Zea mays L. cultivars in nutrient solution using root elongation (24 h) as a parameter showed large genotypic differences in Al resistance among the genetic material evaluated.Callose concentrations in root tips were closely and positively related to Al-induced inhibition of root elongation. Therefore, Al-induced callose formation in root tips appears to be an excellent indicator of Al injury and can be used as a selection criteria for Al sensitivity. In contrast, aluminium concentrations in root tips were not related to Al-induced inhibition of root elongation, nor to Al-induced callose formation. Callose formation was also induced by short-term A1 treatment in root tip protoplasts, and the response of protoplasts clearly reflected the cultivar-specific response to Al of intact roots. This indicates that in maize, Al sensitivity is expressed on the protoplast level.     

Effect of aluminium on the activity of apoplastic acid phosphatase and the exudation of macromolecules by roots and suspension-culture cells ofZea mays L.

Aluminium accumulates predominantly in the root apoplast where it binds to the pectin matrix of the cell wall with its negative charges. In this study, we investigated whether short-term Al treatment (2 h) affects the activity of apoplastic acid phosphatase and the exudation of macromolecules by roots and suspension-culture cells of Zea mays L. The pectin content of the cell cultures was modified by long-term adaptation to NaCl stress or long-term adaptation to the cellulose-synthesis inhibitor 2,6-dichlorbenzonitrile (DCB), and by short-term treatment for up to 15 min with pectolyase. At pH 4.5, neither acid phosphatase activity of commercial enzyme preparations nor of exudates from root-tips and suspension-cells of Zea mays L. were affected directly by Al. However, the exudation and the activity of apoplastic acid phosphatase was reduced to a greater extent by Al cells with high pectin content than in cells with normal pectin content. The strongest reduction of acid phosphatase exudation was observed in pectolyase-treated cells with the lowest pectin content. Al reduced not only the release of acid phosphatase from the suspension cells, but also the release of total proteins and pectins. However, no relationship existed between the magnitude of Al-induced reduction of protein and pectin release and the cell pectin contents. These results support the assumption that Al modifies cell-wall and plasma-membrane transport-properties for macromolecules and the activity of apoplastic enzymes thus modifying Al sensitivity.     

Pectin methylesterase modulates aluminium sensitivity in Zea mays and Solanum tuberosum

Cell suspension cultures of Zeamays L. were adapted to grow under conditions of NaCl stress, which increased the cell‐wall pectin content of these cells by 31% compared with unadapted cells (controls). Both cultures were treated for 5 or 10 min with pectin methylesterase (PME) and afterwards incubated in the presence of Al for 2 h. The different capabilities of the cells to synthesise callose due to pre‐treatment were taken into account by calculating relative Al‐induced callose induction (digitonin=100%). Only in salt‐adapted cells with a degree of methylation of cell‐wall pectin (DM) decreasing from 34% (control) to 13%, did PME treatment enhance total and BaCl₂‐non‐exchangeable Al contents and Al sensitivity as indicated by increased callose formation. In a further step, a wider variation in DM was achieved by subculturing the NaCl‐adapted cells for up to 3 weeks without NaCl supply and adapting them to the cellulose‐synthesis inhibitor 2,6‐dichlorbenzonitrile (DCB). This reduced DM to 26%, while short‐term treatment with pectolyase resulted in the lowest DM (12%). After the 2 h Al treatment, there was a close negative relationship between DM and relative callose formation of Al contents, with the exception of pectolyase‐treated cells. In addition, intact plants of Solanumtuberosum L. genotypes were characterised for their Al sensitivity in hydroponics using root elongation, Al‐induced callose formation and Al contents of root tips as parameters. Based on all three parameters, the transgenic potato mutant overexpressing PME proved to be more Al‐sensitive than the wild type, the Al‐resistant and even the Al‐sensitive potato cultivar. Especially in the root tips (1 cm), Al treatment (2 h, 50 μM) increased the activity of PME more in the Al‐sensitive than in the Al‐resistant genotypes. The presented data emphasise the importance of the DM of the pectin matrix and the activity of PME for the expression of Al toxicity and Al resistance.     

Protein–protein‐interactions in a multiplexed,miniaturized format a functional analysis of Rho GTPase activation and inhibition

A miniaturized, bead‐based protein–protein‐interaction assay was developed to study the interaction of Rho GTPases with regulatory proteins. The setup, which uses only minute amounts of sample, was used to analyze small molecules that inhibit the interaction between Rho GTPases and RhoGDIα. Prenylcysteine analogues and the replacement of GDP by non‐hydrolysable GTP analogues prevented the formation of Rho GTPase‐RhoGDIα complexes in a concentration‐dependent manner.     

Cell wall pectin content modulates aluminium sensitivity of Zea mays (L.) cells grown in suspension culture

The pectin content of the cell walls of maize suspension cells was modified to investigate its role in the expression of aluminium (Al) toxicity. Long‐term adaptation to NaCl or to the cellulose synthesis inhibitor 2,6‐dichlorbenzonitirle (DCB) increased the pectin content by 31 and 86%, respectively. Subculturing salt‐adapted cells for up to 3 weeks without NaCl supply or treatment of cells with pectolyase for up to 15 min reduced pectin contents by up to 46%. Such pre‐cultured cells were incubated for 2 h in presence of Al. There was a close positive correlation between pectin and both total and BaCl₂ non‐exchangeable Al contents. Aluminium‐induced callose formation as an indicator of Al injury was closely positively correlated to the loss of cell viability. In NaCl‐adapted and pectolyase‐treated cells, Al‐induced callose formation was reduced when compared with normal cells. However, there was a close positive relationship between pectin contents and relative callose induction (digitonin‐induced callose formation, reflecting the different capabilities of cells to synthesize callose set to 100%) indicating that cells with higher pectin contents are more Al‐sensitive. The results presented support our view that the binding of Al to the cell wall pectin‐matrix represents an important step in the expression of Al toxicity.