Inhibition of auxin transport and auxin signaling and treatment with far red light induces root coiling in the phospholipase-A mutant ppla-I-1. Significance for surface penetration?

When grown on a non-penetretable at a surface angle of 45°, Arabidopsis roots form wave-like structures and, in wild type rarely, but in certain mutants the tip root even may form circles. These circles are called coils. The formation of coils depends on the complex interaction of circumnutation, gravitropism and negative thigmotropism where - at least - gravitropism is intimately linked to auxin transport and signaling. The knockout mutant of patatin-related phospholipase-AI-1 (pplaI-1) is an auxin-signaling mutant which forms moderately increased numbers of coils on tilted agar plates. We tested the effects of the auxin efflux transport inhibitor NPA (1-naphthylphtalamic acid) and of the influx transport inhibitor 1-NOA (1-naphthoxyacetic acid) which both further increased root coil formation. The pPLAI-1 inhibitors HELSS (haloenol lactone suicide substrate=E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one) and ETYA (eicosatetraynoic acid) which are auxin signaling inhibitors also increased coil formation. In addition, far red light treatment increased coil formation. The results point out that a disturbance of auxin transport and signaling is one potential cause for root coils. As we show that the mutant pplaI-1 penetrates horizontal agar plates better than wild type plants root movements may help penetrating the soil.

Increased endocytosis of fluorescent phospholipid in tobacco pollen in microgravity and inhibition by verapamil.

Gravity sensing in plants occurs in specialised tissues, like in the columella in root tips or the endodermis in shoots. Generally, dense organelles, acting as statoliths, are thought to interact with the cytosekeleton and ion channels in gravitropism. We examined the possibility that tobacco pollen tubes (Nicotiana sylvestris) having an elaborate cytoskeleton could perceive gravity through interaction of the cytoskeleton and the endomembrane system and organelles. Using lipid endocytosis as a quantitative parameter, we show that endocytosis is increased transiently in microgravity within 3 min. This increase is inhibited by the calcium blocker verapamil, suggesting that calcium is lowered in the tip, which is known to increase endocytosis in the pollen tube.

Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity.

Halotolerance is enhanced in carrot callus by sensing hypergravity: influence of calcium modulators and cytochalasin D.

Carrot callus was centrifuged at 10 g and compared to callus growing at 1 g on agar in the presence of increasing sodium chloride concentrations. Growth after 14 days was enhanced in the centrifuged samples versus samples kept at 1 g. This effect was not found when the samples were grown on potassium chloride. At 50 mM NaCl, the calcium ionophore ionomycin was applied to centrifuged and noncentrifuged callus samples. In both experiments, the growth of callus increased with increasing ionomycin concentrations but under 10 g this increase was more enhanced. As inhibitors of calcium influx, lanthanum and gadolinium chloride were chosen in the presence of 50 mM NaCl. Both inhibitors inhibited growth at 1 g at low concentrations of around 2 microM, whereas the centrifuged samples were not or much less so inhibited. We tested an involvement of actin by application of cytochalasin D to callus grown in the presence of 50 mM NaCl. In both types of samples, growth at 1 g and growth at 10 g, cytochalasin D enhanced growth but the effect was clearly stronger at 10 g than at 1 g. As increased halotolerance was only observed in the presence of increased sodium ions, not potassium ions, and as halotolerance is known to be induced by an influx of calcium, the data suggest that a calcium influx induced by hypergravity and possibly modulated by actin caused the observed increase in halotolerance at 10 g.

A cell wall protein down-regulated by auxin suppresses cell expansion in Daucus carota (L.).

We investigated the function of the auxin-regulated cell wall gene DC 2.15, a member of a small gene family, present in Daucus carota (L.) and other plants. Cultured cells derived from carrot hypocotyls transformed by the DC 2.15 cDNA in antisense direction were ten-fold longer than wild-type cells, indicating a function of the corresponding protein in suppression of cell expansion. The analysis of carrot plants expressing the DC2.15 gene in antisense direction showed that the corresponding protein and/or related proteins probably are involved in leaf and vascular bundle development. The antisense plants generally displayed a retarded growth phenotype and delayed greening in comparison to wild-type plants. The asymmetric architecture of the wild-type leaves was degenerated in the DC 2.15 antisense plants and the leaves showed a torsion within and along their major vein. The vascular bundles showed a lowered ratio of the phloem/xylem area in cross sections of the leaf middle vein whereas the bundle sheath and the cambium showed no obvious phenotype. Expression of a promoter-GUS construct was found primarily in vascular bundles of stems, leaves and in the nectar-producing flower discs. The observed pleiotropic antisense phenotype indicates, by loss of function, that one or several related cell wall proteins of this gene family are necessary to realize several complex developmental processes.