A Conditional Mutation in SCD1 Reveals Linkage Between PIN Protein Trafficking, Auxin Transport, Gravitropism, and Lateral Root Initiation.

Auxin is transported in plants with distinct polarity, defined by transport proteins of the PIN-formed (PIN) family. Components of the complex trafficking machinery responsible for polar PIN protein localization have been identified by genetic approaches, but severe developmental phenotypes of trafficking mutants complicate dissection of this pathway. We utilized a temperature sensitive allele of Arabidopsis thaliana SCD1 (stomatal cytokinesis defective1) that encodes a RAB-guanine nucleotide exchange factor. Auxin transport, lateral root initiation, asymmetric auxin-induced gene expression after gravitropic reorientation, and differential gravitropic growth were reduced in the roots of the scd1-1 mutant relative to wild type at the restrictive temperature of 25°C, but not at the permissive temperature of 18°C. In scd1-1 at 25°C, PIN1- and PIN2-GFP accumulated in endomembrane bodies. Transition of seedlings from 18 to 25°C for as little as 20 min resulted in the accumulation of PIN2-GFP in endomembranes, while gravitropism and root developmental defects were not detected until hours after transition to the non-permissive temperature. The endomembrane compartments that accumulated PIN2-GFP in scd1-1 exhibited FM4-64 signal colocalized with ARA7 and ARA6 fluorescent marker proteins, consistent with PIN2 accumulation in the late or multivesicular endosome. These experiments illustrate the power of using a temperature sensitive mutation in the gene encoding SCD1 to study the trafficking of PIN2 between the endosome and the plasma membrane. Using the conditional feature of this mutation, we show that altered trafficking of PIN2 precedes altered auxin transport and defects in gravitropism and lateral root development in this mutant upon transition to the restrictive temperature.

SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis.

To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome.

Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na+,K+-ATPase in rats.

This study demonstrated that chronic aspartame consumption in rats can lead to altered T-maze performance and increased muscarinic cholinergic receptor densities in certain brain regions. Control and treated rats were trained in a T-maze to a particular side and then periodically tested to see how well they retained the learned response. Rats that had received aspartame (250 mg/kg/day) in the drinking water for 3 or 4 months showed a significant increase in time to reach the reward in the T-maze, suggesting a possible effect on memory due to the artificial sweetener. Using [(3)H]quinuclidinyl benzilate (QNB) (1 nM) to label muscarinic cholinergic receptors and atropine (10(-6) M) to determine nonspecific binding in whole-brain preparations, aspartame-treated rats showed a 31% increase in receptor numbers when compared to controls. In aspartame-treated rats, there was a significant increase in muscarinic receptor densities in the frontal cortex, midcortex, posterior cortex, hippocampus, hypothalamus and cerebellum of 80%, 60%, 61%, 65%, 66% and 60%, respectively. The midbrain was the only area where preparations from aspartame-treated rats showed a significant increase in Na(+),K(+)-ATPase activity. It can be concluded from these data that long-term consumption of aspartame can affect T-maze performance in rats and alter receptor densities or enzymes in brain.