Considerable progress has recently been made in understanding adventitious root formation using physiological studies. It is recognized that rooting is a process consisting of distinct phases, each with its own requirements. In this review, the successive phases in the rooting process are described and the possible roles of wounding-related compounds, auxin, ethylene and phenolic compounds during these specific phases are discussed. Recent results are assisting the development of advanced rooting treatments. Molecular studies on rooting are underway and will be essential in revealing the mechanisms underlying adventitious root formation.

Abstract. Gitelman's syndrome is an autosomal recessive renal tubular disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. This disorder results from mutations in the thiazide-sensitive NaCl cotransporter (NCC). To elucidate the functional implications of mutations associated with this disorder, metolazone-sensitive 22 Na ϩ uptake, subcellular localization, and glycosidase-sensitive glycosylation of human NCC (hNCC) were determined in Xenopus laevis oocytes expressing FLAG-tagged wild-type or mutant hNCC. Injection of 10 ng of FLAG-tagged hNCC cRNA resulted in metolazone-sensitive 22

Hereditary primary hypomagnesemia comprises a clinically and genetically heterogeneous group of disorders in which hypomagnesemia is due to either renal or intestinal Mg(2+) wasting. These disorders share the general symptoms of hypomagnesemia, tetany and epileptiformic convulsions, and often include secondary or associated disturbances in calcium excretion. In a large Dutch family with autosomal dominant renal hypomagnesemia, associated with hypocalciuria, we mapped the disease locus to a 5.6-cM region on chromosome 11q23. After candidate screening, we identified a heterozygous mutation in the FXYD2 gene, encoding the Na(+),K(+)-ATPase gamma-subunit, cosegregating with the patients of this family, which was not found in 132 control chromosomes. The mutation leads to a G41R substitution, introducing a charged amino acid residue in the predicted transmembrane region of the gamma-subunit protein. Expression studies in insect Sf9 and COS-1 cells showed that the mutant gamma-subunit protein was incorrectly routed and accumulated in perinuclear structures. In addition to disturbed routing of the G41R mutant, Western blot analysis of Xenopus oocytes expressing wild-type or mutant gamma-subunit showed mutant gamma-subunit lacking a posttranslational modification. Finally, we investigated two individuals lacking one copy of the FXYD2 gene and found their serum Mg(2+) levels to be within the normal range. We conclude that the arrest of mutant gamma-subunit in distinct intracellular structures is associated with aberrant posttranslational processing and that the G41R mutation causes dominant renal hypomagnesemia associated with hypocalciuria through a dominant negative mechanism.

The thiazide-sensitive NaCl cotransporter (NCC) is responsible for the reabsorption of 5% of the filtered load of NaCl in the kidney. Mutations in NCC cause Gitelman syndrome. To gain insight into its regulation, detailed information on the structural composition of its functional unit is essential. Western blot analysis of total membranes of Xenopus laevis oocytes heterologously expressing FLAG-tagged NCC revealed the presence of both complex-(140-kDa) and core (100-kDa)-glycosylated monomers and a broad band of high molecular mass (250 -350-kDa) complexes. Chemical cross-linking with dithiobispropionimidate eliminated the low molecular weight bands and increased the intensity of the high molecular weight bands, indicating that NCC is present in multimeric complexes. Co-expression of HAand FLAG-tagged NCC followed by co-immunoprecipitation demonstrated that these multimers contained at least two complex-glycosylated NCC proteins. The dimeric nature of the multimers was further substantiated by sucrose gradient centrifugation yielding a peak of ϳ310 kDa. A concatameric construct of two NCC polyproteins exhibited significant 22 Na ؉ uptake, indicating that the transporter is functional as a homodimer. A concatamer of partially retarded G980R-and wild type (wt)-NCC displayed normal Na ؉ transport. This demonstrates that G980R-NCC, provided that it reaches the surface, is fully active and that wt-NCC is dominant in its association with this mutant. Conversely a concatamer of fully retarded G741R-and wt-NCC did not reach the cell surface, showing that wt-NCC is recessive in its association with this mutant. Oocytes co-expressing G741R-and wt-NCC did not show G741R staining at the plasma membrane, whereas Na ؉ transport was normal, indicating that wt-NCC dimerizes preferentially with itself. The results are discussed in relation to the recessive nature of NCC mutants in Gitelman syndrome.

Abstract. The thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC), which is expressed on the apical membrane of epithelial cells lining the distal convoluted tubule, is responsible for the reabsorption of 5% to 10% of filtered Na ϩ and Cl Ϫ . To date, functional studies on the structural and regulatory requirements for localized trafficking and ion-transporting activity of NCC have been hampered by lack of a suitable cell system expressing this cotransporter. Reported here is the functional expression of human NCC (hNCC) in a polarized mammalian cell of renal origin-that is, the high-resistance Madin-Darby canine kidney (MDCK) cell. Western blot testing revealed that the cells predominantly expressed the complex glycosylated (approximately 140 kD) form of hNCC. hNCC was present primarily in the apical part of the cell. The functionality of hNCC was demonstrated by the gain of thiazide-sensitive Na ϩ uptake and transepithelial transport activity. Na ϩ uptake was significantly increased after short-term (15 min) treatment with forskolin, whereas cyclic guanosine monophosphate, wortmannin, phorbol 12-myriatate 13-acetate, and staurosporine were without effect. This indicates that hNCC activity is regulated through cyclic adenosine monophosphate, rather than via cyclic guanosine monophosphate, phosphoinositide 3-kinases or protein kinase C. Aldosterone did not alter Na ϩ uptake in the short term (15 min) but significantly increased the transport activity in the long term (16 h). The latter effect of aldosterone was due to an effect on the cytomegalovirus promoter/enhancer driving the expression of hNCC. hNCC-MDCK cells are a good model for the study of the regulation of apical trafficking and ion-transporting activity of hNCC.