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The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, γ-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.

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SummaryA novel Arabidopsis thaliana mutant, named hoc, was found to have an high organogenic capacity for shoot regeneration. The HOC locus may be involved in cytokinin metabolism leading to cytokininoverproduction. In vitro, hoc root explants develop many shoots in the absence of exogenous growth regulators. The mutant displays a bushy phenotype with supernumerary rosettes and with normal phyllotaxy, resulting from precocious axillary meristem development. Genetic and molecular analyses show that the high shoot regeneration and the bushy phenotype are controlled by a recessive single gene, located on chromosome I, next to the GAPB CAPS marker. The mapping data and allelism tests reveal that the hoc mutant is not allelic to other reported Arabidopsis growth-regulator mutants. In darkness the hoc mutant is de-etiolated, with a short hypocotyl, opened cotyledons and true leaves. Growth regulator assays reveal that the mutant accumulates cytokinins at about two-and sevenfold the cytokinin level of wild-type plants in its aerial parts and roots, respectively. Consequently, the elevated amounts of endogenous cytokinins in hoc plants are associated with high organogenic capacity and hence bushy phenotype. Thus hoc is the ®rst cytokinin-overproducing Arabidopsis mutant capable of auto-regenerating shoots without exogenous growth regulators.

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of alpha-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression.

It is usually assumed that aquaporins present in the cellular membranes could be an important route in the control of water flux in plants, but evidence for this hypothesis is scarce. In this paper, we report measurements of the osmotic permeability (P(os) of protoplasts isolated from hypocotyls of wild-type and mutant Arabidopsis thaliana (L.) Heynh. Mutants were affected in their growth and exhibited different sensitivities to the phytohormone, brassinolide. For the two mutants studied (cpd: constitutive photomorphogenesis and dwarfism; bril: brassinosteroid insensitive), hypocotyl length was correlated to P(os) for the protoplasts. Under experimental conditions where hypocotyl growth had ceased, restoration of root, hypocotyl and petiole growth by brassinolide was correlated with an increase in P(os) of the hypocotyl protoplasts. We consider that the increase in Pos of the hypocotyl cells was needed because these cells were part of the transcellular water pathway of the plant. This is the first time, to our knowledge, that brassinolide has been shown to be involved in the modification of the water-transport properties of cell membranes. Our results also emphasize the importance of aquaporins and the transcellular pathway in water transport under normal growth conditions.

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Although cell elongation is a basic function of plant morphogenesis, many of the molecular events involved in this process are still unknown. In this work an extremely dwarf mutant, originally named bul, was used to study one of the main processes of plant development, cell elongation. Genetic analyses revealed that the BUL locus was linked to the nga172 marker on chromosome 3. Recently, after mapping the new dwf7 mutation of Arabidopsis, which is allelic to ste1, it was reported that dwf7 is also linked to the same marker. Sterol analyses of the bull-1 mutant indicated that bul1-1 is defective in the delta 7-sterol-C5-desaturation step leading to brassinosteroid biosynthesis. Considering these findings, we designated our bul mutant as bul1-1/dwf7-3/ste1-4. The bul1-1 mutant was characterized by a very dwarf phenotype, with delayed development and reduced fertility. The mutant leaves had a dark-green colour, which was probably due to continuous stomatal closure. The bul1-1 mutant showed a partially de-etiolated phenotype in the dark. Cellular characterization and rescue experiments with brassinosteroids demonstrated the involvement of the BUL1-1 protein in brassinosteroid-dependent plant growth processes.