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Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

The KNAT2 (for KNOTTED-like from Arabidopsis thaliana 2) homeobox gene is expressed in the vegetative apical meristem. It is also active during flower development, suggesting a function in the structuring of flowers. To investigate its role, we used a DEXAMETHASONE (DEX)-inducible system to generate transgenic plants that overexpressed a fusion of KNAT2 with the hormone binding domain of the glucocorticoid receptor. DEX-induced plants were similar to plants overexpressing the closely related KNAT1 gene, indicating overlapping functions, although we observed differences as well. In particular, KNAT2-GR activation induced ectopic carpel features. First, KNAT2 induced the homeotic conversion of nucellus into carpel-like structures. Second, KNAT2 induced stigmatic papillae on rosette leaves in the ap2-5 background. Third, ectopic expression of the carpel identity gene AGAMOUS (AG) was observed in carpels and ovules. Interestingly, the homeotic conversion was not dependent on AG activity, because it was maintained in the ag-1 ap2-5 double mutant. Therefore, our data indicate that KNAT2 also must activate other carpel regulators. Together, these results suggest that KNAT2 plays a role in carpel development.

The process of organ positioning has been addressed, using the pin-formed 1 (pin1) mutant as a tool. PIN1 is a transmembrane protein involved in auxin transport in Arabidopsis. Loss of function severely affects organ initiation, and pin1 mutants are characterised by an inflorescence meristem that does not initiate any flowers, resulting in the formation of a naked inflorescence stem. This phenotype, combined with the proposed role of PIN1 in hormone transport, makes the mutant an ideal tool to study organ formation and phyllotaxis, and here we present a detailed analysis of the molecular modifications at the shoot apex caused by the mutation. We show that meristem structure and function are not severely affected in the mutant. Major alterations, however, are observed at the periphery of the pin1 meristem, where organ initiation should occur. Although two very early markers of organ initiation, LEAFY and AINTEGUMENTA, are expressed at the periphery of the mutant meristem, the cells are not recruited into distinct primordia. Instead a ring-like domain expressing those primordium specific genes is observed around the meristem. This ring-like domain also expresses a boundary marker, CUP-SHAPED COTYLEDON 2, involved in organ separation, showing that the zone at the meristem periphery has a hybrid identity. This implies that PIN1 is not only involved in organ outgrowth, but that it is also necessary for organ separation and positioning. A model is presented in which PIN1 and the local distribution of auxin control phyllotaxis.