The nitrate assimilatory pathway has been the matter of intensive genetic and molecular analysis over the past decade. Mutants impaired in the expression of nitrate reductase have been characterized in a number of plant species. Molecular analysis of the Nia gene coding for nitrate reductase has been the basis for a three‐domain model of the structure of the enzyme, in agreement with biochemical and genetic data. Mutagenesis and antisense strategies have led to the description of nitrite reductase deficiencies. The molecular analysis of the corresponding Nii genes has provided invaluable information on the structure of nitrite reductase. Recently, a gene involved in nitrate uptake has also been identified. The regulation of the nitrate assimilatory pathway has been investigated. Analysis of the regulation of the pathway at the molecular level has shown evidence for the involvement of nitrate, light and/or sucrose, and reduced nitrogen in the regulation. Surprisingly, no bona fide regulatory mutant specific to this pathway has been identified so far in higher plants. This may reflect the redundancy of regulatory genes. The deregulated expression of one or the other step of the pathway obtained by ectopic expression of the corresponding genes is a new approach to study the physiological role of these regulations. Elements of the pathway have also been successfully used as transposon traps, or negatively selectable markers for other purposes. Finally, the identification at the molecular level of regulatory genes and structural elements involved in transport and storage of nitrate, or in the biosynthesis of cofactors of nitrate and nitrite reductases, will be the goal of the next decade.
Xylanase A from the phytopathogenic bacterium Erwinia chrysanthemi is classified as a glycoside hydrolase family 30 enzyme (previously in family 5) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid β‐d‐xylopyranosyl‐(1→4)‐[4‐O‐methyl‐α‐d‐glucuronosyl‐(1→2)]‐β‐d‐xylopyranosyl‐(1→4)‐d‐xylose as a ligand. The crystal structure of the enzyme–ligand complex was solved at 1.39 Å resolution. The ligand xylotriose moiety occupies subsites −1, −2 and −3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guanidinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear β‐1,4‐xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate. Database Structural and experimental data are available in the Protein Data Bank database under accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2y24 [45].
Most organisms appear to have a molybdenum cofactor consisting of a complex of molybdenum and a pterin derivative. Very little is known about molybdenum cofactor biosynthesis in plants or other eukaryotes, because the instability of the cofactor and its precursors makes it difficult to analyze this pathway. We have isolated two cDNA clones from the higher plant Arabidopsis thaliana encoding genes involved in early steps of molybdenum cofactor biosynthesis. The cDNAs were obtained by functional complementation of two Escherichia coli mutants deficient in single steps of molybdenum cofactor biosynthesis. The two cDNAs, designated Cnx2 and Cnx3, encode proteins of 43 and 30 kDa, respectively. They have significant identity to the E. coli genes, moaA and moaC, involved in molybdenum cofactor biosynthesis. Both genes have N-terminal extensions that resemble targeting signals for the chloroplasts or the mitochondria. Import studies with the translated proteins and purified mitochondria and chloroplasts did not show import of these proteins to either of these organelles. Northern analysis show that Cnx2 is expressed in all organs and strongest in roots. Cnx3 is not expressed in abundant levels in any tissue but roots. For both genes there is no detectable difference in the expression level from plants grown with nitrate or with ammonium. The Cnx2 gene has been mapped to chromosome II. Southern analysis suggests that both genes exist as single copies in the genome.
We constructed and tested a Cre-loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed. The system developed here uses a heat-shock-inducible Cre to excise a DNA fragment flanked by lox sites, thereby generating a constitutive GUS reporter gene under control of the CaMV 35S promoter. Heat-shock-mediated excision of several, independent lines resulted in varying degrees of recombination-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over-expression, or under-expression by antisense, would otherwise affect embryo, seed or seedling viability.
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