In shoots of the garden pea (Pisum sati7um L.), the main (dwarf) mutants, compared with the WT. Metabolism experibioactive gibberellin (GA) is GA 1 , which is synthesised from ments did not provide convincing evidence for feedback regulation of 20-oxidation, possibly because GA 19 appears to be GA 20 by 3-hydroxylation. Gibberellin A 20 is produced from GA 19 , as part of the process known as GA 20-oxidation. metabolised rapidly, even in WT pea shoots. Both 3-hydroxylase and 20-oxidase transcript levels were markedly higher in Because these steps are thought to be negatively regulated by GA 1 , we compared the metabolism of labelled GA 19 and GA 20 the mutants than in isogenic WT lines. The results support previous suggestions that both biosynthetic steps are feedback-in mutants deficient in GA 1 , with that observed in isogenic wild-type (WT) plants. There was a large and specific increase regulated by GA 1 in pea. in the 3-hydroxylation of labelled GA 20 in the GA 1 -deficient

We report the first crystal structure of a plant (Pisum sativum L. cv Oregon sugarpod) mitochondrial nucleoside diphosphate kinase. Similar to other eukaryotic nucleoside diphosphate kinases, the plant enzyme is a hexamer; the six monomers in the asymmetric unit are arranged as trimers of dimers. Different functions of the kinase have been correlated with the oligomeric structure and the phosphorylation of Ser residues. We show that the occurrence of Ser autophosphorylation depends on enzymatic activity. The mutation of the strictly conserved Ser-119 to Ala reduced the Ser phosphorylation to about one-half of that observed in wild type with only a modest change of enzyme activity. We also show that mutating another strictly conserved Ser, Ser-69, to Ala reduces the enzyme activity to 6% and 14% of wild-type using dCDP and dTDP as acceptors, respectively. Changes in the oligomerization pattern of the S69A mutant were observed by cross-linking experiments. A reduction in trimer formation and a change in the dimer interaction could be detected with a concomitant increase of tetramers. We conclude that the S69 mutant is involved in the stabilization of the oligomeric state of this plant nucleoside diphosphate kinase.Nucleoside diphosphate kinases (NDPKs) are ubiquitous enzymes involved in equilibration of the cellular nucleoside triphosphate (NTP, dNTP) pools. They transfer phosphate groups from NTPs to nucleoside diphosphates in the presence of divalent cations, preferably Mg 21 . The reaction involves the formation of a covalent intermediate, whereby the enzyme is phosphorylated at the catalytic His residue. NDPKs have broad substrate specificity and can use both ribo-and deoxyribonucleotides of purines or pyrimidines (Parks et al., 1973). NDPK isoforms can be found within most cellular compartments in eukaryotes. There are eight isoforms in humans and four annotated isoforms in the Arabidopsis genome.Additional roles for NDPKs in processes other than basic metabolism have emerged. This was first observed when decreased expression levels of a nonmetastasis protein, Nm23-H1, correlated with reduced metastasis in certain cancers (Steeg et al., 1988). Nm23-H1 was subsequently revealed to be a NDPK. NDPKs are now thought to be involved in processes such as control of cell proliferation (Cipollini et al., 1997), regulation of transcription (Postel et al., 1993;Ji et al., 1995), and protein phosphotransferase activities in humans and fungi (Engel et al., 1995;Wagner and Vu, 2000;Ogura et al., 2001). Plant NDPKs are also involved in intracellular signaling processes, including phytochrome A response (Choi et al., 1999), UV-B light signaling (Zimmermann et al., 1999), and hormone response (Nato et al., 1997;Novikova et al., 1999). The plant mitochondrial isoform has been implicated in heat-stress response (Escobar Galvis et al., 2001) and cAMP signaling (Knorpp and Håkansson, 1998;Laukens et al., 2001).NDPKs share primary, secondary, and tertiary structural similarity but differ in their quaternary structure. Eukaryotic N...

The gibberellin (GA) economy of young pea (Pisum sativum L.) fruits was investigated using a range of mutants with altered GA biosynthesis or deactivation. The synthesis mutation lh-2 substantially reduced the content of both GA 4 and GA 1 in young seeds. Among the other synthesis mutations, ls-1, le-1 and le-3, the largest reduction in seed GA 1 content was only 1.7-fold (le-1), while GA 4 was not reduced in these mutants, and in fact accumulated in some experiments (compared with the wild type). Mutation sln appeared to block the step GA 20 to GA 29 in young pods and seeds, but not as strongly as in older seeds. Mutations ls-1, le-1 and le-3 markedly reduced pod GA 1 levels, but pod elongation was not aected. After feeds of [ 13 C, 3 H]GA 20 to leaves, the pods contained 13 C, 3 H-labelled GA 20 , GA 1 , GA 29 and GA 81 , and the seeds, [ 13 C, 3 H]GA 20 and [ 13 C, 3 H]GA 29 . These ®ndings are discussed in relation to recent suggestions regarding the role and origin of GA 1 in pea fruits.

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We describe a new mutation, lrs, which reduces internode length in Pisum sativum L. The mutation appears to act by reducing both GA synthesis and the response to GA1. The levels of the 13‐hydroxylated GAs, GA53, GA44, GA19, GA20, GA1, and GA8 in the lrs mutant were greatly reduced compared with the wild‐type. The extent of the reduction in GA1 content in the apical tissues would, at least in part, account for the dwarf phenotype of the mutant. The reduced GA responsiveness of the new mutant was indicated by the inability of applied GA1 to remove the difference in elongation between lrs and LRS plants. The lrs mutant appears to be unique amongst internode length genotypes, possessing characteristics of both GA synthesis and GA response mutants.