Effect of sulfur availability on the integrity of amino acid biosynthesis in plants.

Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.

Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants.

In many higher plants, cellulose synthesis is inhibited by isoxaben and thiazolidinone herbicides such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone. Semidominant mutations at the IXR1 and IXR2 loci of Arabidopsis confer isoxaben and thiazolidinone resistance. Isolation of the IXR1 gene by map-based cloning revealed that it encodes the AtCESA3 isoform of cellulose synthase. The two known mutant alleles contain point mutations that replace glycine 998 with aspartic acid, and threonine 942 with isoleucine, respectively. The mutations occur in a highly conserved region of the enzyme near the carboxyl terminus that is well separated from the proposed active site. Although the IXR1 gene is expressed in the same cells as the structurally related RSW1 (AtCESA1) cellulose synthase gene, these two CESA genes are not functionally redundant.

Positional cloning in Arabidopsis. Why it feels good to have a genome initiative working for you.

Positional (or map-based) cloning techniques are widely used to identify the protein products of genes defined by mutation. In Arabidopsis the information generated by the Genome Initiative is giving this approach a decisive boost. A wealth of sequence polymorphisms and molecular markers is now available and can be exploited for fine mapping with technically simple and robust polymerase chain reaction-based methods. As a result it has become possible to complete positional cloning projects in a short time and with relatively little effort.

The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.

The irregular xylem3 (irx3) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3, and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 (rsw1) plants, irx3 plants show no increase in the accumulation of beta-1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases.

Nitrate Acts as a Signal to Induce Organic Acid Metabolism and Repress Starch Metabolism in Tobacco.

Nia30(145) transformants with very low nitrate reductase activity provide an in vivo screen to identify processes that are regulated by nitrate. Nia30(145) resembles nitrate-limited wild-type plants with respect to growth rate and protein and amino acid content but accumulates large amounts of nitrate when it is grown on high nitrate. The transcripts for nitrate reductase (NR), nitrite reductase, cytosolic glutamine synthetase, and glutamate synthase increased; NR and nitrite reductase activity increased in leaves and roots; and glutamine synthetase activity increased in roots. The transcripts for phosphoenolpyruvate carboxylase, cytosolic pyruvate kinase, citrate synthase, and NADP-isocitrate dehydrogenase increased; phosphoenolpyruvate carboxylase activity increased; and malate, citrate, isocitrate, and [alpha]-oxoglutarate accumulated in leaves and roots. There was a decrease of the ADP-glucose pyrophosphorylase transcript and activity, and starch decreased in the leaves and roots. After adding 12 mM nitrate to nitrate-limited Nia30(145), the transcripts for NR and phosphoenolpyruvate carboxylase increased, and the transcripts for ADP-glucose pyrophosphorylase decreased within 2 and 4 hr, respectively. Starch was remobilized at almost the same rate as in wild-type plants, even though growth was not stimulated in Nia30(145). It is proposed that nitrate acts as a signal to initiate coordinated changes in carbon and nitrogen metabolism.

Identification and purification of translation initiation factor 2 (IF2) from Thermus thermophilus.

Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator RNA to the ribosomal P site. IF2 is a member of the GTP GDP-binding protein superfamily. In the extreme thermophilic bacterium Thermus thermophilus, IF2 was identified as a 66-kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2-mediated binding of formylmethionine-tRNA to 70S ribosomes. The N-terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25 kDa and 40 kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.

Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase.

Although nitrate reductase (NR. EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then decreased by 50-60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40-60% less NR protein and NRA after 3-4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.