NAD-dependent isocitrate dehydrogenase mutants of Arabidopsis suggest the enzyme is not limiting for nitrogen assimilation

NAD-dependent isocitrate dehydrogenase (IDH) is a tricarboxylic acid cycle enzyme that produces 2-oxoglutarate, an organic acid required by the glutamine synthetase/glutamate synthase cycle to assimilate ammonium. Three Arabidopsis (Arabidopsis thaliana) IDH mutants have been characterized, corresponding to an insertion into a different IDH gene (At5g03290, idhv; At4g35260, idhi; At2g17130, idhii). Analysis of IDH mRNA and protein show that each mutant lacks the corresponding gene products. Leaf IDH activity is reduced by 92%, 60%, and 43% for idhv, idhi, and idhii, respectively. These mutants do not have any developmental or growth phenotype and the reduction of IDH activity does not impact on NADP-dependent isocitrate dehydrogenase activity. Soil-grown mutants do not exhibit any alterations in daytime sucrose, glucose, fructose, citrate, ammonium, and total soluble amino acid levels. However, gas chromatography-mass spectrometry metabolic profiling analyses indicate that certain free amino acids are reduced in comparison to the wild type. These data suggest that IDH activity is not limiting for tricarboxylic acid cycle functioning and nitrogen assimilation. On the other hand, liquid culture-grown mutants give a reduced growth phenotype, a large increase in organic acid (citrate is increased 35-fold), hexose-phosphate, and sugar content, whereas ammonium and free amino acids are moderately increased with respect to wild-type cultures. However, no significant changes in 2-oxoglutarate levels were observed. Under these nonphysiological growth conditions, pyridine nucleotide levels remained relatively constant between the wild-type and the idhv line, although some small, but significant, alterations were measured in idhii (lower NADH and higher NADPH levels). On the other hand, soil-grown idhv plants exhibited a reduction in NAD and NADPH content.     

alpha-Aminoadipate as a primary nitrogen source for Saccharomyces cerevisiae mutants.

In contrast to wild-type strains of the yeast Saccharomyces cerevisiae, lys2 and lys5 mutants are able to utilize alpha-aminoadipate as a primary source of nitrogen. Chattoo et al. (B. B. Chattoo, F. Sherman, D. A. Azubalis, T. A. Fjellstedt, D. Mehnert, and M. Ogur, Genetics 93:51-65, 1979) relied on this difference in the effective utilization of alpha-aminoadipate to develop a procedure for directly selecting lys2 and lys5 mutants. In this study we used a range of mutant strains and various media to determine why normal strains are unable to utilize alpha-aminoadipate as a nitrogen source. Our results demonstrate that the anabolism of high levels of alpha-aminoadipate through the biosynthetic pathway of lysine results in the accumulation of a toxic intermediate and, furthermore, that lys2 and lys5 mutants contain blocks leading to the formation of this intermediate.     

A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes

This article provides detailed protocols for collecting pollen and outlines genetic crosses and phenotypic assays that are useful for characterizing mutants that affect pollen development.     

Implications of nitrogen phloem loading for carbon metabolism and transport during Arabidopsis development

Metabolite transport processes and primary metabolism are highly interconnected. This study examined the importance of source-to-sink nitrogen partitioning, and associated nitrogen metabolism for carbon capture, transport and usage. Specifically, Arabidopsis aap8(AMINO ACID PERMEASE 8) mutant lines were analyzed to resolve the consequences of reduced amino acid phloem loading for source leaf carbon metabolism,sucrose phloem transport and sink development during vegetative and reproductive growth phase. Results showed that decreased amino acid transport had a negative effect on sink development of aap8 lines throughout the life cycle, leading to an overall decrease in plant biomass. During vegetative stage, photosynthesis and carbohydrate levels were decreased in aap8 leaves, while expression of carbon metabolism and transport genes, as well as sucrose phloem transport were not affected despite reduced sink strength.However, when aap8 plants transitioned to reproductive phase, carbon fixation and assimilation as well as sucrose partitioning to siliques were strongly decreased. Overall,this work demonstrates that phloem loading of nitrogen has varying implications for carbon fixation, assimilation and source-to-sink allocation depending on plant growth stage. It further suggests alterations in source-sink relationships, and regulation of carbon metabolism and transport by sink strength in a development-dependent manner.     

Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase beta genes.

The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.     

Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants.

We isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up 14C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C12 to C19. However, growth on these alkanes and uptake of [14C]hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up [14C]hexadecane. The addition of small amounts of rhamnolipids restored growth on alkanes and [14C]hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P. aeruginosa.     

Radiation-sensitive Arabidopsis mutants are proficient for T-DNA transformation.

Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure. Received: 23 June 1998 / Accepted: 21 February 1999     

Embryo-lethal mutants of Arabidopsis thaliana: Evidence for gametophytic expression of the mutant genes

Summary Normal and aborted seeds from two recessive embryo-lethal mutants (79A and 124D) of Arabidopsis thaliana were shown to be distributed nonrandomly along the length of heterozygous siliques. Significantly more than half of the aborted seeds in these two mutants were located in the top half of the silique, in the region closest to the stigma surface. Segregation ratios (percent aborted seeds) were unusually low at the base of the silique, and slightly higher than expected at the tip. In contrast, aborted seeds from four other embryo-lethal mutants (87A, 123B, 50B, and 71E) were distributed randomly along the length of the silique. These results suggest that the mutant genes in 79A and 124D are expressed during both the gametophytic (n) and sporophytic (2n) phases of development. These two mutants provide further evidence for the hypothesis that many genes expressed prior to fertilization also perform a critical function during growth and development of the sporophyte. Embryo-lethal mutants of Arabidopsis may therefore be useful in future studies of gametophytic gene expression and the regulation of pollen-tube growth in higher plants.     

Dependence of phytoplankton assimilation quotients on light and nitrogen source: implications for oceanic primary productivity

Photosynthetic production of oxygen by phytoplankton assemblagedominated by Peridinium in Lake Kinneret, Israel, generallyexceeds the molar equivalent rate of carbon assimilation. Carbonassimilation occurs only if oxygenic photosynthesis exceedsa light-dependent threshold. Assimilation quotients (mol C molO₂^–1) are a variable function of irradiance, and typicallyonly about one-half of the photoreductant produced during oxygenicphotosynthesis is used for reduction of carbon dioxide. Mostof the residual oxygenic photoreductant probably is used forlight-dependent reduction of nitrate, which competes with carbondioxide for oxygenic photoreductant. Nitrate is an importantsource of nitrogen for this algal assemblage, and light-dependentnitrate reduction probably is much larger than carbon dioxidereduction at lowest irradiances in the euphotic zone. Oxygenproduction also may be much larger than carbon assimilationat low light levels in other environments where oxidized formsof nitrogen are important nitrogenous nutrients for phytoplankton,as in the lower euphotic zone of the sea, where low rates ofcarbon assimilation by phytoplankton have been thought to beinconsistent with the amount of oxygen that accumulates duringsummer.     

Mutations in Arabidopsis acyl-CoA oxidase genes reveal distinct and overlapping roles in beta-oxidation

Indole-3-butyric acid (IBA) is an endogenous auxin used to enhance rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display enhanced root elongation on inhibitory IBA concentrations but maintain wild-type responses to indole-3-acetic acid, the principle active auxin. A subset of ibr mutants remains sensitive to the stimulatory effects of IBA on lateral root initiation. These mutants are not sucrose dependent during early seedling development, indicating that peroxisomal beta-oxidation of seed storage fatty acids is occurring. We used positional cloning to determine that one mutant is defective in ACX1 and two are defective in ACX3, two of the six Arabidopsis fatty acyl-CoA oxidase (ACX) genes. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third gene, ACX4. Activity assays demonstrated that mutants defective in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, acx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling development, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance but remain sucrose independent. The inability of ACX1, ACX3, and ACX4 to fully compensate for one another in IBA-mediated root elongation inhibition and the ability of ACX2 and ACX5 to contribute to IBA response suggests that IBA-response defects in acx mutants may reflect indirect blocks in peroxisomal metabolism and IBA beta-oxidation, rather than direct enzymatic activity of ACX isozymes on IBA-CoA.     

Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes.

Mutations in the GLN3 gene prevented a normal increase in the NAD-glutamate dehydrogenase and glutamine synthetase levels in glutamate-grown Saccharomyces cerevisiae cells, whereas mutations in the URE2 gene resulted in high levels of these enzymes in glumate- and glutamine-grown cells. A ure2 gln3 double mutant had low levels of glutamate dehydrogenase and glutamine synthetase in cells grown on glutamate and glutamine; thus, gln3 mutations were epistatic to the ure2 mutations. The results suggest that the GLN3 product is capable of promoting increases in enzyme levels in the absence of a functional URE2 product and that the URE2 product antagonizes the GLN3 product. The URE2 and GLN3 genes were also found to regulate the level of arginase activity. This regulation is completely independent of the regulation of arginase by substrate induction. The activities of glutamate dehydrogenase, glutamine synthetase, and arginase were higher in cells grown on glutamate as the nitrogen source than they were in cells grown under a nitrogen-limiting condition. It had previously been shown that the levels of these enzymes can be increased by glutamine deprivation. We propose that the URE2-GLN3 system regulates enzyme synthesis, in response to glutamine and glutamate, to adjust the intracellular concentration of ammonia so as to maintain glutamine at the level required for optimal growth.     

The effect of nitrate assimilation deficiency on the carbon and nitrogen status of Arabidopsis thaliana plants

Carbon (C) and nitrogen (N) metabolism are integrated processes that modulate many aspects of plant growth, development, and defense. Although plants with deficient N metabolism have been largely used for the elucidation of the complex network that coordinates the C and N status in leaves, studies at the whole-plant level are still lacking. Here, the content of amino acids, organic acids, total soluble sugars, starch, and phenylpropanoids in the leaves, roots, and floral buds of a nitrate reductase (NR) double-deficient mutant of Arabidopsis thaliana (nia1 nia2) were compared to those of wild-type plants. Foliar C and N primary metabolism was affected by NR deficiency, as evidenced by decreased levels of most amino acids and organic acids and total soluble sugars and starch in the nia1 nia2 leaves. However, no difference was detected in the content of the analyzed metabolites in the nia1 nia2 roots and floral buds in comparison to wild type. Similarly, phenylpropanoid metabolism was affected in the nia1 nia2 leaves; however, the high content of flavonol glycosides in the floral buds was not altered in the NR-deficient plants. Altogether, these results suggest that, even under conditions of deficient nitrate assimilation, A. thaliana plants are capable of remobilizing their metabolites from source leaves and maintaining the C–N status in roots and developing flowers.     

Overexpression of cytosolic glutamine synthetase. Relation to nitrogen,light, and photorespiration

In plants, ammonium released during photorespiration exceeds primary nitrogen assimilation by as much as 10-fold. Analysis of photorespiratory mutants indicates that photorespiratory ammonium released in mitochondria is reassimilated in the chloroplast by a chloroplastic isoenzyme of glutamine synthetase (GS2), the predominant GS isoform in leaves of Solanaceous species including tobacco (Nicotiana tabacum). By contrast, cytosolic GS1 is expressed in the vasculature of several species including tobacco. Here, we report the effects on growth and photorespiration of overexpressing a cytosolic GS1 isoenzyme in leaf mesophyll cells of tobacco. The plants, which ectopically overexpress cytosolic GS1 in leaves, display a light-dependent improved growth phenotype under nitrogen-limiting and nitrogen-non-limiting conditions. Improved growth was evidenced by increases in fresh weight, dry weight, and leaf soluble protein. Because the improved growth phenotype was dependent on light, this suggested that the ectopic expression of cytosolic GS1 in leaves may act via photosynthetic/photorespiratory process. The ectopic overexpression of cytosolic GS1 in tobacco leaves resulted in a 6- to 7-fold decrease in levels of free ammonium in leaves. Thus, the overexpression of cytosolic GS1 in leaf mesophyll cells seems to provide an alternate route to chloroplastic GS2 for the assimilation of photorespiratory ammonium. The cytosolic GS1 transgenic plants also exhibit an increase in the CO(2) photorespiratory burst and an increase in levels of photorespiratory intermediates, suggesting changes in photorespiration. Because the GS1 transgenic plants have an unaltered CO(2) compensation point, this may reflect an accompanying increase in photosynthetic capacity. Together, these results provide new insights into the possible mechanisms responsible for the improved growth phenotype of cytosolic GS1 overexpressing plants. Our studies provide further support for the notion that the ectopic overexpression of genes for cytosolic GS1 can potentially be used to affect increases in nitrogen use efficiency in transgenic crop plants.     

Expression of Klebsiella pneumoniae nitrogen fixation genes in nitrate reductase mutants of Escherichia coli.

Nitrate reductase (nar) A, B and E mutants of Escherichia coli with plasmids carrying Klebsiella pneumoniae nitrogen fixation (nif) genes reduced acetylene independently of added molybdate, but nar D mutants showed pleiotropic dependence on the concentration of added molybdate for expression of both nar and nif. No complementation of nar mutations by nif occurred; nitrite but not nitrate repressed nif in nar hosts. Derepression of nif occurred in molybdenum-deficient nar D (nif) strains since nitrogenase peptides were present. nifB mutants, thought to have a lesion in the pathway of molybdenum to nitrogenase, as well as nif deletion mutants, had normal nitrate reductase activity.