Marie-Thérèse Leydecker

QUASIMODO1 Encodes a Putative Membrane-Bound Glycosyltransferase Required for Normal Pectin Synthesis and Cell Adhesion in Arabidopsis

Pectins are a highly complex family of cell wall polysaccharides. As a result of a lack of specific mutants, it has been difficult to study the biosynthesis of pectins and their role in vivo. We have isolated two allelic mutants, named quasimodo1 (qua1-1 and qua1-2), that are dwarfed and show reduced cell adhesion. Mutant cell walls showed a 25% reduction in galacturonic acid levels compared with the wild type, indicating reduced pectin content, whereas neutral sugars remained unchanged. Immersion immunofluorescence with the JIM5 and JIM7 monoclonal antibodies that recognize homogalacturonan epitopes revealed less labeling of mutant roots compared with the wild type. Both mutants carry a T-DNA insertion in a gene (QUA1) that encodes a putative membrane-bound glycosyltransferase of family 8. We present evidence for the possible involvement of a glycosyltransferase of this family in the synthesis of pectic polysaccharides, suggesting that other members of this large multigene family in Arabidopsis also may be important for pectin biosynthesis. The mutant phenotype is consistent with a central role for pectins in cell adhesion.

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.

Molybdenum Cofactor Mutants, Specifically Impaired in Xanthine Dehydrogenase Activity and Abscisic Acid Biosynthesis, Simultaneously Overexpress Nitrate Reductase'

The molybdenum cofactor is shared by nitrate reductase (NR), xanthine dehydrogenase (XDH), and abscisic acid (ABA) aldehyde oxidase in higher plants (M. Walker-Simmons, D.A. Kudrna, R.L. Warner [1989] Plant Physiol 90:728–733). In agreement with this, cnx mutants are simultaneously deficient for these three enzyme activities and have physiological characteristics of ABA-deficient plants. In this report we show that aba1 mutants, initially characterized as ABA-deficient mutants, are impaired in both ABA aldehyde oxidase and XDH activity but overexpress NR. These characteristics suggest that aba1 is in fact involved in the last step of molybdenum cofactor biosynthesis specific to XDH and ABA aldehyde oxidase; aba1 probably has the same function as hxB in Aspergillus. The significance of NR overexpression in aba1 mutants is discussed.

Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence

Diurnal variations in nitrate reductase (NR) activity and nitrogen metabolites were examined in wild-type Nicotiana plumbaginifolia and transformants with various degrees of NR deregulation. In the C1 line, NR was only deregulated at the transcriptional level by placing the NR gene under the control of the cauliflower mosaic virus 35S RNA promoter. In the Del8 and S521D lines, NR was additionally deregulated at the posttranslational level either by a deletion mutation in the N-terminal domain or by a mutation of the regulatory phosphorylation site (serine-521). Posttranslational regulation was essential for pronounced diurnal variations in NR activity. Low nitrate content was related to deregulation of NR, whereas the level of total free amino acids was much higher in plants with fully deregulated NR. Abolishing transcriptional and posttranslational regulation (S521D plants) resulted in an increase of glutamine and asparagine by a factor of 9 and 14, respectively, compared with wild type, whereas abolishing transcriptional regulation (C1 plants) only resulted in increases of glutamine and asparagine by factors <2. Among the minor amino acids, isoleucine and threonine, in particular, showed enhanced levels in S521D. Nitrate uptake rates were the same in S521D and wild type as determined with 15N feeding. Deregulation of NR appears to set the level of certain amino acids, whereas diurnal variations were still determined by light/dark. Generally, deregulation of NR at the transcriptional level did not have much influence on metabolite levels, but additional deregulation at the posttranslational level resulted in profound changes of nitrogen metabolite levels.

Cytokinin affects nitrate reductase expression through the modulation of polyadenylation of the nitrate reductase mRNA transcript

Abstract Cytokinin (CK) and low-intensity light effects in modulating nitrate reductase (NR) activity, NR protein and NR encoding mRNAs were studied in tobacco cell suspension cultures. NR activity was strikingly enhanced by CK in dark- as well as in light-grown cells whereas it was less affected by light alone. NR protein accumulation was stimulated by the hormone in the light only; then a CK light-dependent regulation of NR activity was suggested. Light enhanced the steady-state levels of hybridisable total NR mRNA and a light-inductive effect was also observed after transfer from dark to light; this effect was dependent on sucrose supply and was enhanced in CK-supplied cells. NR poly(A) mRNA were assayed in cell poly(A) RNA, purified by oligo(dT)-cellulose chromatography. In growing cells CK enhanced (i) the steady state levels of hybridisable NR poly(A) mRNA and (ii) the ratio of hybridisable NR poly(A) mRNA to total hybridisable NR mRNA. CK-induced accumulation of hybridisable NR poly(A) mRNA resulted in a correlative enhancement of the accumulation of NR protein, provided cells were grown in the light. Overall stimulatory effects of CK on the amounts of bulk poly(A) mRNA and on the mean-size of mRNA poly(A) tails were also observed. It is suggested that CK effect on gene expression involves a modulation of mRNA polyadenylation.

Expression of a deregulated tobacco nitrate reductase gene in potato increases biomass production and decreases nitrate concentration in all organs

We investigated the physiological consequences for nitrogen metabolism and growth of the deregulated expression of an N-terminal-deleted tobacco nitrate reductase in two lines of potato (Solanum tuberosum L. cv Safrane). The transgenic plants showed a higher biomass accumulation, especially in tubers, but a constant nitrogen content per plant. This implies that the transformed lines had a reduced nitrogen concentration per unit of dry weight. A severe reduction in nitrate concentrations was also observed in all organs, but was more apparent in tubers where nitrate was almost undetectable in the transgenic lines. In leaves and roots, but not tubers, this nitrate decrease was accompanied by a statistically significant increase in the level of malate, which acts as a counter-anion for nitrate reduction. Apart from glutamine in tubers, no major changes in amino acid concentration were seen in leaves, roots or tubers. We conclude that enhancement of nitrate reduction rate leads to higher biomass production, probably by allowing a better allocation of N-resources to photosynthesis and C-metabolism.

Role of gibberellins and of the RGA and GAI genes in controlling nitrate assimilation in Arabidopsis thaliana

Abstract Screening of an Arabidopsis cDNA library for complementation of a yeast mutant affected in the regulation of nitrogen metabolism led to the isolation of the RGA and GAI cDNAs that were also known to be involved in plant response to gibberellins. This raised the question of RGA and GAI being also involved in controlling nitrogen metabolism in plants. To address this issue we studied whether loss of function ( rga2 , gai-t6 ) or gain of function mutations ( gai-1 ) in RGA or GAI or in both ( rga24 gai-t6 ) genes had any impact on nitrate assimilation in Arabidopsis plants grown in the greenhouse or in vitro. In addition, as the sole known plant function of RGA and GAI was their implication in gibberellin signal transduction, we analysed the effects of gibberellin treatment on nitrate assimilation of wild-type plants or of the gibberellin-deficient ga1-3 mutant. In most cases, no difference in expression of the NIA1 , NIA2 (encoding nitrate reductase, NR EC 1.6.6.1), NII (encoding nitrite reductase, NiR EC 1.7.7.1), GS1 and GS2 genes (encoding cytosolic and chloroplastic glutamine synthetases EC 6.3.1.2) could be detected in the different genetic backgrounds or after GA treatment of wild-type plants or GA-deficient plants. This absence of effect is also in general supported by similar NR and NiR activities and nitrate or nitrogen content in the different plants. In conclusion, our studies show that RGA, GAI and gibberellins do not act as major factors controlling nitrate assimilation in Arabidopsis at the vegetative stage although they may have, in some instances, an effect on nitrate assimilation genes.

Biochemical characterization of cnx nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

Abstract In cnx mutants of Nicotiana plumbaginifolia , the over-expression of nitrate reductase (NR)-associated NADH cytochrome c reductase (Ccr) activity appears to be correlated with increased levels of NR mRNA. However, in ELISA tests, cnx B, C, D, E and F extracts present low levels of cross-reacting material, whereas cnx A extracts exhibit very high levels, indicating an altered NR structure in all cnx mutants except cnx A, due to the absence of a functional molybdenum cofactor (MoCo). Detection of dimeric NR in all cnx mutants, either by gel filtration or native gel electrophoresis, shows that the cofactor is not required for enzyme dimerization, but probably for the stability of the dimer. Similarities between results obtained with cnx B through F mutants, and those nia mutants impaired in the molybdopterin domain, suggest a related NR structure.

Abscisic-acid in triazine-resistant and susceptible poa annua

Abstract Abscisic-acid (ABA) content in leaves and seeds were determined in two reciprocal hydbrids of cross between a triazine-resistant and a susceptible Poa annua . ABA in leaves appeared to be regulated by at least one major nuclear allele whilst ABA content in seeds appeared to maternally segregate with the cytoplasm. The lower ABA content found in seeds of the resistant parent and the resistant hybrid could be a secondary effect of the mutation that confers the resistance to the triazines.

Methylammonium-resistant mutants ofNicotiana plumbaginifolia are affected in nitrate transport

This work reports the isolation and preliminary characterization ofNicotiana plumbaginifolia mutants resistant to methylammonium.Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up byNicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.