Hoai-Nam Truong

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.

The Arabidopsis ATNRT2.7 Nitrate Transporter Controls Nitrate Content in Seeds

In higher plants, nitrate is taken up by root cells where Arabidopsis thaliana NITRATE TRANSPORTER2.1 (ATNRT2.1) chiefly acts as the high-affinity nitrate uptake system. Nitrate taken up by the roots can then be translocated from the root to the leaves and the seeds. In this work, the function of the ATNRT2.7 gene, one of the seven members of the NRT2 family in Arabidopsis, was investigated. High expression of the gene was detected in reproductive organs and peaked in dry seeds. β-Glucuronidase or green fluorescent protein reporter gene expression driven by the ATNRT2.7 promoter confirmed this organ specificity. We assessed the capacity of ATNRT2.7 to transport nitrate in Xenopus laevis oocytes or when it is expressed ectopically in mutant plants deficient in nitrate transport. We measured the impact of an ATNRT2.7 mutation and found no difference from the wild type during vegetative development. By contrast, seed nitrate content was affected by overexpression of ATNRT2.7 or a mutation in the gene. Finally, we showed that this nitrate transporter protein was localized to the vacuolar membrane. Our results demonstrate that ATNRT2.7 plays a specific role in nitrate accumulation in the seed.

NIT2, the nitrogen regulatory protein of Neurospora crassa, binds upstream of nia, the tomato nitrate reductase gene, in vitro.

SummaryThe nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zincfinger domain/β-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.

Sequence and characterization of two Arabidopsis thaliana cDNAs isolated by functional complementation of a yeast gln3 gdh1 mutant

We have isolated two Arabidopsis thaliana cDNAs by complementation of a yeast gln3 gdh1 strain that is affected in the regulation of nitrogen metabolism. The two clones (RGA1 and RGA2) are homologous to each other and to the SCARECROW (SCR) gene that is involved in regulating an asymmetric cell division in plants. RGA1, RGA2 and SCR share several structural features and may define a new family of genes. RGA1 and RGA2 have been mapped, respectively, to chromosome II and I, and their expression in plant is constitutive.

Deletion analysis of the tobacco Nii1 promoter in Arabidopsis thaliana

Abstract A 1 kb promoter fragment of the Nii1 gene, encoding a tobacco foliar nitrite reductase, has been fused to the β -glucuronidase ( Gus ) or luciferase ( Luc ) reporter genes. These constructs were introduced in Arabidopsis thaliana by in planta infiltration. Analysis of transformants shows that GUS or LUC activities are induced by nitrate. Deletions of the Nii1 promoter fused to the Luc gene have been made in order to delineate cis-sequences necessary for nitrate induction. The Nii1 , Δ C , Δ M and Δ H constructs ending, respectively at −962, −678, −202 and −76 bp before the putative transcription start of the Nii1 gene were fused transcriptionally to the Luc reporter gene. Analysis of LUC activities shows that nitrate induction occurs in the Δ MLuc construct, so promoter sequences required for nitrate induction of the reporter gene are retained in the proximal 200 bp fragment of the promoter. For the smallest construct, nitrate inducibility is maintained in one transformant. Yet, the low level of LUC activity detected in the other transformants does not allow us to assume that nitrate regulatory elements are still present in the Δ HLuc construct. We will discuss these results in relation to those obtained with deletions of other Nia or Nii promoters by different laboratories.

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.

The tomato nia gene promoter functions in fission yeast but not in budding yeast

A fragment comprising 1 kb of the 5′ region and the 81 first nucleotides of the coding region of the tomato nitrate reductase nia gene was placed in translational fusion with the lacZ reporter gene. This construct was introduced in budding and in fission yeast using a derivative of the Saccharomyces cerevisiae/Schizosaccharomyces pombe autonomously replicating vector pUZL. β-galactosidase activity was detected in S. pombe but not in S. cerevisiae. Primer extension experiments show that in fission yeast transcripts are initiated at the same starting point as in tomato, indicating for the first time that a plant promoter can be correctly recognized in fission yeast.

Molecular Genetics of Nitrate Assimilation in Solanaceous Species

Nitrate can be used as the sole nitrogen source to sustain growth, in both microorganisms and higher plants. In plants, two successive enzymatic steps reduce nitrate to ammonium, generally in the leaves. First, nitrate is converted into nitrite in a two electron transfer reaction catalysed by nitrate reductase (NR, EC1. 6. 6. 1), a cytoplasmic enzyme. Nitrite is then translocated to the chloroplast, where it is reduced to ammonium by nitrite reductase (NiR, EC1. 7. 7.1) (for review see Wray 1988, Solomonson and Barber 1990). Ammonium is subsequently incorporated into the amino acid pool through glutamine and glutamate biosynthesis (Guerrero et al 1981).