Expression of the Arabidopsis thaliana invertase gene family

Two new loci have been found to be clustered with five other genes for the nitrate assimilation pathway in the Chlamydomonas reinhardtii genome. One gene, located close to the 3′-end of the high-affinity nitrate transporter (HANT) gene Nrt2;2, corresponds to the nitrite reductase (NiR) structural gene Nii1. This is supported by a number of experimental findings: (i) NiR-deficient mutants have lost Nii1 gene expression; (ii) Nii1 mRNA accumulation is co-regulated with the expression of other structural genes of the nitrate assimilation pathway; (iii) nitrite (nitrate) utilization ability is recovered in the NiR mutants by functional complementation with a wild-type Nii1 gene; (iv) the elucidated NII1 amino acid sequence is highly similar to that of the cyanobacterial and higher-plant enzyme, and contains the predicted domains for plastidic ferredoxin-NiRs. Thus, the mutant phenotype and the mRNA sequence and expression of the Nii1 gene have been unequivocally related. Accumulation of mRNA for the second locus identified, Lde1 (light-dependent expression), was not regulated by nitrogen, but like nitrate-assimilation clustered genes, its expression was down-regulated in the dark. Received: 27 November 1997 / Accepted: 19 January 1998     

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max)

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 μM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots. Received: 22 November 1997 / Accepted: 26 January 1998     

Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii

Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci. Received: 6 October 1997 / Accepted: 30 December 1997     

Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii

Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci.     

Expression studies of Nrt2:1Np, a putative high‐affinity nitrate transporter: evidence for its role in nitrate uptake

Expression of the gene Nrt2Np, which encodes a putative high-affinity nitrate transporter of Nicotiana plumbaginifolia was studied under variable physiological conditions. Nrt2Np is rapidly induced by very low nitrate concentrations and repressed by reduced nitrogen metabolites. Furthermore, Nrt2Np is expressed in coordination with other genes involved in nitrate assimilation (Nia, Nii). A deficiency in nitrate reductase activity, which is accompanied by high internal nitrate concentration and low levels of nitrogen metabolites, e.g. glutamine, leads to an overexpression of Nrt2Np, showing that high nitrate concentration per se does not repress Nrt2Np expression. By investigating plants with altered nitrate uptake properties, we showed a correlation between Nrt2 mRNA accumulation and 15N nitrate influx rates, providing the first evidence that the expression of Nrt2 correlates with the rate of nitrate uptake. In situ hybridization revealed a tissue-specific expression pattern. Nrt2Np mRNA accumulation is localized throughout all layers of the root tip, being highest in epidermal and endodermal cells. However, in mature root tissue, Nrt2 expression was detected mainly in the lateral root primordia and in the epidermis.     

Expression of CKS1At in Arabidopsis thaliana indicates a role for the protein in both the mitotic and the endoreduplication cycle

Although endoreduplication is common in plants, little is known about the mechanisms regulating this process. Here, we report the patterns of endoreduplication at the cellular level in the shoot apex of Arabidopsis thaliana L. Heynh. plants grown under short-day conditions. We show that polyploidy is developmentally established in the pith, maturing leaves, and stipules. To investigate the role of the cell cycle genes CDC2aAt, CDC2bAt, CYCB1;1, and CKS1At in the process of endoreduplication, in-situ hybridizations were performed on the vegetative shoot apices. Expression of CDC2aAt, CDC2bAt, and CYCB1;1 was restricted to mitotically dividing cells. In contrast, CKS1At expression was present in both mitotic and endoreduplicating tissues. Our data indicate that CDC2aAt, CDC2bAt, and CYCB1;1 only operate during mitotic divisions, whereas CKS1At may play a role in both the mitotic and endoreduplication cycle. Received: 11 May 1998 / Accepted: 29 September 1998     

Genetic analysis of mutant clones of Chlamydomonas reinhardtii defective in potassium transport

Mutant clones of Chlamydomonas reinhardtii defective for potassium transport were isolated and characterized. Of the four genes identified, three –TRK1, TRK2 and TRK3– encode high-affinity transport functions, and one gene, HKR1, encodes a low-affinity transport function. Characterization of the potassium dependence of recombinants possessing two mutant trk alleles suggests that the protein products of TRK2 and TRK3 interact functionally, and that TRK1 may serve a regulatory function. The mutant clone defective for a low-affinity potassium transporter was isolated by mutagenizing trk2-1 cells, which lack a functional high-affinity transporter, and screening surviving cells for dependence on very high potassium concentrations. The hkr1 phenotype is expressed only in the presence of trk2-1. Received: 24 August 1998 / Accepted: 16 November 1998     

Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana

Putative high-affinity nitrate (NO3-) transporter genes, designated Nrt2;1At and Nrt2;2At, were isolated from Arabidopsis thaliana by RT-PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30-73% identities with other eukaryotic high-affinity NO3- transporters. Both genes were induced by NO3-, but Nrt2;1At gene expression was not apparent in 2- and 5-day-old plants. By 10 days, and thereafter, Nrt2;1At gene expression in roots was substantially higher than for the Nrt2;2At gene. Root Nrt2;1At expression levels were strongly correlated with inducible high-affinity 13NO3- influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation of Nrt2;1At expression was mediated by NH4+, gln and other amino acids.     

Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae

Root NO3- uptake and expression of two root NO3- transporter genes (Nrt2;1 and Nrt1) were investigated in response to changes in the N- or C-status of hydroponically grown Arabidopsis thaliana plants. Expression of Nrt2;1 is up-regulated by NO3 - starvation in wild-type plants and by N-limitation in a nitrate reductase (NR) deficient mutant transferred to NO3- as sole N source. These observations show that expression of Nrt2;1 is under feedback repression by N-metabolites resulting from NO3- reduction. Expression of Nrt1 is not subject to such a repression. However, Nrt1 is over-expressed in the NR mutant even under N-sufficient conditions (growth on NH4NO3 medium), suggesting that expression of this gene is affected by the presence of active NR, but not by N-status of the plant. Root 15NO3- influx is markedly increased in the NR mutant as compared to the wild-type. Nevertheless, both genotypes have similar net 15NO3- uptake rates due to a much larger 14NO3- efflux in the mutant than in the wild-type. Expressions of Nrt2;1 and Nrt1 are diurnally regulated in photosynthetically active A. thaliana plants. Both increase during the light period and decrease in the first hours of the dark period. Sucrose supply prevents the inhibition of Nrt2;1 and Nrt1 expressions in the dark. In all conditions investigated, Nrt2;1 expression is strongly correlated with root 15NO3- influx at 0.2 mM external concentration. In contrast, changes in the Nrt1 mRNA level are not always associated with similar changes in the activities of high- or low-affinity NO3- transport systems.     

The response of lazy-2 tomato seedlings to curvature-inducing magnetic gradients is modulated by light

Shoots of the lazy-2 mutant of tomato (Lycopersicon esculentum Mill., cv. Ailsa Craig) exhibit negative gravitropism in the dark, but respond positively gravitropically in (red) light. In order to test whether high-gradient magnetic fields (HGMFs) exert only ponderomotive effects on amyloplasts or affect other physiological processes, we induced magnetophoretic curvature in wild-type (WT) and lazy-2 mutant seedlings. Straight hypocotyls of 4-d-old plants were selected and the tips of their hooks were placed in an HGMF near the edge of a magnetized ferromagnetic wedge [grad (H²/2) ≈ 10⁹–10¹⁰ Oe²/cm] and mounted on a 1-rpm clinostat. After 4 h in the dark, 85% of WT hypocotyls and 67% of mutant hypocotyls curved toward the wedge. When the seedlings were exposed to red light for 1 h prior to and during the application of the HGMF, 78% of the WT seedlings curved toward the magnetic gradient, but the majority of the lazy-2 seedlings (75%) curved away from the stronger field area. Intracellular amyloplast displacement in the HGMF was similar for both varieties and resembled the displacement after horizontal reorientation. The WT showed a distinct graviresponse pattern depending on the orientation of the hook, even after excision of the apex. Application of HGMFs to decapitated hypocotyls resulted in curvature consistent with that obtained after horizontal reorientation. After light exposure, decapitated lazy-2 seedlings did not respond positively gravitropically. The data imply that the lazy-2 mutants perceive the displacement of amyloplasts in a similar manner to the WT and that the HGMF does not affect the graviresponse mechanism. The study demonstrates that ponderomotive forces due to HGMFs are useful for the analysis of the gravity-sensing mechanism in plants. Received: 31 August 1998 / Accepted: 6 October 1998     

Carbohydrates modulate the expression of the sunflower cytochrome c gene at the mRNA level

Changes in the steady-state mRNA levels of the gene encoding cytochrome c were analyzed after feeding carbohydrates to detached leaves of sunflower (Helianthus annuus L.). Glucose, fructose and sucrose promoted an increase in mRNA levels, which was not observed with mannitol and other metabolites such as glycerol or acetate. The increase in mRNA levels was proportionally higher in dark-treated leaves. The effect of sugars could be mimicked by compounds that are phosphorylated by hexokinase but not further metabolized, such as mannose or 2-deoxyglucose. This may indicate that hexokinase is involved in the induction of the cytochrome c gene by carbohydrates. The presence of potassium phosphate had no significant effect on the induction by sugars. Our results indicate that the modulation of the expression of nuclear genes encoding mitochondrial components should be added to the list of known effects of carbohydrates on respiration. Received: 5 February 1998 / Accepted: 22 April 1998     

Differential expression of triplicate phosphoribosylanthranilate isomerase isogenes in the tryptophan biosynthetic pathway of Arabidopsis thaliana (L.) Heynh.

Phosphoribosylanthranilate isomerases (PAI) in the tryptophan biosynthetic pathway of Arabidopsis thaliana are encoded by a gene family. Expression patterns of each individual PAI isogene were investigated by analyzing expression of translation-fusions of promoter-β-glucuronidase (GUS) chimeras in transgenic plants. Quantification and histochemical staining of GUS activities expressed in PAI transgenic plants demonstrated that, first, expression of the three PAI isogenes was differentially regulated under normal growth conditions. Both PAI1 and PAI3 showed approximately 10-fold stronger expression than PAI2. Second, PAI isogenes differentially responded to environmental stresses such as ultraviolet irradiation and the abiotic elicitor silver nitrate. PAI2 displayed a stronger response to stresses than the other two PAI isogenes. Third, each individual PAI isogene was differentially expressed in a tissue- and cell-type-specific manner. Fourth, expression of PAI isogenes was coordinated to meet the requirement for normal growth and development of A. thaliana. Deletion of PAI1 is partially responsible for abnormal growth and development in the PAI deletion mutant trp6 as well as strong blue fluorescence in young leaves under ultraviolet irradiation. Received: 15 June 2000 / Accepted: 16 August 2000     

Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearence, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thalianatt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds. Received: 2 October 1998 / Accepted: 10 October 1998     

The isolation and characterisation of a mutant of the C4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C₄ leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F₂ plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO₂ assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C₄ enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C₄ photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME. Received: 2 April 1998 / Accepted: 7 May 1998     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998