Marc Vauterin

THE ARABIDOPSIS THALIANA DHDPS GENE ENCODING DIHYDRODIPICOLINATE SYNTHASE,KEY ENZYME OF LYSINE BIOSYNTHESIS, IS EXPRESSED IN A CELL-SPECIFIC MANNER

Lysine synthesis in prokaryotes, some phycomycetes and higher plants starts with the condensation of L-aspartate-β-semialdehyde (L-ASA) and pyruvate into dihydrodipicolinic acid. The enzyme that catalyses this step, dihydrodipicolinate synthase (DHDPS), is inhibited by the end-product lysine and is therefore thought to have a regulatory control on lysine synthesis. We have cloned and sequenced an Arabidopsis thaliana DNA fragment containing 900 bases upstream of the dhdps coding sequence. A transcriptional fusion of this fragment with the β-glucuronidase reporter gene (uidA, Gus) was used to study the transcription properties of this promoter fragment (DS). No lysine-induced repression on transcription could be detected. Expression of DS-Gus activity in transformed Arabidopsis thaliana and Nicotiana tabacum was found to be cell type-specific. In the vegetative parts of the plant, GUS activity was located in meristems and young vasculature of roots, in vasculature of stem and leaves and in the meristems of young shoots. In flowers, high expression was found in the carpels, style, stigma, developing embryos, tapetum of young anthers and pollen. We demonstrated that the Arabidopsis DS promoter can direct its cell type-specific expression in a heterologous plant, Nicotiana tabacum. The importance of transcriptional regulation of the dhdps gene, and in more general genes involved in amino acid biosynthesis, is discussed.

Sequence analysis, transcriptional and posttranscriptional regulation of the rice VDAC family

The voltage-dependent anion-selective channel (VDAC) is a mitochondrial outer membrane ion channel. Different isoforms exist in plants but information about their specific role remains to be established. Our purpose is to find out the structural features common to three rice VDAC isoforms and to investigate their (post)transcriptional regulation in response to an osmotic stress. Two new cDNAs encoding mitochondrial VDAC from rice (Oryza sativa) were isolated, sequenced and characterized: a phylogenetic reconstruction permitted identification of orthologues in Poaceae and computer-based analyses predicted 18 transmembrane beta-strands, one amphipathic alpha-helix and two different phosphorylation motifs. The expression of three rice vdac genes was investigated. Northern blot analyses indicated that they were expressed in all plant tissues. There was a differential expression of osvdac1 and osvdac3, whereas osvdac2 was homogeneously expressed in all tissues. No change in vdac expression was observed under an osmotic stress. However, a fast-enhanced expression of vdac was observed in roots during the recovery period after stress release. This enhanced expression is not correlated to the amount of VDAC protein detected in roots suggesting a posttranscriptional regulation.

Arabidopsis loss-of-function mutant in the lysine pathway points out complex regulation mechanisms

In plants, the amino acids lysine, threonine, methionine and isoleucine have L‐aspartate‐β‐semialdehyde (ASA) as a common precursor in their biosynthesis pathways. How this ASA precursor is dispersed among the different pathways remains vague knowledge. The proportional balances of free and/or protein‐bound lysine, threonine, isoleucine and methionine are a function of protein synthesis, secondary metabolism and plant physiology. Some control points determining the flux through the distinct pathways are known, but an adequate explanation of how the competing pathways share ASA in a fine‐tuned amino acid biosynthesis network is yet not available. In this article we discuss the influence of lysine biosynthesis on the adjacent pathways of threonine and methionine. We report the finding of an Arabidopsis thaliana dihydrodipicolinate synthase T‐DNA insertion mutant displaying lower lysine synthesis, and, as a result of this, a strongly enhanced synthesis of threonine. Consequences of these cross‐pathway regulations are discussed.

Isolation of a poplar and anArabidopsis thaliana dihydrodipicolinate synthase cDNA clone

A poplar DHDPS cDNA clone has been isolated by functional rescue of thedapA-deficient AT997 mutant ofEscherichia coli. By sequence comparison between the poplar and maize DHDPS cDNAs, two oligonucleotides were designed to perform polymerase chain reaction (PCR) onArabidopsis thaliana genomic DNA. The PCR fragment was subsequently used to isolate anArabidopsis DHDPS genomic and cDNA clone.

Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase

A cDNA clone encoding a monofunctional aspartate kinase (AK, ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe. Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A. thaliana bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH). By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC). The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue. The presence of two homologous genes in A. thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp. 287–294 [this issue]). This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA. The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product.

Metabolic engineering of a complex biochemical pathway: The lysine and threonine biosynthesis as an example

The nutritional quality of crop plants is determined by their content in essential amino acids provided in food for humans or in feed for monogastric animals. Amino acid composition of crop–based diets can be improved via manipulation of the properties of key enzymes of amino acid biosynthetic pathways by mutation and transformation. We focused on the aspartate-derived amino acid pathway producing four essential amino acids: lysine, threonine, isoleucine and methionine. Genes encoding aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS) that operate as key genes of the aspartate pathway have been cloned from Arabidopsis. Genetic and molecular studies revealed that at least five different ak genes are represented. Some of them were characterized in terms of gene and promoter structure, developmental expression and regulatory properties. In the case of dhdps, two quite identical genes have been identified and characterized at expression level. Mutated genes encoding a fully feedback-insensitive form of the DHDPS enzyme were obtained from Nicotiana sylvestris and Arabidopsis. Several chimeric constructs harbouring this mutated allele under the control of constitutive or seed-specific promoters were transferred via Agrobacterium or biolistics in various plant species. In all cases, lines with significant increase of free lysine content were obtained in vegetative organs, but the impact of the transgene in seeds is limited due to the presence of an active catabolic enzyme, lysine ketoreductase. These results show that, although dealing with a complex, highly regulated pathway, the overexpression of a single gene encoding a feedback-insensitive form of the key enzyme DHDPS exerts a significant effect on the carbon flux through the aspartate pathway towards lysine production.

Analysis of Dihydrodipicolinate Synthase Promoter Activity in Arabidopsis thaliana by In-Planta Transformation

The present study was carried out to analyze the dihydrodipicolinate synthase (dhdps) gene promoter activity by tracing the GUS expression in tissues and in organs of Arabidopsis thaliana by in planta transformation. The Agrobacterium construct pBI101 used in the studies consists of the reporter gene gus under the control of Arabidopsis thaliana dhdps promoter with 3’ nos controlling sequences and nptll gene under the control of nos promoter and nos terminator. GUS expression in transformed Arabidopsis thaliana was found to be cell type-specific and expressed mainly in the fast growing tissues, where the protein synthesis is high. The histochemical analysis results indicate that the GUS expression was mainly observed in root meristem (elongation zone), emerging lateral roots and in the leaf vascular tissues. In reproductive organs, the GUS expression was observed in anthers, pollen grains and young immature embryos. Southern blot analysis results of T2, progeny showed the presence of a single integration locus for both the nptll and dhdps promoter.The segregation analysis results showed that the kanamycin resistance gene has not followed the normal Mendelian inheritance.This might be due to the methylation of the nptll gene in some of the transformants.

Biochemical Characterisation and Cloning of α-Kafirin Gene from Sorghum (Sorghum bicolor L Moench)

Seed storage proteins from naturally occurring lysine-rich cultivars namely IS 217O2, CVS 365, G 1058, G 205 and CVS 549 were analyzed biochemically, immunologically and compared with a low-lysine cultivar (White Martin) and a chemically induced high-lysine mutant (P7210). Protein fractionation studies indicated that the high lysine cultivars contained 25% less kafirin and an increased alcohol insoluble reduced glutelin without affecting the total protein content. SDS-PAGE analysis of total kafirin showed the absence of 25.3 kD and 25.9 kD a-kafirin proteins in lysine-rich cultivars IS 217O2, CVS 365 and G 1058, while in G 205 only the 25.9 kD protein was absent compared to low-lysine cultivar White Martin. A genomic clone λGK5 encoding an a-kafirin has been isolated from cv White Martin genomic library using pSKR3 as hybridizing probe and sequenced. Transient expression studies by particle bombardment of immature seeds of sorghum allowed to detect β-glucuronidase (GUS) activity only in endosperm cells confirming that the α-kafirin gene promoter is functional and tissue specific.

Manipulating Essential Amino Acid Metabolism in Plants

The nutritional quality of crop plants is determined by their content in essential amino acids provided as proteins in foods for humans or in feed for monogastric animals. Plant proteins are often deficient in some of the ten essential amino acids which are required in human and animal diet. In general cereals are deficient in lysine and legumes in the sulphur amino acids, methionine and cysteine. Besides conventional genetic means, various strategies have been developed with the goal to improve the amino acid imbalance of crop-based diets. Modifying genes encoding storage proteins aiming at correcting their nutritional deficiency or expressing genes encoding nutritionally superior storage proteins were presented as possible strategies in order to improve protein quality, especially in seeds (J.E. Habben, B.A. Larkins, 1996).