Profiling candidate genes involved in wax biosynthesis in Arabidopsis thaliana by microarray analysis

Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic compounds. We found in the available database 282 candidate genes that may play a role in wax synthesis, regulation and transport. To identify the most interesting candidates, we measured the level of expression of 204 genes in the aerial parts of 15-day-old Arabidopsis seedlings by performing microarray experiments. We showed that only 25% of the putative candidates were expressed to significant levels in our samples, thus significantly reducing the number of genes which will be worth studying using reverse genetics to demonstrate their involvement in wax accumulation. We identified a beta-keto acyl-CoA synthase gene, At5g43760, which is co-regulated with the wax gene CER6 in a number of conditions and organs. By contrast, we showed that neither the fatty acyl-CoA reductase genes nor the wax synthase genes were expressed in 15-day-old leaves and stems, raising questions about the identity of the enzymes involved in the acyl-reduction pathway that accounts for 20% of the total wax amount.     

Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-d-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis

The temperature-sensitive mutant of Arabidopsis , chs5 , developed chlorotic leaves at restrictive temperatures (15°C), but almost normal green leaves at permissive temperatures (22°C). At the restrictive temperature, the chs5 mutation blocked the accumulation of chlorophylls and carotenoids. A temperature-shift analysis revealed that the manifestation of the chlorotic phenotype occurred in young leaf tissues, but did not in mature leaf tissues. Genetic and sequence analysis demonstrated that the chs5 mutation was caused by a single-base change in the coding region of a recently identified CLA1 gene. The CLA1 gene exhibited a high sequence similarity to the genes encoding 1-deoxy- d -xylulose 5-phosphate synthase (DXS) localized to the non-mevalonate pathway, which was recently discovered in bacteria and higher plants. In addition, the application of 1-deoxy- d -xylulose, the free sugar of 1-deoxy- d -xylulose 5-phosphate, rescues the defect in the chs5 mutant. These results indicated that the chlorotic phenotype of the chs5 mutant was caused by a defect in DXS activity and that DXS functions preferentially at an early stage of leaf cell development. A transiently expressed green fluorescent protein fused with the CLA1 transit peptide was localized within the chloroplasts in the green cultured cells of tobacco, which suggests that the putative localization of the non-mevalonate pathway is in plastids.     

Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism

The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compounds in Arabidopsis thaliana. By combining homology and sequence motif searches with knowledge regarding the phylogenetic distribution of pathways of isoprenoid metabolism across species, candidate genes for these pathways in A. thaliana were obtained. A detailed analysis of the vicinity of chromosome loci for genes of isoprenoid metabolism in A. thaliana provided evidence for the clustering of genes involved in common pathways. Multiple sequence alignments were used to estimate the number of genes in gene families and sequence relationship trees were utilized to classify their individual members. The integration of all these datasets allows the generation of a knowledge-based metabolic map of isoprenoid metabolic pathways in A. thaliana and provides a substantial improvement of the currently available gene annotation.     

Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion

UDP-L-rhamnose is required for the biosynthesis of cell wall rhamnogalacturonan-I, rhamnogalacturonan-II, and natural compounds in plants. It has been suggested that the RHM2/MUM4 gene is involved in conversion of UDP-D-glucose to UDP-L-rhamnose on the basis of its effect on rhamnogalacturonan-I-directed development in Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 and RHM3, can be found in the A. thaliana genome. Here we present direct evidence that all three RHM proteins have UDP-D-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and UDP-4-keto-L-rhamnose 4-keto-reductase activities in the cytoplasm when expressed in the yeast Saccharomyces cerevisiae. Functional domain analysis revealed that the N-terminal region of RHM2 (RHM2-N; amino acids 1-370) has the first activity and the C-terminal region of RHM2 (RHM2-C; amino acids 371-667) has the two following activities. This suggests that RHM2 converts UDP-d-glucose to UDP-L-rhamnose via an UDP-4-keto-6-deoxy-D-glucose intermediate. Site-directed mutagenesis of RHM2 revealed that mucilage defects in MUM4-1 and MUM4-2 mutant seeds of A. thaliana are caused by abolishment of RHM2 enzymatic activity in the mutant strains and furthermore, that the GXXGXX(G/A) and YXXXK motifs are important for enzymatic activity. Moreover, a kinetic analysis of purified His(6)-tagged RHM2-N protein revealed 5.9-fold higher affinity of RHM2 for UDP-D-glucose than for dTDP-D-glucose, the preferred substrate for dTDP-D-glucose 4,6-dehydratase from bacteria. RHM2-N activity is strongly inhibited by UDP-L-rhamnose, UDP-D-xylose, and UDP but not by other sugar nucleotides, suggesting that RHM2 maintains cytoplasmic levels of UDP-D-glucose and UDP-L-rhamnose via feedback inhibition by UDP-L-rhamnose and UDP-D-xylose.     

Functional analysis of the tubulin-folding cofactor C in Arabidopsis thaliana

The biogenesis of microtubules comprises several steps, including the correct folding of alpha- and beta-tubulin and heterodimer formation. In vitro studies and the genetic analysis in yeast revealed that, after translation, alpha- and beta-tubulin are processed by several chaperonins and microtubule-folding cofactors (TFCs) to produce assembly-competent alpha-/beta-tubulin heterodimers. One of the TFCs, TFC-C, does not exist in yeast, and a potential function of TFC-C is thus based only on the biochemical analysis. In this study and in a very recently published study by Steinborn and coworkers, the analysis of the Arabidopsis porcino (por) mutant has shown that TFC-C is important for microtubule function in vivo. The predicted POR protein shares weak amino acid similarity with the human TFC-C (hTFC-C). Our finding that hTFC-C under the control of the ubiquitously expressed 35S promoter can rescue the por mutant phenotype shows that the POR gene encodes the Arabidopsis ortholog of hTFC-C. The analysis of plants carrying a GFP:POR fusion construct showed that POR protein is localized in the cytoplasm and is not associated with microtubules. While, in por mutants, microtubule density was indistinguishable from wild-type, their organization was affected.     

A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana

Background

Multiple pathway databases are available that describe the human metabolic network and have proven their usefulness in many applications, ranging from the analysis and interpretation of high-throughput data to their use as a reference repository. However, so far the various human metabolic networks described by these databases have not been systematically compared and contrasted, nor has the extent to which they differ been quantified. For a researcher using these databases for particular analyses of human metabolism, it is crucial to know the extent of the differences in content and their underlying causes. Moreover, the outcomes of such a comparison are important for ongoing integration efforts.

Results

We compared the genes, EC numbers and reactions of five frequently used human metabolic pathway databases. The overlap is surprisingly low, especially on reaction level, where the databases agree on 3% of the 6968 reactions they have combined. Even for the well-established tricarboxylic acid cycle the databases agree on only 5 out of the 30 reactions in total. We identified the main causes for the lack of overlap. Importantly, the databases are partly complementary. Other explanations include the number of steps a conversion is described in and the number of possible alternative substrates listed. Missing metabolite identifiers and ambiguous names for metabolites also affect the comparison.

Conclusions

Our results show that each of the five networks compared provides us with a valuable piece of the puzzle of the complete reconstruction of the human metabolic network. To enable integration of the networks, next to a need for standardizing the metabolite names and identifiers, the conceptual differences between the databases should be resolved. Considerable manual intervention is required to reach the ultimate goal of a unified and biologically accurate model for studying the systems biology of human metabolism. Our comparison provides a stepping stone for such an endeavor.     

Jasmonate is involved in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana

Coronatine-inducible tyrosine aminotransferase (TAT), which catalyses the transamination from tyrosine to p-hydroxyphenylpyruvate, is the first enzyme of a pathway leading via homogentisic acid to plastoquinone and tocopherols, the latter of which are known to be radical scavengers in plants. TAT can be also induced by the octadecanoids methyl jasmonate (MeJA) and methyl-12-oxophytodienoic acid (MeOPDA), as well as by wounding, high light, UV light and the herbicide oxyfluorfen. In order to elucidate the role of octadecanoids in the process of TAT induction in Arabidopsis thaliana (L.) Heynh., the jasmonate-deficient mutant delayed dehiscence (dde1) was used, in which the gene for 12-oxophytodienoic acid reductase 3 is disrupted. The amount of immunodetectable TAT was low. The enzyme was still fully induced by coronatine as well as by MeJA although induction by the latter was to a lesser extent and later than in the wild type. Treatment with MeOPDA, wounding and UV light, however, had hardly any effects. Tocopherol levels that showed considerable increases in the wild type after some treatments were much less affected in the mutant. However, starting levels of tocopherol were higher in non-induced dde1 than in the wild type. We conclude that jasmonate plays an important role in the signal transduction pathway regulating TAT activity and the biosynthesis of its product tocopherol.     

l-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis

l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l-galactono-1,4-lactone (K(m) = 0.17 mm, k(cat) = 134 s(-1)), l-gulono-1,4-lactone (K(m) = 13.1 mm, k(cat) = 4.0 s(-1)) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen (k(ox) = 6 x 10(2) m(-1).s(-1)). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l-galactono-1,4-lactone and l-gulono-1,4-lactone and released their FAD cofactor more easily than wild-type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed.     

Visualization of plastid movement in the pollen tube of Arabidopsis thaliana

Organelle dynamics in the plant male gametophyte has received attention for its importance in pollen tube growth and cytoplasmic inheritance. We recently revealed the dynamic behaviors of plastids in living Arabidopsis pollen grains and tubes, using an inherent promoter-driven FtsZ1–green fluorescent protein (GFP) fusion. Here, we further monitored the movement of pollen tube plastids with an actin1 promoter-driven, stroma-targeted yellow fluorescent protein (YFP). In elongating pollen tubes, most plastids localized to the tube shank, where they displayed either retarded and unsteady motion, or fast, directional, and long-distance movement along the tube polarity. Efficient plastid tracking further revealed a population of tip-forwarding plastids that undergo a fluctuating motion(s) before traveling backward. The behavior of YFP-labeled plastids in pollen basically resembled that of FtsZ1–GFP-labeled plastids, thus validating the use of FtsZ1–GFP for simultaneous visualization of the stroma and the plastid-dividing FtsZ ring.     

Sparse graphical Gaussian modeling of the isoprenoid gene network in Arabidopsis thaliana

We present a novel graphical Gaussian modeling approach for reverse engineering of genetic regulatory networks with many genes and few observations. When applying our approach to infer a gene network for isoprenoid biosynthesis in Arabidopsis thaliana, we detect modules of closely connected genes and candidate genes for possible cross-talk between the isoprenoid pathways. Genes of downstream pathways also fit well into the network. We evaluate our approach in a simulation study and using the yeast galactose network.