Critical analysis of protein signaling networks involved in the regulation of plant secondary metabolism: focus on anthocyanins

Anthocyanin biosynthesis in Arabidopsis is a convenient and relatively simple model for investigating the basic principles of secondary metabolism regulation. In recent years, many publications have described links between anthocyanin biosynthesis and general defense reactions in plants as well as photomorphogenesis and hormonal signaling. These relationships are complex, and they cannot be understood intuitively. Upon observing the lacuna in the Arabidopsis interactome (an interaction map of the factors involved in the regulation of Arabidopsis secondary metabolism is not available), we attempted to connect various cellular processes that affect anthocyanin biosynthesis. In this review, we revealed the main signaling protein modules that regulate anthocyanin biosynthesis. To our knowledge, this is the first reconstruction of a network of proteins involved in plant secondary metabolism.     

Sensing trehalose biosynthesis in plants

A most unexpected finding in research on plant carbohydrate metabolism is the recent discovery that angiosperms encode genes whose products are involved in trehalose metabolism. The presence and functionality of such genes has been elegantly shown by expressing Arabidopsis-derived trehalose phosphate synthase and trehalose phosphate phosphatase genes in yeast mutants lacking these enzymatic activities. Homologue sequences have now been cloned from a number of different plant species suggesting that the capacity to synthesise trehalose is ubiquitous in angiosperms. Except for Myrothamnus flabellifolius, trehalose biosynthesis has never been observed in tissues of higher plants, probably due to the presence of high levels of trehalase activity. The function of trehalose metabolism in plants is still a mystery. One of the postulated functions of trehalose metabolism in yeast is in the control of glucose repression and a similar function in sugar sensing can be proposed for plants as well.     

Protein ubiquitination in plant peroxisomes

Protein ubiquitination regulates diverse cellular processes in eukaryotic organisms,from growth and development to stress response.Proteins subjected to ubiquitination can be found in virtually all subcellular locations and organelles,including peroxisomes,singlemembrane and highly dynamic organelles ubiquitous in eukaryotes.Peroxisomes contain metabolic functions essential to plants and animals such as lipid catabolism,detoxification of reactive oxygen species(ROS),biosynthesis of vital hormone...     

Local auxin production: a small contribution to a big field

Auxin is a plant growth regulator involved in diverse fundamental developmental responses. Much is now known about auxin transport, via influx and efflux carriers, and about auxin perception and its role in gene regulation. Many developmental processes are dependent on peaks of auxin concentration and, to date, attention has been directed at the role of polar auxin transport in generating and maintaining auxin gradients. However, surprisingly little attention has focussed on the role and significance of auxin biosynthesis, which should be expected to contribute to active auxin pools. Recent reports on the function of the YUCCA flavin monooxygenases and a tryptophan aminotransferase in Arabidopsis have caused us to look again at the importance of local biosynthesis in developmental processes. Many alternative and redundant pathways of auxin synthesis exist in many plants and it is emerging that they may function in response to environmental cues.     

Brassinosteroids fine-tune secondary and primary sulfur metabolism through BZR1-mediated transcriptional regulation

For adaptation to ever-changing environments,plants have evolved elaborate metabolic systems coupled to a regulatory network for optimal growth and defense. Regulation of plant secondary metabolic pathways such as glucosinolates(GSLs) by defense phytohormones in response to different stresses and nutrient deficiency has been intensively investigated, while how growth-promoting hormone balances plant secondary and primary metabolism has been largely unexplored. Here, we found that growth-promotin...     

Expression of a Brassica Isopropylmalate Synthase Gene in Arabidopsis Perturbs Both Glucosinolate and Amino Acid Metabolism

Isopropylmalate synthase (IPMS) is a key enzyme in the biosynthesis of the essential amino acid leucine, and thus primary metabolism. In Arabidopsis, the functionally similar enzyme, methythiolalkylmalate synthase (MAM), is an important enzyme in the elongation of methionine prior to glucosinolate (GSL) biosynthesis, as part of secondary metabolism. We describe the cloning of an IPMS gene from Brassica, BatIMS, and its functional characterisation by heterologous expression in E. coli and Arabidopsis. Over expression of BatIMS in Arabidopsis resulted in plants with an aberrant phenotype, reminiscent of mutants in GSL biosynthesis. Metabolite analyses showed that these plants had both perturbed amino acid metabolism and enhanced levels of GSLs. Microarray profiling showed that BatIMS over expression caused up regulation of the genes for methionine-derived GSL biosynthesis, and down regulation of genes involved in leucine catabolism, in addition to perturbed expression of genes involved in auxin and ethylene metabolism. The results illustrate the cross talk that can occur between primary and secondary metabolism within transgenic plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C–C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.     

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli

Summary In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.     

Current aspects of auxin biosynthesis in plants

Auxin is an important plant hormone essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most studied auxin in plants, and its biosynthesis pathway has been investigated for over 70 years. Although the complete picture of auxin biosynthesis remains to be elucidated, remarkable progress has been made recently in understanding the mechanism of IAA biosynthesis. Genetic and biochemical studies demonstrate that IAA is mainly synthesized from l-tryptophan (Trp) via indole-3-pyruvate by two-step reactions in Arabidopsis. While IAA is also produced from Trp via indole-3-acetaldoxime in Arabidopsis, this pathway likely plays an auxiliary role in plants of the family Brassicaceae. Recent studies suggest that the Trp-independent pathway is not a major route for IAA biosynthesis, but they reveal an important role for a cytosolic indole synthase in this pathway. In this review, I summarize current views and future prospects of IAA biosynthesis research in plants.     

Roles of YUCCAs in auxin biosynthesis and drought stress responses in plants

Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.¹ The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.     

Interplay Between Abscisic Acid and Jasmonic Acid and its Role in Water-oxidative Stress in Wild-type, ABA-deficient, JA-deficient, and Ascorbate-deficient Arabidopsis Plants

The interplay between jasmonic acid (JA) and abscisic acid (ABA) in plant responses to water stress and in water-stress-enhanced oxidative stress was investigated in Arabidopsis thaliana plants subjected to water stress by water deprivation. For this purpose a drought assay was conducted using Arabidopsis mutants impaired in ABA (aba2), JA (aos), and ascorbate (vtc1) biosynthesis. Our results show an interaction between ABA and JA during their biosynthesis. Moreover, the coordinated action of ABA and JA protected wild-type, aba2, and aos plants from the effects of stress. However, this effect was not observed in the vtc1 mutant, which showed a distinct decrease in the F _v/F _m ratio, concomitant with a marked fall in relative water content (RWC), despite high endogenous concentrations of JA and ABA. This finding indicates the relevance of ascorbate metabolism in plant acclimation to stress. Despite the interaction between the two phytohormones, drought-associated stomatal closure is regulated mainly by ABA and weakly by JA, whereas JA plays a role in the formation of antioxidants regulating ascorbate and glutathione metabolism. A time course analysis revealed the relevance of plant age and stress duration in the responses of the mutants compared to wild-type plants. Here we discuss the relationship between ABA, JA, ascorbate, and glutathione in plants under water stress.     

Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9, affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, α-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known α-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium. Received: 21 October 1996 / Accepted: 23 September 1997     

The role of hexokinase in plant sugar signal transduction and growth and development

Previous studies have revealed a central role of Arabidopsis thaliana hexokinases (AtHXK1 and AtHXK2) in the glucose repression of photosynthetic genes and early seedling development. However, it remains unclear whether HXK can modulate the expression of diverse sugar-regulated genes. On the basis of the results of analyses of gene expression in HXK transgenic plants, we suggest that three distinct glucose signal transduction pathways exist in plants. The first is an AtHXK1-dependent pathway in which gene expression is correlated with the AtHXK1-mediated signaling function. The second is a glycolysis-dependent pathway that is influenced by the catalytic activity of both AtHXK1 and the heterologous yeast Hxk2. The last is an AtHXK1-independent pathway in which gene expression is independent of AtHXK1. Further investigation of HXK transgenic Arabidopsis discloses a role of HXK in glucose-dependent growth and senescence. In the absence of exogenous glucose, plant growth is limited to the seedling stage with restricted true leaf development even after a 3-week culture on MS medium. In the presence of glucose, however, over-expressing Arabidopsis or yeast HXK in plants results in the repression of growth and true leaf development, and early senescence, while under-expressing AtHXK1 delays the senescence process. These studies reveal multiple glucose signal transduction pathways that control diverse genes and processes that are intimately linked to developmental stages and environmental conditions.     

Evolutionary analysis of aspartate aminotransferases

Aspartate aminotransferase isoenzymes are located in both the cytosol and organelles of eukaryotes, but all are encoded in the nuclear genome. In the work described here, a phylogenetic analysis was made of aspartate aminotransferases from plants, animals, yeast, and a number of bacteria. This analysis suggested that five distinct branches are present in the aspartate aminotransferase tree. Mitochondrial forms of the enzyme form one distinct group, bacterial aspartate aminotransferase formed another, and the plant and vertebrate cytosolic isoenzymes each formed a distinct group. Plant cytosolic isozymes formed a further group of which the plastid sequences were a member. The yeast mitochondrial and cytosolic aspartate aminotransferases formed groups separate from other members of the family. Correspondence to: C.J. Marshall     

A new member of plant CS-lyases. A cystine lyase from Arabidopsis thaliana

Cystine lyases catalyze the breakdown of l-cystine to thiocysteine, pyruvate, and ammonia. Until now there are no reports of the identification of a plant cystine lyase at a molecular level, and it is not clear what biological role this class of enzymes have in plants. A cystine lyase was isolated from Brassica oleracea (L.), and partial amino acid sequencing allowed the corresponding full-length cDNA (BOCL3) to be cloned. The deduced amino acid sequence of BOCL3 showed highest homology to the deduced amino acid sequences of several Arabidopsis thaliana genes annotated as tyrosine aminotransferase-like, including a coronatine, jasmonic acid, and salt stress-inducible gene, CORI3 (78.8% identity), and the unidentified rooty/superroot1 gene (44.8% identity). A full-length expressed sequence tag clone of CORI3 was obtained and recombinant CORI3 was synthesized in Escherichia coli. Isolated recombinant CORI3 catalyzed a cystine lyase reaction, but no aminotransferase reactions. The present study identifies, for the first time, a cystine lyase from plants at a molecular level and redefines the functional assignment of the only functionally identified member of a group of A. thaliana genes annotated as tyrosine aminotransferase-like.