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...     

Isolation and expression analysis of the isopropylmalate synthase gene family of Arabidopsis thaliana

Isopropylmalate synthase (IPMS) is the first enzyme in the leucine biosynthetic pathway. It is the branch point in the biosynthesis of leucine and the other branched-chain amino acids. IPMS is also regulated by negative feedback inhibition by the end-product leucine. There are four highly homologous loci within the Arabidopsis thaliana genome, which contain sequences that code for IPMS. Through library screening and RT-PCR the expression patterns of three of these loci namely IMS1, IMS2, and IMS3 have been isolated and then characterized. cDNAs of IMS2 and IMS3 lacking the 5' chloroplast leader sequence were able to complement a leucine auxotroph of E. coli.     

From amino acid to glucosinolate biosynthesis: protein sequence changes in the evolution of methylthioalkylmalate synthase in Arabidopsis

Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the k(cat)/K(m) for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism.     

A <Emphasis Type="Italic">Sordaria macrospora</Emphasis> mutant lacking the <Emphasis Type="Italic">leu1</Emphasis> gene shows a developmental arrest during fruiting body formation

Developmental mutants with defects in fruiting body formation are excellent resources for the identification of genetic components that control cellular differentiation processes in filamentous fungi. The mutant pro4 of the ascomycete Sordaria macrospora is characterized by a developmental arrest during the sexual life cycle. This mutant generates only pre-fruiting bodies (protoperithecia), and is unable to form ascospores. Besides being sterile, pro4 is auxotrophic for leucine. Ascospore analysis revealed that the two phenotypes are genetically linked. After isolation of the wild-type leu1 gene from S. macrospora, complementation experiments demonstrated that the gene was able to restore both prototrophy and fertility in pro4. To investigate the control of leu1 expression, other genes involved in leucine biosynthesis specifically and in the general control of amino acid biosynthesis (“cross-pathway control”) have been analysed using Northern hybridization and quantitative RT-PCR. These analyses demonstrated that genes of leucine biosynthesis are transcribed at higher levels under conditions of amino acid starvation. In addition, the expression data for the cpc1 and cpc2 genes indicate that cross-pathway control is superimposed on leucine-specific regulation of fruiting body development in the leu1 mutant. This was further substantiated by growth experiments in which the wild-type strain was found to show a sterile phenotype when grown on a medium containing the amino acid analogue 5-methyl-tryptophan. Taken together, these data show that pro4 represents a novel mutant type in S. macrospora, in which amino acid starvation acts as a signal that interrupts the development of the fruiting body. Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00438-005-0021-8     

Molecular modeling of the effects of mutant alleles on chalcone synthase protein structure

Chalcone synthase (CHS) catalyzes the first committed step in flavonoid biosynthesis, a major pathway of plant secondary metabolism. An allelic series for the Arabidopsis CHS locus, tt4, was previously characterized at the gene, protein, and end-product levels. In an effort to deduce the molecular basis for the observed phenotypes, homology models were generated for five of the tt4 proteins based on the crystal structure of CHS2 from Medicago. Molecular dynamics simulations provided insights into how even those substitutions that are not in close spatial proximity to key functional residues may still alter the architecture and dynamic movement of the enzyme, with dramatic effects on enzyme function. Simulations carried out at different temperatures pointed to optimized positioning of key residues in the active site or dimerization domain, rather than enhancement of overall structure, as underlying the higher activity of two temperature-sensitive variants at lower temperatures. Extending this type of analysis to account for protein–protein interactions may offer additional insights into the mechanisms by which single amino-acid substitutions can affect diverse aspects of protein function.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users     

A redox‐active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis

We report a detailed functional characterization of an Arabidopsis isopropylmalate dehydrogenase (AtIPMDH1) that is involved in both glucosinolate biosynthesis and leucine biosynthesis. AtIPMDH1 shares high homology with enzymes from bacteria and yeast that are known to function in leucine biosynthesis. In plants, AtIPMDH1 is co‐expressed with nearly all the genes known to be involved in aliphatic glucosinolate biosynthesis. Mutation of AtIPMDH1 leads to a significant reduction in the levels of free leucine and of glucosinolates with side chains of four or more carbons. Complementation of the mutant phenotype by ectopic expression of AtIPMDH1, together with the enzyme’s substrate specificity, implicates AtIPMDH1 in both glucosinolate and leucine biosynthesis. This functional assignment is substantiated by subcellular localization of the protein in the chloroplast stroma, and the gene expression patterns in various tissues. Interestingly, AtIPMDH1 activity is regulated by a thiol‐based redox modification. This work characterized an enzyme in plants that catalyzes the oxidative decarboxylation step in both leucine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabolism). It provides evidence for the hypothesis that the two pathways share a common origin, and suggests a role for redox regulation of glucosinolate and leucine synthesis in plants.     

Improved drought tolerance without undesired side effects in transgenic plants producing trehalose

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.     

Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis

To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.     

Engineering isoflavone metabolism with an artificial bifunctional enzyme

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

蓝灰链霉菌中Aco类信号分子合酶ScyA1全局性功能的研究北大核心

【目的】研究蓝灰链霉菌中Aco类群感效应信号分子合酶基因scy A1缺失对细胞生理代谢和调控网络造成的广泛性扰动,揭示Scy A1对细胞生命活动的全局性调控作用。【方法】通过测定细胞干重确定scy A1缺失对液体发酵条件下细胞生长的影响。采用RNA-Seq比较分析探究蓝灰链霉菌scy A1突变株和野生型NMWT1发酵培养3d和6d全基因组范围内的显著差异表达基因。【结果】scy A1缺失不影响液体发酵条件下细胞生物量的积累。比较转录组分析显示scy A1缺失后,糖酵解和三羧酸循环途径基因表达表现出双向显著差异变化趋势;L-半乳糖形成UDP-葡萄糖途径、戊糖磷酸途径、氨基酸(L-缬氨酸、L-异亮氨酸和L-色氨酸)合成途径和嘌呤核苷酸降解途径相关基因均表现出显著上调趋势。众多次级代谢生物合成基因簇、保守转录调控因子和菌丝体结构性蛋白组分编码基因表达显著下调,而少数则表达水平显著上调。【结论】Scy A1广泛影响了菌株的初级代谢、次级代谢、保守调控因子和菌丝体结构相关基因的表达。总之,本研究丰富了我们对Aco类群感效应信号分子合酶功能的认知。     

Glucosinolate biosynthesis: role of MAM synthase and its perspectives

Glucosinolates, synthesized by the glucosinolate biosynthesis pathway, are the secondary metabolites used as a defence mechanism in the Brassicaceae plants, including Arabidopsis thaliana. The first committed step in the pathway, catalysed by methylthioalkylmalate (MAM) synthase (EC: 2.3.3.17), is to produce different variants of glucosinolates. Phylogenetic analyses suggest that possibly MAM synthases have been evolved from isopropylmalate synthase (IPMS) by the substitutions of five amino acid residues (L143I, H167L, S216G, N250G and P252G) in the active site of IPMS due to point mutations. Considering the importance of MAM synthase in Brassicaceae plants, Petersen et al. (2019) made an effort to characterise the MAM synthase (15 MAM1 variants) in vitro by single substitution or double substitutions. In their study, the authors have expressed the variants in Escherichia coli and analysed the amino acids in the cultures of E. coli in vivo. Since modifying the MAM synthases by transgenic approaches could increase the resistance of Brassicaceae plants for enhancing the defence effect of glucosinolates and their degraded products; hence, MAM synthases should be characterized in detail in vivo in A. thaliana along with the structural analysis of the enzyme for meaningful impact and for its imminent use in vivo.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A common F-box gene regulates the leucine homeostasis of Medicago truncatula and Arabidopsis thaliana

The F-box domain is a conserved structural protein motif that most frequently interacts with the SKP1 protein, the core of the SCFs (SKP1-CULLIN-F-box protein ligase) E3 ubiquitin protein ligases. As part of the SCF complexes, the various F-box proteins recruit substrates for degradation through ubiquitination. In this study, we functionally characterized an F-box gene (MtF-box) identified earlier in a population of Tnt1 retrotransposon-tagged mutants of Medicago truncatula and its Arabidopsis thaliana homolog (AtF-box) using gain- and loss-of-function plants. We highlighted the importance of MtF-box in leaf development of M. truncatula. Protein–protein interaction analyses revealed the 2-isopropylmalate synthase (IPMS) protein as a common interactor partner of MtF-box and AtF-box, being a key enzyme in the biosynthesis pathway of the branched-chain amino acid leucine. For further detailed analysis, we focused on AtF-box and its role during the cell division cycle. Based on this work, we suggest a mechanism for the role of the studied F-box gene in regulation of leucine homeostasis, which is important for growth.

     

Two callose synthases,GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility

Callose, a β-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1–12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.     

Vitamin E biosynthesis genes in rice: Molecular characterization, expression profiling and comparative phylogenetic analysis

Vitamin E comprises four tocopherols and four tocotrienols, collectively termed tocochromanols that play an essential role as antioxidants in humans, animals and photosynthetic organisms and are also believed to play a role in modulation of signal transduction and gene expression pathways. In rice and Populus genome, we have identified 7 and 11 tocochromanol biosynthesis genes, respectively. A detailed study of domain organization and phylogenetic analysis of these genes in rice, Arabidopsis and other plants has revealed the presence of homologous genes. Expression profiling of rice and Populus genes has been done by full-length cDNA and EST-based analysis. In rice, real-time PCR analysis was done to reveal the light-regulated expression pattern. Microarray-based expression analysis in different rice tissues and developmental stages revealed expression of these genes in almost all plant tissues/organs. Under abiotic stress conditions, expression of gene coding for HPPD enzyme, that regulates pathway flux, was also found to be increased. This information is expected to be helpful for further functional characterization of tocochromanol biosynthesis genes in different plant tissues under diverse growth conditions.     

Cation induced differential effect on structural and functional properties of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> α-Isopropylmalate synthase

Background  

α-isopropylmalate synthase (MtαIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtαIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.     

A valine-resistant mutant ofArabidopsis thaliana displays an acetolactate synthase with altered feedback control

A valine-resistant mutant line, VAL-2, ofArabidopsis thaliana (L.) Heynh. was identified by screening M 2 populations of ethylmethane-sulfonate-mutagenized seeds. The resistance was found to be due to a single, dominant, nuclear gene mutation. Assay of acetolactate synthase (ALS) indicated that the valine resistance in this mutant is caused by decreased sensitivity of ALS to the branched-chain amino acids, valine, leucine andisoleucine. A two fold decrease in apparentK _m value for pyruvate of the mutant ALS enzyme was detected compared with that of the wild type. The sensitivity of the ALS enzyme to sulfonylurea, imidazolinone and triazolopyrimidine herbicides was not altered in the mutant. At the plant growth level the mutant was also resistant to valine plus leucine, but was sensitive to leucine orisoleucine alone. The mutant gene,var1, maps, or is very closely linked, toCSR1, the gene encoding acetolactate synthase inArabidopsis.Abbreviations ALS acetolactate synthase - BCAA branched-chain amino acid - CS chlorsulfuron - IM imidazolinone - SU sulfonylurea - TP triazolopyrimidine We thank Dr. George W. Haughn for providing Arabidopsis lines MSU12, MSU15, MSU21, MSU22 and MSU23. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K., K.W. is grateful for a University of Saskatchewan Graduate Scholarship.