Tamara Gigolashvili

Disruption of adenosine-5'-phosphosulfate kinase in Arabidopsis reduces levels of sulfated secondary metabolites

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5′-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 3′-phosphoadenosine 5′-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.

MYB34, MYB51, and MYB122 distinctly regulate indolic glucosinolate biosynthesis in Arabidopsis thaliana.

The MYB34, MYB51, and MYB122 transcription factors are known to regulate indolic glucosinolate (IG) biosynthesis in Arabidopsis thaliana. To determine the distinct regulatory potential of MYB34, MYB51, and MYB122, the accumulation of IGs in different parts of plants and upon treatment with plant hormones were analyzed in A. thaliana seedlings. It was shown that MYB34, MYB51, and MYB122 act together to control the biosynthesis of I3M in shoots and roots, with MYB34 controlling biosynthesis of IGs mainly in the roots, MYB51 regulating biosynthesis in shoots, and MYB122 having an accessory role in the biosynthesis of IGs. Analysis of glucosinolate levels in seedlings of myb34, myb51, myb122, myb34 myb51 double, and myb34 myb51 myb122 triple knockout mutants grown in the presence of abscisic acid (ABA), salicylic acid (SA), jasmonate (JA), or ethylene (ET) revealed that: (1) MYB51 is the central regulator of IG synthesis upon SA and ET signaling, (2) MYB34 is the key regulator upon ABA and JA signaling, and (3) MYB122 plays only a minor role in JA/ET-induced glucosinolate biosynthesis. The myb34 myb51 myb122 triple mutant is devoid of IGs, indicating that these three MYB factors are indispensable for IG production under standard growth conditions.

The Plastidic Bile Acid Transporter 5 Is Required for the Biosynthesis of Methionine-Derived Glucosinolates in Arabidopsis thaliana

Aliphatic glucosinolate biosynthesis is highly compartmentalized, requiring import of 2-keto acids or amino acids into chloroplasts for side chain elongation and export of the resulting compounds into the cytosol for conversion into glucosinolate. Aliphatic glucosinolate biosynthesis in Arabidopsis thaliana is regulated by three R2R3-MYB transcription factors, the major player being High Aliphatic Glucosinolate 1 (HAG1/MYB28). Here, we show that BAT5, which belongs to the putative bile acid transporter family, is the only member of this family that is transactivated by HAG1/MYB28, HAG2/MYB76, and HAG3/MYB29. Furthermore, two isopropylmalate isomerases genes, IPMI1 and IPMI2, and the isopropylmalate dehydrogenase gene, IPMDH1, were identified as targets of HAG1/MYB28 and the corresponding proteins localized to plastids, suggesting a role in plastidic chain elongation reactions. The BAT proteins also localized to plastids; however, only mutants defective in BAT5 function contained strongly reduced levels of aliphatic glucosinolates. The bat5 mutant chemotype was rescued by induced overexpression of BAT5. Feeding experiments using 2-keto acids and amino acids of different chain length suggest that BAT5 is a plastidic transporter of (chain-elongated) 2-keto acids. Mechanical stimuli and methyl jasmonate transiently induced BAT5 expression in inflorescences and leaves. Thus, BAT5 was identified as the first transporter component of the aliphatic glucosinolate biosynthetic pathway.

Specific and coordinated control of indolic and aliphatic glucosinolate biosynthesis by R2R3-MYB transcription factors in Arabidopsis thaliana

Five members of subgroup 12 R2R3-MYB transcription factors, namely MYB51, MYB122, MYB28, MYB29 and MYB76, are novel regulators of glucosinolate biosynthesis in Arabidopsis thaliana. Overexpression of MYB51 and MYB122 led to an increased accumulation of tryptophan-derived indolic glucosinolates whereas MYB28, MYB29 and MYB76 overexpression lines showed an increase in methionine-derived aliphatic glucosinolates. Likewise, disruption of the corresponding genes caused a significant downregulation of indolic and aliphatic glucosinolates, respectively. Expression analysis of promoter-GUS fusions revealed promoter activities at the sites of glucosinolate synthesis and accumulation. Indolic glucosinolate regulators were mainly found in vegetative organs and roots, whereas aliphatic glucosinolate regulators were preferentially expressed in generative organs. Mechanical stimuli such as touch or wounding induced a transient expression of the regulators and overexpression of MYB28 and MYB51 reduced insect performance demonstrating the role of these transcription factors in plant biotic responses. The subgroup 12 R2R3-MYB transcription factors interdependently control the response to biotic challenges. For the regulation of methionine-derived glucosinolates, the coordinated activation of MYB28, MYB76 and MYB29 is required, whereas MYB51, MYB122 and the sixth member of subgroup 12 R2R3-MYB transcription factors, the previously described ATR1/MYB34, are involved in the regulation of tryptophan-derived glucosinolates. Because these two pathways are reciprocally inhibiting each other, a metabolic balance between both biosynthetic pathways can be accomplished in plants exposed to continuous biotic challenges.

Transporters in plant sulfur metabolism.

Sulfur is an essential nutrient, necessary for synthesis of many metabolites. The uptake of sulfate, primary and secondary assimilation, the biosynthesis, storage, and final utilization of sulfur (S) containing compounds requires a lot of movement between organs, cells, and organelles. Efficient transport systems of S-containing compounds across the internal barriers or the plasma membrane and organellar membranes are therefore required. Here, we review a current state of knowledge of the transport of a range of S-containing metabolites within and between the cells as well as of their long distance transport. An improved understanding of mechanisms and regulation of transport will facilitate successful engineering of the respective pathways, to improve the plant yield, biotic interaction and nutritional properties of crops.

The Arabidopsis Thylakoid ADP/ATP Carrier TAAC Has an Additional Role in Supplying Plastidic Phosphoadenosine 5′-Phosphosulfate to the Cytosol

This study shows that Arabidopsis thaliana TAAC is a plant PAPS transporter (PAPST1). Its functional characterization and the analysis of corresponding mutants demonstrate that TAAC/PAPST1 connects plastidic PAPS synthesis and cytosolic sulfation reactions. In contrast with the known animal PAPS antiporters that are members of the nucleotide-sugar transporter family, TAAC/PAPST1 belongs to the mitochondrial carrier family. 3′-Phosphoadenosine 5′-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3′-phosphoadenosine 5′-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide-sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines.

Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non‐root‐hair cells, but not in the mesophyll of leaves. Two T‐DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.

Arabidopsis Phosphoglycerate Dehydrogenase1 of the Phosphoserine Pathway Is Essential for Development and Required for Ammonium Assimilation and Tryptophan Biosynthesis

Biosynthesis of the amino acid serine occurs mainly via photorespiration in plants. This work shows, however, that locally restricted serine biosynthesis via the alternative phosphoserine pathway is required for proper embryo development and leaf initiation, highlighting the importance of cellular resolution when analyzing metabolic pathways. In plants, two independent serine biosynthetic pathways, the photorespiratory and glycolytic phosphoserine (PS) pathways, have been postulated. Although the photorespiratory pathway is well characterized, little information is available on the function of the PS pathway in plants. Here, we present a detailed characterization of phosphoglycerate dehydrogenases (PGDHs) as components of the PS pathway in Arabidopsis thaliana. All PGDHs localize to plastids and possess similar kinetic properties, but they differ with respect to their sensitivity to serine feedback inhibition. Furthermore, analysis of pgdh1 and phosphoserine phosphatase mutants revealed an embryo-lethal phenotype and PGDH1-silenced lines were inhibited in growth. Metabolic analyses of PGDH1-silenced lines grown under ambient and high CO2 conditions indicate a direct link between PS biosynthesis and ammonium assimilation. In addition, we obtained several lines of evidence for an interconnection between PS and tryptophan biosynthesis, because the expression of PGDH1 and PHOSPHOSERINE AMINOTRANSFERASE1 is regulated by MYB51 and MYB34, two activators of tryptophan biosynthesis. Moreover, the concentration of tryptophan-derived glucosinolates and auxin were reduced in PGDH1-silenced plants. In essence, our results provide evidence for a vital function of PS biosynthesis for plant development and metabolism.

bHLH05 Is an Interaction Partner of MYB51 and a Novel Regulator of Glucosinolate Biosynthesis in Arabidopsis

Protein-protein interaction studies of R2R3 MYB transcription factors regulating glucosinolate biosynthesis and the analysis of multiple loss-of-function mutants and gain-of-function alleles demonstrated the specific role of an associated transcription factor complex in the transcriptional regulation of glucosinolate biosynthesis. By means of yeast (Saccharomyces cerevisiae) two-hybrid screening, we identified basic helix-loop-helix transcription factor05 (bHLH05) as an interacting partner of MYB51, the key regulator of indolic glucosinolates (GSLs) in Arabidopsis (Arabidopsis thaliana). Furthermore, we show that bHLH04, bHLH05, and bHLH06/MYC2 also interact with other R2R3-MYBs regulating GSL biosynthesis. Analysis of bhlh loss-of-function mutants revealed that the single bhlh mutants retained GSL levels that were similar to those in wild-type plants, whereas the triple bhlh04/05/06 mutant was depleted in the production of GSL. Unlike bhlh04/06 and bhlh05/06 mutants, the double bhlh04/05 mutant was strongly affected in the production of GSL, pointing to a special role of bHLH04 and bHLH05 in the control of GSL levels in the absence of jasmonic acid. The combination of two specific gain-of-function alleles of MYB and bHLH proteins had an additive effect on GSL levels, as demonstrated by the analysis of the double MYB34-1D bHLH05D94N mutant, which produces 20-fold more indolic GSLs than bHLH05D94N and ecotype Columbia-0 of Arabidopsis. The amino acid substitution D94N in bHLH05D94N negatively affects the interaction with JASMONATE-ZIM DOMAIN protein, thereby resulting in constitutive activation of bHLH05 and mimicking jasmonic acid treatment. Our study revealed the bHLH04, bHLH05, and bHLH06/MYC2 factors as novel regulators of GSL biosynthesis in Arabidopsis.

A transposon-based activation-tagging population in Arabidopsis thaliana (TAMARA) and its application in the identification of dominant developmental and metabolic mutations

A population of 9471 stable activation‐tagged lines was generated by transposable element mediated activation tagging mutagenesis in Arabidopsis (TAMARA) using the maize En/Spm transposon system. Based on DNA gel blot and flanking sequence analysis, this population contains approximately 6000 independent transposon insertions. A greenhouse‐based screen identified six dominant or semi‐dominant activation tagged mutants with obvious developmental alterations, among these a new pistillata mutant allele. In addition, a subset of 1500 lines was screened by a HPLC based high‐throughput method for dominant activation tagged mutants with enhanced contents of phenolic compounds. One dominant activation tagged mutant (hpc1‐1D) was isolated showing accumulation of a particular compound due to the upregulation of an R2R3‐MYB transcription factor.