New Insights on the Regulation of Glucosinolate Biosynthesis via COP1 and DELLA Proteins in Arabidopsis Thaliana.

The biosynthesis of defensive secondary metabolites, such as glucosinolates (GSLs), is a costly process, which requires nutrients, ATP, and reduction equivalents, and, therefore, needs well-orchestrated machinery while coordinating defense and growth. We discovered that the key repressor of light signaling, the CONSTITUTIVE PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYTOCHROME A-105 (COP1/SPA) complex, is a crucial component of GSL biosynthesis regulation. Various mutants in this COP1/SPA complex exhibited a strongly reduced level of GSL and a low expression of jasmonate (JA)-dependent genes. Furthermore, cop1, which is known to accumulate DELLA proteins in the dark, shows reduced gibberellin (GA) and JA signaling, thereby phenocopying other DELLA-accumulating mutants. This phenotype can be complemented by a dominant gain-of-function allele of MYC3 and by crossing with a mutant having low DELLA protein levels. Hence, SPA1 interacts with DELLA proteins in a yeast two-hybrid screen, whereas high levels of DELLA inhibit MYC function and suppress JA signaling. DELLA accumulation leads to reduced synthesis of GSL and inhibited growth. Thus, the COP1/SPA-mediated degradation of DELLA not only affects growth but also regulates the biosynthesis of GSLs.

Dissecting the Role of SAL1 in Metabolizing the Stress Signaling Molecule 3'-Phosphoadenosine 5'-Phosphate in Different Cell Compartments.

Plants possess the most highly compartmentalized eukaryotic cells. To coordinate their intracellular functions, plastids and the mitochondria are dependent on the flow of information to and from the nuclei, known as retrograde and anterograde signals. One mobile retrograde signaling molecule is the monophosphate 3'-phosphoadenosine 5'-phosphate (PAP), which is mainly produced from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the cytosol and regulates the expression of a set of nuclear genes that modulate plant growth in response to biotic and abiotic stresses. The adenosine bisphosphate phosphatase enzyme SAL1 dephosphorylates PAP to AMP in plastids and the mitochondria, but can also rescue sal1 Arabidopsis phenotypes (PAP accumulation, leaf morphology, growth, etc.) when expressed in the cytosol and the nucleus. To understand better the roles of the SAL1 protein in chloroplasts, the mitochondria, nuclei, and the cytosol, we have attempted to complement the sal1 mutant by specifically cargoing the transgenic SAL1 protein to these four cell compartments. Overexpression of SAL1 protein targeted to the nucleus or the mitochondria alone, or co-targeted to chloroplasts and the mitochondria, complemented most aspects of the sal1 phenotypes. Notably, targeting SAL1 to chloroplasts or the cytosol did not effectively rescue the sal1 phenotypes as these transgenic lines accumulated very low levels of SAL1 protein despite overexpressing SAL1 mRNA, suggesting a possibly lower stability of the SAL1 protein in these compartments. The diverse transgenic SAL1 lines exhibited a range of PAP levels. The latter needs to reach certain thresholds in the cell for its impacts on different processes such as leaf growth, regulation of rosette morphology, sulfate homeostasis, and glucosinolate biosynthesis. Collectively, these findings provide an initial platform for further dissection of the role of the SAL1-PAP pathway in different cellular processes under stress conditions.

Regulation of glucosinolate biosynthesis.

Glucosinolates are secondary defense metabolites produced by plants of the order Brassicales, which includes the model species Arabidopsis and many crop species. In the past 13 years, the regulation of glucosinolate synthesis in plants has been intensively studied, with recent research revealing complex molecular mechanisms that connect glucosinolate production with responses to other central pathways. In this review, we discuss how the regulation of glucosinolate biosynthesis is ecologically relevant for plants, how it is controlled by transcription factors, and how this transcriptional machinery interacts with hormonal, environmental, and epigenetic mechanisms. We present the central players in glucosinolate regulation, MYB and basic helix-loop-helix transcription factors, as well as the plant hormone jasmonate, which together with other hormones and environmental signals allow the coordinated and rapid regulation of glucosinolate genes. Furthermore, we highlight the regulatory connections between glucosinolates, auxin, and sulfur metabolism and discuss emerging insights and open questions on the regulation of glucosinolate biosynthesis.

PAPST2 Plays Critical Roles in Removing the Stress Signaling Molecule 3'-Phosphoadenosine 5'-Phosphate from the Cytosol and Its Subsequent Degradation in Plastids and Mitochondria.

The compartmentalization of PAPS (the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate) synthesis (mainly in plastids), PAPS consumption (in the cytosol), and PAP (the stress signaling molecule 3'-phosphoadenosine 5'-phosphate) degradation (in plastids and mitochondria) requires organellar transport systems for both PAPS and PAP. The plastidial transporter PAPST1 (PAPS TRANSPORTER1) delivers newly synthesized PAPS from the stroma to the cytosol. We investigated the activity of PAPST2, the closest homolog of PAPST1, which unlike PAPST1 is targeted to both the plastids and mitochondria. Biochemical characterization in Arabidopsis thaliana revealed that PAPST2 mediates the antiport of PAP, PAPS, ATP, and ADP. Strongly increased cellular PAP levels negatively affect plant growth, as observed in the fry1 papst2 mutant, which lacks the PAP-catabolizing enzyme SALT TOLERANCE 1 and PAPST2. PAP levels were specifically elevated in the cytosol of papst2 and fiery1 papst2, but not in papst1 or fry1 papst1 PAPST1 failed to complement the papst2 mutant phenotype in mitochondria, because it likely removes PAPS from the cell, as demonstrated by the increased expression of phytosulfokine genes. Overexpression of SAL1 in mitochondria rescued the phenotype of fry1 but not fry1 papst2 Therefore, PAPST2 represents an important organellar importer of PAP, providing a piece of the puzzle in our understanding of the organelle-to-nucleus PAP retrograde signaling pathway.

AP2 transcription factor CBX1 with a specific function in symbiotic exchange of nutrients in mycorrhizal Lotus japonicus.

The arbuscular mycorrhizal (AM) symbiosis, a widespread mutualistic association between land plants and fungi, depends on reciprocal exchange of phosphorus driven by proton-coupled phosphate uptake into host plants and carbon supplied to AM fungi by host-dependent sugar and lipid biosynthesis. The molecular mechanisms and cis-regulatory modules underlying the control of phosphate uptake and de novo fatty acid synthesis in AM symbiosis are poorly understood. Here, we show that the AP2 family transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), a WRINKLED1 (WRI1) homolog, directly binds the evolutionary conserved CTTC motif that is enriched in mycorrhiza-regulated genes and activates Lotus japonicus phosphate transporter 4 (LjPT4) in vivo and in vitro. Moreover, the mycorrhiza-inducible gene encoding H+-ATPase (LjHA1), implicated in energizing nutrient uptake at the symbiotic interface across the periarbuscular membrane, is coregulated with LjPT4 by CBX1. Accordingly, CBX1-defective mutants show reduced mycorrhizal colonization. Furthermore, genome-wide-binding profiles, DNA-binding studies, and heterologous expression reveal additional binding of CBX1 to AW box, the consensus DNA-binding motif for WRI1, that is enriched in promoters of glycolysis and fatty acid biosynthesis genes. We show that CBX1 activates expression of lipid metabolic genes including glycerol-3-phosphate acyltransferase RAM2 implicated in acylglycerol biosynthesis. Our finding defines the role of CBX1 as a regulator of host genes involved in phosphate uptake and lipid synthesis through binding to the CTTC/AW molecular module, and supports a model underlying bidirectional exchange of phosphorus and carbon, a fundamental trait in the mutualistic AM symbiosis.

The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis.

Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.

Sulfur deficiency-induced repressor proteins optimize glucosinolate biosynthesis in plants.

Glucosinolates (GSLs) in the plant order of the Brassicales are sulfur-rich secondary metabolites that harbor antipathogenic and antiherbivory plant-protective functions and have medicinal properties, such as carcinopreventive and antibiotic activities. Plants repress GSL biosynthesis upon sulfur deficiency (-S); hence, field performance and medicinal quality are impaired by inadequate sulfate supply. The molecular mechanism that links -S to GSL biosynthesis has remained understudied. We report here the identification of the -S marker genes sulfur deficiency induced 1 (SDI1) and SDI2 acting as major repressors controlling GSL biosynthesis in Arabidopsis under -S condition. SDI1 and SDI2 expression negatively correlated with GSL biosynthesis in both transcript and metabolite levels. Principal components analysis of transcriptome data indicated that SDI1 regulates aliphatic GSL biosynthesis as part of -S response. SDI1 was localized to the nucleus and interacted with MYB28, a major transcription factor that promotes aliphatic GSL biosynthesis, in both yeast and plant cells. SDI1 inhibited the transcription of aliphatic GSL biosynthetic genes by maintaining the DNA binding composition in the form of an SDI1-MYB28 complex, leading to down-regulation of GSL biosynthesis and prioritization of sulfate usage for primary metabolites under sulfur-deprived conditions.

Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase.

Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.

Regulation of Pathogen-Triggered Tryptophan Metabolism in Arabidopsis thaliana by MYB Transcription Factors and Indole Glucosinolate Conversion Products.

MYB34, MYB51, and MYB122 transcription factors are known as decisive regulators of indolic glucosinolate (IG) biosynthesis with a strong impact on expression of genes encoding CYP79B2 and CYP79B3 enzymes that redundantly convert tryptophan to indole-3-acetaldoxime (IAOx). This intermediate represents a branching point for IG biosynthesis, and pathways leading to camalexin and indole-carboxylic acids (ICA). Here we investigate how these MYBs affect the pathogen-triggered Trp metabolism. Our experiments indicated that these three MYBs affect not only IG production but also constitutive biosynthesis of other IAOx-derived metabolites. Strikingly, the PENETRATION 2 (PEN2)-dependent IG-metabolism products, which are absent in myb34/51/122 and pen2 mutants, were indispensable for full flg22-mediated induction of other IAOx-derived compounds. However, gene induction and accumulation of ICAs and camalexin upon pathogen infection was not compromised in myb34/51/122 plants, despite strongly reduced IG levels. Hence, in comparison with cyp79B2/B3, which lacks all IAOx-derived metabolites, we found myb34/51/122 an ideal tool to analyze IG contribution to resistance against the necrotrophic fungal pathogen Plectosphaerella cucumerina. The susceptibility of myb34/51/122 was similar to that of pen2, but much lower than susceptibility of cyp79B2/B3, indicating that MYB34/51/122 contribute to resistance toward P. cucumerina exclusively through IG biosynthesis, and that PEN2 is the main leaf myrosinase activating IGs in response to microbial pathogens.

Trichoderma volatiles effecting Arabidopsis: from inhibition to protection against phytopathogenic fungi.

Trichoderma species are present in many ecosystems and some strains have the ability to reduce the severity of plant diseases by activating various defense pathways via specific biologically active signaling molecules. Hence we investigated the effects of low molecular weight volatile compounds of Trichoderma asperellum IsmT5 on Arabidopsis thaliana. During co-cultivation of T. asperellum IsmT5 without physical contact to A. thaliana we observed smaller but vital and robust plants. The exposed plants exhibit increased trichome numbers, accumulation of defense-related compounds such as H2O2, anthocyanin, camalexin, and increased expression of defense-related genes. We conclude that A. thaliana perceives the Trichoderma volatiles as stress compounds and subsequently initiates multilayered adaptations including activation of signaling cascades to withstand this environmental influence. The prominent headspace volatile of T. asperellum IsmT5 was identified to be 6-pentyl-α-pyrone (6PP), which was solely applied to A. thaliana to verify the growth and defense reactions. Most noticeable is that A. thaliana preexposed to 6PP showed significantly reduced symptoms when challenged with Botrytis cinerea and Alternaria brassicicola, indicating that defense-activated plants subsequently became more resistant to pathogen attack. Together, these results support that products that are based on Trichoderma volatiles have the potential being a useful biocontrol agent in agriculture.

The role of MYB34, MYB51 and MYB122 in the regulation of camalexin biosynthesis in Arabidopsis thaliana.

The phytoalexin camalexin and indolic glucosinolates share not only a common evolutionary origin and a tightly interconnected biosynthetic pathway, but regulatory proteins controlling the shared enzymatic steps are also modulated by the same R2R3-MYB transcription factors. The indolic phytoalexin camalexin is a crucial defense metabolite in the model plant Arabidopsis. Indolic phytoalexins and glucosinolates appear to have a common evolutionary origin and are interconnected on the biosynthetic level: a key intermediate in the biosynthesis of camalexin, indole-3-acetaldoxime (IAOx), is also required for the biosynthesis of indolic glucosinolates and is under tight control by the transcription factors MYB34, MYB51, and MYB122. The abundance of camalexin was strongly reduced in myb34/51 and myb51/122 double and in triple myb mutant, suggesting that these transcription factors are important in camalexin biosynthesis. Furthermore, expression of MYB51 and MYB122 was significantly increased by biotic and abiotic camalexin-inducing agents. Feeding of the triple myb34/51/122 mutant with IAOx or indole-3-acetonitrile largely restored camalexin biosynthesis. Conversely, tryptophan could not complement the low camalexin phenotype of this mutant, which supports a role for the three MYB factors in camalexin biosynthesis upstream of IAOx. Consistently expression of the camalexin biosynthesis genes CYP71B15/PAD3 and CYP71A13 was not negatively affected in the triple myb mutant and the MYBs could not activate pCYP71B15::uidA expression in trans-activation assays with cultured Arabidopsis cells. In conclusion, this study reveals the importance of MYB factors regulating the generation of IAOx as precursor of camalexin.

Classic myrosinase-dependent degradation of indole glucosinolate attenuates fumonisin B1-induced programmed cell death in Arabidopsis.

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1-induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1-induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col-0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase ß-thioglucoside glucohydrolase (TGG)-deficient double mutant tgg1 tgg2, rather than atypical myrosinase-deficient mutant pen2-2, is more sensitive to FB1 than Col-0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG-dependent IGS hydrolysis is involved in FB1-induced PCD. Indole-3-acetonitrile (IAN) and indole-3-carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS-scavenging ability. Despite the involvement of indole-3-acetic acid (IAA) in restricting FB1-induced PCD, feeding of IAN and I3C attenuated FB1-induced PCD in the IAA receptor mutant tir1-1 just as in Col-0. Taken together, our results indicate that TGG-catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA-independent way.

Update on the role of R2R3-MYBs in the regulation of glucosinolates upon sulfur deficiency.

To balance the flux of sulfur (S) into glucosinolates (GSL) and primary metabolites plants exploit various regulatory mechanisms particularly important upon S deficiency (-S). The role of MYB34, MYB51 and MYB122 controlling the production of indolic glucosinolates (IGs) and MYB28, MYB29, and MYB76 regulating the biosynthesis of aliphatic glucosinolates (AGs) in Arabidopsis thaliana has not been fully addressed at -S conditions yet. We show that the decline in the concentrations of GSL during S depletion does not coincide with the globally decreased transcription of R2R3-MYBs. Whereas the levels of GSL are diminished, the expression of MYB34, MYB51, MYB122, and MYB28 is hardly changed in early phase of S limitation. Furthermore, the mRNA levels of these MYBs start to raise under prolonged S starvation. In parallel, we found that SLIM1 can downregulate the MYBs in vitro as demonstrated in trans-activation assays in cultured Arabidopsis cells with SLIM1 as effector and ProMYB51:uidA as a reporter construct. However, in vivo, only the mRNA of MYB29 and MYB76 correlated with the levels of GSL at -S. We propose that the negative effect of SLIM1 on GSL regulatory genes can be overridden by a "low GSL signal" inducing the transcription of MYBs in a feedback regulatory loop. In accordance with this hypothesis, the expression of MYB34, MYB51, MYB122, and CYP83B1 was further induced in cyp79b2 cyp79b3 mutant exposed to -S conditions vs. cyp79b2 cyp79b3 plants grown on control medium. In addition, the possible role of MYBs in the regulation of essential S assimilation enzymes, in the regulation of GSL biosynthesis upon accelerated termination of life cycles, in the mobilization of auxin and lateral root formation at S deficiency is discussed.

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.

bHLH05 is an interaction partner of MYB51 and a novel regulator of glucosinolate biosynthesis in Arabidopsis.

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.

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.

Arabidopsis phosphoglycerate dehydrogenase1 of the phosphoserine pathway is essential for development and required for ammonium assimilation and tryptophan biosynthesis.

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.

Glucosinolates are produced in trichomes of Arabidopsis thaliana.

Glucosinolates (GS) are important plant secondary metabolites in plant resistance to herbivores, bacteria, and fungi, which have been shown to be accumulating in different organs and tissue types at varying concentrations. There are more than 200 GS species found in order Brassicales and presence of these compounds is well documented on organ-specific but not on cell-specific level. We used UPLC/ESI-QTOF-MS to measure the presence of GS and qRT-PCR to analyse the expression of GS biosynthetic and regulatory genes in isolated Arabidopsis thaliana trichomes. Trichomes of Arabidopsis are shown to synthesize chemoprotective aliphatic glucosinolates (AGS) and indolic glucosinolates (IGS), which are known for their biological activities against fungi, bacterial pathogens, or herbivores. UPLC/ESI-QTOF-MS analysis of various IGS mutants reveal increased or decreased levels of IGS in trichomes of gain- and loss-of-function mutants correspondingly. Using pMYB51/HIG1-uidA and pMYB28/PMG1/HAG1-uidA reporter plants we demonstrate that production of these important compounds is activated in trichomes of leaves or inflorescences in response to wounding. Since trichomes represent the first interface in plant-environment interactions, the possible role of GS containing trichomes in plant defense or signaling is discussed.

The Arabidopsis thylakoid ADP/ATP carrier TAAC has an additional role in supplying plastidic phosphoadenosine 5'-phosphosulfate to the cytosol.

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.

Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network in Arabidopsis thaliana.

Glucosinolates are plant secondary metabolites involved in responses to biotic stress. The final step of their synthesis is the transfer of a sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) onto a desulfo precursor. Thus, glucosinolate synthesis is linked to sulfate assimilation. The sulfate donor for this reaction is synthesized from sulfate in two steps catalyzed by ATP sulfurylase (ATPS) and adenosine 5'-phosphosulfate kinase (APK). Here we demonstrate that R2R3-MYB transcription factors, which are known to regulate both aliphatic and indolic glucosinolate biosynthesis in Arabidopsis thaliana, also control genes of primary sulfate metabolism. Using trans-activation assays we found that two isoforms of APK, APK1, and APK2, are regulated by both classes of glucosinolate MYB transcription factors; whereas two ATPS genes, ATPS1 and ATPS3, are differentially regulated by these two groups of MYB factors. In addition, we show that the adenosine 5'-phosphosulfate reductases APR1, APR2, and APR3, which participate in primary sulfate reduction, are also activated by the MYB factors. These observations were confirmed by analysis of transgenic lines with modulated expression levels of the glucosinolate MYB factors. The changes in transcript levels also affected enzyme activities, the thiol content and the sulfate reduction rate in some of the transgenic plants. Altogether the data revealed that the MYB transcription factors regulate genes of primary sulfate metabolism and that the genes involved in the synthesis of activated sulfate are part of the glucosinolate biosynthesis network.