Ricarda Jost

Analysis of the Arabidopsis O-Acetylserine(thiol)lyase Gene Family Demonstrates Compartment-Specific Differences in the Regulation of Cysteine Synthesis

Cys synthesis in plants takes place in plastids, cytosol, and mitochondria. Why Cys synthesis is required in all compartments with autonomous protein biosynthesis and whether Cys is exchanged between them has remained enigmatic. This question was addressed using Arabidopsis thaliana T-DNA insertion lines deficient in the final step of Cys biosynthesis catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL). Null alleles of oastlA or oastlB alone showed that cytosolic OAS-TL A and plastid OAS-TL B were completely dispensable, although together they contributed 95% of total OAS-TL activity. An oastlAB double mutant, relying solely on mitochondrial OAS-TL C for Cys synthesis, showed 25% growth retardation. Although OAS-TL C alone was sufficient for full development, oastlC plants also showed retarded growth. Targeted affinity purification identified the major OAS-TL–like proteins. Two-dimensional gel electrophoresis and mass spectrometry showed no compensatory changes of OAS-TL isoforms in the four mutants. Steady state concentrations of Cys and glutathione and pulse-chase labeling with [35S]sulfate indicated strong perturbation of primary sulfur metabolism. These data demonstrate that Cys and also sulfide must be sufficiently exchangeable between cytosol and organelles. Despite partial redundancy, the mitochondria and not the plastids play the most important role for Cys synthesis in Arabidopsis.

Characterization of TCTP, the Translationally Controlled Tumor Protein, from Arabidopsis thaliana

The translationally controlled tumor protein (TCTP) is an important component of the TOR (target of rapamycin) signaling pathway, the major regulator of cell growth in animals and fungi. TCTP acts as the guanine nucleotide exchange factor of the Ras GTPase Rheb that controls TOR activity in Drosophila melanogaster. We therefore examined the role of Arabidopsis thaliana TCTP in planta. Plant TCTPs exhibit distinct sequence differences from nonplant homologs but share the key GTPase binding surface. Green fluorescent protein reporter lines show that Arabidopsis TCTP is expressed throughout plant tissues and developmental stages with increased expression in meristematic and expanding cells. Knockout of TCTP leads to a male gametophytic phenotype with normal pollen formation and germination but impaired pollen tube growth. Silencing of TCTP by RNA interference slows vegetative growth; leaf expansion is reduced because of smaller cell size, lateral root formation is reduced, and root hair development is impaired. Furthermore, these lines show decreased sensitivity to an exogenously applied auxin analog and have elevated levels of endogenous auxin. These results identify TCTP as an important regulator of growth in plants and imply a function of plant TCTP as a mediator of TOR activity similar to that known in nonplant systems.

Molecular and biochemical analysis of the enzymes of cysteine biosynthesis in the plant Arabidopsis thaliana

Summary. Among the amino acids produced by plants cysteine plays a special role as a mediator between assimilatory sulfate reduction and provision of reduced sulfur for cell metabolism. Part of this characteristic feature is the presence of cysteine synthesis in plastids, mitochondria and cytosol. Plants are the major source of reduced sulfur for human and animal nutrition. Cysteine biosynthesis deserves special attention, since reduced sulfur is channelled from cysteine into many sulfur-containing compounds in food and feed. Recent investigations are reviewed that focus on structure and regulation of cysteine synthesis in the model plant Arabidopsis thaliana. These data indicate that cysteine synthesis is not just an intermediate reaction step but that it is part of a regulatory network that mediates between inorganic sulfur supply and the demand for reduced sulfur during plant growth and in response to environmental changes.

Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana.

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.

Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis thaliana

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up- or down-regulated more than 2–fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfur-related genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply- and demand-driven regulation of the sulfate assimilation pathway.

Root Architecture Responses: In Search of Phosphate

Root development alters in response to phosphate availability, which affects the production of cluster roots in a limited number of species. Soil phosphate represents the only source of phosphorus for plants and, consequently, is its entry into the trophic chain. This major component of nucleic acids, phospholipids, and energy currency of the cell (ATP) can limit plant growth because of its low mobility in soil. As a result, root responses to low phosphate favor the exploration of the shallower part of the soil, where phosphate tends to be more abundant, a strategy described as topsoil foraging. We will review the diverse developmental strategies that can be observed among plants by detailing the effect of phosphate deficiency on primary and lateral roots. We also discuss the formation of cluster roots: an advanced adaptive strategy to cope with low phosphate availability observed in a limited number of species. Finally, we will put this work into perspective for future research directions.

Low levels of ribosomal RNA partly account for the very high photosynthetic phosphorus-use efficiency of Proteaceae species

Abstract Proteaceae species in south-western Australia occur on phosphorus- (P) impoverished soils. Their leaves contain very low P levels, but have relatively high rates of photosynthesis. We measured ribosomal RNA (rRNA) abundance, soluble protein, activities of several enzymes and glucose 6-phosphate (Glc6P) levels in expanding and mature leaves of six Proteaceae species in their natural habitat. The results were compared with those for Arabidopsis thaliana. Compared with A. thaliana, immature leaves of Proteaceae species contained very low levels of rRNA, especially plastidic rRNA. Proteaceae species showed slow development of the photosynthetic apparatus (‘delayed greening’), with young leaves having very low levels of chlorophyll and Calvin–Benson cycle enzymes. In mature leaves, soluble protein and Calvin–Benson cycle enzyme activities were low, but Glc6P levels were similar to those in A. thaliana. We propose that low ribosome abundance contributes to the high P efficiency of these Proteaceae species in three ways: (1) less P is invested in ribosomes; (2) the rate of growth and, hence, demand for P is low; and (3) the especially low plastidic ribosome abundance in young leaves delays formation of the photosynthetic machinery, spreading investment of P in rRNA. Although Calvin–Benson cycle enzyme activities are low, Glc6P levels are maintained, allowing their effective use.

Phosphorus nutrition of phosphorus-sensitive Australian native plants: threats to plant communities in a global biodiversity hotspot

South-western Australia harbours a biodiversity hotspot on severely phosphorus-impoverished soils. Threats include eutrophication due to phosphorus enrichment, due to increased fire frequency and spraying with phosphite to reduce the impacts of the introduced pathogen Phytophthora cinnamomi. We propose a strategy to work towards alternatives to phosphite for pathogen management.

Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate

BackgroundIn plants, the uptake from soil and intercellular transport of inorganic phosphate (Pi) is mediated by the PHT1 family of membrane-spanning proton : Pi symporters. The Arabidopsis thaliana AtPHT1 gene family comprises nine putative high-affinity Pi transporters. While AtPHT1;1 to AtPHT1;4 are involved in Pi acquisition from the rhizosphere, the role of the remaining transporters is less clear.ResultsPi uptake and tissue accumulation studies in AtPHT1;8 and AtPHT1;9 knock-out mutants compared to wild-type plants showed that both transporters are involved in the translocation of Pi from the root to the shoot. Upon inactivation of AtPHT1;9, changes in the transcript profiles of several genes that respond to plant phosphorus (P) status indicated a possible role in the regulation of systemic signaling of P status within the plant. Potential genetic interactions were found among PHT1 transporters, as the transcript profile of AtPHT1;5 and AtPHT1;7 was altered in the absence of AtPHT1;8, and the transcript profile of AtPHT1;7 was altered in the Atpht1;9 mutant. These results indicate that AtPHT1;8 and AtPHT1;9 translocate Pi from the root to the shoot, but not from the soil solution into the root.ConclusionAtPHT1;8 and AtPHT1;9 are likely to act sequentially in the interior of the plant during the root-to-shoot translocation of Pi, and play a more complex role in the acclimation of A. thaliana to changes in Pi supply than was previously thought.

Biochemical Characterization of Two Wheat Phosphoethanolamine N-Methyltransferase Isoforms with Different Sensitivities to Inhibition by Phosphatidic Acid

In plants the triple methylation of phosphoethanolamine to phosphocholine catalyzed by phosphoethanolamine N-methyltransferase (PEAMT) is considered a rate-limiting step in the de novo synthesis of phosphatidylcholine. Besides being a major membrane phospholipid, phosphatidylcholine can be hydrolyzed into choline and phosphatidic acid. Phosphatidic acid is widely recognized as a second messenger in stress signaling, and choline can be oxidized within the chloroplast to yield the putative osmoprotectant glycine betaine. Here we describe the cloning and biochemical characterization of a second wheat PEAMT isoform that has a four times higher specific activity than the previously described WPEAMT/TaPEAMT1 enzyme and is less sensitive to product inhibition by S-adenosyl homocysteine, but more sensitive to inhibition by phosphocholine. Both enzymes follow a sequential random Bi Bi mechanism and show mixed-type product inhibition patterns with partial inhibition for TaPEAMT1 and a strong non-competitive component for TaPEAMT2. An induction of TaPEAMT protein expression and activity is observed after cold exposure, ahead of an increase in gene expression. Our results demonstrate direct repression of in vitro enzymatic activities by phosphatidic acid for both enzymes, with TaPEAMT1 being more sensitive than TaPEAMT2 in the physiological concentration range. Other lipid ligands identified in protein-lipid overlays are phosphoinositide mono- as well as some di-phosphates and cardiolipin. These results provide new insights into the complex regulatory circuits of phospholipid biosynthesis in plants and underline the importance of head group biosynthesis in adaptive stress responses.