Thomas Rausch

Ectopic expression of a tobacco invertase inhibitor homolog prevents cold-induced sweetening of potato tubers.

We have transformed potato with Nt-inhh cDNA, encoding a putative vacuolar homolog of a tobacco cell wall invertase inhibitor, under the control of the CaMV 35S promoter. In transgenic tubers, cold-induced hexose accumulation was reduced by up to 75%, without any effect on potato tuber yield. Processing quality of tubers was greatly improved without changing starch quantity or quality, an important prerequisite for the biotechnological use of Nt-inhh for potato transformation.

cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial gamma-glutamylcysteine synthetase isoform.

In roots of Brassica juncea L. cadmium (Cd) exposure (25 µM) induces a massive formation of phytochelatins (PCs), which is accompanied by an only moderate decrease (−20%) of the putative PC precursor glutathione (GSH). As PC formation in roots could be the result of local GSH de novo synthesis and/or depend on GSH import from the shoot, we have analyzed the expression of the enzymes involved in GSH synthesis in the root, namely OAS(thiol)lyase (OAS-TL; catalysing the last step in Cys biosynthesis), γ-glutamylcysteine synthetase (γ-ECS), and glutathione synthetase (GSHS). cDNA clones were isolated from a cDNA library prepared from heavy metal exposed roots. Protein sequences from cDNA clones encoding OAS-TL, γ-ECS, and GSHS, all exhibited putative mitochondrial targeting sequences, however, for OAS-TL also two putative cytosolic isoforms were isolated. Furthermore, we have cloned several metallothionein cDNAs of the MT2 group. Northern blot analysis with coding region probes revealed that in roots of Cd-exposed plants transcript amounts for OAS-TL and GSHS were only moderately increased, whereas γ-ECS mRNA showed a stronger increase. Expression analysis with 3′-UTR probes indicated that among the putative mitochondrial OAS-TL, γ-ECS and GSHS isoforms only γ-ECS was up-regulated in response to Cd exposure. Conversely, transcripts for MT2 appeared to be slightly reduced. The results indicate that in roots Cd-induced PC synthesis correlates with a moderate increase of expression of genes involved in GSH synthesis, the change for γ-ECS being most pronounced.

Early Salt Stress Effects on the Differential Expression of Vacuolar H+-ATPase Genes in Roots and Leaves of Mesembryanthemum crystallinum

In Mesembryanthemum crystallinum, the salt stress-induced metabolic switch from C3 photosynthesis to Crassulacean acid metabolism is accompanied by major changes in gene expression. However, early effects of salt exposure (i.e. prior to Crassulacean acid metabolism induction) on genes coding for vacuolar transport functions have not yet been studied. Therefore, the expression of vacuolar H+-ATPase genes was analyzed in different organs of 4-week-old plants stressed with 400 mM NaCl for 3, 8, or 24 h. Partial cDNAs for the subunits A, B, and c were cloned and used as homologous probes for northern blot analysis. In control plants, the mRNA levels for the different subunits showed organ-specific differences. In fully expanded leaves, subunit c mRNA was very low but increased transiently during the light period. Plant organs also differed in their salt-stress response. In roots and young leaves, mRNA levels for all three subunits increased about 2-fold compared to control plants, whereas in fully expanded leaves only subunit c mRNA responded to salt. The results indicate that the expression of vacuolar H+-ATPase genes does not always involve a fixed stoichiometry of mRNAs for the different subunits and that the mRNA level for subunit c is particularly sensitive to developmental and environmental changes.

Salt stress responses of higher plants : The role of proton pumps and Na+/H+-antiporters

In salt-stressed higher plants NaCl may either be excluded from the cells or sequestered into the vacuole. Different pathways may dominate in different plants and different organs of the same plant. The proteins involved in salt transport across the plasma membrane and the tonoplast i.e. proton pumps and Na + /H + -antiporters have been identified. Progress in cloning of the P-type H + -ATPase, the V-type H + -ATPase, and the vacuolar H + -PP i ase has provided important tools for the study of the molecular mechanisms involved in ion sequestration. However, not a single plant has as yet been studied in sufficient detail to allow a comprehensive evaluation of the relative importance of individual transport processes for the salt tolerance of an intact plant. This review summarizes our present as yet limited knowledge and identifies promissing areas for future research.

The R2R3-MYB Transcription Factors MYB14 and MYB15 Regulate Stilbene Biosynthesis in Vitis vinifera

This study reports the identification and functional characterization of two stress-inducible R2R3-MYB–type transcription factors, termed MYB14 and MYB15, which regulate the stilbene biosynthetic pathway in grapevine. Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB–type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

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.

Structural basis for the redox control of plant glutamate cysteine ligase

Glutathione (GSH) plays a crucial role in plant metabolism and stress response. The rate-limiting step in the biosynthesis of GSH is catalyzed by glutamate cysteine ligase (GCL) the activity of which is tightly regulated. The regulation of plant GCLs is poorly understood. The crystal structure of substrate-bound GCL from Brassica juncea at 2.1-Å resolution reveals a plant-unique regulatory mechanism based on two intramolecular redox-sensitive disulfide bonds. Reduction of one disulfide bond allows a β-hairpin motif to shield the active site of B. juncea GCL, thereby preventing the access of substrates. Reduction of the second disulfide bond reversibly controls dimer to monomer transition of B. juncea GCL that is associated with a significant inactivation of the enzyme. These regulatory events provide a molecular link between high GSH levels in the plant cell and associated down-regulation of its biosynthesis. Furthermore, known mutations in the Arabidopsis GCL gene affect residues in the close proximity of the active site and thus explain the decreased GSH levels in mutant plants. In particular, the mutation in rax1-1 plants causes impaired binding of cysteine.

The N‐terminal pro region mediates retention of unprocessed type‐I PME in the Golgi apparatus

The pectin matrix of the cell wall, a complex and dynamic network, impacts on cell growth, cell shape and signaling processes. A hallmark of pectin structure is the methylesterification status of its major component, homogalacturonan (HGA), which affects the biophysical properties and enzymatic turnover of pectin. The pectin methylesterases (PMEs), responsible for de-esterification, encompass a protein family of more than 60 isoforms in the Arabidopsis genome. The pivotal role of PME in the regulation of pectin properties also requires tight control at the post-translational level. Type-I PMEs are characterized by an N-terminal pro region, which exhibits homology with pectin methylesterase inhibitors (PMEIs). Here, we demonstrate that the proteolytic removal of the N-terminal pro region depends on conserved basic tetrad motifs, occurs in the early secretory pathway, and is required for the subsequent export of the PME core domain to the cell wall. In addition, we demonstrate the involvement of AtS1P, a subtilisin-like protease, in Arabidopsis PME processing. Our results indicate that the pro region operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, pro-protein processing could constitute a post-translational mechanism regulating PME activity.

Identification of pollen‐expressed pectin methylesterase inhibitors in Arabidopsis

Pectin methylesterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell wall. Previous work indicated that plant PMEs may be subject to post‐translational regulation. Here, we report the analysis of two proteinaceous inhibitors of PME in Arabidopsis thaliana (AtPMEI1 and 2). The functional analysis of recombinant AtPMEI1 and 2 proteins revealed that both proteins are able to inhibit PME activity from flowers and siliques. Quantitative RT‐PCR analysis indicated that expression of AtPMEI1 and 2 mRNAs is tightly regulated during plant development with highest mRNA levels in flowers. Promotor::GUS fusions demonstrated that expression is mostly restricted to pollen.

In seedlings of the heavy metal accumulator Brassica juncea Cu2+ differentially affects transcript amounts for γ-glutamylcysteine synthetase (γ-ECS) and metallothionein (MT2)

© 1997 Federation of European Biochemical Societies.