Michael Hotze

Bio: Michael Hotze is an academic researcher from University of Freiburg. The author has contributed to research in topics: Catharanthus roseus & Complementary DNA. The author has an hindex of 8, co-authored 9 publications receiving 632 citations.

Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase.

Abstract: Summary The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deoxyloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.

Vitamin-B12-independent methionine synthase from a higher plant (Catharanthus roseus). Molecular characterization, regulation, heterologous expression, and enzyme properties.

Abstract: Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in L-methionine biosynthesis, and it also serves to regenerate the methyl group of S-adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharunthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50 % identity with the cobalamin-independent methionine synthase from Escherichia coli (MetE) and 41 % identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require S-adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plant extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.

Vitamin‐B12‐Independent Methionine Synthase from a Higher Plant (Catharanthus Roseus)

Abstract: Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in l-methionine biosynthesis, and it also serves to regenerate the methyl group of 5-adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50 % identity with the cobalamin-independent methionine synthase from Escherichia coli (MetE) and 41 % identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require 5-adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plant extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.

The tomato genome sequence provides insights into fleshy fruit evolution

Abstract: Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera1 and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium2, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.

Selenium in higher plants.

Abstract: Plants vary considerably in their physiological response to selenium (Se). Some plant species growing on seleniferous soils are Se tolerant and accumulate very high concentrations of Se (Se accumulators), but most plants are Se nonaccumulators and are Se-sensitive. This review summarizes knowledge of the physiology and biochemistry of both types of plants, particularly with regard to Se uptake and transport, biochemical pathways of assimilation, volatilization and incorporation into proteins, and mechanisms of toxicity and tolerance. Molecular approaches are providing new insights into the role of sulfate transporters and sulfur assimilation enzymes in selenate uptake and metabolism, as well as the question of Se essentiality in plants. Recent advances in our understanding of the plant's ability to metabolize Se into volatile Se forms (phytovolatilization) are discussed, along with the application of phytoremediation for the cleanup of Se contaminated environments.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Abstract: We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Sulfur Assimilation in Photosynthetic Organisms: Molecular Functions and Regulations of Transporters and Assimilatory Enzymes

Abstract: Sulfur is required for growth of all organisms and is present in a wide variety of metabolites having distinctive biological functions. Sulfur is cycled in ecosystems in nature where conversion of sulfate to organic sulfur compounds is primarily dependent on sulfate uptake and reduction pathways in photosynthetic organisms and microorganisms. In vascular plant species, transport proteins and enzymes in this pathway are functionally diversified to have distinct biochemical properties in specific cellular and subcellular compartments. Recent findings indicate regulatory processes of sulfate transport and metabolism are tightly connected through several modes of transcriptional and posttranscriptional mechanisms. This review provides up-to-date knowledge in functions and regulations of sulfur assimilation in plants and algae, focusing on sulfate transport systems and metabolic pathways for sulfate reduction and synthesis of downstream metabolites with diverse biological functions.