Elisabeth A. Mudd

Binding and glutathione conjugation of porphyrinogens by plant glutathione transferases

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.

A 125 kDa RNase E/G-like protein is present in plastids and is essential for chloroplast development and autotrophic growth in Arabidopsis

Endoribonuclease E (RNase E) is a regulator of global gene expression in Escherichia coli and is the best studied member of the RNase E/G ribonuclease family. Homologues are present in other bacteria but the roles of plant RNase E/G-like proteins are not known. Arabidopsis thaliana contains a single nuclear gene (At2g04270) encoding a product with the conserved catalytic domain of RNase E/G-like proteins. At2g04270 and the adjacent At2g04280 gene form converging transcription units with a ∼40 base overlap at their 3’ ends. Several translation products were predicted from the analyses of At2g04270 cDNAs. An antibody raised against a recombinant A. thaliana RNase E/G-like protein recognized a 125 kDa protein band in purified chloroplast preparations fractionated by SDS-PAGE. The 125 kDa RNase E/G-like protein was detected in cotyledons, rosette and cauline leaves. T-DNA insertions in exon 6 or intron 11 of At2g04270 result in loss of the 125 kDa band or truncation to a 110 kDa band. Loss of At2g04270 function resulted in the arrest of chloroplast development, loss of autotrophic growth, and reduced plastid ribosomal, psbA and rbcL RNA levels. Homozygous mutant plants were pale-green, contained smaller plastids with fewer thylakoids and shorter granal stacks than wild-type chloroplasts, and required sucrose at all growth stages following germination right up to flowering and setting seeds. Recombinant A. thaliana RNase E/G-like proteins rescued an E. coli RNase E mutant and cleaved an rbcL RNA substrate. Expression of At2g04270 was highly correlated with genes encoding plastid polyribonucleotide phosphorylase, S1 RNA-binding, and CRS1/YhbY domain proteins.

Visualisation of plastids in endosperm, pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO, rice FtsZ and maize ferredoxin III proteins

The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.

A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera

Hepatitis C virus (HCV) is a major disease agent affecting approximately 3% of the worlds population. Expression in plant chloroplasts enables low-cost production of the conserved HCV core protein used in diagnostic tests to combat virus spread in developing countries with high infection rates. The bactericidal activity of the 21 kDa precore protein hinders cloning the core gene in plastid expression cassettes, which are active in bacteria due to the similarities between bacterial and plastid promoters and ribosome binding sites. This was overcome by using a topology-dependent expression cassette containing tandem rrn and psbA plastid promoters, whose activity was shown to be dependent on temperature. The viral core gene and a codon-optimised gene encoding a C-terminal truncated 16 kDa core polypeptide were expressed in tobacco chloroplasts. The codon-optimised gene increased monocistronic core mRNA levels by at least 2-fold and core polypeptides by over 5-fold, relative to the native viral gene. Expression of the 16 kDa core polypeptide was stable in leaves of different ages. Anti-core antibodies in HCV-infected human sera were detected by the 16 kDa core polypeptide in total leaf protein fractionated on Western blots providing a first step towards developing a chloroplast-based HCV diagnostic method.

Growth of Transplastomic Cells Expressing d-Amino Acid Oxidase in Chloroplasts Is Tolerant to d-Alanine and Inhibited by d-Valine

Dual-conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. High-yield expression of recombinant products and maternal inheritance of plastid-encoded traits make plastids attractive sites for modification. Here, we describe the cloning and expression of a dao gene encoding d-amino acid oxidase from Schizosaccharomyces pombe in tobacco (Nicotiana tabacum) plastids. The results provide genetic evidence for the uptake of d-amino acids into plastids, which contain a target that is inhibited by d-alanine. Importantly, this nonantibiotic-based selection system allows the use of cheap and widely available d-amino acids, which are relatively nontoxic to animals and microbes, to either select against (d-valine) or for (d-alanine) cells containing transgenic plastids. Positive/negative selection with d-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment, where it can be used to provide tolerance against potential d-alanine-based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop d-valine-based methods to manage the spread of transgenic plastids tagged with dao.

Excision of plastid marker genes using directly repeated DNA sequences.

Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

Homologous recombination allows efficient isolation of marker-free transplastomic plants.

Gene transfer technologies allow the rapid introduction of new traits into plants. The technology is reliant on bacterial marker genes for cloning trait genes in Escherichia coli plasmids, and plant marker genes for identifying transformed plant cells. Marker genes have served their purpose once transgenic plants are obtained but they are often retained (Fig 1A). Antibiotic resistance genes are commonly used as selectable markers and their use in transgenic research has been criticised due to the theoretical risk of their acquisition by pathogenic bacteria. Within the European Economic Community, directive 2001/18/EC requires the gradual elimination of antibiotic resistance markers, which might have adverse effects on human health and the environment, from genetically manipulated organisms by the end of 2004 for commercial releases and the end of 2008 for research purposes. Compliance with the directive requires the development of efficient procedures for removing antibiotic resistance genes from transgenic crops or the use of alternative marker genes. A gene excision strategy has the advantages of eliminating the need for risk assessments on marker genes in transgenic crops and allowing multiple trait genes to be combined in the same plant by repeat transformations with the most efficient marker gene. Removing excess foreign DNA from transgenic crops has the additional benefit of focusing attention on the important trait genes.