No abstract

Plastid transformation is routine in tobacco (Nicotiana tabacum) but 100-fold less frequent in Arabidopsis (Arabidopsis thaliana), preventing its use in plastid biology. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetylcoenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC b-carboxylase subunit. We bombarded ACC2-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (aadA) gene and gfp, encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.

No abstract

SUMMARY We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.

Plant NADP-malic enzyme (NADP-ME, EC 1.1.1.40) participates in a large number of metabolic pathways, but little is known about the NADP-ME family in woody plants or trees. Here, we characterized the tree Populus trichocarpa NADP-ME (PtNADP-ME) family and the properties of the family members. Five NADP-ME genes (PtNADP-ME1-PtNADP-ME5) were found in the genome of Populus. Semi-quantitative RT-PCR analysis show that the transcription levels of PtNADP-ME1 in lignified stems and roots are clearly higher than in other tissues, and PtNADP-ME2, PtNADP-ME3, PtNADP-ME4 and PtNADP-ME5 are broadly expressed in various tissues. PtNADP-ME gene expression was found to respond to salt and osmotic stresses, and NaCl salts upregulated the transcripts of putative plastidic ones (PtNADP-ME4 and PtNADP-ME5) significantly. Further, the NADP-ME activities of Populus seedlings increased at least two-fold under NaCl, mannitol and PEG treatments. Also, the expression of PtNADP-ME2 and PtNADP-ME3 increased during the course of leaf wounding. Each recombinant PtNADP-ME proteins were expressed and purified from Escherichia coli, respectively. Coomassie brilliant blue and NADP-ME activity staining on native polyacrylamide gels showed different oligomeric states of the recombinant PtNADP-MEs in vitro. Noticeably, the cytosolic PtNADP-ME2 aggregates as octamers and hexadecamers while the plastidic PtNADP-ME4 resembles hexamers and octamers. The four PtNADP-ME proteins except for PtNADP-ME1 have high activities on native polyacrylamide gels including different forms for PtNADP-ME2 (octamers and hexadecamers) or for PtNADP-ME4 (hexamers and octamers). High concentrations of NADP substrate decreased the activities of all PtNADP-MEs slightly, while the malate had no effect on them. The kinetic parameters (V (max), K (m), K (cat), and K (cat)/K (m)) of each isoforms were summarized. Our data show the different effects of metabolites (influx into tricarboxylic acid cycle or Calvin cycle) on the activity of the individual PtNADP-ME in vitro. According to phylogenetic analysis, five PtNADP-MEs are clustered into cytosolic dicot, plastidic dicot, and monocot and dicot cytosolic groups in a phylogenetic tree. These results suggest that woody Populus NADP-ME family have diverse properties, and possible roles are discussed.