A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

Abstract

A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

Rapid and efficient in vitro regeneration of transplastomic potato (Solanum tuberosum L.) plants after particle bombardment

“…Besides, the recovery of homoplasmic plant lines is another big obstacle for this technology (Ahmad et al, 2016). Current attempts to streamline this process include introducing high throughput cloning methods for chloroplast expression vector construction (Gottschamel et al, 2013;Vafaee et al, 2014) and finding new selectable markers (Bellucci et al, 2015). Nevertheless, it has proven extremely difficult to extend the transplastomic technology to field crops.…”

Perspectives on protein biopolymers: miniaturized flow field-flow fractionation-assisted characterization of a single-cysteine mutated phaseolin expressed in transplastomic tobacco plants

“…The PCR product was digested with NdeI/NotI and cloned into pCR2.1- [40], to obtain the pCR2.1--P* intermediate plasmid, in which phaseolin is under the control of the plastidial psbA -P* cassette was excised from pCR2.1-R-P* by EcoRV/NotI digestion and subcloned into pLD-CTV tobacco plastid vector [41], generating pLD-CTV-P*. Homoplasmic transplastomic plants were obtained by particle bombardment into tobacco leaves of gold microprojectiles coated with pLD-CtV-P* as described [42]. Transplastomic plants were grown at 25°C in 16 h of light in axenic conditions before being transferred to the greenhouse for seed production.…”

Selectable Markers and Reporter Genes for Engineering the Chloroplast of Chlamydomonas reinhardtii

“…This is particularly an issue when employing herbicide-insensitive variants of endogenous chloroplast enzymes as markers, rather than novel enzymes that metabolise the herbicide to a non-toxic product. An example of this is a mutant form of the bacterial hemL gene that encodes a gabaculine-insensitive variant of the enzyme glutamate 1-semialdehyde aminotransferase (GSA) [50]. In photosynthetic eukaryotes, GSA is localised in the chloroplast and plays a central role in chlorophyll synthesis.…”

“…Gabaculine-mediated inhibition of GSA results in chlorosis and cell death in plants and algae, including C. reinhardtii [84]. When the hemL gene was over-expressed in the tobacco chloroplast, the plants became insensitive to gabaculine [50], but attempts to use hemL for direct selection were unsuccessful. Other possible markers include bacterial genes encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme involved in the biosynthesis of aromatic amino acids.…”