Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Salt stress increases the expression of p5cs gene and induces proline accumulation in cactus pear.

Proline (Pro) is one of the most accumulated osmolytes in salinity and water deficit conditions in plants. In the present study, we measured the Pro content, the activity and the expression level of delta 1-pyrroline-5-carboxylate synthetase (P5CS: gamma-glutamyl kinase, EC 2.7.2.11 and glutamate-5-semialdehyde dehydrogenase, EC 1.2.1.41), a key regulatory enzyme involved in the biosynthesis of Pro, in cactus pear (Opuntia streptacantha) subjected to 6, 9 and 11 days of salt stress. Treatment with NaCl of O. streptacantha young plants resulted in a decrease in the cladode thickness and root length, and in a significant and gradual accumulation of Pro in young cladodes, in a time- and concentration-dependent manner. P5CS activity, studied as gamma-glutamyl kinase, was reduced at all times as a consequence of salt treatment, except at the sixth day at 75 and 150mM of NaCl, where a slight increase was observed. We isolated an open reading frame (ORF) fragment of p5cs gene. The deduced amino acid sequence of the P5CS protein exhibited 90.4% of identity with the P5CS protein from Mesembryanthemum crystallinum. RT-PCR analysis revealed that the Osp5cs gene of O. streptacantha was induced by salt stress at 9 and 11 days of treatment. Furthermore, ABA-induced Osp5cs gene expression was observed in cladodes of cactus pear young plants. We observed an evident correlation between the transcript up-regulation and the Pro accumulation under salt stress; however, these results do not parallel with the changes in P5CS enzymatic activity. This Pro accumulation might function as an osmolyte for the intracellular osmotic adjustment and might be playing a critical role in protecting photosynthetic activity in O. streptacantha plants under salt stress.