BackgroundDrought is a major constraint for plant growth and crop productivity that is receiving an increased attention due to global climate changes. Chloroplasts act as environmental sensors, however, only partial information is available on stress-induced mechanisms within plastids. Here, we investigated the chloroplast response to a severe drought treatment and a subsequent recovery cycle in tomato through physiological, metabolite and proteomic analyses.ResultsUnder stress conditions, tomato plants showed stunted growth, and elevated levels of proline, abscisic acid (ABA) and late embryogenesis abundant gene transcript. Proteomics revealed that water deficit deeply affects chloroplast protein repertoire (31 differentially represented components), mainly involving energy-related functional species. Following the rewatering cycle, physiological parameters and metabolite levels indicated a recovery of tomato plant functions, while proteomics revealed a still ongoing adjustment of the chloroplast protein repertoire, which was even wider than during the drought phase (54 components differentially represented). Changes in gene expression of candidate genes and accumulation of ABA suggested the activation under stress of a specific chloroplast-to-nucleus (retrograde) signaling pathway and interconnection with the ABA-dependent network.ConclusionsOur results give an original overview on the role of chloroplast as enviromental sensor by both coordinating the expression of nuclear-encoded plastid-localised proteins and mediating plant stress response. Although our data suggest the activation of a specific retrograde signaling pathway and interconnection with ABA signaling network in tomato, the involvement and fine regulation of such pathway need to be further investigated through the development and characterization of ad hoc designed plant mutants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-0971-0) contains supplementary material, which is available to authorized users.

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55(gag)) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55(gag) expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55(gag) sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5'-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7-8% TSP, equivalent to 312-363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55(gag) polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55(gag) polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.

BackgroundBiofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels.ResultsThe selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher in the whole 24–72 h range.ConclusionsThe very high production level of thermophilic and hyperthermophilic enzymes, their stability and bioconversion efficiencies described in this study demonstrate that plastid transformation represents a real cost-effective production platform for cellulolytic enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0569-z) contains supplementary material, which is available to authorized users.