Plant bioreactors as a sustainable approach for useful compounds production: innovation and applied research perspectives.

Section I) Plants have always been a source of metabolic compounds useful for human life. Today, different genetic engineering techniques allow us to modify both whole plants and in vitro cultured material for several purposes. These span from the elicitation of bioactive molecules that are already produced by these organisms to de novo biosynthesis of metabolites and recombinant proteins. This section presents three strategies currently being pursued for the production in plants of antioxidant, immunomodulatory compounds and with interesting structural properties for the production of biomaterials. They are based either on stable or transient genetic transformation of plant cells from (Chapter I) Malus domestica and (Chapter II, III) Nicotiana tabacum. In Chapter I, calli derived from apple pulp have been transformed with the bHLH transcription factor Sn (TF) from Zea mays. This TF interacts with MYB and WD40 proteins to upregulate the biosynthesis of anthocyanins and/or proanthocyanidins in different plant species and growing conditions. In Chapter II, tobacco leaf protoplasts were transiently transformed with the human enzyme IDO1, responsible for immunomodulation response. By promoting the degradation of tryptophan to produce kynurenines, molecules capable to modulate the immune system, this enzyme exerts a powerful immunoregulatory action. In Chapter III a vacuolar storage protein from common bean (Phaseolus vulgaris), called phaseolin, was investigated. This protein was engineered adding a Cysteine at C-terminal tail, allowing it to form inter-chain disulfide bridges and complex structures: the production capacity of this biopolymer was investigated both in plant and in Escherichia coli, in order to compare distinct bioreactor systems. Section II) The indefinite growth capacity and organogenesis in plants are guaranteed by meristems, tissues highly regulated by intercellular signaling pathways, where cells are in continuous division and differentiation. In shoots apical meristem of Arabidopsis thaliana, a differentiation-promoting peptide obtained by the proteolytic maturation of the protein CLAVATA3(CLV3) establishes an autoregulatory negative-feedback loop against the expression of cell-promoting transcription factor WUSCHEL. The shoot apical meristem remains alive for the entire plant life, maintaining a perfect balance between the continuous loss of daughter cells due to organogenesis. Nowadays, CLV3 is considered a secretory protein, matured by secreted proteases at the extracellular level into the active dodecapeptide form: this maturated fragment behaves as a ligand of CLV1/CLV2 receptor complex. Recent studies in tobacco suggest that the protein's maturation process could not occur in the apoplast but probably through the intracellular ERAD (Endoplasmic Reticulum Associated Degradation) process, which is naturally used by cells to degrade unfolded proteins. To confirm this hypothesis, we studied the expression of the fusion protein in both Nicotiana tabacum plants and callus-cell suspension cultures by analyzing in a steady-state way the localisation of the fusion protein CLV3-GFP. To further investigate the protein maturation process, it has been studied the possible role of ubiquitination in CLV3 maturation process, as it is a key starting point in ERAD. A CLV3-YFP transgenic cell strain of Chlamydomonas reinhardtii was produced, to be able to follow the maturation process of this protein in an organism that doesn’t show any meristem organization and CLV3 expression. The obtained results suggest a different destiny for CLV3: different genetic and biochemical strategies were carried out to shed light on this maturation process, which follows different ways in different biosystems.