Artificial activation of APETALA1 using recombinant proteins and cell penetrating peptides

Abstract

This thesis reports a novel method for the artificial activation of reproductive development in Arabidopsis. In this way, reproductive development was triggered by an exogenous delivery of the recombinant florigen protein, Flowering Locus T (FT), at the shoot apical meristem in order to artificially activate Apetala 1 (AP1), the major floral homeotic gene. This was assessed by large phenotyping experiments after protein treatment. In addition, a transgenic reporter was created, using the promoter of AP1 and GFP to assess the artificial activation of AP1.

Furthermore, this thesis reports the action of cell penetrating peptide uptake (namely R9, Tat, TP10 and pVEC) into plant tissue. An in-depth investigation into R9 uptake revealed mechanistic details in particular the initial interaction of R9 with the cell wall to create a high local peptide concentration in proximity to the membrane. Further studies reported the successful uptake of R9 into mesophyll cell culture, protoplasts and apical meristems, including the development of a novel quantitative assessment for R9 uptake into root tips.

Tool development notably included the covalent attachment of R9 and Tat to FT and Sortagging, a novel labeling method enabling the fluorescent tracking of proteins in planta using confocal microscopy. Superior uptake was reported for FT-Tat over FT-R9 in cell culture and FT-Tat uptake was reported in the shoot apical meristem. This is the first example of covalent CPP-mediated delivery of a functional protein to the apical tissue.

This study reports progress en route to developing a universal tool for flowering time control in agriculture. It is the first reported example of an exogenous macromolecular treatment with a recorded biological effect in planta and as such is a novel approach to target and control AP1 expression.