Understanding the molecular mechanisms responsible for the genetic regulation of fleshy fruits is a major objective of fruit researchers. Fleshy fruits are divided into two main categories depending on whether the ripening process is controlled or not by the gaseous phytohormone "ethylene". Climacteric fruits such as tomato, apple, pear, and banana are characterized by a ripening-associated burst of respiration and the production of ethylene. By contrast, non-climacteric fruits, such as grape, orange, strawberry and pineapple lack this ethylene-associated respiratory peak. The signaling pathways that drive the ripening process in non-climacteric fruits remain elusive although the combined actions of ABA (Abscisic Acid), auxin, and sugar are suspected to be instrumental in the ripening initiation. In our lab, we use grape berry and more specifically the uneven development of a grape cluster to characterize the influence of these different plant growth regulators with the objective to identify the regulatory candidate genes that trigger the ripening initiation. Our scientific question is focused on the role of auxin during the ripening onset (Figure 2). We use the microvine (Chaib et al., 2010, Torregrosa et al., 2019), a suitable system for genetic (short life cycle, ever flowering, and easy to transform through Agrobacterium tumefaciens) to validate the function several candidate genes associated with Auxin, but also those that seems to interact with other regulators of plant growth regulators such as sugar and ABA.
- Using the microvine, we are currently validating the function of several genes involved in the auxin signaling (VitviARF4), and other ARF4 "partners". We have conducted gain and loss-of-function studies on these different genes using a conditional expression system the Plant Gene Switch System (Martinez-Andujar et al., 2011 - Figure 3). This expression system enables us to decide when to induce the transgene (Ox and microRNA-driven Knock Down), which present values when one wants to understand gene function in a spatial and temporal context.
- We also develop a research project focused on the influence of seed development the ripening. Previous works in the lab have demonstrated a relationship between seed mass relative to berry mass (Seed Index) with the timing of of ripening initiation (Gouthu and Deluc, 2015; Vondras et al., 2017). We have demonstrated an indirection relationship between the Seed Index and the entry of individual grape berries to the ripening regardless of the timing of anthesis (Vondras et al., 2016). High seed index berries delays the ripening while Low seed index berries are advanced. During the berry maturation phase, seeds experience a significant growth activity that is associated with the embryo differentiation and development. We infer that a seed-driven auxin export to the pericarp explain the delay in berry with high seed weight. To answer this hypothesis, we have carried out two main projects aimed to understand the influence of seed on the ripening process. In the first research project, we carried out a genome-wide study to compare the transcriptional ripening programs of seed, pulp, and skin tissues of berries with high and low SI from berry maturation (pre-ripening phase) to berry maturity (harvest). Our research objective here is mainly oriented towards gene discovery to identify the main regulatory "hubs" of the ripening program influenced by the seed. The second project is to understand how seed development and embryo differentiation affects the movement and transport of auxin from the seed to the pericarp. For this, we are using the microvine as system to express fluorescent proteins (DR5:V2 and R2D2) that report on the auxin input and concentration in cells (Liao et al., 2015).
- The specific interplay between ABA, auxin and sugar is a critical component of the ripening initiation but the effects of genetic regulatory processes like epigenetic regulation are also explored. Some preliminary works in the lab indicate a potential interaction between Auxin signaling and a class of epigenetic factors (Demethylase: Jumonji Class). We are currently working in the development of transgenic lines using dCAs9 fused to either transcriptional activator or repressor under conditional expression (Lowder et al., 2015). Our objective is to spatially and temporally characterize three Jumonji genes at different transitional phases of grapevine growth (root, berries, shoot).