Team:Valencia UPV/Design
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BIOLOGICAL DESIGN
HUMAN-PLANT
As far as communication is concerned, a bidirectional channel must be established. In our project, we evidence that is possible to deliver stimuli light orders into plants. An optogenetic circuit was designed to establish a reliable communication channel between humans and plants. We can control both plant development and behavior with the aim of helping to achieve a future sustainable agriculture
One of the main challenges in plant platforms is using light to precisely control cellular behavior. Recently, it started to be addressed and ChatterPlant aims to contribute to this knowledge. In order to control in time and space protein expression levels accurately, light irradiation can be used due to its easy manipulation and precision.
Therefore, we resolved designing a light-inducible system based on red/far-red light in order to control the genetic expression on any desired protein (Müller, Naumann, Weber & Zurbriggen, 2015). Therefore, we will be able to regulate plants at genetic level accurately, contributing to the development of a more efficient and sustainable agriculture in our world.
OPTOGENETIC SWITCH
Control of gene expression is carried out using a red/far-red light-switch (Müller et al., 2014), allowing us to activate the genetic circuit with red light (660 nm) and turn it off by subsequent illumination with far-red light (740 nm). The molecular response of this optogenetic switch is based on the interaction between phytochrome-interacting factor 6 (PIF6) and phytochrome B (PhyB).
The photoreceptor (PhyB) was isolated from Arabidopsis thaliana and it can integrate light signals, as well as it participates in the control of floral induction and germination. Despite this, this protein was found to be useful as a tool for Plant Synthetic Biology. As a photosensor protein, is able to change its conformation to the active form when receiving red photons (660 nm). Once in an activated state, it will be able to interact specifically with the transcription factor PIF6 (Khanna, 2004). Bounded complex allows expression of the desired coding sequence under control of operator site Etr8 (an E-responsive operator motif) and minimal promoter (minCMV).
In order to allow any protein expression, PIF6 is fused to a DNA-binding domain (E), which binds to the operator site (Etr8) in the construction we intend to express. Equally, PhyB is linked to a nuclear location sequence (NLS) and an activator domain (VP64), that has the ability to act as a strong transcriptional activator of a gene. This ON state can up-regulate the transcription of the next element of the circuit sparking off the signaling pathway. Likewise, absorption of a far-red photon (740 nm) converts PhyB into the inactive form, leading to dissociation from PIF6 and turning the circuit in an OFF state. (Ni, Tepperman & Quail, 1999). Once genetic pathway is inactivated, gene transcription returns to its basal expression. Plants can produce wanted transcripts in proper time, avoiding continuously even unnecessary expression (i.e. decreasing cell-burden effects).
Figure 1: Graphic design of human-plant circuit. a) The transcriptional factor, PIF6, is fused to a DNA-binding domain (E) and PhyB is fused to the activator domain (VP64) and a nuclear location sequence (NLS). Both genetic expressions are controlled by plant strong promoters. b) When irradiated with 660nm light, PhyB changes its conformation and this complex is recruited to PIF6 at the promoter site. The polymerase III will recognize the activation domain and the transcription will begin. Only upon absorption of a far-red photon the interaction between PhyB and PIF6 is terminated, resulting in a shut-off of gene expression.
We proposed using Flowering Locus T (FT) because of its ability of inducing flowering process when it is activated. This protein is constitutively expressed in plant cotyledons and leaves, and it involves a remarkable and conservable role in plants. In response to inductive long days Flowering Locus T protein (i.e. phosphatidylethanolamine-binding protein) acts in the shoot apex to induce target meristem identity genes such as APETALA1 (AP1) (Notaguchi, Daimon, Abe & Araki, 2009) and initiates floral morphogenesis. Bearing that in mind, we propose to regulate FT transcriptional rate in order to help improving the current agriculture.
However, a modular and standard system was designed in order to reach all users’ necessities. Circuit’s modularity allows to select easily the light-regulated cellular process by changing the output module. Therefore, we are able to induce almost any response on plant (e.g. flowering induction, climate conditions protection, organoleptic features enhancement). Essentially, we could easily personalize any desired element to provide the maximum possible control over the plant.
LIGHT APPLICATION SITE
Since white light presents all visible wavelengths, controlling system activation/inactivation becomes almost impossible. We resolved this issue by plant root-specific promoter so optogenetic circuit can be activated when roots (which do not need light and are usually in dark conditions) are illuminated by red/far red light. This strategy guarantees almost a total root-specific regulated transcription, preventing interferences in circuit signaling and making our system more accurate, safe and efficient.
TRANSPORT FROM ROOTS
Since our genetic circuit will be activated in roots, proteins of the desired regulated element will be only synthesized there. Unfortunately, many proteins are not capable to transport themselves to other parts of the plant so, a systemic movement strategy along the plant becomes necessary to fulfil is metabolic role.
One of the main strategy that is still being developed is the use of mRNAs as long-distance signaling molecules because of its ability to deliver a signal in its non-functional form (Spiegelman, Golan & Wolf, 2013). Therefore, the translation of the desired element occurs specifically at the target site. Nevertheless, long-distance movement of transcripts precise much more control and to date, the complete understanding of the molecular machine underlying the mRNA trafficking has not clearly been reported to provide a definitive proof for this notion. For instance, it has been shown that protein CmPP16 from Cucurbita maxima possess properties similar to those of viral movement proteins. However it might not behave similar in other species (Xoconostle-Cázares, 1999). Equally with AtABCG14, critical for cytokinin translocation (Ko et al., 2014)
Despite that, there are recent evidence of different molecules that can act as potential components of transport long-distance mRNAs. One is the presence of translocatable RNA-binding proteins (RBPs) in the phloem (Pallas & Gómez, 2013) while another possibility is cell-penetrating peptides (CPPs) that transport hydrophilic macromolecules into cells. Here, application of CPPs for macromolecule delivery has been successfully demonstrated for plant cells even though further characterization is needed to determine which structural features influence its function (Chugh, Eudes & Shim, 2010).
Considering that mRNA transport approach is not the most suitable approach yet, we resolved to use plant viral vectors to amplify and transport any desired molecule. This strategy involves two main advantages in our system. First, the protein production is faster, and its yield is higher due to its ability to auto replicate itself. The second one is related to viral vector’s systemic movement ability. Thus, any desired protein can be transported from roots to the aerial part of the plant in an efficient way. These reasons encouraged us to work with viral vector strategy although we know that our main future challenge is understanding the mechanisms of mRNA import and transport in order to enhance and promote ChatterPlant’s possibilities.
WHY AN ON-OFF SYSTEM?
Constitutive expression of transgenes in plants often leads to pleiotropic undesirable phenotypes, limiting Plant SynBio applications in real world (Yi et al, 2010). Different transgenic plants show abnormal development (Romero et al, 1997), growth retardation or severe reduction in seed production (Liu et al, 1998) during their development. Accurate control of transgene expression allows to optimize development and behavior of transgenic plants without giving up improved features of the organism.
An ON-OFF optogenetic circuit offers us the possibility to activate and deactivate transgene expression depending on plant status, avoiding interferences with plant development and decreasing metabolic charge in cells.
Maintaining essential mineral micronutrients and vitamins levels of staple food crops allows to ensure access to affordable food for people with nutritional deficiencies. Biofortification born as a whole of techniques used to improve nutritional content of staple food crops through modern biotechnology, agronomic practices or conventional plant breeding.
As an example, phytoene synthase (PSY) is involved in carotenoids biosynthesis. (Rodríguez-Villalón, Gas and Rodríguez-Concepción, 2009). Carotenoids are isoprenoids essential for plant life and human health, working as antioxidant molecules. Epidemiological studies correlates lack of carotenoid consumption with different diseases related with oxidative stresses and chronic disorders. Induced expression of PSY-encoding genes in transgenic plants results in increased carotenoid levels. However, constitutive expression of PSY-gene causes dwarfism in transgenic tomatoes, interfering in both behavior and development of the plant.
