Engineering Plant Cell Fates and Functions for Agriculture and Industry.

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation. Many of the mutations that underlie these changes have been identified in key regulators of cellular identity and function. As key determinants of yield, these regulators are key targets for synthetic biology approaches to engineer new forms and functions. However, our understanding of many plant developmental programs and cell-type specific functions is still incomplete. In this Perspective, we discuss how advances in cellular genomics together with synthetic biology tools such as biosensors and DNA-recording devices are advancing our understanding of cell-specific programs and cell fates. We then discuss advances and emerging opportunities for cell-type-specific engineering to optimize plant morphology, responses to the environment, and the production of valuable compounds.

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana.

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

CIPK-B is essential for salt stress signalling in Marchantia polymorpha.

Calcium signalling is central to many plant processes, with families of calcium decoder proteins having expanded across the green lineage and redundancy existing between decoders. The liverwort Marchantia polymorpha has fast become a new model plant, but the calcium decoders that exist in this species remain unclear. We performed phylogenetic analyses to identify the calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) network of M. polymorpha. We analysed CBL-CIPK expression during salt stress, and determined protein-protein interactions using yeast two-hybrid and bimolecular fluorescence complementation. We also created genetic knockouts using CRISPR/Cas9. We confirm that M. polymorpha has two CIPKs and three CBLs. Both CIPKs and one CBL show pronounced salt-responsive transcriptional changes. All M. polymorpha CBL-CIPKs interact with each other in planta. Knocking out CIPK-B causes increased sensitivity to salt, suggesting that this CIPK is involved in salt signalling. We have identified CBL-CIPKs that form part of a salt tolerance pathway in M. polymorpha. Phylogeny and interaction studies imply that these CBL-CIPKs form an evolutionarily conserved salt overly sensitive pathway. Hence, salt responses may be some of the early functions of CBL-CIPK networks and increased abiotic stress tolerance required for land plant emergence.