ABSTRACT
Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make heterologous gene expression more predictable in this testbed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S×2) provided the highest yield of proteins although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia ETHYLENE RESPONSE FACTOR 1 (MpERF1) and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER (MpC2HDZ) genes drove expression to higher levels across all tissues without growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed the polycistronic RUBY betalain synthesis cassette to demonstrate coordinated expression of metabolic enzymes. A heat-shock inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing toolkit for gene expression in Marchantia and provide new resources for the Marchantia research community.
ABSTRACT
The hornworts are a small group of land plants, consisting of only 11 families and approximately 220 species. Despite their small size as a group, their phylogenetic position and unique biology are of great importance. Hornworts, together with mosses and liverworts, form the monophyletic group of bryophytes that is sister to all other land plants (Tracheophytes). It is only recently that hornworts became amenable to experimental investigation with the establishment of Anthoceros agrestis as a model system. In this perspective, we summarize the recent advances in the development of A. agrestis as an experimental system and compare it with other plant model systems. We also discuss how A. agrestis can help to further research in comparative developmental studies across land plants and to solve key questions of plant biology associated with the colonization of the terrestrial environment. Finally, we explore the significance of A. agrestis in crop improvement and synthetic biology applications in general.
ABSTRACT
The rising population and the ongoing climate crisis call for improved means to monitor and optimise agriculture. A promising approach to tackle current challenges in food production is the early diagnosis of plant diseases through non-invasive methods, such as the detection of volatiles. However, current devices for detection of multiple volatiles are based on electronic noses, which are expensive, require complex circuit assembly, may involve metal oxides with heating elements, and cannot easily be adapted for some applications that require miniaturisation or limit front-end use of electronic components. To address these challenges, a low-cost optoelectronic nose using chemo-responsive colorimetric dyes drop-casted onto filter paper has been developed in the current work. The final sensors could be used for the quantitative detection of up to six plant volatiles through changes in colour intensities with a sub-ppm level limit of detection, one of the lowest limits of detection reported so far using colorimetric gas sensors. Sensor colouration could be analysed using a low-cost spectrometer and the results could be processed using a microcontroller. The measured volatiles could be used for the early detection of plant abiotic stress as early as two days after exposure to two different stresses: high salinity and starvation. This approach allowed a lowering of costs to GBP 1 per diagnostic sensing paper. Furthermore, the small size of the paper sensors allows for their use in confined settings, such as Petri dishes. This detection of abiotic stress could be easily achieved by exposing the devices to living plants for 1 h. This technology has the potential to be used for monitoring of plant development in field applications, early recognition of stress, implementation of preventative measures, and mitigation of harvest losses.
ABSTRACT
Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering.
