The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.

The phloem is a highly specialized vascular tissue that forms a fundamentally important transport and signaling pathway in plants. It is therefore a system worth protecting. The main function of the phloem is to transport the products of photosynthesis throughout the whole plant, but it also transports soluble signaling molecules and propagates electrophysiological signals. The phloem is constantly threatened by mechanical injuries, phloem-sucking pests and parasites, and the spread of pathogens, which has led to the evolution of efficient defense mechanisms. One such mechanism involves structural phloem proteins, which are thought to facilitate sieve element occlusion following injury and to defend the plant against pathogens. In leguminous plants, specialized structural phloem proteins known as forisomes form unique mechanoproteins via sophisticated molecular interaction and assembly mechanisms, thus enabling reversible sieve element occlusion. By understanding the structure and function of forisomes and other structural phloem proteins, we can develop a toolbox for biotechnological applications in material science and medicine. Furthermore, understanding the involvement of structural phloem proteins in plant defense mechanisms will allow phloem engineering as a new strategy for the development of crop varieties that are resistant to pests, pathogens and parasites.

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Forisome structures are fine-tuned by the subunit composition Forisomes are large, spindle-shaped protein complexes that are present in the sieve elements of papilionoid legumes.1,2 In their spindle-shaped conformation, forisomes facilitate the transport of photoassimilates along the sieve tube lumen, but when the vascular system is wounded they change into a pluglike/expanded conformation that causes occlusion to prevent leakage. After tissue regeneration, forisomes can revert to their spindle-shaped conformation, thereby providing a reversible sieve tube plugging mechanism. The conformational change is calcium-dependent and can also be induced ex vivo by nonphysiological pH values.3 The forisome subunits in M. truncatula were identified by mass spectrometry following the purification of native forisomes. 4 The MtSEO-F1 protein sequence (formerly Mtfor1) was used for homology searches and transcriptional analysis, resulting in the characterization of a large sieve element occlusion (SEO) protein family spanning a wide range of dicot plants. 5,6 The group 1 SEO proteins are only present in papilionoid legumes and these represent forisome subunits, whereas members of groups 5 and 6 represent the common P-proteins in most dicot species that probably cause irreversible sieve element occlusion. [5][6][7][8] In our recent study, we expressed group 1 SEO-F genes from different legumes in a heterologous background to study their impact on forisome assembly, including the DpSEO-F1 gene from the basal papilionoid species D. panamensis. 9 The D. panamensis lineage diverged from lineages of the Old World clade, i.e., Canavalia gladiata, Lotus japonicus, and Medicago truncatula, more than 50 million years ago. The artificial protein bodies produced by expressing SEO-F genes from Lotus japonicus , , Vicia faba (VfSEO-F1), and M. truncatula ) appeared longer and thinner than wild-type forisomes, whereas the artificial forisome composed of DpSEO-F1 was similar in geometry to its native counterpart. We also found that forisome geometry was affected by coexpression, e.g., MtSEO-F2 was able to widen the forisome body Forisomes are specialized multimeric protein complexes found only in the papilionoid legumes. they undergo a reversible conformational change in response to phloem injury to enable the occlusion of sieve tubes, thus preventing the loss of photoassimilates. the individual subunits are designated by the letters SEo-F (sieve element occlusion by forisomes) and are part of the larger SEo protein family, which also includes the typical P-proteins found in most dicots and some monocots. When specific SEo-F subunits from different species are expressed in a heterologous background, they self-assemble into fully-functional artificial forisomes. however, with the exception of basal species such as Dipteryx panamensis, the geometry of these artificial forisomes differs from that of their native counterparts. Studies involving SEo-F proteins from the model legume Medicago truncatula have shown that a combination of 3 of the 4 ...