Cell type-specific control and post-translational regulation of specialized metabolism: opening new avenues for plant metabolic engineering.

Although plant metabolic engineering enables the sustainable production of valuable metabolites with many applications, we still lack a good understanding of many multi-layered regulatory networks that govern metabolic pathways at the metabolite, protein, transcriptional and cellular level. As transcriptional regulation is better understood and often reviewed, here we highlight recent advances in the cell type-specific and post-translational regulation of plant specialized metabolism. With the advent of single-cell technologies, we are now able to characterize metabolites and their transcriptional regulators at the cellular level, which can refine our searches for missing biosynthetic enzymes and cell type-specific regulators. Post-translational regulation through enzyme inhibition, protein phosphorylation and ubiquitination are clearly evident in specialized metabolism regulation, but not frequently studied or considered in metabolic engineering efforts. Finally, we contemplate how advances in cell type-specific and post-translational regulation can be applied in metabolic engineering efforts in planta, leading to optimization of plants as metabolite production vehicles.

Expression Patterns and Functional Characterization of Arabidopsis Galactose Oxidase-Like Genes Suggest Specialized Roles for Galactose Oxidases in Plants.

Galactose oxidases (GalOxs) are well-known enzymes that have been identified in several fungal species and characterized using structural and enzymatic approaches. However, until very recently, almost no information on their biological functions was available. The Arabidopsis (Arabidopsis thaliana) gene ruby particles in mucilage (RUBY) encodes a putative plant GalOx that is required for pectin cross-linking through modification of galactose (Gal) side chains and promotes cell-cell adhesion between seed coat epidermal cells. RUBY is one member of a family of seven putative GalOxs encoded in the Arabidopsis genome. To examine the function(s) of GalOxs in plants, we studied the remaining six galactose oxidase-like (GOXL) proteins. Like RUBY, four of these proteins (GOXL1, GOXL3, GOXL5 and GOXL6) were found to localize primarily to the apoplast, while GOXL2 and GOXL4 were found primarily in the cytoplasm. Complementation and GalOx assay data suggested that GOXL1, GOXL3 and possibly GOXL6 have similar biochemical activity to RUBY, whereas GOXL5 only weakly complemented and GOXL2 and GOXL4 showed no activity. Members of this protein family separated into four distinct clades prior to the divergence of the angiosperms. There have been recent duplications in Brassicaceae resulting in two closely related pairs of genes that have either retained similarity in expression (GOXL1 and GOXL6) or show expression divergence (GOXL3 and RUBY). Mutant phenotypes were not detected when these genes were disrupted, but their expression patterns suggest that these proteins may function in tissues that require mechanical reinforcements in the absence of lignification.

RUBY, a Putative Galactose Oxidase, Influences Pectin Properties and Promotes Cell-To-Cell Adhesion in the Seed Coat Epidermis of Arabidopsis.

Cell-to-cell adhesion is essential for establishment of multicellularity. In plants, such adhesion is mediated through a middle lamella composed primarily of pectic polysaccharides. The molecular interactions that influence cell-to-cell adhesion are not fully understood. We have used Arabidopsis (Arabidopsis thaliana) seed coat mucilage as a model system to investigate interactions between cell wall carbohydrates. Using a forward-genetic approach, we have discovered a gene, RUBY PARTICLES IN MUCILAGE (RUBY), encoding a protein that is annotated as a member of the Auxiliary Activity 5 (AA5) family of Carbohydrate-Active Enzymes (Gal/glyoxal oxidases) and is secreted to the apoplast late in the differentiation of seed coat epidermal cells. We show that RUBY is required for the Gal oxidase activity of intact seeds; the oxidation of Gal in side-chains of rhamnogalacturonan-I (RG-I) present in mucilage-modified2 (mum2) mucilage, but not in wild-type mucilage; the retention of branched RG-I in the seed following extrusion; and the enhancement of cell-to-cell adhesion in the seed coat epidermis. These data support the hypothesis that RUBY is a Gal oxidase that strengthens pectin cohesion within the middle lamella, and possibly the mucilage of wild-type seed coat epidermal cells, through oxidation of RG-I Gal side-chains.

Analysis of Monosaccharides from Arabidopsis Seed Mucilageand Whole Seeds Using HPAEC-PAD.

Arabidopsis seed coat epidermal cells deposit a significant quantity of mucilage, composed of the cell wall components pectin, hemicellulose, and cellulose, into the apoplast during development. When mature seeds are hydrated, mucilage extrudes to form a gelatinous capsule around the seed. Determining the monosaccharide composition of both extruded mucilage and whole seeds is an essential technique for characterizing seed coat developmental processes and mutants with altered mucilage composition. This protocol covers growth of plants to produce seeds suitable for analysis, extraction of extruded mucilage using water and sodium carbonate (used for mutants with impaired mucilage release), and extraction of alcohol insoluble residue (AIR) from whole seeds. The prepared polysaccharides are then hydrolyzed using sulfuric acid, which hydrolyses all polysaccharides including cellulose. Sensitive and reproducible quantification of the resulting monosaccharides is achieved using high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD).

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins.

Interactions between cell wall polymers are critical for establishing cell wall integrity and cell-cell adhesion. Here, we exploit the Arabidopsis (Arabidopsis thaliana) seed coat mucilage system to examine cell wall polymer interactions. On hydration, seeds release an adherent mucilage layer strongly attached to the seed in addition to a nonadherent layer that can be removed by gentle agitation. Rhamnogalacturonan I (RG I) is the primary component of adherent mucilage, with homogalacturonan, cellulose, and xyloglucan constituting minor components. Adherent mucilage contains rays composed of cellulose and pectin that extend above the center of each epidermal cell. CELLULOSE SYNTHASE5 (CESA5) and the arabinogalactan protein SALT-OVERLY SENSITIVE5 (SOS5) are required for mucilage adherence through unknown mechanisms. SOS5 has been suggested to mediate adherence by influencing cellulose biosynthesis. We, therefore, investigated the relationship between SOS5 and CESA5. cesa5-1 seeds show reduced cellulose, RG I, and ray size in adherent mucilage. In contrast, sos5-2 seeds have wild-type levels of cellulose but completely lack adherent RG I and rays. Thus, relative to each other, cesa5-1 has a greater effect on cellulose, whereas sos5-2 mainly affects pectin. The double mutant cesa5-1 sos5-2 has a much more severe loss of mucilage adherence, suggesting that SOS5 and CESA5 function independently. Double-mutant analyses with mutations in MUCILAGE MODIFIED2 and FLYING SAUCER1 that reduce mucilage release through pectin modification suggest that only SOS5 influences pectin-mediated adherence. Together, these findings suggest that SOS5 mediates adherence through pectins and does so independently of but in concert with cellulose synthesized by CESA5.