Designer Glycans

Hemicelluloses are important dietary fibers and a key component of lignocellulosic biomass. Despite numerous observations for fluorescently tagged cellulose synthases, the subcellular journeys and biochemical activities of intracellular cellulose synthase-like enzymes such as β-mannan synthases (ManS) remain largely unexplored. This study identifies C-terminal fluorescent protein tags that maintain ManS activity in yeast to accelerate the Design, Build, Test, Learn cycles for polysaccharide biosynthesis. Using the Amorphophallus konjac ManS as a case study, we demonstrate that the enzyme colocalizes with a known yeast marker for the Golgi apparatus despite the toxic effects of plant glucomannan accumulation in Pichia pastoris. The ManS first transmembrane domain was found to be critical for the punctate localization of the enzyme, its overall expression level and its function. Additionally, we explored how fluorescently tagged ManS is influenced by genetic or chemical perturbations of native yeast cell wall components, such as reducing protein mannosylation and severely disrupting β-1,3-glucans. Finally, we identified alternative feeding strategies and episomal vectors for Pichia, which were extended to Saccharomyces cerevisiae, to accelerate hemicellulose research. We propose that expanding the Plant MoClo-compatible plasmid repertoire is essential to swiftly prototype carbohydrate-active enzymes in yeast before proceeding with more time-intensive analyses in plants. Requiring only hours or days instead of weeks or months for plant transformation/regeneration, our yeast prototyping strategies can derisk the bioengineering of carbohydrate-active enzymes.

Plant breeding and genetics play a major role in the adaptation of plants to meet human needs. The current requirement to make agriculture more sustainable can be partly met by a greater reliance on biological nitrogen fixation (BNF) by symbiotic diazotrophic microorganisms that provide crop plants with ammonium. Select accessions of the cereal crop sorghum (Sorghum bicolor (L.) Moench) form mucilage-producing aerial roots that harbor nitrogen-fixing bacteria. Breeding programs aimed at developing sorghum varieties that support diazotrophs will benefit from a detailed understanding of the genetic and environmental factors contributing to aerial root formation. A genome-wide association study (GWAS) of the sorghum minicore, a collection of 242 landraces, and 30 accessions from the sorghum association panel (SAP) was conducted in Florida and Wisconsin and under two fertilizer treatments to identify loci associated with the number of nodes with aerial roots and aerial root diameter. Sequence variation in genes encoding transcription factors that control phytohormone signaling and root system architecture showed significant associations with these traits. In addition, the location had a significant effect on the phenotypes. Concurrently, we developed F2 populations from crosses between bioenergy sorghums and a landrace that produced extensive aerial roots to evaluate the mode of inheritance of the loci identified by the GWAS. Furthermore, the mucilage collected from aerial roots contained polysaccharides rich in galactose, arabinose, and fucose, whose composition displayed minimal variation among 10 genotypes and two fertilizer treatments. These combined results support the development of sorghums with the ability to acquire nitrogen via BNF.

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Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate–sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.

Wound healing is a fundamental property of plants and animals that requires recognition of cellular damage to initiate regeneration. In plants, wounding activates a defense response via the production of jasmonic acid and a regeneration response via the hormone auxin and several ethylene response factor (ERF) and NAC domain-containing protein (ANAC) transcription factors. To better understand how plants recognize damage and initiate healing, we searched for factors upregulated during the horticulturally relevant process of plant grafting and found four related DNA binding with one finger (DOF) transcription factors, HIGH CAMBIAL ACTIVITY2 (HCA2), TARGET OF MONOPTEROS6 (TMO6), DOF2.1, and DOF6, whose expression rapidly activated at the Arabidopsis graft junction. Grafting or wounding a quadruple hca2, tmo6, dof2.1, dof6 mutant inhibited vascular and cell-wall-related gene expression. Furthermore, the quadruple dof mutant reduced callus formation, tissue attachment, vascular regeneration, and pectin methylesterification in response to wounding. Wcalluscallus found that activation of DOF gene expression after wounding required auxin, but hormone treatment alone was insufficient for their induction. However, modifying cell walls by enzymatic digestion of cellulose or pectin greatly enhanced TMO6 and HCA2 expression, whereas genetic modifications to the pectin or cellulose matrix using the PECTIN METHYLESTERASE INHIBITOR5 overexpression line or korrigan1 mutant altered TMO6 and HCA2 expression. Changes to the cellulose or pectin matrix were also sufficient to activate the wound-associated ERF115 and ANAC096 transcription factors, suggesting that cell-wall damage represents a common mechanism for wound perception and the promotion of tissue regeneration.Graphical abstract

Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes are guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis. Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles in cell wall polysaccharide biosynthesis. Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. IQD9 as well as a previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein act to maintain cellulose synthase velocity. Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in cell wall biosynthesis. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.

Hemicellulosic polysaccharides built of b-1,4-linked mannose units have been found throughout the plant kingdom and have numerous industrial applications. Here, I review recent advances in the biosynthesis and modification of plant b-mannans. These matrix polymers can associate with cellulose bundles to impact the mechanical properties of plant fibers or biocomposites. In certain algae, mannan microfibrils even replace cellulose as the dominant structural component of the cell wall. Conversely, patterned galactoglucomannan found in Arabidopsis thaliana seed mucilage significantly modulates cell wall architecture and abiotic stress tolerance despite its relatively low content. I also discuss the subcellular requirements for b-mannan biosynthesis, the increasing number of carbohydrate-active enzymes involved in this process, and the players that continue to be puzzling. I discuss how cellulose synthase-like enzymes elongate (gluco)mannans in orthogonal hosts and highlight the discoveries of plant enzymes that add specific galactosyl or acetyl decorations. Hydrolytic enzymes such as endo-b-1,4-mannanases have recently been involved in a wide range of biological contexts including seed germination, wood formation, heavy metal tolerance, and defense responses. Synthetic biology tools now provide faster tracks to modulate the increasingly-relevant mannan structures for improved plant traits and bioproducts.

Abstract Manganese (Mn2+) is essential for a diversity of processes, including photosynthetic water splitting and the transfer of glycosyl moieties. Various Golgi-localized glycosyltransferases that mediate cell wall matrix polysaccharide biosynthesis are Mn2+ dependent, but the supply of these enzymes with Mn2+ is not well understood. Here, we show that the BIVALENT CATION TRANSPORTER 3 (BICAT3) localizes specifically to trans-cisternae of the Golgi. In agreement with a role in Mn2+ and Ca2+ homeostasis, BICAT3 rescued yeast (Saccharomyces cerevisiae) mutants defective in their translocation. Arabidopsis (Arabidopsis thaliana) knockout mutants of BICAT3 were sensitive to low Mn2+ and high Ca2+ availability and showed altered accumulation of these cations. Despite reduced cell expansion and leaf size in Mn2+-deficient bicat3 mutants, their photosynthesis was improved, accompanied by an increased Mn content of chloroplasts. Growth defects of bicat3 corresponded with an impaired glycosidic composition of matrix polysaccharides synthesized in the trans-Golgi. In addition to the vegetative growth defects, pollen tube growth of bicat3 was heterogeneously aberrant. This was associated with a severely reduced and similarly heterogeneous pectin deposition and caused diminished seed set and silique length. Double mutant analyses demonstrated that the physiological relevance of BICAT3 is distinct from that of ER-TYPE CA2+-ATPASE 3, a Golgi-localized Mn2+/Ca2+-ATPase. Collectively, BICAT3 is a principal Mn2+ transporter in the trans-Golgi whose activity is critical for specific glycosylation reactions in this organelle and for the allocation of Mn2+ between Golgi apparatus and chloroplasts.

SummaryArabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes is guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis (SCE).Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles for cell wall polysaccharide biosynthesis and cortical microtubule (MT) organization.Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. Double mutant analyses revealed that their closest paralogs (IQD10 and KLCR2, respectively) are not required for mucilage biosynthesis. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. Similar to the previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein, IQD9 is required to maintain the velocity of cellulose synthases.Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in guiding the distribution of cell wall polysaccharides. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.

Background The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past five years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes.Results Here, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made β-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana.Conclusion The results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as β-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins.