Compensatory Guaiacyl Lignin Biosynthesis at the Expense of Syringyl Lignin in 4CL1-Knockout Poplar.

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula × alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1 Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.

Tubulins, rhythms and cell walls in poplar leaves: it's all in the timing.

Plant cell walls exhibit architectural and compositional changes throughout their development and in response to external cues. While tubulins are involved in cell wall biogenesis, much remains unknown about the scope of their involvement during the orchestration of this resource-demanding process. A transgenic approach coupled with cell wall compositional analysis, RNA-seq and mining of publicly available diurnal gene expression data was used to assess the involvement of tubulins in poplar leaf cell wall biogenesis. Leaf cell walls of transgenic poplar lines with constitutive overexpression of α-tubulin (TUA) exhibited an increased abundance of homogalacturonan, along with a reduction in xylose. These changes were traced to altered expression of UDP-glucuronic acid decarboxylase (GADC) in the transgenic leaves. A model is postulated by which altered diurnal control of TUA through its constitutive overexpression led to a metabolic tradeoff affecting cellular utilization of GADC substrate UDP-glucuronic acid. While there were no effects on cellulose, hemicellulose or lignin abundance, subtle effects on hemicellulose composition and associated gene expression were noted. In addition, expression and enzymatic activity of pectin methylesterase (PME) decreased in the transgenic leaves. The change is discussed in a context of increased levels of PME substrate homogalacturonan, slow stomatal kinetics and the fate of PME product methanol. Since stomatal opening and closing depend on fundamentally contrasting microtubule dynamics, the slowing of both processes in the transgenic lines as previously reported appears to be directly related to underlying cell wall compositional changes that were caused by tubulin manipulation.

A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors.

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

Kingella kingae Expresses Four Structurally Distinct Polysaccharide Capsules That Differ in Their Correlation with Invasive Disease.

Kingella kingae is an encapsulated gram-negative organism that is a common cause of osteoarticular infections in young children. In earlier work, we identified a glycosyltransferase gene called csaA that is necessary for synthesis of the [3)-ß-GalpNAc-(1→5)-ß-Kdop-(2→] polysaccharide capsule (type a) in K. kingae strain 269-492. In the current study, we analyzed a large collection of invasive and carrier isolates from Israel and found that csaA was present in only 47% of the isolates. Further examination of this collection using primers based on the sequence that flanks csaA revealed three additional gene clusters (designated the csb, csc, and csd loci), all encoding predicted glycosyltransferases. The csb locus contains the csbA, csbB, and csbC genes and is associated with a capsule that is a polymer of [6)-α-GlcpNAc-(1→5)-ß-(8-OAc)Kdop-(2→] (type b). The csc locus contains the cscA, cscB, and cscC genes and is associated with a capsule that is a polymer of [3)-ß-Ribf-(1→2)-ß-Ribf-(1→2)-ß-Ribf-(1→4)-ß-Kdop-(2→] (type c). The csd locus contains the csdA, csdB, and csdC genes and is associated with a capsule that is a polymer of [P-(O→3)[ß-Galp-(1→4)]-ß-GlcpNAc-(1→3)-α-GlcpNAc-1-] (type d). Introduction of the csa, csb, csc, and csd loci into strain KK01Δcsa, a strain 269-492 derivative that lacks the native csaA gene, was sufficient to produce the type a capsule, type b capsule, type c capsule, and type d capsule, respectively, indicating that these loci are solely responsible for determining capsule type in K. kingae. Further analysis demonstrated that 96% of the invasive isolates express either the type a or type b capsule and that a disproportionate percentage of carrier isolates express the type c or type d capsule. These results establish that there are at least four structurally distinct K. kingae capsule types and suggest that capsule type plays an important role in promoting K. kingae invasive disease.

Novel rhamnogalacturonan I and arabinoxylan polysaccharides of flax seed mucilage.

The viscous seed mucilage of flax (Linum usitatissimum) is a mixture of rhamnogalacturonan I and arabinoxylan with novel side group substitutions. The rhamnogalacturonan I has numerous single nonreducing terminal residues of the rare sugar l-galactose attached at the O-3 position of the rhamnosyl residues instead of the typical O-4 position. The arabinoxylan is highly branched, primarily with double branches of nonreducing terminal l-arabinosyl units at the O-2 and O-3 positions along the xylan backbone. While a portion of each polysaccharide can be purified by anion-exchange chromatography, the side group structures of both polysaccharides are modified further in about one-third of the mucilage to form composites with enhanced viscosity. Our finding of the unusual side group structures for two well-known cell wall polysaccharides supports a hypothesis that plants make a selected few ubiquitous backbone polymers onto which a broad spectrum of side group substitutions are added to engender many possible functions. To this end, modification of one polymer may be accompanied by complementary modifications of others to impart functions to heterocomposites not present in either polymer alone.

Detection and identification of rhamnogalacturonan lyase activity in intercellular spaces of expanding cotton cotyledons.

Rhamnogalacturonan lyase (RG lyase) activity has been detected and its relative activity measured in vivo during the expansion of cotton (Gossypium hirsutum L.) cotyledons. Rhamnogalacturonan (RG) oligomers labeled with a fluorescent tag were injected into the intercellular spaces of cotton cotyledons and, after incubation, the digested substrate was rinsed out. Enzyme digestion products were detected and identified by capillary zone electrophoresis. Rhamnogalacturonan lyase products were identified as such by co-migration with the digestion products of linear RG oligomers when the oligomers were treated with fungal RG lyase but not when treated with fungal RG hydrolase. In addition, reaction of plant RG lyase digestion products of RG oligomers with I(2)/KI, which selectively removes unsaturated galactopyranosyluronic acid (GaLap) residues formed at the non-reducing end of the oligomer, converted the plant digestion products into RG oligomers that co-migrated with fungal RG hydrolase products. The activity of the enzyme in the intercellular spaces of cotton cotyledons is very low and could be detected most easily when not >0.03 nmol of substrate was injected in a approximately 0.7-cm(2) area and incubated in vivo for 2-6 h. Rhamnogalacturonan lyase activity was the highest in rapidly expanding 3- to 4-day-old cotyledons and gradually decreased during the slow-down in expansion over the next 2-3 days. The RG lyase activity was also detected when the APTS (8-aminopyrene-1,3,6-trisulfonic acid, trisodium salt)-labeled substrates were introduced into intercellular spaces by infiltration instead of injection, indicating that the activity was not induced by wounding or released into the apoplast by cell damage. An exo-RG galacturonohydrolase activity was also found, but RG hydrolase and exo-RG rhamnohydrolase were not detected.