Acetylation of homogalacturonan and rhamnogalacturonan-I is catalyzed by a suite of trichome birefringence-like proteins.

Plant cell wall polysaccharides, including xylan, mannan, xyloglucan, and pectins, are often acetylated and members of the domain of unknown function 231 (DUF231)/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases mediating the acetylation of xylan, mannan, and xyloglucan. However, little is known about the O-acetyltransferases responsible for pectin acetylation. In this report, we biochemically characterized a suite of Arabidopsis DUF231/TBL proteins for their roles in pectin acetylation. We generated 24 TBL recombinant proteins in mammalian cells and demonstrated that 10 of them were able to transfer acetyl groups from acetyl-CoA onto the pectins homogalacturonan (HG) or rhamnogalacturonan-I (RG-I), and thus were named pectin O-acetyltransferase 1 to 10 (POAT1 to 10). It was found that POAT2,4,9,10 specifically acetylated HG and POAT5,6 acetylated RG-I, whereas POAT1,3,7,8 could act on both HG and RG-I. The acetylation of HG and RG-I by POATs was further corroborated by hydrolysis with pectin acetylesterases and by nuclear magnetic resonance spectroscopy. In addition, mutations of the conserved GDS and DXXH motifs in POAT3 and POAT8 were shown to lead to a loss of their ability to acetylate HG and RG-I. Furthermore, simultaneous RNA interference downregulation of POAT1,3,6,7,8 resulted in reduced cell expansion, impaired plant growth, and decreased pectin acetylation. Together, our findings indicate that these POATs are pectin O-acetyltransferases involved in acetylation of the pectin polysaccharides HG and RG-I.

Suppressing the rhamnogalacturonan lyase gene FaRGLyase1 preserves RGI pectin degradation and enhances strawberry fruit firmness.

Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the "hairy" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.

Identification of intracellular activation mechanism of rhamnogalacturonan-I type polysaccharide purified from Panax ginseng leaves in macrophages and roles of component sugar chains on activity.

This study aimed to investigate the mechanisms underlying intracellular signaling pathways in macrophages in relation to the structural features of rhamnogalacturonan (RG) I-type polysaccharide (PGEP-I) purified from Panax ginseng leaves. For this investigation, we used several specific inhibitors and antibodies against mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and pattern recognition receptors (PRRs). Furthermore, we investigated the roles of component sugar chains on immunostimulating activity through a sequential enzymatic and chemical degradation steps. We found that PGEP-I effectively induced the phosphorylation of several MAPK- and NF-κB-related proteins, such as p38, cJun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p65. Particularly, immunocytochemistry analysis confirmed the PGEP-I-induced translocation of p65 into the nucleus. Furthermore, the breakdown of PGEP-I side chains and main chain during sequential enzymatic and chemical degradation reduced the PGEP-I-induced macrophage cytokine secretion activity. IL-6, TNF-α, and NO secreted by macrophages are associated with several signaling pathway proteins such as ERK, JNK, and NF-κB and several PRRs such as dectin-1, CD11b, CD14, TLR2, TLR4, and SR. Thus, these findings suggest that PGEP-I exerts potent macrophage-activating effects, which can be attributed to its typical RG-I structure comprising arabinan, type II arabinogalactan, and rhamnose-galacturonic acid repeating units in the main chain.

Cellulose assembles into helical bundles of uniform handedness in cell walls with abnormal pectin composition.

Plant cells and organs grow into a remarkable diversity of shapes, as directed by cell walls composed primarily of polysaccharides such as cellulose and multiple structurally distinct pectins. The properties of the cell wall that allow for precise control of morphogenesis are distinct from those of the individual polysaccharide components. For example, cellulose, the primary determinant of cell morphology, is a chiral macromolecule that can self-assemble in vitro into larger-scale structures of consistent chirality, and yet most plant cells do not display consistent chirality in their growth. One interesting exception is the Arabidopsis thaliana rhm1 mutant, which has decreased levels of the pectin rhamnogalacturonan-I and causes conical petal epidermal cells to grow with a left-handed helical twist. Here, we show that in rhm1 the cellulose is bundled into large macrofibrils, unlike the evenly distributed microfibrils of the wild type. This cellulose bundling becomes increasingly severe over time, consistent with cellulose being synthesized normally and then self-associating into macrofibrils. We also show that in the wild type, cellulose is oriented transversely, whereas in rhm1 mutants, the cellulose forms right-handed helices that can account for the helical morphology of the petal cells. Our results indicate that when the composition of pectin is altered, cellulose can form cellular-scale chiral structures in vivo, analogous to the helicoids formed in vitro by cellulose nano-crystals. We propose that an important emergent property of the interplay between rhamnogalacturonan-I and cellulose is to permit the assembly of nonbundled cellulose structures, providing plants flexibility to orient cellulose and direct morphogenesis.

Characterization of a novel strain of Aspergillus aculeatinus: From rhamnogalacturonan type I pectin degradation to improvement of fruit juice filtration.

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.

Transport of UDP-rhamnose by URGT2, URGT4, and URGT6 modulates rhamnogalacturonan-I length.

Rhamnogalacturonan-I biosynthesis occurs in the lumen of the Golgi apparatus, a compartment where UDP-Rhamnose and UDP-Galacturonic Acid are the main substrates for synthesis of the backbone polymer of pectin. Recent studies showed that UDP-Rha is transported from the cytosol into the Golgi apparatus by a family of six UDP-rhamnose/UDP-galactose transporters (URGT1-6). In this study, analysis of adherent and soluble mucilage (SM) of Arabidopsis thaliana seeds revealed distinct roles of URGT2, URGT4, and URGT6 in mucilage biosynthesis. Characterization of SM polymer size showed shorter chains in the urgt2 urgt4 and urgt2 urgt4 urgt6 mutants, suggesting that URGT2 and URGT4 are mainly involved in Rhamnogalacturonan-I (RG-I) elongation. Meanwhile, mutants in urgt6 exhibited changes only in adherent mucilage (AM). Surprisingly, the estimated number of RG-I polymer chains present in urgt2 urgt4 and urgt2 urgt4 urgt6 mutants was higher than in wild-type. Interestingly, the increased number of shorter RG-I chains was accompanied by an increased amount of xylan. In the urgt mutants, expression analysis of other genes involved in mucilage biosynthesis showed some compensation. Studies of mutants of transcription factors regulating mucilage formation indicated that URGT2, URGT4, and URGT6 are likely part of a gene network controlled by these regulators and involved in RG-I synthesis. These results suggest that URGT2, URGT4, and URGT6 play different roles in the biosynthesis of mucilage, and the lack of all three affects the production of shorter RG-I polymers and longer xylan domains.