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.

Pectin Methylesterases Modulate Plant Homogalacturonan Status in Defenses against the Aphid Myzus persicae.

Because they suck phloem sap and act as vectors for phytopathogenic viruses, aphids pose a threat to crop yields worldwide. Pectic homogalacturonan (HG) has been described as a defensive element for plants during infections with phytopathogens. However, its role during aphid infestation remains unexplored. Using immunofluorescence assays and biochemical approaches, the HG methylesterification status and associated modifying enzymes during the early stage of Arabidopsis (Arabidopsis thaliana) infestation with the green peach aphid (Myzus persicae) were analyzed. Additionally, the influence of pectin methylesterase (PME) activity on aphid settling and feeding behavior was evaluated by free choice assays and the Electrical Penetration Graph technique, respectively. Our results revealed that HG status and HG-modifying enzymes are significantly altered during the early stage of the plant-aphid interaction. Aphid infestation induced a significant increase in total PME activity and methanol emissions, concomitant with a decrease in the degree of HG methylesterification. Conversely, inhibition of PME activity led to a significant decrease in the settling and feeding preference of aphids. Furthermore, we demonstrate that the PME inhibitor AtPMEI13 has a defensive role during aphid infestation, since pmei13 mutants are significantly more susceptible to M. persicae in terms of settling preference, phloem access, and phloem sap drainage.

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage.

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1 These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.

New steps in mucilage biosynthesis revealed by analysis of the transcriptome of the UDP-rhamnose/UDP-galactose transporter 2 mutant.

Upon imbibition, epidermal cells of Arabidopsis thaliana seeds release a mucilage formed mostly by pectic polysaccharides. The Arabidopsis mucilage is composed mainly of unbranched rhamnogalacturonan-I (RG-I), with low amounts of cellulose, homogalacturonan, and traces of xylan, xyloglucan, galactoglucomannan, and galactan. The pectin-rich composition of the mucilage and their simple extractability makes this structure a good candidate to study the biosynthesis of pectic polysaccharides and their modification. Here, we characterize the mucilage phenotype of a mutant in the UDP-rhamnose/galactose transporter 2 (URGT2), which exhibits a reduction in RG-I and also shows pleiotropic changes, suggesting the existence of compensation mechanisms triggered by the lack of URGT2. To gain an insight into the possible compensation mechanisms activated in the mutant, we performed a transcriptome analysis of developing seeds using RNA sequencing (RNA-seq). The results showed a significant misregulation of 3149 genes, 37 of them (out of the 75 genes described to date) encoding genes proposed to be involved in mucilage biosynthesis and/or its modification. The changes observed in urgt2 included the up-regulation of UAFT2, a UDP-arabinofuranose transporter, and UUAT3, a paralog of the UDP-uronic acid transporter UUAT1, suggesting that they play a role in mucilage biosynthesis. Mutants in both genes showed changes in mucilage composition and structure, confirming their participation in mucilage biosynthesis. Our results suggest that plants lacking a UDP-rhamnose/galactose transporter undergo important changes in gene expression, probably to compensate modifications in the plant cell wall due to the lack of a gene involved in its biosynthesis.

GoSAMTs are required for pectin methyl-esterification and mucilage release in seed coat epidermal cells.

Introduction: GoSAMTs play a role in the methylation of polysaccharides synthesized by the Golgi. Pectin homogalacturonan (HG) methyl-esterification is essential for the proper function of this polysaccharide in cell walls. In order to better understand the role of GoSAMTs in HG biosynthesis, we analyzed mucilage methyl-esterification in gosamt mutants. Methods: To determine the function of GoSAMT1 and GoSAMT2 in HG methyl-esterification we utilized epidermal cells of seed coats, as these structures produce mucilage, which is a pectic matrix. We evaluated differences in seed surface morphology and quantified mucilage release. We measured methanol release, and used antibodies and confocal microscopy to analyze HG methyl-esterification in mucilage. Results: We observed morphological differences on the seed surface and delayed, uneven mucilage release in gosamt1-1gosamt2-1 double mutants. We also found changes in the distal wall length indicating abnormal cell wall breakage in this double mutant. Using methanol release and immunolabeling, we confirmed that GoSAMT1 and GoSAMT2 are involved in HG methyl-esterification in mucilage. However, we did not find evidence of decreasing HG in the gosamt mutants. Confocal microscopy analyses detected different patterns in the adherent mucilage and a greater number of low-methyl-esterified domains near the seed coat surface, which correlates with a greater number of "egg-box" structures in this region. We also detected a shift in the partitioning between the Rhamnogalacturonan-I soluble and adherent layers of the double mutant, which correlated with increased amounts of arabinose and arabinogalactan-protein in the adherent mucilage. Discussion: The results show that the HG synthesized in gosamt mutant plants is less methyl esterified, resulting in more egg-box structures, which stiffen the cell walls in epidermal cells and change the rheological properties of the seed surface. The increased amounts of arabinose and arabinogalactan-protein in adherent mucilage, also suggests that compensation mechanisms were triggered in the gosamt mutants.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

Functional Interchangeability of Nucleotide Sugar Transporters URGT1 and URGT2 Reveals That urgt1 and urgt2 Cell Wall Chemotypes Depend on Their Spatio-Temporal Expression.

Nucleotide sugar transporters (NSTs) are Golgi-localized proteins that play a role in polysaccharide biosynthesis by transporting substrates (nucleotide sugars) from the cytosol into the Golgi apparatus. In Arabidopsis, there is an NST subfamily of six members, called URGTs, which transport UDP-rhamnose and UDP-galactose in vitro. URGTs are very similar in protein sequences, and among them, URGT1 and URGT2 are highly conserved in protein sequence and also showed very similar kinetic parameters toward UDP-rhamnose and UDP-galactose in vitro. Despite the similarity in sequence and in vitro function, mutants in urgt1 led to a specific reduction in galactose in rosette leaves. In contrast, mutants in urgt2 showed a decrease in rhamnose content in soluble mucilage from seeds. Given these specific and quite different chemotypes, we wonder whether the differences in gene expression could explain the observed differences between the mutants. Toward that end, we analyzed whether URGT2 could rescue the urgt1 phenotype and vice versa by performing a promoter swapping experiment. We analyzed whether the expression of the URGT2 coding sequence, controlled by the URGT1 promoter, could rescue the urgt1 rosette phenotype. A similar strategy was used to determine whether URGT1 could rescue the urgt2 mucilage phenotype. Expression analysis of the swapped genes, using qRT-PCR, was similar to the native URGT1 and URGT2 genes in wild-type plants. To monitor the protein expression of the swapped genes, both URGTs were tagged with green fluorescent protein (GFP). Confocal microscopy analyses of the swapped lines containing URGT2-GFP showed fluorescence in motile dot-like structures in rosette leaves. Swapped lines containing URGT1-GFP showed fluorescence in dot-like structures in the seed coat. Finally, the expression of URGT2 in urgt1 mutants rescued galactose reduction in rosette leaves. In the same manner, the expression of URGT1 in urgt2 mutants recovered the content of rhamnose in soluble mucilage. Hence, our results showed that their expression in different organs modulates the role in vivo of URGT1 and URGT2. Likely, this is due to their presence in different cellular contexts, where other proteins, acting in partnership, may drive their functions toward different pathways.