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

Abstract

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.

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

“…The analysis of urgt2-2 plants revealed an almost complete reduction of URGT2 expression in seeds and rosette leaves, whereas URGT1 transcript levels in urgt2-2 remained unaltered. As was previously described, the monosaccharide composition of urgt2-2 showed a reduction on the total sugar content of SM due to a reduction of Rha and GalA monosaccharides, the main components of the RG-I polymer filling the mucilage pocket of Arabidopsis seeds (Rautengarten et al, 2014;Ralet et al, 2016;Parra-Rojas et al, 2019). The reduction in Rha and GalA monosaccharides was also accompanied by an increase of Xyl content, a result that was previously related to the polymerization of RG-I and the xylan ramifications presented in SM (Fabrissin et al, 2019;Parra-Rojas et al, 2019).…”

“…Indeed, URGT proteins have a range of identities from 43.1 to 93.1% and sequence similarities from 60.5 to 96.7% ( Figure 1A ). URGT1 and URGT2 have highly conserved protein sequences (89.4% identity and 93.4% similarity; Figure 1A and Supplementary Figure 1 ); however, the mutants on each of these genes lead to different phenotypes ( Rautengarten et al, 2014 ; Parra-Rojas et al, 2019 ). Indeed, urgt1 mutant plants show a decrease in the content of galactose in rosette leaves, whereas the content of rhamnose remains invariable.…”

“…The quantification and normalization procedures were done using the equation described in Saez-Aguayo et al (2017) . EF1aA4 ( North et al, 2007 ), Clathrin adaptor complex subunit (AT5G46630), and Seed reference gene (At4g12590) ( Hong et al, 2010 ) were used as reference genes, and all primers used in this study were described in Saez-Aguayo et al (2017) and Parra-Rojas et al (2019) . The reaction was performed on RNA extracted from three biological replicates and was analyzed via qRT-PCR with three technical replicates each.…”

“…Clade I contains the UDP-rhamnose (UDP-Rha) and UDP-galactose (UDP-Gal) transporters (URGTs), a subfamily composed of six transporters, named URGT1–URGT6, which are able to transport UDP-Rha and UDP-Gal in vitro ( Rautengarten et al, 2014 ). Mutations in URGT1 and URGT2 led to observable phenotypes associated with defects in pectin composition in vivo ( Rautengarten et al, 2014 ; Parra-Rojas et al, 2019 ). urgt2 mutants exhibited a reduction in mucilage content with a decrease of rhamnose (Rha) and galacturonic acid (GalA) amounts, which are the basic units of the rhamnogalacturonan I (RG-I) polymer, the main component of the soluble mucilage (SM) layer, suggesting that URGT2 is involved in the synthesis of the mucilage RG-I polymer ( Rautengarten et al, 2014 ; Takenaka et al, 2018 ; Parra-Rojas et al, 2019 ).…”

“…Mutations in URGT1 and URGT2 led to observable phenotypes associated with defects in pectin composition in vivo ( Rautengarten et al, 2014 ; Parra-Rojas et al, 2019 ). urgt2 mutants exhibited a reduction in mucilage content with a decrease of rhamnose (Rha) and galacturonic acid (GalA) amounts, which are the basic units of the rhamnogalacturonan I (RG-I) polymer, the main component of the soluble mucilage (SM) layer, suggesting that URGT2 is involved in the synthesis of the mucilage RG-I polymer ( Rautengarten et al, 2014 ; Takenaka et al, 2018 ; Parra-Rojas et al, 2019 ). In contrast to urgt2 , urgt1 mutants did not exhibit an SM phenotype but showed a small reduction of galactose (Gal) content in rosette leaves ( Rautengarten et al, 2014 ).…”