Players in pectin production: rhamnose transporters affect the length of rhamnogalacturonan-I

Pectin polymers are structurally complex polysaccharides that form a gel-like matrix that surrounds hemicelluloses and cellulose microfibrils in the plant cell wall (Carpita and Gibeaut, 1993). Difficulties in extracting intact polymers from plant cells hamper the research on pectin structure (Atmodjo et al., 2013), but the Arabidopsis seed coat mucilage is a suitable system to study pectin in vivo (Haughn and Western, 2012). The three major types of polymers in pectin are homogalacturonan, an unbranched homopolymer of α-(1,4)-linked galacturonic acid (GalA), and rhamnogalacturonans (RGs) I and II. RG-I and RG-II are composed of the repeating disaccharide unit (1,4)-α-GalA-(1,2)-α-Rha (rhamnose; Rha; (Mohnen, 2008). Rha residues in RG-I can be decorated with oligosaccharides, including galactans and arabinans (Atmodjo et al., 2013). Rha is a key component in plant cell walls and, in some cases, conjugated to specialized metabolites such as alkaloids, triterpenoids, and anthocyanins (Jiang et al., 2021).

Pectin polymers are produced by conjugation of UDP-sugar monomers. Unlike UDP-GalA, which can be synthesized in both the cytosol and the Golgi apparatus, UDP-Rha is exclusively synthesized in the cytosol. Translocation of UDP-Rha to the Golgi is required to initiate biosynthesis of RG-I, making this transport a critical step for cell wall biosynthesis (Temple et al., 2016). A study by Rautengarten et al. (2014) reported that six UDP-rhamnose/UDP-galactose transporters (URGTs) from Arabidopsis thaliana are able to transport Rha and GalA in vitro. In the latest issue of Plant Physiology, Saez-Aguayo et al. (2021) describe a comprehensive functional analysis of the six URGT family members in A. thaliana in order to puzzle out the in planta contribution of each URGT in Rha transport and the consequences of impairing Rha flux in RG-I biosynthesis.

Expression analysis of URGT1-6 highlighted that URGT1, URGT2, and URGT4 transcript abundance coincides with the timing of polysaccharide accumulation in seed coat epidermal cells, which produce the pectin-rich seed coat mucilage. Mutant lines of urgt2, urgt4, or urgt6 showed reduced levels of Rha and RG-I in the soluble layer of seed coat mucilage. Additionally, molecular complementation of the corresponding genes rescued the chemotypes and restored the content of RG-I. Taken together, these results confirmed the engagement of URGT2, URGT4, and URGT6 in biosynthesis of RG-I.

Since the expression profiles indicated a potential redundancy among the transporters, the authors generated double and triple mutants. Compared to wild type, the triple urgt mutant displayed a more than 36% decrease of GalA and Rha in the mucilage around the seeds. Additionally, the triple urgt mutant showed alterations of other sugars and especially xylose (Xyl), for which an increase up to 130% was observed in the soluble mucilage layer. Although double mutants were not affected so drastically, a similar pattern was observed. The additive effects observed in these crosses suggested that these transporters contribute synergistically to the biosynthesis of mucilage pectin.

Reduced availability of Rha also had an impact on the interactions among the synthesized RG-I polymers, which in turn affected the structure of the adherent mucilage layer. Double and triple mutants exhibited an increased permeability compared to wild type, possibly due to less densely packed polysaccharides, which altered the porosity of the adherent mucilage layer.

Notably, unbalancing the flux of Rha and GalA caused both quantitative and qualitative changes in RG-I biosynthesis. Compared to wild type, the triple urgt mutant had almost 67% shorter RG-I polymers and more than double the amount of RG-I molecules in soluble mucilage. This result was attributed to the lower availability of Rha, which caused the elongation of RG-I to terminate prematurely, followed by a subsequent reinitiation of a new RG-I polymer. Glycosidic linkage analysis suggested also that the RG-I polymer is less substituted in the triple urgt mutant and possibly the accompanying hemicellulose xylan chains are longer. However, further studies are needed to verify whether the xylan moieties are altered.

Unexpectedly, the triple urgt mutant also exhibited altered expression levels of four genes implicated in Rha and Xyl biosynthesis. In general, expression of these genes was delayed in the mutant compared to wild type, since these genes were expressed at lower levels at the beginning and higher levels toward the end of seed development. How the reduction of URGT transcripts results in misregulation of the expression levels of genes related to sugar synthesis remains to be addressed.

To place these genes into the known regulatory network of seed mucilage synthesis, the expression levels of URGT2, URGT4, and URGT6 were studied in mutants of the transcription factors GLABRA2 (GL2), TRANSPARENT TESTA GLABRA2 (TTG2), MYB61, and LEUNIG_HOMOLOG (LUH)/MUCILAGE-MODIFIED 1 (MUM1), regulators of synthesis of mucilage polysaccharides in the Arabidopsis seed coat (Golz et al., 2018). This analysis indicated that the four transcription factors positively regulate the expression levels of URGT2 and URGT4 but MUM1 represses the expression levels of URGT6.

The work of Saez-Aguayo et al. (2021) sheds light on Rha transport from the cell cytosol into the Golgi apparatus by validating the functional role of each URGT in planta. Analysis of a series of combinatorial mutants enabled the authors to bypass the redundancy of the transporters revealing that decreasing Rha accumulation has both qualitative and quantitative impacts in the assembly of RG-I polymers, therefore expanding our understanding of the pectin biosynthetic framework.

Conflict of interest statement. The authors declare no conflict of interest.