Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds

Abstract

All cells of terrestrial plants are fortified by walls composed of crystalline cellulose microfibrils and a variety of matrix polymers. Xylans are the second most abundant type of polysaccharides on Earth. Previous studies of Arabidopsis (Arabidopsis thaliana) irregular xylem (irx) mutants, with collapsed xylem vessels and dwarfed stature, highlighted the importance of this cell wall component and revealed multiple players required for its synthesis. Nevertheless, xylan elongation and substitution are complex processes that remain poorly understood. Recently, seed coat epidermal cells were shown to provide an excellent system for deciphering hemicellulose production. Using a coexpression and sequence-based strategy, we predicted several MUCILAGE-RELATED (MUCI) genes that encode glycosyltransferases (GTs) involved in the production of xylan. We now show that MUCI21, a member of an uncharacterized clade of the GT61 family, and IRX14 (GT43 protein) are essential for the synthesis of highly branched xylan in seed coat epidermal cells. Our results reveal that xylan is the most abundant xylose-rich component in Arabidopsis seed mucilage and is required to maintain its architecture. Characterization of muci21 and irx14 single and double mutants indicates that MUCI21 is a Golgi-localized protein that likely facilitates the addition of xylose residues directly to the xylan backbone. These unique branches seem to be necessary for pectin attachment to the seed surface, while the xylan backbone maintains cellulose distribution. Evaluation of muci21 and irx14 alongside mutants that disrupt other wall components suggests that mucilage adherence is maintained by complex interactions between several polymers: cellulose, xylans, pectins, and glycoproteins.

Figure 1.

Phylogenetic tree of GT61 proteins from five species. Branches are annotated with National Center for Biotechnology Information Reference sequences, except for the known AtXYLT, OsXAT2, OsXAT3, and OsXAX1 enzymes. Clades are named as first reported (Anders et al., 2012). MUCI21 belongs to the hitherto uncharacterized clade B of the GT61 family. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap values indicate the reliability of the nodes.

Figure 2.

Overview of MUCI21 and IRX14 expression and mutations. A, MUCI21 and IRX14 are expressed in the seed coat (Winter et al., 2007; Belmonte et al., 2013). B, Insertions and qRT-PCR primers (arrowheads) used. C, Transcription in wild-type (WT) siliques stages based on embryo shape (two biological replicates each). All values are relative to the first MUCI21 sample. D, Mutant siliques at the linear stage have reduced expression (arrows; Student’s t test, P < 0.05) relative to the wild type. Data in C and D show the means + sd (normalized to UBQ5) of three technical replicates.

Figure 3.

muci21 and irx14 mutants have seed mucilage defects. A to H, Seeds hydrated in water were stained with RR. The 35S:MUCI21-sYFP transgene rescued muci21-1 staining defects (D) but not the irx14-2 phenotype (H). The muci21 and irx14 mutants produced normal amounts of total mucilage (I) but had severe decreases in Xyl content (J). Letters in J indicate significant changes between the mutants and wild type (WT; Student’s t test, P < 10^–5). Bars = 200 µm.

Figure 4.

MUCI21 decorates xylan backbones produced by IRX14. A, Mucilage Xyl linkages from two independent experiments. B, Abundance of unbranched xylan labeled by LM10 (McCartney et al., 2005) in mucilage extracts. Epitopes were not detected (ND) in the wild type (WT) and irx14 after background correction. C, Proposed structure of mucilage xylan, with Xyl units colored according to the legend in A. D, Composition of total mucilage extracts. Letters show that 35S:MUCI21-sYFP rescued the Xyl defect of muci21-1, while muci21 irx14 double mutants were similar to irx14 (Student’s t test, P < 10^–3). Data show means + sd of at least three biological replicates, except only two for irx14-2 in A and the double mutant in D.

Figure 5.

MUCI21-sYFP proteins are localized in the Golgi. Arabidopsis cells expressing MUCI21-sYFP (A), the free sYFP tag (B), or no transgene (C). A to C, sYFP (yellow), chloroplast intrinsic fluorescence (blue), and transmitted light (gray) signals overlaid in Fiji. E to H, After BFA treatment, MUCI21-sYFP and the Wave22Y Golgi marker aggregated in large bodies (red arrows). H to J, MUCI21-sYFP and ST-RFP Golgi punctae colocalized (white arrows). Bars = 50 (A–G) and 10 µm (H–J).

Figure 6.

Mucilage is easily detached from muci21 and irx14 seeds. A to F, Nonadherent mucilage (arrows) released by seeds hydrated directly in RR. G to L, The majority of irx14 seeds, and some muci21 seeds, floated (arrows) after 20 min of contact with water. Similar seed quantities were added to each tube. Bars = 2 mm.

Figure 7.

Comparison of muci21 and irx14 with other loss-of-adherence mutants. A, Dimensions of seeds and their RR-stained mucilage capsules. B, Rha and GalA easily detached from the mutant seeds. Data show means + sd of five biological replicates. The a and b indicate significant changes between the wild type and the mutants (Student’s t test, P < 0.05).

Figure 8.

Cellulose structure in mutants with impaired mucilage adherence. A to L, S4B staining of cellulose in mucilage. Signals were visualized with the Orange Hot look-up table, using the calibration bar shown in C. All mutants displayed ray-like regions (arrows) except sos5. Magnified panels correspond to the seeds above them. Bars = 200 (A–C and G–I) and 25 µm (D–F and J–L).

Figure 9.

muci21 and irx14 mucilage has impaired RG I and xylan distribution. Optical sections show unbranched RG I labeled by CCRC-M36 (A–F) or xylan bound by CCRC-M139 (G–L) in green. Cellulose was counterstained with S4B (magenta). As xylan epitopes were less abundant than RG I, green intensity was increased in postacquisition (G–L). Bars = 200 (A–C and G–I) and 25 µm (D–F and J–L).