Dissecting the Genetic Basis for Seed Coat Mucilage Heteroxylan Biosynthesis in Plantago ovata Using Gamma Irradiation and Infrared Spectroscopy

Abstract

Seeds from the myxospermous species Plantago ovata release a polysaccharide-rich mucilage upon contact with water. This seed coat derived mucilage is composed predominantly of heteroxylan (HX) and is utilized as a gluten-free dietary fiber supplement to promote human colorectal health. In this study, a gamma-irradiated P. ovata population was generated and screened using histological stains and Fourier Transform Mid Infrared (FTMIR) spectroscopy to identify putative mutants showing defects in seed coat mucilage HX composition and/or structure. FTMIR analysis of dry seed revealed variation in regions of the IR spectra previously linked to xylan structure in Secale cereale (rye). Subsequent absorbance ratio and PCA multivariate analysis identified 22 putative mutant families with differences in the HX IR fingerprint region. Many of these showed distinct changes in the amount and subtle changes in structure of HX after mucilage extrusion, while 20% of the putative HX mutants identified by FTMIR showed no difference in staining patterns of extruded mucilage compared to wild-type. Transcriptional screening analysis of two putative reduced xylan in mucilage (rxm) mutants, rxm1 and rxm3, revealed that changes in HX levels in rxm1 correlate with reduced transcription of known and novel genes associated with xylan synthesis, possibly indicative of specific co-regulatory units within the xylan biosynthetic pathway. These results confirm that FTMIR is a suitable method for identifying putative mutants with altered mucilage HX composition in P. ovata, and therefore forms a resource to identify novel genes involved in xylan biosynthesis.

A small-scale fractionation pipeline for rapid analysis of seed mucilage characteristics

“…To validate this screen, a subset of the three lowest yielding (252-7, 768-9, and 1064-5) and three highest (743-4, 1072-12, and 776-5) mutants were selected for further analysis. Expanded mucilage architecture has been used previously to visually screen for altered mucilage phenotypes in mutants of Arabidopsis [4,57,58] and Plantago [10]. Here we show variation in expanded mucilage architecture between the mutants (Fig.…”

“…P. ovata varieties were grown in field trials conducted in 2017 and 2018 in Kununurra, Western Australia. Gamma-irradiated P. ovata mutants used for germplasm screening were obtained from a glasshouse-grown population described previously [10].…”

“…This pipeline has utility for rapid screening of mucilage yield traits in a germplasm set, demonstrated here using gamma-irradiated P. ovata mutants generated in a previous study [10]. Total mucilage yield data (a pooling of CWE, HWE and IAE fractions) was obtained for a subset of 206 randomly-selected glasshouse-grown P. ovata mutants (Fig.…”

“…The striking similarities in the phenotypes of the mew mutants suggests that they may contain mutant alleles. Importantly, mew mutant 252-7 has already been identified as a putative reduced mucilage xylan mutant [10] and the characterisation of this class of mutant is ongoing [11]. Mutant 776-5 represents a previously unseen class of P. ovata mucilage mutant [10,11,59].…”

“…The mucilage of Arabidopsis thaliana has often been used as a proxy for studying cell wall biosynthesis [1][2][3][4][5][6][7][8]. More recently other myxospermous species like Linum usitatissimum and Plantago ovata have also been adopted as genetic models [9][10][11][12][13][14] revealing the utility that novel systems can have in unravelling complex synthetic pathways. Furthermore, these novel model systems have the added benefit of being directly commercially-relevant.…”