# Hidden Xyloglucan Architecture of the Pollen Intine in Gagea lutea Revealed by Sequential Enzymatic Unmasking

## TL;DR

This study reveals hidden xyloglucan structures in pollen walls using enzyme treatments to improve detection.

## Contribution

A novel enzymatic approach was developed to unmask concealed xyloglucan epitopes in monocot pollen walls.

## Key findings

- Xyloglucan epitopes in the pollen intine became more detectable after sequential enzymatic digestion.
- Enzymatic treatment primarily enhanced epitope accessibility rather than increasing polymer abundance.
- The study provides a framework for interpreting digestion-dependent changes in pollen wall architecture.

## Abstract

The inner pollen wall is essential for hydration and initiation of pollen tube growth, but its molecular organisation is difficult to resolve because many wall components are embedded within the matrix and therefore hard to detect by immunolabelling. Here, we examined mature pollen grains of Gagea lutea using fluorescence microscopy and transmission electron microscopy, combined with highly specific antibodies recognising defined polysaccharide epitopes (xyloglucan, heteroxylan, heteromannan and xylan). We compared untreated sections with sections subjected to a stepwise enzymatic treatment (pectate lyase followed by endo-β-mannanase) aimed at removing selected matrix constraints, particularly pectins, and thereby increasing epitope accessibility. Xyloglucan-related epitopes were detected in the intine and became markedly more detectable and more continuous after sequential digestion. Quantitative fluorescence measurements confirmed that the largest increases occurred only after the combined digestion steps, supporting the interpretation that the treatment primarily enhanced epitope accessibility rather than increasing polymer abundance. Several other epitopes remained below the detection threshold under the conditions used; therefore, non-labelling should be interpreted as non-detection rather than definitive absence. Overall, these results provide an improved framework for mapping concealed wall architecture in monocot pollen.

The organisation of hemicelluloses within the pollen intine of many monocots remains inadequately characterised, partly due to the masking of epitopes within complex wall matrices. In this study, mature pollen grains of Gagea lutea (L.) Ker-Gawl. were analysed using immunofluorescence and immunogold technique with a variety of monoclonal antibodies that target xyloglucan (LM15, LM24, LM25, CCRC-M48), heteroxylan (LM10, LM11), heteromannan (LM21, LM22), and xylan (CCRC-M138). Semithin sections of LR White were examined both untreated and following a sequential enzymatic pretreatment, which included alkaline de-esterification followed by treatment with pectate lyase (RbPel1A) and endo-β-mannanase 5A. In untreated pollen, xyloglucan-related epitopes were identified within the intine, accompanied by additional intracellular labelling for LM15, and LM25; while for LM24 signal was only to the intine ring. Conversely, CCRC-M48 exhibited a more punctate distribution. Neither xylan- nor mannan-related epitopes were detected in the wall or intracellularly. The enzymatic digestion significantly altered the detectability of epitopes, resulting in an increase in continuous wall labelling within the intine across multiple probes. These findings indicate that enzymatic modification of pectic and mannan components has a considerable impact on the apparent distribution of hemicellulose epitopes within the pollen wall of G. lutea. Together, these results expand the still limited in situ immunolocalisation evidence base for hemicellulose-related epitopes in pollen, and provide a practical framework for interpreting digestion-dependent changes primarily in terms of epitope accessibility within the intine matrix.