Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes

Abstract

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

“…In the second approach, mycelium was stained with the recently developed chitosan‐specific fluorescent probe OGA488 (Mravec et al . , 2014). In this case, much stronger staining of hyphae was observed (Figure 1a), which is perhaps attributable to the superior penetrability of the OGA488 probe compared with the antibody.…”

“…Chitosan has also been shown to localize to hyphal septa in Neurospora crassa (Mravec et al . , 2014), suggesting that this may be a conserved feature of the septal cell wall in filamentous fungi.…”

“…Samples (mycelial pellets and coverslips that were overgrown with hyphae or had previously been inoculated with conidia) were washed briefly with 25 mM MES (pH 5.6) and incubated with OGA488, a generous gift from William Willats (Mravec et al . , 2014; diluted 1/1000 in 25 mM MES) for 15 min on ice. This was followed by 2–3 brief washes with 25 mM MES, after which the samples were viewed using the C‐Apochromat 40×/1.2 water corrected objective lens of a Zeiss LSM 510 Meta confocal microscope, at 488‐nm excitation and emission collected with an LP505 filter.…”

Chitin distribution in the Oithona digestive and reproductive systems revealed by fluorescence microscopy

“…WGA-FITC staining is widely used for chitin detection by fluorescence, in a liquid medium containing lysed cells or directly on whole organisms [25][26][27][28]. On copepods, WGA-FITC was used only once before; but after dissolution of the soft tissues which did not allow the investigation of the internal anatomy [29]. In the present study, the WGA-FITC and DAPI staining was used to provide a new insight into the internal anatomy and chitin content of Oithona nana and Oithona similis with a focus on their digestive and reproductive systems.…”

Chitin distribution in the Oithona digestive and reproductive systems revealed by fluorescence microscopy

“…WGA-FITC staining is widely used for chitin detection by fluorescence, in a liquid medium containing lysed cells or directly on whole organisms [25][26][27][28]. On copepods, WGA-FITC was used only once before; but after dissolution of the soft tissues which did not allow the investigation of the internal anatomy [29]. In the present study, the WGA-FITC and DAPI staining was used to provide a new insight into the internal anatomy and chitin content of Oithona nana and Oithona similis with a focus on their digestive and reproductive systems.…”