Comprehensive Leaf Cell Wall Analysis Using Carbohydrate Microarrays Reveals Polysaccharide-Level Variation between Vitis Species with Differing Resistance to Downy Mildew

Abstract

The cell wall acts as one of the first barriers of the plant against various biotic stressors. Previous studies have shown that alterations in wall polysaccharides may influence crop disease resistance. In the grapevine family, several native species (e.g., Chinese wild grapevine) show a naturally higher resistance to microbial pathogens than cultivated species (e.g., Vitis vinifera), and this trait could be inherited through breeding. Despite the importance of the cell wall in plant immunity, there are currently no comprehensive cell wall profiles of grapevine leaves displaying differing resistance phenotypes, due to the complex nature of the cell wall and the limitations of analytical techniques available. In this study, the cutting-edge comprehensive carbohydrate microarray technology was applied to profile uninfected leaves of the susceptible cultivar (Vitis vinifera cv. "Cabernet Sauvignon"), a resistant cultivar (Vitis amurensis cv. "Shuanghong") and a hybrid offspring cross displaying moderate resistance. The microarray approach uses monoclonal antibodies, which recognize polysaccharides epitopes, and found that epitope abundances of highly esterified homogalacturonan (HG), xyloglucan (with XXXG motif), (galacto)(gluco)mannan and arabinogalactan protein (AGP) appeared to be positively correlated with the high resistance of Vitis amurensis cv. "Shuanghong" to mildew. The quantification work by gas chromatography did not reveal any significant differences for the monosaccharide constituents, suggesting that polysaccharide structural alterations may contribute more crucially to the resistance observed; this is again supported by the contact infrared spectroscopy of cell wall residues, revealing chemical functional group changes (e.g., esterification of pectin). The identification of certain wall polysaccharides that showed alterations could be further correlated with resistance to mildew. Data from the use of the hybrid material in this study have preliminarily suggested that these traits could be inherited and may be applied as potential structural biomarkers in future breeding work.

Keywords: cell wall; grapevine; monoclonal antibodies; oomycete; polysaccharide microarray.

Figure 1

Grapevine downy mildew symptoms on leaf disks with different sporulation density observed at 7 days post-inoculation. “Cab”: Vitis vinifera cv. “Cabernet sauvignon”; “SH”: Vitis amurensis cv. “Shuanghong”; “Cross”: the hybrid from “Cab” and “SH”.

Figure 2

Comprehensive Microarray Polymer Profiling (CoMPP) analysis of CDTA extract of grapevine leaf cell wall AIR samples. The heatmap shows the relative abundance of plant cell wall glycan-associated structural motifs. The highest signal was set as 100, and others were adjusted accordingly; the color intensity is correlated to the mean spot signal. A cut-off (<5) was applied.

Figure 3

PCA score plot (A) and loading plot (B) of the CDTA (pectin-rich) extract from AIR sourced from grapevine leaves.

Figure 4

Comprehensive Microarray Polymer Profiling (CoMPP) analysis of NaOH extract of grapevine leaf cell wall AIR samples. The heatmap showed the relative abundance of plant cell wall glycan-associated structural motifs. The highest signal was set as 100, and others were adjusted accordingly; the color intensity is correlated to the mean spot signal. A cut-off (<5) was applied.

Figure 5

PCA score plot (A) and loading plot (B) of the NaOH (hemicellulose-rich) extract from AIR sourced from grapevine leaves.

Figure 6

The monosaccharide composition of AIR sourced from grapevine leaves. The composition is expressed in relative mol% of seven main monosaccharides present in grapevine leaf cell wall. Ara: arabinose; Rha: rhamnose; Xyl: xylose; GalA: galacturonic acid; Man: mannose; Gal: galactose; Glc: glucose. Error bars represent the standard derivation of the mean value of three biological repeat.

Figure 7

Fourier transform infrared (FT-IR) spectra generated from the AIR samples sourced from grapevine leaves.