Investigating grapevine cell walls

Wine quality is strongly connected in various ways to the cell wall properties of the grapevine tissues. We have implemented methods to analytically profile cell wall components in tobacco (used for plant-pathogen studies) and grapevine (leaves and berries). These methods have been very useful in various industry-relevant research objectives. This article briefly describes the following:

• How these methods work.
• How they have been applied in fundamental studies in understanding grapevine disease resistance to fungal pathogens.
• How it is useful in developing ripening biomarkers and assessing progression of berry maturation in wine and table grapes.
• How these approaches provide useful information on the effectiveness of enzyme maceration approaches and the impact on final wine composition.

Why is studying grapevine cell walls important in grape and wine research? Plant cell walls are complex, interconnected structures of polysaccharides (pectin, cellulose and hemicellulose), cell wall proteins and polyphenols, and play an important role in many agricultural and industrial processes such as fruit ripening, plant-pathogen interactions and as substrates in bio-refineries[1]. In the grapevine, the cell walls of the leaves are the first line of defence against pathogenic attack[2]. Fungal diseases such as grey rot (Botrytis cinerea) and mildew (e.g. Plasmopara viticola) usually first infect the young leaves and then spread to the berries. These pathogens secrete enzymes that degrade the cell walls of these tissues in order to gain access to the vine nutrients. To understand the details of the pathogen attack and the disease resistance mechanisms of the plant which will enable us to develop effective disease preventative methods, we need knowledge on the composition and three-dimensional architecture of the grape berry and leaf cell walls. On another level a portion of the proteins, polysaccharides and polyphenols from the cell walls of the grape berry end up in the wine[3]. Arabinogalactan-proteins (AGPs) and rhamnogalacturonan-II (RG-II) polymers are the main grape-derived polysaccharides in wine[4], but arabinans and homogalacturonans (HG) can also be present[5]. These polysaccharides together with the polyphenols such as hydroxycinnamic acids, anthocyanins and tannins contribute towards the mouthfeel, astringency, bitterness, aroma, colour and colour stabilisation, tannin aggregation and precipitation, protein haze formation and aroma retention of must and wine[6] and are therefore dominant role players in determining wine quality. At present the exact location and specific cell wall polymer interaction of every phenolic compound is unknown, but we do know that the proanthocyanidins are predominantly bound to the pectin polymers and to a lesser degree to the xyloglucan and cellulose[7]. Other polyphenols are found in the vacuole of the grape skin cells and thus require a disrupted cell wall to be released during the fermentation. Due to this relationship between the cell wall and polyphenols, it follows that the extractability of phenolic compounds during the wine fermentation depends on the recalcitrance of the cell wall, but also on the grape cultivar (low extraction from thick skinned cultivars, e.g. Mourvèdre), the degree of ripeness (higher extraction from riper grapes) and the winemaking practices used (skin contact and use of maceration enzymes)[8 – 10]. Given the crucial importance of grapevine-derived polysaccharides in disease resistance and in the quality of the wine produced, it is therefore of critical value to develop methods to trace and assess these carbohydrate polymers in the vine and in the grape berry through maceration and fermentation to the final wine.

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How do we study grapevine (berry and leaf) cell walls?

To analyse grape-derived cell wall polysaccharides we have developed and applied a combination of high-throughput techniques including monosaccharide compositional analysis, comprehensive microarray polymer profiling (CoMPP) analysis, FT-IR spectroscopy and oligosaccharide mass fingerprinting, as well as more in-depth approaches using chemical and enzymatic fractionation methods (Figure 1). Before cell walls can be analysed, it has to be extracted from the plant tissue which involves mechanical grinding and treatment with a series of organic solvents and the final alcohol insoluble residue (AIR) obtained, represents the cell walls. This material can now be used in various ways:

a) We can break it down with acids to see the different sugars (e.g. glucose etc.) that make up the polymers;

b) We can probe with monoclonal antibodies (mAbs) and carbohydrate binding modules (CBMs) to identify specific types of polysaccharides (e.g. pectins, the gummy ‘sticky’ stuff that clogs filters and presses during winemaking);

c) We can use infra-red (IR) spectroscopic tools with chemometric methods for a quick overall picture of the polymers; and

d) We can use more detailed analytical and fractionation methods to understand the more intricate complexity of the cell walls (e.g. using enzymes with liquid chromatography such as HPAEC or mass spectrometry).

A useful schematic of how we proceed with our studies is provided (Figure 1), as well as a table showing the benefits and drawbacks of the different methods (Table 1). Studying grape cell walls is like a scientific detective story; we get pieces of information from the different methods which we add together to form a more complete picture. This picture provides a hypothesis on certain cell wall characteristics which we are able to test under different conditions (e.g. disease studies; berry ripening progression and wine processing with enzymes). The results of our testing allow us to make more accurate predictions and develop a new understanding which can be used by the industry to implement better practises in the future. In the last section we describe how these methods have been put to good use in helping to understand industry-relevant grape and wine problems and questions.

Deciphering the grapevine cell wall

The methods described have been optimised and used extensively over the last five years at the IWBT. In the first study[11] we confirmed the accuracy of these profiling methods for determining the polysaccharide composition of tobacco leave cell walls (tobacco is the model plant on which to perform plant-pathogen studies) and that it can be used as a high-throughput screening method. Enzymatic oligosaccharide fingerprinting methods were used to determine the specific structure and diversity of the arabinoxyloglucan species found in tobacco. A follow-up study[12] investigated how the wall-associated grapevine gene VvPGIP1 (Polygalacturonase-Inhibiting Protein) which can protect plants against fungal infection, changed the cellulose-xyloglucan network when expressed in tobacco. This gave a new understanding on disease resistance mechanisms. Another study[13] established the baseline cell wall profiles for grapevine leaves which is necessary for further investigations on grapevine disease resistance. We did extensive studies on the changes happening in the grape berry cell wall (Crimson Seedless and Cabernet Sauvignon) during ripening[14] and specific cell wall polymers were identified that can serve as ripening biomarkers. Cell wall ripening changes were also part of the topic of another study on Pinotage grapes[15], but the focus was on the Pinotage berry skin and the cell wall dynamics due to maceration in the presence and absence of maceration enzymes. De-pectination and cell wall unravelling due to enzyme treatment were observed and the impact of the grape ripeness was also demonstrated. Our most recent study investigated the polysaccharides released into the wine, as well as those left over in the pressed Cabernet Sauvignon grape cell walls[16] and suggested the existence of an enzyme-resistant pectin layer that shields the xyloglucan network in the skin cells. This has important implications for understanding the interaction between the polysaccharide degrading enzymes and the grape tissues.

Acknowledgements

This work was supported by grants from the South African Table Grape Industry (SATI), Wine Industry Network for Expertise and Technology (Winetech) [Winetech grants: Cell Wall Platform (IWBT-P 09/01)], the South African Technology and Human Resources for Industry Programme (THRIP) and the Claude Leon Foundation. The Central Analytical Facility (CAF) of Stellenbosch University is gratefully thanked for technical support, as well as Prof. Emile van Zyl (Department of Microbiology, Stellenbosch University) for providing access to the HPAEC-PAD instrument. The CoMPP analysis is done in collaboration with Prof. William Willats and Dr. Jonatan Fangel from the University of Copenhagen.

– For more information, contact Anscha Zietsman at jjv@sun.ac.za.

References

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