PerspectivePlant Biology

A Baroque Residue in Red Wine

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Science  26 Oct 2001:
Vol. 294, Issue 5543, pp. 795-797
DOI: 10.1126/science.1066242

The walls of higher plants contain small amounts of a mysterious polysaccharide known as rhamnogalacturonan II (RGII). RGII is thought to be the most complex polysaccharide on Earth, and its presence and strong conservation in all higher plants suggest that it is important for the structure or growth of plant cell walls. The study by O'Neill et al. (1) on page 846 of this issue convincingly shows, 23 years after its discovery (2), that RGII is essential for plant growth and that minor changes in its structure cause growth defects.

More than 300 years ago, Robert Hooke pointed his primitive microscope at a slice of cork and discovered the cellular basis of organisms. Sadly, since then, plant cell walls, which formed the compartments he actually observed, have never been considered particularly entertaining structures. Indeed, the word wall itself evokes something dull and rigid, built only to enclose, support, divide, and protect. However, a closer look reveals just how erroneous this view is. Walls of growing plant cells are extremely sophisticated composite materials made of dynamic networks of polysaccharides, protein, and phenolic compounds. Cellulose microfibrils with a tensile strength comparable to that of steel provide the plant with a load-bearing framework. These microfibrils are rigid wires made of crystalline arrays of β-1,4-linked chains of glucose residues, which are extruded from little hexameric spinnerets in the plant cell plasma membrane and surround the growing cell like the hoops around a barrel. Because cellulose microfibrils constrain turgor-driven cell expansion in one preferential direction, they control the shape of plant cells and ultimately that of the plants themselves. Hemicelluloses, such as xyloglucans, are tethered by hydrogen bonds to cellulose and form cross-links that may control the separation of the cellulose microfibril hoops. The cellulose-hemicellulose network is embedded in a matrix of complex galacturonic acid-rich pectic polysaccharides (including RGII) that form a hydrated gel inside the wall and provide a dynamic operating environment for cell wall processes.

Most of the components of the pectic matrix are notable for their heterogeneity, but intriguingly, RGII is highly conserved. During the growth of plant cells, which can extend hundreds and even thousands of times their original length, the synthesis, deposition, assembly, and remodeling of wall polysaccharides must be carefully coordinated. All of this must occur while the plant maintains its resistance to the extreme tension (several hundreds of megapascals) on the relatively thin cell wall generated by the turgor pressure within the solute-filled cell. The assembly and extension of the wall is in part mediated by wall-associated enzymes, which cleave, rearrange, or cross-link polysaccharides. Plants have evolved unique strategies to fine-tune the activity of these extracellular enzymes indirectly, for example, by modifying the extracellular pH or the redox state of the plant cell or by generating localized oxygen radicals. Pectic polysaccharides are also thought to be important in this control, because they can influence the porosity of the cell wall and hence the mobility of the wall enzymes.

RGII is a remarkable molecule (see the figure). It is composed of 11 kinds of sugar monomers, and it is thought that at least 21 enzymes are dedicated to the construction of all the linkages between the sugar residues. This molecule must adopt a well-defined three-dimensional conformation, as suggested by its marked resistance to enzymatic degradation. As a result, large amounts of RGII can be found in fermented products such as wine. Why do all higher plants invest so much effort in producing this baroque structure in their walls? The first hint came from the observation that RGII exists as a dimer that is cross-linked by a borate diester (see the figure). Boron is an essential micronutrient of plants, and its deficiency not only causes the disappearance of RGII dimers but also leads to growth inhibition associated with dramatic changes in cell wall architecture. These findings suggest, but do not prove, that the absence of RGII dimers causes growth defects. To demonstrate a causal relationship, plant mutants with specifically altered RGII would be needed.

Missing links.

RGII, an extremely complex polysaccharide, is present in the cell walls of all higher plants. It is composed of 11 kinds of sugar monomers and can form dimers through boron (B), which forms diesters with apiose residues (purple). Small changes in RGII interfere with dimer formation in the Arabidopsis mutant mur1 (1). The growth defect in this plant mutant is a result of this change in RGII. [Reprinted with permission from (3); copyright American Society of Plant Biologists]

In the absence of such specific mutants, O'Neill et al. (1) took advantage of the known pleiotropic mutant mur1 in the model plant Arabidopsis thaliana. The altered gene in this mutant codes for the enzyme GDP-D-mannose-4,6-dehydratase, which is required for the synthesis of GDP-L-fucose. This activated sugar is the substrate used for the fucosylation of RGII. The mutant plants are dwarfed and have more fragile cell walls. Watering the mutant with fucose restores normal growth. The problem is that fucose is not only found in RGII. Two other types of polymer are also fucosylated: xyloglucans and the glycan side chains of many proteins. But which altered polysaccharides are responsible for the growth defect in mur1? To resolve this whodunit, the authors commandeered observations reported for two other mutants. Indeed, mutant cgl1 entirely lacks fucose in its glycoproteins and surprisingly does not show an observable phenotype. Similarly, mur2 mutants synthesize xyloglucans with only 2% of the normal amount of fucose and still grow normally. This left the authors with RGII. They next showed that mur1 mutants contained normal amounts of RGII, which, as expected, lacked terminal fucose and 2-O-Me-L-Fuc, a fucose derivative. These residues were replaced, respectively, by L-galactose and 2-O-Me-L-Gal in the mutant, probably because the enzyme that adds fucose and 2-O-Me-L-Fuc to the RGII backbone also can incorporate L-galactose and its derivative. Interestingly, as a result of these modest changes, only half the amount of RGII dimer was formed in the mur1 mutants. In vitro studies led to the conclusion that the “mutant” RGII dimerizes more slowly and has a reduced stability at low pH. It also requires higher concentrations of boron to dimerize. This also turned out to be true in vivo, because watering the plants with boron restored the formation of RGII dimers. The most exciting observation of this study is that this treatment also restored normal growth to mur1 plants, thus demonstrating unequivocally that RGII dimer formation is essential for normal plant growth.

As usual, these observations raise more questions than they answer. Why is RGII dimerization so important? RGII is covalently attached to a backbone composed of a linear chain of galacturonic acids, and dimerization promotes the formation of a cross-linked pectin network. This network controls the porosity of the cell wall, as shown by the increased pore size of cell walls of boron-starved cell cultures. It will be interesting to see whether the altered pore size also affects the accessibility of polysaccharide-modifying enzymes to their substrates, which could be a mechanism by which reduced boron cross-linking affects growth of mur1 plants. Does RGII cross-linking control normal plant growth? A reduction in pore size has been observed upon transition from the growth phase to the stationary phase in cell cultures. It is not known, however, whether RGII is involved in this change. What is the three-dimensional structure of this molecule? Insights come from molecular modeling, and attempts to crystallize this polysaccharide are under way. Maybe these approaches will explain its unusual stability. Finally, it will also be interesting to know whether this complex molecule carries out tasks other than those related to its ability to form dimers. Next time you drink a glass of red wine, rich in RGII, why not reflect on these intriguing questions.

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