Assembling chimeras: BC16 helps to reshape protein GPI lipid anchors, thereby tuning cell wall mechanics in rice

The world depicted in Greek mythology is nothing short of remarkable. One of the most fanciful and bewildering of its inhabitants is Chimera, a creature composed of incongruous parts. The sole duty of this beast was to terrorize the local peasants. In biology, on the other hand, chimeras (artificially produced chimeric or hybrid organisms and molecules) have a rather benign purpose, serving as tools helping to solve a wide range of problems. Such hybrid entities, however, are also natural components of living systems.

One example is proteins carrying lipid moieties, such as glycosylphosphatidylinositol-anchored proteins (GPI-APs). The C-terminal GPI modification allows proteins to associate with membrane lipid bilayers and anchor on the external surface of the plasma membrane. The class of GPI-anchored proteins in plants is vast (more than 200 in Arabidopsis) and essential––abolishing GPI modification leads to embryo lethality. However, very few GPI-APs have been characterized to date, and their role remains poorly understood. Cell wall modification is recognized as a function of GPI-APs. Thus, Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR PROTEIN 1 (PMEI1), which adjusts pectin methylesterification status (De Caroli et al., 2011). Another plant GPI-AP involved in regulating cell wall properties is a rice (Oryza sativa) COBRA-like protein, BRITTLE CULM 1 (BC1) that binds cellulose and affects its crystallinity, thereby tuning cell wall mechanics (Liu et al., 2013). Mutants for BC1 have fragile internodes (hence, brittle culm) and are affected in growth. Despite the quantity and importance of GPI-APs in plants, they remain as cryptic as the mythological Chimera. We know little about how they are assembled and what guides them to their plasma membrane locations. A new study (Xu et al., 2022) sheds some light on these questions.

Screening rice mutants for the fragile internode phenotype, the authors identified a gene involved in the cell wall assembly that they named BC16. To scrutinize the underlying cause of bc16 phenotype, the authors focused on analyzing fiber cells and xylem vessels, two important load-bearing tissues of the rice culm. Examination by scanning electron microscopy and transmission electron microscopy revealed decreased cell wall thickness in both tissues.

Similar to its yeast and mammalian homologs, BC16 was predicted to have several transmembrane domains and, as visualized through green fluorescent protein (GFP) fusions in Nicotiana benthamiana and rice protoplasts, resided in the endoplasmic reticulum and the Golgi apparatus (see Figure). The high sequence and structural similarity to Gup1, the yeast (Saccharomyces cerevisiae) GPI lipid acyltransferase, pointed to a possible functional analogy. To probe this assumption, the authors performed a complementation assay using S. cerevisiae mutant Gup1 with compromised cell wall that is unable to grow in the presence of a cell wall dye calcofluor white (CFW). Surprisingly, the growth of Gup1 mutant on a medium containing CFW was fully restored by complementation with BC16. To determine the extent of biochemical similarity between BC16 and its yeast GUP1 homolog, Xu and colleagues compared GPI lipid profiles of the wild type (WT), Gup1 mutant, and the BC16 complemented strain. Strikingly, the complementation with BC16 nearly perfectly restored the WT GPI lipid profile, suggesting that BC16 has acyltransferase activity.

Figure.

The role of BC16 in regulating cellulose assembly and wall mechanical strength. BC16 functions as an O-acyltransferase that remodels the GPI lipid tail in plants. BC16 resides in the endoplasmic reticulum (ER) and Golgi apparatus (Golgi). BC1 is a GPI-AP modified by BC16. The GPI-anchored BC1 proteins are delivered to the plasma membrane lipid nanodomains, where cellulose synthase complex (CSC) is also present. BC1 and CSC together facilitate assembly of cellulosic nanofibers. Adapted from Xu et al. (2022, Figure 7).

To test if BC16 has this biochemical function in plants, the authors assessed GPI-AP lipid profiles of WT and bc16 internodes. Surprisingly, the GPI lipid distribution of WT was similar to that of yeast and bc16 mutation inflicted disturbances analogous to those in Gup1 yeast mutant.

To test if BC16 is involved in GPI modification of BC1, the authors expressed GFP-tagged BC1 in WT and bc16 rice protoplasts. Indeed, the residence of BC1 at the plasma membrane was strongly compromised in bc16 protoplasts. Interestingly, BC1 turned out to localize to plasma membrane nanodomains enriched in a detergent-resistant membrane fraction. In the absence of BC16, the abundance of BC1 was significantly decreased.

To understand how bc16 mutation affects cell wall structure, the authors observed the deposition of cellulosic nanofibers using atomic force microscopy. The analysis revealed that nanofiber arrays tend to be less aligned in bc16 and to form thinner nanofibers than those of WT. The phenotype was even more pronounced in bc1 plants. Assessing the mechanical properties through atomic force microscopy and tissue stretching, Xu and co-workers found that bc16 fiber cells and internodes demonstrate significantly higher rigidity than those of WT.

The cell wall exists in dynamic crosstalk with the cellular interior through its contact with the plasma membrane, but we know fairly little of how this crosstalk operates. Having the affinity for all cellular components in question, GPI-APs and other molecules of a chimeric nature might be important mediators of this interaction. Therefore, understanding how they are assembled and guided toward the plasma membrane is crucial. The study by Xu and colleagues sets us toward this insight.