Researchers discover key functions of plasma membrane–cell wall adhesion in rice and critical genes involved

In a study published in Nature Plants, a research team led by Prof. Chao Daiyin from the Center of Excellence for Molecular Plant Sciences of the Chinese Academy of Sciences identified a novel protein family in plants named as GAPLESS, and revealed that members of this family mediate the adhesion between the cell wall and the plasma membrane at the Casparian strip (CS), a barrier in the root endodermis. This adhesion plays a critical role in controlling nutrient transport and the growth development of rice.

Living organisms require barriers to control the transport and exchange of matters. In animals, gut epithelial cells are tightly anchored through a series of cell membrane fusion proteins, forming "tight junctions" to seal the intercellular space to control the intake of nutrients while preventing their leakage. The root is an organ of plants for water and nutrient uptake and transport, and it structurally and functionally resembles the animal gut. Root endodermal cells, resembling gut epithelial cells, function in controlling water/nutrient transport and preventing water/nutrient leak.

However, due to the presence of the cell wall, plant root endodermal cells cannot establish direct tight junctions like gut epithelial cells. Plants have developed a unique cell wall structure called the CS that prevents free diffusion of water and nutrients. Named after its discoverer Robert Caspary, this structure is composed of highly hydrophobic lignin, which prevents the free diffusion of water-soluble substances.

In 1935, American scientist Bryant discovered that the plasma membrane next to the CS, so called CS domain (CSD), tightly adheres to the CS, resembling tight junctions in animals. Such an adhesion is incredibly strong and cannot even be separated by high osmotic treatments that tear cells apart. This adhesion has been speculated to play an important role in plants. Due to the difficulty of research and lack of evidence, this speculation has not been confirmed, and the molecular basis and adhesion mechanism it produces have remained unclear.

In this study, the researchers first identified a series of genes specifically expressed in the root endodermis of rice through bioinformatics analysis. Among them, three previously uncharacterized genes are highly similar in sequences, and the proteins they encoded have a C-terminal non-conserved structure rich in glycine (G), alanine (A), and proline (P), and a conserved lectin domain (LE) and a secretion signal peptide (SS) at the N-terminus. The researchers named the family GAPLESS. These proteins are widely present in plants and specifically expressed in unsuberized cells of the endodermis.

When the genes encoding the three GAPLESS proteins were individually knocked out, only the mutant gapless1 exhibited obvious growth and ion homeostasis defects such as reduced plant height, decreased tillering, lower individual yield, reduced potassium ion levels, and increased calcium ion levels. However, the double mutant gapless1/2 showed extremely severe growth and ion homeostasis phenotypes with a yield less than 3% of the wild type and potassium ion content only 10% of the wild type. These results indicated the importance of these proteins and suggested functional redundancy of these genes.

Subsequent research showed that the barrier function of the root endodermis of the single mutant gapless1 to prevent the free diffusion of water-soluble molecules decreased, while that in the double mutant gapless1/2 was even more severely impaired. However, further investigation revealed that their CS lignin accumulation was not defective; instead, the CS in the gapless1/2 double mutant was wider and stronger. This suggested that their barrier defects are not caused by CS defects.

As the adhesion of CS and CSD is necessary for establishing the endodermal cell barrier, the researchers used electron microscopy to observe this structure in wild-type and mutant plants. The results showed that the adhesion of CS and CSD weakened in the gapless1 single mutant, and about half of the adhesion was completely lost in the gapless1/2 double mutant with even the remaining adhesion severely narrowed. These findings indicated that the GAPLESS protein family is necessary for CS and CSD adhesion and this cell adhesion is crucial for nutrient balance and growth development in rice.

Further investigation revealed that the GAPLESS proteins are specifically localized in CS and close to the OsCASP1 protein, which is specifically localized in the CSD. A series of experimental evidence showed that the GAPLESS protein can strongly interact with OsCASP1, forming a robust GAPLESS-OsCASP complex. Therefore, the GAPLESS protein embedded in the CS and OsCASP embedded in CSD to form a stable complex, adhering the CS to the cell membrane, thereby preventing the free diffusion of water and ions in the root.

The elucidation of the molecular features and mechanism of action of GAPLESS not only refreshes understanding of the function of cell wall proteins, but also expands the knowledge of cell adhesion mechanisms in multicellular organisms.

This study unravels the century-old mystery of the molecular mechanism underlying the formation of the CS-cell membrane adhesion and confirms the importance of this tight adhesion for plant nutrient balance and growth development. It has significant implications for improving the efficiency of mineral nutrient utilization and deciphering plant salt and drought tolerance mechanisms, since the CS plays a vital role in plant selective absorption and response to stresses like drought and salinity.