Unprecedented glycan nanocompartments sustain plant vessel wall patterning and xylem robustness

Unprecedented glycan nanocompartments sustain plant vessel wall patterning and xylem robustness
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Polysaccharides embedded in plant cell wall represent the most abundant nature resource on earth and a renewable raw material that can be converted into numerous valuable applications. Plant cell wall surrounding every plant cell is a highly organized polysaccharide network. Its structural complexity is dedicated to satisfy various requirements in plant growth and development. Although studies in more than twenty years’ plant functional genomics era have identified many crucial genes for cell wall synthesis and remodeling, there is almost no knowledge about how wall synthesis is connected to wall structure. A major barrier is cell wall heterogeneities in chemistry and structure.

 Cell wall heterogeneity basically originates from unevenly deposition of plant cell wall components, similar to compartmentalization mediated by, for example clustering or phase separation of lipids and proteins, which is essential for efficient organization of biological processes in all living cells. Saccharides are an important class of biomacromolecules that are capable of compartmentalization; however, our knowledge about how these saccharide-based compartments form, and what their biological functions are, is very limited. Xylem vessel cells of the plant vasculature display various polysaccharide compartments. How cell wall matrices self-organize to support compartmentalization is unknown.

 Our article entitled “Xylan-based nanocompartments orchestrate plant vessel wall patterning” was published in Nature Plants on Mar 22, 2022 (https://doi.org/10.1038/s41477-022-01113-1). This study identified unprecedented xylan-based nanoclusters and revealed that this compartment structure is mostly produced by the de novo xylan synthase IRREGULAR XYLEM (IRX)10 and five homologues from glycosyltransferase (GT) 47 family. We also outlined how the xylan nanocompartments maintain the coherence of pit patterns and vessel robustness. This work provides new insights into cell wall compartmentalization and self-organization, placing molecular mechanisms of xylan synthesis and compartmentalization in direct context of plant physiology.

 Here, we would like to share some difficult experiences and exciting moments, which might evoke in-depth thinking and wide discussion in research data practices.

 Our lab has identified a dozen of genes involved in cell wall biosynthesis in rice, a monocot model crop bearing a different cell wall type from the dicot plants. Nevertheless, which wall component contributes mostly to plant cell wall construction and deconstruction is one of our long-term concerns. Recent advances in nuclear magnetic resonance and mass spectrometry identified xylan as a crosslinking polymer that coats cellulosic nanofibrils via flattened twofold ribbon conformer and interacts with hydrophobic lignin via threefold screw conformer, establishing this polymer a crucial role in cell wall coherence. Those discoveries verify our hypothesis that xylan is an important determinant for cell wall heterogeneity and cell wall structure. However, whether xylan can assemble into dedicated nanostructures to boost cell physiology is unclear.

 Actually, xylan synthesis machinery has been debated since 2009 while the genes from GT47 and GT43 families, referred to as IRX10, IRX9, IRX14, and their homologs, have been found implicated in xylan backbone elongation. How xylan synthesis is initiated is still unknown. We have been looking for xylan synthases in rice for almost a decade. In several times of in vitro enzymatic activity tests, we found that rice IRX10 could produce xylooligomers without an acceptor, indicating an unprecedented de nova synthetic machinery. This serendipity prompted us to take about eight years to validate this new activity. We carried out a lot of tests, such as adjusting culture conditions and reaction parameters, establishing reliable methodologies to examine the reaction products, and comparing the affinities to different substrates. We further performed time-course catalytic activity assays and site-directed mutation experiments. A series of in vitro biochemical evidence demonstrated rice IRX10 as a de novo xylan synthase.

 Unlike Arabidopsis that has two IRX10 proteins, rice has six IRX10-related proteins. Generating single and higher-order rice mutants for IRX10 and its five homologs using CRISPR/Cas9 genome editing approach was arduous and took about several years. The higher-order mutants we finally obtained are quadruple mutants. We failed to obtain a quintuple or sextuple mutant, suggesting that completely blocking IRX10-related functions may lead to plant lethality. To acquire a genetic evidence that xylan synthesis is completely eliminated in the mutant cells, we developed a microscale cell wall composition analytical method suitable for detecting a few cells. We chose metaxylem vessels for examination as IRX10 is highly expressed in this cell type. The trace xylan amounts in ten vessel cells of a triple mutant represents an important genetic data to confirm the initiation activity of IRX10.

 This clue further guided us to look into xylem vessel cells that have highly organized cell wall compartments, such as spiral, reticulate, scalariform, and pitted patterns. These microcompartments dictate physiological functions, including robust water transport and prevention of air embolism and invasion of pathogens. We performed immunolabeling and visualization of xylan in the metaxylem vessels isolated from rice internodes undergoing secondary wall deposition. Super-resolution confocal microscopy showed that xylan patterns in these vessels mirrored those of xylan synthesis in a developmental trajectory. More importantly, we observed fascinating xylan-rich nanostructures at pit edges of vessel walls, which directed us to investigate vessel wall patterning and vessel robustness in the mutants. The wall topography around pit boundary shown by atomic force microscopy further impressed us. Owing to the reduced level of xylan-nanoclusters, in the irx10 mutants, cellulosic nanofibrils are disorganized and extended outside the pit boundary and across the pits, resulting in reticulate-like patterns that compromise vessel robustness, water transport, xylem integrity, and stem height. The xylan nanostructures at pit boundary are analogous to merrow edges of buttonholes to sustain coherence of heterogeneous structures. We further found xylan nanodomains also present in the vessels of a dicot plant Arabidopsis, implying conservativeness and importance of this glycan structure in various plant species.

In summary, through this study, we acknowledge the importance of applying interdisciplinary advanced technologies, as it can approach innovation research data unavailable with the conventional approaches. Furthermore, although solidifying serendipitous discoveries to convincing data is quite difficult, the outputs would be exciting. All those are the experiences we want to share.

a, Z-plane of a pit in wild-type xylem vessel wall probed using xylan-recognized antibodies (green). b, AFM images of vessel walls, showing disorganized cellulosic nanofibrils in the mutant. c, Model for formation xylan nanoclusters in pitted vessel cells by xylan synthase (XS). The embedded figure is mass spectrography of the reaction products catalyzed by IRX10 without an acceptor substrate.

Fig. 1| Xylan-based nanocompartments govern vessel wall patterning. a, Z-plane of a pit in wild-type xylem vessel wall probed using xylan-recognized antibodies (green). b, AFM images of vessel walls, showing disorganized cellulosic nanofibrils in the mutant. c, Model for formation xylan nanoclusters in pitted vessel cells by xylan synthase (XS). The embedded figure is mass spectrography of the reaction products catalyzed by IRX10 without an acceptor substrate.

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