A comparative meta-proteomic pipeline for the identification of plasmodesmata proteins and regulatory conditions in diverse plant species.

BACKGROUND: A major route for cell-to-cell signalling in plants is mediated by cell wall-embedded pores termed plasmodesmata forming the symplasm. Plasmodesmata regulate the plant development and responses to the environment; however, our understanding of what factors or regulatory cues affect their structure and permeability is still limited. In this paper, a meta-analysis was carried out for the identification of conditions affecting plasmodesmata transport and for the in silico prediction of plasmodesmata proteins in species for which the plasmodesmata proteome has not been experimentally determined. RESULTS: Using the information obtained from experimental proteomes, an analysis pipeline (named plasmodesmata in silico proteome 1 or PIP1) was developed to rapidly generate candidate plasmodesmata proteomes for 22 plant species. Using the in silico proteomes to interrogate published transcriptomes, gene interaction networks were identified pointing to conditions likely affecting plasmodesmata transport capacity. High salinity, drought and osmotic stress regulate the expression of clusters enriched in genes encoding plasmodesmata proteins, including those involved in the metabolism of the cell wall polysaccharide callose. Experimental determinations showed restriction in the intercellular transport of the symplasmic reporter GFP and enhanced callose deposition in Arabidopsis roots exposed to 75-mM NaCl and 3% PEG (polyethylene glycol). Using PIP1 and transcriptome meta-analyses, candidate plasmodesmata proteins for the legume Medicago truncatula were generated, leading to the identification of Medtr1g073320, a novel receptor-like protein that localises at plasmodesmata. Expression of Medtr1g073320 affects callose deposition and the root response to infection with the soil-borne bacteria rhizobia in the presence of nitrate. CONCLUSIONS: Our study shows that combining proteomic meta-analysis and transcriptomic data can be a valuable tool for the identification of new proteins and regulatory mechanisms affecting plasmodesmata function. We have created the freely accessible pipeline PIP1 as a resource for the screening of experimental proteomes and for the in silico prediction of PD proteins in diverse plant species.

Emerging models on the regulation of intercellular transport by plasmodesmata-associated callose.

The intercellular transport of molecules through membranous channels that traverse the cell walls-so-called plasmodesmata-is of fundamental importance for plant development. Regulation of plasmodesmata aperture (and transport capacity) is mediated by changes in the flanking cell walls, mainly via the synthesis/degradation (turnover) of the (1,3)-ß-glucan polymer callose. The role of callose in organ development and in plant environmental responses is well recognized, but detailed understanding of the mechanisms regulating its accumulation and its effects on the structure and permeability of the channels is still missing. We compiled information on the molecular components and signalling pathways involved in callose turnover at plasmodesmata and, more generally, on the structural and mechanical properties of (1,3)-ß-glucan polymers in cell walls. Based on this revision, we propose models integrating callose, cell walls, and the regulation of plasmodesmata structure and intercellular communication. We also highlight new tools and interdisciplinary approaches that can be applied to gain further insight into the effects of modifying callose in cell walls and its consequences for intercellular signalling.

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils.

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.

Immunofluorescence Detection of Callose in Plant Tissue Sections.

The accumulation of the cell wall component callose at plasmodesmata (PD) is crucial for the regulation of symplastic intercellular transport in plants. Here we describe protocols to fluorescently image callose in sectioned plant tissue using monoclonal antibodies. This protocol achieves high-resolution images by the fixation, embedding, and sectioning of plant material to expose internal cell walls. By using this protocol in combination with high-resolution confocal microscopy, we can detect PD callose in a variety of plant tissues and species.

Altering arabinans increases Arabidopsis guard cell flexibility and stomatal opening.

Stomata regulate plant water use and photosynthesis by controlling leaf gas exchange. They do this by reversibly opening the pore formed by two adjacent guard cells, with the limits of this movement ultimately set by the mechanical properties of the guard cell walls and surrounding epidermis.1,2 A body of evidence demonstrates that the methylation status and cellular patterning of pectin wall polymers play a core role in setting the guard cell mechanical properties, with disruption of the system leading to poorer stomatal performance.3-6 Here we present genetic and biochemical data showing that wall arabinans modulate guard cell flexibility and can be used to engineer stomata with improved performance. Specifically, we show that a short-chain linear arabinan epitope associated with the presence of rhamnogalacturonan I in the guard cell wall is required for full opening of the stomatal pore. Manipulations leading to the novel accumulation of longer-chain arabinan epitopes in guard cell walls led to an increase in the maximal pore aperture. Using computational modeling combined with atomic force microscopy, we show that this phenotype reflected a decrease in wall matrix stiffness and, consequently, increased flexing of the guard cells under turgor pressure, generating larger, rounder stomatal pores. Our results provide theoretical and experimental support for the conclusion that arabinan side chains of pectin modulate guard cell wall stiffness, setting the limits for cell flexing and, consequently, pore aperture, gas exchange, and photosynthetic assimilation.

Cell Wall Polymer Composition and Spatial Distribution in Ripe Banana and Mango Fruit: Implications for Cell Adhesion and Texture Perception.

Banana (Musa acuminata) and mango (Mangifera indica) are two of the most popular fruits eaten worldwide. They both soften during ripening but their textural attributes are markedly different. This study aimed to elucidate the molecular mechanism underpinning textural differences between banana and mango. We used a novel combination of methods at different scales to analyse the surface properties of fruit cells and the potential contribution of cells and cell wall components to oral processing and texture perception. The results indicated that cell separation occurred easily in both organs under mild mechanical stress. Banana cells showed distinctively elongated shapes with distinct distribution of pectin and hemicellulose epitopes at the cell surface. In contrast, mango had relatively spherical cells that ruptured during cell separation. Atomic force microscopy detected soft surfaces indicative of middle lamella remnants on banana cells, while mango cells had cleaner, smoother surfaces, suggesting absence of middle lamellae and more advanced cell wall disassembly. Comparison of solubilized polymers by cell wall glycome analysis showed abundance of mannan and feruylated xylan in separation exudate from banana but not mango, but comparable levels of pectin and arabinogalactan proteins. Bulk rheology experiments showed that both fruits had similar apparent viscosity and hence might be extrapolated to have similar "oral thickness" perception. On the other hand, oral tribology experiments showed significant differences in their frictional behavior at orally relevant speeds. The instrumental lubrication behavior can be interpreted as "smooth" mouthfeel for mango as compared to "astringent" or "dry" for banana in the later stages of oral processing. The results suggest that cell wall surface properties contribute to lubricating behavior associated with textural perception in the oral phase.

Interactions between callose and cellulose revealed through the analysis of biopolymer mixtures.

The properties of (1,3)-ß-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-ß-glucan and cellulose, in ionic liquid solution and hydrogels, as proxies to understand the physico-mechanical properties of callose. We show that after callose addition the stiffness of cellulose hydrogels is reduced at a greater extent than predicted from the ideal mixing rule (i.e., the weighted average of the individual components' properties). In contrast, yield behaviour after the elastic limit is more ductile in cellulose-callose hydrogels compared with sudden failure in 100% cellulose hydrogels. The viscoelastic behaviour and the diffusion of the ions in mixed ionic liquid solutions strongly indicate interactions between the polymers. Fourier-transform infrared analysis suggests that these interactions impact cellulose organisation in hydrogels and cell walls. We conclude that polymer interactions alter the properties of callose-cellulose mixtures beyond what it is expected by ideal mixing.

Stomatal Opening Involves Polar, Not Radial, Stiffening Of Guard Cells.

It has long been accepted that differential radial thickening of guard cells plays an important role in the turgor-driven shape changes required for stomatal pore opening to occur [1-4]. This textbook description derives from an original interpretation of structure rather than measurement of mechanical properties. Here we show, using atomic force microscopy, that although mature guard cells display a radial gradient of stiffness, this is not present in immature guard cells, yet young stomata show a normal opening response. Finite element modeling supports the experimental observation that radial stiffening plays a very limited role in stomatal opening. In addition, our analysis reveals an unexpected stiffening of the polar regions of the stomata complexes, both in Arabidopsis and other plants, suggesting a widespread occurrence. Combined experimental data (analysis of guard cell wall epitopes and treatment of tissue with cell wall digesting enzymes, coupled with bioassay of guard cell function) plus modeling lead us to propose that polar stiffening reflects a mechanical, pectin-based pinning down of the guard cell ends, which restricts increase of stomatal complex length during opening. This is predicted to lead to an improved response sensitivity of stomatal aperture movement with respect to change of turgor pressure. Our results provide new insight into the mechanics of stomatal function, both negating an established view of the importance of radial thickening and providing evidence for a significant role for polar stiffening. Improved stomatal performance via altered cell-wall-mediated mechanics is likely to be of evolutionary and agronomic significance.

Stomatal Function Requires Pectin De-methyl-esterification of the Guard Cell Wall.

Stomatal opening and closure depends on changes in turgor pressure acting within guard cells to alter cell shape [1]. The extent of these shape changes is limited by the mechanical properties of the cells, which will be largely dependent on the structure of the cell walls. Although it has long been observed that guard cells are anisotropic due to differential thickening and the orientation of cellulose microfibrils [2], our understanding of the composition of the cell wall that allows them to undergo repeated swelling and deflation remains surprisingly poor. Here, we show that the walls of guard cells are rich in un-esterified pectins. We identify a pectin methylesterase gene, PME6, which is highly expressed in guard cells and required for stomatal function. pme6-1 mutant guard cells have walls enriched in methyl-esterified pectin and show a decreased dynamic range in response to triggers of stomatal opening/closure, including elevated osmoticum, suggesting that abrogation of stomatal function reflects a mechanical change in the guard cell wall. Altered stomatal function leads to increased conductance and evaporative cooling, as well as decreased plant growth. The growth defect of the pme6-1 mutant is rescued by maintaining the plants in elevated CO2, substantiating gas exchange analyses, indicating that the mutant stomata can bestow an improved assimilation rate. Restoration of PME6 rescues guard cell wall pectin methyl-esterification status, stomatal function, and plant growth. Our results establish a link between gene expression in guard cells and their cell wall properties, with a corresponding effect on stomatal function and plant physiology.