Proteins Among the Polysaccharides

Abstract

Charles Darwin recognized the power of the root cap as a model for plant signalling and behavior, and used it to explore the ways plants sense and respond to diverse stimuli. Over ensuing decades, various groups have reported tantalizing clues regarding the role of a complex extracellular matrix that ensheaths the tip region housing the apical and root cap meristems. In the course of characterizing root tip resistance to infection and injury and the role border cells play in this phenomenon, we confirmed and extended early- and mid-20^th century studies reporting enzyme activities secreted from the root cap. Multidimensional protein analysis revealed, in fact, that >100 proteins are actively synthesized and secreted from the root cap and border cells. This ‘root cap secretome’ appears to be a critical component of root tip resistance to infection. We have developed a microscopic assay to quantify the protein-based extracellular response to dynamic changes in environmental conditions including hydroponic culture, and present the results here. This tool provides a simple, direct measure that can be used to explore the ways border cells may function in the manner of white blood cells to trap, immobilize and neutralize threats to the growing root tip.

In a process similar to the shedding of ‘buccal’ cells into the mammalian oral cavity, root caps of most higher plants naturally can shed thousands of healthy ‘border’ cells into the external environment each day.^4,5 Like buccal cells ensheathed in saliva, populations of border cells are contained within a mucous-like material.^1,2,6,7 This material-historically termed root cap ‘slime’ or ‘mucilage’-has been found by several groups to be comprised of a high molecular weight polysaccharide, with a small amount of protein (95% and 5%, respectively).^1,8–10 Previously, we used two-dimensional electrophoresis to characterize root cap and border cell proteins in pea and were surprised to find that within a 1 h period of exposure to ³⁵S-labelled methionine, >100 proteins are synthesized and exported into the extracellular environment.¹¹ The protein activities within this ‘root cap secretome’ appear to be critical to the capacity of the root cap extracellular material to protect the root tip from infection.¹² When these proteins are degraded the mucilage disintegrates, as described below, suggesting for the first time that the minority protein component of the root cap slime layer is a key structural component.

In summary, multidimensional protein analysis confirmed that the root cap secretome includes a mixture of ∼120 proteins including defense and signalling enzymes as well as structural proteins like actin.¹² When the proteins are solubilized in situ using a broad spectrum protease, the root tip of pea completely loses its resistance to infection by the pea pathogen, Nectria haematococca. Normally, only 3% of inoculated roots of a susceptible host develop a lesion at the root tip even under conditions highly conducive to infection.¹³ After protease treatment, however, frequency of infection was increased to 100%.¹² Every inoculated root tip became necrotic. This surprising result reveals that, despite being a minor physical component of the extracellular matrix, the secretome is a major functional component.

India ink can be used to visualize bacterial and fungal cells, whose extracellular capsules exclude penetration of the carbon particles.^14,15 Here we report that the contours of the entire slime-mucilage ‘blob’ (Fig. 1), with border cells embedded within the matrix (Fig. 1, arrows) can be visualized using this simple assay. Surprisingly, the same protease treatment used to solubilize the secretome and eliminate root tip resistance to infection¹² also eliminates the matrix seen with India ink. These data indicate that protein is a key structural as well as functional component of the matrix, such that solubilizing the 5% of the matrix that is protein causes the entire structure to disintegrate.

Figure 1.

India ink assay to visualize root cap ‘mucilage’. A root tip (white arrow) was placed onto a glass slide and India ink was added. After addition of a cover slip, a clear delineation can be seen (white triangles) where ink fails to penetrate due to the presence of an impermeable layer. Border cells (black arrows) are present throughout the boundary layer. Addition of proteinase K, under conditions that destroy root tip resistance to infection, destroyed the boundary layer, resulting in an unbroken field of black when ink is added (not shown). Magnification: bar = 1 mm.

As previously shown in a study of genotype-specific responses of Phaseolus vulgarus border cells to aluminum,¹⁶ the India ink assay revealed that individual border cells exhibit species-specific dynamic responses to microbial challenge (Fig. 2). Control pea border cells, washed to remove soluble material, are surrounded by a ca 5 µm wide capsule around the cell periphery (Fig. 2A). When incubated with N. haematococca conidia, a marked increase in the size of the capsule occurred (Fig. 2B). In contrast, when incubated with proteinase K, the capsule virtually disappeared (Fig. 2C). Control corn border cell capsules are slightly larger than those of pea (Fig. 2D). After cocultivation with Pseudomonas aeruginosa, the capsule increased by several-fold and bacterial cells could be seen enmeshed within the layer (Fig. 2E, arrows). Remarkably, a dramatic, >50-fold increase in capsule size occurred on border cells of corn cocultivated in hydroponic culture for 7–10 days with a gram-positive bacterium (Bacillus sp) found as a seed-borne epiphyte (Fig. 2F).

Figure 2.

Dynamics of border cell capsule induction, and solubilization by proteinase K. India ink was used to visualize the boundary (triangles) of (A–C) pea and corn (D–F) border cell capsules. (A) Control pea border cell capsule; (B) Increased capsule size in response to N. haematococca conidia; (C) Effect of protease treatment on the the border cell capsule. The cell remains viable, as can been seen by the intact nucleus (arrow), but the capsule is nearly eliminated. (D) Control corn border cell; (E) Increased capsule occurring in response to cocultivation with Pseudomonas aeruginosa. Trapped bacteria can be seen within the capsule (arrows); (F) Massive capsule around a single corn border cell after hydroponic culture for >1 week. Magnification: bar = 15 µm.

Brinkmann et al.¹⁷ reported that, in mammalian systems, white blood cells (neutrophils) produce extracellular structures containing antimicrobial proteins. According to the authors, these neutrophil extracellular traps (NETs) ‘appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.’ The trapping of pathogenic bacteria within the border cell capsule was reported previously.¹⁸ Knudson¹⁹ reported that border cells of pea and corn survive for months in hydroponic culture, and the potential impact of such massive capsules on microbial survival over time will be of interest. Our results support the premise, as others have suggested, that the root cap extracellular matrix is a dynamic conduit for plant signalling and behavior responses.^20,21 Recognition of the critical role the extracellular proteome plays in its function will provide a context to dissect how root caps perceive and respond to incoming signals to control root growth and development.

A note on terminology: Perhaps it might be time to consider that a more dignified term than root cap ‘slime’ would more accurately represent this dynamic component of plant root systems. If the extracellular matrix functions, as in neutrophils, to kill bound bacteria and fungi, then border cell extracellular trap (BET) might work. Border cell ‘capsule,’ in the meantime, accurately conveys the concept of a functional unit surrounding individual cells, and highlights the likely functional parallels with microbial cells.^14,15 The capsule of Bacillus anthracis cells, for example, also includes integral proteins—the ‘S-layer’—which underlie surface receptors that control pathogen-host recognition.¹⁴ However, root cap capsule seems a bit redundant. We would be interested in the views of our colleagues: Stick with slime? Suggestions for an alternative name?

Addendum to: Wen F, VanEtten HD, Tsaprailis G, Hawes MC. Extracellular Proteins in Pea Root Tip and Border Cell Exudates. Plant Physiol. 2007;143:773–783. doi: 10.1104/pp.106.091637.