A project undertaken at Australian National University, and supervised by David Collings
While the ability for plant roots to grow downwards through soil is fundamental in anchoring the plant and in providing water and nutrition, the mechanisms that allow the roots to penetrate a hard substrate remain poorly characterised. Our study focuses on one specific plant anatomical feature, the phi thickening, and asks whether and how these structures might strengthen the root and aid soil penetration.
Figure 1.
Phi thickenings are narrow bands of reinforced wall that encircle root cortical cells and which are coordinated between adjacent cells to form a single scaffold that surrounds the root’s vascular tissue. These structures were first described in the late 1800s, and occur in multiple angiosperm and gymnosperm families. Despite being present in agriculturally and horticulturally important plant species such as Brassica oleracea (broccoli, cauliflower and cabbage), Brassica napus (canola) and Malus domestica (apple), they have not been shown to have any specific function. A role for phi thickenings in mechanically strengthening the growing root tip has long hypothesised but the concept remains untested.
Figure 2.
Our study uses Brassica roots to investigate phi thickenings at the cellular, molecular and biomechanical levels, and is based on an agar plate system in which we can rapidly induce phi thickenings with low concentrations of the hormone jasmonic acid. Alternatively, if we induce thickenings with an osmotic shock, their development can be inhibited by ibuprofen which, in plants, blocks the biochemical pathway through which jasmonic acid is formed. This ability to regulate thickening development is critical for our experiments as it allows us to study the pathways that lead to their development and their function.
We use confocal fluorescence microscopy to investigate phi thickenings (Figure 1). Basic fuchsin only stains the weakly lignified endodermis in controls (A) but strongly labels phi thickenings (arrows) in jasmonic acid-treated roots (B). Root cross (lower images), showing lignin stained with basic fuchsin (red) and cell walls stained with calcofluor (cyan), also demonstrate that phi thickenings (arrows) form in response to jasmonic acid.
Confocal microscopy is currently being used to survey phi thickenings in other plant families, and has revealed that these structures are essentially identical in a wide range of plants. Does this mean that phi thickenings have been conserved during evolution, or are thickenings an example of the convergent evolution of a structure in response to evolutionary pressure? This question can only be answered through understanding the cellular and molecular pathways that lead to thickening formation. To investigate the molecular genetics of phi thickening formation in Brassica, we make use of jasmonic acid’s ability to induce phi thickenings. Because different Brassica cultivars show markedly different responses to jasmonic acid, we are collaborating with colleagues at the NSW Department of Primary Industries to screen Brassica cultivar collections and breeding populations. This work allows us to begin the mapping of genes associated with phi thickening formation. Understanding this genetic variability within cultivars will be important for breeding roots that have better penetration into the soil.
Our ability to control phi thickening induction allows testing of the biomechanical properties of roots. We are developing a two-phase culture system in which roots initially grow through a soft, upper agar layer and are then challenged when they strike the low hard agar layer (Figure 2). Does the presence of phi thickenings, induced by the presence of jasmonic acid in the low, help the root penetrate this harder layer? Preliminary data suggests that this is indeed the case, and this is the first direct evidence of a function for phi thickenings since they were discovered more than 150 years ago. Further experimentation is currently being conducted to confirm this mechanical role. Additionally, in collaboration with Professor Volker Nock and Dr Ayelen Tayagui (University of Canterbury, Christchurch New Zealand), we will develop a microfluidic cell system with which to measure the forces that the growing root can exert on the substrate.