Analysis of a novel mechanism that regulates microtubule severing in

The growth and shape of a plant cell is determined by the direction of cell expansion. This expansion is remarkable as plant cells can expand up to 1000 times their original size. For many cells, such as those in the stem or root, expansion needs to occur in a single direction (i.e. upwards) for proper plant growth to occur. To achieve this directional expansion, plant cells need to organise their cell wall and particularly cellulose a very strong fibrillar polymer that has a big influence of cell wall mechanical properties. Cellulose is normally deposited perpendicular to the direction of cell expansion. A protein scaffold, referred to as the microtubule network that is found within the cells, dictates the organisation of cellulose within the wall. Microtubules are able to adopt different patterns. Expansion in a single direction requires a pattern of aligned microtubules whereas expansion in several directions results from a net-like or mesh pattern of microtubules.
Our previous work has found that the microtubules organise themselves into an aligned configuration by cutting away any unaligned microtubules. Without this cutting, microtubules instead adopt a net-like configuration. The cutting machinery recognises unaligned microtubules by only cutting those that crossover existing microtubules. There is increasing evidence that microtubule rearrangements that are essential for many aspects of normal plant growth depend upon microtubule severing. An enzyme called katanin carries out microtubule severing. Katanin mutants have only "net-like" arrays.
We have recently found that plants containing defects in a second protein, called SPIRAL2 (SPR2), that fail to form net-like arrays in cells that normally adopt this pattern. Instead they form a predominantly aligned array. Whereas a katanin mutant does not cut microtubules, a spiral2 mutant shows high rates of microtubule severing. This suggests that the amount of cutting at microtubule crossovers ultimately determines how the microtubules will be organised. It also points to SPIRAL2 being an important factor that modifies the activity of katanin. The SPIRAL2 proteins is present in all cells, but in some cell types it remains attached to microtubules crossover points, while it is constantly moving along microtubules in other cell types allowing severing of microtubules. Stationary binding of SPR2 at crossover points is what appears to prevent severing. We would now like to know more about what controls the activity of SPR2 and understand how it is able to recognise and bind to microtubule crossover points, what determines SPR2 behaviour, i.e. whether SPR2 binds to microtubule crossover points or moves along microtubules and what other factors it need to help it regulate microtubule severing and microtubule array alignment.
Our proposed work will probe what determine SPR2 localisation and mobility. Not only will this work answer important fundamental questions about cell growth and microtubule patterning (including identifying the underlying mechanism that gives rise to twisted growth), but will potentially give us the ability to predictably alter microtubule patterns. In the future this may allow us to manipulate plant development and engineers more efficient canopies or stronger shorter stems. It may also provide a means of increasing plant biomass either for better crop yields or to generate material for bioenergy production.

In plants, the switch from isotropic to anisotropic (directional) cell expansion coincides with a massive change in the organisation of the cortical microtubule array from a net-like to an aligned configuration. The amount of severing by the enzyme katanin has been shown to be a critical factor for determining array configuration and katanin mutants only have net-like arrays, consistent with severing being a major driver for alignment. Microtubule severing occurs at microtubule crossovers and represents a means of removing unaligned microtubules. Recently we have identified that the previously characterised, microtubule-binding protein, SPIRAL2 (SPR2) is a key regulator of severing. spr2 mutants promote aligned arrays even in pavement cells that normally exhibit net-like configurations. These aligned arrays correlate with increased severing by katanin and demonstrate the latent severing capacity of all cells. The difference in severing is attributed to differences in SPR2 mobility and localisation. In pavement cells SPR2 remains stationary and attached to microtubule crossover points where it is able to prevent severing. In contrast, in petiole cells with aligned arrays, SPR2 is largely mobile and moving along the microtubules exposing microtubule crossover sites to the action of katanin. The proposed work will explore in detail, the mechanism by which SPR2 is able to regulate severing. This will involve using a range of techniques, including live-cell imaging, protein chemistry and genetics to analyse the domain structure of the SPR2 protein and the role of phosphorylation in regulating its activity, determining how SPR2 is able to recognise and bind to microtubule crossover sites and identifying and functionally characterise SPR2 binding partners that facilitate its function as a key regulator of microtubule organisation.

The academic community is likely to benefit from this research since so much of plant development and consequently crop productivity is dependent upon cell expansion. Since this work focuses on one of the fundamental processes that underlie the process of cell expansion, it will impact on a large number of areas of plant science. Furthermore there is increasing interesting in using systems biology and computational approaches to understand plant development and morphology since MT organization is fundamental to all plant morphology so understanding the mechanisms that regulate plant microtubule organization is essential for a proper understanding of this process, and also for generating proper mechanistic models of plant development.
Dwarfing genes such as gai form the foundation of the green revolution and the development of high yielding crops. More recently brassinosteroid mutants have been show to increase canopy area by a process that involves decreased cell expansion. Although we have no direct plans to undertake this work during the course of this project, being able to regulate cell expansion would clearly have huge beneficial consequences. If we were able to better understand cell expansion it should be possible to generate tissue specific alterations that would give optimal height and canopy without affecting seed development and hence yield. In the long term this will help with food security issues and help meet the increasing demands for food in a rapidly changing global climate. It is also possible that better regulation of microtubule organization and cell shape may lead to increased plant biomass that may be useful to meet the demands of the growing plant biofuel industry.