Understanding ice formation in plants: finding new routes to freezing tolerance (PlantIce).

In many countries, including the UK, crops suffer severe damage due to freezing conditions during the winter. This has become worse with climate change as unpredictable late or early frosts hit plants at vulnerable growth stages and when they have not been acclimatised to winter conditions.

Plants are damaged and killed by frost not as a direct result of the low temperature but because of the effect of ice forming. Much of the damage caused by ice is a consequence of water no longer being available to the plant's cells; they become dehydrated as if the plant was experiencing drought conditions. However, some plant species manage to prevent ice from forming even when the temperature drops below zero degrees. Complex factors determine whether or not they are able to achieve this and thus avoid the consequences of freezing. These factors are clearly worth pursuing if we are to produce crops that are more resilient to low winter temperatures.

Over the past 2 decades research effort has been targeted towards identifying the genes that allow some plants to survive freezing conditions. This has met with some success in understanding how some - but not other - plants can cope with ice but has seldom led to successful commercial exploitation. Perhaps surprisingly, little effort has focused on identifying the features of plants that determine whether or not these damaging ice crystals will form.

Our proposal addresses this gap in our current knowledge by looking at the outer barrier of the plant cell, the cell wall. Most ice forms first around the cell wall and within the spaces between the walls of one cell and its neighbour; hence, this is the most important place to look. If some plants have "the right kind" of cell walls to prevent ice forming and others do not, we need to understand more about the features of a cell wall that make it the "right kind" and thus less prone to ice crystals. In addition, we already have good evidence from our own research and that of others that the composition of the cell wall can influence plant survival of freezing conditions. Via this project we will identify the features of the cell wall that determine whether and how ice crystals start to form and continue to grow and spread. To this end, we will combine state-of-the-art computer simulations, to tell us which cell wall chemical components or structural features encourage or discourage the formation of ice, with novel imaging techniques that will track ice as it forms and spreads in plants.

Building on the microscopic understanding of ice formation we will achieve by this blended methodology, we will investigate genetic mutants of the model plant species Arabidopsis that are known to present specific alterations in their cell wall. By examining how they respond to sub-zero temperatures, we will discover which of these altered cell wall properties are linked with ice avoidance and freezing tolerance. In particular, we will measure how well whole plants and individual cells survive freezing conditions if they have cell walls with altered strength or porosity; two features that are thought to be important in determining freezing tolerance. Our work will identify genes that promote freezing tolerance through their effects on modifying the cell wall. Once we have identified the genes and characteristics that are important for Arabidopsis plants to survive freezing conditions, we can apply this knowledge to crops, focusing immediately on crop genes that we know confer the same characteristics.

Ultimately the knowledge gained through this work will lead to breeding, or creation through genetic modification, of crops with genes that confer improved resilience to freezing conditions.

The aim of this proposal is to understand the role of plant cell wall constituents, particularly pectins, in mediating and/or preventing extracellular ice formation, which plays a key role in determining the freezing tolerance of plants.

The two main objectives of this project are: (1) to discover the impact of cell wall pectin cross-linking on ice nucleation and growth in plant tissues (WP1) and (2) to reveal the mechanism whereby pectin cross-linking influences plant freezing tolerance (WP2).

To this end, we will use molecular dynamics simulations to investigate the atomistic details of how modified and cross-linked pectins affect ice nucleation and growth and high-speed cryo microscopy to visualise how ice forms and spreads between particular cell types and under different conditions. Then, we will use cryo microscopy and infra-red thermal imaging of Arabidopsis genetic mutants to reveal how specific changes to the cell wall mediated by single genes influence ice growth within and between tissues. The structural properties of cell walls in the mutants examined will be characterised with respect to their mechanical strength and porosity, via a confocal micro-extensometer as well as by a fluorescence quenching method relying on the ability of less porous cell walls to exclude large molecules. Under all conditions, the sensitivity of wild type and mutant plant tissues to freezing and thawing will be assessed by measuring ion conductivity in electrolyte leakage assays. The ability of plants to survive freezing events will be monitored through visual assessment of re-growth after the freezing event and subsequent return to normal temperatures.

As a whole, this ambitious programme of work will enable the rational design of genetic modifications specifically tailored to enhance freezing tolerance in plants, building on the novel microscopic insight that we will deliver by our complementary methodologies, bringing together experiments and simulations.