Understanding how frost-tolerant wheat varieties cope with the cold has taken a step forward with the identification of a biochemical pathway unique to frost-tolerant varieties.

The finding by a GRDC-invested CSIRO project opens the door to new possibilities for phenotyping.

Lead researcher Dr Rudy Dolferus explains that frost tolerance relates to the composition of lipids in the cell membranes: “Cell membranes are made of lipids, which are types of fats. At different temperatures, different lipids behave differently. Think of it like butter – if you put butter in the fridge, it goes solid, but at room temperature it is softer. If you add vegetable oils to the butter and then put it in the fridge, it won’t set as hard.

“This is basically what is going on in the cell membranes. The composition and proportions of different fats and lipids in the cell membrane are very important when the temperatures get cold.”

Low temperatures, high stakes

It is estimated that the Australian wheat industry loses $360 million annually due to direct and indirect losses from chilling and frost. Cold spells during flowering in early spring can lead to 30 to 50 per cent yield losses, while severe frost events can lead to even-more-costly losses. Low, but above freezing, temperatures (chilling) occur more frequently in the field than actual frost events and the grain yield losses (sterility) associated with low temperatures do not receive as much attention compared to more obvious tissue damage caused by frosts.

“Despite the seriousness of the damage caused by chilling and frost to wheat crops, we still know little about how these stresses affect the plant,” Dr Dolferus says. “Lack of knowledge about the effect of low above-zero and below-zero temperatures on the physiology of the wheat plant further complicate accurate field phenotyping.”

Dr Dolferus and his colleagues used temperature-controlled cabinets to investigate cold tolerance mechanisms in the frost-tolerant variety Young and the frost-sensitive variety Wyalkatchem.

“The variability and unpredictability of chilling and frost events in the field makes it one of the hardest stresses to tackle via field experimentation,” Dr Dolferus says.

“The chilling period occurring before frost events is critically important for plants. This is when they try to adapt and establish an acclimation response to protect themselves against future frost damage.”

The project aims to find markers that would allow researchers to discriminate genetic variation in cold acclimation and improve phenotyping for frost tolerance.

Wheat plants in a CSIRO frost trial. Researchers are trying to understand the genetic basis of how plants lose water when they are affected by frost. Photo: Rudy Dolferus

“We need to be able to determine what discriminates frost-tolerant from sensitive wheat lines and how we can identify the genetic variation,” Dr Dolferus says.

“Understanding what traits could be targeted to improve chilling and frost tolerance will enable breeders to target specific traits.”

Cell membranes crucial

In an earlier part of the project, the researchers profiled the biochemical changes in the different wheat lines, determining that the tolerant line increases the concentration of unsaturated versus saturated lipids in the cell membranes at lower temperatures.

“We now know what is happening biochemically. We have done that first step on measuring the biochemical properties – the lipids themselves. But also, running parallel to that work, at the genome level we have used gene expression profiling to identify the genes involved in that process.

“Now that we know that the lipids are different, we can look at the genes in that process. We can use lipid profiling to identify the genetic loci. Once they are identified we can identify molecular markers that will make it easier and cheaper to screen for frost tolerance.”

Plant cells, like all living cells, have a cell membrane. But plant cells also have a rigid cell wall, which is made of cellulose and gives plants their structural strength. Dr Dolferus says that while there may be other factors that contribute to frost tolerance, the lipid concentrations in membranes are important.

“All components to make the cell wall grow need to pass through the cell membrane,” Dr Dolferus explains. “When it gets cold, the cell membrane becomes solid. Maintaining membrane fluidity is important to maintain membrane functionality and to support cell wall synthesis. When membranes solidify at low temperatures, building blocks for strengthening the cell wall cannot pass and the whole tissue structure is weakened and damaged.

“When sensitive wheat lines are frosted, they lose water because the membrane is broken and the water leaks out. This is why frosted plants often look wilted … frost-sensitive plants lose water.”

The researchers are now trying to discover the genetic basis of that. What are the genes that switch on to change the lipid concentration in the cell membranes? Dr Dolferus says plant hormones are likely involved, as well as other downstream factors that regulate gene expression.

A wheat head that has been frosted. Research into frost is helping set up technology for breeding work. Photo: Rudy Dolferus

It is a complex equation to be unravelled: “There are several lipids involved in the adaptation to low temperatures. There’s not only one lipid but a spectrum of lipids that achieves improved membrane fluidity at low temperatures. And the genes involved could vary from one variety to another. There may be one expressed much higher and others expressed not as highly in a tolerant or sensitive line. There may be genes that are expressed in tolerant and not in sensitive lines.

“In the next year, we will identify which genes are involved and we will get a better idea about the regulatory mechanism and what genes are involved at that level. We will also proceed to identify the genetic loci responsible for the lipid composition changes we see. And then we can focus on identifying the genetic markers. Once we achieve that, it will be easier for the breeders.”

Dr Dolferus says that knowing which genes cause the changes in lipid concentrations that provide the frost tolerance will allow DNA markers to be associated with those loci. Those markers would help breeders make sure they did not lose that trait when breeding for other characteristics.

“Without identifiable markers it is down to pure luck whether we get the cold tolerance genes carrying through from one generation to the next,” he says. “If we use molecular tools to identify these genes it will be a lot easier.

“If you don’t know where the loci are, then you run the risk of breeding them out. For example, cold-tolerance genes could be located close to known flowering time or disease sensitivity loci. Selecting for certain alleles for these (or other) traits could accidentally get rid of cold-tolerance characteristics – a process called linkage drag. The work we are doing is pre-breeding. We are setting up the technologies for the breeding level. We are using the technologies and tools that breeders can use down the track. That will be the next stage.”

Dr Dolferus says the most important thing for all stressors is knowing how to phenotype. “What we have done now is find something that is objectively telling us this is what a cold-tolerant plant needs. Based on this knowledge we can now start to look for germplasm and genetic variation with a better lipid composition that will allow us to improve the threshold of cold tolerance even higher. We can look at germplasm lines that are better adapted to cold. This is what we can do once we can reliably work out the phenotype and how to phenotype. Once we know the markers we can do even more.”

He says all of the genes that are involved in tolerances are members of complex gene families. Members of these gene families can react to different stressors. This is why at the biochemical level, heat, cold and drought stressors can be similar but at the genetic level different genes may be involved.

More information: Dr Rudy Dolferus, 02 6246 5010, rudy.dolferus@csiro.au