Frost’s chilling impacts on wheat yields have long fascinated Dr Rudy Dolferus.

At his CSIRO Agriculture & Food laboratories in Canberra, Dr Dolferus recently translated years of meticulous biological sleuthing into the ability to reliably detect cold-tolerant wheat lines, using newly developed metabolic markers.

The breakthrough markets are now being used to screen for better cold tolerance in diverse wheat populations.

The most reliable and specific among the new markers detect differences in the membrane fluidity of plant cells following exposure to cold.

Dr Dolferus explains that membrane fluidity changes rapidly after a single exposure to cold in a response that is highly specific for this stress. The change also persists following the stress event, making the markers suited to field trials where delays may occur between collecting flag leaves and testing them.

In cold tolerant lines, the trait is meditated by unsaturated lipids that allow cell membranes to remain fluid, whereas sensitive lines have membranes that Dr Dolferus compares to butter:

“When you put butter in the fridge it goes rock-solid,” he says.

“That also happens with membranes in plants.

“Without the unsaturated lipids, the membrane loses fluidity and, as it hardens, it loses functionality and can be damaged by frost quite easily.”

The development of metabolic markers for membrane fluidity is an important milestone in frost research.

Work is now underway to validate and scale-up the use of the metabolic markers to screen for cold tolerance in field trials.

Also underway are efforts to reveal the genetic underpinnings to the membrane fluidity trait in order to develop DNA markers for cold tolerance for use by wheat breeders.

The foundations

The marker development work hinges on Dr Dolferus’ prior discovery that yield losses from cold exposure in wheat occur primarily during flowering and are due to the loss of pollen fertility.

While that trait is useful, Dr Dolferus found that pollen sterility can be induced by other environmental stress, including drought.

He then developed ‘controlled environment (CE) phenotyping’ in order to grow wheat in a controlled environment that limits stress exposure specifically to cold and tests for pollen fertility and spike grain number.

The accuracy of this phenotyping method was demonstrated when it accurately preproduced the same cold-tolerance rankings of wheat lines obtained by the National Frost Initiative in field trials.

With this phenotyping method came the ability to look for biological differences between cold-tolerant and sensitive wheat lines, as typified by Young and Wyalkatchem respectively.

Initially this involved screening several mapping populations to identify DNA markers linked to cold tolerance.

When the resulting quantitative trait loci (QTL) data proved somewhat fuzzy, Dr Dolferus turned his attention to profiling metabolic differences between tolerant and sensitive lines.

Metabolic markers

The marker breakthrough was achieved using ‘metabolomics technology’, which profiled the entire complement of metabolites in leaves during acclimation to cold in Young and Wyalkatchem.

Among the observed differences are metabolites associated with osmotic protection to cold stress and differences in water loss response: Young accumulates mannitol and citrulline, which are osmotic protectants, while the contrasting accumulation in Wyalkatchem of sucrose and ABA is correlated with its sensitivity to chilling.

Of all the differences detected, however, it was lipid metabolites that most excited Dr Dolferus.

“The most significant metabolites are the unsaturated lipids that affect membrane fluidity because they are very specific to cold stress,” he says.

Now, the membrane fluidity markers are being validated in field trials of cold-tolerant wheat lines previously selected using CE phenotyping.

This proof-of-concept work got underway in 2019 at CSIRO’s Ginnindera Experiment Station near Canberra.

Field trials will also be carried out in 2020 to test metabolic markers to screen entire mapping populations.

Validation studies will also encompass field trials undertaken by Dr Ben Biddulph at the Department of Primary Industries and Regional Development (DPIRD) in Western Australia.

Mapping back to the genome

Moving forward, the metabolic markers permit the wheat genome to be re-interrogated, this time for the genetic underpinning of a highly cold-specific trait.

He is taking a holistic route based on a clever marriage of metabolomics, genomics based on three mapping populations and ‘transcriptomics’ (gene expression) technology.

To Dr Dolferus, data from these technologies are pieces of a jigsaw puzzle: QTL data point to segments of the gargantuan wheat genome to focus on and transcriptomics identifies genes whose expression changes with cold stress.

The frame in which to assemble the pieces correctly is provided by the wheat genome sequence.

Already, transcriptomics has been used to profile cold acclimation differences in Wyalkatchem and Young leaf tissue, finding that spike development under chilling conditions is controlled by a balance of two hormones: ABA and auxin.

“The sensitive Wyalkatchem line induces a stress response with clear involvement of ABA and ethylene responses while the tolerant line Young mainly showed an auxin-mediated growth reaction,” Dr Dolferus says.

Downstream of these key regulatory events, he can see genes expressed that are related to the membrane of fluidity trait and lipid metabolism.

By aligning all three data sets, he is working his way towards DNA markers that are specific for cold tolerance and can be used in breeding programs to make further genetic gain. This amounts to new capability in an otherwise difficult area of research.

“Chilling and frost tolerance is a challenging stress that has proven difficult to study under field contributions due to the variability of the field environment and unpredictability of frost events.

“With CE phenotyping and the metabolic work, we attempted to find a way around the impasse.

“We hope that from the metabolite work, we can move to DNA markers because they are easier and possibly cheaper for breeders to work with.”

The project involves a dedicated CSIRO team that includes GRDC post-doctoral fellow Dr Olive Onyemaobi and experimental scientists Xiaomei Wallace and Sue Kleven, collaboratively with Ute Roessner and Bo Eng Cheong (University of Melbourne), Matthew Hayden and Kerrie Forrest (La Trobe University) and AGRF.

More information: Rudy Dolferus, rudy.dolferus@csiro.au