Tune-up for key wheat evolution enzyme

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Engineering more efficient photosynthesis in cereal crops is a key strategy among pre-breeders globally to lift yields

Photo of Emmanuel Young, Yu Zhou, Vinuri Wijedasa, Associate Professor Spencer Whitney, Tim Rhodes, Sally Buck and Ding Yuan
(From left) Emmanuel Young (PhD student), Yu Zhou (PhD student), Vinuri Wijedasa (research officer), Associate Professor Spencer Whitney, Tim Rhodes (PhD student), Sally Buck (PhD student) and Ding Yuan (PhD student).
PHOTO: Natalia Bateman Vargas

The rise in importance of photosynthesis as a yield trait has elevated the importance of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), the rate-limiting enzyme that fixes atmospheric carbon dioxide into energy-rich sugars.

In many ways, Rubisco is fundamental to life on Earth. Understandably, the enzyme is complex, composed of 16 carefully arranged protein subunits that are encoded by two sets of genes. Assembling these subunits correctly, however, does not occur spontaneously, but requires a battalion of auxiliary proteins – called chaperones – to fold, align and arrange the subunits correctly.

The slightest disruption to the assembly process – including from genetic variation that normally drives natural selection – can easily result in inactive Rubisco forms.

This intolerance to changes in the Rubisco gene-chaperone system is so extreme that scientists now believe it has stunted its evolution and hamstrung photosynthesis with several inefficient anachronisms.

One example in C3 plants (which include wheat) is the inability of Rubisco to distinguish between carbon dioxide and oxygen, even though using oxygen results in a futile photorespiration reaction rather than sugar-producing photosynthesis.

The problem is that Rubisco’s structure became fixed at a time when there was little oxygen in the atmosphere and the enzyme did not require an in-built specificity for carbon dioxide.

As oxygen levels rose, Rubisco was unable to acquire that specificity, leaving plants with no option but to ramp up Rubisco levels or concentrate carbon dioxide levels inside the leaf to compensate for the loss of efficiency. Due to this ‘ramping up’, Rubisco is today considered the most abundant enzyme on the planet.

Pre-breeders worldwide are itching to apply selective pressure on Rubisco to develop more efficient variants that can be used to drive crop yield gains. Capitalising on this potential, however, hits the same problem faced by evolution: Rubisco variants that promise improved efficiencies generally fail to fold and assemble correctly. Instead, gains generally tend to be due to genetic changes (either evolutionary or through selective breeding) that bump up the amount of Rubisco made by the plant.

In 2018, after decades of trying, researchers have finally developed new tools able to give Rubisco a more definitive molecular tune-up.

Central to these advances is research undertaken at the Australian National University (ANU) led by Associate Professor Spencer Whitney.

“Rubisco has been a tough nut to crack,” he says. “I have been studying it since 1990. It’s an academic obsession.

“Based on our recent research findings with non-plant forms of Rubisco, I think an increase of 10 to 30 per cent in wheat Rubisco efficiency is feasible in the near term with minimum fuss. And we may get even more.”

Revving up Rubisco activity

The approach taken by Associate Professor Whitney dates back a decade when he compared Rubisco enzyme performance in a collection of Australian wheat varieties and found something peculiar.

Infographic of Plant Rubisco
FIGURE 1 Plant Rubisco is a large protein complex comprising a core of four pairs of large (L) subunits (green shading) that house 8 catalytic sites in total. The L8 core is capped either end by 4 small subunits (grey shading) that stabilise the complex.

As expected, given Rubisco’s stunted evolutionary history, no major differences were observed in the enzyme’s activity from different wheat species … at least, not when it was measured at 25°C, which is the standard for laboratory testing.

Wheat, however, is not grown at a standard temperature. When the scientists looked across a range of temperature profiles, subtle variations in wheat Rubisco activity were detected, with some being more efficient at higher or lower temperature conditions.

Associate Professor Whitney saw in this trait variation a rare and unexpected opportunity to optimise wheat productivity by building on this variation to optimise Rubisco function under differing environmental conditions.

“Only now are the sophisticated experimental tools available to pinpoint the underpinning secrets to this diversity and make further improvements,” he says.

Normally, the genetics that underlie trait variation can be identified, validated and used to drive gains in a breeding program. That was not the case with wheat Rubisco. As Associate Professor Whitney explains: “When we compared Rubisco with different temperature kinetics, we did see differences in both sets of genes that encode the Rubisco subunits. However, we could not implicate them with the changes responsible for the observed functional differences in Rubisco.”

In other words, it was not possible to identify precisely what breeders should select for to improve the performance of wheat cultivars. Now, that limitation is being lifted with the development of a new and rather extraordinary technique.

Rather than dealing with the complication of working with plants to narrow down the causes of the temperature trait, Associate Professor Whitney is using bacterial cells as a proxy. Given the ease of introducing genes into bacteria, the new method involves expressing Rubisco genes sourced from Australian wheat varieties in the bacteria alongside genes encoding the most important chaperones.

This synthetic approach allows bacteria to make plant Rubisco. “By being able to express wheat Rubisco in E. coli we can unequivocally implicate genetic changes that cause the observed functional differences in Rubisco’s temperature kinetics,” Associate Professor Whitney says. “We are quite excited about that.”

This validation is the precursor to using the bacterial system as a form of laboratory-based evolution in which mutated versions of the Rubisco-chaperone system can be quickly screened to obtain a 10 to 30 per cent gain in Rubisco activity for use by the grains industry.

Accelerated evolution

To accelerate Rubisco evolution in the laboratory simply requires the inclusion of one additional gene into the bacteria that makes the cell’s survival dependent on Rubisco activity.

The additional gene diverts the bacteria’s own sugar metabolism to make the starting substance Rubisco uses to begin sugar synthesis. The substance, called ribulose-1,5-bisphosphate (RuBP) is toxic to bacteria, putting the cells under extreme selection pressure to remove it or die. By supplying active Rubisco to the cells, the toxic RuBP is converted into useable sugars and the bacteria grow.

More efficient forms of Rubisco also stand out because they allow the bacteria to grow at faster rates.

The genetic evolution process in the laboratory is a rapid and simple process. It involves amplifying the Rubisco genes to create a library of hundreds of thousands of randomly mutated variants that introduce one to five amino acid changes in the Rubisco sequence. These are then screened in the bacteria to weed out non-functional forms and highlight the Rubisco variants with improved efficiency.

“The exciting thing about this system is that it allows both Rubisco and its chaperones to be altered and then tested for higher-functioning variants,” Associate Professor Whitney says. “But it involves a balancing act between being able to change Rubisco and ensuring it can fold in the target plant species.”

At ANU, the targeted evolution of Rubisco was first trialled using simpler forms of Rubisco sourced from microorganisms.

In 2018, however, a graduate from the Associate Professor’s laboratory, Dr Robert Wilson, helped establish the technique using plant Rubisco for the first time. The work was undertaken in the laboratory of Dr Manajit Hayer-Hartl at the Max Planck Institute of Biochemistry in Germany.

The jury is still out on how much gain can be made to Rubisco efficiency through accelerated evolution. The method does run the risk of merely detecting changes to the assembly process (and, therefore, the amount of Rubisco produced) rather than improving the enzyme’s actual catalytic activity. Nonetheless, researchers agree that a new chapter has opened on Rubisco research.

Associate Professor Whitney is ideally situated to exploit breakthroughs to advance the productivity of the Australian wheat industry. He is capitalising on that advantage by modelling the likely impact on wheat productivity of improved Rubisco activity in collaboration with Dr Alex Woo and Professor Graham Hammer of the University of Queensland through the Australian Research Council Centre of Excellence for Translational Photosynthesis.

“As part of the APSIM model they have climate data which is relevant for estimating productivity impacts from more efficient forms of Rubisco at different locations,” Associate Professor Whitney says. “This will be pivotal in selecting changes to Rubisco that will be useful to wheat breeders and growers.”

More information

Spencer Whitney
spencer.whitney@anu.edu.au