Identifying the best test for the job


Crop plants growing in an experimental glasshouse facility

Lancaster scientists are helping to speed up world-wide research into the enzyme that controls photosynthesis, supporting efforts to boost crop production.

Researchers around the world are working to increase the productivity of staple crops by finding ways to improve photosynthesis – the process by which plants turn sunlight and carbon dioxide (CO2) into plant matter.

A main focus of research is rubisco, the enzyme that controls photosynthesis, because while it is the most plentiful enzyme in the world, it is inefficient and works slowly.

“If we can identify plants with faster rubisco activity then we can find ways to speed up the process of fixing CO2,” explains Dr Cristina Sales, lead author of a new paper that compares the cost, ease of use and precision of four methods of testing the efficiency of rubisco.

“Methods are very important: if you don’t have consistency of methods between researchers you can’t compare the results properly,” explains Cristina. Her paper is the first of a new type of ‘technical innovation’ paper published in the Journal of Experimental Botany, which explores advances in research techniques.

Cristina came up with the idea for the study when she was working as a post-doctoral researcher with the Photosynthesis Group at the Lancaster Environment Centre. She was testing for rubisco activity in wheat, using a radioactive test that uses 14C, which can only be done in a few laboratories around the world, with costly equipment and appropriate safety precautions.

“It’s a very precise test, but it is also expensive and more laborious, so you can’t do a lot of tests at once. This was a problem for me because I had more than 300 wheat plants I needed to compare.”

She decided to find out how the radioactive 14C test performed in comparison to three alternative microplate-based tests, which are carried out with a spectrophotometer, a routine piece of equipment available in many laboratories.

Cristina and her colleagues discovered that the microplate tests were all less accurate than the radioactive 14C test, measuring up to 30% lower levels of rubisco activity. However, they had advantages: they were cheaper, required just a quarter of the sample size of the 14C test; and could test a lot of samples at the same time. Even better, the cheapest test was also the one that correlated most closely with the 14C test.

What their study does, Cristina argues, is compare these tests for the first time and so help scientists choose the best test for the job.

“While the microplate method is not as precise, it is suitable for making comparisons between large numbers of plants to assess variation in rubisco. In some cases, however, it will be important to be precise and accurate in determining exact values of rubisco activity, then you should use the 14C-radioactive test.”

The researchers came up with protocols for each test, step by step instructions on how to carry it out, what equipment and chemicals they used and in what quantities. These protocols are published on an open access website – protocols.io - alongside the journal article, so that other researchers can do similar experiments using exactly the same methods.

“In biological science, a robust and reproducible protocol is essential if you are to rely on the results,” says Cristina, who has recently taken up a post at the University of Cambridge.

Dr Elizabete Carmo-Silva, a co-author from Lancaster’s Photosynthesis Group, said: “We are very proud of this work, we hope it will be useful to the community and help us make faster progress towards identifying promising targets to optimise crop production, and contribute to future food security, agricultural sustainability and climate resilience.”

Read the paper - Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays.

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