In the quest to increase crop productivity, scientists for the Realizing Increased Photosynthetic Efficiency (RIPE) project focused on Rubisco, one of the most abundant enzymes on Earth. Rubisco plays a vital role in photosynthesis but has several inefficiencies, which limit the rate of photosynthesis and present a barrier to crop productivity. In the recently published book chapter, the researchers from Lancaster University looked at how these obstacles can be overcome.

The chapter, Radiometric determination of Rubisco activation state and quantity in leaves, is part of the latest volume of Methods in Enzymology, and provides a protocol for using radiometric assays to measure Rubisco’s activation state and quantity in plant leaves. The text is intended as a robust, practical guide for newcomers to plant biochemistry who aim to work with Rubisco. It offers a detailed procedural outline that can be adapted to various plant species, making it a versatile resource for labs around the world.

“We have conducted previous studies using the absorbance of light to determine Rubisco activity, but they have been shown to underestimate the activity of Rubisco,” said Caty Ashton, lead author on the chapter and a senior biochemistry technician for RIPE at Lancaster. “The use of radiometric methods tend to give more reliable results due to the direct tracking of Rubisco’s activity, whereas alternative methods work by tracking secondary reactions.”

According to Ashton, using methods such as measuring light absorbance to determine Rubisco activity often fails to capture data as accurately as what can be captured from radiometric analysis. In this case, the radiometric method offers a more robust analysis, allowing researchers to observe Rubisco activity under various conditions.

“The activity of Rubisco changes really quickly in response to light intensity changes, so if samples are shaded whilst being collected, the results of the assay will be misleading,” said Ashton. “Fluctuations in light intensity can cause Rubisco activity levels to decline within seconds, so we like to use a specialized light rig sampling system designed to simulate consistent sunlight in a controlled chamber. However, as we discuss in the book chapter, careful sampling of plants can still allow consistent results.”

In the chapter, authors from the Carmo-Silva lab offer best practices for collecting samples. For example, exposing the leaf samples to a stable light and then freezing them in liquid nitrogen eliminates the variability caused by light changes occurring up to 45 minutes before sampling. This enables scientists to more accurately understand the activity of Rubisco and its response to variables other than light.

The research supporting this work is part of RIPE, an international research project that aims to increase global food production by developing food crops that turn the sun’s energy into food more efficiently with support from Gates Agricultural Innovations. Within RIPE, the Carmo-Silva group are focused on understanding the regulation of Rubisco activity, which involves exploring Rubisco’s interactions with regulatory proteins and other small molecules and ions found within the chloroplast of leaves. Understanding these mechanisms enables the group to establish targets that could allow enhanced photosynthetic rates in future research.

The team’s research has direct implications for crop improvement, particularly for crops like cowpea, a staple crop in many parts of the world and a focus of the RIPE project. By revealing how different environmental conditions affect Rubisco, the radiometric method could guide breeding programs and agricultural practices, helping farmers select crop varieties better suited to their environments.

Looking ahead, Ashton sees this work as a stepping-stone toward larger-scale applications such as guiding researchers in selecting which crop varieties are worth testing in the field. 

“I hope our book chapter can be used by other researchers to allow a greater understanding of Rubisco function in plants, which is one of the key targets for improving the efficiency of photosynthesis and, therefore, crop yields,” said Ashton.

Further reading

Ashton recommends the following as further reading for those interested in learning about other relevant studies done on Rubisco:

●     Amaral, J., Lobo, A. K., Carmo-Silva, E., & Orr, D. J. (2024). Purification of rubisco from leaves. Photosynthesis: Methods and Protocols (pp. 417–426). New York, NY: Springer US. Sales, C. R. G., Silva, A., & Carmo-Silva, E. (2020).

●     Protocols from Sales et al. (2020) Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays. Protocols.io. https://doi.org/10.17504/protocols.io.bf8djrs6.

●     Taylor, S. H., Gonzalez-Escobar, E., Page, R., Parry, M. A., Long, S. P., & Carmo-Silva, E. (2022). Faster than expected Rubisco deactivation in shade reduces cowpea photosynthetic potential in variable light conditions. Nature Plants, 8(2), 118-124.

 

This feature story was written by David Hong, RIPE’s science communication intern during the Fall 2024 semester