A convenient and simple system for in vivo 13CO2 -labelling in plant leaves: Enhancing accessibility for tracer studies in photosynthesis

Tracer experiments using 13CO2 is a valuable tool for studying the metabolic flux of carbon during photosynthesis, providing precise insights into carbon assimilation and distribution within plant systems. However, the complexity of designing and setting up a 13CO2 delivery system is a significant factor limiting the use of tracer experiments in many research laboratories. In the present work, we report a simple 13CO2 delivery system that can be used in conjunction with an Infrared gas analyser (IRGA) for short duration in vivo 13C labelling in plant leaves. Briefly, 13CO2 is released in an external airtight container upon acidification of NaH13CO3 and subsequently introduced into the leaf chamber of an IRGA. A defined concentration of 13CO2 ({+/-} 60 ppm for about 2 minutes) can be generated by using an appropriate volume and concentration of NaH13CO3 and HCl. The functionality of the 13CO2 labelling system was assessed by feeding 13CO2 to the tobacco leaf for two different time intervals (30 secs and 2 min) under controlled environment conditions. The amino acids fractions from both labelled and unlabelled control plants were analysed using liquid chromatography-mass spectrometry (LC-MS) followed by determination of changes in 13C enrichment. The results showed that after 30 seconds, only two amino acids, serine and alanine, exhibited a significant increase in 13C enrichment, with values of 8.9% and 8.3%, respectively, compared to the unlabelled control. In contrast, after 2 minutes, a significant increase in 13C enrichment was observed in serine (24.1%), glycine (13.8%), aspartate (13.5%), and alanine (25.9%) as compared to the unlabelled control. The results, therefore, confirmed the workability of the system for feeding 13CO2 to a plant leaf in a time dependent manner. In addition to its simpler configuration and better control over environmental conditions, the major advantage of this system is its portability, allowing users to conduct 13CO2 experiments on plants growing in their natural habitat while minimizing physiological and metabolic alterations.