Due to the difficulty of separating net CO2 and O2 fluxes from gross fluxes, photosynthesis has primarily been studied in a bubble, essentially ignoring interactions with respiration and even photorespiration. Much has been learned from the “bubble” approach, but efforts to use these data to predict basic things such as growth and carbon sequestration have largely been unsuccessful. Stable isotopes provide a powerful, and in my case, non-destructive approach for probing in vivo metabolic function. The benefit of my tunable diode laser – infra red gas analyzer (TDL-IRGA ) approach is that it has greatly simplified the methodology for isotopic gas exchange in addition to increasing the frequency of data collection (Barbour et al. 2007).

1. • Photorespiratory metabolism: It is well known that when you measure gas exchange immediately after putting a high-light adapted leaf in the dark, you can measure two pulses of CO2 from the leaf before steady state respiration is achieved. The first pulse, within 30s, is associated with photorespiration and the second, around 5-15 minutes, is called light-enhanced dark respiration (LEDR). The magnitude of this burst is indicative of the amount of photorespiration while the isotopic signature reflects a combination of enzymatic fractionation and the isotopic composition of the substrate for the released CO2. Photorespiratory metabolism was thought to be well understood, but in the last couple years, mutants of Arabidopsis have been used to suggest a new cytosolic component of the photorespiratory pathway and an undescribed portion of the pathway that can alter the ratio of O2 consumption to CO2 production. We are using the isotopic signature of the photorespiratory burst and other techniques to track and describe the activity of the new components of the photorespiratory pathway.
   

1. • Light enhanced dark respiration: We have also examined the isotopic composition of LEDR, which has led to my discovery (and subsequent NSF grant) that phospho-enolpyruvate carboxylase (PEPc) activity in C3 plants is the source of the malate being decarboxylated during the LEDR response (Barbour 2007, Barbour and Hanson 2009).  Quantifying LEDR may provide a new in vivo approach to assessing PEPc activity, and this is important because recent papers have questioned the accuracy of assaying leaf extracts for PEPc activity. Since LEDR is believed to be caused by an imbalance between photosynthesis and respiration, LEDR can be used as an in vivo diagnostic tool to identify the conditions that generate metabolic imbalances. There is a great deal of uncertainty regarding the regulation of respiration in a leaf during the day and LEDR and related transient fluxes may be useful for discovering large changes in daytime respiratory activity.
   

1. • Other topics: My work on photorespiration and LEDR just touches the surface of what I plan to achieve using high frequency, semi-automated, measurements of leaf isotope and trace gas exchange that are now practical using my TDL system. Essentially, any pathway that generates or consumes a gas (e.g. CO2 or hydrocarbons) can be monitored and perturbed to study metabolism in vivo. For example, we have acquired an N2O laser to study nitrogen metabolism of endophytic fungi.
   

1. • New directions in imaging of plant function: In spring 2010, UNM opened a cyclotron capable of generating 11CO2 and 15O2, along with other radioisotopes. These positron emitters allow in vivo, real-time, non-destructive imaging of plant function (including methods to view root function while plants are still potted) due to their high energy and short half-life.  They also provide a method for labeling metabolites that is 100 million times more sensitive than 14C, though analyses must be rapid. A pioneering US scientist in the use of positron emitters for studying plant function, Dr. Richard Ferrieri (Brookhaven National Labs), visited my lab and we will soon begin collecting  data in this area.