Mesophyll conductance (g ) is an important factor limiting rates of C photosynthesis. However, its role in C photosynthesis is poorly understood because it has been historically difficult to estimate. We use two methods to derive the temperature responses of g in C species. The first (Δ O) combines measurements of gas exchange with models and measurements of O discrimination. The second method (in vitro V ) derives g by retrofitting models of C photosynthesis and C discrimination with gas exchange, kinetic constants and in vitro V measurements. The two methods produced similar g for Setaria viridis and Zea mays. Additionally, we present the first temperature response (10-40°C) of C g in S. viridis, Z. mays and Miscanthus × giganteus. Values for g at 25°C ranged from 2.90 to 7.85 μmol m s Pa . Our study demonstrated that: the two described methods are suitable to calculate g in C species; g values in C are similar to high-end values reported for C species; and g increases with temperature analogous to reports for C species and the response is species specific. These results improve our mechanistic understanding of C photosynthesis.

ORCID IDs: 0000-0003-4009-7700 (R.A.B.); 0000-0001-7184-5113 (A.G.).The photosynthetic assimilation of CO 2 in C 4 plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C 4 biochemical models of leaf CO 2 exchange in response to changes in CO 2 availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C 4 plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO 3 2 , and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C 4 plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO 3 2 limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C 3 species and the C 4 plant S. viridis.

Carbonic anhydrase (CA) catalyzes the first biochemical step of the carbon-concentrating mechanism of C4 plants, and in C4 monocots it has been suggested that CA activity is near limiting for photosynthesis. Here, we test this hypothesis through the characterization of transposon-induced mutant alleles of Ca1 and Ca2 in maize (Zea mays). These two isoforms account for more than 85% of the CA transcript pool. A significant change in isotopic discrimination is observed in mutant plants, which have as little as 3% of wild-type CA activity, but surprisingly, photosynthesis is not reduced under current or elevated CO2 partial pressure (pCO2). However, growth and rates of photosynthesis under subambient pCO2 are significantly impaired in the mutants. These findings suggest that, while CA is not limiting for C4 photosynthesis in maize at current pCO2, it likely maintains high rates of photosynthesis when CO2 availability is reduced. Current atmospheric CO2 levels now exceed 400 ppm (approximately 40.53 Pa) and contrast with the low-pCO2 conditions under which C4 plants expanded their range approximately 10 million years ago, when the global atmospheric CO2 was below 300 ppm (approximately 30.4 Pa). Thus, as CO2 levels continue to rise, selective pressures for high levels of CA may be limited to arid climates where stomatal closure reduces CO2 availability to the leaf.

Spring geophytes produce larger storage organs and present delayed leaf senescence under lower growth temperature. Bulb and leaf carbon metabolism were investigated in Erythronium americanum to identify some of the mechanisms that permit this improved growth at low temperature. Plants were grown under three day/night temperature regimes: 18/14 °C, 12/8 °C, and 8/6 °C. Starch accumulated more slowly in the bulb at lower temperatures probably due to the combination of lower net photosynthetic rate and activation of a ‘futile cycle’ of sucrose synthesis and degradation. Furthermore, bulb cell maturation was delayed at lower temperatures, potentially due to the delayed activation of sucrose synthase leading to a greater sink capacity. Faster starch accumulation and the smaller sink capacity that developed at higher temperatures led to early starch saturation of the bulb. Thereafter, soluble sugars started to accumulate in both leaf and bulb, most probably inducing decreases in fructose-1,6-bisphosphatase activity, triose-phosphate utilization in the leaf, and the induction of leaf senescence. Longer leaf life span and larger bulbs at lower temperature appear to be due to an improved equilibrium between carbon fixation capacity and sink strength, thereby allowing the plant to sustain growth for a longer period of time before feedback inhibition induces leaf senescence.