Photosynthetic Characteristics of C3-C4 Intermediate Flaveria

Four species of the genus Flaveria, namely F. anomala, F. liaris, F. pubescens, and F. ramosissima, were identified as intermediate C3-C4 plants based on leaf anatomy, photosynthetic CO2 compensation point, 02 inhibition of photosynthesis, and activities of C4 enzymes. F. anomala and F. rawosissina exhibit a distinct Kranz-like leaf anatomy, similar to that of the C4 species F. trinervia while the other C3-C4 intermediate Flaveria species possess a less differentiated Kranz-like leaf anatomy. Photosynthetic CO2 compensation points of these intermediates at 30°C were very low relative to those of C3 plants, ranging from 7 to 14 microliters per liter. In contrast to C3 plants, net photosynthesis by the intermediates was not sensitive to 02 concentrations below 5% and decreased relatively slowly with increasing 02 concentration. Under similar conditions, the percentage inhibition of photosynthesis by 21% 02 varied from 20% to 25% in the intermediates compared with 28% in Lycopersicon esculentW, a typical C3 species. The inhibition of carboxylation efficiency by 21% 02 varied from 17% for F. ramosissima to 46% for F. anomala and were intermediate between the C4 (2% for F. trinervia) and C3 (53% for L. escudentun) values. The intermediate Flaveria species, especially F. ramosissima, have substantial activities of the C4 enzymes, phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, NADP-malic enzyme, and NADP-malate dehydrogenase, indicating potential for C4 photosynthesis. It appears that these Flaveria species may be true biochemical C3-C4 intermediates. All available evidence suggests that C4 plants have evolved from ancestors possessing the C3 pathway of photosynthesis and this has occurred independently many times in taxonomically diverse groups (3, 21). At present, the precise evolutionary transition, at the anatomical, physiological, and biochemical levels, from a C3 to a C4 plant is not clear. It is generally believed that studies of C3-C4 intermediate species might provide insight into the evolution of C4 photosynthesis. In addition, since most of the world's important crops are C3 plants, there has been considerable interest in improving their productivity by screening for mutants with reduced rates of photorespiration or by incorporating C4 characteristics into C3 plants (3, 19, 20). Thus, the search for naturally ' This research was supported in part by a grant from Washington State University Graduate School and gift funds from Monsanto Agricultural Products Company, St. Louis, MO. 'Present address: Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder, CO 80309. occurring CrC4 intermediates and the study of their anatomical, physiological, and biochemical characteristics are of importance to both theoretical and applied disciplines of plant biology. Since 1975, naturally occurring species intermediate between C3 and C4 plants have been found in the genera Panicum (6), Mollugo (22), and Moricandia (2). The intermediate nature of these species is based on Kranz-like leaf anatomy, low photosynthetic CO2 compensation point, and a reduced level ofphotorespiration. Most recently, two species of Flaveria (F. anomala and F. pubescens) have also been identified as C3-C4 intermediates based on low CO2 compensation point at 21% 02 (1). In the present study, we examined the leaf anatomy, photosynthetic response to C02, sensitivity of net photosynthesis to 02, and activity ofkey enzymes in C3 and C4 photosynthesis of several species of Flaveria, a genus apparently having C3, C4 and C3-C4 intermediate species (1, 21). MATERIALS AND METHODS Plant Material and Growth Conditions. Plants of Flaveria anomala Robinson, F linearis Lag., F pubescens Rydb., F. ramosissima Klatt, F trinervia Mohr, and Lycopersicon esculentum Mill (C3) were obtained by germinating the seeds on top of fime soil in peat pots which were placed in trays and watered by absorption or on moist filter paper in Petri dishes. After seedlings reached I to 3 cm in height, they were transplanted into larger pots filled with a mixture of peat and sand, and maintained in a growth chamber under a daily regime of 14 h of light at 27°C and 8 h of darkness at 22°C. Light was provided by a combination of fluorescent and incandescent lamps, giving a photosynthetic photon flux density of 80 nE/cm2 .s at plant height. Plants were watered with dilute nutrient solution three times a week. Young and newly expanded leaves from 2to 4-month-old plants were used for experiments. Leaf Anatomy. Samples (approximately 4 mm2) of tissue were cut from young, fully expanded leaves and vacuum infiltrated with cold fixative (2% depolymerized paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate, pH 7.0). After 2 h, the tissue was washed with buffer, dehydrated in a graded ethanol series, and embedded in 'L.R. White' embedding medium according to the supplier's instructions (Polysciences, Inc.). Sections were cut at 2.5-,pm thickness and stained with the periodic acid-Schiff reaction for insoluble carbohydrate (12). Gas Exchange Measurements. CO2 and water vapor exchange of intact individual leaves were measured with an open IR gas analysis system as described in a previous paper (18). Leaf temperatures were maintained at 30 + 0.5°C using a peltier-cooled heat exchanger. A photosynthetic photon flux density of 180 nE/ 2 cm . s within the leaf chamber was provided by a combination of