C(4) photosynthesis is one of the most convergent evolutionary phenomena in the biological world, with at least 66 independent origins. Evidence from these lineages consistently indicates that the C(4) pathway is the end result of a series of evolutionary modifications to recover photorespired CO(2) in environments where RuBisCO oxygenation is high. Phylogenetically informed research indicates that the repositioning of mitochondria in the bundle sheath is one of the earliest steps in C(4) evolution, as it may establish a single-celled mechanism to scavenge photorespired CO(2) produced in the bundle sheath cells. Elaboration of this mechanism leads to the two-celled photorespiratory concentration mechanism known as C(2) photosynthesis (commonly observed in C(3)-C(4) intermediate species) and then to C(4) photosynthesis following the upregulation of a C(4) metabolic cycle.

Plants using the C 4 photosynthetic pathway have greater water use efficiency (WUE) than C 3 plants of similar ecological function. Consequently, for equivalent rates of photosynthesis in identical climates, C 4 plants do not need to acquire and transport as much water as C 3 species. Because the structure of xylem tissue reflects hydraulic demand by the leaf canopy, a reduction in water transport requirements due to C 4 photosynthesis should affect the evolution of xylem characteristics in C 4 plants. In a comparison of stem hydraulic conductivity and vascular anatomy between eight C 3 and eight C 4 herbaceous species, C 4 plants had lower hydraulic conductivity per unit leaf area ( K L ) than C 3 species of similar life form. When averages from all the species were pooled together, the mean K L for the C 4 species was 1.60, which was only one-third of the mean K L of 4.65determined for the C 3 species. The differences in K L between C 3 and C 4 species corresponded to the two-to three-fold differences in WUE observed between C 3 and C 4 plants. In the C 4 species from arid regions, the difference in K L was associated with a lower hydraulic conductivity per xylem area, smaller and shorter vessels, and less vulnerable xylem to cavitation, indicating the C 4 species had evolved safer xylem than the C 3 species. In the plants from resource-rich areas, such as the C 4 weed Amaranthus retroflexus , hydraulic conductivity per xylem area and xylem anatomy were similar to that of the C 3 species, but the C 4 plants had greater leaf area per xylem area. The results indicate the WUE advantage of C 4 photosynthesis allows for greater flexibility in hydraulic design and potential fitness. In resource-rich environments in which competition is high, an existing hydraulic design can support greater leaf area, allowing for higher carbon gain, growth and competitive potential. In arid regions, C 4 plants evolved safer xylem, which can increase survival and performance during drought events.

Higher water use efficiency (WUE) in C4 plants may allow for greater xylem safety because transpiration rates are reduced. To evaluate this hypothesis, stem hydraulics and anatomy were compared in 16 C3, C3-C4 intermediate, C4-like and C4 species in the genus Flaveria. The C3 species had the highest leaf-specific conductivity (KL) compared with intermediate and C4 species, with the perennial C4 and C4-like species having the lowest KL values. Xylem-specific conductivity (KS) was generally highest in the C3 species and lower in intermediate and C4 species. Xylem vessels were shorter, narrower and more frequent in C3-C4 intermediate, C4-like and C4 species compared with C3 species. WUE values were approximately double in the C4-like and C4 species relative to the C3-C4 and C3 species. C4-like photosynthesis arose independently at least twice in Flaveria, and the trends in WUE and KL were consistent in both lineages. These correlated changes in WUE and KL indicate WUE increase promoted KL decline during C4 evolution; however, any involvement of WUE comes late in the evolutionary sequence. C3-C4 species exhibited reduced KL but little change in WUE compared to C3 species, indicating that some reduction in hydraulic efficiency preceded increases in WUE.

Xylem structure and function is proposed to reflect an evolutionary balance between demands for efficient movement of water to the leaf canopy and resistance to cavitation during high xylem tension. Water use efficiency (WUE) affects this balance by altering the water cost of photosynthesis. Therefore species of greater WUE, such as C(4) plants, should have altered xylem properties. To evaluate this hypothesis, we assessed the hydraulic and anatomical properties of 19 C(3) and C(4) woody species from arid regions of the American west and central Asia. Specific conductivity of stem xylem ( K(s) ) was 16%-98% lower in the C(4) than C(3) shrubs from the American west. In the Asian species, the C(3) Nitraria schoberi had similar and Halimodendron halodendron higher K(s) values compared with three C(4) species. Leaf specific conductivity ( K(L); hydraulic conductivity per leaf area) was 60%-98% lower in the C(4) than C(3) species, demonstrating that the presence of the C(4) pathway alters the relationship between leaf area and the ability of the xylem to transport water. C(4) species produced similar or smaller vessels than the C(3) shrubs except in Calligonum, and most C(4) shrubs exhibited higher wood densities than the C(3) species. Together, smaller conduit size and higher wood density indicate that in most cases, the C(4) shrubs exploited higher WUE by altering xylem structure to enhance safety from cavitation. In a minority of cases, the C(4) shrubs maintained similar xylem properties but enhanced the canopy area per branch. By establishing a link between C(4) photosynthesis and xylem structure, this study indicates that other phenomena that affect WUE, such as atmospheric CO(2) variation, may also affect the evolution of wood structure and function.

The whole-plant CO 2 compensation point (C plant ) is the minimum atmospheric CO 2 level required for sustained growth. The minimum CO 2 requirement for growth is critical to understanding biosphere feedbacks on the carbon cycle during low CO 2 episodes; however, actual values of C plant remain difficult to calculate. Here, we have estimated C plant in tobacco by measuring the relative leaf expansion rate at several low levels of atmospheric CO 2 , and then extrapolating the leaf growth vs. CO 2 response to estimate CO 2 levels where no growth occurs. Plants were grown under three temperature treatments, 19/15, 25/20 and 30/25 1C day/night, and at CO 2 levels of 100, 150, 190 and 270 lmol CO 2 mol À1 air. Biomass declined with growth CO 2 such that C plant was estimated to be approximately 65 lmol mol À1 for plants grown at 19/15 and 30/25 1C. In the first 19 days after germination, plants grown at 100 lmol mol À1 had low growth rates, such that most remained as tiny seedlings (canopy size o1 cm 2 ). Most seedlings grown at 150 lmol mol À1 and 30/25 1C also failed to grow beyond the small seedling size by day 19. Plants in all other treatments grew beyond the small seedling size within 3 weeks of planting. Given sufficient time (16 weeks after planting) plants at 100 lmol mol À1 eventually reached a robust size and produced an abundance of viable seed. Photosynthetic acclimation did not increase Rubisco content at low CO 2 . Instead, Rubisco levels were unchanged except at the 100 and 150 lmol mol À1 where they declined. Chlorophyll content and leaf weight per area declined in the same proportion as Rubisco, indicating that leaves became less expensive to produce. From these results, we conclude that the effects of very low CO 2 are most severe during seedling establishment, in large part because CO 2 deficiency slows the emergence and expansion of new leaves. Once sufficient leaf area is produced, plants enter the exponential growth phase and acquire sufficient carbon to complete their life cycle, even under warm conditions (30/25 1C) and CO 2 levels as low as 100 lmol mol À1 .