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- MATERIALS AND METHODS
Plants using the C4 photosynthetic pathway have greater water use efficiency (WUE) than C3 plants of similar ecological function. Consequently, for equivalent rates of photosynthesis in identical climates, C4 plants do not need to acquire and transport as much water as C3 species. Because the structure of xylem tissue reflects hydraulic demand by the leaf canopy, a reduction in water transport requirements due to C4 photosynthesis should affect the evolution of xylem characteristics in C4 plants. In a comparison of stem hydraulic conductivity and vascular anatomy between eight C3 and eight C4 herbaceous species, C4 plants had lower hydraulic conductivity per unit leaf area (KL) than C3 species of similar life form. When averages from all the species were pooled together, the mean KL for the C4 species was 1.60 × 10−4 kg m−1 s−1 MPa−1, which was only one-third of the mean KL of 4.65 × 10−4 kg m−1 s−1 MPa−1 determined for the C3 species. The differences in KL between C3 and C4 species corresponded to the two- to three-fold differences in WUE observed between C3 and C4 plants. In the C4 species from arid regions, the difference in KL was associated with a lower hydraulic conductivity per xylem area, smaller and shorter vessels, and less vulnerable xylem to cavitation, indicating the C4 species had evolved safer xylem than the C3 species. In the plants from resource-rich areas, such as the C4 weed Amaranthus retroflexus, hydraulic conductivity per xylem area and xylem anatomy were similar to that of the C3 species, but the C4 plants had greater leaf area per xylem area. The results indicate the WUE advantage of C4 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, C4 plants evolved safer xylem, which can increase survival and performance during drought events.
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- MATERIALS AND METHODS
Two opposing evolutionary selection pressures act upon xylem structure and function (Zimmermann 1983; Tyree, Davis & Cochard 1994). The benefit derived from enhanced photosynthesis selects for efficient xylem consisting of relatively long and wide vessels that rapidly supply water to transpiring leaves. Wide vessels enhance conducting efficiency because flow capacity increases with the fourth-power of the conduit radius; longer vessels reduce hydraulic resistance within the xylem by reducing the number of inter-vessel pits that water must cross while flowing from roots to leaves (Sperry et al. 2002; Tyree & Zimmermann 2002). The cost of efficient xylem is a greater risk of catastrophic xylem failure, caused when high tension in xylem conduits cavitates the water column. To minimize the probability of xylem failure, an opposing selection pressure favours safer xylem characterized by reduced flow capacity and shorter, narrower and mechanically stronger vessels (Tyree et al. 1994; Wagner, Ewers & Davis 1998; Hacke & Sperry 2001). In addition, efficient xylem is more vulnerable to catastrophic failure because there is less redundancy in the conducting tissue than in safe xylem (Tyree & Sperry 1989). Cavitation of large vessels increases the probability of catastrophic xylem failure relative to cavitation in small vessels, because the loss of function in a large vessel represents a much greater loss of total transport capacity (Comstock & Sperry 2000; Hacke & Sperry 2001). In contrast to efficient xylem, evolution of safer xylem with less flow capacity requires a reduction in water use by the leaf canopy because the capacity of the stem to re-supply water lost in transpiration is reduced (Hubbard, Bond & Ryan 1999; Brodribb & Feild 2000; Hubbard et al. 2001; Sperry et al. 2002). Reduction in water use usually occurs via stomatal closure or a decline in leaf area, both of which reduce whole-plant photosynthetic capacity (Brodribb & Feild 2000; Salleo et al. 2000; Davis et al. 2002; Sperry et al. 2002).
As a consequence of the evolutionary pressures selecting for efficiency or safety, xylem structure should reflect the balance between water supply and potential canopy evaporation in environments where freezing is not an issue (Tyree et al. 1994). The optimal solution is predicted to occur when the xylem structure in a plant provides just enough flow capacity to meet the highest transpiration rate a leaf canopy normally exhibits during a growing season (Tyree & Sperry 1989; Tyree 2003). In arid environments, the balance between safety and efficiency is weighted towards safety features, reflecting adaptations to low soil water supply and high xylem tension. In mesic environments in which competition for light is critical, the balance would shift towards hydraulic efficiency in order to support a larger leaf canopy. The evolutionary balance would also reflect unique aspects of the environment or plants, such as average humidity and temperature, soil water and nutrient status and allocation between roots and shoots. Differences in water use efficiency (WUE) could also affect xylem structure and function because WUE affects the balance between safety and efficiency (Sperry et al. 2002). In this regard, innovation of novel metabolic pathways that increase WUE, such as C4 photosynthesis, should also influence xylem characteristics of plants.
C4 plants have two- to four-fold greater WUE than C3 plants and therefore have substantially lower transpiration rates, assuming equivalent growth form and environmental conditions (Osmond, Björkman & Anderson 1980; Pearcy & Ehleringer 1984; Long 1999; Sage & Pearcy 2000). For example, the C4Atriplex species, A. rosea, A. expansa and A. serenana, have two to three times the WUE of the C3Atriplex species, A. triangularis, A. hortensis and A. heterosperma (Osmond et al. 1980). By evolving C4 photosynthesis, plants might shift the optimal balance between xylem efficiency and safety, such that the lower water requirements of the C4 leaf canopy might allow for safer xylem than present in their C3 ancestors. Alternatively, for the same amount of xylem, C4 plants could exploit the benefits of higher WUE not by increasing safety, but instead by supporting a greater leaf area per unit of xylem tissue and thereby improving overall carbon gain. In either case, C4 species should have a lower leaf specific conductivity (KL; stem hydraulic conductivity relative to the leaf area supported by the stem) compared with similar C3 plants.
Many studies have compared hydraulic properties of xylem from a wide range of species (reviewed in Hacke & Sperry 2001). Few, however, have examined hydraulic properties in C4 plants, and none to our knowledge has specifically compared xylem hydraulics of C3 and C4 species of similar life forms, ecological requirements or phylogenetic affinity. The leafy CAM plant Clusia uvitana has been observed to have KL values that are 1/3 to 1/30 of a range of tropical C3 species, indicating that the photosynthetic pathway can alter xylem function (Zotz, Tyree & Cochard 1994). In woody C3 species differing in habitat, differences in WUE have been inversely correlated with hydraulic conductivity and xylem efficiency (Pockman & Sperry 2000; Sobrado 2000; Sperry et al. 2002), which is consistent with the hypothesis that the higher C4 WUE could promote a drop in KL. For example, in a comparison of three Venezuelan mangrove species, WUE was lowest in Rhizophora mangle, intermediate in Laguncularia racemosa and highest in Avicennia germinans; KL was highest in R. mangle and lowest in A. germinans (Sobrado 2000). In the Great Basin desert in Utah, stems of the C4 shrubs Atriplex canescens and Atriplex confertifolia were more cavitation-resistant than the stems of the co-occurring C3 shrubs Chrysothamnus nauseosus and Chrysothamnus viscidiflorus (Hacke, Sperry & Pittermann 2000; Sperry & Hacke 2002). Although this work was not designed to compare photosynthetic pathway effects on xylem properties, the data from Atriplex and Chrysothamnus spp. is consistent with the possibility that C4 photosynthesis promotes evolutionary changes in the hydraulic pathway.
In the study described here, we evaluated whether differences in photosynthetic pathway affect xylem structure and function using 16 species of herbaceous plants having similar taxonomic and/or ecological distribution. Hydraulic properties and anatomical characteristics of the stem xylem were measured in eight C3 and eight C4 species segregated into four functional groups (Table 1). Group 1 consisted of four co-occurring annual species common in disturbed habitats such as old fields, cultivated lands and severely degraded habitats (wastelands). All were from the Chenopodiaceae/Amaranthaceae taxonomic complex. Group 2 consisted of five annual Atriplex species from the family Chenopodiaceae. Many of these species are from coastal habitats where wave action and sand movement create new habitat. The third group included two phylogenetically related annuals from the Sesuvioideae subfamily of the family Aizoaceae (Bittrich & Hartmann 1988; Kubitzki, Rohwer & Bittrich 1993). Group 4 consisted of trailing herbs from tropical coastal strands and roadsides of northern Australia. Because environment and phylogeny can influence xylem properties (Wagner, Ewers & Davis 1998; Sperry & Hacke 2002), we have compared these four groups to provide enough information to allow for broad inferences regarding the effects of photosynthetic pathway. If photosynthetic pathway or WUE alters xylem properties, we hypothesized that leaf specific conductivity will be lower in the C4 species relative to the C3 species within a comparison group.
Table 1. Functional group, species names, photosynthetic type, family, habitat, and collection site or seed source of the species used for this study. Species with geographic coordinates given were collected by F. Kocacinar or R. Sage
|Functional group Species||Ph. type||Family||Habitat||Collection site/seed source |
|Group 1: weedy annuals|
| Kochia scoparia L.||C4||Chenopodiaceae||Waste, disturbed land||Toronto, ON (43°65′ N 79°38′ W)|
| Amaranthus retroflexus L.||C4||Amaranthaceae||Waste, cultivated land||Toronto, ON (43°65′ N 79°38′ W)|
| Chenopodium album L.||C3||Chenopodiaceae||Waste, cultivated land||Toronto, ON (43°65′ N 79°38′ W)|
| Chenopodium botrys L.||C3||Chenopodiaceae||Waste, cultivated land||Toronto, ON (43°65′ N 79°38′ W)|
|Group 2: annual Atriplex|
| Atriplex texana S. Wats.||C4||Chenopodiaceae||Arid land, roadsides||Marathon, TX (30°10′ N 103°15′ W)|
| Atriplex rosea L.||C4||Chenopodiaceae||Waste, disturbed land||Australian National University|
| Atriplex hortensis L.||C3||Chenopodiaceae||Waste, disturbed land||Free University Botanical Garden, The Netherlands|
| Atriplex triangularis Willd.||C3||Chenopodiaceae||Saline wetlands||Pigeon Point, CA (37°9′ N 122°25′ W)|
| Atriplex littoralis L.||C3||Chenopodiaceae||European coastal strands||Antwerp Botanical Garden, Belgium|
|Group 3: Sesuvioideae annuals|
| Trianthema portulacastrum L.||C4||Aizoaceae||Arid land ephemeral||St. George, UT (37°12′ N 113°35′ W)|
| Sesuvium verrucosum L.||C3||Aizoaceae||Ephemeral saline wetlands||L. Lahonton, NV (39°19′ N 119°9′ W)|
|Group 4: coastal-strand herbs|
| Boerhavia dominii Meikle & Hewson||C4||Nyctaginaceae||Waste land, saline marshes||Darwin, Australia (12°15′ S 131°10′ E)|
| Boerhavia coccinea Miller||C4||Nyctaginaceae||Waste land, coastal strands||Darwin, Australia (12°15′ S 131°10′ E)|
| Tribulus eichlerianus (K. L. Wilson)||C4||Zygophyllaceae||Waste land, coastal strands||Darwin, Australia (12°15′ S 131°10′ E)|
| Canavalia rosea (Sw.) DC.||C3||Fabaceae||Coastal strands, beaches||Darwin, Australia (12°15′ S 131°10′ E)|
| Ipomoea pes-caprae (L) R. Br.||C3||Convolvulaceae||Coastal strands, beaches||Darwin, Australia (12°15′ S 131°10′ E)|
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- MATERIALS AND METHODS
C4 plants are widely noted for having superior WUE than C3 plants, and this is often presumed to increase carbon gain and drought tolerance. As a secondary consequence of the superior WUE, we hypothesized that C4 plants have modified xylem structure and function to improve hydraulic safety and/or enhance photosynthetic potential by allowing a larger leaf area per unit of xylem. In either case, the key index for changes in functional xylem traits is leaf specific conductivity, KL. In all comparison groups, the C4 species consistently exhibited lower KL than the corresponding C3 species, demonstrating shifts in the relationship between hydraulic transport capacity and leaf water use. These differences were apparent in the two groups in which species had close taxonomic affinity (groups 2 and 3), and in the two groups in which species were less related but shared identical ecological habitats (groups 1 and 4). Differences in KL between the species reflected differences in inherent WUE typically observed between the C3 and C4 pathways. C4 plants are commonly noted to have a WUE that is two to four times greater than ecologically similar C3 plants (Osmond et al. 1980; Larcher 1995), which corresponds to the three-fold difference in mean KL observed between the C3 and C4 species examined in this study. In the specific case of C. album and A. retroflexus, WUE at 10 mbar vapour pressure difference between leaf and air was previously measured to be 7–10 mmol CO2 mol−1 H2O in the C4 species grown at high soil nitrogen; WUE in C. album was 3–5 mmol CO2 mol−1 H2O under identical conditions (Sage & Pearcy 1987b). These differences correspond to the two fold difference in KL measured here between C. album and A. retroflexus.
Differences in KL can reflect either changes in xylem structure or the amount of leaf area produced relative to xylem tissue. Both were apparent in our samples. The C4 plants Amaranthus retroflexus, Atriplex rosea and T. portulacastrum exhibited similar Ks, Kls and xylem anatomy to the C3 species in their respective study groups. In contrast to their C3 counterparts, these C4 species supported substantially higher leaf areas per unit xylem, thus causing the lower values of KL and conduit efficiency. Amaranthus retroflexus supported two and three times higher leaf area per unit xylem than the C3C. album and C. botrys, respectively. Atriplex rosea had a mean leaf area per xylem area of 62.5 cm2 mm−2, which was twice that of the C3 plants A. hortensis (33.2 cm2 mm−2) and A. triangularis (30.5 cm2 mm−2), and three-fold more than the C3A. littoralis (21.0 cm2 mm−2). Similarly, for the same xylem area, T. portulacastrum supported twice the leaf area as the C3S. verrucosum (72 versus 34 cm2 leaf area mm−2 xylem area, respectively; despite twice the number of vessels per area in S. verrucosum). The other C4 species appear to have exploited the greater WUE primarily by enhancing xylem safety rather than leaf area, as indicated by relatively low Ks, Kls and anatomical values. Kochia scoparia and A. texana in the first two groups, and the three C4 species from the fourth group (B. dominii, B. coccinea and T. eichlerianus) produced shorter and narrower vessels compared to their respective C3 counterparts. Consistently, K. scoparia and B. coccinea had less vulnerable xylem than C. album and I. pes-caprae, their respective C3 counterparts in the comparison.
In habitats with abundant water and nutrients, high shoot growth is promoted and light availability becomes the main limiting resource that determines competitive outcomes (Bloom et al. 1985; Bazzaz 1996; Hutchings 1997). In these environments, strong competitive interactions above ground provide the evolutionary selection pressure that could favour allocation to greater leaf area, but only as long as the hydraulic pathway is able to support the leaf canopy. The greater WUE of C4 photosynthesis should relax the hydraulic demands of the canopy and allow for greater leaf area. This hypothesis is supported by the greater leaf area observed in Amaranthus retroflexus, Atriplex rosea and T. portulacastrum. Amaranthus retroflexus is one of the world's worst weeds, growing in highly productive agricultural fields and other resource-rich sites such as old fields and livestock pens (Paul & Elmore 1984; Holm et al. 1997). It often grows amongst Chenopodium spp. in weedy situations and the two are considered be competitors in these sites (Pearcy, Tumosa & Williams 1981). Atriplex rosea similarly grows in old field and abandoned lots, where it often competes with C3Atriplex and Chenopodium species (Holm et al. 1997). Neither of these species are very drought tolerant, although the Atriplex spp. are tolerant of moderate salinity (Osmond et al. 1980). Trianthema portulacastrum and S. verrucosum are also fast-growing annuals, but the main characteristic of their habitat is the ephemeral presence of water. They grow on river flood-plains, recently exposed mudflats and where water puddles following heavy rain, typically in arid regions and on soils with some levels of salinity (Wayne 1993; Sage, personal observation). High rates of growth appear valuable in order to set seed before soil water is depleted and salinity stress becomes extreme. In this regard, the greater leaf canopy supported by T. portulacastrum should lead to greater carbon gain, seed yield and potential fitness.
On drought-prone soils, relaxation of hydraulic requirements due to greater WUE could be exploited by increasing xylem safety. Improved safety would allow plants to maintain photosynthesis at lower leaf water potentials without compromising the hydraulic pathway. This could allow for longer growing seasons, as leaves could remain active later into a dry season (Osmond et al. 1980), or alternatively, leaves could reduce the level of stomatal closure during low humidity periods, such as occurs during midday (Schulze & Hall 1982; Sperry, Alder & Eastlack 1993; Sperry 1995). Comparisons of C3 and C4 responses in arid communities support these hypotheses. In Death Valley in California, C4Atriplex hymenelytra maintained daily leaf conductance and CO2 uptake two to three times higher than co-occurring C3Larrea divaricata during the dry season (Osmond et al. 1980). In the Great Basin desert of North America, the C4 shrub Atriplex confertifolia maintains its leaf canopy in late summer conditions that cause die-back in sympatric Chrysothamnus and Ceratoides species (Osmond et al. 1980; Sperry & Hacke 2002). In the Negev desert, C4Hammada (=Haloxylon) scoparia maintains gas exchange under more extreme conditions of atmospheric and soil drought than the sympatric C3 plants Zygophyllum dumosum and Artemisia herba-alba (Schulze et al. 1980). This was also the case in C3 and C4 annual Atriplex species grown in a common garden under non-irrigated conditions in coastal California. Atriplex rosea (C4) maintained activity toward the end of the dry season whereas A. triangularis (C3) died early in the dry season (Nobs et al. 1972).
Most of the comparisons of C3 and C4 plants have focused on the direct advantages of the C4 pathway such as the suppression of photorespiration, enhanced photosynthetic potential in warm climates, and greater water, nitrogen and radiation use efficiency. Differences in resource use efficiency facilitate secondary evolution that relieves constraints that may occur elsewhere in the system such as in the hydraulic pathway (Bloom et al. 1985). The secondary evolutionary response to the WUE advantage in C4 plants is pronounced and complements the direct benefits of the C4 pathway by allowing C4 plants to develop adaptive traits to a greater degree than may be possible in C3 plants. For example, in resource-rich environments, the ability to carry more leaf area per stem allows for greater light capture, productive potential and competitive ability (Potter & Jones 1977; Sage & Pearcy 1987a). This explains in part the ability of C4 species to exhibit greater yields than C3 species, and to become aggressive weeds (Brown 1999). By contrast, in arid environments, the secondary advantages of WUE could allow C4 species to occur in drier soils and maintain function at drier periods of the year.
The high productivity of C4 plants has led to attempts to engineer C4 photosynthesis into crops such as rice (Häusler et al. 2002). Our results indicate the benefits of engineering C4 plants do not end with the insertion of C4 photosynthesis into leaves. To fully exploit the benefits of C4 photosynthesis, bioengineers should eventually consider modifying xylem properties to optimize KL. As the crop yield is directly related to canopy area (Gifford & Evans 1981) shifts in KL will also be needed to maximize the productive potential of the C4 pathway.
In conclusion, the results here demonstrate the WUE advantage of C4 plants lead to more than just greater rates of carbon gain or water savings. As in many economic enterprises, efficiencies realized in one part of the system can allow resources to be re-allocated to functions that address the next most critical environmental challenge (Bloom et al. 1985). In arid environments, this may be greater safety. In resource-rich environments, this could be competitive potential, as indexed by leaf area production or increased fecundity. In either case, it is apparent that evolution within the hydraulic pathway in response to the advent of C4 photosynthesis enhances the ability of these plants to address environmental challenges.