Physiologia Plantarum 153: 454–466. 2015
© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Photosynthetic, hydraulic and biomass properties in closely related C3 and C4 species Ferit Kocacinara,b,* of Forestry, Department of Forest Engineering, Division of Plant Physiology, Kahramanmara¸s Sütçü ˙Imam Üniversitesi, Kahramanmara¸s 46100, Turkey b Department of Bioengineering and Sciences, Kahramanmara¸s Sütçü ˙Imam Üniversitesi, Kahramanmara¸s 46100, Turkey a Faculty
Correspondence *Corresponding author, e-mail: [email protected] Received 28 January 2014; revised 30 April 2014 doi:10.1111/ppl.12240
In plants, most water is absorbed by roots and transported through vascular conduits of xylem which evaporate from leaves during photosynthesis. As photosynthesis and transport processes are interconnected, it was hypothesized that any variation in water transport demand influencing water use efficiency (WUE), such as the evolution of C4 photosynthesis, should affect xylem structure and function. Several studies have provided evidence for this hypothesis, but none has comprehensively compared photosynthetic, hydraulic and biomass allocation properties between C3 and C4 species. In this study, photosynthetic, hydraulic and biomass properties in a closely related C3 Tarenaya hassleriana and a C4 Cleome gynandra are compared. Light response curves, measured at 30∘ C, showed that the C4 C. gynandra had almost twice greater net assimilation rates than the C3 T. hassleriana under each increasing irradiation level. On the contrary, transpiration rates and stomatal conductance were around twice as high in the C3 , leading to approximately 3.5 times higher WUE in the C4 compared with the C3 species. The C3 showed about 3.3 times higher hydraulic conductivity, 4.3 times greater specific conductivity and 2.6 times higher leaf-specific conductivity than the C4 species. The C3 produced more vessels per xylem area and larger vessels. All of these differences resulted in different biomass properties, where the C4 produced more biomass in general and had less root to shoot ratio than the C3 species. These results are in support of our previous findings that WUE, and any changes that affect WUE, contribute to xylem evolution in plants.
Introduction During photosynthesis, plants transpire large quantities of water to the atmosphere in exchange for CO2 acquisition. Therefore, over evolutionary time there should be a balance in plants for this exchange under each specific environmental niche. This balance is best reflected in the xylem structure and functional properties, where safety vs efficiency features play important roles in plant distribution, competition and survival (Tyree and Zimmermann 2002, Sperry 2003). Under
marginal lands, such as semiarid, arid and saline habitats, the balance between safety and efficiency is shifted toward safety traits, reflecting adaptations to water stress and high xylem tension. In humid and resource-rich environments, however, where fast growth and above ground allocation is more beneficial to plants, the evolutionary balance is shifted toward hydraulically efficient xylem needed to feed this greater leaf area and speedy growth. In an invasive species Solidago canadensis, for example, specific hydraulic conductivity of stem (Ks ) was about twofold and leaf-specific hydraulic conductivity
Abbreviations – DW, dry weight; FW, fresh weight; gs, stomatal conductance; PNUE, photosynthetic nitrogen-use efficiency; PPFD, photosynthetic active photon flux density; VF, vessel frequency; VPD, vapor pressure deficit; WUE, water use efficiency.
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(KL ) was about 1.5 times higher at the humid than at the drier site (Nolf et al. 2014). Also, vulnerability of the xylem to drought-induced embolism decreased, and safety margins increased along the transect from humid to drier sites. This indicates a trade-off between hydraulic safety and efficiency and may help explain invasive potential of this species (Nolf et al. 2014). Other intrinsic evolutionary traits such as high root to shoot ratio, better and more efficient stomatal regulation, higher water storage capacity and C4 biochemical pathway with higher water use efficiency (WUE) could also affect this balance by affecting xylem structure and function (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). Species with the C4 biochemical pathway concentrate CO2 around Rubisco, either in bundle sheath cells or in deeper site of elongated mesophyll cells, thereby significantly increasing carboxylation efficiency and reducing photorespiration. The rate and the efficiency of assimilation are higher in C4 than C3 plants under low atmospheric CO2 , and under high light, temperature and vapor pressure deficit (VPD), suggesting these factors as important evolutionary selective agents (Osborne and Sack 2012). All of these factors significantly increase photorespiration and reduce WUE of plants. Because the C4 photosynthetic pathway has independently evolved over 60 times in at least 19 families (Sage et al. 2011), this multiple convergent evolution provides excellent tool to study and understand the repeated evolution of C4 associated traits in closely related C3 and C4 species (Way 2012). Evolution of the C4 photosynthetic pathway has significantly increased WUE in C4 plants, where C4 plants exhibit two to four times higher instantaneous WUE than ecologically and taxonomically similar C3 plants (Osmond et al. 1982, Pearcy and Ehleringer 1984, Long 1999, Kocacinar et al. 2008, Vogan and Sage 2011). The discovery of the strong coordination between hydraulic and photosynthetic processes in plants, (Hubbard et al. 1999, Brodribb and Feild 2000, Hubbard et al. 2001, Brodribb et al. 2002, Franks and Brodribb 2005, Kocacinar et al. 2008), provided new venues for comparative studies. Since then, several studies comparing ecologically and taxonomically similar C3 and C4 plants have shown that WUE influences xylem structure and hydraulic function to the leaf canopy (Kocacinar and Sage 2003, Kocacinar 2004, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). In all comparisons, C4 plants exhibit lower KL than C3 plants of similar functional type, ecological habitat and phylogenetic distance (Kocacinar and Sage 2003, Kocacinar 2004, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008).
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For example, in a comparison of KL and vascular anatomy between eight C3 and eight C4 herbaceous species, the C3 species consistently showed higher KL , longer and wider vessels than the C4 (Kocacinar and Sage 2003). The KL differences between C3 and C4 species generally corresponded to the twofold to fourfold differences in WUE between C3 and C4 plants. In the comparison group of woody perennial plants adapted to arid habitats, where xylem safety traits provide a priority for adaptation and a competitive advantage, the lower KL of C4 plants was associated with changes in xylem structure that increase hydraulic safety, whereas in the comparison group of annual species from more humid habitats, the lower KL was associated with greater leaf area per unit xylem in C4 compared with C3 species (Kocacinar and Sage 2003, Kocacinar and Sage 2004). The hydraulic and xylem pattern differences between C3 and C4 plants are present in both herbaceous (Kocacinar and Sage 2003) and woody species (Kocacinar and Sage 2004), and between species of similar ecological habit and close taxonomic affinity (Kocacinar et al. 2008). Moreover, these trends were present in a statistically robust comparative analysis in the genus Flaveria (Kocacinar et al. 2008), where the phylogeny is well established based on chloroplastic trnl-F, nuclear internal transcribed spacer (ITS) and external transcribed sequence (ETS) (McKown et al. 2005). Among the 16 C3 , C3 –C4 intermediate, C4 -like and C4 Flaveria species compared, the C3 species had the highest KL compared with the intermediate, C4 -like and C4 species (Kocacinar et al. 2008). Ks was also generally high in the C3 and low in the intermediate and C4 species. The differences were also evident in the xylem structure, where xylem vessels were shorter, narrower and higher in number per xylem area in C3 –C4 intermediate, C4 -like and C4 species compared with C3 species (Kocacinar et al. 2008). The differences in these patterns were more pronounced as C4 -cycle activity increased in the C3 –C4 intermediates and moving from intermediates to C4 -like and C4 species. In Flaveria, three main groups of intermediates are classified with varying degree of C4 anatomy and physiology: type I, type II and C4 -like intermediates, each with increasing degree of C4 traits (Sage 2004, McKown and Dengler 2007, Kocacinar et al. 2008, Gowik and Westhoff 2011, Vogan and Sage 2011). However, WUE is not enhanced in type I and type II intermediates, where C4 -like intermediates show similar values to full C4 species (Kocacinar et al. 2008, Vogan and Sage 2011). There was also a strong correlation between KL and Ks and between KL contrasts and WUE contrasts using the Flaveria phylogeny of McKown et al. (2005). These studies provided strong evidence that WUE and xylem structure and functional properties 455
are significantly different between C3 and C4 species, but no study has comprehensively compared photosynthetic, hydraulic and biomass properties between close lineages of C3 and C4 species. In this study, hydraulics, photosynthesis, growth and WUE were compared between close lineages of C3 Tarenaya hassleriana and C4 Cleome gynandra, the most closely related C4 model to Arabidopsis (Brown et al. 2005, Voznesenskaya et al. 2007). Tarenaya hassleriana, formerly known as Cleome hassleriana and sometimes incorrectly referred as Cleome spinosa, and C. gynandra species are both from the family Cleomaceae, the sister family to Brassicaceae which includes Arabidopsis thaliana and Brassica crops (Bhide et al. 2014). The genome of T. hassleriana has been recently sequenced and can serve for understanding of gene, genome and trait evolution (Cheng et al. 2013). This study could serve as a physiological model for understanding the hydraulic advantages of C4 photosynthesis. If the results of this study show the same trend as all the previous studies in this field, then this can offer a potential for work on the genetic and genomic basis of altered WUE and hydraulics, because genes can be mapped from these two species onto the Arabidopsis reference genome. If we can discover genes that control WUE and hydraulics, then we will be able to engineer them in crop plants – either in rapeseed or rice, which is being engineered to use C4 , or in other crops where we may wish to change hydraulic conductance. This will be important to realize, for example, the full potential of C4 rice to deliver improved resource-use efficiency.
Materials and methods Plant material and growth conditions The seeds of the C4 species C. gynandra L. and the C3 species T. hassleriana (Chodat) H.H. Iltis were provided by Prof. Rowan F. Sage from the University of Toronto, Toronto, Ontario, Canada. The seeds of each species were collected from several different individual plants grown in an outdoor rooftop common garden in Toronto. The untreated seeds were kept at 4∘ C until sown in pots. Plants were grown from seeds in 10 l pots filled with a mix of 50% organic matter, 25% topsoil and 25% sand. The plants were grown outdoors in a common garden under natural conditions from April until June in 2009 at the Kahramanmara¸s Sütcü ˙Imam University, Kahramanmara¸s, Turkey. There was no precipitation effect as the common garden was covered, only on top, with a built transparent plastic cover. The average minimum and maximum temperatures were 15–20 and 30–35∘ C, respectively, during the growth period. 456
All plants were watered regularly as needed, two to three times a week, and fertilized once a week with a full-strength Hoagland’s solution (Sage and Pearcy 1987a). Gas exchange measurements and WUE Gas exchange measurements were conducted on randomly selected six plants from each species using a GFS-3000 portable gas exchange and fluorescence system (Heinz Walz GmbH, Effeltrich, Germany). Measurements were started at 10:00 h (mid-morning) and lasted until 16:00 h (mid-afternoon). All measurements were conducted indoor on a laboratory bench. A light response curve for each individual plant was constructed using measurements taken in the dark and under gradually increasing photosynthetic active photon flux density (PPFD) up to 2000 μmol m−2 s−1 , at a leaf temperature of 30∘ C, 380 μmol ambient CO2 and cuvette relative humidity of approximately 55%. For each light response curve, a plant was brought inside the laboratory and a fully expanded leaf from the tip was selected and enclosed into the leaf chamber to dark adapt for at least 30 min before measurements started. When a next level PPFD was set, assimilation rate increased immediately and reached a steady state after about 15–20 min. The progress was monitored for 5–10 min using the GFS-3000 software with a live-graph and several measurements were taken and averaged for each assimilation rate under each PPFD level. After a last measurement was taken at 2000 μmol m−2 s−1 PPFD, the leaf temperature was increased to 40∘ C and a new measurement was taken at this temperature in order to boost the difference between the C3 and the C4 species under elevated temperature. Under each light level, net photosynthesis, transpiration and stomatal conductance were measured, and WUE (mmol CO2 mol−1 H2 O) was calculated as net assimilation rate divided by transpiration rate. During all measurements, cuvette relative humidity was set to 55%, which was similar to the ambient humidity where plants were grown. This relative humidity corresponded to VPD of 1.5–2.2 and 2.3–2.9 kPa under 30 and 40∘ C leaf temperatures, respectively, in T. hassleriana and VPD of 2.0–2.4 and 3.3–3.6 kPa in C. gynandra. Stem hydraulic properties and xylem anatomy Hydraulic conductivity (Kh ) was measured on six different plants from each species as described previously in detail (Kocacinar and Sage 2003). All the stems sampled for hydraulic measurements were main stems of plants and had a complete and healthy leaf canopy to avoid leaf loss impacting on KL . Kh , Ks , and KL were assessed Physiol. Plant. 153, 2015
on stem segments ranging from 0.5 to 1.5 cm in diameter and from 8 to 10 cm in length. Stem segments were cut under water and connected to tubing filled with filtered (0.2 μm) tap water for all hydraulic measurements. Perfusion water was changed frequently to reduce the possibility of microbial growth in the tubing and the container hung above the segments. Although the stem segments were cut under water, in order to remove any possible natural embolism and to measure maximum Kh , the stem segments were first flushed with perfusion water under high pressure (150–200 kPa), generated by a pressure chamber (Model 1000, PMS Instrument Company, Albany, NY). Following a high pressure perfusion usually for 10–15 min, the flow through the stem segments was generated gravimetrically using a hydraulic head of between 5 and 20 kPa (Dryden and Van Alfen 1983). A plot of flow vs pressure was generated for each sample, taking at least three independent measurements under three different pressures. Kh was then calculated as the slope of the pressure-flow plot multiplied by segment length. However, differences in Kh could arise from different allocation strategies such as allocation to stem diameter, conducting xylem area, conduit diameters or xylem porosity (Kocacinar and Sage 2004). Therefore, variation in the xylem allocation was accounted for by dividing Kh by the total xylem cross-sectional area, determined using free-hand sections and a light microscope (Eclipse 90i, Nikon Instruments Europe B.V., Amstelveen, The Netherlands), to determine Ks . KL was calculated as Kh divided by total leaf area distal to the stem segment. Leaf area was measured using a LI-COR 3100 area meter (LI-COR Inc. Lincoln, NE). Xylem area, number of vessels per xylem area [vessel frequency (VF)], and conduit diameters were measured on stem cross-sections that had been previously used for Kh determination. For each species, six of the stem segments were fixed in FAA (formalin, acetic acid and alcohol), rinsed with tap water, free hand sectioned, stained with an aqueous solution containing 0.05% toluidine blue O (TBO, Sigma-Aldrich Chemie Gmbh, Munich, Germany) (O’Brien et al. 1964), and examined under the light microscope. Images were analyzed with an imaging software (NIS-Elements, Microscope Imaging Software, Nikon Instruments Europe B.V. Amstelveen, The Netherlands). The vessel diameters were measured as the average of the lengths of major and minor axes of the conduits. Maximum vessel diameter (MVD) was measured on the largest vessel from each cross-section. Vessel mean diameter (VMD) was determined as the mean of all the vessels in a randomly chosen part of the xylem area from each cross-section. VF was determined as the number of vessels per xylem area on randomly chosen sectors from each cross-section.
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Growth properties Five plants from each species were randomly selected for growth analysis at approximately 60 days old, before flowering had commenced. Stem height was measured from the soil surface to tip of the stem using a ruler, and root collar diameter was determined on the same stems at the soil surface using a digital caliper. Shoot fresh weights (FWs) were immediately determined after excising the shoots from the soil surface. Shoot dry weights (DWs) were measured after oven drying samples for 48 h at 70∘ C. Roots were carefully washed, dried of excess water with paper towels and weighed for fresh root mass. Roots were then dried for 48 h at 70∘ C then weighed again for root dry mass. Data analysis Data were statistically tested using SIGMASTAT software (SYSTAT software, Richmond, CA). Mean values of all parameters between the C3 and the C4 species were compared using a one-way ANOVA followed by least significance difference (LSD) test (P < 0.05). VF and vessel diameters data were log-transformed, because these data are not normally distributed and failed the normality test.
Results Gas exchange properties and WUE The C4 C. gynandra had significantly higher net photosynthetic rate than the C3 T. hassleriana under each PPFD (Fig. 1A). The difference in net photosynthetic rate between the C4 and the C3 was widened as PPFD increased up to 2000 μmol m−2 s−1 . At PPFD of 2000 μmol m−2 s−1 , net assimilation rate was almost two times greater in the C4 than the C3 species measured at leaf temperature of 30∘ C (Fig. 1A). When leaf temperature was raised to 40∘ C, assimilation rate declined in the C3 but increased in the C4 , leading to approximately 2.5 times greater values in the C4 than the C3 species, 33.8 vs 13.9 μmol CO2 m−2 s−1 , respectively (Fig. 1A). However, transpiration rates were significantly higher in the C3 than the C4 under each PPFD (Fig. 1B). Transpiration rates were almost 10 times higher in the dark, (2.6 vs 0.3 mmol H2 O m−2 s−1 ), and 2 times greater at maximum PPFD of 2000 μmol m−2 s−1 , (11.9 vs 5.9 mmol H2 O m−2 s−1 ), in the C3 than the C4 species, respectively (Fig. 1B). When leaf temperature was raised from 30 to 40∘ C, transpiration rates increased significantly in both species and were 1.8 times more in the C3 than the C4 (Fig. 1B). Stomatal conductance increased in both species as PPFD increased to 2000 μmol m−2 s−1 , however, it was almost 2.5 times greater in the C3 compared 457
Fig. 1. Net photosynthetic rate (A), transpiration rate (B), stomatal conductance (C) and WUE, (D) of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ) as a function of increasing PPFD. Measurements were taken at 380 μmol ambient CO2 , 55% relative humidity and 30∘ C leaf temperature. After the last measurement, leaf temperature was increased to 40∘ C and a new measurement was taken. Each point is the mean of six different plants. Vertical lines represent ±1 SE of the mean.
with the C4 under each PPFD level (Fig. 1C). Raising leaf temperature to 40∘ C at 2000 μmol m−2 s−1 PPFD significantly increased stomatal conductance in both species, but the difference was much greater in the C3 than the C4 species (Fig. 1C). WUE values reached a maximum at 500 PPFD and remained constant as PPFD increased from 500 to 2000 μmol quanta m−2 s−1 in both species (Fig. 1D). Under each of the PPFD levels above 500, WUE was on average 1.5 mmol CO2 mol−1 H2 O in the C3 T. hassleriana and significantly higher, about 3.5 times higher (5.0 mmol CO2 mol−1 H2 O) in the C4 C. gynandra species (Fig. 1D). When leaf temperature was raised to 40∘ C at 2000 PPFD, WUE was reduced by 40% in the C4 , from 5 to 3 mmol CO2 mol−1 H2 O, and by 50% in the C3 species, from 1.4 to 0.7 mmol CO2 mol−1 H2 O (Fig. 1D). Stem hydraulic properties and xylem anatomy Stem hydraulic conductivity was significantly higher, about 3.3 times more, in the C3 than the C4 species (P = 0.004) (Fig. 2A). Although the C4 had slightly higher mean xylem cross sectional area (19.6 mm2 ) than 458
the C3 (15.5 mm2 ), the difference was not significant (P = 0.145), therefore, Ks was also significantly higher in the C3 than the C4 species (P < 0.001) (Fig. 2B). Ks was about 0.6 kg m−1 s−1 MPa−1 in the C4 and approximately 4.3 times more, 2.6 kg m−1 s−1 MPa−1 , in the C3 plants (Fig. 2B). Even though the C3 produced slightly more leaf area than the C4 , the difference was not significant (P = 0.14, Fig. 2C). When leaf area was accounted for, KL was 2.6 times higher in the C3 than the C4 species (Fig. 2D). KL was 1.2 × 10−4 kg m−1 s−1 MPa−1 in the C4 and 3.1 × 10−4 kg m−1 s−1 MPa−1 in the C3 species (Fig. 2D). The significant differences in the xylem hydraulics between the C3 and the C4 were probably a result of the differences in xylem anatomical properties. The C3 produced more vessels per xylem area and significantly larger vessels than the C4 Cleome species (Table 1 and Fig. 3). Maximum and mean vessel diameters were significantly higher in the C3 compared with the C4 Cleome species (Table 1). Differences in vessel number and size were also obvious in representative xylem cross-section micrographs (Fig. 3).
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Fig. 2. Hydraulic conductivity (Kh ) (A), specific conductivity (Ks ) (B), total leaf area per stem (C) and leaf specific conductivity (KL ) (D), of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ). Bars are means (±1 SE) of six different plants. Different letters represent significant differences (P < 0.05). Table 1. Xylem anatomical parameters of the C3 Tarenaya hassleriana and the C4 Cleome gynandra species. Maximum vessel diameter (MVD) was measured on the largest vessel from each cross-section. Vessel mean diameter (VMD) was determined as the mean of all the vessels in part of the xylem from each cross-section. VF was determined as the number of vessels in part of the xylem from each cross-section. Different letters represent significant differences (P < 0.05). Values are means (±1 SE) of six samples for each species. Species T. hassleriana C. gynandra
Photo. type
MVD (μm)
VMD (μm)
VF (no. mm−2 )
C3 C4
73.9 ± 1.5a 63.2 ± 3.4b
66.1 ± 1.3a 58.0 ± 3.3b
108 ± 9.0a 66 ± 8.0b
Growth properties Differences in photosynthetic and hydraulic properties were reflected in growth properties and biomass allocation differences between the C3 and the C4 species (Figs Physiol. Plant. 153, 2015
4 and 5). Shoot FW was significantly higher in the C4 than the C3 species (Fig. 4A). Shoot FWs were 66.4 g per plant in the C4 and 39.8 g per plant, 40% less, in the C3 species (Fig. 4A). Although the difference was not significant (P = 0.056), the C3 produced about 45% less shoot DW than the C4 species; 5.1 g shoot DW per plant in the C3 and 9.4 g per plant in the C4 , respectively (Fig. 4A). Even though, root FW and DW were not significantly different between the two species, the C3 produced 30% less root DW (1.0 g per plant) than the C4 species (1.4 g per plant) (Fig. 4B). Whole plant FW and DW were, respectively, 37 and 44% less in the C3 than the C4 species (Fig. 4C). Root to shoot ratio was significantly higher in the C3 than the C4 species (Fig. 4D). Both root collar diameter and stem height were significantly higher in the C4 compared with the C3 species (Fig. 5A, B). 459
Fig. 3. Representative cross-sections of stem xylem of the C3 Tarenaya hassleriana (left) and the C4 Cleome gynandra (right). Note vessel size and number differences between the two species. The scale bar in the right bottom micrograph is 200 μm and applies to both species.
Discussion Gas exchange properties and WUE In most cases, C4 plants have superior photosynthetic rates, higher nitrogen, water and irradiation use efficiencies compared with C3 species of similar ecological habitat or taxonomic affinity (Osmond et al. 1982, Ehleringer and Pearcy 1983, Pearcy and Ehleringer 1984, Wong et al. 1985, Sage and Pearcy 1987a, 1987b, Long 1999, von Caemmerer and Furbank 1999, Sage and Pearcy 2000, Kocacinar et al. 2008, Vogan and Sage 2011, Cristiano et al. 2012). In this study, gas-exchange properties under increasing PPFD levels, stem hydraulics including xylem anatomy and growth properties were all investigated in taxonomically close C4 C. gynandra and C3 T. hassleriana species. The C4 species showed almost two times more net assimilation rates especially under increasing PPFD levels than its counterpart C3 species (Fig. 1A). Besides this superiority in net assimilation rates, the C4 made more efficient use of high irradiation than the C3 ; for example, the C3 reached its maximum assimilation rates at PPFD of 1000 μmol quanta m−2 s−1 , whereas the C4 continued to increase its assimilation rate up to 2000 μmol quanta m−2 s−1 (Fig. 1A). Moreover, under saturating 2000 μmol quanta m−2 s−1 PPFD, raising the leaf temperature from 30 to 40∘ C increased net photosynthetic rate in the C4 by about 14%, but decreased it in the C3 by almost 15% (Fig. 1A). Net photosynthetic rate was 2.4 times higher in the C4 than the C3 at leaf temperature of 40∘ C and 2000 μmol quanta m−2 s−1 PPFD. The superiority in net assimilation rates, irradiation use and high temperature optima in plants utilizing C4 biochemistry was recognized even before the discovery of the C4 biochemical pathway (Hesketh 1963, Hesketh and Moss 1963, Murata and Iyama 1963, El-Sharkaway and Hesketh 1964). Following the discovery of C4 biochemistry in the late 1960s, 460
especially under high temperature and in heat adapted species, many studies have reported higher rates of photosynthesis at all light levels or higher quantum efficiency in C4 plants compared with similar C3 species (Osmond et al. 1980, Pearcy and Ehleringer 1984). Previously, it was also reported that C4 photosynthesis is characterized to be light saturated only at very high light intensities as evidenced by C4 Flaveria bidentis wild-type plants compared with transgenic F. bidentis with reduced amount of Rubisco (Furbank et al. 1996, Siebke et al. 1997, von Caemmerer and Furbank 1999). Transgenic F. bidentis plants saturated at much lower irradiances. Superior net photosynthetic rates of C4 were also evident in a comparison between the cold adapted co-occurring C3 Calamogrostis canadensis and C4 Muhlenbergia glomerata grasses native to high latitudes (Kubien and Sage 2004). The C4 M. glomerata showed about 40 and 80% more net photosynthesis rate, measured at leaf temperatures of 20 and 30∘ C, respectively, when compared with the co-occurring C3 C. canadensis, both grown at warm conditions (26/22∘ C) (Kubien and Sage 2004). The difference in net assimilation rates between C4 and C3 plants under increasing both PPFD and leaf temperature is partly due to the difference in photorespiration between these two functional groups. The C4 grass Panicum maximum showed no apparent increase in photorespiration and CO2 compensation concentration as PPFD raised from near darkness to 2000 μmol quanta m−2 s−1 and from 15 to 40∘ C leaf temperature compared with significantly increased values in the C3 grass Festuca arundinacea (Brown and Morgan 1980). In this study, quantum yield for CO2 uptake, calculated as the slope of the response curve relating net CO2 uptake to incident photon flux at unsaturated light of 250 μmol quanta m−2 s−1 , quantum yield not corrected for leaf absorbance and assuming all photons are absorbed, was 0.03 μmol CO2 per μmol photons for Physiol. Plant. 153, 2015
Fig. 4. Shoot fresh and dry weight (A), root fresh and dry weight (B), whole plant fresh and dry weight (C) and root to shoot ratio of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ). Bars are means (±1 SE) of five plants per species. Different letters represent significant difference between species, where in some cases P values are given to compare the means.
the C4 C. gynandra and 0.02 μmol CO2 per μmol photons for the C3 T. hassleriana. The C4 fixed 50% more carbon per photon compared with the C3 species measured at 30∘ C leaf temperature, 380 ppm ambient CO2 and 55% relative humidity. Similar results were reported for the heat adapted C4 Tidestromia oblongifolia and the C3 Larrea divaricata measured at leaf temperature of 40∘ C, where quantum yield (the slope of light response curve) was calculated as 0.03 for the C4 and 0.02 for the C3 at low light intensities (Osmond et al. 1980). Ehleringer and Pearcy (1983) surveyed the quantum yields of large number of C3 and C4 species and reported higher values than found in this study, ranging from 0.046 to 0.055 mol CO2 per mol photons for the C3 dicots and 0.052 to 0.069 mol CO2 per mol photons for the C4 species. In this survey, however, quantum yield calculations were done at very low light intensities, below 20 μmol quanta m−2 s−1 , and
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were corrected for different leaf absorbances, which resulted in higher values than reported here in this study. The C3 species T. hassleriana lost a significantly greater amount of water to the atmosphere as evidenced by very high rates of transpiration and stomatal conductance under each photon flux density compared with the C4 species C. gynandra. Transpiration and stomatal conductance rates were at least two and three times higher, respectively, in the C3 than the C4 species. In a phylogenetically controlled analysis, Taylor et al. (2012) also showed that C4 grasses had significantly lower maximum stomatal conductance rates (gmax ) than their C3 relatives. To account for differences in habitat, C4 species showed lower gmax than C3 species in both mesic and dry environments, demonstrating intrinsic stomatal traits differences between the two groups (Taylor et al. 2012). In a comparison between co-occurring species of C3 Chenopodium album and C4 Amaranthus retroflexus, 461
Fig. 5. Root collar diameter (A) and stem height (B) of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ). Bars are means (±1 SE) of five plants per species. Different letters represent significant differences (P < 0.05).
Pearcy et al. (1981) reported three times higher leaf conductance under low (17∘ C) and moderate (25∘ C) growth temperatures and two times more leaf conductance under high growth temperature (34∘ C) in Chenopodium relative to Amaranthus. Similar results were also reported for a number of C3 and C4 species grown under different nutrition, light and ambient CO2 regimes (Wong et al. 1985). One striking example is the difference in gas exchange properties between the C4 Zea mays and the C3 Gossypium hirsutum, grown under various nitrate regimes. For the same photosynthetic rate, G. hirsutum exhibited two times higher leaf conductance than Z. mays under each nitrate treatment (Wong et al. 1985). In this study, the significant differences in transpiration rates and stomatal conductance between the C3 and the C4 species resulted in a 3.5 times higher photosynthetic WUE in the C4 plants (Fig. 1D). WUE averaged about 5.0 and 1.4 mmol CO2 per mol H2 O in the C4 and the C3 plants, respectively. Caldwell et al. (1977) compared photosynthetic WUE for the C4 Atriplex confertifolia and the C3 Ceratoides lanata, growing together either in mixed communities or in mono-specific stands next to each other in a cold desert area in Utah. They found that the two species did not show any significant WUE differences during the cool spring when soil water was ample. However, during the hot summer the C4 Atriplex showed superior values; estimated annual average WUE for growth of 4.3 and 2.9 mg DW g−1 H2 O in A. confertifolia and C. lanata, respectively (Caldwell et al. 1977). Similar field results were also reported for the Death Valley, California evergreen drought-tolerant shrubs C4 Atriplex hymenelytra and C3 L. divaricata (Osmond et al. 1980). The WUE of A. hymenelytra was two to three times higher than L. divaricata under water stress down 462
to −4.6 MPa. In a comparison among C3 , C3 –C4 intermediate and C4 grass species under different nitrogen nutrition, the C4 P. maximum exhibited superior apparent photosynthesis especially under higher leaf nitrogen concentration and two times higher WUE at all leaf nitrogen levels compared with both the C3 –C4 intermediate Panicum milioides and the C3 F. arundinacea species, which showed similar values (Bolton and Brown 1980). The superiority of photosynthetic capacity, quantum yield and WUE of C4 has been suggested to provide the greatest adaptive advantage under xeric, hot, saline and open high light environments (Pearcy and Ehleringer 1984). Numerous studies confirm a greater C4 abundance in warmer, open, high light and tropical environments (Osmond et al. 1982, Hattersley 1983, Pearcy and Ehleringer 1984, Sage et al. 1999). Stem hydraulic properties and xylem anatomy Because of the well-established co-ordination between hydraulic conductance and stomatal conductance, therefore, photosynthesis in plants (Hubbard et al. 1999, Brodribb and Feild 2000, Hubbard et al. 2001, Brodribb et al. 2002, Kocacinar et al. 2008), it was hypothesized that any innovation during the evolution of plants, such as evolving C4 photosynthesis with superior WUE, would have affected water transport demand, namely, hydraulic properties of the xylem. In this study, significant hydraulic properties differences between the C4 and the C3 species supported this hypothesis once more. The C3 T. hassleriana showed almost 3.3, 4.3 and 2.6 times higher Kh , Ks and KL , respectively, compared with the C4 C. gynandra species. These results are consistent with the results of previous large comparisons with 51
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C3 and C4 species of close taxonomic affinity, similar ecological habitat and growth form (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). Within the previous 51 comparisons including the one in this study, KL was consistently lower in C4 compared with C3 species of a similar life form and taxonomic group, within any comparison group not a single C3 species showed lower KL . Associated with the differences in KL and WUE, C4 species in these previous comparisons had vessels that were similar to, or shorter and narrower than the corresponding C3 species (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). Here in this comparison, the C4 also showed significantly narrower conduit diameters but almost twice higher VF per xylem area than the C3 species (Table 1 and Fig. 3). In Flaveria, significant phylogenetic relationship between WUE and KL was previously established (Kocacinar et al. 2008). The results from Flaveria and other earlier studies by Kocacinar and Sage (2003, 2004) including the results found in this study show strong support for the hypothesis that C4 evolution changed the transport demand of the leaf canopy and resulted in safer xylem and lower KL . Where the xylem anatomy properties were found to be comparable between the C4 and the C3 species in a comparison group, C4 plants seemed to utilize the WUE advantage by having larger leaf canopy especially in species adapted to mesic habitats. In comparisons of species adapted to more xeric habitats, where xylem safety maintenance is more crucial for survival and persistence than having larger leaf canopy, C4 plants utilized WUE advantage by xylem modification mainly having shorter and narrower but more frequent conduits which confer safer xylem in C4 compared with C3 species (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). One recent study proposed the opposite situation in closely related C3 and C4 PACMAD clade grasses (Osborne and Sack 2012). The authors argued that the low stomatal conductance (gs) values in C4 species save the hydraulic system from embolism, especially as leaf temperature increases in open environments, and stomata are stimulated to remain open under low CO2 . Their model further demonstrated that the low gs values in C4 also decrease stomatal sensitivity to hydraulic feedbacks, allowing stomata to remain open and photosynthesis to continue under drought, but only if C4 species have similar or higher whole-plant hydraulic conductance relative to C3 species, and thus a higher hydraulic supply relative to demand. However, the reason for why a species should have a high hydraulic supply relative to demand needs further explanation. The reason for this discrepancy may
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be due to differences between monocots and dicots or something not accounted for in the model. Growth properties One other objective of this study was to test whether higher photosynthetic capacity and WUE would reflect any differences in growth, biomass and allocation properties in C4 compared with C3 species. The C4 plants, in general, produced higher biomass compared with the C3 plants. For example, the C4 Cleome had higher shoot, root and whole plant FW and DW, although some of the parameters not being significantly different, than the counterpart C3 species (Fig. 4). As C4 plants have reduced water demand for photosynthesis, they would acquire less water from the soil, therefore, invest less into the root system. This can be seen from reduced root to shoot ratio in the C4 compared with the C3 . Moreover, the C4 plants produced thicker and taller stems relative to the C3 . In an earlier study, Pearcy et al. (1981) compared relative growth rates of C4 A. retroflexus and C3 C. album grown under three temperature regimes. They found that under cool growth temperature (17/14∘ C, day/night), Chenopodium had higher growth rate, however, under warm growth temperature (34/28∘ C), Amaranthus showed higher growth rate whereas under moderate growth temperature of 25/18∘ C, the two species produced the same growth rates (Pearcy et al. 1981). They also found that the competitive abilities in mixtures showed similarities to the photosynthetic performances as such Amaranthus having an advantage at high temperatures and Chenopodium at low temperatures. However, in contrast to temperature, growth of the plants under limited water supply had no effect on the competitive interactions, concluding that the presence of the C4 pathway alone was not sufficient to produce a competitive advantage over the C3 species under water limited conditions. In a more recent phylogenetically controlled study, Taylor et al. (2010) compared leaf physiology and growth in multiple lineages of C3 and C4 NAD-ME and NADP-ME subtypes grasses sampled from a monophyletic clade. They found that although C4 species had lower stomatal conductance and water potential deficits, and higher WUE, photosynthetic rates and photosynthetic nitrogen-use efficiency (PNUE), there were no systematic differences in biomass partitioning and growth rate between the photosynthetic types (Taylor et al. 2010). The reason for this is probably that the comparison was complicated by the growth habit, annual or perennial, and different evolutionary adaptation to habitats where the species are collected. The statistical analysis showed that there were significant differences 463
between annual and perennial growth habit (Taylor et al. 2010). In another comparison of very closely related C3 and C4 subspecies of Alloteropsis semialata, photosynthesis was higher and unaffected by nitrogen (N) treatments in the C4 than the C3 subspecies, and the C4 produced more biomass at high N levels, diverting a higher portion of growth into inflorescences and corms but less into roots and leaves compared with C3 subspecies (Ripley et al. 2008). Higher PNUE of the C4 was linked with greater investment in sexual reproduction and storage, and the avoidance of N-limitations on leaf growth, suggesting advantages of the C4 in disturbed and infertile environments. In conclusion, the results from this study show, once more, superior carbon gain and water savings of C4 plants resulting in secondary consequences that decreased hydraulic transport of the xylem and increased biomass production relative to similar C3 species. The differences in hydraulic properties between C3 and C4 photosynthesis is very important to consider when engineering C4 into C3 photosynthesis. If the genes controlling hydraulic structure and function in plants can be identified using recent technological approaches such as next-generation sequencing technologies to study the transcriptome of a species without a sequenced genome, then these genes can be engineered into C3 crops. The hydraulic advantage of C4 could be important to realize, for example, the full potential of C4 rice to deliver improved resource-use efficiency. The results also indicate that any factor affecting WUE of plants may contribute to xylem evolution over time. One important factor affecting WUE is changes in atmospheric CO2 concentration over geological time that may have contributed significantly to xylem evolution. Acknowledgements – This research was supported by a grant, no. 106O384, from the Scientific and Technological Research Council of Turkey (TUB˙ITAK) to F. K. The author thanks Dr Colin P. Osborne for comments on this manuscript.
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© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
Photosynthetic, hydraulic and biomass properties in closely related C3 and C4 species Ferit Kocacinara,b,* of Forestry, Department of Forest Engineering, Division of Plant Physiology, Kahramanmara¸s Sütçü ˙Imam Üniversitesi, Kahramanmara¸s 46100, Turkey b Department of Bioengineering and Sciences, Kahramanmara¸s Sütçü ˙Imam Üniversitesi, Kahramanmara¸s 46100, Turkey a Faculty
Correspondence *Corresponding author, e-mail: [email protected] Received 28 January 2014; revised 30 April 2014 doi:10.1111/ppl.12240
In plants, most water is absorbed by roots and transported through vascular conduits of xylem which evaporate from leaves during photosynthesis. As photosynthesis and transport processes are interconnected, it was hypothesized that any variation in water transport demand influencing water use efficiency (WUE), such as the evolution of C4 photosynthesis, should affect xylem structure and function. Several studies have provided evidence for this hypothesis, but none has comprehensively compared photosynthetic, hydraulic and biomass allocation properties between C3 and C4 species. In this study, photosynthetic, hydraulic and biomass properties in a closely related C3 Tarenaya hassleriana and a C4 Cleome gynandra are compared. Light response curves, measured at 30∘ C, showed that the C4 C. gynandra had almost twice greater net assimilation rates than the C3 T. hassleriana under each increasing irradiation level. On the contrary, transpiration rates and stomatal conductance were around twice as high in the C3 , leading to approximately 3.5 times higher WUE in the C4 compared with the C3 species. The C3 showed about 3.3 times higher hydraulic conductivity, 4.3 times greater specific conductivity and 2.6 times higher leaf-specific conductivity than the C4 species. The C3 produced more vessels per xylem area and larger vessels. All of these differences resulted in different biomass properties, where the C4 produced more biomass in general and had less root to shoot ratio than the C3 species. These results are in support of our previous findings that WUE, and any changes that affect WUE, contribute to xylem evolution in plants.
Introduction During photosynthesis, plants transpire large quantities of water to the atmosphere in exchange for CO2 acquisition. Therefore, over evolutionary time there should be a balance in plants for this exchange under each specific environmental niche. This balance is best reflected in the xylem structure and functional properties, where safety vs efficiency features play important roles in plant distribution, competition and survival (Tyree and Zimmermann 2002, Sperry 2003). Under
marginal lands, such as semiarid, arid and saline habitats, the balance between safety and efficiency is shifted toward safety traits, reflecting adaptations to water stress and high xylem tension. In humid and resource-rich environments, however, where fast growth and above ground allocation is more beneficial to plants, the evolutionary balance is shifted toward hydraulically efficient xylem needed to feed this greater leaf area and speedy growth. In an invasive species Solidago canadensis, for example, specific hydraulic conductivity of stem (Ks ) was about twofold and leaf-specific hydraulic conductivity
Abbreviations – DW, dry weight; FW, fresh weight; gs, stomatal conductance; PNUE, photosynthetic nitrogen-use efficiency; PPFD, photosynthetic active photon flux density; VF, vessel frequency; VPD, vapor pressure deficit; WUE, water use efficiency.
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(KL ) was about 1.5 times higher at the humid than at the drier site (Nolf et al. 2014). Also, vulnerability of the xylem to drought-induced embolism decreased, and safety margins increased along the transect from humid to drier sites. This indicates a trade-off between hydraulic safety and efficiency and may help explain invasive potential of this species (Nolf et al. 2014). Other intrinsic evolutionary traits such as high root to shoot ratio, better and more efficient stomatal regulation, higher water storage capacity and C4 biochemical pathway with higher water use efficiency (WUE) could also affect this balance by affecting xylem structure and function (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). Species with the C4 biochemical pathway concentrate CO2 around Rubisco, either in bundle sheath cells or in deeper site of elongated mesophyll cells, thereby significantly increasing carboxylation efficiency and reducing photorespiration. The rate and the efficiency of assimilation are higher in C4 than C3 plants under low atmospheric CO2 , and under high light, temperature and vapor pressure deficit (VPD), suggesting these factors as important evolutionary selective agents (Osborne and Sack 2012). All of these factors significantly increase photorespiration and reduce WUE of plants. Because the C4 photosynthetic pathway has independently evolved over 60 times in at least 19 families (Sage et al. 2011), this multiple convergent evolution provides excellent tool to study and understand the repeated evolution of C4 associated traits in closely related C3 and C4 species (Way 2012). Evolution of the C4 photosynthetic pathway has significantly increased WUE in C4 plants, where C4 plants exhibit two to four times higher instantaneous WUE than ecologically and taxonomically similar C3 plants (Osmond et al. 1982, Pearcy and Ehleringer 1984, Long 1999, Kocacinar et al. 2008, Vogan and Sage 2011). The discovery of the strong coordination between hydraulic and photosynthetic processes in plants, (Hubbard et al. 1999, Brodribb and Feild 2000, Hubbard et al. 2001, Brodribb et al. 2002, Franks and Brodribb 2005, Kocacinar et al. 2008), provided new venues for comparative studies. Since then, several studies comparing ecologically and taxonomically similar C3 and C4 plants have shown that WUE influences xylem structure and hydraulic function to the leaf canopy (Kocacinar and Sage 2003, Kocacinar 2004, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). In all comparisons, C4 plants exhibit lower KL than C3 plants of similar functional type, ecological habitat and phylogenetic distance (Kocacinar and Sage 2003, Kocacinar 2004, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008).
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For example, in a comparison of KL and vascular anatomy between eight C3 and eight C4 herbaceous species, the C3 species consistently showed higher KL , longer and wider vessels than the C4 (Kocacinar and Sage 2003). The KL differences between C3 and C4 species generally corresponded to the twofold to fourfold differences in WUE between C3 and C4 plants. In the comparison group of woody perennial plants adapted to arid habitats, where xylem safety traits provide a priority for adaptation and a competitive advantage, the lower KL of C4 plants was associated with changes in xylem structure that increase hydraulic safety, whereas in the comparison group of annual species from more humid habitats, the lower KL was associated with greater leaf area per unit xylem in C4 compared with C3 species (Kocacinar and Sage 2003, Kocacinar and Sage 2004). The hydraulic and xylem pattern differences between C3 and C4 plants are present in both herbaceous (Kocacinar and Sage 2003) and woody species (Kocacinar and Sage 2004), and between species of similar ecological habit and close taxonomic affinity (Kocacinar et al. 2008). Moreover, these trends were present in a statistically robust comparative analysis in the genus Flaveria (Kocacinar et al. 2008), where the phylogeny is well established based on chloroplastic trnl-F, nuclear internal transcribed spacer (ITS) and external transcribed sequence (ETS) (McKown et al. 2005). Among the 16 C3 , C3 –C4 intermediate, C4 -like and C4 Flaveria species compared, the C3 species had the highest KL compared with the intermediate, C4 -like and C4 species (Kocacinar et al. 2008). Ks was also generally high in the C3 and low in the intermediate and C4 species. The differences were also evident in the xylem structure, where xylem vessels were shorter, narrower and higher in number per xylem area in C3 –C4 intermediate, C4 -like and C4 species compared with C3 species (Kocacinar et al. 2008). The differences in these patterns were more pronounced as C4 -cycle activity increased in the C3 –C4 intermediates and moving from intermediates to C4 -like and C4 species. In Flaveria, three main groups of intermediates are classified with varying degree of C4 anatomy and physiology: type I, type II and C4 -like intermediates, each with increasing degree of C4 traits (Sage 2004, McKown and Dengler 2007, Kocacinar et al. 2008, Gowik and Westhoff 2011, Vogan and Sage 2011). However, WUE is not enhanced in type I and type II intermediates, where C4 -like intermediates show similar values to full C4 species (Kocacinar et al. 2008, Vogan and Sage 2011). There was also a strong correlation between KL and Ks and between KL contrasts and WUE contrasts using the Flaveria phylogeny of McKown et al. (2005). These studies provided strong evidence that WUE and xylem structure and functional properties 455
are significantly different between C3 and C4 species, but no study has comprehensively compared photosynthetic, hydraulic and biomass properties between close lineages of C3 and C4 species. In this study, hydraulics, photosynthesis, growth and WUE were compared between close lineages of C3 Tarenaya hassleriana and C4 Cleome gynandra, the most closely related C4 model to Arabidopsis (Brown et al. 2005, Voznesenskaya et al. 2007). Tarenaya hassleriana, formerly known as Cleome hassleriana and sometimes incorrectly referred as Cleome spinosa, and C. gynandra species are both from the family Cleomaceae, the sister family to Brassicaceae which includes Arabidopsis thaliana and Brassica crops (Bhide et al. 2014). The genome of T. hassleriana has been recently sequenced and can serve for understanding of gene, genome and trait evolution (Cheng et al. 2013). This study could serve as a physiological model for understanding the hydraulic advantages of C4 photosynthesis. If the results of this study show the same trend as all the previous studies in this field, then this can offer a potential for work on the genetic and genomic basis of altered WUE and hydraulics, because genes can be mapped from these two species onto the Arabidopsis reference genome. If we can discover genes that control WUE and hydraulics, then we will be able to engineer them in crop plants – either in rapeseed or rice, which is being engineered to use C4 , or in other crops where we may wish to change hydraulic conductance. This will be important to realize, for example, the full potential of C4 rice to deliver improved resource-use efficiency.
Materials and methods Plant material and growth conditions The seeds of the C4 species C. gynandra L. and the C3 species T. hassleriana (Chodat) H.H. Iltis were provided by Prof. Rowan F. Sage from the University of Toronto, Toronto, Ontario, Canada. The seeds of each species were collected from several different individual plants grown in an outdoor rooftop common garden in Toronto. The untreated seeds were kept at 4∘ C until sown in pots. Plants were grown from seeds in 10 l pots filled with a mix of 50% organic matter, 25% topsoil and 25% sand. The plants were grown outdoors in a common garden under natural conditions from April until June in 2009 at the Kahramanmara¸s Sütcü ˙Imam University, Kahramanmara¸s, Turkey. There was no precipitation effect as the common garden was covered, only on top, with a built transparent plastic cover. The average minimum and maximum temperatures were 15–20 and 30–35∘ C, respectively, during the growth period. 456
All plants were watered regularly as needed, two to three times a week, and fertilized once a week with a full-strength Hoagland’s solution (Sage and Pearcy 1987a). Gas exchange measurements and WUE Gas exchange measurements were conducted on randomly selected six plants from each species using a GFS-3000 portable gas exchange and fluorescence system (Heinz Walz GmbH, Effeltrich, Germany). Measurements were started at 10:00 h (mid-morning) and lasted until 16:00 h (mid-afternoon). All measurements were conducted indoor on a laboratory bench. A light response curve for each individual plant was constructed using measurements taken in the dark and under gradually increasing photosynthetic active photon flux density (PPFD) up to 2000 μmol m−2 s−1 , at a leaf temperature of 30∘ C, 380 μmol ambient CO2 and cuvette relative humidity of approximately 55%. For each light response curve, a plant was brought inside the laboratory and a fully expanded leaf from the tip was selected and enclosed into the leaf chamber to dark adapt for at least 30 min before measurements started. When a next level PPFD was set, assimilation rate increased immediately and reached a steady state after about 15–20 min. The progress was monitored for 5–10 min using the GFS-3000 software with a live-graph and several measurements were taken and averaged for each assimilation rate under each PPFD level. After a last measurement was taken at 2000 μmol m−2 s−1 PPFD, the leaf temperature was increased to 40∘ C and a new measurement was taken at this temperature in order to boost the difference between the C3 and the C4 species under elevated temperature. Under each light level, net photosynthesis, transpiration and stomatal conductance were measured, and WUE (mmol CO2 mol−1 H2 O) was calculated as net assimilation rate divided by transpiration rate. During all measurements, cuvette relative humidity was set to 55%, which was similar to the ambient humidity where plants were grown. This relative humidity corresponded to VPD of 1.5–2.2 and 2.3–2.9 kPa under 30 and 40∘ C leaf temperatures, respectively, in T. hassleriana and VPD of 2.0–2.4 and 3.3–3.6 kPa in C. gynandra. Stem hydraulic properties and xylem anatomy Hydraulic conductivity (Kh ) was measured on six different plants from each species as described previously in detail (Kocacinar and Sage 2003). All the stems sampled for hydraulic measurements were main stems of plants and had a complete and healthy leaf canopy to avoid leaf loss impacting on KL . Kh , Ks , and KL were assessed Physiol. Plant. 153, 2015
on stem segments ranging from 0.5 to 1.5 cm in diameter and from 8 to 10 cm in length. Stem segments were cut under water and connected to tubing filled with filtered (0.2 μm) tap water for all hydraulic measurements. Perfusion water was changed frequently to reduce the possibility of microbial growth in the tubing and the container hung above the segments. Although the stem segments were cut under water, in order to remove any possible natural embolism and to measure maximum Kh , the stem segments were first flushed with perfusion water under high pressure (150–200 kPa), generated by a pressure chamber (Model 1000, PMS Instrument Company, Albany, NY). Following a high pressure perfusion usually for 10–15 min, the flow through the stem segments was generated gravimetrically using a hydraulic head of between 5 and 20 kPa (Dryden and Van Alfen 1983). A plot of flow vs pressure was generated for each sample, taking at least three independent measurements under three different pressures. Kh was then calculated as the slope of the pressure-flow plot multiplied by segment length. However, differences in Kh could arise from different allocation strategies such as allocation to stem diameter, conducting xylem area, conduit diameters or xylem porosity (Kocacinar and Sage 2004). Therefore, variation in the xylem allocation was accounted for by dividing Kh by the total xylem cross-sectional area, determined using free-hand sections and a light microscope (Eclipse 90i, Nikon Instruments Europe B.V., Amstelveen, The Netherlands), to determine Ks . KL was calculated as Kh divided by total leaf area distal to the stem segment. Leaf area was measured using a LI-COR 3100 area meter (LI-COR Inc. Lincoln, NE). Xylem area, number of vessels per xylem area [vessel frequency (VF)], and conduit diameters were measured on stem cross-sections that had been previously used for Kh determination. For each species, six of the stem segments were fixed in FAA (formalin, acetic acid and alcohol), rinsed with tap water, free hand sectioned, stained with an aqueous solution containing 0.05% toluidine blue O (TBO, Sigma-Aldrich Chemie Gmbh, Munich, Germany) (O’Brien et al. 1964), and examined under the light microscope. Images were analyzed with an imaging software (NIS-Elements, Microscope Imaging Software, Nikon Instruments Europe B.V. Amstelveen, The Netherlands). The vessel diameters were measured as the average of the lengths of major and minor axes of the conduits. Maximum vessel diameter (MVD) was measured on the largest vessel from each cross-section. Vessel mean diameter (VMD) was determined as the mean of all the vessels in a randomly chosen part of the xylem area from each cross-section. VF was determined as the number of vessels per xylem area on randomly chosen sectors from each cross-section.
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Growth properties Five plants from each species were randomly selected for growth analysis at approximately 60 days old, before flowering had commenced. Stem height was measured from the soil surface to tip of the stem using a ruler, and root collar diameter was determined on the same stems at the soil surface using a digital caliper. Shoot fresh weights (FWs) were immediately determined after excising the shoots from the soil surface. Shoot dry weights (DWs) were measured after oven drying samples for 48 h at 70∘ C. Roots were carefully washed, dried of excess water with paper towels and weighed for fresh root mass. Roots were then dried for 48 h at 70∘ C then weighed again for root dry mass. Data analysis Data were statistically tested using SIGMASTAT software (SYSTAT software, Richmond, CA). Mean values of all parameters between the C3 and the C4 species were compared using a one-way ANOVA followed by least significance difference (LSD) test (P < 0.05). VF and vessel diameters data were log-transformed, because these data are not normally distributed and failed the normality test.
Results Gas exchange properties and WUE The C4 C. gynandra had significantly higher net photosynthetic rate than the C3 T. hassleriana under each PPFD (Fig. 1A). The difference in net photosynthetic rate between the C4 and the C3 was widened as PPFD increased up to 2000 μmol m−2 s−1 . At PPFD of 2000 μmol m−2 s−1 , net assimilation rate was almost two times greater in the C4 than the C3 species measured at leaf temperature of 30∘ C (Fig. 1A). When leaf temperature was raised to 40∘ C, assimilation rate declined in the C3 but increased in the C4 , leading to approximately 2.5 times greater values in the C4 than the C3 species, 33.8 vs 13.9 μmol CO2 m−2 s−1 , respectively (Fig. 1A). However, transpiration rates were significantly higher in the C3 than the C4 under each PPFD (Fig. 1B). Transpiration rates were almost 10 times higher in the dark, (2.6 vs 0.3 mmol H2 O m−2 s−1 ), and 2 times greater at maximum PPFD of 2000 μmol m−2 s−1 , (11.9 vs 5.9 mmol H2 O m−2 s−1 ), in the C3 than the C4 species, respectively (Fig. 1B). When leaf temperature was raised from 30 to 40∘ C, transpiration rates increased significantly in both species and were 1.8 times more in the C3 than the C4 (Fig. 1B). Stomatal conductance increased in both species as PPFD increased to 2000 μmol m−2 s−1 , however, it was almost 2.5 times greater in the C3 compared 457
Fig. 1. Net photosynthetic rate (A), transpiration rate (B), stomatal conductance (C) and WUE, (D) of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ) as a function of increasing PPFD. Measurements were taken at 380 μmol ambient CO2 , 55% relative humidity and 30∘ C leaf temperature. After the last measurement, leaf temperature was increased to 40∘ C and a new measurement was taken. Each point is the mean of six different plants. Vertical lines represent ±1 SE of the mean.
with the C4 under each PPFD level (Fig. 1C). Raising leaf temperature to 40∘ C at 2000 μmol m−2 s−1 PPFD significantly increased stomatal conductance in both species, but the difference was much greater in the C3 than the C4 species (Fig. 1C). WUE values reached a maximum at 500 PPFD and remained constant as PPFD increased from 500 to 2000 μmol quanta m−2 s−1 in both species (Fig. 1D). Under each of the PPFD levels above 500, WUE was on average 1.5 mmol CO2 mol−1 H2 O in the C3 T. hassleriana and significantly higher, about 3.5 times higher (5.0 mmol CO2 mol−1 H2 O) in the C4 C. gynandra species (Fig. 1D). When leaf temperature was raised to 40∘ C at 2000 PPFD, WUE was reduced by 40% in the C4 , from 5 to 3 mmol CO2 mol−1 H2 O, and by 50% in the C3 species, from 1.4 to 0.7 mmol CO2 mol−1 H2 O (Fig. 1D). Stem hydraulic properties and xylem anatomy Stem hydraulic conductivity was significantly higher, about 3.3 times more, in the C3 than the C4 species (P = 0.004) (Fig. 2A). Although the C4 had slightly higher mean xylem cross sectional area (19.6 mm2 ) than 458
the C3 (15.5 mm2 ), the difference was not significant (P = 0.145), therefore, Ks was also significantly higher in the C3 than the C4 species (P < 0.001) (Fig. 2B). Ks was about 0.6 kg m−1 s−1 MPa−1 in the C4 and approximately 4.3 times more, 2.6 kg m−1 s−1 MPa−1 , in the C3 plants (Fig. 2B). Even though the C3 produced slightly more leaf area than the C4 , the difference was not significant (P = 0.14, Fig. 2C). When leaf area was accounted for, KL was 2.6 times higher in the C3 than the C4 species (Fig. 2D). KL was 1.2 × 10−4 kg m−1 s−1 MPa−1 in the C4 and 3.1 × 10−4 kg m−1 s−1 MPa−1 in the C3 species (Fig. 2D). The significant differences in the xylem hydraulics between the C3 and the C4 were probably a result of the differences in xylem anatomical properties. The C3 produced more vessels per xylem area and significantly larger vessels than the C4 Cleome species (Table 1 and Fig. 3). Maximum and mean vessel diameters were significantly higher in the C3 compared with the C4 Cleome species (Table 1). Differences in vessel number and size were also obvious in representative xylem cross-section micrographs (Fig. 3).
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Fig. 2. Hydraulic conductivity (Kh ) (A), specific conductivity (Ks ) (B), total leaf area per stem (C) and leaf specific conductivity (KL ) (D), of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ). Bars are means (±1 SE) of six different plants. Different letters represent significant differences (P < 0.05). Table 1. Xylem anatomical parameters of the C3 Tarenaya hassleriana and the C4 Cleome gynandra species. Maximum vessel diameter (MVD) was measured on the largest vessel from each cross-section. Vessel mean diameter (VMD) was determined as the mean of all the vessels in part of the xylem from each cross-section. VF was determined as the number of vessels in part of the xylem from each cross-section. Different letters represent significant differences (P < 0.05). Values are means (±1 SE) of six samples for each species. Species T. hassleriana C. gynandra
Photo. type
MVD (μm)
VMD (μm)
VF (no. mm−2 )
C3 C4
73.9 ± 1.5a 63.2 ± 3.4b
66.1 ± 1.3a 58.0 ± 3.3b
108 ± 9.0a 66 ± 8.0b
Growth properties Differences in photosynthetic and hydraulic properties were reflected in growth properties and biomass allocation differences between the C3 and the C4 species (Figs Physiol. Plant. 153, 2015
4 and 5). Shoot FW was significantly higher in the C4 than the C3 species (Fig. 4A). Shoot FWs were 66.4 g per plant in the C4 and 39.8 g per plant, 40% less, in the C3 species (Fig. 4A). Although the difference was not significant (P = 0.056), the C3 produced about 45% less shoot DW than the C4 species; 5.1 g shoot DW per plant in the C3 and 9.4 g per plant in the C4 , respectively (Fig. 4A). Even though, root FW and DW were not significantly different between the two species, the C3 produced 30% less root DW (1.0 g per plant) than the C4 species (1.4 g per plant) (Fig. 4B). Whole plant FW and DW were, respectively, 37 and 44% less in the C3 than the C4 species (Fig. 4C). Root to shoot ratio was significantly higher in the C3 than the C4 species (Fig. 4D). Both root collar diameter and stem height were significantly higher in the C4 compared with the C3 species (Fig. 5A, B). 459
Fig. 3. Representative cross-sections of stem xylem of the C3 Tarenaya hassleriana (left) and the C4 Cleome gynandra (right). Note vessel size and number differences between the two species. The scale bar in the right bottom micrograph is 200 μm and applies to both species.
Discussion Gas exchange properties and WUE In most cases, C4 plants have superior photosynthetic rates, higher nitrogen, water and irradiation use efficiencies compared with C3 species of similar ecological habitat or taxonomic affinity (Osmond et al. 1982, Ehleringer and Pearcy 1983, Pearcy and Ehleringer 1984, Wong et al. 1985, Sage and Pearcy 1987a, 1987b, Long 1999, von Caemmerer and Furbank 1999, Sage and Pearcy 2000, Kocacinar et al. 2008, Vogan and Sage 2011, Cristiano et al. 2012). In this study, gas-exchange properties under increasing PPFD levels, stem hydraulics including xylem anatomy and growth properties were all investigated in taxonomically close C4 C. gynandra and C3 T. hassleriana species. The C4 species showed almost two times more net assimilation rates especially under increasing PPFD levels than its counterpart C3 species (Fig. 1A). Besides this superiority in net assimilation rates, the C4 made more efficient use of high irradiation than the C3 ; for example, the C3 reached its maximum assimilation rates at PPFD of 1000 μmol quanta m−2 s−1 , whereas the C4 continued to increase its assimilation rate up to 2000 μmol quanta m−2 s−1 (Fig. 1A). Moreover, under saturating 2000 μmol quanta m−2 s−1 PPFD, raising the leaf temperature from 30 to 40∘ C increased net photosynthetic rate in the C4 by about 14%, but decreased it in the C3 by almost 15% (Fig. 1A). Net photosynthetic rate was 2.4 times higher in the C4 than the C3 at leaf temperature of 40∘ C and 2000 μmol quanta m−2 s−1 PPFD. The superiority in net assimilation rates, irradiation use and high temperature optima in plants utilizing C4 biochemistry was recognized even before the discovery of the C4 biochemical pathway (Hesketh 1963, Hesketh and Moss 1963, Murata and Iyama 1963, El-Sharkaway and Hesketh 1964). Following the discovery of C4 biochemistry in the late 1960s, 460
especially under high temperature and in heat adapted species, many studies have reported higher rates of photosynthesis at all light levels or higher quantum efficiency in C4 plants compared with similar C3 species (Osmond et al. 1980, Pearcy and Ehleringer 1984). Previously, it was also reported that C4 photosynthesis is characterized to be light saturated only at very high light intensities as evidenced by C4 Flaveria bidentis wild-type plants compared with transgenic F. bidentis with reduced amount of Rubisco (Furbank et al. 1996, Siebke et al. 1997, von Caemmerer and Furbank 1999). Transgenic F. bidentis plants saturated at much lower irradiances. Superior net photosynthetic rates of C4 were also evident in a comparison between the cold adapted co-occurring C3 Calamogrostis canadensis and C4 Muhlenbergia glomerata grasses native to high latitudes (Kubien and Sage 2004). The C4 M. glomerata showed about 40 and 80% more net photosynthesis rate, measured at leaf temperatures of 20 and 30∘ C, respectively, when compared with the co-occurring C3 C. canadensis, both grown at warm conditions (26/22∘ C) (Kubien and Sage 2004). The difference in net assimilation rates between C4 and C3 plants under increasing both PPFD and leaf temperature is partly due to the difference in photorespiration between these two functional groups. The C4 grass Panicum maximum showed no apparent increase in photorespiration and CO2 compensation concentration as PPFD raised from near darkness to 2000 μmol quanta m−2 s−1 and from 15 to 40∘ C leaf temperature compared with significantly increased values in the C3 grass Festuca arundinacea (Brown and Morgan 1980). In this study, quantum yield for CO2 uptake, calculated as the slope of the response curve relating net CO2 uptake to incident photon flux at unsaturated light of 250 μmol quanta m−2 s−1 , quantum yield not corrected for leaf absorbance and assuming all photons are absorbed, was 0.03 μmol CO2 per μmol photons for Physiol. Plant. 153, 2015
Fig. 4. Shoot fresh and dry weight (A), root fresh and dry weight (B), whole plant fresh and dry weight (C) and root to shoot ratio of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ). Bars are means (±1 SE) of five plants per species. Different letters represent significant difference between species, where in some cases P values are given to compare the means.
the C4 C. gynandra and 0.02 μmol CO2 per μmol photons for the C3 T. hassleriana. The C4 fixed 50% more carbon per photon compared with the C3 species measured at 30∘ C leaf temperature, 380 ppm ambient CO2 and 55% relative humidity. Similar results were reported for the heat adapted C4 Tidestromia oblongifolia and the C3 Larrea divaricata measured at leaf temperature of 40∘ C, where quantum yield (the slope of light response curve) was calculated as 0.03 for the C4 and 0.02 for the C3 at low light intensities (Osmond et al. 1980). Ehleringer and Pearcy (1983) surveyed the quantum yields of large number of C3 and C4 species and reported higher values than found in this study, ranging from 0.046 to 0.055 mol CO2 per mol photons for the C3 dicots and 0.052 to 0.069 mol CO2 per mol photons for the C4 species. In this survey, however, quantum yield calculations were done at very low light intensities, below 20 μmol quanta m−2 s−1 , and
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were corrected for different leaf absorbances, which resulted in higher values than reported here in this study. The C3 species T. hassleriana lost a significantly greater amount of water to the atmosphere as evidenced by very high rates of transpiration and stomatal conductance under each photon flux density compared with the C4 species C. gynandra. Transpiration and stomatal conductance rates were at least two and three times higher, respectively, in the C3 than the C4 species. In a phylogenetically controlled analysis, Taylor et al. (2012) also showed that C4 grasses had significantly lower maximum stomatal conductance rates (gmax ) than their C3 relatives. To account for differences in habitat, C4 species showed lower gmax than C3 species in both mesic and dry environments, demonstrating intrinsic stomatal traits differences between the two groups (Taylor et al. 2012). In a comparison between co-occurring species of C3 Chenopodium album and C4 Amaranthus retroflexus, 461
Fig. 5. Root collar diameter (A) and stem height (B) of the C4 Cleome gynandra ( ) and the C3 Tarenaya hassleriana ( ). Bars are means (±1 SE) of five plants per species. Different letters represent significant differences (P < 0.05).
Pearcy et al. (1981) reported three times higher leaf conductance under low (17∘ C) and moderate (25∘ C) growth temperatures and two times more leaf conductance under high growth temperature (34∘ C) in Chenopodium relative to Amaranthus. Similar results were also reported for a number of C3 and C4 species grown under different nutrition, light and ambient CO2 regimes (Wong et al. 1985). One striking example is the difference in gas exchange properties between the C4 Zea mays and the C3 Gossypium hirsutum, grown under various nitrate regimes. For the same photosynthetic rate, G. hirsutum exhibited two times higher leaf conductance than Z. mays under each nitrate treatment (Wong et al. 1985). In this study, the significant differences in transpiration rates and stomatal conductance between the C3 and the C4 species resulted in a 3.5 times higher photosynthetic WUE in the C4 plants (Fig. 1D). WUE averaged about 5.0 and 1.4 mmol CO2 per mol H2 O in the C4 and the C3 plants, respectively. Caldwell et al. (1977) compared photosynthetic WUE for the C4 Atriplex confertifolia and the C3 Ceratoides lanata, growing together either in mixed communities or in mono-specific stands next to each other in a cold desert area in Utah. They found that the two species did not show any significant WUE differences during the cool spring when soil water was ample. However, during the hot summer the C4 Atriplex showed superior values; estimated annual average WUE for growth of 4.3 and 2.9 mg DW g−1 H2 O in A. confertifolia and C. lanata, respectively (Caldwell et al. 1977). Similar field results were also reported for the Death Valley, California evergreen drought-tolerant shrubs C4 Atriplex hymenelytra and C3 L. divaricata (Osmond et al. 1980). The WUE of A. hymenelytra was two to three times higher than L. divaricata under water stress down 462
to −4.6 MPa. In a comparison among C3 , C3 –C4 intermediate and C4 grass species under different nitrogen nutrition, the C4 P. maximum exhibited superior apparent photosynthesis especially under higher leaf nitrogen concentration and two times higher WUE at all leaf nitrogen levels compared with both the C3 –C4 intermediate Panicum milioides and the C3 F. arundinacea species, which showed similar values (Bolton and Brown 1980). The superiority of photosynthetic capacity, quantum yield and WUE of C4 has been suggested to provide the greatest adaptive advantage under xeric, hot, saline and open high light environments (Pearcy and Ehleringer 1984). Numerous studies confirm a greater C4 abundance in warmer, open, high light and tropical environments (Osmond et al. 1982, Hattersley 1983, Pearcy and Ehleringer 1984, Sage et al. 1999). Stem hydraulic properties and xylem anatomy Because of the well-established co-ordination between hydraulic conductance and stomatal conductance, therefore, photosynthesis in plants (Hubbard et al. 1999, Brodribb and Feild 2000, Hubbard et al. 2001, Brodribb et al. 2002, Kocacinar et al. 2008), it was hypothesized that any innovation during the evolution of plants, such as evolving C4 photosynthesis with superior WUE, would have affected water transport demand, namely, hydraulic properties of the xylem. In this study, significant hydraulic properties differences between the C4 and the C3 species supported this hypothesis once more. The C3 T. hassleriana showed almost 3.3, 4.3 and 2.6 times higher Kh , Ks and KL , respectively, compared with the C4 C. gynandra species. These results are consistent with the results of previous large comparisons with 51
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C3 and C4 species of close taxonomic affinity, similar ecological habitat and growth form (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). Within the previous 51 comparisons including the one in this study, KL was consistently lower in C4 compared with C3 species of a similar life form and taxonomic group, within any comparison group not a single C3 species showed lower KL . Associated with the differences in KL and WUE, C4 species in these previous comparisons had vessels that were similar to, or shorter and narrower than the corresponding C3 species (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). Here in this comparison, the C4 also showed significantly narrower conduit diameters but almost twice higher VF per xylem area than the C3 species (Table 1 and Fig. 3). In Flaveria, significant phylogenetic relationship between WUE and KL was previously established (Kocacinar et al. 2008). The results from Flaveria and other earlier studies by Kocacinar and Sage (2003, 2004) including the results found in this study show strong support for the hypothesis that C4 evolution changed the transport demand of the leaf canopy and resulted in safer xylem and lower KL . Where the xylem anatomy properties were found to be comparable between the C4 and the C3 species in a comparison group, C4 plants seemed to utilize the WUE advantage by having larger leaf canopy especially in species adapted to mesic habitats. In comparisons of species adapted to more xeric habitats, where xylem safety maintenance is more crucial for survival and persistence than having larger leaf canopy, C4 plants utilized WUE advantage by xylem modification mainly having shorter and narrower but more frequent conduits which confer safer xylem in C4 compared with C3 species (Kocacinar and Sage 2003, Kocacinar and Sage 2004, Kocacinar and Sage 2005, Kocacinar et al. 2008). One recent study proposed the opposite situation in closely related C3 and C4 PACMAD clade grasses (Osborne and Sack 2012). The authors argued that the low stomatal conductance (gs) values in C4 species save the hydraulic system from embolism, especially as leaf temperature increases in open environments, and stomata are stimulated to remain open under low CO2 . Their model further demonstrated that the low gs values in C4 also decrease stomatal sensitivity to hydraulic feedbacks, allowing stomata to remain open and photosynthesis to continue under drought, but only if C4 species have similar or higher whole-plant hydraulic conductance relative to C3 species, and thus a higher hydraulic supply relative to demand. However, the reason for why a species should have a high hydraulic supply relative to demand needs further explanation. The reason for this discrepancy may
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be due to differences between monocots and dicots or something not accounted for in the model. Growth properties One other objective of this study was to test whether higher photosynthetic capacity and WUE would reflect any differences in growth, biomass and allocation properties in C4 compared with C3 species. The C4 plants, in general, produced higher biomass compared with the C3 plants. For example, the C4 Cleome had higher shoot, root and whole plant FW and DW, although some of the parameters not being significantly different, than the counterpart C3 species (Fig. 4). As C4 plants have reduced water demand for photosynthesis, they would acquire less water from the soil, therefore, invest less into the root system. This can be seen from reduced root to shoot ratio in the C4 compared with the C3 . Moreover, the C4 plants produced thicker and taller stems relative to the C3 . In an earlier study, Pearcy et al. (1981) compared relative growth rates of C4 A. retroflexus and C3 C. album grown under three temperature regimes. They found that under cool growth temperature (17/14∘ C, day/night), Chenopodium had higher growth rate, however, under warm growth temperature (34/28∘ C), Amaranthus showed higher growth rate whereas under moderate growth temperature of 25/18∘ C, the two species produced the same growth rates (Pearcy et al. 1981). They also found that the competitive abilities in mixtures showed similarities to the photosynthetic performances as such Amaranthus having an advantage at high temperatures and Chenopodium at low temperatures. However, in contrast to temperature, growth of the plants under limited water supply had no effect on the competitive interactions, concluding that the presence of the C4 pathway alone was not sufficient to produce a competitive advantage over the C3 species under water limited conditions. In a more recent phylogenetically controlled study, Taylor et al. (2010) compared leaf physiology and growth in multiple lineages of C3 and C4 NAD-ME and NADP-ME subtypes grasses sampled from a monophyletic clade. They found that although C4 species had lower stomatal conductance and water potential deficits, and higher WUE, photosynthetic rates and photosynthetic nitrogen-use efficiency (PNUE), there were no systematic differences in biomass partitioning and growth rate between the photosynthetic types (Taylor et al. 2010). The reason for this is probably that the comparison was complicated by the growth habit, annual or perennial, and different evolutionary adaptation to habitats where the species are collected. The statistical analysis showed that there were significant differences 463
between annual and perennial growth habit (Taylor et al. 2010). In another comparison of very closely related C3 and C4 subspecies of Alloteropsis semialata, photosynthesis was higher and unaffected by nitrogen (N) treatments in the C4 than the C3 subspecies, and the C4 produced more biomass at high N levels, diverting a higher portion of growth into inflorescences and corms but less into roots and leaves compared with C3 subspecies (Ripley et al. 2008). Higher PNUE of the C4 was linked with greater investment in sexual reproduction and storage, and the avoidance of N-limitations on leaf growth, suggesting advantages of the C4 in disturbed and infertile environments. In conclusion, the results from this study show, once more, superior carbon gain and water savings of C4 plants resulting in secondary consequences that decreased hydraulic transport of the xylem and increased biomass production relative to similar C3 species. The differences in hydraulic properties between C3 and C4 photosynthesis is very important to consider when engineering C4 into C3 photosynthesis. If the genes controlling hydraulic structure and function in plants can be identified using recent technological approaches such as next-generation sequencing technologies to study the transcriptome of a species without a sequenced genome, then these genes can be engineered into C3 crops. The hydraulic advantage of C4 could be important to realize, for example, the full potential of C4 rice to deliver improved resource-use efficiency. The results also indicate that any factor affecting WUE of plants may contribute to xylem evolution over time. One important factor affecting WUE is changes in atmospheric CO2 concentration over geological time that may have contributed significantly to xylem evolution. Acknowledgements – This research was supported by a grant, no. 106O384, from the Scientific and Technological Research Council of Turkey (TUB˙ITAK) to F. K. The author thanks Dr Colin P. Osborne for comments on this manuscript.
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