High C3 photosynthetic capacity and high intrinsic water use efficiency underlies the high productivity of the bioenergy grass Arundo donax

Arundo donax has attracted interest as a potential bioenergy crop due to a high apparent productivity. It uses C3 photosynthesis yet appears competitive with C4 grass biomass feedstock’s and grows in warm conditions where C4 species might be expected to be that productive. Despite this there has been no systematic study of leaf photosynthetic properties. This study determines photosynthetic and photorespiratory parameters for leaves in a natural stand of A. donax growing in southern Portugal. We hypothesise that A. donax has a high photosynthetic potential in high and low light, stomatal limitation to be small and intrinsic water use efficiency unusually low. High photosynthetic rates in A. donax resulted from a high capacity for both maximum Rubisco (Vc,max 117 μmol CO2 m−2 s−1) and ribulose-1:5-bisphosphate limited carboxylation rate (Jmax 213 μmol CO2 m−2 s−1) under light-saturated conditions. Maximum quantum yield for light-limited CO2 assimilation was also high relative to other C3 species. Photorespiratory losses were similar to other C3 species under the conditions of measurement (25%), while stomatal limitation was high (0.25) resulting in a high intrinsic water use efficiency. Overall the photosynthetic capacity of A. donax is high compared to other C3 species, and comparable to C4 bioenergy grasses.

Giant reed (Arundo donax L.) has attracted interest as a potential bioenergy crop, due to a high apparent productivity and suitability as an accepted feedstock for cellulosic fuel production 1 . It is found throughout the Mediterranean climate zones of the world and has been cultivated in Asia, southern Europe and North Africa for over a thousand years. It is a rhizomatous perennial herbaceous grass that produces stems of ca. 2-3 cm diameter that may grow to heights of 3-6 m. It is found in moist grasslands and wetlands although it is able to thrive across a wide range of soil types, while also being tolerant of mild drought and salinity, and is tolerant of water-logged soils [2][3][4] . In climates with warm winters it is evergreen but dies back in climates with freezing winter temperatures. Although there are related species within the genus, molecular analysis suggests that A. donax is monophyletic in origin and its spread has not involved hybridisation with the related species. These analyses also suggest that it originated in west Asia and then spread to the Mediterranean region 5 . The high productivity of A. donax is achieved in warm growing conditions despite the fact that it uses C3 photosynthesis, which might be expected to be a competitive disadvantage compared to C4 species under such growing conditions. Side-by-side trials in central Italy over 11 years showed an average yield of above-ground dry biomass of 28.7 t ha −1 yr −1 for the highly productive C4 perennial grass Miscanthus x giganteus Deu et Greef, but 37.7 t ha −1 yr −1 for A. donax 6 . Although this appears a relatively low yield for M. x giganteus under irrigated conditions in the Mediterranean 7 , the reported yield for A. donax is high by any measure. A. donax in central and southern Italy showed average dry matter yield over 3 years of up to 74 t ha −1 yr −1 , which exceeds the highest yields reported for M. x giganteus 8 . Similarly, in a multi-year study in Alabama A. donax achieved an average yield of 35.5 t ha −1 yr −1 compared to 23.5 t ha −1 yr −1 for switchgrass (Panicum virgatum L.) 9 . In a comparison with C4 sweet sorghum (Sorghum bicolor L.) in northern Italy, light penetration into an A. donax canopy studied over a three year period was found to be 60% higher and the efficiency of conversion of intercepted radiation into biomass was ca. 60% higher. This might be explained by its more effective distribution of light through the canopy, if it is coupled with a similar efficiency of leaf photosynthesis 10,11 . However, yields may be considerably lower on marginal land. On a sandy loam with limited nutrient availability A. donax required three years to accumulate 20 t ha −1 , and build a rhizome mass of 16 t ha −1 4 , by contrast to M. x giganteus which has proved significantly more productive on marginal sites 12 . While M. x giganteus was found particularly vulnerable to damage by water-logging of soils during autumn and winter, A. donax appeared to thrive in these situations. This tolerance of water-logging also allows A. donax to spread along watercourses from rhizome fragments where M. x giganteus cannot 13 . Its ability to survive anaerobic soil may explain its ability to tolerate high levels of reduced ions of heavy metals 14 . A. donax is clearly a resilient, but also very productive species. However, there is little fundamental knowledge available on how it is able to realise this productivity. Understanding this demands knowledge of leaf photosynthesis as well as canopy microclimate.
Leaf photosynthetic rates of CO 2 uptake (A) measured in A. donax growing on an estuary in S. Africa were between 20 and 37 μ mol m −2 s −1 15 . By combining modulated chlorophyll fluorescence and gas exchange, photorespiration was estimated to decrease A by 30%. Although water potential declined to − 2.1 MPa around noon on sunny days, stomatal conductance (g s ) and A changed little. Such low leaf water potential would normally be expected to at least cause partial stomatal closure in most species 15 . The rates of stomatal conductance reported are at the upper end of those found in other C3 species but not much greater than those of the productive C4 bioenergy grasses M. x giganteus and P. virgatum, as well as productive modern cultivars of maize (Zea mays L.) 16,17 .
In summary, A. donax achieves and exceeds the productivities normally associated with C4 perennial grasses in warm climates. For a plant which forms a high leaf area index (LAI) monocultures in nature and as a crop, it would be expected to have a high photosynthetic capacity at both high and low light intensity under warm conditions. The high photosynthetic capacity is needed in the context of a dense canopy where shaded leaves need to achieve high efficiency, as well as those in full sun, and where all leaves need to be efficient under the low light conditions of dawn, dusk and cloudy days. Yet it is a C3 species, growing in warm conditions where only a C4 species might be expected to be photosynthetically efficient 18 . The limited published data suggests that A. donax may indeed have high light-saturated leaf photosynthetic rates, which may also be supported by high leaf conductances. High leaf conductances will allow a higher CO 2 concentration at ribulose-1:5-bisphosphate carboxylase/ oxygenase (Rubisco), so increasing the rate of carboxylation and decreasing photorespiration, but at the expense of water loss and water use efficiency 19 .
To better define the photosynthetic capacity of A. donax and understand how this may explain its high productivity this study aimed to define key in vivo properties. These were: J max , the maximum whole chain electron transport rate supporting ribulose-1:5-bisphosphate (RuBP) regeneration, V c,max the maximum rate of carboxylation that can be supported by Rubisco, the light-saturated rate of CO 2 assimilation (A sat ) and the maximum quantum yield of CO 2 assimilation (Φ CO2,max ) defined by the initial slope of the response of A to absorbed photon flux (αI). Concurrent measurement of water vapour flux and modulated chlorophyll fluorescence were used to determine g s , stomatal limitation to CO 2 uptake (l), intercellular CO 2 concentration (c i ), and the whole chain electron transport rate (J). These parameters were determined for leaves in a natural stand of A. donax growing in a dry valley in S. Portugal. These are used to test the hypotheses that A. donax has a high photosynthetic potential in both high and low light, that stomatal limitation may be unusually small and thus intrinsic leaf water use efficiency poor. The in vivo measures: J max , V c,max , A sat and Φ CO2,max , are key to parameterization of the steady-state biochemical model of C3 photosynthesis 20 which underlies most models of crop and ecosystem productivity 21 . This study therefore also serves as a key resource for parameterizing production models of this emerging crop.

Materials and Methods
Field location. The study was undertaken on a naturally occurring A. donax stand located at Quinta de São Pedro -Centro de estudos (Armadas, 2815-786 Sobreda, Portugal 38°38′40.6″ N 9°11′ 34.5″ W, altitude 85 m). A mixed 5 acre site with a disturbed dry valley community of annuals, patches of native macchia and scrub, pine woodland, introduced Acacia woodland and a few planted ornamentals on a Cambisol soils 22 . Meteorological assessment. Relative humidity, air temperature and irradiance were measured over the sampling period using a Weather Station (WS-GP2 with GP2 Data Logger, Delta-T Devices Ltd, Cambridge, UK) (Fig. S1). Relative Humidity and air temperature were used to calculate vapour pressure deficit (VPD), as a virtual channel within the GP2. Soil temperature and soil water content were measured within the stand over the sampling period using soil moisture sensors (SM300 with GP2 Data Logger, Delta-T Devices Ltd, Cambridge, UK).

Stomatal Conductance and
In situ Chlorophyll Fluorescence. Measurements of stomatal conductance were made with a diffusion porometer (AP4, Delta-T Devices Ltd, Cambridge, UK). All measurements of conductance and photosynthesis were made on the most recently expanded leaf, as judged by ligule emergences. Readings were taken on several leaves, locating the porometer at three points along each leaf: 1) the base just above the ligule, 2) the midpoint between ligule to tip; and 3) the area just below the leaf tip. Both the Scientific RepoRts | 6:20694 | DOI: 10.1038/srep20694 adaxial and abaxial surfaces were measured at these points at midday and mid-afternoon following the procedures of Monteith 23 , and Potter 24 . Modulated chlorophyll fluorescence was monitored to estimate the operating efficiency of PSII (F q ′ /F m ′ ) 25,26 on fully emerged leaves at midday and late afternoon using a fluorometer (MONITORING-PAM, Heinz Walz GmbH, Effeltrich, Germany) (Fig. S1).
Photosynthetic intercellular-CO 2 response curves. Leaves were sampled pre-dawn from the A. donax stand. Leaves were excised just above the ligule, and immediately re-cut under water and then kept in cool low-light conditions (20 °C and 20 μ mol m −2 s −2 ) until use. The response of net leaf CO 2 uptake rate (A) to external CO 2 concentration (c a ) was assessed on five different leaves. Leaves were placed in the leaf cuvette of a portable open gas exchange system (LCPro-SD with 6.25 cm 2 cuvette; ADC BioScientific Ltd. Herts, England; or LI6400XT with 6 cm 2 cuvette; LI-COR Biosciences, Lincoln, Nebraska, USA). Air temperature was controlled at 25 o C, leaf temperature 27 o C and VPD at 0.96 kPa. To fully induce photosynthesis before measurements commenced, leaves were first allowed to equilibrate at a photon flux (I) of 500 μ mol m −2 s −1 and reference c a of 400 μ mol mol −1 until A had reached a stable value, the I was then increase to 1500 μ mol m −2 s −1 . When A had reached a stable value at I = 1500 μ mol m −2 s −1 c a was then changed to the following levels in sequence, 300, 250, 200, 150, 100, 50, 400, 600, 900, 1200 and 1500 μ mol mol −1 . The leaf remained at each level until a stable A could be determined. Potential Rubisco carboxylation (V c,max ) and electron transport through photosystem II (J max ) were determined from the responses of A sat to c i . For each leaf values of A located above the transition between Rubisco-limited and RuBP-or electron transport-limited photosynthesis in the A/c i response were used to solve for J max using the equations representing RuBP regeneration-limited A 20 . Values were temperature adjusted to 25 o C using the temperature response equations of Bernacchi et al. (2001) 27 for the Rubisco-limited, and Bernacchi et al. (2003) 28 for the RuBP regeneration limited portions of the A vs c i curves. Stomatal limitation (l) at the current ambient c a of 400 μ mol mol −1 was derived using the method outlined in Long and Bernacchi (2003) 29 .
Photosynthetic light response curves. Sampling and measurements of A vs. I were performed in parallel with A vs. c i curves. Leaves were placed in the leaf cuvette of an integrated open gas exchange system and modulated chlorophyll fluorometer (GFS-3000FL, LED-Array/PAM-Fluorometer 3055-FL and 3080-O2 Oxygen sensor. Heinz Walz GmbH, Effeltrich, Germany). Leaves were adapted to an incident photon flux of 1500 μ mol m −2 s −1 prior to measurements with the [CO 2 ] at 400 μ mol mol −1 and leaf temperature controlled at 25 o C. The VPD of the air entering the gas exchange system was maintained between 0.8 and 1.1 kPa. Photosynthetic light response curves were then obtained at ambient (21%) and low (2%) oxygen concentrations. Photon flux was varied in a step-wise manner, either starting from the photon flux at which photosynthesis had been induced, after a steady-state A was obtained or alternating between different photon fluxes, in each case waiting for a steady-state to be obtained. Photon flux levels were 2000, 1500, 1000, 700, 500, 350, 250, 150, 75, 45 μ mol m −2 s −1 . At each light level, once a new steady state was reached gas exchange rates were recorded. Simultaneously, F q ′ /F m ′ was determined via modulated chlorophyll fluorescence and application of a saturating flash as previously described 25,26,30 .
The light response of CO 2 assimilation rate is described by a four-parameter non-rectangular hyperbola, according to Marshall and Biscoe 31 : where A is the CO 2 assimilation rate (μ mol m −2 s −1 ), Φ max is the apparent maximum quantum yield (mol CO 2 (mol photons) −1 ), I is the incident photon flux (μ mol m −2 s −1 ), A sat is the CO 2 assimilation rate at saturating photon flux (μ mol m −2 s −1 ), θ is the curve convexity (dimensionless) and R d is mitochondrial respiration in the light (μ mol m −2 s −1 ). The light compensation point (LCP) was determined as the incident photon flux (I) where A = 0 as predicted from the fitted curve. The quantum yield of CO 2 assimilation, Φ CO2 , was determined according to Genty 30 : where A is corrected for respiratory loss (R d ) and divided by the absorbed photon flux (αI), with α as the leaf absorbance. The method used to determine absorbance under the chamber lighting conditions is described later. The parameters Φ CO2 and F q ′ /F m ′ have been shown to be linearly related under conditions where photorespiration is suppressed 30,32,33 . Under ambient (21%) and low (2%) oxygen atmosphere, the relationship can be used to assess the possible operation of electron acceptors other than CO 2 , e.g. photorespiration or active oxygen production, which are manifest as an increased F q ′ /F m ′ relative to its corresponding Φ CO2
The relation of the quantum yield of CO 2 assimilation (Ф CO2 ) after Valentini 35 to the operating efficiency of PSII (F q ′ /F m ′ ) under non-photorespiratory conditions can be described as a linear relationship 30 , where b is the y axis intercept and k is the slope of the relationship between F q ′ /F m ′ and Ф CO2 : Assuming 4 electrons are necessary for the fixation of a CO 2 molecule, this can be expressed as: Scientific RepoRts | 6:20694 | DOI: 10.1038/srep20694 where Φ e− is the quantum yield of total electron flow, which can be rewritten as: This relationship is assumed to hold in the presence and in the absence of photorespiration, where in the presence of photorespiration it can be used to calculate the total electron flow, which is the sum of electron flow to the reactions of carboxylation (J C ) and oxygenation (J O ) 35 : J C can be calculated by assuming 4 electrons are needed per CO 2 for carboxylation: where R p is the amount of CO 2 released by photorespiration. J O can be calculated by assuming that 8 electrons are needed per CO 2 released in photorespiration: Combining the equations for J T , J C and J O then gives: Leaf transmission and reflectance. Immediately following completion of the gas exchange measurements leaf absorbance (α) was measured from 400 to 700 nm, with a dual-channel spectrometer and integrating spheres (SpectroClip-JAZ-TR, Ocean Optics, Oxford, UK). Six spectral measurements of leaf transmittance and reflectance were made per leaf. The average transmittance (τ ) and reflectance (R) for each leaf was used to determine α = (1 − R − τ ). Total absorbance for the PAR spectrum was calculated. Total absorbed light was also calculated by combining the percentage of actinic light emitted by the blue (470 nm) and red (640 nm) LEDs in the leaf gas exchange chamber with α for the peak wavelength of the two LED types 36 . The apparent maximum quantum yield (Φ max ) was then recalculated to give the maximum absolute quantum yield, i.e. the net number of CO 2 molecules absorbed by the leaf per photon absorbed:

10
CO max max 2 Intrinsic leaf water use efficiency. Intrinsic leaf water use efficiency (LWUE) was calculated from gas exchange of CO 2 and H 2 O as the ratio of CO 2 assimilation (A) over stomatal conductance (g s ) under 21% O 2 at photon fluxes of 1000 and 2000 μ mol m −2 s −1 . Contrary to the water use efficiency calculated from A over transpiration (E) 37 , A/g s is considered to be more realistic and comparable between studies, as it is not influenced by changes in leaf to air VPD in the leaf chamber 38 .

Results
The light response of A. donax for A at high and low O 2 showed an increase of A when O 2 was lowered, compared to that at 21% O 2 , indicating the reduction of A in air due to photorespiration (Fig. 1a). At both O 2 concentrations, A did not reach a plateau, even at high photon flux. Both the light response of g s and c i were lowered when O 2 concentration was reduced (Fig. 1b). The response of g s to light largely scaled with A, while c i reached a plateau above a photon flux of 600 μ mol m −2 s −1 . From the response of A to light, parameters were derived ( Table 1). The  (Fig. 2.). However, the electron flow to carboxylation (J C ) increased further with increasing photon flux and did not appear to reach a plateau (Fig. 2). It was estimated that ca. 25% of electron flow is accounted for by J O under saturating light intensity. The ratio of CO 2 production by photorespiration, R p , to assimilation (A sat ) was 0.216.

Table 1. Parameters derived from photosynthetic light response curves calculated with incident (I) and absorbed photon flux (αI); replication and conditions of measurement as given in
in slope of the relation, representing the apparent electron demand to assimilate one CO 2 , at ambient and low O 2 concentration can be attributed to the presence or absence of photorespiration. The response of A to c i for A. donax leaves at saturating light intensity was determined at ambient O 2 concentration (Fig. 4). Subsequently, parameters were derived to describe the A/c i response and the stomatal limitation ( Table 2). The maximum velocity of carboxylation by Rubisco (V c,max ), was 117.8 μ mol m −2 s −1 and the maximum rate of electron transport (J max ) was 213.7 μ mol m −2 s −1 . The CO 2 compensation point, Γ , was 44.2 μ mol mol −1 and the limitation to A imposed by stomata and leaf boundary layer, l, was 0.25 (Table 2).  Soil water content between 20 cm and 40 cm soil depth averaged 0.14 m 3 m −3 (+ /− 0.01) across the sampling area over the sampling period, with an average soil temperature of 21 o C (+ /− 0.9). Stomatal conductance of A. donax, measured on ad-and abaxial surfaces of leaves on three different positions along the leaf blade, were highest at noon (12:00) and declined in the afternoon (16:00, Table 3). The highest stomatal conductance was observed in the middle of the leaf at both time points and both surfaces. The greatest decline in stomatal conductance between noon and afternoon was observed in the middle of the leaf, on both adaxial and abaxial surfaces (Table 3). Even though there was a great decline in stomatal conductance, the operating efficiency of PSII (F q ′ /F m ′ ) at noon and in the afternoon in the field did not decrease as much (Table 3). This implies that stomatal conductance was still not greatly limiting for photosynthesis during the afternoon.  Table 2. Parameters derived from photosynthetic intracellular CO 2 (c i ) response curves; replication and conditions of measurement as given in Fig. 4. mean, (SE), n = 5.

Discussion
Light-saturated capacity for photosynthetic CO 2 uptake is determined by the maximum rate of carboxylation (V c,max ) and the maximum rate of electron transport, (J max ), depending on the inter-cellular CO 2 concentration (c i ) 20 . Averaged across 109 C3 species, V c,max was 64 μ mol m −2 s −1 and J max 134 μ mol m −2 s −1 39 , this compares to values found here for A. donax of 117.8 and 213.7 μ mol CO 2 m −2 s −1 , respectively, which are almost double the C3 average. These values are for a natural and unfertilized stand. Typically in C3 crops these values will increase with nitrogen status 40 (Fig. 1), the photorespiratory rate of CO 2 release (R p ) would be 6.3 μ mol m −2 s −1 , following the equations of Farquhar et al. 20 . This calculated rate, based on properties of spinach, is very close to the rate derived here for A. donax by combining fluorescence and gas exchange measurements (Fig. 2) via the equations of Valentini et al. 35 . This suggests that as a proportion of net photosynthesis, photorespiratory loss of carbon is just as great in A. donax as in other C3 species.
Is the rate of CO 2 assimilation in limiting light high in A. donax? Light limited photosynthesis is determined by the efficiency with which the leaf can absorb incident light (α), and the maximum absolute quantum yield of CO 2 assimilation (Φ CO2,max ), i.e. the initial slope of the response of A to I, corrected for absorptance (α). The value is therefore the maximum ratio of net absorbed CO 2 molecules to absorbed photons. The Φ CO2,max determined for A. donax here in normal air at 25 °C was 0.056 mol mol −1 . Osborne and Garrett 49 similarly measured Φ CO2,max by combining gas exchange and integrating sphere measurements across a range of C3 herbage grasses and cereals, covering different ploidy levels and cultivars in normal air and at 25 °C. They reported an average Φ CO2,max of 0.051, with a range of 0.047 to 0.055 across these C3 grasses. So this does place the values here for A. donax at the upper end of this range and 8% higher than the average. The realized efficiency of light limited photosynthesis will be the product of Φ CO2,max and the absorptance of the leaf (α). In surveying a wide range of healthy leaves of C3 species from tropical to polar habitats, Long et al. 50 , found an average α of 0.80, and a range across species from 0.65 to 0.90, similarly measured in an integrating sphere. By comparison, the α for A. donax of 0.89 therefore appears at the upper end of this range and 14% higher above the average. The product of the indicated above average Φ CO2,max and α therefore represents a 24% increase in CO 2 uptake per unit incident light, under light-limiting conditions. Does A. donax achieve high photosynthetic rates by minimizing stomatal limitation? The stomatal limitation imposed on photosynthesis, l, for A. donax found in this study (0.25 or 25%) was higher than for other C3 plants, which varied from 0.137 to 0.217 [51][52][53][54] . This indicates that A. donax does not achieve its high photosynthetic rates through a high stomatal conductance. Indeed c i /c a at full sunlight (2000 μ mol m −2 s −1 ) as a measure of the balance between stomatal conductance and assimilation was 0.64. This is almost 11% less than the average c i /c a of 0.72 for a range of C3 species 19 . A lower c i /c a requires a lower stomatal conductance relative to the photosynthetic rate, indicating a higher leaf level water use efficiency for a given leaf-air water vapour pressure deficit than the average for C3 species. So, while stomatal conductance may appear high (Fig. 1b), the low values of l and c i /c a in fact show that conductance is low relative to the high rates of leaf CO 2 uptakes for a C3 plant. Nevertheless its high light-saturated and light-limited photosynthetic rates can still only be achieved at the expense of considerable transpiration. The relatively high stomatal conductance maintained into the late afternoon (Table 3) suggests that the plant may be able to tap deep water resources even in its native Mediterranean in the late summer. The LWUE of A. donax in this study (62.9 to 66.0 μ mol mol −1 ) was higher than generally found for herbaceous species (43 μ mol mol −1 ) and more similar to LWUE found for evergreen shrubs and deciduous trees (64 and 66 μ mol mol −1 , respectively) 55 . However, the LWUE of A. donax was still much lower than for C4 species such as Miscanthus and switchgrass (115 and 107 μ mol mol −1 ) 16 . The high productivity of A. donax does resemble and exceeds that of C4 plants, however its LWUE is much lower and clearly C3-like. This supports the notion that, as discussed above, A. donax is able to achieve its high photosynthetic rates with substantial transpiration, but is still more efficient than most C3 species. This is interesting when considering the diversity of habitat distribution that A. donax is found in, which ranges from very wet loam to relatively dry sandy soils.
Overall this study has found that the photosynthetic capacity of A. donax in full sunlight is high compared to other C3 species, and comparable to C4 bioenergy grasses. This is not the result of lower photorespiratory rates, but rather a high capacity for both RuBP-limited and RuBP-saturated photosynthesis, as evidenced by values of both V c,max and J max that are near double the average for C3 species. High photosynthetic rates were not achieved through a high stomatal conductance, in fact stomatal limitation was found to be greater not less than in other C3 species. Light-limited photosynthesis, which will determine carbon uptake during periods of low light flux, around dawn and dusk, and during heavy cloud, as well as in the lower canopy may be aided by relatively high maximum quantum yields of CO 2 assimilation and high leaf absorptances.