CO2 availability influences hydraulic function of C3 and C4 grass leaves.

Atmospheric CO2 (ca) has increased since the last glacial period, increasing photosynthetic water use efficiency and improving plant productivity. Evolution of C4 photosynthesis at low ca led to decreased stomatal conductance (gs), which provided an advantage over C3 plants that may be reduced by rising ca. Using controlled environments, we determined how increasing ca affects C4 water use relative to C3 plants. Leaf gas exchange and mass per area (LMA) were measured for four C3 and four C4 annual, crop-related grasses at glacial (200 µmol mol-1), ambient (400 µmol mol-1), and super-ambient (640 µmol mol-1) ca. C4 plants had lower gs, which resulted in a water use efficiency advantage at all ca and was broadly consistent with slower stomatal responses to shade, indicating less pressure on leaf water status. At glacial ca, net CO2 assimilation and LMA were lower for C3 than for C4 leaves, and C3 and C4 grasses decreased leaf hydraulic conductance (Kleaf) similarly, but only C4 leaves decreased osmotic potential at turgor loss. Greater carbon availability in C4 leaves at glacial ca generated a different hydraulic adjustment relative to C3 plants. At current and future ca, C4 grasses have advantages over C3 grasses due to lower gs, lower stomatal sensitivity, and higher absolute water use efficiency.

Introduction

C4 photosynthetic pathways have evolved as solutions to photosynthetic inefficiencies linked with the oxygenation reaction of Rubisco (Sage, 2004; Sage ). Because of its potential for greater efficiency, engineered C4 photosynthesis has been proposed as a potential solution for improving global food security (von Caemmerer ), and C4 crops are leading contenders as sources of renewable biomass energy (Byrt ). Our understanding of C4 photosynthesis as an ecological adaptation is continuing to develop rapidly (Edwards and Smith, 2010; Lundgren ; Atkinson ; Watcharamongkol ). New insights into the timing and sequence of C4 evolution from phylogenetic studies have renewed debate about its expected physiological advantages (Edwards and Still, 2008; Edwards ; Christin , 2014; Sage ). Some C4 lineages probably arose during the Oligocene (~30 million years ago) but most arose over the last 20 million years, during the subsequent Miocene (Christin , 2011; Vicentini ; Besnard ; but see Kadereit ). During this period, ‘icehouse’ conditions of globally cooler temperatures and drier climates were linked with atmospheric CO2 concentrations (ca) lower than present day (Pagani ). Atmospheric CO2 has increased since the last glacial period, and consequent increases in photosynthetic water use efficiency have been associated with declines in water stress and improvements in plant productivity (Polley ; Mayeux ). It has been speculated that in addition to impacts on photosynthetic performance (Ehleringer ), hydraulic function in C3 and C4 plants was differentially affected at low atmospheric CO2 (Osborne and Sack, 2012) because of greater stomatal opening in C3 plants resulting in greater water stress (Polley ). It is also expected that under future, high CO2 climates, the combination of CO2 fertilization and improved water use efficiency will continue to influence the relative performance of C3 and C4 plants (Ghannoum ; Ainsworth and Long, 2005; Leakey, 2009). To establish whether C4 plants gain hydraulic advantages because of relatively small increases in stomatal conductance (gs) responding to ca (Osborne and Sack, 2012), it is important to verify relative stomatal responses experimentally and investigate their impact on physiological function, including hydraulic properties.

Photosynthesis in C4 leaves is characterized by biochemical pumps that initially combine phosphoenolpyruvate (PEP) and CO2 to form C4 acids and subsequently transfer those acids, release CO2 in the presence of Rubisco, and recycle PEP (Edwards ; Sage, 2004). The initial biochemical step used to form C4 acids is highly efficient, and a high CO2 concentration at the site of Rubisco carboxylation minimizes photorespiration in C4 plants. Therefore, leaf internal CO2 concentrations (ci) are lower for CO2 compensation and photosynthetic saturation, and quantum yield can be greater (Pearcy and Ehleringer, 1984). Importantly for plant hydraulics, photosynthetic water use efficiency is consequently high (Pearcy and Ehleringer, 1984; Long, 1999). A central question has been whether this improved water use efficiency provides advantages for C4 plants over C3 plants in habitats with restricted water availability (Osmond ; Hattersley, 1983; Pearcy and Ehleringer, 1984). Recent comparative studies of the numerous C4 lineages in the grass family have supported the idea that their evolution and maintenance were often linked with improved performance in drier or more open habitats compared with C3 sister groups (Osborne and Freckleton, 2009; Edwards and Smith, 2010; Christin and Osborne, 2014). Osborne and Sack (2012) proposed that improved hydraulic safety, afforded by the evolution of lower gs among C4 species (Osmond ; Taylor , 2012), might have increased the potential of C4 grasses to colonize drier habitats when ca was lower than it is today. They also noted that gs is usually higher at glacial ca compared with ambient ca, but the increase in gs is less at glacial ca in C4 plants than in C3 plants. Using steady-state models of coupled photosynthesis and plant hydraulics, they showed that lower gs could have protected C4 plants from loss of hydraulic conductivity and allowed net CO2 assimilation (A) to be maintained as soil dried at low ca. They therefore proposed that in addition to biochemical advantages supporting higher A at low ci, protection of hydraulic function was an important advantage to C4 grasses at low ca.

Importantly, the models that Osborne and Sack (2012) used to predict hydraulic performance in C3 and C4 species at glacial ca did not predict potential adjustments to co-ordination of leaf gas exchange and hydraulic function at low ca. Although evidence suggests that in non-woody species, decreased gs at elevated ca is associated with less negative leaf water potentials, lower hydraulic conductivity, and greater resistance to embolism, little is known about the influence of ca on co-ordination between photosynthetic capacity and hydraulic function (Domec ). Changing irradiance results in parallel changes in leaf hydraulic conductance (Kleaf) and photosynthetic capacity of woody C3 plants, optimizing leaf hydraulic function (Brodribb and Jordan, 2011; Carins Murphy ). In contrast, adjustment to high vapour pressure deficit (VPD) is linked with closure of stomata to protect hydraulic function (Carins Murphy ). In the case of ca, hydraulic demand is influenced by changes in gs that compensate for carbon availability (Franks ). Because the economics of leaf structure–function relationships may depend on ca, it is likely that ca has complex effects on co-ordination between Kleaf and gs. For instance, smaller, more densely packed stomata are sometimes observed at low ca (Woodward, 1987; Woodward and Bazzaz, 1988; Franks and Beerling, 2009), which may increase the sensitivity of gs to VPD (Franks and Beerling, 2009; Drake ), serving a protective function. Conversely, higher anatomical maxima for gs observed at low ca in sunflower, which were a result of larger, more densely packed stomata, were linked with greater xylem-specific conductivity, but the phloem ratio and hydraulic safety were decreased (Rico ). Photosynthetic type may further affect the impact of ca on the relationships between hydraulic supply and demand because the carbon assimilation advantage provided by C4 photosynthesis may support additional flexibility in hydraulic adjustment. At ambient CO2, relative to C3 species, C4 dicots maintain A at relatively lower g, and either increase hydraulic safety by decreasing xylem conduit diameter, or display greater leaf area for similar investments in stem xylem supply (Kocacinar and Sage, 2003, 2004; Kocacinar ).

In grasses, leaf hydraulic performance is particularly important: leaves contribute 50–72% of resistance along whole-plant hydraulic pathways (Meinzer ; Martre ). The relative sensitivities of Kleaf and gs are also crucial in determining water use strategies among grasses. Both C3 and C4 grasses have been reported to show routine diurnal declines in leaf hydraulic conductivity when stomata do not close sufficiently to protect hydraulic function (Neufeld ; Holloway-Phillips and Brodribb, 2011). Susceptibility to declines in conductivity is variable both among species and among cultivars (Holloway-Phillips and Brodribb, 2011), and nocturnal root pressure and refilling of embolized vessels facilitates recovery from diurnal stress in some grass species (McCully, 1999; Holloway-Phillips and Brodribb, 2011; Cao ; Gleason ). Protection against runaway declines in Kleaf can be provided by stomatal closure (Brodribb and Holbrook, 2003), and fast stomatal responses are considered a key characteristic of grasses (Hetherington and Woodward, 2003; Franks and Farquhar, 2007). Faster stomatal responses to light can improve intrinsic water use efficiency (iWUE=A/gsw, where gsw is gs for water) by producing a better match between rapid photosynthetic responses and the slower stomata, which may improve overall water use efficiency, resulting in greater conservation of soil water and thereby decreased hydraulic stress (Lawson and Blatt, 2014).

Our goal was to determine whether growth ca had different impacts on leaf function in selected C3 and C4 annual grasses comparable with crop species. We predicted that to support increased transpiration at low ca, Kleaf would increase and turgor loss points would decrease to compensate for increased hydraulic demand. In addition, we determined whether rates of stomatal closure, responding to low light, increased at low ca. We anticipated that leaf mass per area (LMA) would decrease in plants with carbon limitation at low ca, and that decreases in iWUE and the extent of carbon limitation imposed by low ca would be greater for C3 than for C4 species (Osborne and Sack, 2012). We therefore expected that leaf physiological responses to a range of ca would be larger in C3 than in C4 grasses. Plants were grown in CO2 concentrations that represented: some of the lowest ca conditions (~200 µmol mol−1) that occurred in the glacial period during which C4 grass lineages diversified (Edwards ); ambient ca (400 µmol mol−1); and super-ambient ca (640 µmol mol−1).

Materials and methods

Growth conditions

Plants were grown in walk-in climate-controlled growth chambers (Biochambers, Winnipeg, Manitoba) equipped with additive CO2, and CO2 scrubber equipment. Three ca treatments were imposed: glacial (cGLA), 204 ± 27 μmol mol−1; ambient (cAMB), 408 ± 11 μmol mol−1; and super-ambient (cSUP) 640 ± 2 μmol mol−1 (mean ±SD; 72 daily means). The cAMB and cSUP treatments were rotated between cabinets 1 week prior to the first measurements, during the fourth week after sowing. The cGLA treatment was maintained in a single cabinet throughout the experiment because of the technical demands of obtaining a stable CO2 concentration at glacial ca. Growing conditions were set to a night-time temperature of 18 °C and a daytime temperature of 26 °C, resulting in a daily mean temperature of 22 °C (mean ±SD for 72 daily means: cGLA, 21.9 ± 0.23; cAMB, 22.1 ± 0.21; and cSUP, 22.1 ± 0.28). Temperatures and light levels were ramped daily in two even steps between 06.00 h and 08.00 h and between 18.00 h and 20.00 h (14 h light:10 h dark). Light was supplied by HID lamps, which provided photosynthetic photon flux density (PPFD) at the top of the canopy that varied within each cabinet between 300 μmol m−2 s−1 and 650 μmol m−2 s−1; daily quantum inputs were ~21 mol m−2 d−1 (mean ±SD for 72 daily means: cGLA, 21.3 ± 1.8; cAMB, 21.5 ± 1.9; and cSUP, 21.5 ± 2.0). Mean daily values for relative humidity ranged from 60% to 83% (mean ±SD for 72 daily means: cGLA, 77 ± 8; cAMB, 78 ± 1; and cSUP, 76 ± 6), providing VPDs of ~0.74 kPa under daytime conditions, and ~0.46 kPa during the night.

Plant material

Our study plants were eight annual grass species, four C3 and four C4, used as food crops or close relatives of species used as food crops. In Poacaeae, all C4 grasses belong to a clade referred to as PACMAD (Aliscioni ); within PACMAD two C4 crop species have been domesticated from wild relatives in the Chloridoideae (teff and finger millet), and several from the Panicoideae subfamily (Christin ). Grasses with C3 photosynthesis used as grain crops originate in the subfamilies Pooideae and Oryzoideae, which belong to a separate clade currently referred to as BEP (Kellogg, 1998; Aliscioni ). Relevant Chloridoideae species could not be obtained, so we only used C4 grasses from the Panicoideae. Sorghum bicolor (great millet), Setaria italica (foxtail millet), and Digitaria exilis (fonio millet) represent independent evolutionary origins of the NADP-malic enzyme (NADP-ME) subtype of C4 photosynthesis (Aliscioni ); Panicum miliaceum (proso millet) represents the NAD-ME C4 subtype (Giussani ; Aliscioni ). C3 species were Panicum bisulcatum and Steinchisma laxa (two wild relatives from Panicoideae), Triticum turgidum (durum wheat, Pooideae), and Oryza sativa ssp. japonica (rice, Ehrhartoideae; Table 1).

Table 1.

Sources and phylogenetic placement of study species

Species	Photosynthetic typea	Phylogenetic placementa	Accession (source)
Triticum turgidum L. ssp. durum	C3	Pooideae	AUS-26564 /PERSIA128 (Tony Condon, CSIRO Agriculture, ACT)
Oryza sativa L. ssp. japonica Kato	C3	Ehrhartoideae	IAC1131 (Brian Atwell, Macquarie University, Sydney NSW)
Panicum bisulcatum Thunb.	C3	Panicoideae: Paniceae	(Ghannoum laboratory)
Steinchisma laxa (Sw.) Zuloaga	C3	Panicoideae: Paspaleae	(Ghannoum laboratory)
Sorghum bicolor (L.) Moench	C4	Panicoideae: Andropogoneae	Tx623 (Alan Cruickshank, Department of Agriculture and Fisheries, Hermitage Research Facility, Warwick QLD)
Setaria italica (L.) P.Beauv.	C4	Panicoideae: Paniceae: Cenchrinae	AusTRCF 108040 (AusPGRIS: Tropical Crops and Forages Collection)
Digitaria exilis (Kippist) Stapf	C4	Panicoideae: Paniceae: Anthephorinae	AusTRCF 108024/PDE7 (AusPGRIS: Tropical Crops and Forages Collection)
Panicum miliaceum L.	C4	Panicoideae: Paniceae: Panicinae	(Ghannoum laboratory)

Aliscioni .

Plants were grown from seed in Osmocote Professional Seed Raising & Cutting Mix (Scotts Australia Pty Ltd, Bella Vista, NSW) in 0.55 litre plastic square tubes (Garden City Plastics, Somersby NSW: top dimension 70 × 70 mm, 160 mm deep). Seeds were sown directly into six pots and germinated under the different CO2 treatments; the number of plants per pot and the size of plants varied depending on the species. To allow for balanced sampling and to account for within-cabinet variability, at germination, pots were arranged into a fully randomized blocked design with one pot from every species in each block. The pots were checked daily and watered as necessary to prevent surface drying. To minimize root binding, roots were allowed to grow out of pots into a layer of wetted Scoria. To minimize nutrient limitation, plants were fed with a complete fertilizer (Thrive All Purpose Soluble Plant Food, Yates, Auckland, New Zealand) every 2–3 weeks during the course of the experiment.

Steady-state gas exchange and stomatal response to PPFD

We measured gas exchange using six LI-6400XT photosynthesis systems (LI-COR Inc., Lincoln NE, USA) equipped with CO2 mixers (LI-6400-01) and 2 × 3 cm red-blue LED light sources (LI-6400-02B). Pairs of LI-6400XT machines were randomly allocated to the three ca treatments and were rotated every 2 d: each pair of machines was used to measure two of the six blocks in every cabinet over the course of the experiment. Measurements were made under the growth conditions. To minimize disruption of ca treatments, the cuvette and integrated gas analysers of the LI-6400XT were placed inside the growth chambers and consoles outside the growth chambers (growth chambers were opened briefly before and after switching leaves). Measurements were conducted on the mid-section of individual, recently expanded leaves inserted parallel to the long axis of the 2 × 3 cm chamber, and leaf areas were calculated as cuvette length×average leaf width, measured to the nearest 0.5 mm with a ruler. Leaves were allowed to come to steady state [showing no systematic trends with a coefficient of variation (CV) <0.1 over a 5 min period] at a PPFD of 500 μmol m−2 s−1 (growth light levels) and cuvette CO2 concentrations matched to ca at the time of measurement: (cGLA, 184 ± 4 μmol mol−1; cAMB, 406 ± 5 μmol mol−1; and cSUP, 647 ± 6 μmol mol−1; mean ±SD ≥43 leaves, CV for individual leaves <5%). Relative humidity was maintained at ~70% and block temperature at 26 °C, resulting in leaf VPDs of 1 ± 0.07 kPa (mean ±SD, n=137 leaves; CV for individual leaves <8.1%). An auto-program (logging every 10 s) was used to record initial steady-state values for gas exchange (A, gsw, and iWUE), followed by the response of g to a step-change decrease in light availability from 500 μmol m−2 s−1 to 100 μmol m−2 s−1 PPFD. The rate of stomatal response to PPFD (Δgsw/Δt) was characterized based on the magnitude (Δgsw) and duration (Δt) of the initial decrease in gsw.

Leaf hydraulic conductance and LMA

Because C3 and C4 species differed in the response of A to ca between cGLA and cAMB, we determined Kleaf in those treatments using the evaporative flux method (Sack ). Cut stems were transported to the lab in water, where flag or second-leaf laminas were excised and, using parafilm, were sealed onto parafilm-wrapped, cylindrical plastic rods. The rod and leaf were submerged, and the leaf re-cut and positioned in water-filled Tygon tubing linked to a reservoir of de-gassed Milli-Q water on a balance (CPA225D, Sartorius, Göttingen, Germany; 10 µg accuracy). The seal was tested by pressure from a 100 ml syringe that was used to fill and empty the system and was attached to the Tygon tubing using Luer fittings. Leaves were supported using fishing line stretched across a wooden frame, with their adaxial surface uppermost and parallel with the meniscus in the reservoir. Transpiration was induced using a desk fan and a lamp (leaf surface PPFD 100–150 μmol m−2 s−1). Every second, output from the balance was logged to a computer and plotted to determine when transpiration (E, mol m−2 s−1) obtained a steady state for 5 min. At steady state, the temperature of the abaxial surface of the leaf (Tleaf) and air (Tair, shaded) were established using two type-K thermocouples and a Pico TC-08, then the leaf was immediately sheathed in plastic and cut at its base. After 15 min equilibration, a Scholander pressure bomb (PMS 1505D with grass compression gland; PMS Instrument Company, Albany, OR, USA) was used to determine water potential, which estimated the leaf hydrostatic gradient (ΔΨ). Leaf area for these leaves (and an extra set collected from the cSUP treatment) was determined using a Canon LiDE 510 flatbed scanner and ImageJ software (Abràmoff ). We calculated Kleaf as E/(area×ΔΨ).

LMAs (g m−2) were calculated using dry masses determined after a minimum of 48 h drying at 65 °C.

Pressure–volume relationships

Pressure–volume (P–V) relationships were determined using bench-drying. On the morning of measurement, attached flag leaves were sealed into plastic bags containing exhaled breath and were allowed to equilibrate for a minimum of 40 min to quench transpiration and ensure high turgor. Leaves sheathed in this manner were subsequently excised at the base of the lamina and moved to the laboratory. Initially, leaves remained sealed in plastic between measurements of fresh mass (FM, g) and water potential (Ψ; Scholander pressure bomb). As water potential declined, leaves were occasionally removed from the plastic for short periods to increase the rate of drying. A minimum of 20 min equilibration was ensured between pressure bomb measurements. At the conclusion of FM and Ψ measurements, leaves were dried for a minimum of 48 h at 65 °C to determine dry mass (DM, g). The turgid mass (TM, g) was estimated by extrapolation of the initial linear FM–Ψ relationship (Kubiske and Abrams, 1990) and used to calculate relative water contents (RWCS) for entire leaves as (FM−DM)/(TM−DM), and leaf dry matter content (LDMC=DM/TM).

We optimized parameter selection for P–V relationships of individual leaves by minimizing the absolute difference between estimates of osmotic potential at full turgor (π0, MPa) obtained below (π0,1) and above (π0,2) turgor loss, comparing all possible combinations that could be fit for each leaf within our data set. First, below-turgor loss fits for 1/Ψ=a(1−RWC)+π0,1 (linear regression with slope a, and y-intercept π0,1) were obtained from all sequences representing at least three of the smallest RWC values and excluding two or more of the highest RWC values. Next, the x-intercept of the below-turgor loss relationship (apoplastic fraction, af) was used to establish the RWC of the symplasm [RWCS=(RWC−af)/(1−af)]. Then the osmotic potential, π=1/[a(1−RWCS)+π0,1] (MPa) and turgor pressure, ΨP=Ψ−π (MPa) were derived for the complementary above-turgor loss data. Finally, the bulk modulus of elasticity (ε, MPa) and π0,2 were obtained from linear regression of ΨP= −ε(1–RWCS)−π0,2. Turgor loss point characteristics were calculated for the pair of linear relationships where [π0,1−π0,2] was smallest, and 0

Statistical analysis

Statistics were calculated using R Language and Environment (R Core Team, 2016; https://www.R-project.org/). We fit linear models (lm) of species×ca responses, which we minimized using the Akaike information criterion (AIC). Where species effects were significant (or marginally so), we used linear contrasts to compare species means by photosynthetic type. Tests of photosynthetic type effects are approximate because the C3/C4 comparisons were not phylogenetically independent, and only a small number of species were included in our experiment. To adjust for heteroskedasticity, loge transformation was applied to values for g, iWUE, and Kleaf. Heteroskedasticity in Δgsw/Δt and P–V parameters could not be eliminated using transformation. For these parameters, we applied non-parametric Kruskal–Wallis tests, and present median values.

Results

Impact of ca on steady-state leaf gas exchange

iWUE was more responsive to the difference between cGLA and cAMB than the difference between cAMB and cSUP (Fig. 3A; especially on a relative scale, Table 2). At cGLA, iWUE was 33–74% lower (mean 56%) than at cAMB, while iWUE at cSUP was 4–56% higher (mean 30%) than at cAMB. Differences in iWUE were significant for comparisons between cGLA and cSUP in every species (Tukey’s HSD, P≤0.0016), and between cGLA and cAMB in every species except T. turgidum (Tukey’s HSD, P≤0.0001; T. turgidum, P=0.11); differences between cAMB and cSUP were not significant (P≥0.077).

Fig. 3.

Response of leaf mass per area (LMA, individual upper canopy leaf lamina) to growth ca (GLA, ~200 μmol mol−1; AMB, ~400 μmol mol−1; SUP, ~640 μmol mol−1). Values are the mean ±SE (n=5–6) for eight crop and crop-related annual grass species, plotted by photosynthetic type: C3 (filled: circles, Triticum turgidum; squares, Oryza sativa; diamonds, Steinchisma laxa; triangles, Panicum bisulcatum) or C4 (open: circles, Sorghum bicolor; squares, Digitaria exilis; diamonds, Setaria italica; triangles, Panicum miliaceum).

Table 2.

Impact of photosynthetic type on leaf gas exchange relative to ambient CO2 (cAMB ~400 μmol mol−1), at glacial CO2 (cGLA ~200 μmol mol−1), and at super-ambient CO2 (cSUP ~640 μmol mol−1)

Photosynthetic type(C4 subtype)	Species	iWUE(µmol mol−1)		Net CO2 assimilation(µmol m−2 s−1)		Stomatal conductance(mol m−2 s−1)
		c GLA:cAMB	c SUP:cAMB	c GLA:cAMB	c SUP:cAMB	c GLA:cAMB	c SUP:cAMB
C3	T. turgidum	0.66	1.35	0.51	1.07	0.77	0.79
C3	O. sativa	0.37	1.3	0.63	1.2	1.72	0.93
C3	S. laxa	0.36	1.32	0.64	1.16	1.78	0.88
C3	P. bisulcatum	0.26	1.28	0.58	0.81	2.21	0.63
C4 (NADP-ME)	S. bicolor	0.4	1.39	0.95	1.05	2.34	0.76
C4 (NADP-ME)	D. exilis	0.53	1.04	0.87	0.92	1.65	0.88
C4 (NADP-ME)	S. italica	0.42	1.13	0.93	0.94	2.23	0.83
C4 (NAD-ME)	P. miliaceum	0.51	1.56	0.93	1.11	1.83	0.71
Kruskal–Wallis P C3/C4(df=1)		NS	NS	*	NS	NS	NS

*P<0.05.

C4 species showed much larger absolute decreases in iWUE than C3 species (Fig. 1A), linked with a marginally significant contrasts term for photosynthetic type×ca (t113, P=0.048). At every ca, iWUE was always higher in C4 species (range 51–232 μmol mol−1) compared with C3 species (13–68 μmol mol−1; Fig. 1A). Relative changes in iWUE were not significantly different between C3 and C4 grasses, but this comparison was strongly influenced by C3T. turgidum (Table 2). Triticum turgidum showed only a small reduction in iWUE from cAMB to cGLA and, surprisingly, decreased gsw at cGLA compared with cAMB (Fig. 1B). The remaining three C3 grasses showed larger relative decreases in iWUE (63–74%) than any of the four C4 grasses (47–60%; Table 2).

Fig. 1.

Response of steady-state leaf gas exchange to growth ca (GLA, ~200 μmol mol−1; AMB, ~400 μmol mol−1; SUP, ~640 μmol mol−1), measured at growth PPFD (500 μmol m−2 s−1) and moderate vapour pressure deficit. (A) Intrinsic water use efficiency (iWUE); (B) net CO2 assimilation rate (A); and (C) stomatal conductance to water (gsw). Values are the mean ±SE (n=5–6) for eight crop and crop-related annual grass species, plotted by photosynthetic type: C3 (filled: circles, Triticum turgidum; squares, Oryza sativa; diamonds, Steinchisma laxa; triangles, Panicum bisulcatum) or C4 (open: circles, Sorghum bicolor; squares, Digitaria exilis; diamonds, Setaria italica; triangles, Panicum miliaceum).

Under the light conditions provided by our controlled-environment cabinets, which were non-saturating for photosynthesis (maximum 650 μmol m−2 s−1 PPFD), A was not higher in C4 species compared with C3 species at cAMB and cSUP (Fig. 1B), so the higher iWUE of C4 species at those CO2 concentrations was primarily due to lower gsw (Fig. 1C). Higher iWUE among C4 grasses at cGLA was primarily due to smaller relative reductions in A between cAMB and cGLA for C4 grasses (5–13%) compared with C3 grasses (36–49%); over the same range, relative increases in gsw were comparable for C3 and C4 species (C3 excluding T. turgidum, +72–121%; T. turgidum −23%; C4, +65–134%; Table 2).

Impact of ca on dynamic leaf gas exchange

The rate of decrease in gsw responding to a step decrease in PPFD from 500 μmol m−2 s−1 to 100 μmol m−2 s−1 (Δgsw/Δt) was generally greater for C3 than C4 grasses (Fig. 2). This was broadly consistent with the higher steady-state gsw of C3 species at a PPFD of 500 μmol m−2 s−1 (Fig. 1C). A significant ca×species interaction was detected (F14,113, P<0.0001), associated with significant photosynthetic-type effects on the average response between cGLA and cAMB (t113, P=0.041), and between cGLA and cSUP (t113, P<0.0001). All C4 species increased Δgsw/Δt with decreasing ca, but only one C3 species (P. bisulcatum) showed a similar trend (Fig. 2). Pairwise tests for responses of Δgsw/Δt within species were significant only when comparing cGLA and cSUP of three species: P. bisulcatum (C3, P<0.001), S. bicolor (C4 NADP-ME, P<0.001), and P. miliaceum (C4 NAD-ME, P=0.004). Within their respective photosynthetic types, these three species showed the greatest values for iWUE and lowest values for gsw at cSUP, and the greatest decreases in iWUE from cSUP to cGLA (Fig. 1A, C).

Fig. 2.

Effect of growth ca (GLA, ~200 μmol mol−1; AMB, ~400 μmol mol−1; SUP, ~640 μmol mol−1) on rate of stomatal response to shade: a step-change decrease in PPFD from 500 μmol m−2 s−1 to 100 μmol m−2 s−1 (Δgsw/Δt) at a steady vapour pressure deficit. Values are the mean ±SE (n=5–6) for eight crop and crop-related annual grass species, plotted by photosynthetic type: C3 (filled: circles, Triticum turgidum; squares, Oryza sativa; diamonds, Steinchisma laxa; triangles, Panicum bisulcatum) or C4 (open: circles, Sorghum bicolor; squares, Digitaria exilis; diamonds, Setaria italica; triangles, Panicum miliaceum).

Impact of ca on LMA

At cSUP, LMA values for flag leaves of C3 and C4 species were similar (Fig. 3); however, the response of LMA to ca differed among the eight species (Fig. 3; F14,102, P=0.0004). None of the C4 species exhibited significant changes in LMA in response to ca (Tukey’s HSD, P>0.72). In the C3 species, LMA was similar across the three ca treatments for S. laxa, but T. turgidum, P. bisulcatum, and O. sativa all showed significant reductions in LMA from either cSUP to cGLA (T. turgidum and P. bisulcatum, Tukey’s HSD P≤0.034) or from cAMB to cGLA (O. sativa, P=0.039; Fig. 3). The contrasts term for photosynthetic type×ca, which was statistically significant (t-test102, P=0.014), was therefore broadly associated with less sensitivity of LMA to ca among the C4 species. Conservation of LMA across CO2 treatments in most C4 species was linked with proportionate decreases in mass and area of the flag leaves as ca was reduced. Among C3 species, decreases in LMA arose because flag leaf mass decreased with ca from cSUP to cGLA, and flag leaf area decreased from cSUP to cAMB, but leaf areas were often similar at cGLA and cAMB (Fig. 4).

Fig. 4.

Effect of cGLA (black symbols, versus grey for cAMB and cSUP) on proportionality between leaf mass and area for individual upper canopy leaves (log–log allocation plots). Crop and crop-related annual grass species are plotted by photosynthetic type: C3 (filled: circles, Triticum turgidum; squares, Oryza sativa; diamonds, Steinchisma laxa; triangles, Panicum bisulcatum) or C4 (open: circles, Sorghum bicolor; squares, Digitaria exilis; diamonds, Setaria italica; triangles, Panicum miliaceum). Points are means (n=4–6); SE is omitted for clarity. Lines have slope=1, and the intersect is the mean value at cAMB.

Response of Kleaf and P–V characteristics to decreases in ca from cAMB to cGLA

There were no significant species×ca effects on Kleaf (species×caF7,64, P=0.814); however, on average, Kleaf was lower in plants grown at cGLA (F1,64, P=0.005). The exception was D. exilis, a C4 species with small leaves, for which measurement errors were large (Fig. 5A).

Fig. 5.

Response of leaf hydraulic conductance (Kleaf) to growth at glacial (cGLA, ~200 μmol mol−1: dark shading) versus ambient (cAMB, ~400 μmol mol−1: light shading) CO2. Values are the mean ±SE (n=5) for eight crop and crop-related annual grass species differing in photosynthetic type. C3: Tturg, Triticum turgidum; Osati, Oryza sativa; Slaxa, Steinchisma laxa; Pbisu, Panicum bisulcatum. C4: Sbico, Sorghum bicolor; Dexil, Digitaria exilis; Sital, Setaria italica; Pmili, Panicum miliaceum.

In the P–V analysis, the response of LDMC to ca was consistent with that of LMA measured during determination of Kleaf. LDMC was not significantly different between the photosynthetic types at either ca, but was lower at cGLA among C3 and not C4 leaves (Table 3). LDMC decreased by 4–11% in C3 grasses grown at cGLA, but C4 species showed no adjustment to cGLA or increased LDMC by ≤3% at cGLA. This difference in average LDMC responses to ca was statistically significant when comparing C3 and C4 species (Table 3). Despite these differences in LDMC responses between C3 and C4 species, we found no evidence for significant effects of photosynthetic type on the response of ε or RWCTLP to ca. In contrast, the median πTLP differed between C3 and C4 grasses at cAMB but not at cGLA, linked with a significant effect of photosynthetic type (Table 3). At cAMB, πTLP was less negative among C4 species (C4, −0.72 MPa to −0.87 MPa; C3, −0.94 MPa to −1.36 MPa). This difference was eliminated at cGLA because only C4 grasses decreased πTLP to more negative values (C4, −0.79 MPa to −1.27 MPa; C3, −0.91 MPa to −1.21 MPa; Table 2).

Table 3.

Impact of growth ca on pressure–volume curve characteristics and leaf dry matter content (medians, n=3–5; cGLA, glacial CO2 ~200 μmol mol−1; cAMB, ambient CO2 ~400 μmol mol−1)

Photosynthetictype(C4 subtype)	Species	Modulus ofelasticity(ε, MPa)			Osmotic potential at turgor loss(πTLP, MPa)			RWCat turgor loss(RWCTLP, %)			Leaf dry matter content(LDMC, %)
		c GLA	c AMB	Difference in median: cGLA−cAMB	c GLA	c AMB	Difference in median: cGLA−cAMB	c GLA	c AMB	Difference in median: cGLA−cAMB	c GLA	c AMB	Difference in median: cGLA−cAMB
C3	T. turgidum	11	9.4	1.6	−1.17	−1.31	0.14	94.8	94.5	~0	17	26	−8*
C3	O. sativa	5.4	5.7	−0.3	−1.21	−1.36	0.15	94.2	89.4	4.8	24	28	−4
C3	S. laxa	8	7.1	0.9	−1.13	−1.01	−0.12	93.3	92.3	1	18	29	−11*
C3	P. bisulcatum	4.1	6.2	−2.1	−0.91	−0.94	0.04	89.4	93.9	−4.5	26	32	−6†
C4 (NADP-ME)	S. bicolor	7.7	5.1	2.6	−1.03	−0.82	−0.2	97	97	0	26	23	3
C4 (NADP-ME)	D. exilis	5.9	9.4	−3.5	−0.79	−0.72	−0.08	92.1	93.6	−1.5	19	19	0
C4 (NADP-ME)	S. italica	8.1	7.4	0.7	−1.27	−0.87	−0.41**	93.2	95.7	−2.5	28	26	2
C4 (NAD-ME)	P. miliaceum	6.5	7.3	−0.8	−1	−0.85	−0.18	94.8	95.8	−1	31	29	2
Kruskal–Wallis P species (df=7)		NS	NS	–	**	***	–	NS	*	–	**	**	–
Kruskal-Wallis P C3/C4 (df=1)		NS	NS	NS	NS	*	*	NS	NS	NS	NS	NS	*

*P<0.05; **0.0140% from cGLA treatment that led to a median of 22% and difference in median of 10%

Discussion

We exposed C3 and C4 grasses to atmospheric CO2 concentrations ranging from levels that occurred during the last 30 million years, when C4 lineages evolved and diversified, to those that could be experienced in the coming centuries. Across the range of ca, we expected that C4 species would maintain an iWUE advantage and show smaller physiological adjustments. Our results broadly support this expectation: the absolute response of gsw to ca was greater among C3 than among C4 grasses; and, as ca decreased from cAMB to cGLA, A, LDMC, and LMA declined more among C3 than among C4 species. Investigation of leaf hydraulic function at cAMB and cGLA showed that at cGLA, Kleaf decreased in both C3 and C4 species; and πTLP of C4 leaves became more negative, hence more similar to πTLP of C3 leaves, which did not adjust. Assaying the stomatal response to shade showed that higher steady-state gsw of C3 species was linked with more rapid adjustment of gsw to match A. Rates of stomatal closure were slightly more similar for C3 and C4 species at low ca, driven by strong responses of species that achieved high iWUE at elevated ca. These new findings are consistent with the hypothesis that carbon limitation is an important factor influencing leaf hydraulic function at different atmospheric [CO2]. Although there was substantial variation among species, photosynthetic type affected how leaf dry matter was deployed and how leaf turgor characteristics responded to cGLA.

Gas exchange responses to ca

Steady-state gas exchange measurements provided the expected outcomes: gs usually increased as ca decreased (Osborne and Sack, 2012; Franks ); high gs of C3 grasses was associated with greater gs responses to ca; and low gsw of C4 leaves resulted in higher iWUE at all levels of ca. Importantly, A declined for C3 but not C4 grasses at cGLA. Greater A among some C3 species compared with C4 species at cAMB and cSUP suggested that C4 photosynthetic performance may have been limited by PPFD, so the iWUE advantage to C4 species may underestimate advantages to C4 species that could arise at higher irradiances (Osmond ).

C3 grass leaves generally closed their stomata more quickly than C4 leaves in response to shade. The higher steady-state gsw of C3 leaves may partially explain this difference between the photosynthetic types, but closer inspection of the data shows that Δgsw/Δt did not parallel the steady-state gsw for species within each photosynthetic type. Interestingly, among C4 species, the rate of gsw responses to light was slightly, but consistently, greater at cGLA compared with cAMB and cSUP. This decreased the difference in Δgsw/Δt between C3 and C4 species. However, a more striking trend, that probably underpinned the subtle difference in relative performance based on photosynthetic type, was that species with higher iWUEs showed greater changes in Δgsw/Δt in response to decreasing ca. At cSUP, species with high iWUE showed some of the slowest stomatal responses to shade. Because faster stomatal responses are consistent with improved water use efficiency (Lawson and Blatt, 2014), this suggests that transpiration is regulated less tightly at high ca, supporting the overarching hypothesis that increasing ca minimizes the costs associated with hydraulic stress (Polley ). It also suggests that characteristics producing high iWUE in the steady state may be costly in low-ca-like scenarios that increase transpiration. For example, high iWUE is likely to be facilitated by high rates of internal diffusion, which are linked with decreases in cell wall dry matter (Onoda ) and might increase vulnerability to changes in leaf water status.

Among-species variation was an important feature of our gas exchange results. This is consistent with previous studies, which have indicated that the degree to which grass stomata protect against decreases in hydraulic conductance varies even among genotypes (Neufeld ; Holloway-Phillips and Brodribb, 2011). Among C3 species in our study, only that with the highest iWUE, P. bisulcatum, showed a clear negative association between ca and the stomatal response to shade. At the other extreme, T. turgidum showed exceptionally high steady-state gsw and slow stomatal responses to shade in all three ca treatments, suggesting high transpiration irrespective of leaf water status, a strategy that can maximize CO2 uptake at a cost to hydraulic conductance (Holloway-Phillips and Brodribb, 2011). The apparent lack of stomatal regulation in T. turgidum compared with other C3 species is important to note because iWUE for this species did not decrease at cGLA, contradicting the otherwise consistent trend towards greater decreases in iWUE among C3 compared with C4 species.

Impact of glacial ca on LMA and hydraulic characteristics

LMA decreased at cGLA among C3 but not C4 grasses. This finding is consistent with observed differences in A, results from a meta-analysis addressing variation in LMA (Poorter ), and more recent comparisons using species and ca treatments similar to those chosen for our experiment (Pinto ). Further evidence is needed, however, before this result can be generalized as a photosynthetic type effect. LMA responses can, for example, be modified by temperature (Pinto ). It is also important to note that the C4 and two of the wild C3 species included in our experiment were drawn from one subfamily of the Poaceae: Panicoideae, a broadly mesic-adapted clade (Taub, 2000; Osborne, 2008; Edwards and Smith, 2010; Visser ). We expect leaf functional traits to reflect adaptations to habitat, and some major C4 lineages are adapted to drier environments than those favoured by the Panicoideae (Taub, 2000; Edwards and Smith, 2010). In addition, LMA responses to ca (Pinto ) and leaf size (Liu ) differ between the Chloridoideae and Panicoideae grass subfamilies. While further work will be needed to establish whether the patterns we observed are general across grass lineages, our findings are directly relevant to crop and crop-related annual grass species from mesic habitats. Taken together with the gas exchange results, differences in LMA indicate that cGLA was linked with greater carbon limitation in C3 grasses compared with their C4 relatives. This is important because differences in carbon supply affecting plant size and allocation at the whole-plant level have previously been highlighted as central to functional contrasts between C3 and C4 plants (Long, 1999; Atkinson ), and influence the mechanisms by which plants acclimate to hydraulic stress (Maseda and Fernández, 2006).

C3 and C4 grasses showed similar Kleaf, and Kleaf decreased at cGLA. The finding that there was no clear difference in Kleaf between photosynthetic types is consistent with a previous comparison using the high pressure flow meter technique, applied to predominantly perennial, North American prairie grasses (Ocheltree ). Both of these results are surprising because the clearest anatomical differences between C3 and C4 grass lineages are in the ratio of bundle sheath to mesophyll (Hattersley, 1984; Dengler ; Christin ; Griffiths ; Lundgren ). Increases in this ratio should decrease hydraulic resistance external to the xylem (Buckley ), supporting the hypothesis that differences in leaf hydraulic properties could affect responses to stress imposed by low ca and/or water availability (Osborne and Sack, 2012; Griffiths ). It is possible that other aspects of C4 leaf anatomy or function counteract positive effects of increased bundle sheath ratios on Kleaf in C4 grasses. It is also important to note that C3 and C4 grasses often show similar average mesophyll cell sizes at ambient CO2 (Lundgren ), and the cross-sectional area of vascular relative to chlorenchyma tissues does not necessarily change with photosynthetic type (Dengler ).

The evidence we found for decreased Kleaf at cGLA was surprising, because xylem conductivity generally increases with declining ca to support increased gsw (Rico ; Domec ). Previous in situ measurements of transpiration and leaf water potential in sunflower plants grown at ca similar to cGLA and cAMB showed the expected result: that Kleaf measured at ambient CO2 increased for plants grown at cGLA, minimizing the impact of increased gsw on ΔΨ (Simonin ). A decrease in xylem conductivity, linked with smaller conduits in water-stressed tissue that would increase redundancy among conducting elements (Comstock and Sperry, 2000), might contribute to decreases in Kleaf for leaves grown at cGLA. However, this is not consistent with the decrease in LMA that we observed and, since transpiration was driven using moderate levels of light, we expect that the primary source of hydraulic resistance was exterior to the xylem (Ocheltree ).

The values of Kleaf were low compared with other recently published estimates for similar species [S. bicolor, 19–38 mmol m−2 s−2 MPa−1 (Ocheltree ); O. sativa cultivars, 7.1–8.7 mmol m−2 s−2 MPa−1 (Xiong )], but are within the range reported in the literature for grasses (~0.44–51 mmol m−2 s−2 MPa−1; Holloway-Phillips and Brodribb, 2011; Ocheltree , ; Liu and Osborne, 2015; Xiong ) and may be a consequence of moderate PPFD during growth and measurements (Cochard ; Ocheltree ). Further experimentation and comparison of methods is needed for measurements of Kleaf in grasses. We need to understand why measurements of Kleaf produce similar values for C3 and C4 species; to establish whether Kleaf responses to ca correspond to changes in hydraulic vulnerability; and to determine the anatomical basis of adjustments to Kleaf, especially given evidence for declining LDMC and LMA among C3 species at cGLA. It will also be important to measure Kleaf at different [CO2]; as in the study of Kleaf responses to ca that used sunflower (Simonin ), we measured Kleaf at ambient [CO2].

Effects of ca on P–V characteristics also provide motivation for further investigation of photosynthetic type×ca responses. As leaf size decreased at cGLA, C4 grasses maintained LDMC and C3 grasses did not. In parallel, πTLP of C4 grasses became more negative at cGLA, while πTLP of C3 grasses did not change. This is an important result because πTLP is a powerful indicator of physiological responses that is expected to integrate smaller changes in, for example, π0 and ε (Bartlett ). The decrease in LDMC shown by C3 leaves grown at cGLA is consistent with both lower A and LMA, and previous evidence that C3 leaves decrease mesophyll cell volume and total non-structural carbohydrates as ca declines (Poorter ). Maintenance of LDMC and more negative πTLP in C4 grasses therefore might be linked with solute accumulation at cGLA. Presumably, decreases in πTLP of C4 leaves at low ca would support maintenance of turgor in the presence of larger ΔΨ induced by higher gsw (Franks, 2006; Simonin ); however, we do not know how leaf-level changes were integrated with adjustments in root and stem properties. The lack of an adjustment in πTLP by C3 grasses grown at cGLA might be associated with maintenance of leaf water status if root and stem xylem hydraulic conductivity increased or xylem solute concentrations decreased.

Conclusions

We predicted that gas exchange would show greater absolute responses to ca in C3 compared with C4 grass leaves, especially in terms of the positive relationship between iWUE and ca. We also predicted that low iWUE at cGLA would be linked with changes in leaf hydraulic properties. We found that while the iWUE advantage of some C4 grass leaves increased in absolute terms at cSUP, co-ordination among leaf traits was more strongly affected by cGLA than by cSUP. These experimental results broadly support predicted smaller impacts of cGLA on performance of C4 grasses (Osborne and Sack, 2012), and suggest that iWUE advantages to C4 species will continue to be important in future. A finding with potential importance for crop improvement programmes is that as ca increases, pressure on plants to improve iWUE through rapid stomatal responses to shade may be reduced, particularly for species capable of achieving high iWUE. These results highlight the need for continued efforts to establish how hydraulics and photosynthetic performance are co-ordinated, both within leaves and at the scale of whole plants. The mechanistic basis of these responses still needs to be better understood to predict the physiological implications of C4 photosynthesis, both under past glacial climates and as they will affect performance in a future high CO2 world.