Yield, growth and grain nitrogen response to 
elevated CO2 in six lentil (Lens culinaris) 
cultivars grown under Free Air CO2 
Enrichment (FACE) in a semi-arid 
environment
Authors: M. Bourgault, J. Brand, S. Tausz-Posch, R.D. 
Armstrong, G.L. O’Leary, G.J. Fitzgerald, & M. Tausz
NOTICE: this is the author’s version of a work that was accepted for publication in European 
Journal of Agronomy. Changes resulting from the publishing process, such as peer review, 
editing, corrections, structural formatting, and other quality control mechanisms may not be 
reflected in this document. Changes may have been made to this work since it was submitted 
for publication. A definitive version was subsequently published in European Journal of 
Agronomy, VOL# 87, (July 2017), DOI# 10.1016/j.eja.2017.05.003.
Bourgault, Maryse, J. Brand, S. Tausz-Posch, R.D. Armstrong, G.L. O'Leary, G.J. Fitzgerald, and 
M. Tausz. "Yield, growth and grain nitrogen response to elevated CO2 in six lentil (Lens culinaris) 
cultivars grown under Free Air CO2 Enrichment (FACE) in a semi-arid environment." European 
Journal of Agronomy 87 (Juy 2017): 50-58. DOI: 10.1016/j.eja.2017.05.003.
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Yield, growth and grain nitrogen response to elevated CO2 in six lentil (Lens
culinaris) cultivars grown under Free Air CO2 Enrichment (FACE) in a semi-
arid environment
M. Bourgaulta,d,⁎, J. Brandb, S. Tausz-Poscha,e, R.D. Armstrongb, G.L. O’Learyb, G.J. Fitzgeraldb,
M. Tauszc,f
a Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, 4 Water St, Creswick, VIC 3363, Australia
b Agriculture Victoria,Grains Innovation Park, 110 Natimuk Rd, Horsham, VIC, 3401, Australia
c Faculty of Science, The University of Melbourne, 4 Water St, Creswick, VIC, 3363, Australia
d Northern Agricultural Research Center, Montana State University, 3710 Assinniboine Rd, Havre, MT, 59501-8214, USA
e School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
f Birmingham Institute of Forest Research, University of Birmingham, Birmingham B15 2TT, UK
A R T I C L E I N F O
Keywords:
Source-sink relationships
Physiological pre-breeding
Climate change adaptation
Terminal drought
A B S T R A C T
Atmospheric CO2 concentrations ([CO2]) are predicted to increase from current levels of about 400 ppm to reach
550 ppm by 2050. The direct benefits of elevated [CO2] (e[CO2]) to plant growth appear to be greater under low
rainfall conditions, but there are few field (Free Air CO2 Enrichment or FACE) experimental set-ups that directly
address semi-arid conditions. The objectives of this study were to investigate the following research questions: 1)
What are the effects of e[CO2] on the growth and grain yield of lentil (Lens culinaris) grown under semi-arid
conditions under FACE? 2) Does e[CO2] decrease grain nitrogen in lentil? and 3) Is there genotypic variability in
the response to e[CO2] in lentil cultivars? Elevated [CO2] increased yields by approximately 0.5 t ha−1 (relative
increase ranging from 18 to 138%) by increasing both biomass accumulation (by 32%) and the harvest index (by
up to 60%). However, the relative response of grain yield to e[CO2] was not consistently greater under dry
conditions and might depend on water availability post-flowering. Grain nitrogen concentration was
significantly reduced by e[CO2] under the conditions of this experiment. No differences were found between
the cultivars selected in the response to elevated [CO2] for grain yield or any other parameters observed despite
well expressed genotypic variability in many traits of interest. Biomass accumulation from flowering to maturity
was considerably increased by elevated [CO2] (a 50% increase) which suggests that the indeterminate growth
habit of lentils provides vegetative sinks in addition to reproductive sinks during the grain-filling period.
1. Introduction
Atmospheric CO2 concentrations ([CO2]) have been increasing from
about 280 ppm to 406 ppm from the pre-industrial era until now
(January 2017; www.co2.earth; last accessed 23 February 2017). If
global greenhouse gas emissions remain at the 2010 level, then atmo-
spheric CO2 concentrations ([CO2]) should reach 550 ppm by 2050
(IPCC, 2014). This increase in the substrate of photosynthesis has direct
implications for plant metabolism, such as increased growth and yield,
at least in C3 plants and in the absence of changes in temperature and
rainfall patterns (Ainsworth and Long 2005; Ziska et al., 2012).
Elevated [CO2] (e[CO2]) also reduces stomatal conductance, leading
to higher transpiration efficiency (Leakey et al., 2009; Tausz-Posch
et al., 2013) and potentially increased crop water productivity under
conditions of water stress (Deryng et al., 2016; Gifford, 1979). In
environments prone to severe terminal drought (where the crop
progressively runs out of water with no resupply), there are however
concerns that the early increases in leaf area and biomass accumulation
under e[CO2] might negate the water savings from higher transpiration
efficiency, potentially leading to an earlier onset of drought. This could
reduce post-flowering growth and translocation of assimilates and
therefore reduce the yield response to e[CO2]. There are few FACE
experimental set-ups that directly address this type of water stress, and
experimental data to support or disprove this theory is sparse (Deryng
et al., 2016).
Higher growth and grain yield are often associated with decreased
MARK
grain quality, especially in cereals, where decreases in grain nitrogen
concentration ([N]) and therefore protein concentrations, raise con-
cerns about nutrition and product quality (Jablonski et al., 2002; Myers
et al., 2014). Decreased grain [N] under e[CO2] is preceded by
decreases in [N] in vegetative biomass, particularly in leaves (Leakey
et al., 2009). There are several hypotheses to explain this decrease in
tissue [N] (reviewed by Taub and Wang, 2008). The dilution hypothesis
contends that N supply fails to keep up with the increased demand from
stimulated biomass growth. Legumes, with their nitrogen-fixing sym-
bionts, would be able to overcome such limitation because the
additional carbohydrate acquired under e[CO2] could feed the N-fixing
symbiosis (Rogers et al., 2009). Indeed, field grown soybean did not
show decreases in leaf [N] from about mid-season onwards, once the N-
fixing symbiosis had established (Rogers et al., 2006). Studies in
chambers or high rainfall agro-ecosystems suggested that, in contrast
to cereals, legume grains maintain protein concentrations under e[CO2]
(Jablonski et al., 2002; Taub et al., 2008). As N-fixation is more
sensitive to water stress than biomass accumulation and leaf expansion
(Serraj et al., 1998), in semi-arid environments N-fixation is likely
interrupted by water stress and therefore biomass dilution of [N] might
occur. Some recent reports have found small but significant decreases in
grain protein in legumes which might be partially explained by water
stress (Lam et al., 2012 in chickpea (Cicer arietinum); Bourgault et al.,
2016 in field pea (Pisum sativum)).
It might be possible to take advantage of rising atmospheric [CO2]
by selecting for greater CO2 responsiveness in crop breeding programs
either directly or by selecting traits that are associated with a greater
response (Ainsworth et al., 2008; Tausz et al., 2013; Ziska et al., 2012).
Intraspecific variability in the response to e[CO2] − a prerequisite for
this approach − has been reported in soybean (Glycine max): Bishop
et al. (2015) have found consistent differences in grain yield response to
e[CO2] among 18 cultivars grown under Free Air CO2 Enrichment
(FACE) and suggested this is a heritable trait. They also suggested that
high e[CO2] response is related to a greater harvest index and a short
stature. Similarly, Bunce (2008) investigated variability in the response
to e[CO2] of 4 common bean (Phaseolus vulgaris) cultivars and
suggested that greater grain yield response was associated with the
ability to produce more pods under e[CO2]. Similarly, Ziska et al.
(2001) showed that greater yield response in soybean was related to the
ability of some cultivars to increase the seed production on auxiliary
branches. In contrast, studies reporting a lack of response to e[CO2] in
one cultivar have often related this to limitations in the ability to use
the additional carbohydrates. For example, Sicher et al. (2010) found
that a dwarf cultivar of soybean did not show yield increases under e
[CO2]. Grains of this cultivar were 75% smaller and had lower oil seed
content than grains of normal cultivar. Taken together, these studies
suggest that the response to e[CO2] might depend on both the capacity
of the plant to utilise the additional carbohydrates produced under e
[CO2] and the effective translocation of resources to grains later in yield
formation.
Lentil (Lens culinaris) is one of the oldest cultivated crops in the
world and its production has more than quadrupled since the 1960s
reaching a global production of over 5 million tonnes in 2013
(FAOSTAT, 2016). It is well-known as a nutritious grain and forms
the basis of many traditional Asian and Middle Eastern recipes
(Raghuvanshi and Singh, 2009). In Australia, it is grown as a winter
crop under non-irrigated conditions, and therefore frequently subjected
to terminal drought conditions. Further, the crop is often exposed to
low temperatures during the vegetative stage and high temperature
stress by pod filling (Materne and Siddique, 2009). Substantial efforts in
breeding lentils in Australia are recent and expanding genetic varia-
bility is still seen as a major activity (Siddique et al., 2013). Intraspe-
cific variability in many traits of interest is adequate and could be used
in breeding programs (Erskine et al., 2009), although there is little
published information on drought tolerance characteristics and no
information on potential intraspecific variability in the response to e
[CO2] in lentils.
In this study, we grew a range of lentil lines over three seasons in
the Australian Grains FACE (AGFACE) facility. AGFACE is located in the
south-eastern Australian grain cropping belt, with a typical semi-arid
Mediterranean climate, making it a representative site for significant
areas of global lentil production (Materne and Siddique, 2009).
Therefore, this experimental set-up allowed us to address the following
research questions:
• What are the effects of e[CO2] on the growth and grain yield of lentil
(Lens culinaris) grown under realistic drought conditions under Free
Air CO2 Enrichment (FACE)?
• Does e[CO2] decrease grain [N] in lentil?
• Is there genotypic variability in the response to e[CO2] in lentil
cultivars?
2. Materials and methods
2.1. Experimental site and growing conditions
The Australian Grains Free Air CO2 Enrichment (AGFACE) facility is
located near Horsham, Victoria (36°45′07″S 142°06′52″E, 127 m above
sea level). The site is cracking clay soil (Vertosol) with approximately
35% clay content at the surface increasing to 60% at 1.4 m depth. Long
term average (based on 1981–2010 period) annual rainfall is 435 mm,
with approximately 320 mm falling during the winter growing season
(from May to November inclusive). Average maximum and minimum
temperatures are 17.6 °C and 5.3 °C respectively during the season,
with July being the coldest month (Bureau of Meteorology, 2016).
Maximum and minimum temperatures as well as rainfall data during
the 2013–2015 growing seasons were recorded by an on-site weather
station (MEA Premium Weather Station 103, Measurement Engineering
Australia, Magill, SA, Australia; Fig. 1). Soil moisture was also
monitored in 2014 and 2015, on a weekly basis in two cultivars to a
depth of 1 m with a PR2 Profile Probe (Delta-T Devices, Cambridge,
UK) (Fig. 2).
Elevated CO2 levels (target 550 μmol mol−1 air) were maintained
during daylight hours by injecting pure CO2 into the air on the upwind
side from horizontal stainless-steel tubes so the gas would be carried
across the ring. The tubes were positioned about 150 mm above the
canopy and raised following the growth of the crop. Concentrations
were maintained within 90% target (495–605 μmol mol−1 air) for
93–98% of the time. More details on the site and the CO2 exposure
equipment are given in Mollah et al. (2009).
Plots were sown on 5 June 2013, 12 May 2014 and 26 May 2015.
Plots were treated according to local practice with pre-emergence
herbicides (simazine, dimethenamid-P, trifluralin, isoxaflutole, and/or
glyphosate) prior to sowing and with haloxyfob approximately
4–8 weeks after emergence if required. In 2014, the insecticide
dimetoate was also used to control aphids. Superphosphate was placed
with the seed at sowing at 9 kg P ha−1 and 11 kg S ha−1 each year. No
nitrogen fertilizer was added, but seeds were inoculated with granular
pea and lentil inoculant in 2013 and 2014 (Nodulator, BASF
Corporation, Research Triangle Park, NC, USA), and peat-based inocu-
lant in 2015 (NoduleN, New Edge Microbials Pty Ltd, Albury, NSW,
Australia).
2.2. Experimental design
In each year (2013–2015), there were 4 ambient and 4 elevated CO2
octagonal main plots organised in bays each containing one ambient
and one e[CO2] plot. The design within each plot differed from year to
year. In 2013, plots were 8 m in diameter and split in half for a plus/
minus supplemental irrigation treatment. Six cultivars were organised
in sub-plots of 2 rows (0.55 m) by 4 m in each half. However, because
the supplemental irrigation treatment was omitted during this season
and emergence was uneven, the ‘best’ sub-plot (by emergence) per
cultivar per ring was chosen for the harvest sampling. In 2014, plots
were 4 m in diameter. The same six cultivars were organised in sub-
plots of 4 rows (0.976 m) by 2 m length. In 2015, lentil sub-plots were
included in predominantly wheat main plots, on adjacent edge sub-
plots (6 rows or 1.5 m by 4 m length). Only PBA Ace and 05H010L-
07HS3010 were grown in 2015.
2.3. Plant material
Five lentil cultivars and one breeding line were selected to account
for the genetic diversity present in the Australian collection. The
cultivars Boomer and PBA Giant are large-seeded green lentil types,
while PBA Ace, PBA Jumbo, Nipper and 05H010L-07HS3010 are red
lentils. Nipper is a small-seeded lentil, PBA Ace and 05H010L-
07HS3010 are medium sized and PBA Jumbo is a large-seeded red
lentil. The breeding line 05H010L-07HS3010 (abbreviated HS3010 in
figures) also showed large biomass accumulation in favourable yield
conditions, but with a smaller harvest index than commercial lines (M.
Rodda, personal communication).
2.4. Measurements
Non-destructive measurements of canopy reflectance, from which
Normalised Difference Vegetation Index (NDVI) was calculated, were
taken with a CropCircle ACS-210 with GeoSCOUT GLS-400 data logger
(Holland Scientific, Lincoln, NE, USA) as an indirect measurement of
crop canopy development (Fitzgerald, 2010; Perry et al., 2012). These
were taken approximately every week (2014) or fortnightly (2015)
from the vegetative stage to the pod filling stage in 2014 and 2015.
Destructive samplings were conducted at flowering (2014 and 2015
only) and maturity stages. These correspond to the full bloom (R2) and
full maturity (R8) stages (Erskine et al., 1990). At the flowering stage,
aboveground biomass was harvested from a quadrat (central 2 rows by
Fig. 1. Weather data recorded on-site in the 2013–2015 growing seasons in AGFACE (Horsham, Victoria, Australia) with arrows showing relevant events.
0.25 m in 2014 or 0.12 m2; central 4 rows by 0.30 m in 2015 or
0.29 m2). A subsample was separated into leaves and stems. All samples
were dried at 65–70 °C for at least 72 h and weighed for above-ground
biomass determination. Leaf and stem samples were then ground and
analysed for [N] by LECO Tru Mac Elemental Analyser (LECO
Corporation, St. Joseph, MI, USA). At maturity in 2013, two 0.50 m
rows were harvested (corresponding to 0.275 m2). In 2014, a quadrat of
the central 2 rows by 0.20 m (0.10 m2) was harvested and in 2015, the
quadrat was 4 rows by 0.30 m (0.29 m2), also excluding the two outside
rows. Samples were dried at 40 °C for 72 h, then weighed for total
above-ground biomass. Fully-formed pods (even if empty) were sepa-
rated and counted, then threshed. Grains were weighed and seed count
was also recorded. In 2013 and 2015, pods and seeds were determined
on a sub-sample but in 2014 the samples were small enough that the
pods and seeds were determined on the entire sample. Grain and straw
were ground and analysed for [N] as described above. Post-flowering
biomass accumulation, as a measure of an indeterminate growth habit,
was calculated by subtracting the biomass accumulation at flowering
from the total biomass accumulation at the maturity stage.
2.5. Statistical analysis
The experiment was designed as a complete block split-plot design.
Years, [CO2] treatments as the main plot (i.e ‘rings’), and cultivars (the
sub-plot) were considered fixed effects. Blocks and ring number (nested
within blocks) were considered random effects. For yield and final
biomass data, the variance was heterogeneous and correlated to means,
so a log-transformation was performed before the statistical analysis.
Dependent variables were analysed using a mixed model analysis
procedure using the AsREML algorithm (Butler et al., 2009; Gilmour
et al., 1995 Gilmour et al., 1995; AsREML-R, VSN International, Hemel
Hempstead, UK) as implemented in the R language (R Core Team,
2013). While the [CO2] treatments are the same as rings within an
individual year, having both a fixed term and a random term decouples
the physical locations from the treatments across the three years, and
ensures that the stratification of treatments is properly imposed in the
analysis with AsREML as recommended by Butler et al. (2007); (Section
8.2). Wald tests were performed for the fixed effects. For repeated
measures of NDVI during the course of the season, we performed a
multivariate analysis with NDVI values for each date as dependent
variables based on the same model as described above, but for each
year separately, and with an antedependence variance-covariance
structure to account for the correlation of residuals from measurements
done on the same plots as suggested by Wolfinger (1996) and Smith
et al. (2007).
3. Results
3.1. Grain yield and yield components
Grain yield increased in response to e[CO2] and there was a wide
range of relative responses between years (32, 138, and 18% in 2013,
2014 and 2015 respectively) (Fig. 3A). The absolute CO2 response in
grain yield appeared to be constant at approximately 0.5 t ha−1
regardless of yield potential. This increase in yield was associated with
an increase in total above-ground biomass measured at maturity (by
Fig. 2. Plant available water (mm) in lentil plots in the 2014 and 2015 growing seasons in AGFACE (Horsham, Victoria, Australia).
Means are presented as averages ± standard errors over four replicates, two cultivars and two CO2 levels. No significant differences between CO2 levels were detected in any of the
readings.
Fig. 3. Effects of elevated [CO2] on grain yield (A), final above-ground biomass (B) and harvest index (C) for six lentil cultivars in AGFACE (2013–2015).
Means are presented as averages ± standard errors over four replicates. Interactions were not significant unless indicated in the figure. Significance levels: p > 0.10 “NS”, p < 0.05
“*”, p < 0.01 “**”, p < 0.001 “***”.
32%; Fig. 3B), harvest index (by 16%; Fig. 3C) and in the number of
pods per m2 (by 55%; Table 1). Harvest index increased relatively more
under e[CO2] with low values under ambient conditions (Fig. 3C): in
2014, HI increased by 60% compared to 7 and−3% in 2013 and 2015
respectively, and this is confirmed by a significant year by [CO2]
interaction.
There were large differences in grain yield and yield components
between years (Table 1), reflecting the variable climate typical of the
region. Elevated [CO2] consistently increased the number of pods per
m2, but the effect on seed weight and on the number of seeds per pod
was dependent on the year. In 2013 and 2015, when the seed weight
was relatively high under ambient [CO2] (a[CO2]), e[CO2] had no
effect, but it increased seed weight in 2014 under terminal drought
conditions (Table 1). Similarly, the number of seeds per pod, on
average, were increased by e[CO2] in 2014 and 2015, but not in
2013, although the cultivars PBA Ace and PBA Giant showed a positive
response to e[CO2] in all three years (Table 1).
Cultivar differences were also significant for most of these para-
meters, except for biomass accumulation which was similar for all
cultivars. Expression of cultivar differences was consistent with cultivar
characterisations. For example, 05H010L-07HS3010 showed lower
grain yield and a low harvest index (Fig. 3A,C), while the cultivar
Nipper showed small grain size (Table 1). No cultivar by [CO2]
interactions were detected for these parameters, which suggests all
cultivars responded similarly to e[CO2].
3.2. Growth
Above-ground biomass at flowering was increased by e[CO2] by
18% (Fig. 4). The green lentil cultivar PBA Giant showed the highest
biomass accumulation at this growth stage with 05H010L-07HS3010
the smallest. Biomass accumulation from flowering to maturity was
increased substantially under e[CO2] (a 50% increase, Fig. 4). However,
year main effects were not significant for these parameters, despite
contrasting water availability in 2014 and 2015.
The Normalised Difference Vegetation Index (NDVI) as an indirect
measure of crop canopy development showed significant [CO2] effects
with the NDVI values being greater under e[CO2] at every time point
Table 1
Effects of elevated [CO2] on yield components for six lentil cultivars in AGFACE (2013–2015).
2013 2014 2015
Ambient [CO2] Elevated [CO2] Ambient [CO2] Elevated [CO2] Ambient [CO2] Elevated [CO2]
Cultivar 100-seed weight (g)
Nipper 3.00 2.97 1.84 1.91
PBA Ace 3.89 4.06 2.19 2.62 4.47 3.92
05H010L-07HS3010 3.86 3.83 2.44 3.17 4.32 3.87
PBA Jumbo 4.28 4.38 2.93 3.05
Boomer 5.58 5.46 3.18 4.14
PBA Giant 6.13 6.08 3.35 3.77
Cultivar means 4.46 4.47 2.7 3.1 4.40 3.90
Significance Year ***
[CO2] NS Ambient [CO2] 4.04
Cultivar *** Elevated [CO2] 3.98
Year × [CO2] ***
Year × Cultivar ***
Year × [CO2] × Cultivar NS
Pods (# m−2)
Nipper 8666 11462 3273 6075
PBA Ace 6403 9150 1921 2976 4646 4357
05H010L-07HS3010 6338 12108 978 3153 2929 3061
PBA Jumbo 9036 10742 2682 6047
Boomer 6172 9905 1647 2177
PBA Giant 5029 6341 1627 3217
Cultivar means 6941 9951 2021 3941 3788 3709
Significance Year ***
[CO2] ** Ambient [CO2] 4624
Cultivar *** Elevated [CO2] 6114
Year × [CO2] NS
Year × Cultivar NS
Year × [CO2] × Cultivar NS
Seed per pod (#)
Nipper 1.42 1.33 1.05 1.23
PBA Ace 1.42 1.50 1.31 1.53 0.94 1.06
05H010L-07HS3010 1.11 0.94 0.96 1.13 0.78 0.82
PBA Jumbo 1.24 1.10 1.13 1.15
Boomer 1.16 0.77 0.88 1.31
PBA Giant 1.14 1.18 0.88 1.01
Cultivar means 1.25 1.14 1.04 1.23 0.86 0.94
Significance Year ***
[CO2] NS Ambient [CO2] 1.03
Cultivar *** Elevated [CO2] 1.04
Year × [CO2] ***
Year × Cultivar **
Year × [CO2] × Cultivar *
p > 0.10 “NS”.
* p < 0.05.
** p < 0.01.
*** p < 0.001.
(Fig. 5). Time by cultivar interactions were also detected in both years
(not shown). In 2014 there were large differences between cultivars at
the beginning of measurements, with PBA Ace showing the highest crop
canopy cover, and 05H010L-07HS3010 showing the lowest but catch-
ing up later in the season. In 2015, these same cultivars started off at
the same point, but PBA Ace stabilised at higher NDVI values than
05H010L-07HS3010. There was also a significant [CO2] by cultivar
interaction in 2014 where 05H010L-07HS3010 showed lower NDVI
values under ambient conditions compared to other cultivars while it
reached similar levels under e[CO2] (not shown). However, emergence
was lower in 2014 than in 2015 which might explain part of the crop
canopy cover differences.
3.3. Above-ground nitrogen concentration and content at flowering
Above-ground nitrogen concentration ([N]) at flowering decreased
by 4% under e[CO2](Fig. 6). This was due to a decrease in leaf [N] as
stem [N] was unaffected by the [CO2] treatment. There were also
cultivar differences with PBA Jumbo showing the highest [N] concen-
tration in both leaves and stems while PBA Giant showed the lowest
concentration (Fig. 6). Total N content in the above-ground biomass at
the flowering stage increased under e[CO2] (not shown) because of
greater biomass under e[CO2], but cultivar differences were marginally
significant (with 05H010L-07HS3010 showing the smallest N accumu-
lation). Again, no [CO2] by cultivar interactions were found for these
parameters.
Fig. 4. Biomass accumulation at flowering and from flowering to maturity in six lentil cultivars in AGFACE (2014–2015).
Means are presented as averages ± standard errors over four replicates, and for graphs on the left, 8 year-cultivar combinations (i.e two years for PBA Ace and 05H010L-
07HS3010–shortened as *HS3010, one year for others; left side n = 32; right side n = 4 or 8). The CO2 main effects were significant for both parameters, while the cultivar main effects
were significant for biomass accumulation at flowering. The year main effects were not significant for either parameter presented here. A flowering sampling was not collected in 2013.
Fig. 5. Time course of the Normalized Difference Vegetation Index (NDVI) values in six lentil cultivars in AGFACE (2014–2015).
Each point is presented ± its standard error and represents the average over four replicates (n = 4). The [CO2] main effect was significant in both years.
3.4. Nitrogen concentration and content in grains and stubble
Grain [N] was slightly but significantly decreased by 2% with e
[CO2] and this was consistent between cultivars (Fig. 6). The small-
seeded red lentil cultivar Nipper had the highest grain [N], while the
large red lentil PBA Jumbo had the lowest. Stubble [N] was lower by
22% under e[CO2] (Fig. 6), but there was a trend for total N in the
stubble to be higher under e[CO2] (p = 0.09) because of higher stubble
biomass (64.5 compared to 81.4 kg ha−1 under e[CO2]). There were
significant differences between cultivars in the stubble [N] with green
lentils exhibiting lower [N] in the stubble than red lentils, but no
differences in the response to e[CO2] between cultivars were found
(Fig. 6). The nitrogen harvest index (nitrogen content in grains divided
by the amount of nitrogen in above-ground biomass) was increased
under e[CO2], but only at lower levels with a similar pattern to the
grain harvest index (Fig. 3C).
4. Discussion
Grain yield increases (18, 32 and 138%) reported here with e[CO2]
are relatively large compared to previous FACE studies in two years out
of three. Other FACE studies with legumes include the SoyFACE
Fig. 6. Nitrogen concentration at flowering and maturity in six lentil cultivars in AGFACE (2013–2015).
Means are presented as averages ± standard errors over four replicates, 14 year-cultivar combinations for biomass above-ground [N] at flowering and grain [N] (left side n = 56, right
side n = 8 or 12) or 8 year-cultivar combination for leaf[N], stem[N] and stubble [N] (left side n = 32; right side n = 4 or 8). The [CO2] main effects were significant for all parameter
except stem [N] and the cultivar main effects were significant for all parameters. Year main effects were also significant, but without interactions with CO2 levels. The CO2 by cultivar
interaction was not significant for any of the parameters presented here.
experiment (Illinois, USA), where soybean yields increased on average
by 15% (Morgan et al., 2005) and from 0 to 22% depending on the
cultivar in a different experiment (Bishop et al., 2015). The mini-FACE
array at the Chinese Academy of Agricultural Sciences (Changping,
China) also investigated soybean and showed that one cultivar did not
respond while the other showed a 26% increase in yield with e[CO2]
(Hao et al., 2012). Mungbean (Vigna radiata) was also grown at the
Chinese mini-FACE and showed yield increases of 14% (Gao et al.,
2015). Finally, the Australian Grains Free Air CO2 Enrichment (AGFA-
CE) site (Horsham, Australia) investigated field peas from 2010 to
2012, producing average grain yield responses of 26% (Bourgault et al.,
2016).
The relative increase in yields under e[CO2] may be greater with
decreasing water availability (Gifford, 1979), as e[CO2] improves plant
water use efficiency (Leakey et al., 2009). Comparisons between FACE
experiments for the response to e[CO2] are difficult because, apart from
climatic differences, many additional factors potentially contribute to
differences in response − species, soils, genotypes, nutritional levels,
etc. However, in this present study, we had three very contrasting years.
In 2013, there were frequent rainfall events during the entire season. In
2014, the season started relatively wet, but the crop experienced a
terminal drought, while in 2015, drought was relieved after flowering
with several irrigation events. The relative response for these years was
32%, 138% and 18% respectively and the absolute response in grain
yield appeared to be more or less constant at approximately 0.5 t ha−1
regardless of yield potential. As demonstrated by Gifford (1979) in a
controlled environment study, it is possible that a crop failure under a
[CO2] might result in some small yield under e[CO2]. In this case, the
relative response to e[CO2] becomes infinite, although the absolute
response might be small. Are relative responses then appropriate to
evaluate grain yield response to e[CO2] when yields under a[CO2] are
close to a crop failure? Regardless, we also observed a greater response
in 2013, a wet year, compared to 2015, so the existence of water stress
might not be enough to promote a greater response to e[CO2]. A greater
response to e[CO2] might only be expected under conditions of terminal
drought, when water saved from growth under e[CO2] is then available
during pod filling. Further investigations are warranted to confirm this
as we could not detect differences in soil moisture data between a[CO2]
and e[CO2] to support this hypothesis. However, Manschadi et al.
(2006) suggested that water savings below detection thresholds could
have substantial effects on grain yield.
Biomass accumulation between flowering and maturity was sub-
stantially increased by e[CO2] (50%) and substantially more stimulated
than biomass accumulation from planting to flowering (18%). Lentil is
an indeterminate crop that can continue to produce biomass including
vegetative structures past flowering. The relatively larger increase in
biomass post flowering suggests that the indeterminate growth habit
provides vegetative sinks in addition to reproductive sinks. There were
similar increases in biomass gain after flowering in both 2014 and
2015, despite large differences in soil water availability at this stage of
the crop development (Fig. 2). However, there were large differences in
harvest index (7, 60 and −3% for 2013, 2014 and 2015 respectively),
translating to differences in grain yield. To our knowledge, there is only
one other report of e[CO2] increasing harvest index in crops: Fitzgerald
et al. (2016) reported a significant increase in wheat harvest index, but
only for one year and one site (Walpeup in Victoria, Australia, but not
AGFACE at Horsham). Field studies that reported data on the harvest
index of legumes include Bishop et al. (2015) who showed an average
reduction of 5% in harvest index in soybean and Bourgault et al. (2016)
who showed no change from e[CO2] in field peas. Other AGFACE
studies with w5 heat performed on the same site also showed no change
in harvest index (Tausz-Posch et al., 2012; Tausz-Posch et al. 2015).
Manderschied and Weigel (1997), who investigated historical cultivars
that differed in harvest index, also showed that there was no change in
HI with e[CO2] for any of the cultivars tested. Improvements in water
use efficiency, especially if this leads to increased water availability at
the pod filling stage, could explain the increase in harvest index
observed in lentil, but again, we could not detect these differences in
soil moisture data.
The decrease in leaf and grain [N] in lentils in this study appears
contrary to what is commonly reported in the literature (Jablonski
et al., 2002; Myers et al., 2014; Rogers et al., 2006), although the
decrease in grain [N], though significant, was relatively small. In
nitrogen fixing legumes, the additional assimilates could help feed the
N-fixing symbiosis and thereby avoid decreases in leaf and grain [N]
(Rogers et al., 2009). In SoyFACE, field grown soybean did not show
decreases in leaf [N] under e[CO2] from about mid-season onwards,
once the N-fixing symbiosis had established (Roger et al., 2006). In this
present study, we observed poor nodulation in 2014 which could
explain decreases in tissue [N] as the lentils may have depended on soil
nitrogen, but nodulation was adequate in 2015. Yet, both leaf and grain
[N] were depressed regardless of nodulation levels. This decrease in
tissue [N] could potentially be explained by a number of hypotheses
(reviewed by Taub and Wang, 2008), including the natural dilution of
[N] with growth (Coleman et al., 1993) or higher photosynthetic
efficiency leading to reduced nitrogen allocations to leaves (Moore
et al., 1999). This is difficult to ascertain in this study because of the
limited number of destructive samplings. Other hypotheses include
decreased transpiration flow (McGrath and Lobell, 2013), or inhibition
of nitrate assimilation (Bloom et al., 2014), although in the context of
this study, the latter is not immediately apparent, as both poor and well
nodulated crops showed the same decrease in tissue [N].
The identification of consistent genotypic variability in the response
to e[CO2] is a prerequisite to use this information in breeding programs.
Unlike the studies by Bishop et al. (2015) in soybean and Bunce (2008)
in common bean, we could not detect differences between the cultivars
tested in their response to e[CO2] for grain yield or other parameters
observed. Traits related to source-sink relationships are candidate traits
that might impact the response to e[CO2] (Tausz et al., 2013). Traits
such as a determinate growth habit (Ainsworth et al., 2004), small
seeds (Sicher et al., 2010) and low harvest index (Bishop et al., 2015)
could limit the capacity of plants to utilise the additional assimilates
provided by e[CO2]. In this study, we used genotypic variability in
some of these traits: 1) the cultivar Nipper had the expected small seeds
with a grain size of 2.8 g per 100 seeds, while the breeding line PBA
Giant had a grain size of 5.1 g per 100 seeds (Table 1); 2) the breeding
line 05H0101L-07HS3010 had the lowest harvest index compared to
PBA whereas Jumbo which had the highest (Fig. 3C), and 3) the
cultivar PBA Giant tended to have the lowest biomass accumulation
post-flowering, while the Boomer had the highest (Fig. 4). Yet, all of
these cultivars showed similar benefits from e[CO2] for grain yield.
While these traits might be related to sink limitation, they might not be
sufficient to cause sink limitation and lower the response to e[CO2]. We
therefore suggest the lentil cultivars selected were not sink-limited
under e[CO2]. Whitehead et al. (2014) have found that seed yield
improvement in lentils has been associated with higher biomass and
occurred independently of improvements in harvest index. It is possible
that grain yield in lentil is source-limited, in which case, it might be
worth looking for genotypic variability in photosynthetic assimilation
rates.
5. Conclusion
In this study, e[CO2] increased yields by approximately 0.5 t ha−1
(relative increase ranging from 18 to 138%) by increasing both biomass
accumulation (by 32%) and the harvest index (by up to 60%). The
response of grain yield and harvest index varied with season, with the
greatest response in both parameters observed during a terminal
drought. Biomass accumulation post-flowering was increased consider-
ably by e[CO2] (a 50% increase), suggesting that the indeterminate
growth habit of lentil provides vegetative sinks in addition to repro-
ductive sinks. Grain yield in lentil appears to be source limited, and
traits associated with carbon assimilation might be better candidates to
improve response to e[CO2] as we were not able to detect differences in
the response to elevated [CO2] for grain yield in cultivars that differed
in biomass accumulation, seed size or harvest index. Further research is
warranted to explain decreases in tissue [N] in well nodulated lentil
crops grown under e[CO2] and to explain why lentil appears to be the
only crop to increase harvest index under e[CO2].
Acknowledgements
Research at the Australian Grains Free Air Carbon dioxide
Enrichment (AGFACE) facility is jointly run by the Victorian
Government and the University of Melbourne and receives substantial
additional funding from the Australian Commonwealth Department of
Agriculture and Water Resources (DAFWR) and the Grains Research
and Development Corporation (GRDC). We wish to acknowledge the
crucial contributions of Mahabubur Mollah (AGFACE research engi-
neer) and Russel Argall (senior technical officer) and their team in
running and maintaining the AGFACE facility, as well as Samuel Henty,
Shahnaj Parvin and other team members from the University of
Melbourne for technical help.
References
Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2
enrichment (FACE)? A meta-analytic review of the responses of photosynthesis,
canopy properties and plant production to rising CO2. New Phytol. 165, 351–372.
Ainsworth, E.A., Rogers, A., Nelson, R., Long, S.P., 2004. Testing the source-sink
hypothesis of down-regulation f photosynthesis in elevated [CO2] in the field with
single gene substitutions in Glycine max. Agric. Forest Meteorol. 122, 85–94.
Ainsworth, E., Beier, C., Calfapietra, C., Ceulemans, R., Durand-Tardif, M., Farquhar,
G.D., Godbold, D.L., Hendry, G.R., Hickler, T., Kaduk, J., Karnosky, D.F., Kimball, B.,
Körner, C., Koornneef, M., Lafarge, T., Leakey, A.D., Lewin, K.F., Long, S.P.,
Manderscheid, R., McNeil, D.L., Mies, T.A., Miglieta, F., Morgan, J.A., Nagy, J.,
Norby, R.J., Norton, R.M., Percy, K.E., Rogers, A., Soussana, J.-F., Stitt, M., Weigel,
H.-J., White, J.W., 2008. Next generation of elevated [CO2] experiments with crops: a
critical investment for feeding the future world. Plant. Cell Environ. 31, 1317–1324.
Bishop, K.R., Betzelberger, A.M., Long, S.P., Ainsworth, E.A., 2015. Is there potential to
adapt soybean (Glycine max Merr.) to future [CO2]? An analysis of the yield response
of 18 genotypes in free- air CO2 enrichment. Plant. Cell Environ. 38, 1765–1774.
Bloom, A.J., Burger, M., Kimball, B., Ointer Jr., P.J., 2014. Nitrate assimilation is
inhibited by elevated CO2 in field-grown wheat. Nat. Clim. Change 4, 477–480.
Bourgault, M., Brand, J., Tausz, M., Fitzgerald, G.J., 2016. Yield: growth and grain
nitrogen response to elevated CO2 of five field pea (Pisum sativum L.) cultivars in a
low rainfall environment. Field Crop Res.h 196, 1–9.
Bunce, J.A., 2008. Contrasting responses of seed yield to elevated carbon dioxide under
field conditions within Phaseolus vulgaris. Agric. Ecosyst. Environ. 128, 219–224.
Bureau of Meteorology, 2016. Climate Data Online Database (Horsham Polkemmet Rd
VIC). Australian Government. http://www.bom.gov.au/climate/data/. (Accessed 18
January 2016).
Butler, D.G., Cullis, B.R., Gilmour, A.R., Gogel, B.J., 2007. ASReml-R reference manual
Queensland Department Primary Industries and Fisheries. Toowoomba, QLD,
Australia 148 pp.
Coleman, J.S., McConnaughay, K.D.M., Bazzaz, F.A., 1993. Elevated CO2 and plant
nitrogen-use: is reduced tissue nitrogen concentration size dependent? Oecologia 93,
195–200.
Deryng, D., Elliott, J., Folberth, C., Müller, C., Pugh, T.A.M., Boote, K.J., Conway, D.,
Ruane, A.C., Gerten, D., Jones, J.W., Khabarov, N., Olin, S., Schaphoff, S., Schmid, E.,
Yang, H., Rosenzweig, C., 2016. Regional disparities in the beneficial effects of rising
CO2 concentrations on crop water productivity. Nat. Clim. Change 6 (8), 786–790.
http://dx.doi.org/10.1038/nclimate2995.
Erskine, W., Muehlbauer, F.J., Sarker, A., Shama, A., 2009. Chapter 1: introduction. In:
Erskine, W., Muehlbauer, F.J., Sarker, A., Shama, A. (Eds.), The Lentil: Botany,
Production and Uses. CAB International Cambridge, MA, USA, pp. 1–3.
FAOSTAT, 2016. Food and Agriculture Organisation of the United Nations Statistics
Division. http://faostat3.fao.org/home/E. (Accessed 5 February 2016).
Fitzgerald, G.J., Tausz, M., O’Leary, G., Mollah, M.R., Tausz-Posch, S., Seneweera, S.,
Mock, I., Löw, M., Partington, D.L., McNeil, D., Norton, R.M., 2016. Elevated
atmospheric [CO2] can dramatically increase wheat yields in semi-arid environments
and buffer against heat waves. Global Change Biol. 22 (6), 2269–2284.
Fitzgerald, G.J., 2010. Characterizing vegetation indices derived from active and passive
sensors. Int. J. Remote Sens. 31 (16), 4335–4348.
Gao, J., Seneweera, S., Li, P., Zong, Y.-Z., Dong, Q., Lin, E., Hao, X., 2015. Leaf
photosynthesis and yield components of mung bean under fully open-air elevated
[CO2]. J. Integr. Agric. 14 (5), 977–983.
Gifford, R.M., 1979. Growth and yield of CO2-enriched wheat under water-limited
conditions. Aus. J. Plant Physiol. 6, 367–378.
Gilmour, A.R., Thompson, R., Cullis, B.R., 1995. Average information REML: An efficient
algorithm for variance component estimation in linear mixed models. Biometrics 51,
1440–1450.
Hao, X.Y., Han, X., Lam, S.K., Wheeler, T., Ju, H., Wang, H.R., Li, Y.C., Lin, E.D., 2012.
Effects of fully open-air [CO2] elevation on leaf ultrastructure, photosynthesis, and
yield of two soybean cultivars. Photosynthetica 50, 362–370.
Intergovernmental Panel on Climate Change (IPCC), 2014. Climate Change 2014:
Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment
Report of the Intergovernmental Panel on Climate Change. In: Pachauri, R.K., Meyer,
L.A. (Eds.), IPCC, Geneva Switzerland (151 pp).
Jablonski, L.M., Wang, X., Curtis, P., 2002. Plant reproduction under elevated CO2
conditions: a meta-analysis of reports on 79 crops and wild species. New Phytol. 156,
9–26.
Lam, S.K., Chen, D., Norton, R., Armstrong, R., 2012. Does phosphorus stimulate that
effect of elevated [CO2] on growth and symbiotic nitrogen fixation of grain and
pasture legumes? Crop Pasture Sci. 63, 53–62.
Leakey, A.D.B., Ainsworth, E.A., Bernacchi, C.J., Rogers, A., Long, S.P., Ort, D.R., 2009.
Elevated CO2 effects on plant carbon, nitrogen, and water relations: six important
lessons from FACE. J. Exp. Bot. 60 (10), 2859–2876.
Manderschied, R., Weigel, H.J., 1997. Photosynthetic and growth responses of old and
modern spring wheat cultivars to atmospheric CO2 enrichment. Agric. Ecosyst.
Environ. 64, 65–73.
Manschadi, A.M., Christopher, J., deVoil, P., Hammer, G.L., 2006. The role of root
architectural traits in adaptation of wheat to water-limited environments. Funct.
Plant Biol. 33, 823–837.
McGrath, J.M., Lobell, D.B., 2013. Reduction of transpiration and altered nutrient
allocation contribute to nutrient decline of crops grown in elevated CO2
concentrations. Plant Cell Environ. 36, 697–705.
Mollah, M., Norton, R., Huzzey, J., 2009. Australian grains free-air carbon dioxide
enrichment (AGFACE) facility: design and performance. Crop Pasture Sci. 60,
697–707.
Moore, B.D., Cheng, S.-H., Sims, D., Seemann, J.R., 1999. The biochemical and molecular
basis for photosynthetic acclimation to elevated CO2. Plant Cell Environ. 22,
567–582.
Morgan, P.B., Bollero, G.A., Nelson, R.L., Dohleman, F.G., Long, S.P., 2005. Smaller than
predicted increase in aboveground net primary production and yield of field-grown
soybean under fully open-air [CO2] elevation. Global Change Biol. 11, 1856–1865.
Myers, S.S., Zanobetti, A., Klogg, I., Huybers, P., Leakey, A.D.B., Bloom, A.J., Carlisle, E.,
Dietterich, L.E., Fitzgerald, G., Hasegawa, T., Holbrook, N.M., Nelson, R.L., Ottman,
M.J., Raboy, V., Sakai, H., Sartor, K.A., Schwartz, J., Seneweera, S., Tausz, M., Usui,
Y., 2014. Increasing CO2 threatens human nutrition. Nature 510, 139–143.
Perry, E.M., Fitzgerald, G.J., Poole, N., Craig, S., Whitlock, A., 2012. NDVI from active
optical sensors as a measure of canopy cover and biomass. Int. Arch. Photogramm.
XXXIX-B8, 317–319.
R Core Team, 2013. R: A Language and Environment for Statistical Computing. R
Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/.
Raghuvanshi, R.S., Singh, D.P., 2009. Chapter 25: food preparation and use. In: Erskine,
W., Muehlbauer, F.J., Sarker, A., Shama, A. (Eds.), The Lentil: Botany, Production
and Uses. CAB International, Cambridge MA USA, pp. 408–424.
Rogers, A., Gibon, Y., Stitt, M., Morgan, P.B., Bernacchi, C.J., Ort, D.R., Long, S.L., 2006.
Increased C availability at elevated carbon dioxide concentration improves N
assimilation in a legume. Plant Cell Environ. 29, 1651–1658.
Rogers, A., Ainsworth, E.A., Leakey, A.D.B., 2009. Will elevated carbon dioxide
concentration amplify the benefits of nitrogen fixation in legumes? Plant Physiol.
151, 1009–1016.
Serraj, R., Sinclair, T.R., Allen, L.H., 1998. Soybean nodulation and N2 fixation response
to drought under carbon dioxide enrichment. Plant. Cell Environ. 21, 491–500.
Sicher, R., Bunce, J., Matthews, B., 2010. Differing responses to carbon dioxide
enrichment by a dwarf and a normal-sized soybean cultivar may depend on sink
capacity. Can. J. Plant Sci. 90, 257–264.
Siddique, K.H.M., Erskine, W., Hobson, K., Knights, E.J., Leonforte, A., Khan, T.N., Paull,
J.G., Redden, R., Materne, M., 2013. Cool-season grain legume improvement in
Australia – use of genetic resources. Crop Pasture Sci. 64, 347–360.
Smith, A.B., Stringer, J.K., Wei, X., Cullis, B.R., 2007. Varietal selection for perennial
crops where data relate to multiple harvests from a series of field trials. Euphytica
175, 253–266.
Taub, D.R., Wang, X., 2008. Why are nitrogen concentrations in plant tissues lower under
elevated CO2? A critical examination of the hypotheses. J. Integr. Plant Biol. 50 (11),
1365–1374.
Tausz, M., Tausz-Posch, S., Norton, R.M., Fitzgerald, G.J., Nicolas, M.E., Seneweera, S.,
2013. Understanding crop physiology to select breeding targets and improve crop
management under increasing atmospheric CO2 concentrations. Environ. Exp. Bot.
88, 71–80.
Tausz-Posch, S., Seneweera, S., Norton, R.M., Fitzgerald, G.J., Tausz, M., 2012. Can a
wheat cultivar with high transpiration efficiency maintain its yield advantage over a
near-isogenic cultivar under elevated CO2? Field Crops Res. 133, 160–166.
Tausz-Posch, S., Norton, R.M., Seneweera, S., Fitzgerald, G.J., Tausz, M., 2013. Will intra-
specific differences in transpiration efficiency be maintained in a high CO2 world? A
FACE study. Physiol. Plant. 148, 232–245.
Tausz-Posch, S., Dempsey, R.W., Seneweera, S., Norton, R.M., Fitzgerald, G., Tausz, M.,
2015. Does a freely tillering wheat cultivar benefit more from elevated CO2 than a
restricted tillering cultivar in a water-limited environment? Eur. J. Agron. 64, 21–28.
Whitehead, S.J., Summerfield, R.J., Muehlbauer, F.J., Coyne, C.J., Ellis, R.H., Wheeler,
T.R., 2014. Crop improvement and the accumulation and partitioning of biomass and
nitrogen in lentil. Crop Sci. 40, 110–120.
Wolfinger, 1996. Heterogeneous variance-covariance structures for repeated measures.
Journal of Agric. Biol. Environ. Stat. 1, 362–389.
Ziska, L.H., Bunce, J.A., Caufield, F.A., 2001. Rising atmospheric carbon dioxide and seed
yield of soybean genotypes. Crop Sci. 41, 385–391.
Ziska, L.H., Bunce, J.A., Shimono, H., Gealy, D.R., Baker, J.T., Newton, P.C.D., Reynolds,
M., Jagadish, K.S.V., Zhu, C., Howden, M., Wilson, L.T., 2012. Food security and
climate change: on the potential to adapt global crop production by active selection
to rising atmospheric carbon dioxide. Proc. R. Soc. B 279, 4097–4105.