Introduction
The natural abundance analysis of stable carbon isotopes in plants has become
an essential tool for studying plant-environmental interactions, plant
metabolism, carbon allocation, and biosphere–atmosphere exchanges of carbon
fluxes (Dawson et al., 2002; Bowling et al., 2008; Tcherkez et al., 2011;
Cernusak et al., 2013). Understanding processes and factors controlling
carbon isotope compositions in different plant organs, which are not
homogenous (Leavitt and Long, 1986), is crucial to the successful
applications of this tool (Hobbie and Werner, 2004). The primary determinant
of plant-carbon isotope compositions is the photosynthetic discrimination
against the heavier carbon isotope 13C. This primary discrimination
process has been relatively well understood, and detailed theoretical models
relating the discrimination to environmental forcing conditions, leaf
physiology, and biochemistry have been developed (Farquhar et al., 1982;
Farquhar and Cernusak, 2012; Gu and Sun, 2014). However, other processes must
also influence plant-carbon isotope compositions as heterotrophic plant
organs (e.g., stems, roots, seeds and fruits) in C3 plant species have
been found to be generally enriched in 13C as compared to the
autotrophic organ (leaves) that supplies them with carbohydrates (Craig,
1953; Leavitt and Long, 1982; Ehleringer et al., 1987; Hobbie and Werner,
2004; Badeck et al., 2005; Cernusak et al., 2009). In contrast to the
relatively well-understood photosynthetic carbon isotope discrimination,
processes controlling the observed heterotrophic 13C enrichment in
C3 plant species remain unclear even though the phenomenon was first
reported 60 years ago (Craig, 1953).
Cernusak et al. (2009) and Ghashghaie and Badeck (2014) summarized more than
half a dozen of nonexclusive processes that may explain the heterotrophic
13C enrichment in C3 plant species. These processes generally
belong to two broad groups. Group I processes involve the occurrence of
contrasting biochemical and metabolic fractionations between autotrophic and
heterotrophic organs, for example, 13C-enriched autotrophic vs.
13C-depleted heterotrophic mitochondrial respirations, low autotrophic
vs. high heterotrophic CO2 fixation by phosphoenolpyruvate (PEP)
carboxylase, and low autotrophic vs. high heterotrophic loss rates of
13C-depleted volatile organic compounds, surface waxes and other
products from secondary plant metabolism. Group II processes involve the
utilization of contrasting organ-building photoassimilates, which in turn may
be a result of a number of processes, including preferential export of
13C-enriched nighttime sucrose to heterotrophic organs, reduced
photosynthetic discrimination against 13C due to developmental shifts in
exporting mature leaves, and asynchronous growth of autotrophic vs.
heterotrophic organs in contrasting environmental conditions. Although the
term post-photosynthetic discrimination or post-carboxylation discrimination
has been often used to refer the processes included in both groups, some of
the processes in Group II cannot be strictly considered as occurring post
photosynthesis or carboxylation. Nearly all processes outlined above have
supporting as well as opposing evidences from observational and experimental
studies (Cernusak et al., 2009). Thus it remains a challenge to identify
cause(s) for the 60-year-old puzzle of heterotrophic 13C enrichment.
It is important to overcome this challenge as many fundamental issues in a
variety of scientific disciplines ranging from plant physiology to global
carbon cycle studies depend on a precise knowledge of plant-carbon isotope
compositions. Towards this goal, we have identified two areas that require
strengthening in the studies of heterotrophic 13C enrichment. First,
there is a need for systemic whole-plant studies. Although heterotrophic
13C enrichment in C3 plant species has been reported widely, most
previous studies have been done by comparing heterotrophic organs
independently and on a piecemeal basis with leaves. This lack of systemic,
whole-plant studies is not conducive to understanding the mechanism of
heterotrophic 13C enrichment, because to achieve this understanding, one
must first have a comprehensive picture of the enrichment (or depletion)
across all organs of the same plant.
Second, whether and how nutrients affect heterotrophic 13C enrichment
needs to be investigated. Nutrients, particularly nitrogen (N) and
phosphorous (P), control leaf photosynthetic capacity (Field and Mooney,
1986; Domingues et al., 2010), which in turn affects the drawdown of CO2
along stomatal and mesophyll diffusional pathways. It has been shown that
leaf N content is positively correlated with leaf δ13C and negatively with carbon isotope discrimination (Sparks and Ehleringer, 1997;
Livingston et al., 1999; Duursma and Marshall, 2006; Cernusak et al., 2007).
This relationship is consistent with the expectation that higher leaf
photosynthetic capacity associated with higher leaf N leads to a sharper
drawdown of CO2 along the diffusional pathways (Cernusak et al., 2007,
2013), resulting in an expected pattern according to the photosynthetic
isotope discrimination equations (Farquhar et al., 1982; Farquhar and
Cernusak, 2012; Gu and Sun, 2014). To our knowledge, hitherto there has been no
effort to systematically investigate how plant nutrients might affect
heterotrophic 13C enrichment compared to leaves. A lack of such an
effort is not justifiable because plant nutrients play important roles in
many of the processes discussed in Cernusak et al. (2009) and Ghashghaie and
Badeck (2014). Thus it would not be surprising if certain relationships exist
between plant nutrients and heterotrophic 13C enrichment. An
identification of such relationships will greatly assist the illumination of
the underlining cause(s) of heterotrophic 13C enrichment.
Therefore, the objective of the present study was to gain insight into the
longstanding puzzle of heterotrophic 13C enrichment by jointly
addressing the two deficiencies identified above. We conducted systematic and
simultaneous analyses of carbon isotope ratios and N and P contents with
excavated whole architectures of Nitraria tangutorum Bobrov, a
C3 shrub species endemic to northwestern deserts in China. These
analyses were complemented with investigations of seasonal variations in leaf
carbon isotope ratios on intact plants of the same species, thus enabling the
analyses of carbon isotope compositions of different heterotrophic organs in
a dynamic reference framework. N. tangutorum is interesting because
it has an exceptional capability of controlling landscape evolution by fixing
sands and building sand dunes known as nebkha or coppice dunes around its
extensive shoot and root systems (Baas and Nield, 2007; Lang et al., 2013; Li
et al., 2013). This characteristic makes it relatively easy to excavate the
whole plant including roots for isotope and nutrient analyses, although to
our knowledge, this species has never been investigated for heterotrophic
13C enrichment.
We will report, for the first time, that variations in 13C enrichment in
different heterotrophic organs strongly depend on their N contents,
indicating a role of a within-organ N-mediated process in heterotrophic
13C enrichment. We will also show that the observed N–heterotrophic
13C enrichment relationship is most parsimoniously explained through the
respiratory CO2 refixation by PEP carboxylase. Future studies on
heterotrophic 13C enrichment should investigate isotopic effects of N
content and CO2 refixation in different plant organs. Direct
measurements of PEP carboxylase activity will be essential.
Flowers (top, 10 June 2009, Minqin), fruits (middle, 18
July 2009, Minqin) and nebkha (bottom, 3 August 2010, Dengkou) of Nitraria tangutorum Bobrov.
Pictures courtesy of Jianmin Chu, Research Institute of Forestry, Chinese
Academy of Forestry.
Materials and methods
Biological and environmental characteristics of
Nitraria tangutorum Bobrov
Nitraria tangutorum Bobrov (Fig. 1) is a spiny shrub species in
the Nitraria genus of the Zygophyllaceae family. Species in
the Nitraria genus are generally xerophytes, widely distributed in
the Middle East, Central Asia, and in the northwestern regions of China. N. tangutorum, however, is endemic to the northwestern regions of China,
including northeastern Tibet, Gansu, Qinghai, Xinjiang, western Inner
Mongolia, western Ningxia, and northern Shaanxi. It is a pioneer species and
has high tolerance for drought, heat, and salts. N. tangutorum plays
an important ecological role in combating desertification due to its
exceptional capabilities in forming phytogenic nebkha dunes which prevent or
slow down the movement of sands. According to Li and Jiang (2011) and Li et
al. (2013), the process of forming a nebkha typically starts when occasional
ample moisture allows a seed to germinate inside clay cracks in dried-up flat
beds of previous rivers or lakes. As the resulting ortet grows, it intercepts
aeolian sands and the plant enters into a clonal reproductive stage. When
branches are buried by sands, layering occurs and adventitious roots are
formed. Under appropriate sand-burial depth and sufficient moisture, ramets
are developed from axillary buds in the layering and a clonal colony is
formed. If aeolian sand supply is not interrupted, repetitive layering and
ramet development will enlarge the colony and further increase its capacity
to intercept aeolian sands; thereby, a phytogenic nebkha dune is formed (Fig. 1c).
The height of a N. tangutorum nebkha ranges from 1 to 3 m and some can reach 5 m. The
base of a nebkha often has the shape of an ellipse with the major axis
parallel to the local prevailing wind direction. The formation of nebkhas
alters local micro-environments and provides habitats for other desert
species. Li and Jiang (2011) described in detail the biological and
environmental characteristics of species in the Nitraria genus with a focus on N. tangutorum.
Study sites
The field work was carried out at two desert locations. The first study site
was within an experimental area (40∘24′ N, 106∘43′ E)
managed by the Experimental Center of Desert Forestry of the Chinese Academy
of Forestry. This site is located in Dengkou County, Inner Mongolia
Autonomous Region, China. Dengkou County is at the junction between the Hetao
Plain and Ulan Buh Desert of the Mongolian Plateau in the middle reaches of
the Yellow River. The mean annual temperature is 8.84∘ and the mean
annual precipitation is 147 mm, with 77.5 % of annual rainfall occurring
from June to September (1983–2012 averages). The mean annual potential
evaporation is 2381 mm (Li et al., 2013). The soils in the study region in
general are sandy soil and gray-brown desert soil (Cambic Arenosols and Luvic
Gypsisols in FAO (Food and Agriculture Organization of the United Nations) taxonomy). The N. tangutorum nebkhas at the study
site are formed on clay soils deposited by the Yellow River. Although the
plant community is dominated by N. tangutorum, xerophytic species
such as semi-shrub Artemisia ordosica, perennial grass
Psammochloa villosa, and annual species Agriophyllum squarrosum and Corispermum mongolicum can also be found.
The second study site was the Gansu Minqin Desert Ecosystem Research Station
(38∘34′ N, 102∘58′ E), Minqin County, Gansu Province,
China. Minqin County is located in the lower reaches of Shiyang River,
surrounded by the Badain Jaran Desert in the west and north and the Tengger
Desert in the east. The mean annual temperature is 8.87∘ and the mean
annual precipitation is 117 mm, with 73.1 % of annual rainfall occurring
from June to September (1983–2012 averages). The mean annual potential
evaporation is 2643 mm (Du et al., 2010). Thus the second study site is
somewhat drier than the first site, but with similar annual mean temperatures.
The soil at the Minqin site is similar to that at the Dengkou site with sandy
soil in the nebkhas and gray-brown desert soil between nebkhas. The native
vegetation in the study area is usually dominated by shrubs and semi-shrubs
with species such as N. tangutorum and Calligonum mongolicum. Experimental plots used in this study contained semi-fixed
nebkha dunes developed by the growth of N. tangutorum. Typically in
dry years, N. tangutorum is the only species growing in the nebkhas
although in wet years, annual species such as Agriophyllum squarrosum and Corispermum mongolicum can also be found. Because
the Minqin site is drier than the Dengkou site, the nebkhas at the Minqin
site are generally smaller and less populated with plants than at the Dengkou
site. The rooting depth is deeper at the Minqin site than at the Dengkou site
(Table 1).
Main geometrical and biometrical characteristics of the nebkhas
excavated in this study.
Nebkha
Dengkou-1
Dengkou-2
Dengkou-3
Minqin-1
Minqin-2
Minqin-3
Major axis (m)
13.6
9.9
3.65
4
4.6
6.4
Minor axis (m)
8.38
5.9
3.24
3.5
2.9
4.6
Height (m)
2.02
1.38
0.57
0.35
0.44
0.8
Plant cover (%)
80
70
80
11
15
7
Below-plain rooting depth (cm)
<60
<40
<40
<80
<80
<80
Leaf biomass (g C m-2 and %)
62.9 (10)
93.7 (12)
85.1 (11)
12.7 (6)
23.0 (11)
11.0 (9)
Stem biomass (g C m-2 and %)
159.7 (25)
169.3 (22)
213.3 (28)
35.2 (16)
70.0 (34)
22.2 (19)
In-sand root biomass (g C m-2 and %)
289.9 (45)
370.6 (47)
214.7 (28)
92.0 (41)
34.9 (17)
51.9 (44)
Blow-plain root biomass (g C m-2 and %)
137.7 (21)
148.7 (19)
260.8 (34)
84.5 (38)
80.5 (39)
32.5 (28)
Total biomass (g C m-2 and %)
650.2 (100)
782.3 (100)
773.9 (100)
224.4 (100)
208.3 (100)
117.6 (100)
Excavation of Nitraria tangutorum nebkhas
In August 2012, we excavated three nebkhas at each study site. The
geometrical and biometrical characteristics of the six nebkhas were
summarized in Table 1. At the Dengkou site, the three nebkhas were excavated
in a sampling area of 40 m × 40 m. At the Minqin site, nebkhas
were generally much smaller. To ensure availability for analyses of
sufficient biomass materials at this site, particularly the fine roots (see
below), three sampling areas each with a dimension of 30 m × 30 m
were established and three nebkhas from each sampling area were tentatively
excavated. Two nebkhas from one sampling area and one from another were
determined to have sufficient amount of fine roots for analyses and were
therefore excavated fully.
We excavated the nebkhas by carefully teasing away the sands from the mounds
to expose the root architecture of N. tangutorum with particular attention paid to the
preservation of fine roots. The roots of a N. tangutorum can be found inside the sand
mounds as well as inside the clay layer that generally forms a plain on
which the sand mounds rest. We therefore also excavated any roots inside the
clay layer to a depth until no more roots could be found.
We separated the whole plant biomass into leaves, stems, in-sand roots and
below-plain roots. The in-sand roots, which were roots found inside the
nebkha sands but above the plain formed by the underlying clay layer, were
further separated into in-sand fine roots (diameter ≤ 2 mm) and
in-sand coarse roots (diameter > 2 mm). The same root diameter threshold
was used to separate the below-plain roots, which were found inside the clay
layer under the nebkha sands. Furthermore, the below-plain fine and coarse
roots were grouped in a 20 cm depth increment from the plain surface. We did
not separate the in-sand fine and coarse roots into layers, because a nebkha
has a cone shape on top, making a layer hard to define. Also we did not use a
simple “below-ground” group, because “ground” is not well defined in a
nebkha-populated landscape, and because there are large physical and chemical
differences between sands and clay, which may affect the isotope compositions
of roots growing in them. Litter was rarely found on the nebkhas, presumably
because strong winds at the study sites can easily blow away any litter
produced. However, woody debris from dead ramets was present inside the sand
mounds and was collected during excavation. Thus for each nebkha, we
differentiated the following categories of N. tangutorum biomass:
the autotrophic organ of leaves, the heterotrophic organs of stems, in-sand
fine roots (ISFR), in-sand coarse roots (ISCR), below-plain fine roots (BPFR)
in 20 cm depth increments, and below-plain coarse roots (BPCR) in 20 cm
increments, and the heterotrophic woody debris (WD). Nutrient contents and
carbon isotope compositions were measured separately for each category.
Measurements of nutrient contents and carbon isotope compositions
with excavated biomass
All categories of N. tangutorum biomass (leaves, stems, ISFR,
ISCR, BPFR in 20 cm increments, BPCR in 20 cm increments, and WD) from each
excavated nebkha were dried to constant weight (60∘, 48 h). The dry
weight of biomass was determined with 0.01 g accuracy on an analytical scale.
The biomass carbon stocks were expressed relative to the base area of the
nebkha which was assumed to be an ellipse. The fraction of each component was
also calculated.
Dried materials were randomly selected from each biomass category and ground
to 80 mesh. The resultant powder was separated into six duplicates. Three
duplicates were analyzed for carbon (C), nitrogen (N) and phosphorous (P)
contents and the remaining three for isotope compositions. The C, N, and P
contents were measured in the EnvironmentalChemistry Analysis Laboratory in
the Institute of Geographic Sciences and Natural Resources Research, the
Chinese Academy of Sciences, Beijing, China. Total sample carbon and N were
measured with the vario MACRO cube (Elementar Company, Germany). The
analytical precision was better than 0.5 % relative standard deviation
(RSD). Total P was measured with the ICP-OES OPTIMA 5300DV (PE, USA). The
analytical precision was better than 2 % RSD.
The carbon isotope compositions were analyzed at the Stable Isotope Ratio
Mass Spectrometer Laboratory of the Chinese Academy of Forestry (SIRMSL,
CAF), Beijing, China. The instrument used was a Delta V Advantage Mass
Spectrometer (Thermo Fisher Scientific, Inc., USA) coupled with an elemental
analyzer (FlashEA 1112; HT Instruments, Inc., USA) in the continuous flow
mode. Isotope compositions were expressed using the delta notation (δ)
in parts per thousand (‰): δ13C
(‰) = [(Rsample) / (Rstandard)-1] × 1000, where R is the ratio of 13C to 12C. The
measurement applied the IAEA-600 standard (caffeine) relative to V-PDB
(Vienna PeeDee formation belemnite limestone). The analytical precision was
better than 0.1‰, based on replicate measurements of the reference
standard.
Measurements of seasonal variations in leaf δ13C and Ci / Ca ratio
Photosynthetic carbon isotope discrimination depends on environmental
conditions (Farquhar et al., 1982; Farquhar and Cernusak, 2012; Gu and Sun,
2014); consequently, leaf carbon isotope ratio δ13C may change
seasonally, potentially making the autotrophic–heterotrophic differences
in carbon isotope compositions time dependent. Thus in addition to the
isotopic and nutrient analyses for samples from the excavated plant
materials, we also measured seasonal variations in leaf carbon isotope
compositions and ratios of leaf intercellular airspace (Ci) to
ambient (Ca) CO2 concentrations on nearby un-excavated
nebkhas at both the Dengkou and Minqin study sites. Four samples of leaves
were taken in each month from May to September of 2012 at both sites and
analyzed for carbon isotope ratios at the SIRMSL of CAF. The seasonal
variations in Ci / Ca ratios were measured with a
Li-6400 portable photosynthetic system (Li-Cor Environmental Sciences,
Lincoln, NE, USA) each month from June to September of 2012 at the Dengkou
site with 24–28 samples per month and from July to September of 2011 at the
Minqin site with 16 samples per month. The chamber environment (temperature,
light, and relative humidity) was kept close to ambient conditions at the
time of measurement. Seasonal variations in leaf nutrient contents were not
measured. The measurements of seasonal variations in leaf δ13C
provide a dynamic reference framework for examining the δ13C values
of heterotrophic organs, while the independent measurements of seasonal
variations in Ci / Ca ratios allow us to
determine whether the seasonal patterns in leaf δ13C are
consistent with our current understanding of the photosynthetic carbon
isotope discrimination (Farquhar et al., 1982).
The difference in carbon isotope compositions between
leaves and heterotrophic organs of Nitraria tangutorum Bobrov, which
is measured by Δ13Corganin Eq. (1) and averaged
across the nebkhas excavated at the same study site (Dengkou or Minqin).
Negative values indicate 13C enrichment in heterotrophic organs compared
to leaves. Upper-case letters denote analysis-of-variance (ANOVA) results within a study site (i.e.,
comparing Δ13Corgan among different organs at the same
site) and lower case letters between the two sites (i.e.,
comparing Δ13Corgan of the same organ between the two
sites). IS stands for in-sand, FR for fine root, and for CR coarse root. 1, 2, 3, and 4
in front of FR or CR stand for 0–20, 20–40, 40–60, and 60–80 cm,
respectively, below the plains on which nebkhas rest. Woody debris (WD) from dead ramets is
also included in the figure. No ANOVA results for 3FR and 3CR at the Dengkou
site as there was only one nebkha having roots between 40 and 60 cm. No roots
were found below 60 cm at the Dengkou site.
Quantification of heterotrophic 13C
enrichment and statistical analyses
We quantified the difference in carbon isotope composition between the
leaves (autotrophic) and a heterotrophic organ with the following
expression:
Δ13Corgan=RleafRorgan-1×1000=δ13Cleaf/1000+1δ13Corgan/1000+1-1×1000=δ13Cleaf-δ13Corgan1+δ13Corgan/1000.
Thus a value of Δ13Corgan<0 indicates an enrichment
of 13C in a heterotrophic organ relative to the leaves while Δ13Corgan>0 indicates heterotrophic depletion. The values of
δ13CLeaf used to calculate Δ13Corgan came
from leaves harvested from N. tangutorum of the excavated nebkhas,
not from those for seasonal patterns. The use of Δ in Eq. (1) makes
the relationship between autotrophic and heterotrophic organs analogous to
that between reactants and products (Farquhar et al., 1989), which is
appropriate for the purpose of this study. A great advantage of introducing
Δ13Corgan is that heterotrophic 13C enrichment can
be compared not only among the organs of the same plant, but also across
different plants at the same site or at different sites which may differ in
autotrophic isotopic signatures. Thus the use of Δ13Corgan facilitates the identification of general
patterns.
Two-way ANOVA analyses (organ by site) were performed with SPSS (Ver.17.0).
C, N, and P contents, δ13C, Δ13Corgan,
C / N ratios, N / P ratios, and C / P ratios were analyzed for
differences between organs and between study sites. Tukey post hoc tests were
used to determine pairwise differences for significant effects (P<0.05).
Regression analyses were used to determine the relationship between the
heterotrophic 13C enrichment and nutrient contents.
Results
Variations in Δ13Corgan among plant organs and
between study sites
At both the Dengkou and Minqin study sites, the values of Δ13Corgan for all heterotrophic organs examined were
significantly smaller than zero, indicating that without any exception, the
heterotrophic organs were enriched in 13C compared to the leaves
(Fig. 2). However, there were considerable variations in
Δ13Corgan among the heterotrophic organs at both study
sites and between the heterotrophic organs across the study sites. Stems were
less enriched (closer to zero) than roots at both sites. At the Dengkou site,
the most enriched organ was the coarse roots inside the nebkha sands. At the
Minqin site, the most enriched part was the fine roots inside the nebkha
sands although the difference between the coarse and fine roots inside the
sands was not significant. At the Dengkou site, the coarse roots were
consistently more enriched than the corresponding fine roots both inside the
nebkha sands and below the plains. In contrast, at the Minqin site, the coarse
roots were less enriched than the corresponding fine roots except for the
roots deep into the plains (40–80 cm) where the coarse roots were more
enriched. However at both sites, the statistical power of the coarse–fine
root isotope differences were low as they were not significant at the
significance level of 0.05. At the Dengkou site, the woody debris was more
enriched than the stems but less enriched than the roots while at the Minqin
site, it was less enriched than either the stems or the roots. In all biomass
categories investigated, the Dengkou site was more enriched than the Minqin
site, particularly in below-plain roots and in woody debris.
Variations in nutrient concentrations among plant organs and between
sites
There are considerable variations in nutrient contents among plant organs and
between sites (Fig. 3). At both the Dengkou and Minqin sites, leaves appeared
to have the lowest C (Fig. 3a) but highest N (Fig. 3b) and P (Fig. 3c)
contents. At both sites, stems tended to have lower N contents than roots
either inside the sand dunes or below the plains under the sand dunes; in
contrast, P contents in stems were within the variations of P contents in
roots. At the Dengkou site, roots inside the sand dunes had lower N contents
than roots below the plain; at the Minqin site, the coarse roots inside the
sand dunes had lower N than either coarse or fine roots below the plain while
the fine roots inside the sand dunes had N within the variations of those of
coarse and fine roots below the plain. At the Dengkou site, the fine roots
appeared to have higher P than coarse roots but the differences diminished
from inside sands to below plain. There were no clear patterns on root P at
the Minqin site. Woody debris had N contents similar to stems at both sites
and tended to have significantly less P contents than leaves, stems or roots.
Between the two study sites, the leaves had lower C but higher N and P
contents at the Dengkou site than at the Minqin site, but the difference is
not significant at the significance level of 0.05. In contrast, heterotrophic
organs at the Dengkou site tended to have significantly higher N and P
contents than at the Minqin site. This contrast suggests that N. tangutorum may be able to maintain nutrient contents in leaves for
photosynthesis at the expense of stems and roots.
Carbon (C) (a), nitrogen (N) (b) and phosphorous (P)
content (c) of different organs of Nitraria tangutorum Bobrov, at the Dengkou and Minqin study
sites. Symbols and letters denoting ANOVA results are explained in Fig. 2.
Carbon (C) to nitrogen (N) (a), N to phosphorous (P) (b)
and C to P mass ratios (c) of different organs of Nitraria tangutorum Bobrov, at the Dengkou
and Minqin study sites. Symbols and letters denoting ANOVA results are
explained in Fig. 2.
Consistent with the variations in C, N and P contents, there were also
substantial variations in the ratios of C / N (Fig. 4a), N / P
(Fig. 4b) and C / P (Fig. 4c) among plant organs and between sites. For
the live biomass (leaves, stems, and roots), the ratios of C / N ranged
from about 11 to 30, N / P from 20 to 40 and C / P from 300 to 700.
As expected, leaves had the lowest C / N and C / P ratios at both
sites. Leaves also had the lowest N / P ratios except for stems. Overall,
the Dengkou site had lower ratios of C / N and C / P but higher
ratios of N / P than the Minqin site, particularly for roots below the
plain.
Relationships between 13C enrichment and
nutrient contents
The observed large variations in 13C enrichment and nutrient contents
among heterotrophic organs and between study sites give us an opportunity to
examine whether 13C enrichment in heterotrophic organs relative to
leaves could be affected by their nutrient contents. We found that across the
two study sites and across the heterotrophic organs, Δ13Corgan was significantly correlated with the N content
(Fig. 5b), the C / N ratio (Fig. 5d), and the N / P ratio (Fig. 5e)
in the heterotrophic organs. The correlation was negative for N content and
N / P ratio but positive for C / N ratio, indicating that higher
heterotrophic N contents resulted in larger heterotrophic 13C enrichment
relative to leaves. The C / N ratio explained a higher percentage
(52 %) of variance in Δ13Corgan than did the N
content or the N / P ratio (44 and 42 %, respectively). No
significant effect of heterotrophic organ C content (Fig. 5a), P content
(Fig. 5c), or C / P ratio (Fig. 5f) on Δ13Corgan
were found.
We did not have enough independent samples to look at how leaf N contents
might affect the heterotrophic 13C enrichment. However, we examined the
relationship between Δ13Corgan and organ nutrient
contents normalized by the corresponding leaf nutrient contents (i.e., the
ratio of heterotrophic to corresponding leaf nutrient values). The normalized
heterotrophic N contents explained somewhat less variance with reduced
statistical power compared to the un-normalized values (compare Supplement
Fig. S1 to Fig. 5), suggesting that it is the absolute N contents of the
heterotrophic organs, not their relative departure from the corresponding
leaf N contents, that affect the heterotrophic 13C enrichment.
Nutrient dependence of the difference in carbon isotope
compositions between leaves and heterotrophic organs of Nitraria tangutorum Bobrov, which is measured by Δ13Corgan in Eq. (1) and averaged across the nebkhas excavated at the same study site.
Negative values indicate 13C enrichment in heterotrophic organs compared
to leaves. Changes of Δ13Corgan as a function of organ
contents of carbon (C) (a), nitrogen (N) (b) and phosphorous (P) (c) and of
organ mass ratios of C to N (d), N to P (e), and C to P (f). The two arrows
in (b) indicate values for woody debris from dead ramets at each study site
while in (d) indicates an outlier caused by measurements in phosphorous
content (see the outlier in c and f).
Seasonal changes in the ratios of leaf carbon isotopes (a)
and intercellular (Ci) to ambient (Ca) CO2
concentrations of Nitraria tangutorum Bobrov at the Dengkou and
Minqin study sites (b). For comparison, the biomass-averaged isotope ratios of
roots from the excavated nebkhas are also shown in (a).
Seasonal variations in leaf δ13C and
Ci/Ca ratios
At both Dengkou and Minqin sites, leaf δ13C of N. tangutorum decreased from May to September (Fig. 6a), indicating progressive
depletion in the heavier carbon isotope in leaves as the season progressed.
Meanwhile, the Ci / Ca ratio increased from the
early to late growing season (Fig. 6b). Thus the relationship between the
seasonal patterns in leaf δ13C and
Ci / Ca ratios is consistent with the prediction
by the leaf photosynthetic carbon isotope discrimination models (Farquhar et
al., 1982; Farquhar and Cernusak, 2012; Gu and Sun, 2014). However, the
differences in leaf δ13C between the two sites cannot be entirely
explained by the differences in the Ci / Ca
ratios. In all months examined, the Ci / Ca
ratios at the Dengkou site were consistently higher than at the Minqin site.
If the Ci / Ca ratios were the only factor
controlling the leaf δ13C, then the Dengkou site should have
consistently lower leaf δ13C (higher
Ci / Ca ratios increase discrimination against
13C during photosynthesis). To the contrary, the Dengkou site had higher
leaf δ13C than the Minqin site in May, June and July; only in
August and September, the difference in leaf δ13C was consistent
with the effect of the difference in Ci / Ca
ratios between the two sites (although the difference in leaf δ13C
between the two sites were still not significant).
Interestingly, the leaf δ13C in May and June was close to the
biomass-weighted average of root δ13C at both study sites,
suggesting that the initial building materials of new leaves might have
largely come from stored carbon in roots.
Discussion
A major finding from this study is that the N content of a heterotrophic
organ, expressed either as a fraction of total dry biomass or as a ratio of C
to N or N to P, is strongly correlated with this organ's enrichment in
13C relative to leaves with higher N concentrations corresponding to
larger enrichment. Because this relationship is caused by variations among
heterotrophic organs, and because normalizing the heterotrophic N content by
the corresponding leaf N content did not improve or even worsen this
relationship, the process responsible for it must reside inside the
heterotrophic organs themselves. Further, this process must be mediated by N.
What N-mediated process could be responsible for the positive N–13C
enrichment relationship among heterotrophic organs? A parsimonious candidate
is the respiratory CO2 refixation by PEP carboxylase. CO2 from the
respiration of heterotrophic organs may dissolve into water and be hydrated
into HCO3- which is then fixed by PEP carboxylase into oxaloacetate.
Both the dissolution of CO2 into water and the fixation of HCO3- by PEP carboxylase discriminate slightly against 13C. However, the
hydration process fractionates strongly in favor of 13C and causes it to
concentrate in HCO3-. Consequently, the CO2 refixation by PEP
carboxylase has a net fractionation of 5.7‰ in favor of 13C
relative to the gaseous CO2 (Farquhar, 1983; Melzer and O'Leary, 1987;
Farquhar et al., 1989). Thus the respiratory CO2 refixation by PEP
carboxylase should lead to a depletion of 13C in CO2 escaped to
outside compared to the original substrates for respiration while
heterotrophic organs should be 13C-enriched due to the addition of
organic materials from PEP carboxylase activities. Previous studies have
reported high PEP carboxylase activities in heterotrophic organs of a variety
of C3 plant species (Melzer and O'Leary 1987; Berveiller and Damesin
2008; Gessler et al., 2009, 2014). If increased N content increases the
respiratory CO2 refixation in heterotrophic organs, then it should also
increase 13C enrichment in these organs. Berveiller et al. (2010) showed
that CO2 refixation rates of Fagus sylvatica stems increased as
stem N content increased, which provides a direct support for the hypothesis
that CO2 refixation by PEP carboxylase is a process responsible for our
observed positive relationship between N and 13C enrichment in
heterotrophic organs.
Observed respiration rates of leaves, stems and roots tend to increase with
increased N contents (Reich et al., 2008). This does not necessarily contradict
the PEP carboxylase hypothesis suggested above. The actual respiration rates
of these organs may increase so much with increased N contents that the
increase cannot be offset by the increased refixation rates by PEP
carboxylase. Consequently, the observed rates of CO2 evolved from
heterotrophic organs may still increase even though the refixation rates
have increased with increased N contents.
The PEP carboxylase hypothesis does imply that the CO2 escaped to
outside from the heterotrophic organs are depleted in 13C compared to
the substrates utilized for respiration. As summarized in the review of
Ghashghaie and Badeck (2014), most isotopic studies on root respiration have
found that CO2 evolved from roots are depleted in 13C compared with
bulk root material, in contrast to leaf dark respiration, which is generally
enriched. For stem respiration, however, more contradictory results have been
reported. Wingate et al. (2010) showed that CO2 evolved from stems of
Pinus pinaster was more depleted in 13C when compared to the currently
measured net CO2 flux by photosynthetic branches or with the phloem
water-soluble organic matter and wood cellulose. Gessler et al. (2009) also
found that the respiration of stems as well as roots of Ricinus communis was depleted in 13C relative to the assumed respiratory
substrates. This latter study was particularly relevant to this present study
because the authors determined that the depletion was caused by a strong
refixation of respiratory CO2 catalyzed by PEP carboxylase. In contrast
to these studies, Damesin and Lelarge (2003) reported that stem respiration
of Fagus sylvatica was enriched in 13C compared with the total
organic matter, while Kodama et al. (2008) showed that CO2 evolved from
the stem of Pinus sylvestris had higher or similar δ13C
values compared to that of phloem exudate organic matter, depending on
respiration rates. More studies are needed to determine whether carbon
isotope fractionations of stem respiration depend on species, ages, or
environments. Also, the dissolution and hydration of respiratory CO2 may
decouple in location from the fixation of HCO3- by PEP carboxylase
if there is a strong transpiration stream in xylem, with isotopic
consequences. For example, respiratory CO2 can be dissolved and hydrated
in roots and stems but the HCO3- molecules formed can be carried up
in xylem transpiration streams (Aubrey and Teskey, 2009; Angert and Sherer,
2011; Bloemen et al., 2013; Trumbore et al., 2013) and fixed by PEP
carboxylase in branches, which will serve to redistribute isotope signatures
among different parts of the plant body.
Additional studies are also needed to determine whether there are other
causes for the observed heterotrophic N–13C enrichment relationship.
For example, if different organ N contents are associated with chemical
compounds with different isotope signatures or different “fragmentation
fractionation” (enzymatic reaction of substrate molecules with heterogeneous
13C distribution; Tcherkez et al., 2004; Hobbie and Werner 2004), one
may expect organ N contents to be correlated with organ isotope signatures,
potentially leading to the observed relationship. Another possibility to
consider is that atmospheric δ13C has been decreasing since the
Industrial Revolution due to the emission of 13C-depleted fossil
CO2. If a heterotrophic organ contains a higher fraction of carbon with
an old age, then its bulk δ13C would be higher. Stems and roots
should contain more old carbon than leaves do. We do not have data to
quantify this possibility. However, a qualitative reasoning led us to doubt
that a general decreasing trend in atmospheric δ13C can explain the
observed heterotrophic N–13C enrichment relationship. Although we do
not know the ages of the six nebkhas excavated, atmospheric N deposition has
probably been increasing during the lifetime of these nebkhas. Therefore,
younger tissues should contain lower δ13C and higher N, which would
imply a negative N–13C enrichment relationship, opposite to what we
observed. Therefore the positive heterotrophic N–13C enrichment
relationship most likely has a phytogenic (rather than an atmospheric)
origin.
It is important to clarify that our suggestion that the process responsible
for the positive heterotrophic N–13C enrichment relationship resides in
heterotrophic organs does not imply that the cause(s) for heterotrophic
enrichment of 13C relative to leaves resides entirely in heterotrophic
organs. In fact, to explain the full magnitude of the observed heterotrophic
enrichment (2 ‰), about 35 % (100 × 2/5.7) of the
carbon of heterotrophic organs has to have cycled through PEP carboxylase
once, which appears to be surprisingly large for C3 plants (Hobbie et
al., 2003). Also, our finding that the δ13C of leaves in the early
growing season was close to the mean isotope ratio of roots, but decreased as
the season progressed, indicates that processes inside leaves must also
contribute to the overall isotope differences between leaves and
heterotrophic organs, if the leaf samples for reference are from middle to
late growing seasons. The reference leaf samples in our calculation of
Δ13Corgan were from middle growing seasons (August).
Therefore, the progressive seasonal depletion in foliar 13C increased
the magnitude of the obtained Δ13Corgan. Furthermore,
processes such as preferential loading into phloem of the heavier isotope and
loss of depleted outer bark materials should also affect the overall
autotrophic–heterotrophic isotope differences (Cernusak et al., 2009;
Ghashghaie and Badeck, 2014). While these processes may boost the overall
magnitude of heterotrophic 13C enrichment, they cannot explain its
relationship with N content among heterotrophic organs.
It is likely that leaf N also plays an important role in determining 13C
enrichment in heterotrophic organs relative to leaves. We do not have enough
leaf-level data to examine this issue in depth but findings from previous
studies allow us to speculate about what this role might be. As discussed
early, leaf N content is positively correlated with leaf δ13C
because higher leaf N increases leaf photosynthetic capacity, which results
in decreased Ci / Ca ratios and thus reduced
discrimination against 13C during photosynthesis (Sparks and Ehleringer,
1997; Livingston et al., 1999; Duursma and Marshall, 2006; Cernusak et al.,
2007, 2013). However, a positive relationship between leaf N and leaf
δ13C does not necessarily mean that higher leaf N will reduce the
degree of heterotrophic enrichment in 13C compared to leaves as
heterotrophic organs use photosynthetic products from leaves. An interesting
pathway for leaf N to influence heterotrophic 13C enrichment may lie in
the relationship between leaf N and dark respiration. It is known that leaf
dark respiration scales with leaf N (Reich et al., 2008). It is also known
that leaf dark respiration is enriched in 13C, contrary to respirations
of stems and roots (Ghashghaie and Badeck, 2014). Thus higher leaf N may
actually increase the depletion of 13C in leaves relative to
heterotrophic organs. Consequently one may expect that N in autotrophic and
heterotrophic organs of plants contributes to the isotope difference between
these two types of organs in the same direction but through fundamentally
different mechanisms.
Our analyses benefitted from the large variations in nutrient contents and
heterotrophic 13C enrichment both across plant organs and between sites,
allowing any relationship (if exists) between these two sets of variables to
be seen clearly. The large variations across plant organs are a validation of
our systemic, whole-plant sampling strategy. The large between-site
differences in organ nutrient contents likely reflect a site difference in
soil fertility. The soil of the vegetated area at Dengkou contained
0.024 ± 0.006 % N (Jia, 2010) while at Minqin the value was
0.01 ± 0.001 % (Song et al., 2012), explaining the generally higher
plant organ N contents at Dengkou than at Minqin. Soil P contents have not
been measured at either site. However, we suspect that soil at Dengkou was
also richer in P than at Minqin, as plant organs generally contained higher P
contents at the former than latter site. The cross-organ variations in
nutrient contents were larger at Dengkou than at Minqin, possibly because
poorer soil nutrient availability limited organ nutrient content variations
at the latter site. Correspondingly, the range of heterotrophic 13C
enrichment was also wider at Dengkou than at Minqin. Both the cross-organ and
between-site variations contributed the observed relationship between the N
content and heterotrophic 13C enrichment. However, even within the same
site, a pattern between N content and heterotrophic 13C enrichment can
be clearly seen, particularly at the Dengkou site. Further, the patterns of
the two sites appear to be consistent with each other and form a single
relationship. This consistency suggests that the same mechanism operates at
the two sites to generate a unified dependence of 13C enrichment on N
content across heterotrophic plant organs.
The lack of a clear relationship between P content and heterotrophic 13C
enrichment (Fig. 5c and Fig. S1c) is interesting. In plants, proteins, which
are rich in N, must be maintained with an allocation of a certain fraction of
total body P to ribosomal ribonucleic acid (rRNA) (Niklas et al., 2005; Elser
et al., 2010). Thus the N and P contents are generally positively correlated,
and the measurements from Minqin and Dengkou are not exceptions (Fig. S2). So
why is there is a clear dependence of heterotrophic 13C enrichment on N
but not on P? It could be that the relationship of heterotrophic 13C
enrichment with P is considerably weaker than that with N, and our data were
not sensitive enough to detect it.
The relationship of heterotrophic 13C enrichment with the N / P
ratio (Fig. 5e and S1e) is broadly similar to that with N (Fig. 5b and S1b),
suggesting that the relationship of heterotrophic 13C enrichment with
the N / P ratio is largely due to the effect of N rather than to the
ratio itself. However, some level of direct dependence of the enrichment on
the N / P ratio cannot be ruled out. Niklas et al. (2005) and Elser et
al. (2010) integrated biological stoichiometry and metabolic scaling
theories, which led them to suggest that growth rates and plant sizes should
be related to N / P ratios. These authors' analyses focused on individual
plants while our study is on plant organs. However, if the N / P ratio
affects fractionating metabolic processes of plant organs, it is conceivable
that the N / P ratio can also affect the 13C enrichment (or
depletion) of this organ relative to leaves.