Introduction
Molecular paleoclimate research has made great strides in recent years, establishing a series of paleoclimate indicators and discussing their applicability, helping us to better understand the history of paleoclimatic changes (
Eglinton and Eglinton, 2008;
Pu et al., 2018;
Zang et al., 2018; Liu and An, 2020).
n-Alkanes are essential components of plant leaf waxes, acting as an important protection against water loss, and are not easy to degrade in sediments because of their unique molecular structures (
Eglinton and Hamilton, 1967;
Koch and Ensikat, 2008). The hydrogen isotope composition of leaf wax
n-alkanes (
d2H
alk) has been widely investigated and has been verified as a robust paleohydrological tool for paleoenvironmental reconstruction (
Sachse et al., 2012;
Sessions, 2016).
Besides the hydrological significance, the ecological potential of
d2H
alk values has gradually attracted attention (
Cormier et al., 2018 and
2019). In the quite limited studies, it is common to observe a
2H-enrichment of bulk biomass or
n-alkanes in the obligate heterotrophic plants (
Ziegler, 1995;
Cormier et al., 2018 and
2019). However, in our recent study, we find a
2H-depletion of
n-alkanes in a holoparasitic plant species (
Cuscuta chinensis). The genera
Cuscuta (dodder) is distributed worldwide and contains nearly 200 species (
Mishra, 2009). All species of
Cuscuta are annual obligate parasites and uptake nutrients from their hosts via haustorium.
Cuscuta species are devoid of proper leaves and roots and exhibit very little to no photosynthetic activity. As compared to the heterotrophic species investigated in
Cormier et al. (2019), which grows on the roots of hosts or aids by fungi,
Cuscuta species are stemparasitical. Here we report the molecular, carbon, and hydrogen isotope compositions of
n-alkanes in specimens of
C. chinensis and its hosts, intending to elucidate the possible reason responding for the
2H-depletion in the obligate heterotrophic plant species.
Materials and methods
During a field trip of late July 2018, eight pairs of
C. chinensis and hosts were collected along a small mountain stream and in a nearby talus in Zigui County (30°53′N, 110°49′E), Hubei Province, China. These hosts belong to dwarf tree/shrub varieties (
Sapium sebiferum,
Alangium chinense,
Debregeasia orientalis,
Boehmeria nivea,
Alchornea davidii) and veins (
Parthenocissus dalzielii,
Vitis amurensis,
Ampelopsis delavayana). Zigui County is located in the Three Gorges, with steep slopes near the Yangtze River and its tributaries. This region is dominated by subtropical monsoon, with annual precipitation of 1490 mm and a yearly temperature of 17°C–19°C (
Li et al., 2019). Under such a climate,
C. chinensis begins to grow in mid-April and is about one month later than its hosts.
The aboveground part of
C. chinensis (mainly stems) and leaves of hosts were used for lipid extraction. Detailed information on the lipid extraction, fractionation, and analysis procedures is identical to that of
Zhao et al. (2018). Briefly, plant samples (ca. 2 g dry weight) were freeze-dried, ground, and ultrasonically extracted with CH
2Cl
2/MeOH (9:1, V/V). Then the extract was fractionated into aliphatic, aromatic, and polar fractions using silica gel column chromatography, and
n-alkanes were eluted in the aliphatic fraction.
n-Alkanes were quantified using a Shimadzu GC-2010 gas chromatograph (GC) equipped with a flame ionization detector. The
d13C values of individual
n-alkanes were determined by using a Finnigan Trace GC attached to a Finnigan Delta Plus XP isotope ratio mass spectrometer, while the
d2H values of individual
n-alkanes were analyzed by using a Trace GC-thermal conversion-isotope ratio mass spectrometer (Thermo Delta V advantage). All
d13C and
d2H values were reported in the notation (‰) relative to the VPDB standard and VSMOW standard, respectively.
Results
n-Alkanes in these plant samples mainly range from C23 to C33, showing a strong odd over even predominance (Fig. 1, Table 1). Among the hosts, three (D. orientalis, B. nivea, and V. amurensis) have a Cmax (the homolog with the highest concentration) of C31, while C29 dominates in the other hosts. Unlike the hosts, distributions of n-alkanes in Cuscuta are consistent, with the C29 taken>75% of the total concentration. Additionally, the concentrations of n-alkanes in Cuscuta are unexpectedly high, even higher than that in their hosts, with a maximum concentration of up to 1521.9 mg/g dry weight.
Since n-C29 is abundant in both hosts and Cuscuta, this homolog is selected for the comparison of compound-specific carbon and hydrogen isotope compositions (Fig. 2). The d13C values of C29 (d13C29) in Cuscuta range from -30.7‰ to -36.4‰, which are less negative than those in their hosts (-33.2‰ to -37.1‰). The difference of d13C29 in the paired host-Cuscuta has a mean value of 1.8‰. Besides, the d2H values of C29 (d2H29) in Cuscuta vary between -225‰ and -244‰, which are negative than those in their hosts (-176‰ to -200‰). The difference of d2H29 in the paired host-Cuscuta averages -48‰. Furthermore, the d13C29 values in Cuscuta are strongly correlated with those in their hosts (R2 = 0.73, p<0.01), whereas the d2H29 values do not correlate between the host-Cuscuta pairs.
Discussion
As an important component of leaf epicuticle, leaf waxes are commonly attributed to maintaining water balance (
Eglinton and Hamilton, 1967;
Koch and Ensikat, 2008). For parasitic plants, the transpiration rates are relatively high (
Ehleringer et al., 1986). Herein the accumulation of a higher concentration of
n-alkanes in the aboveground of
Cuscuta may prevent water loss and reduce evaporation. It is a little strange that the specimens of
Cuscuta in this study are dominated by a single homolog (C
29), which is uncommon in autotrophic plants (Bush and McInerney, 2013). To date, data of
n-alkane distributions in parasitic plants are quite limited. Thus, it is not easy to explain why
Cuscusta preferentially synthesizes
n-C
29 alkane. More works are required to investigate additional parasitic specimens and to explore their alkane synthesizing genes.
The host is the only carbon source for
Cuscuta, which absorbs nutrients (sucrose, water, etc.) that the host transports from leaves to stems. The
d13C values of parasite biomass are almost identical to those of the hosts (Ziegler, 1995). In this study, the
d13C
29 values in all
Cuscuta specimens are less negative than those in their hosts. The differences of
d13C
29 values between
Cuscuta and hosts in seven of the eight pairs>1.5‰, far larger than the instrumental error (Fig. 2). Such a
13C-enrichment of C
29 in
Cuscuta probably results from the carbon isotope fractionation among plant organs. In plants, sucrose is the main form of nutrient transport that provides energy (
O’Leary, 1981). Along with the transport, the
d13C values of sucrose increase from leaves to stems and to roots (
Gleixner et al., 1993;
Badeck et al., 2005). Therefore, the less negative
d13C
29 values in
Cuscuta than those in its hosts probably result from the uptake of carbon nutrient from stems rather than from leaves. Such a close connection between carbon in the heterotrophic plant and its host is further supported by the strong correlation of
d13C
29 values between
Cuscuta and its host.
Different from
d13C
29,
d2H
29 values are lower in
Cuscuta than in its hosts. During the synthesis of
n-alkanes in plants, there are three major influences which determine the
d2H values of
n-alkanes: 1) the
d2H values of plant’s source water (
Sachse et al., 2012); 2) influence of evapotranspiration on leaf water (
Kahmen et al., 2013); 3) the
d2H values of the precursor molecule produced by the decomposition of sucrose and the associated central metabolic pathways (
Newberry et al., 2015;
Cormier et al., 2018). Plant life forms and species differences may result in different
d2H
29 signatures in the host plants, which could be inherited into the heterotrophs (
Sachse et al., 2012). For the more negative
d2H
29 in
Cuscuta than in its hosts, we speculate the following two main reasons. First,
Cuscuta absorbs water from the host stem, which is less affected by evapotranspiration than the host leaves. In contrast, the host leaves utilize
2H-enriched leaf water for
n-alkane synthesis (
Kahmen et al., 2013).
Cormier et al. (2019) observed that
d2H values of leaf water in the host (
Hedera helix) were about 15‰ higher than those of the parasite (
Orobranche hederae).
Alternatively, the
2H-depletion of C
29 in
Cuscuta may result from the different growth periods between
Cuscuta and its host, which leads to the different
d2H value of the raw material for the synthesis of
n-alkanes. In broad-leaved angiosperms, leaf waxes are normally synthesized in the early stage of leaf development (e.g.,
Kahmen et al., 2011; Tipple, 2013). In this case, the leaves of hosts tend to uptake stored
2H-enriched sugars for the synthesis of
n-alkanes (
Newberry et al., 2015).
Cuscuta occupies when the leaves of hosts have been matured. Herein the heterotrophic plants can use the newly formed photosynthetic products, which is
2H depleted relative to the stored carbohydrates (
Newberry et al., 2015;
Cormier et al., 2018).
The pattern of
2H-depletion of
n-alkanes in
Cuscuta is quite different from the report of
Cormier et al. (2019), which observes a consistent
2H-enrichment. Such a contrary may result from the species differences. The specimens in
Cormier et al. (2019) belong to root parasites or mycoheterotrophic plants. The latter acquires solutes from hosts via fungi. In contrast,
Cuscuta is a stem parasitic plant in this study. We could not exclude the influence of environmental conditions. This study was conducted in a humid subtropical climate, while
Cormier et al. (2019) investigated specimens from tropical rainforest or temperate forest. Anyway, this study highlights that there are diverse
2H patterns in parasite-host pairs, and consolidates the (paleo)ecological potential of leaf wax
d2H values. Certainly more works are required to explore the distributions of
n-alkanes from more parasitic plants and from diverse geographical settings, particularly under greenhouse conditions, to elucidate the relationship between
n-alkanes and environmental factors. It is worthy of inferring whether the molecular and isotopic signatures can be transferred to sedimentary archives.
Conclusions
By comparing the molecular, carbon and hydrogen isotope compositions of leaf wax n-alkanes in parasitic plants and their hosts, the main findings are as follows:
1) the concentration of n-alkanes is quite high in Cuscuta and is preferentially dominated by C29 n-alkane. Cuscuta may synthesize more n-alkanes to keep the plant hydrated.
2) the d13C29 values in Cuscuta are more positive than those in its hosts, possibly because of the carbon isotope fractionation of sucrose from leaves to stem in hosts.
3) the d2H29 values in Cuscuta are more negative than those in its hosts, and the reasons may result from the utilization of stem water with less influence from evapotranspiration, and/or the use of newly synthesized carbohydrates which is 2H-depleted relative to stored sugars.
The above results infer the importance of plant nutrient status on the molecular and isotopic compositions of leaf waxes, which is worthy of exploring further whether this process affects the paleo applications of leaf wax ratios in ancient archives.
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