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Carbohydrates and secondary compounds of alpine tundra shrubs in relation to experimental warming



It is critical to understand the sensitivity, response direction and magnitude of carbohydrates and secondary compounds to warming for predicting the structure and function of the tundra ecosystem towards future climate change.


Open-top chambers (OTCs) were used to passively increase air and soil temperatures on Changbai Mountain alpine tundra. After seven years’ continuous warming (+ 1.5 °C), the vegetation coverage, nonstructural carbohydrates (soluble sugars and starch) and secondary compounds (total phenols, flavonoids and triterpenes) of leaves and roots in three dominant dwarf shrubs, Dryas octopetala var. asiatica, Rhododendron confertissimum and Vaccinium uliginosum, were investigated during the growing season. Warming did not significantly affect the concentrations of carbohydrates but decreased total phenols for the three species. Carbohydrates and secondary compounds showed significantly seasonal pattern and species-specific variation. No significant trade-off or negative relationship between carbohydrates and secondary compounds was observed. Compared to Dr. octopetala var. asiatica, V. uliginosum allocated more carbon on secondary compounds. Warming significantly increased the coverage of Dr. octopetala var. asiatica, did not change it for V. uliginosum and decreased it for Rh. confertissimum. Rh. confertissimum had significantly lower carbohydrates and invested more carbon on secondary compounds than the other two species.


Enhanced dominance and competitiveness of Dr. octopetala var. asiatica was companied by increased trend in carbohydrate concentrations and decreased ratio of secondary compounds to total carbon in the warming OTCs. We, therefore, predict that Dr. octopetala var. asiatica will continue to maintain dominant status, but the competition ability of V. uliginosum could gradually decrease with warming, leading to changes in species composition and community structure of the Changbai tundra ecosystem under future climate warming.


Global air temperature has been predicted to increase continuously, with the most marked increase in alpine and tundra regions [1]. During the past decades from 1950 to 2010, Changbai Mountain alpine tundra experienced significant increases in both temperature and precipitation and decreases in frost and ice days. The warming rate of growing season is 0.0239 °C y-1, higher than that of the average earth surface temperature. The ice day decreased 0.2245 d y-1 and the increased rate of potential evapotranspiration is 0.6312 mm y-1 [2]. In addition, it is hard to see the snow on the top of the Changbai Mountain in summer. Temperature change alone, and in combination with other environmental changes, will inevitably have considerable impacts on ecosystems [3]. Tundra vegetation on the Changbai Mountain has changed significantly over the last decades, showing that the abundance of shrubs decreased, whereas the grasses’ abundance continuously significantly increased [4]. For instance, the grasses that previously either occurred in the mountain birch forests at a lower elevation or were only occasionally observed in the tundra have extensively invaded the Changbai Mountain alpine shrub tundra, and the tundra vegetation is currently co-dominated by shrubs and six herb species (Calamagrostis angustifolia, Geranium baishanense, Ligularia jamesii, Sanguisorba parviflora, S. stipulata, and Saussurea tomentosa) [5, 6]. Jin et al. stated very recently that the Changbai Mountain shrub tundra will be replaced by a grass tundra, and which is mainly regarded as a consequence of continuous air warming [7].

Strong and sensitive reactions in phenology, photosynthesis, growth, leaf nutrients, and carbon contents and components of alpine and arctic plants to climate warming, including positive and negative responses, have been observed [8,9,10,11,12]. A question that remains unclear is whether species that respond negatively to warming will have disadvantages and thus must escape from the community under future climate warming. For example, it has been observed that two moss species completely disappeared, and the abundance of three dwarf shrub species decreased, but the abundance of forb and grass species did not change after four years’ warming in an alpine plant community, southwestern Norway [13]. Two previously dominant shrubs, Rhododendron chrysanthum and Vaccinium uliginosum, on the west-facing slope of Changbai Mountain alpine tundra showed different responses to climate change over the last decades. V. uliginosum significantly decreased its abundance and changed its previously even distribution pattern into a patch distribution, while Rh. chrysanthum did not [7]. Whether such decreased abundance of some species or disappearance of moss is related to physiologically negative responses to climate change, and/or whether the change in community structure can be explained by physiological competition relationships among species are poorly understood [13].

Competitive relationship among co-existing species is affected by many factors. The competitive ability of a species is influenced by available nonstructural carbohydrates (NSCs, mainly sugars and starch) and secondary metabolism and vice versa [14, 15]. Competition may affect the distribution and allocation pattern of carbon between growth and defense. NSCs and secondary compounds support plant physiological processes of vegetative and reproductive growth, maintenance, storage, and defense, hence, they may be allocated for various purposes, affecting competition or trade-offs between NSCs and secondary compounds at an individual level [16]. For example, more investment of carbon to plant growth could lower plant concentrations of carbon-based secondary compounds [17]. Growth on tundra is limited by low temperature. According to hypotheses of resource based defence, alpine plants might invest more carbon in defence. When temperature increases, increased resource availability will prioritize growth and spend less on defence [17]. The relative growth rate of four trembling aspen was negatively related to condensed tannin content [14]. The carbon allocation to defensive compounds decreased for tomato due to competition [18]. The competition led to a decrease in starch concentration for two Larix species, but soluble sugars and total NSCs (TNC) concentrations were not directly influenced by competition [15].

Changes in environmental conditions will alter the allocation of photosynthetic fixed carbon between primary and secondary compounds and the responses might be highly species specific. Aerts et al. observed that the carbon concentration of three co-existing sub-arctic dwarf shrubs – Empetrum hermaphroditum, Andromeda polifolia and V. uliginosum responded significantly differently to experimental warming [19]. Starch reserves increased across 14 tree species along a natural temperature gradient from lowland to the alpine treeline [20]. According to a simulated warming experiment, elevated temperature did not significantly change the concentrations of soluble sugars and starch in needles, bark and wood of Larix decidua grown at the alpine treeline [21]. The carbon-based secondary compound concentrations of Tofieldia pusilla were decreased by warming, while Saussurea alpina, Carex vaginata, V. uliginosum, Selaginella selaginoides in the same growth environment did not respond to the warming treatment [17]. The tannin concentrations of Cassiope tetragona and V. vitis-idaea leaves were increased by warming [22]. We wonder, therefore, whether a synchronized change or a trade-off between NSCs for growth and the secondary compounds for defense exists in tundra plants under global warming.

We used a 7-year experimentally warming site (+ 1.5 °C in the air temperature and + 0.8 °C in the soil temperature at 10 cm depth) in the alpine tundra on the north-facing slope of Changbai Mountain to study whether changes in plant coverage are associated with changes in carbon resource availability and allocation between growth and defense. We measured NSCs (soluble sugars, starch) and carbon-based secondary compounds (total phenols, flavonoids and triterpenes) in three dominated shrub species (Dryas octopetala var. asiatica, V. uliginosum, Rh. confertissimum) which have shown marked variation in their coverage responses to warming over time (see Table 1). We hypothesize that (1) a decrease in the coverage of species implies negative responses of NSCs to warming, and vice versa; (2) the levels of NSCs and carbon-based secondary compounds of the three species respond to warming differently, showing a significant interaction between species and warming; and (3) there is a trade-off between NSCs and secondary compounds to warming. We aimed to assess the effects of warming on NSCs and secondary compounds in dominant shrubs on alpine tundra, to investigate the link between the change in vegetation coverage and NSCs as well as secondary compounds, and thus to understand whether the plant carbon-physiological parameters can be used as useful indicators to predict vegetation change (winner or loser) under global warming or environmental changes.

Table 1 The average coverage of three shrub (Dryas octopetala var. asiatica, Vaccinium uliginosum and Rhododendron confertissimum) species in the warming open-top chambers and the control plots investigated before and after seven years’ warming treatment (unit: %; n=8). Different lowercase letters indicated significant difference in coverage among years. The statistical results of the effects of warming on coverage was shown by P value for each species (** P < 0.01, *** P ≤ 0.001, ns P > 0.05.)


Responses of species coverage to warming

Experimental warming significantly increased the coverage of Dr. octopetala var. asiatica (P < 0.01), but it highly significantly decreased (P < 0.001) the coverage of Rh. confertissimum (Table 1). The coverage of V. uliginosum was not markedly changed by warming (Table 1). After seven years of warming treatment, the coverage of Rh. confertissimum in the warming OTCs was very small, less than 5% (Table 1).

Leaf nonstructural carbohydrates

Warming treatment had no marked effects on leaf soluble sugars, starch and TNC concentrations, as well as the ratio of sugars to starch, but all the four traits varied over time and were significantly species-specific (Table 2). Generally, the concentrations of soluble sugars were relatively higher, and the starch concentration was lower in both Dr. octopetala var. asiatica and V. uliginosum (Fig. 1).

Table 2 The statistical results of the effects of treatment (warming and control), sampling dates (July, August, September) and species (Dryas octopetala var. asiatica, Rhododendron confertissimum and Vaccinium uliginosum) on the soluble sugars, starch and total nonstructural carbohydrate (TNC) (sugar + starch) concentrations, the ratio of soluble sugars to starch, and secondary compounds (flavonoids, total phenols, triterpene) concentrations in leaves using three-way ANOVA
Fig. 1
figure 1

Concentrations of soluble sugars, starch, TNC (sugar + starch) and the ratio of sugars to starch in leaves of Dryas octopetala var. asiatica, Vaccinium uliginosum and Rhododendron confertissimum grown in the warming open-top chambers (black bars) and the control plots (white bars) measured during the growing season. Error bars are one standard deviation (n = 6). The effects of treatment (T) and sampling date (D) on all parameters were analyzed by two-way ANOVA. * P < 0.05, ** P < 0.01, *** P ≤ 0.001, ns P > 0.05

Starch and TNC concentrations of Dr. octopetala var. asiatica in the warming OTCs were 11% and 4% higher than those in the control across the whole growing season although the difference was not statistically significant. The soluble sugars of Rh. confertissimum in the warming decreased by 17% in July, 34% in August and 18% in September compared to the control, but starch concentration did not change with warming across the whole growing season (Fig. 1). We observed that warming decreased starch concentration of V. uliginosum leaves by 14% in July, 12% in August and 21% in September, on average 15% decline across the whole growing season (P = 0.003). Therefore, the ratio of sugars to starch of V. uliginosum leaves in the warming was higher than that in the control, but the difference was not significant (Fig. 1).

Soluble sugars, starch and TNC in leaves of the three species showed a pronounced seasonal variation for both the warming OTCs and the controls. Soluble sugars and the ratio of sugars to starch increased with time, reaching the maximum values for the three species in September when the temperature was relatively lower (Fig. 1). The starch concentrations of Rh. confertissimum in the warming OTCs were relatively stable during the whole growing season (Fig. 1). The seasonal trend of starch in Dr. octopetala var. asiatica and V. uliginosum showed an opposite trend of soluble sugars, and therefore, the TNC was relatively stable with time. However, the TNC concentrations of Rh. confertissimum leaves increased with time, reflecting the seasonal pattern of soluble sugars (Fig. 1).

The concentrations of soluble sugars and TNC did not differ between Dr. octopetala var. asiatica and V. uliginosum (P > 0.05) but significantly higher than Rh. confertissimum (P = 0.007). The soluble sugars concentrations of Dr. octopetala var. asiatica and V. uliginosum were approx. 1.9 times that of Rh. confertissimum. There was no significant difference in starch concentration among the three species, ranging from 101.2 to 136.9 mg g-1 when all data were pooled. The TNC concentration had a similar trend to soluble sugars (Fig. 1). The TNC concentration of Dr. octopetala var. asiatica and V. uliginosum was approx. 1.5 times that of Rh. confertissimum.

Carbon-based secondary compounds in leaves

Warming significantly affected the concentrations of total phenols (P < 0.001) and flavonoids of Rh. confertissimum (P = 0.026). Warming decreased the total phenols concentrations by 16% for Rh. confertissimum, 21% for Dr. octopetala var. asiatica and 7% for V. uliginosum across the growing season. On average, Rh. confertissimum in the warming OTCs had 14% higher flavonoids concentration than the controls across the whole growing season.

Total phenols and triterpenes showed pronounced seasonal fluctuation, but different species had different patterns (Fig. 2). Total phenols and triterpenes of Dr. octopetala var. asiatica grown in the warming OTCs decreased with time, but the controls had the highest values in August. V. uliginosum had relatively higher total phenols and triterpenes in September. The flavonoids were stable during the growing season for the three species.

Fig. 2
figure 2

Concentrations of flavonoids, total phenol and triterpenes in leaves of Dryas octopetala var. asiatica, Rhododendron confertissimum and Vaccinium uliginosum grown in the warming open-top chambers (black bars) and the control plots (white bars) measured during the growing season. Error bars are one standard deviation (n = 6). The effects of treatment (T) and sampling date (D) on all parameters were analyzed by two-way ANOVA. * P < 0.05, ** P < 0.01, *** P ≤ 0.001, ns P > 0.05

There were significant differences in flavonoids, total phenols and triterpenes concentrations among the three species (Table 2). Rh. confertissimum had the lowest concentrations of flavonoids, total phenols and triterpenes compared to Dr. octopetala var. asiatica and V. uliginosum (Fig. 2). The total phenols concentration of V. uliginosum were 1.4 times higher than Dr. octopetala var. asiaticfrom and Rh. confertissimum in the warming and approx. 1.2 times higher in the controls. V. uliginosum had 1.4 and 2.4 times higher triterpenes concentration than Dr. octopetala var. asiaticfrom and Rh. confertissimum across the whole growing season, with the highest difference mainly occurring in September (Table 2, Fig. 2).

Nonstructural carbohydrates in roots

No significant effects of warming on soluble sugars, starch and TNC concentrations and the ratio of sugars to starch in roots were observed for the three species, but there were significant differences among species (Fig. 3). V. uliginosum roots had 24% higher starch concentration and 12% higher TNC concentration compared to Dr. octopetala var. asiatica and Rh. confertissimum. The TNC concentration in Dr. octopetala var. asiatica roots was very close to Rh. confertissimum (Fig. 3).

Fig. 3
figure 3

Soluble sugars, starch, total carbohydrate concentrations (TNC) and the ratio of sugars to starch in roots of Dryas octopetala var. asiatica, Vaccinium uliginosum and Rhododendron confertissimum from the warming open-top chambers (black bars) and the controls (white bars) measured at the end of the growing season. (mg g−1 ± SD) (n = 6). The effects of treatment (T) and species (S) on all parameters were analyzed by two-way ANOVA. ** P < 0.01, *** P ≤ 0.001, ns P > 0.05


Warming effects on carbohydrates and their possible effects on coverage

Experimental warming in tundra regions often causes increased photosynthesis and growth rate [23,24,25]. In line with our hypothesis 1, Dr. octopetala var. asiatica positively responded to OTC-warming in tissue NSCs, plant coverage, photosynthesis and single leaf size [25], indicating that warming makes Dr. octopetala var. asiatica to fix more carbon, to grow fast, and then to occupy more space and have stronger competitiveness. The soluble sugars of Rh. confertissimum negatively responded to warming (Fig. 1), and decreases in the coverage of Rh. confertissimum have already been recorded during the experimental period (Table 1), consistent with the hypothesis 1. The coverage of V. uliginosum grown in the warming OTCs and the control plots did not significantly change during the whole experimental period (Table 1). Jin et al. (2019) found that V. uliginosum on the west-facing slope of Changbai Mountain alpine tundra showed a patch distribution deviating from the previously normal distribution [7]. Our research field was located on the north-facing slope whose microenvironment (e.g. lower temperature) should be different from the west-facing slope (higher temperature) of Changbai Mountain alpine tundra. The starch storage of V. uliginosum responded negatively to the warming (Fig. 1), so we predict that the distribution of V. uliginosum on the north-facing slope might decrease in the future or gradually show a patch distribution like on the west-facing slope.

The responses of carbohydrates to warming in tundra shrubs were found to be species-specific in the present study which is consistent with our hypothesis 2 (Table 2). Similar results were also observed by other studies [26, 27]. Warming increased NSCs concentrations in Himantormia lugubris but decreased them in Polytrichastrum alpinum, Pinus sylvestris, Pseudotsuga menziesii and Picea mariana [27,28,29,30]. No significant change in TNC was observed in Salix Polaris, Carex vaginata, Saussurea alpine, Selaginella selaginoides, V. uligonosum, Usnea antarctica, U. aurantiaco-atra, Sanionia uncinata, Quercus robur and Q. petraea leaves in response to warming [17, 24, 26, 27]. Different species have different sensitivity to warming which can affect photosynthesis, the distribution and utilization of photosynthate, and growth etc.

Stored carbohydrates in roots can be used by plants for defense and regrowth, or as a buffer under insufficient carbon production [31,32,33,34,35]. Warming did not significantly affect root carbohydrate concentrations of the three species, which is consistent with previous findings that there were non-significant responses of root carbohydrates at the end of the growing season to warming for alpine plants (Elymus nutans, Euphrasia regelii and Swertia mussotii) on the Tibetan Plateau [10], for grapevines in Barossa Valley of Australia [33], for Pinus taeda and P. ponderosa [36]. V. uliginosum roots had higher starch and TNC concentrations compared to Dr. octopetala var. asiatica and Rh. confertissimum, which might be related to that V. uliginosum is a deciduous species. Deciduous plants need more energy and nutrition for new leaves sprouting in the next spring, especially on alpine tundra regions.

The accumulation of NSCs is one of the cryoprotective mechanisms [37]. The sugar-starch system in plants adjusts the ratio of sugar to starch in response to low temperature or other stressors [38]. At high elevations, a higher sugar-starch ratio reflects that plants are subjected to lower temperatures, sometimes positively correlating with cold stress [39]. We found that the ratios of soluble sugars to starch in the warming OTCs were lower than those in the control plots for all the three species, indicating that warming can affect sugar-starch relationship. The three species grown in the warming OTCs hydrolyze less starch against freezing which might be beneficial to growth.

Time-dependent NSC levels and their possible effects on coverage

Marked seasonal patterns of NSCs concentrations for the three species were observed, which is in agreement with earlier findings of the seasonal NSCs fluctuation with soluble sugars concentrations reaching a higher level at the end of the growing season in various tree species [40,41,42]. Soluble sugars, serving as osmotic adjustment and signal substances, play an important role against cold [41, 43]. The starch concentrations peaked in July or August (the active growing season) and decreased in September, indicating that starch hydrolyzes to soluble sugars towards end-season [42]. The temporal variation in the level of NSCs also illustrates that temperature might affect the proportions of carbohydrate component.

Wintergreen Dr. octopetala var. asiatica and deciduous V. uliginosum are typical alpine and arctic dwarf shrubs, especially Dr. octopetala var. asiatica generally dominating community [44]. Evergreen Rh. confertissimum has a small distribution area compared to the two others, only in tundra regions. Generally, deciduous trees require more abundant carbohydrates for vegetative or reproductive growth before the new leaves grow [34, 45]. V. uliginosum had similar concentrations of TNC as Dr. octopetala var. asiatica, but V. uliginosum did not show the same obvious advantages in coverage, photosynthesis, leaf size and growth as Dr. octopetala var. asiatica did [25], indicating that V. uliginosum might allocate some carbohydrates to the growth of new organs. The roots of V. uliginosum had significantly higher starch and TNC concentrations than Dr. octopetala var. asiatica and Rh. confertissimum. Starch, different from soluble sugars, is inactive and accumulates as a storage compound. V. uliginosum, as a deciduous species, is assumed to store high amounts of carbohydrates over harsh winter to support leaf flush in spring [45]. The concentrations of TNC in V. uliginosum and Dr. octopetala var. asiatica were significantly higher than those of Rh. confertissimum that had significantly decreased coverage. Therefore, TNC concentrations are related to the species coverage, which is consistent with our hypothesis 1.

The NSCs responses of Dr. octopetala var. asiatica to warming support the idea that carbon allocation is a key factor for determining dominance. After five years of warming by open-top chambers in the alpine region of southwestern Norway, the carbohydrates storage of Dr. octopetala increased [46]. Dr. octopetala var. asiatica showed increased trend in the carbohydrate concentrations in the present study and significantly increased leaf size [25], indicating that the total carbohydrate contents (leaf biomass or size × concentration) have been stimulated. The stimulation and accumulation of carbohydrates are conducive to dominance, expansion and improvement of competitiveness, which may further lead to changes in community composition and structure of alpine and tundra ecosystems under future climate change [44].

Responses of secondary compounds and trade-off with NSCs

Deciduous species probably have lower concentrations of secondary compounds compared to evergreen plants [47]. Higher concentrations of secondary compounds in perennial leaves could be a greater need for defending herbivorous predator due to longer life span [48]. However, we found that the deciduous V. uliginosum had the highest absolute concentrations of total secondary compounds compared to the other two species. One of the reasons is probably related to that V. uliginosum produces delicious fruits which might need more secondary compounds to defend animals, especially triterpenes.

We observed that Rh. confertissimum allocated relatively more carbon to defense than V. uliginosum and Dr. octopetala var. asiatica based on the ratio of the sum of secondary compounds to total carbon (secondary compounds + TNC). Herms and Mattson (1992) found that increased investment in secondary defense is accompanied by decreased growth, plant size and competitive ability [16]. Thus, more carbon is allocated to secondary compounds in expense of growth, dominance and competition. Warming may alter interspecific competitive relationships and community structure because of the changes in carbon allocation pattern and defense abilities [46].

Total phenols concentrations were decreased by warming in the present study, similar to the results of Holopainen et al. [49]. Phenolic compounds originate from the shikimic acid pathway which is related to the carbohydrate metabolisms [50, 51] and antioxidative potential of plants [52]. Tundra ecosystem is generally characterized by simultaneous stresses such as low temperature, high UV radiation, low nutrient availability which could make plants to produce high levels of secondary compounds or allocate more proportion carbon to the secondary compounds [46, 53]. Thus, alleviation of low temperature by warming is expected to decrease the contents of secondary compounds. The decreases in the concentrations of secondary compounds for Bistorta vivipara, Dr. octopetala, Salix reticulate, Cassiope tetragona, S. herbacea × Polaris and Tofieldia pusilla have been reported [17, 22, 46]. The decrease in total phenols concentration probably relates to the fewer carbon resources for defense substance or more carbon for growth [17]. The decreased proportion of total phenols of Dr. octopetala var. asiatica was relatively higher than the other two species (Fig. 2) which is consistent with higher NSCs and growth. Hence, Dr. octopetala var. asiatica has strong competitive potential.

Flavonoids concentrations of Rh. confertissimum were increased by warming, but OTC warming did not affect the levels of flavonoids in Dr. octopetala var. asiatica and V. uliginosum. We also found that Rh. confertissimum grown in the warming OTCs had relatively lower soluble sugars and significantly reduced coverage than the controls or the other two species. The three species in the present study changed their defense levels to some extent when experiencing continuous 7 years’ warming, consistent with our hypothesis 2 that there was significant interaction between species and warming on the total phenols and flavonoids.

Inconsistent with our hypothesis 3, no significant trade-off relationship between NSCs and secondary compounds in leaves was observed based on the correlation analysis (Figure not shown). However, the secondary compounds tended to be positively correlated with NSCs for Dr. octopetala var. asiatica while negatively correlated with NSCs for V. uliginosum and Rh. confertissimum. Further research is necessary to continue to examine if long-term warming can result in a trade-off relationship between NSCs and secondary compounds for species with decreased distribution or competitiveness.


We conclude that warming increased NSCs and decreased secondary compound in Dr. octopetala var. asiatica, which makes the species still maintain dominant status in the Changbai Mountain alpine tundra with climate change. The dominance of V. uliginosum could gradually decline with continuous warming. Rh. confertissimum had relatively lower carbohydrates compared to Dr. octopetala var. asiatica and V. uliginosum and increased secondary compounds investment with warming. The coverage of Rh. confertissimum in the OTCs was getting small. All these traits will weaken the competition ability of Rh. confertissimum in tundra community. Thus, we predict that Dr. octopetala var. asiatica will maintain its dominant status, V. uliginosum could gradually decrease its coverage, and Rh. confertissimum might be in danger of disappearance with air warming, leading to changes in species composition and community structure of the Changbai tundra ecosystem under future climate warming.

Materials and Methods

Study site and experimental design

The study was conducted on the north-facing slope of alpine tundra on Changbai Mountain (41°58´- 42°42´N; 127°67´- 128°27´E, 2046 m a.s.l.), northeastern China with typical characteristics of Arctic tundra [54]. Changbai Mountain alpine tundra experienced significant increases in temperature and precipitation and decreases in frost and icing days based on the data from 1950 to 2010 [2]. The mean annual temperature is -7.3 °C and mean annual precipitation is 1373 mm in this region [55]. The mean air temperature during the growing season (June to September) is 5.9 °C, and the highest mean daily temperature is less than 10 °C [2]. The majority falling as rain occurs during the short summer (July and August). A snow-free season lasts from May to September. Soils are characterized by Haplic Cambisol (Humic, Dystric). The total nitrogen, phosphorus and potassium were 3.6 ± 0.19, 1.00 ± 0.06 and 1.64 ± 0.01 mg/g, respectively, and the soil had an organic matter content of 124 ± 9.2 mg/g. The vegetation mainly consisted of Dr. octopetala var. asiatica, V. uliginosum, Rh. confertissimum, Rh. chrysanthum, Sanguisorba parviflora, S. stipulate, Calamagrostis angustifolia, etc.

Open-top chambers (OTCs) were used to increase air and soil temperature according to the criteria of the International Tundra Experiment [56]. Eight OTCs were established near the viewing platform in June 2010, about half a mile away from road, with a steep slope in the middle. The OTCs were hexagonal, 0.45 m high, had inclined sides (60°), enclosed a surface of 1.0 m2, and were made of transparent polycarbonate. The OTCs were left in place year-round. Control plots were established beside each OTC with similar species composition and vegetation coverage. Air and soil temperature, air and soil humidity, radiation in the OTCs and the control plots were logged every 30 minutes during growing seasons (June to September) by Em 50 Data Collection System (Decagon, USA). On average, the OTC increased air and soil temperature at 10 cm depth by 1.5 °C and 0.8 °C (with less increase in air temperature, while decrease in soil temperature during night). The air relative humidity was not significantly changed by warming, but the soil water content was decreased by 0.05 m3 m-3 [25].

Three dominant species growing on Changbai Mountain tundra were selected in the present study. Dr. octopetala var. asiatica is an evergreen dwarf shrub and often forms heath communities on calcareous soils in arctic-alpine environments [46]. V. uliginosum is deciduous dwarf shrub with thin and approximate round leaves. Rh. confertissimum is an evergreen dwarf shrub with relatively thicker leaves. The specimens of the three plants can be found in the Institute of Applied Ecology, Chinese Academy of Sciences with voucher ID C.Y. Li 1962, Y.L. Zhou 1951, and P.Y. Fu 1959.

Sampling leaves and roots

Experimental area and sampling for scientific research were approved by local administrative department that supported the present project. We identified the three plant species. After all samples were dried and ground in the field station, they were stored in the laboratory in Shanghai Institute of Technology.

The leaves of Dr. octopetala var. asiatica, V. uliginosum, Rh. confertissimum were sampled from three plants in each OTC and the control plot on July 13th, August 13th and September 18th, 2017. The roots were only sampled on September 18th because of the destructive sampling. Plants were always randomly sampled away from the chamber sides.

Nonstructural carbohydrates analysis

Dried leaves and roots were ground into fine powders using a small grinder. Extraction and determination of carbohydrates from leaves and roots were based on the Anthrone method [57]. The powdered material was suspended in 80% ethanol and incubated for 30 min at 80°C for extraction of soluble sugars. The supernatant was decanted after centrifugation. Then the residual was resuspended in 80% ethanol and repeated the same procedure twice. The supernatant was in constant volume and then was quantified using anthrone as a reagent at 620 nm. The remaining pellet was kept for starch analysis. The pellet was washed two times with 4.6 mol/L HClO4 to digest starch. The supernatant was quantified using the same method as soluble sugars. TNC were calculated by summing total soluble sugars and starch [58].

Secondary compounds analysis

Due to an insufficient number of root samples, only leaf secondary compounds were measured. Secondary compounds of total phenols, flavonoids and triterpenes were extracted by adding air dried leaf powder to 70% ethanol that was heated to reflux for two hours. The procedure was repeated two times. The filtered extract was condensed with a vacuum evaporator. Total phenols were measured at 760 nm wavelength by the Folinol method [59]. Flavonoids were determined at 510 nm wavelength with Rutin as the standard solution [60]. Triterpenes were measured at 542 nm wavelength with vanillin and glacial acetic acid as the chromogenic reagent [61].

Data analysis

Twenty-four plants were respectively selected in the warming OTCs and the control for each species. The leaves of Rh. confertissimum were not enough to measure all parameters, so every four plants were mixed into one sample. MANOVA was used to test the significance of main effect factors (warming treatment, species and sampling date) and their interactions on carbohydrates and secondary compounds. Due to the overall significant effects of species on parameters studied, repeated measures ANOVAs were used to analyze the effects of treatment (between subject), sampling date (within subject), and their interaction on parameters within each species. Multiple comparisons were used to examine the difference in levels of the parameters among the three species. Paired-samples T test was used to assess the effects of warming treatment on every parameter for each species for each sampling date. Data were analyzed statistically using SPSS 16.0 system (SPSS Inc., Chicago, IL, USA). All tests of statistical significance were conducted at a level of 0.05.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.



Open-top chambers


non-structural carbohydrates


total NSCs


  1. Deslippe JR, Simard SW. Below-ground carbon transfer among Betula nana may increase with warming in Arctic tundra. New Phytol. 2011;192:689–98.

    Article  PubMed  CAS  Google Scholar 

  2. Zong SW, Wu ZF, Du HB. Study on climate change in alpine tundra of the Changbai Mountain in growing season in recent 52 years. Arid Zone Res. 2013;30:41–9.

    Google Scholar 

  3. Li MH, Kräuchi N, Gao XP. Global warming: can existing reserves really preserve current levels of biological diversity? J Integr Plant Biol. 2006;48:255–9.

    Article  Google Scholar 

  4. Zong SW, Xu JW, Wu FZ, Qiao LL, Wang DD, Meng XJ, et al. Analysis on the process and impacts of Deyeuxia angustifolia invasion on the alpine tundra, Changbai Mountain. Acta Eclol Sin. 2014;34:6837–46.

    Google Scholar 

  5. Jin YH, Xu JW, Liu LN, He HS, Tao Y, Zong SW, et al. Spatial distribution pattern and associations of dominant plant species in the alpine tundra of the Changbai Mountains. Sci Geogr Sinica. 2016;36:1212–8.

    Google Scholar 

  6. Jin YH, Zhang YJ, Xu JW, Tao Y, He HS, Guo M, et al. Comparative Assessment of Tundra Vegetation Changes Between North and Southwest Slopes of Changbai Mountains, China, in Response to Global Warming. Chin Geogr Sci. 2018;28:665–79.

    Article  Google Scholar 

  7. Jin Y, Xu J, He H, Li MH, Tao Y, Zhang Y, et al. The Changbai alpine shrub tundra will be replaced by herbaceous tundra under global climate change. Plants. 2019;8:370.

    Article  PubMed Central  Google Scholar 

  8. Arft AM, Walker MD, Gurevitch J, Alatalo JM, Bret-Harte MS, Dale M, et al. Responses of tundra plants to experimental warming: meta-analysis of the International Tundra Experiment. Ecol Monogr. 1999;69:491–511.

    Google Scholar 

  9. Root TL, Price JT, Hall KR, Schneider SH, Rosenzweig C, Pounds JA. Fingerprints of global warming on wild animals and plants. Nature. 2003;421:57–60.

    Article  PubMed  CAS  Google Scholar 

  10. Shi C, Sun G, Zhang H, Xiao B, Ze B, Zhang N, et al. Effects of warming on chlorophyll degradation and carbohydrate accumulation of alpine herbaceous species during plant senescence on the Tibetan Plateau. PlosOne. 2014;9:e107874.

    Article  CAS  Google Scholar 

  11. Gargallo-Garriga A, Sardans J, Pérez-Trujillo M, Oravec M, Urban O, Jentsch A, et al. Warming differentially influences the effects of drought on stoichiometry and metabolomics in shoots and roots. New Phytol. 2015;07:591–603.

    Article  CAS  Google Scholar 

  12. Aspinwall MJ, Drake JE, Campany C, Vårhammar A, Ghannoum O, Tissue DT, et al. Convergent acclimation of leaf photosynthesis and respiration to prevailing ambient temperatures under current and warmer climates in Eucalyptus tereticornis. New Phytol. 2016;212:354–67.

    Article  PubMed  CAS  Google Scholar 

  13. Klanderud K. Species-specific responses of an alpine plant community under simulated environmental change. J Veg Sci. 2008;19:363–72.

    Article  Google Scholar 

  14. Donaldson JR, Kruger EL, Lindroth RL. Competition- and resource-mediated tradeoffs between growth and defensive chemistry in trembling aspen (Populus tremuloides). New Phytol. 2006;169:561–70.

    Article  PubMed  CAS  Google Scholar 

  15. Guo Q, Li J, Zhang Y, Zhang J, Lu D, Korpelainen H, et al. Species-specific competition and N fertilization regulate non-structural carbohydrate contents in two Larix species. For Ecol Manage. 2016;364:60–9.

    Article  Google Scholar 

  16. Herms DA, Mattson WJ. The dilemma of plants: to grow or to defend. Quart Rev Biol. 1992;67:283–335.

    Article  Google Scholar 

  17. Nybakken L, Sandvik SM, Klanderud K. Experimental warming had little effect on carbon-based secondary compounds, carbon and nitrogen in selected alpine plants and lichens. Environ Exp Bot. 2011;72:368–76.

    Article  CAS  Google Scholar 

  18. Stamp N, Bradfield M, Li S, Alexander B. Effect of competition on plant allometry and defense. Am Midl Nat. 2004;151:50–64.

    Article  Google Scholar 

  19. Aerts R, Callaghan TV, Dorrepaal E, Van Logtestijn RSP, Cornelissen JHC. Seasonal climate manipulations result in species-specific changes in leaf nutrient levels and isotopic composition in a sub-arctic bog. Funct Ecol. 2009;23:680–8.

    Article  Google Scholar 

  20. Hoch G, Körner C. Global patterns of mobile carbon stores in trees at the high-elevation tree line. Glob Ecol Biogeogr. 2011;21:861–71.

    Article  Google Scholar 

  21. Streit K, Rinne KT, Hagedorn F, Dawes MA, Saurer M, Hoch G, et al. Tracing fresh assimilates through Larix decidua exposed to elevated CO2 and soil warming at the alpine treeline using compound-specific stable isotope analysis. New Phytol. 2013;197:838–49.

    Article  PubMed  CAS  Google Scholar 

  22. Hansen AH, Jonasson S, Michelsen A, Julkunen-Tiitto R. Long-term experimental warming, shading and nutrient addition affect the concentration of phenolic compounds in arctic-alpine deciduous and evergreen dwarf shrubs. Oecologia. 2006;147:1–11.

    Article  PubMed  Google Scholar 

  23. Wookey PA, Robinson CH, Parsons AN, Welker JM, Press MC. Environmental constraints on the growth and performance of Dryas octopetala ssp. Octopeta at a High Arctic polar semi-desert. Oecologia. 1995;104:567–78.

    Google Scholar 

  24. Dormann CF. Consequences of manipulations in carbon and nitrogen supply for concentration of anti-herbivore defence compounds in Salix polaris. Ecoscience. 2003;10:312–8.

    Article  Google Scholar 

  25. Zhou YM, Deng JF, Tai ZJ, Jiang LF, Han JQ, Meng GL, et al. Leaf anatomy, morphology and photosynthesis of three tundra shrubs after 7-Year experimental warming on Changbai Mountain. Plants. 2019;8:271.

    Article  PubMed Central  Google Scholar 

  26. Li MH, Dobbertin CM, Arend M, Xiao WF, Rigling A. Responses of leaf nitrogen and mobile carbohydrates in different Quercus species/provenances to moderate climate changes. Plant Biol. 2013;15(Suppl 1):177–84.

    Article  PubMed  CAS  Google Scholar 

  27. Casanova-Katny A, Pizarro M, Caballero MM, Cordero R, Zúñiga GE. Non-structural carbohydrate content in cryptogamic Antarctic species after two years of passive warming on the Fildes Peninsula. Czech Polar Rep. 2015;5:88–98.

    Article  Google Scholar 

  28. Zha T, Ryyppö A, Wang KY, Kellomäki S. Effects of elevated carbon dioxide concentration and temperature on needle growth, respiration and carbohydrate status in field-grown Scots pines during the needle expansion period. Tree Physiol. 2001;21:1279–87.

    Article  PubMed  CAS  Google Scholar 

  29. Hobbie EA, Gregg J, Olszyk DM, Rygiewicz PT, Tingey DT. Effects of climate change on labile and structural carbon in Douglas-fir needles as estimated by δ13 and Carea measurements. Glob Chang Biol. 2002;8:1072–84.

    Article  Google Scholar 

  30. Way DA. Sage RF Elevated growth temperatures reduce the carbon gain of black spruce [Picea mariana (Mill.) BSP]. Glob Chang Biol. 2008;14:624–36.

    Article  Google Scholar 

  31. Zhu WZ, Xiang JS, Wang SG, Li MH. Resprouting ability and mobile carbohydrate reserves in an oak shrubland decline with increasing elevation on the eastern edge of the Qinghai-Tibet Plateau. Forest Ecol Manag. 2012;278:118–26.

    Article  Google Scholar 

  32. Kleijn D, Treier UA, Müller-Schärer H. The importance of nitrogen and carbohydrate storage for plant growth of the alpine herb Veratrum album. New Phytol. 2005;166:565–75.

    Article  PubMed  CAS  Google Scholar 

  33. Sadras VO, Moran MA. Asymmetric warming effect on the yield and source:sink ratio of field-grown grapevine. Agric For Meteorol. 2013;173:116–26.

    Article  Google Scholar 

  34. Sperling O, Silva LCR, Tixier A, Théroux-Rancourt G, Zwieniecki MA. Temperature gradients assist carbohydrate allocation within trees. Sci Rep. 2017;7:3265.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Li MH, Jiang Y, Wang A, Li X, Zhu W, Yan CF, et al. Active summer carbon storage for winter persistence in trees at the cold alpine treeline. Tree Physiol. 2018;38:1345–55.

    Article  PubMed  CAS  Google Scholar 

  36. King JS, Thomas RB, Strain BR. Morphology and tissue quality of seedling root systems of Pinus taeda and Pinus ponderosa as affected by varying CO2, temperature, and nitrogen. Plant and Soil. 1997;195:107–19.

    Article  CAS  Google Scholar 

  37. Collins FW, Chandorkar RK. Soluble sugar changes occurring during cold hardening of spring wheat for rye and alfalfa. Can J Plant Sci. 1983;63:415–20.

    Article  Google Scholar 

  38. Li MH, Xiao WF, Wang SG, Cheng GW, Cherubini P, Cai XH, et al. Mobile carbohydrates in Himalayan treeline trees I. Evidence for carbon gain limitation but not for growth limitation. Tree Physiol. 2008;28:1287–96.

    Article  PubMed  CAS  Google Scholar 

  39. Patton AJ, Cunningham SM, Volenec JJ, Reicher ZJ. Differences in freeze tolerance of Zoysiagrasses: II. carbohydrate and proline accumulation. Crop Sci. 2007;47:2170–81.

    Article  CAS  Google Scholar 

  40. Terziev N, Boutelje J, Larsson K. Seasonal fluctuations of low-molecular-weight sugars, starch and nitrogen in sapwood of Pinus sylvestris L. Scand J Forestry Res. 1997;12:216–24.

    Article  Google Scholar 

  41. Wong BL, Baggett KL, Rye AH. Seasonal patterns of reserve and soluble carbohydrates in mature sugar maple (Acer saccharum). Can J Bot. 2003;81:780–8.

    Article  CAS  Google Scholar 

  42. Zhu WZ, Cao M, Wang SG, Xiao WF, Li MH. Seasonal dynamics of mobile carbon supply in Quercus aquifolioides at the upper elevational limit. PLoSONE. 2012;7:e34213.

    Article  CAS  Google Scholar 

  43. Li N, He N, Yu G, Wang Q, Sun J. Leaf non-structural carbohydrates regulated by plant functional groups and climate: Evidences from a tropical to cold-temperate forest transect. Ecol Indic. 2016;62:22–31.

    Article  CAS  Google Scholar 

  44. Klanderud K, Totland Ø. Simulated climate change altered dominance hierarchies and diversity of an alpine biodiversity hotspot. Ecology. 2005;86:2047–54.

    Article  Google Scholar 

  45. Hoch G, Richter A, Körner C. Non-structural carbon compounds in temperate forest trees. Plant Cell Environ. 2003;26:1067–81.

    Article  CAS  Google Scholar 

  46. Nybakken L, Klanderud K, Totland Ø. Simulated environmental change has contrasting effects on defensive compound concentration in three alpine plant species. Arct Antarct Alp Res. 2008;40:709–15.

    Article  Google Scholar 

  47. Lima ALS, Zanella F, Schiavinato MA, Haddad CRB. Nitrogenous compounds, phenolic compounds and morphological aspects of leaves: comparison of deciduous and semideciduous arboreal legumes. Sci Agric. 2006;63:40–5.

    Article  CAS  Google Scholar 

  48. Aerts R. Nutrient use efficiency in evergreen and deciduous species from heatlands. Oecologia. 1990;84:391–7.

    Article  PubMed  Google Scholar 

  49. Holopainen JK, Virjamo V, Ghimire RP, Blande JD, Julkunen-Tiitto R, Kivimäenpää M. Climate change effects on secondary compounds of forest trees in the northern hemisphere. Front Plant Sci. 2018;9:1445.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Seigler DS. Plant Secondary Metabolism. Dordrecht: Kluwer Academic Publishers; 1998.

    Book  Google Scholar 

  51. Lindroth RL. Atmospheric change, plant secondary metabolites, and ecological interactions. In: The Ecology of Plant Secondary Metabolites: From Genes to Global Processes, Iason GR, Dicke M, Hartley SE ed; 2012. p. 120–53.

    Chapter  Google Scholar 

  52. Someya S, Yoshiki Y, Okubo K. Antioxidant compounds from bananas (Musa Cavendish). Food Chem. 2002;79:351–4.

    Article  CAS  Google Scholar 

  53. Thoss V, Shevtsova A, Nilsson M-C. Environmental manipulation treatment effects on the reactivity of water-soluble phenolics in a subalpine tundra ecosystem. Plant and Soil. 2004;259:355–65.

    Article  CAS  Google Scholar 

  54. Qian H. Numerical classification and ordination of plant communities in Mt Changbai. J Appl Ecol. 1990;1:254–63.

    Google Scholar 

  55. Liu QJ, Zhang GC, Xu QQ, Wang YD, Wang HM. Simulation of soil respiration in response to temperature under snowpacks in the Changbai Mountain, China. Chin J Plant Ecol. 2010;34:477–87.

    Google Scholar 

  56. Henry GHR, Molau U. Tundra Plants and Climate Change: the International Tundra Experiment (ITEX). Glob Chang Biol. 1997;3(Suppl 1):1–9.

    Article  Google Scholar 

  57. Seifter S, Dayton S, Novic B, Muntwyler E. The estimation of glycogen with the anthrone reagent. Arch Biochem. 1950;25:191–200.

    PubMed  CAS  Google Scholar 

  58. Li MH, Hoch G, Körner C. Source/sink removal affects mobile carbohydrates in P inus cembra at the Swiss treeline. Trees Struct Funct. 2002;16:331–7.

    Article  CAS  Google Scholar 

  59. Singleton VL, Orthofer R, Lamuela-Raventós R. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol. 1999;299:152–78.

    Article  CAS  Google Scholar 

  60. Wang ZZ, Lin SY, Liu JB, Wang EL, Zhang W. Study on structure identification of flavonoids in Vaccinium uliginosum L. Food Sci. 2007;28:455–7.

    CAS  Google Scholar 

  61. Zong W, Xia WS, Cui BL. Determination of total triterpenes in Lagerstroemia specious L. by thin layer chromatography-spectrophotometry. Food Sci. 2005;26:222–5.

    CAS  Google Scholar 

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We would like to thank Xiuxiu Wang for the field sampling and maintaining the experimental field. We also thank Guanhua Dai and Changbai Moutain Forest Ecosystem Open Research for the assistance in the laboratory measurements.


This research was funded by the National Natural Science Foundation of China (31170461) and by the Open Research Fund Program of the Changbai Mountain Academy of Sciences (201504). The funding bodies played no role in the design of the study, in collection, analysis, and interpretation of the data, and in writing the


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YMZ, MY, JJJ finished lab work. ZJT and DTL collected field data and statistically analyzed the data. YMZ wrote the original draft. YMZ and XM revised the manuscript. All authors contributed to the text of the manuscript. All authors have read and approved the manuscript, and ensure that this is the case.

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Correspondence to Xia Ma.

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Zhou, Y., Yang, M., Tai, Z. et al. Carbohydrates and secondary compounds of alpine tundra shrubs in relation to experimental warming. BMC Plant Biol 22, 482 (2022).

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