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Drought stress enhanced andrographolides contents in Andrographis paniculata
CHNAES-00683; No of Pages 9 Acta Ecologica Sinica xxx (2020) xxx
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Drought stress enhanced andrographolides contents in Andrographis paniculata Xiaoying Chen, Yueying Xie, Kunhua Wei, Zuzai Lan, Cui Li, Ying Li, Xiaoyun Guo ⁎ Guangxi key Laboratory of Medicinal Resources Protection and Genetic Improvement, Guangxi Botanical Garden of Medicinal Plants, No. 189 Changgang Road, Nanning 530023, PR China
a r t i c l e
i n f o
Article history: Received 8 December 2017 Received in revised form 6 January 2020 Accepted 6 February 2020 Available online xxxx Keywords: Andrographis paniculata Drought stress Flooding stress Growth physiology characteristics Andrographolides
a b s t r a c t Andrographis paniculata (AP) is an important medicinal plant in South China, but it is often exposed to drought and flooding stress. Here three drought stresses and two flooding stresses with three treated time were imposed on potted AP to explore the water stress effects. The growth physiology characteristics and major effective ingredients (andrographolide (AG), 14-deoxy-11,12-didehydroandrographolide (DDAG), and total andrographolides (AGs)) accumulations were studied. Results showed that water stress treated time was statistically effective while its interactions with treatment were not, except for a few parameters. Leaf area and stem diameter of water stressed plants were not statistically differentiated from control all the time, but the plant height, root length, dry weight, and root-shoot ratio were significantly affected by severe drought/flooding stress in prolonged treated time. Significant bioactive component changes were only occurred in the longest treated time under severe stresses, especially those of DDAG and AGs. Results also showed that the plant dry weight were relatively well correlated with andrographolides contents when expressed on ‘per plant’ basis. The underlying mechanisms behind these responses were discussed. © 2020 Published by Elsevier B.V. on behalf of Ecological Society of China.
1. Introduction Andrographis paniculata (Burm. f.) Wall. ex Nees (AP), also known as kalmegh in India and Chuanxinlian in Chinese, was originated in India, Sri Lanka, Burma, Indonesia, Thailand, Vietnam, and was introduced into China in 1950s. AP has an extremely bitter taste and is called ‘King of Bitter’. It is traditionally used in Ayurvedic, Unani, Thai and Chinese medicine systems to preserve health, treat infectious diseases and snake bite. Later much more therapeutic effects are found in AP, especially the potential curative effects on cancer and HIV [1–4]. In addition to flavonoids and polyphenols, the pharmaceutical value of AP is often correlated with four major active diterpene lactones: andrographolide (AG), 14-deoxy11,12-didehydroandrographolide (DDAG), neoandrographolide, and 14-deoxyandrographolide [5–7]. Contents of these andrographolides are often used as indicators to represent the medicinal quality of AP, especially the first two andrographolides. Normally the content of AG in AP is higher than DDAG but the price of AG is lower than DDAG; 5 mg
Abbreviations: AP, Andrographis paniculata; AG, andrographolide; DDAG, 14-deoxy11,12-didehydroandrographolide; AGs, total andrographolides; DW, dry weight; SWC, saturated soil water content. ⁎ Corresponding author. E-mail address: [email protected] (X. Guo).
analytical standard (Sigma-Aldrich) AG and DDAG is $99.10 and $285.00 respectively. Nowadays natural resources of AP in China are seldom found because of over-harvesting, but cultivated AP is widely planted in China's Guangxi Zhuang Autonomous Region, Guangdong Province, Fujian Province, and Sichuan Province. Among these places Guangxi has the biggest AP planted areas, and provides almost all the planting seeds to other provinces. AP is becoming an economically important medicinal plant in China. But during the cultivation of AP in China's Guangxi province, short periods of drought often occurs after heavy rain because of the hot and humid climate environment and the poor ability of soil waterholding capacity, and thus AP is often threatened by drought stress and flooding stress. Though few researches have shown the positive water stress effects on the accumulation of andrographolides in planting AP [8,9], the intensity and duration of drought/flooding stresses and the changing patterns of AG, DDAG, and total andrographolides (AGs) under such stresses are not clearly clarified yet. The correlations between water stressed plant growth characters and their bioactive component changes are still vaguely informed as well. To solve these problems, three drought stresses and two flooding stresses with three treated time were imposed on potted AP, and the growth characteristics, major bioactive constituents, and their correlations were studied. The underlying mechanisms behind these responses were also discussed.
https://doi.org/10.1016/j.chnaes.2020.02.003 1872-2032/© 2020 Published by Elsevier B.V. on behalf of Ecological Society of China.
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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2. Materials and methods 2.1. Plant material and growth conditions Andrographis paniculata (Burm. f.) Wall. ex Nees (AP) was planted in Guangxi Botanical Garden of Medicinal Plants, Guangxi Zhuang Autonomous Region, Nanning, Southwest China (22.51°N, 108.19°E). The altitude is 72–113 m above sea level. The climate is subtropical humid monsoon, with distinctive dry (October to March) and wet (April to September) seasons. There is a generally long, hot summer and occasionally short, cold winter. The average annual temperature is 17 °C to 23 °C, while average annual precipitation is 1 250 mm to 2 800 mm. The average annual sunshine duration is about 1 827 h. The frost-free period usually exceeds 300 days a year. 2.2. Water stress treatments Seeds of AP were sown on 13 April 2015, and after 43 days seedlings were transplanted individually to pot (base diameter 10.5 cm, top diameter 16 cm, height 12 cm) outside on 26 May 2015. Each pot contained 1 kg soils, mixed with loam soil, peat soil, and organic fertilizer (3:1:1). The total organic carbon content was 4.24 mg/ g, and total nitrogen content was 2.01 mg/ g, respectively. The soil pH is 6.56. The soil saturated water content (SWC) was 28.15% (w/w). The water stress treatments began when AP grew about 100 days after seedling (13 August), which was just before the flowering time of AP and was proved to be the best harvest time for better yield and andrographolides content [6]. First all plants were irrigated to SWC (‘W1’, saturated moisture flooding stress), then withholding water until the soil water content was 80% (‘CK’, field capacity control), 60% (‘LD’, mild drought stress, i.e., the drought stress was imposed lightly), 40% (‘MD’, moderate drought stress), and 20% (‘SD’, severe drought stress) of SWC, respectively. Another flooding stress is waterlogging (‘W2’, severe flooding stress), by placing each plant pot into a water tank and adjusting the upper water level to 3–4 cm below the potting soil surface [10]. All the six water treatments were replicated 3 times, lasting for 7 days (time1), 12 days (time2), and 17 days (time3), respectively. To ensure experimental homogeneity, 25 healthy plants with almost the same initial plant height were chosen for each treatment. The pot layout was completely randomized. During the water stress experiments, the soil water content maintenance was controlled by weighting the pots and adding the water loss every day. Rainfall was avoided by covering polyethylene film. 2.3. Determination of plant growth physiology parameters At the end of the water stress treatments, predawn leaf water potential was detected using a dew point microvoltmeter (Psypro, Wescor Inc., Logan, UT, USA) [11], and plant growth and morphological parameters (leaf area, plant height, stem diameter, root length, shoot/root/ total dry weight (shoot/root DW and DWs), root-shoot ratio) were measured and calculated [12]. The whole plant material, including shoot and root, were harvested and after oven-dried (60 °C, 96 h.), bioactive component contents were analyzed by HPLC method. The HPLC procedure was as follows: HPLC sample preparation: 1 g dried plant powder was dissolved in 20 mL HPLC grade methanol and then the solution was ultrasonic extracted for 60 min. After filtering and removing the filter residues, the filtrates were evaporated to dryness and then dissolved in methanol. Next the solution was transferred to a 10-mL volumetric flask, diluted with methanol to volume, and mixed. Finally, the solution was filtered through a 0.45 μm microporous filter membrane. HPLC analysis method: The chromatographic column was Dikma Diamonsil ODS-C18 (4.6 * 250 mm, 5 μm) (Dikma Technologies Inc., Beijing, China). The mobile phase was consisted of eluent A and eluent B. Eluent A was a mixture of methanol: acetonitrile solution (4:1, v/v)
while eluent B was 0.2% phosphate water. The detection wavelength was 250 nm. The injection volume was 10 μL. The flow rate was 0.8 mL min−1. The chromatographic column temperature was 25 °C. Under these conditions, the baseline separation of standard control from other components was well achieved, the resolution with the adjacent peaks was greater than 1.5, and the theoretical plate number was no less than 5000. Contents of AG, DDAG and AGs were expressed in two ways: AGC, DDAGC and their sum (AGCs) on percent (‘%’, converted from ‘mg/g dry weight’) basis; and the respective AGP, DDAGP, and AGPs on ‘per plant’ (‘mg/plant’) basis. 2.4. Statistical analysis Data management and statistical analysis were performed using IBM SPSS Statistics 24. The normality of distribution and homogeneity of variance were tested before analysis of variance (ANOVA). Repeated Measures ANOVA was used to analyze the time effects and the interactions between time and water treatments. Multivariate analysis of variance (MANOVA) was used to analyze the effects of water stress on plant physiology parameters in each treated time. Duncan's post-hoc analysis was used to compare the differences of means. Spearman rank correlation analysis was used to test the relationships between plant growth parameters and bioactive components. 3. Results 3.1. Effects of time and their interactions with water treatments Repeated Measures ANOVA was used to analyze the within-subjects effects, namely, time effects and their interactions with water treatments. First, Mauchly's test of sphericity (Table. A.1) was used to judge whether there were relations among the repeatedly measured data. If not (P ≤ 0.05), Greenhouse-Geisser corrected results or MANOVA should be taken next. Table 1 shows that different treated time had significant statistical effects on most measured parameters, except for leaf area, root length, root DW. Moreover, its interactive effects with water treatments were not significant except for leaf water potential, root length, root-shoot ratio, and AGCs. Based on these analysis and our actual results, in the following part, we focused on the multiple comparisons among the six water treatments in each treated time, rather than the pairwise comparisons of the repeatedly measured data in different treated time of each water treatment. 3.2. Effects of water stress on leaf water potential Changes of leaf water potential reflected the plant water status under soil water stress. When AP was water stressed for 7 days (time1), the leaf water potential of SD, MD, W1, and W2 were significantly different from CK and LD. But when the stress time prolonged (time2, time3), dramatic declines of leaf water potential were only appeared in SD, while the other leaf water potentials were not significantly differentiated (Fig. 1). This phenomenon showed that the soil moisture changes did not influence most of the leaf water levels when water stress time extended. It also reflected the well resilience of AP to external water changes after adaptation. 3.3. Effects of water stress on plant growth and morphological parameters Fig. 2 shows the changes of four morphological parameters under water stress. In each treated time, the water stressed leaf areas (Fig. 2A) and the stem diameter (Fig. 2B) were not significantly variated from CK, even if they were all lower than CK when treated the longest time (time3) in present study. And although the plant height (Fig. 2C)
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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was all reduced by MD and SD in every time, only the reductions of SD were significant while no marked differences were detected in other water treatments. The root length variations were different in the three times (Fig. 2D), with no significant changes in time1 and significant higher by W2 in time2 and by SD in time3. In a word, these morphological traits were only dramatically altered by the severe drought/ flooding stress and in prolonged treated time. The plant biomass accumulations were detected by observing changes of plant dry weight under water stressed conditions. Shoot DW was all declined in water stressed plants compared to CK in each treated time, with no statistical changes in time1 and significantly decreases by SD in time2 (30.64%) and time3 (30.53%) (Fig. 3A). Root DW was less changed than shoot DW under drought stress but significantly decreased by W2 in time2 (33.55%) and time3 (33.90%) (Fig. 3B). The total plant dry weight (DWs) was almost unaffected in time1 but significantly decreased by SD in time2 (27.70%) and time3 (25.35%), and the respective non-significant reductions of DWs by MD, W1 and W2 were 16.13%, 7.95%, 17.12% in time2, and 16.58%, 6.25%, 13.51% in time3 (Fig. 3C). These results also suggested severe drought/flooding stress significantly affected plant dry weight. The root-shoot ratio is often used to indicate changes of biomass allocation. Fig. 3D shows that the root-shoot ratio was statistically unchanged in time1 but significantly increased by SD in time2 (17.55%) and time3 (30.40%) and significantly decreased by W2 in time2 (25.88%) and time3 (32.03%). This result implied that AP adjusted its photoassimilate allocation pattern under relatively severe water stress conditions, by transporting more to the below-ground parts in severe drought stress while more to the above-ground parts in severe flooding stress.
3.4. Effects of water stress on therapeutic active components contents The contents of therapeutic active components in medicinal plants can be expressed on ‘%’ basis and on ‘per plant’ basis, although the first representation is commonly used in most studies, the latter is recommended by many scientists because it can specifically describe the changes of active components in individual plant and that's the interesting point. Fig. 4A–C shows the changes of AP's andrographolides contents on ‘%’ basis. AGC showed no statistical differences in time1 and little changes in water stressed plants compared to CK in time2 and time3 (Fig. 4A). DDAGC showed no statistical changes in time1 and time2 but significantly increased by SD in time3 (Fig. 4B). AGCs showed similar change patterns to DDAGC (Fig. 4C). In addition, although the changes were not always being significant, AGC, DDAGC and AGCs were all increased by SD and MD but decreased by W2 in all three times (Fig. 4A–C). In a word, drought stress led to the increase of andrographolides while flooding stress resulted in their decrease when the water stress duration time increased. Specifically, in time3, the increase of DDAGC by SD and MD compared to CK was 53.62%, 23.93% and the decrease by W1 and W2 was 12.31%, 27.28%. And the respective increase of AGCs by SD and MD was 44.02%, 24.35% and the decrease by W1 and W2 was 6.70%, 30.41%.
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Fig. 1. Changes of Andrographis paniculata (AP)’s leaf water potential under different water treatments. ‘SD’, ‘MD’, and ‘LD’ meant that the drought stresses were respectively imposed severely, moderately, and lightly. Their corresponding soil water contents were 20%, 40%, 60% of saturated soil water content (SWC). ‘CK’ was field capacity control, with the soil water content maintaining at around 80% of SWC. ‘W1’ and ‘W2’ were two flooding stresses. ‘W1’ meant the soil water content was saturated soil water content, i.e. 100% of SWC. ‘W2’ meant waterlogging stress, namely, each plant pot was placed into a water tank and the upper water level was adjusted to 3–4 cm below the soil surface. All six water treatments were lasted for 7 days (time1), 12 days (time2), and 17 days (time3), respectively. The same colored lowercase indicates differences of means among the six water treatments in each treated time by Duncan's post-hoc method. The significance level was 0.05.
Fig. 4D–F shows the changes of AP's andrographolides contents on ‘per plant’ basis. AGP showed no statistical differences among water stresses in all treated times (Fig. 4D). DDAGP showed no statistical changes in time1 and time2 but significantly increased by SD in time3 (Fig. 4E). AGPs showed similar change patterns to DDAGP (Fig. 4F). Similarly, in time3, the increase of DDAGP by SD and MD compared to CK was 25.39%, 2.88% and the decrease by W1 and W2 was 13.34%, 27.88%. And the respective increase of AGPs by SD and MD was 20.28%, 2.20% and the decrease by W1 and W2 was 2.71%, 28.20%. To sum up, when AP was water stressed for 17 days (time3), severe drought stress (SD) resulted in a 1.53/1.25 fold increase in DDAGC/ DDAGP and a 1.44/1.20 fold increase in AGCs/AGPs (with 25.35% total biomass loss), and moderate drought stress (MD) resulted in a 1.24/ 1.29 fold increase in DDAGC/DDAGP and a 1.24/1.22 fold increase in AGCs/AGPs (with 16.58% total biomass loss). 3.5. Relationships between plant growth parameters and andrographolides contents under water stressed conditions The plant growth parameters were relatively easy available and cheaper than the data of the andrographolides contents, so we explored their relationships with andrographolides contents under water stressed conditions, trying to find the ones which could represent the andrographolides variations (Table 2). Two plant growth parameters, leaf area and root length, were not significantly correlated to any andrographolide content. And no significant correlations were found between AGC and any growth parameter.
Table 1 Significance (P-value) of univariate tests of within-subjects effects. Source
Leaf water potential
Leaf area
Plant height
Stem diameter
Root length
Shoot DW
Root DW
DWs
Time Time ⁎ treatment
0.026 0.046⁎
0.141 0.141
0.000⁎⁎ 0.612
0.000⁎⁎ 0.655
0.136 0.000⁎⁎
0.000⁎⁎ 0.687
0.090 0.078
0.001⁎⁎ 0.609
Source Time Time ⁎ treatment
Root-shoot ratio 0.001⁎⁎ 0.008⁎⁎
AGC 0.004⁎⁎ 0.642
DDAGC 0.000⁎⁎ 0.572
AGCs 0.000⁎⁎ 0.008⁎⁎
AGP 0.034⁎ 0.887
DDAGP 0.003⁎⁎ 0.239
AGPs 0.001⁎⁎ 0.371
DW: dry weight (g); AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/plant’) basis.
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Fig. 2. Changes of AP's leaf area, stem diameter, plant height, and root length under different water treatments. Other legends are the same as in Fig. 1.
The correlation coefficients between other plant growth parameters with DDAGC/AGCs were very low (b0.30), even if they were significant. Lower correlation coefficients were also found in plant height and stem diameter with DDAGP/AGPs (b0.40). These lower correlations could be ignored because of not much use in practice. However, relatively higher positive correlation coefficients were found in shoot DW, root DW and DWs with AGP/DDAGP/AGPs, especially with DDAGP/AGPs (close to 0.50). These results demonstrated that a close relationship existed between plant dry weight and bioactive contents when expressed on ‘per plant’ basis.
143.7 mM salinity stress applied before AP flowering led to the maximum increases of AG and DDAG. In summary, our study suggested that water stress duration time should be considered when applying different degrees of drought/flooding to AP, and in another respect this also reflected the well adaptability of AP to water stress. Therefore, in the following part, to clarify the water stress effects on AP, we would discuss its responses in the longest treated time (time3), rather than the other two times (time1, time2).
4. Discussion
The dynamics of AP's leaf potential during water stress treatments implied its adapted processes (Fig. 1). In drought stress, the cellular dehydration causes osmotic stress, plant responds to accumulate osmoregulation substances (betaine, proline, soluble sugar, etc.), which decreases the value of leaf potential [13]. And accompanied with the increase of tissue water content, the value of leaf potential also increased. The responses of AP's leaf area, stem diameter, plant height and root length to different water stress treatments revealed that these plant growth and morphological characteristics were not significantly affected except by severe drought/flooding stress (Fig. 2A–D). This growth stability implied the good tolerance of AP to water stress. During the experimental period, AP was still in its fast growing stage, so the water stress could be compensated rapidly and serious damage could be avoided. But when in severe drought stress conditions, the scarcity of water finally changed AP's growth physiology. Besides preventing excessive water deficits by reducing plant water transpiration through stomatal closure, and absorbing much more water from underground by elongating root length, AP adjusted its photosynthetic assimilate distribution strategies and more assimilation were allocated to roots than the
4.1. Water stress effects related to the treated time In our study, repeated measures ANOVA showed that water stress treated time was statistically effective while time and treatment interactions were not, except for a few parameters (Table 1). The time effects of water stress were further proved by plant morphological, dry weight, and bioactive component changes (Figs. 2-4). For example, water stress decreased shoot DW and DWs compared to CK in every treated time, while these decreases were non-significant when treated with short time (7 days, time1) even by severe drought stress (SD), and became significant by SD when stress time prolonged (12 days, time2; 17 days, time3). In the case of bioactive contents, drought (SD, MD) induced increases of AGC, DDAGC, and AGCs were non-significant in time1 and time2, but the increases of DDAGC and AGCs by SD were significant in time3. Similar change patterns were found in DDAGP and AGPs as affected by SD. The needs of relatively longer stress time and higher stress intensity to trigger productive effects in AP were also revealed in salinity stress. For instance, Talei et al. [13] found a 30-day,
4.2. Water stress changed the biomass allocation of AP
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Fig. 3. Changes of AP's shoot DW (shoot dry weight), root DW (root dry weight), DWs (total dry weight), and root-shoot ratio under different water treatments. Other legends are the same as in Fig. 1.
above-ground. The significant increases of root-shoot ratio in the severe drought stress also indicated the allocation pattern changed compared to control. Consistent with this, AP's shoot, root, and total DW was also decreased variously when confronted with water stress (Fig. 3A– C). Farooq et al., [14] thought the plant growth reduction caused by drought stress was a result of impaired mitosis, cell elongation and expansion. To be noticed, waterlogging stress significantly decreased the root DW but not the shoot DW, indicating the influence of flooding stress was more severe on the below-ground rather than the above-ground. Oxygen-depletion is one of the most serious symptoms when plants meet with flooding, roots undergone anaerobic respirations are hard to survive. The smaller root biomass was also found in Islam's research [15]; their study proved that under waterlogging stress the metabolism and carbon budget was more restricted in root than shoot. 4.3. Drought stress enhanced the level of DDAG in AP Researchers found the proper harvest time for this annual herb AP is between the budding stage and early blooming stage, i.e., the transformation of vegetative stage to reproductive stage, which is about 100–120 days after seeding stage [6,16–18]. Studies also proved that during this time, both the dry matter accumulation and total andrographolides production were gradually reached the peak and maintained relative stability [17,18]. Here we applied water stress prior to the final harvest time to get the higher andrographolides production. Our results showed that drought stress (SD, MD) induced considerable increases of DDAG and AGs while flooding stress (W1, W2) resulted in their decreases in AP. Similar results were reported in water
stressed amur corktree (Phellodendron amurense), a pharmaceutical resource plant of traditional Chinese medicine cortex phellodendri [19]. Drought and waterlogging stress inhibited the growth of amur corktree, its height, diameter and biomass were significantly lower than those of the control, but drought (soil water potential was −80 – −60 KPa) increased the contents of three major alkaloids (berberine, jatrorrhizine, palmatine) while waterlogging stress (soil water potential was −20–0 KPa) decreased these alkaloids contents. In hawthorn (Crataegus laevigata and C. monogyna), it was found that water-deficit stress caused the increase of the desired polyphenolics levels while flooding stress led to the decrease of them [20]. We speculated that the main reason was the cell hypoxia environment caused by flooding stress, which restricted the biosynthesis of plant bioactive compounds. The phenomena of drought-stress induced bioactive secondary metabolites accumulation were also observed in other medicinal plants. For example, it was found that drought stress increased the major secondary metabolites, hyperforin concentration in St. John's wort (Hypericum perforatum) leaves [21], total indole alkaloid content in Catharanthus roseus shoots and roots [22,23], artemisinin in annual wormwood (Artemisia annua L., Asteraceae) leaves and whole plants [24], etc. Reasons about this increase were discussed a lot. In summary, this drought-stress induced bioactive components accumulation in medicinal plants is thought to be a response and adaptation to environmental stress and there are several hypotheses explaining it. Kleinwächter and Selmar [25–27] had summarized the phenomena of drought stress-related concentration increases of natural products and proposed the following theory: Reduction of CO2 fixation from water shortage results in the oversupply of reduction equivalents (NADPH + H+), and thus the metabolic processes are shifted to
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Fig. 4. Changes of AP's andrographolides contents on ‘%’ and ‘per plant’ basis under different water treatments. AG: andrographolide; DDAG: 14-deoxy-11,12-didehydroandrographolide; AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/ plant’) basis. Other legends are the same as in Fig. 1.
Table 2 Relationships between plant growth parameters and andrographolides contents under water stressed conditions.
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Leaf area (cm ) Plant height (cm) Stem diameter (cm) Root length (cm) Shoot DW (g) Root DW (g) DWs (g) Root-shoot ratio
AGC (%)
DDAGC (%)
AGCs (%)
AGP (mg/plant)
DDAGP (mg/plant)
AGPs (mg/plant)
−0.264 −0.102 −0.156 0.033 −0.102 0.042 −0.066 0.178
−0.130 −0.249⁎ −0.186 0.162 −0.285⁎⁎
−0.230 −0.236⁎ −0.214⁎
−0.253 0.161 0.189 −0.032 0.368⁎⁎ 0.423⁎⁎ 0.408⁎⁎
−0.139 0.214⁎ 0.366⁎⁎
−0.219 0.231⁎ 0.360⁎⁎
0.000 0.481⁎⁎ 0.499⁎⁎ 0.515⁎⁎
−0.005 0.504⁎⁎ 0.509⁎⁎ 0.538⁎⁎
0.050
0.014
−0.001
−0.134 −0.259⁎ 0.228⁎
0.157 −0.280⁎⁎ −0.132 −0.253⁎ 0.215⁎
DW: dry weight (g); AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/plant’) basis. ⁎ Correlation is significant at the 0.05 level (2-tailed). ⁎⁎ Correlation is significant at the 0.01 level (2-tailed).
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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stress [36].These highly heritable traits could be used for yielding drought tolerant AP with high bioactive contents as well. By the way, several studies suggested that the problem of drought-stress caused growth reduction could be alleviated by adding fertilization [23,45,48,49] or supplying elevated carbon dioxide during the stress [40,50]. This needs further research. Contrary to other researches, in our study, the amount of DDAG in AP was about at least 4-fold higher than AG. This may be explained by the instability of AG. Studies showed that fresh AP had higher AG content but AG was easily converted into DDAG during storage [7]. The other reason was our material handling method before the HPLC determination of the two diterpenoids. We dried AP in an oven at 60 °C for 96 h, but other researches oven-dried the material at relatively low temperature (such as 35–45 °C or shade dried) [51]. Studies also showed that thermal treatment made it easier for AG to transform into DDAG [52]. We also found that shoot DW, root DW and DWs were relatively highly correlated with bioactive contents when expressed on ‘per plant’ basis (Table 2). The higher plant dry weight meant the higher andrographolides contents in AP. This gave us good indicators representing medicinal quality when applying water treatments. The plant weight could be weighed rapidly at low cost, and it could be used for prompt and large-scale selection for high medicinal quality AP after stress.
pathways that consume the surplus reduction equivalents, such as the secondary metabolite biosynthesis of phenols, alkaloids and isoprenoids [25–31]. Their theories were kind of similar to other reducing equivalents accumulation circumstances such as dissipation system of excess light energy. For example, concomitant with chloroplast overreduction by excess light, plants re-oxidizes reduction equivalents through antioxidant system, photorespiration, and violaxanthine cycle to reduce damages [32,33]. The second type of hypothesis believes that drought stress causes the production of reactive oxygen species (ROS), besides antioxidant system, plant develops the secondary metabolites accumulation defense mechanism to protect plants from oxidative stress [21–23,34–36]. However, some scientists consider drought-stress induced ROS as a trigger of signal transduction pathway that promotes the gene expressions of bioactive components in medicinal plants [37,38]. These hypotheses were the most easily accepted interpretations and proved by many researches. The third type of hypothesis relates to the carbon/nutrient balance and growth differentiation balance hypotheses which predict a trade-off between plant growth and defense (though this kind of hypothesis lays emphasis on plant chemical defenses against pathogens and herbivores). When plant growth and development are inhibited by water deficit, the carbon fixed during photosynthesis tends to be allocated to the defense formation of secondary compounds [34,39–42]. However, these hypotheses were often specifically used when plants confronted with biotic stress. In short, reasonable drought stress (proper applying time, duration and intensity) seems to be beneficial for the biosynthesis and accumulation of bioactive secondary metabolites in medicinal plants [24,43–45]. Nonetheless, our results also demonstrated the inevitable biomass loss of AP accompanied with the severe and moderate drought induced increase of DDAG/AGs. And part of this andrographolides increase attributed to the reduction of biomass, as researched by Talei et al. [13] when discussing the salinity induced increase of AG. Therefore, the balance between stress-induced bioactive component enhancement and biomass reduction should be evaluated according to the different harvesting purposes in modern agronomical cultivation of AP [13,23,46]. Furthermore, as revealed by Taeil et al. [47], AG and DDAG exhibited moderate and high intraspecific hybridization heterosis and their high heritability was beneficial for producing higher andrographolide varieties. AG (and proline, net photosynthetic rate) also showed high broad-sense heritability in AP encountered salinity
5. Conclusion Water stress effects on AP related to the treated time, and most of the plant growth and andrographolides changes were significantly affected by severe drought/flooding stress in prolonged treated time. Severe and moderate drought induced increase of DDAG/AGs with inevitable biomass loss. Plant dry weight were relatively highly correlated with bioactive contents when expressed on ‘per plant’ basis. Declaration of Competing Interest None. Acknowledgements This work was supported by China's National Natural Science Foundation [31200305] and Special program for guidig local science and technology development by the central government [Guangxi ZY1949023].
Appendix A. Appendix Table A.1 Mauchly's test of sphericitya. Within subjects effect
Mauchly's W
Approx. chi-square
df
Sig.
Time
0.396 0.979 0.630 0.993 0.925 0.767 0.863 0.774 0.782 0.745 0.910 0.886 0.711 0.831 0.807
10.182 0.236 13.404 0.218 2.269 7.674 4.273 7.427 7.136 5.894 1.880 2.416 5.798 3.140 3.645
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
0.006 0.889 0.001 0.897 0.322 0.022 0.118 0.024 0.028 0.053 0.391 0.299 0.055 0.208 0.162
Leaf water potential Leaf area Plant height Stem diameter Root length Shoot DW Root DW DWs Root-shoot ratio AGC DDAGC AGCs AGP DDAGP AGPs
Epsilonb Greenhouse-Geisser
Huynh-Feldt
Lower-bound
0.624 0.979 0.730 0.993 0.930 0.811 0.880 0.816 0.821 0.797 0.918 0.898 0.776 0.856 0.838
0.951 1.000 0.886 1.000 1.000 0.994 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
0.396 0.979 0.630 0.993 0.925 0.767 0.863 0.774 0.782 0.745 0.910 0.886 0.500 0.500 0.500
Tests the null hypothesis that the error covariance matrix of the orthonormalized transformed dependent variables is proportional to an identity matrix. DW: dry weight (g); AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/plant’) basis. a Design: Intercept + treatment; Within Subjects Design: time. b May be used to adjust the degrees of freedom for the averaged tests of significance. Corrected tests are displayed in the Tests of Within-Subjects Effects table.
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Drought stress enhanced andrographolides contents in Andrographis paniculata Xiaoying Chen, Yueying Xie, Kunhua Wei, Zuzai Lan, Cui Li, Ying Li, Xiaoyun Guo ⁎ Guangxi key Laboratory of Medicinal Resources Protection and Genetic Improvement, Guangxi Botanical Garden of Medicinal Plants, No. 189 Changgang Road, Nanning 530023, PR China
a r t i c l e
i n f o
Article history: Received 8 December 2017 Received in revised form 6 January 2020 Accepted 6 February 2020 Available online xxxx Keywords: Andrographis paniculata Drought stress Flooding stress Growth physiology characteristics Andrographolides
a b s t r a c t Andrographis paniculata (AP) is an important medicinal plant in South China, but it is often exposed to drought and flooding stress. Here three drought stresses and two flooding stresses with three treated time were imposed on potted AP to explore the water stress effects. The growth physiology characteristics and major effective ingredients (andrographolide (AG), 14-deoxy-11,12-didehydroandrographolide (DDAG), and total andrographolides (AGs)) accumulations were studied. Results showed that water stress treated time was statistically effective while its interactions with treatment were not, except for a few parameters. Leaf area and stem diameter of water stressed plants were not statistically differentiated from control all the time, but the plant height, root length, dry weight, and root-shoot ratio were significantly affected by severe drought/flooding stress in prolonged treated time. Significant bioactive component changes were only occurred in the longest treated time under severe stresses, especially those of DDAG and AGs. Results also showed that the plant dry weight were relatively well correlated with andrographolides contents when expressed on ‘per plant’ basis. The underlying mechanisms behind these responses were discussed. © 2020 Published by Elsevier B.V. on behalf of Ecological Society of China.
1. Introduction Andrographis paniculata (Burm. f.) Wall. ex Nees (AP), also known as kalmegh in India and Chuanxinlian in Chinese, was originated in India, Sri Lanka, Burma, Indonesia, Thailand, Vietnam, and was introduced into China in 1950s. AP has an extremely bitter taste and is called ‘King of Bitter’. It is traditionally used in Ayurvedic, Unani, Thai and Chinese medicine systems to preserve health, treat infectious diseases and snake bite. Later much more therapeutic effects are found in AP, especially the potential curative effects on cancer and HIV [1–4]. In addition to flavonoids and polyphenols, the pharmaceutical value of AP is often correlated with four major active diterpene lactones: andrographolide (AG), 14-deoxy11,12-didehydroandrographolide (DDAG), neoandrographolide, and 14-deoxyandrographolide [5–7]. Contents of these andrographolides are often used as indicators to represent the medicinal quality of AP, especially the first two andrographolides. Normally the content of AG in AP is higher than DDAG but the price of AG is lower than DDAG; 5 mg
Abbreviations: AP, Andrographis paniculata; AG, andrographolide; DDAG, 14-deoxy11,12-didehydroandrographolide; AGs, total andrographolides; DW, dry weight; SWC, saturated soil water content. ⁎ Corresponding author. E-mail address: [email protected] (X. Guo).
analytical standard (Sigma-Aldrich) AG and DDAG is $99.10 and $285.00 respectively. Nowadays natural resources of AP in China are seldom found because of over-harvesting, but cultivated AP is widely planted in China's Guangxi Zhuang Autonomous Region, Guangdong Province, Fujian Province, and Sichuan Province. Among these places Guangxi has the biggest AP planted areas, and provides almost all the planting seeds to other provinces. AP is becoming an economically important medicinal plant in China. But during the cultivation of AP in China's Guangxi province, short periods of drought often occurs after heavy rain because of the hot and humid climate environment and the poor ability of soil waterholding capacity, and thus AP is often threatened by drought stress and flooding stress. Though few researches have shown the positive water stress effects on the accumulation of andrographolides in planting AP [8,9], the intensity and duration of drought/flooding stresses and the changing patterns of AG, DDAG, and total andrographolides (AGs) under such stresses are not clearly clarified yet. The correlations between water stressed plant growth characters and their bioactive component changes are still vaguely informed as well. To solve these problems, three drought stresses and two flooding stresses with three treated time were imposed on potted AP, and the growth characteristics, major bioactive constituents, and their correlations were studied. The underlying mechanisms behind these responses were also discussed.
https://doi.org/10.1016/j.chnaes.2020.02.003 1872-2032/© 2020 Published by Elsevier B.V. on behalf of Ecological Society of China.
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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2. Materials and methods 2.1. Plant material and growth conditions Andrographis paniculata (Burm. f.) Wall. ex Nees (AP) was planted in Guangxi Botanical Garden of Medicinal Plants, Guangxi Zhuang Autonomous Region, Nanning, Southwest China (22.51°N, 108.19°E). The altitude is 72–113 m above sea level. The climate is subtropical humid monsoon, with distinctive dry (October to March) and wet (April to September) seasons. There is a generally long, hot summer and occasionally short, cold winter. The average annual temperature is 17 °C to 23 °C, while average annual precipitation is 1 250 mm to 2 800 mm. The average annual sunshine duration is about 1 827 h. The frost-free period usually exceeds 300 days a year. 2.2. Water stress treatments Seeds of AP were sown on 13 April 2015, and after 43 days seedlings were transplanted individually to pot (base diameter 10.5 cm, top diameter 16 cm, height 12 cm) outside on 26 May 2015. Each pot contained 1 kg soils, mixed with loam soil, peat soil, and organic fertilizer (3:1:1). The total organic carbon content was 4.24 mg/ g, and total nitrogen content was 2.01 mg/ g, respectively. The soil pH is 6.56. The soil saturated water content (SWC) was 28.15% (w/w). The water stress treatments began when AP grew about 100 days after seedling (13 August), which was just before the flowering time of AP and was proved to be the best harvest time for better yield and andrographolides content [6]. First all plants were irrigated to SWC (‘W1’, saturated moisture flooding stress), then withholding water until the soil water content was 80% (‘CK’, field capacity control), 60% (‘LD’, mild drought stress, i.e., the drought stress was imposed lightly), 40% (‘MD’, moderate drought stress), and 20% (‘SD’, severe drought stress) of SWC, respectively. Another flooding stress is waterlogging (‘W2’, severe flooding stress), by placing each plant pot into a water tank and adjusting the upper water level to 3–4 cm below the potting soil surface [10]. All the six water treatments were replicated 3 times, lasting for 7 days (time1), 12 days (time2), and 17 days (time3), respectively. To ensure experimental homogeneity, 25 healthy plants with almost the same initial plant height were chosen for each treatment. The pot layout was completely randomized. During the water stress experiments, the soil water content maintenance was controlled by weighting the pots and adding the water loss every day. Rainfall was avoided by covering polyethylene film. 2.3. Determination of plant growth physiology parameters At the end of the water stress treatments, predawn leaf water potential was detected using a dew point microvoltmeter (Psypro, Wescor Inc., Logan, UT, USA) [11], and plant growth and morphological parameters (leaf area, plant height, stem diameter, root length, shoot/root/ total dry weight (shoot/root DW and DWs), root-shoot ratio) were measured and calculated [12]. The whole plant material, including shoot and root, were harvested and after oven-dried (60 °C, 96 h.), bioactive component contents were analyzed by HPLC method. The HPLC procedure was as follows: HPLC sample preparation: 1 g dried plant powder was dissolved in 20 mL HPLC grade methanol and then the solution was ultrasonic extracted for 60 min. After filtering and removing the filter residues, the filtrates were evaporated to dryness and then dissolved in methanol. Next the solution was transferred to a 10-mL volumetric flask, diluted with methanol to volume, and mixed. Finally, the solution was filtered through a 0.45 μm microporous filter membrane. HPLC analysis method: The chromatographic column was Dikma Diamonsil ODS-C18 (4.6 * 250 mm, 5 μm) (Dikma Technologies Inc., Beijing, China). The mobile phase was consisted of eluent A and eluent B. Eluent A was a mixture of methanol: acetonitrile solution (4:1, v/v)
while eluent B was 0.2% phosphate water. The detection wavelength was 250 nm. The injection volume was 10 μL. The flow rate was 0.8 mL min−1. The chromatographic column temperature was 25 °C. Under these conditions, the baseline separation of standard control from other components was well achieved, the resolution with the adjacent peaks was greater than 1.5, and the theoretical plate number was no less than 5000. Contents of AG, DDAG and AGs were expressed in two ways: AGC, DDAGC and their sum (AGCs) on percent (‘%’, converted from ‘mg/g dry weight’) basis; and the respective AGP, DDAGP, and AGPs on ‘per plant’ (‘mg/plant’) basis. 2.4. Statistical analysis Data management and statistical analysis were performed using IBM SPSS Statistics 24. The normality of distribution and homogeneity of variance were tested before analysis of variance (ANOVA). Repeated Measures ANOVA was used to analyze the time effects and the interactions between time and water treatments. Multivariate analysis of variance (MANOVA) was used to analyze the effects of water stress on plant physiology parameters in each treated time. Duncan's post-hoc analysis was used to compare the differences of means. Spearman rank correlation analysis was used to test the relationships between plant growth parameters and bioactive components. 3. Results 3.1. Effects of time and their interactions with water treatments Repeated Measures ANOVA was used to analyze the within-subjects effects, namely, time effects and their interactions with water treatments. First, Mauchly's test of sphericity (Table. A.1) was used to judge whether there were relations among the repeatedly measured data. If not (P ≤ 0.05), Greenhouse-Geisser corrected results or MANOVA should be taken next. Table 1 shows that different treated time had significant statistical effects on most measured parameters, except for leaf area, root length, root DW. Moreover, its interactive effects with water treatments were not significant except for leaf water potential, root length, root-shoot ratio, and AGCs. Based on these analysis and our actual results, in the following part, we focused on the multiple comparisons among the six water treatments in each treated time, rather than the pairwise comparisons of the repeatedly measured data in different treated time of each water treatment. 3.2. Effects of water stress on leaf water potential Changes of leaf water potential reflected the plant water status under soil water stress. When AP was water stressed for 7 days (time1), the leaf water potential of SD, MD, W1, and W2 were significantly different from CK and LD. But when the stress time prolonged (time2, time3), dramatic declines of leaf water potential were only appeared in SD, while the other leaf water potentials were not significantly differentiated (Fig. 1). This phenomenon showed that the soil moisture changes did not influence most of the leaf water levels when water stress time extended. It also reflected the well resilience of AP to external water changes after adaptation. 3.3. Effects of water stress on plant growth and morphological parameters Fig. 2 shows the changes of four morphological parameters under water stress. In each treated time, the water stressed leaf areas (Fig. 2A) and the stem diameter (Fig. 2B) were not significantly variated from CK, even if they were all lower than CK when treated the longest time (time3) in present study. And although the plant height (Fig. 2C)
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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was all reduced by MD and SD in every time, only the reductions of SD were significant while no marked differences were detected in other water treatments. The root length variations were different in the three times (Fig. 2D), with no significant changes in time1 and significant higher by W2 in time2 and by SD in time3. In a word, these morphological traits were only dramatically altered by the severe drought/ flooding stress and in prolonged treated time. The plant biomass accumulations were detected by observing changes of plant dry weight under water stressed conditions. Shoot DW was all declined in water stressed plants compared to CK in each treated time, with no statistical changes in time1 and significantly decreases by SD in time2 (30.64%) and time3 (30.53%) (Fig. 3A). Root DW was less changed than shoot DW under drought stress but significantly decreased by W2 in time2 (33.55%) and time3 (33.90%) (Fig. 3B). The total plant dry weight (DWs) was almost unaffected in time1 but significantly decreased by SD in time2 (27.70%) and time3 (25.35%), and the respective non-significant reductions of DWs by MD, W1 and W2 were 16.13%, 7.95%, 17.12% in time2, and 16.58%, 6.25%, 13.51% in time3 (Fig. 3C). These results also suggested severe drought/flooding stress significantly affected plant dry weight. The root-shoot ratio is often used to indicate changes of biomass allocation. Fig. 3D shows that the root-shoot ratio was statistically unchanged in time1 but significantly increased by SD in time2 (17.55%) and time3 (30.40%) and significantly decreased by W2 in time2 (25.88%) and time3 (32.03%). This result implied that AP adjusted its photoassimilate allocation pattern under relatively severe water stress conditions, by transporting more to the below-ground parts in severe drought stress while more to the above-ground parts in severe flooding stress.
3.4. Effects of water stress on therapeutic active components contents The contents of therapeutic active components in medicinal plants can be expressed on ‘%’ basis and on ‘per plant’ basis, although the first representation is commonly used in most studies, the latter is recommended by many scientists because it can specifically describe the changes of active components in individual plant and that's the interesting point. Fig. 4A–C shows the changes of AP's andrographolides contents on ‘%’ basis. AGC showed no statistical differences in time1 and little changes in water stressed plants compared to CK in time2 and time3 (Fig. 4A). DDAGC showed no statistical changes in time1 and time2 but significantly increased by SD in time3 (Fig. 4B). AGCs showed similar change patterns to DDAGC (Fig. 4C). In addition, although the changes were not always being significant, AGC, DDAGC and AGCs were all increased by SD and MD but decreased by W2 in all three times (Fig. 4A–C). In a word, drought stress led to the increase of andrographolides while flooding stress resulted in their decrease when the water stress duration time increased. Specifically, in time3, the increase of DDAGC by SD and MD compared to CK was 53.62%, 23.93% and the decrease by W1 and W2 was 12.31%, 27.28%. And the respective increase of AGCs by SD and MD was 44.02%, 24.35% and the decrease by W1 and W2 was 6.70%, 30.41%.
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Fig. 1. Changes of Andrographis paniculata (AP)’s leaf water potential under different water treatments. ‘SD’, ‘MD’, and ‘LD’ meant that the drought stresses were respectively imposed severely, moderately, and lightly. Their corresponding soil water contents were 20%, 40%, 60% of saturated soil water content (SWC). ‘CK’ was field capacity control, with the soil water content maintaining at around 80% of SWC. ‘W1’ and ‘W2’ were two flooding stresses. ‘W1’ meant the soil water content was saturated soil water content, i.e. 100% of SWC. ‘W2’ meant waterlogging stress, namely, each plant pot was placed into a water tank and the upper water level was adjusted to 3–4 cm below the soil surface. All six water treatments were lasted for 7 days (time1), 12 days (time2), and 17 days (time3), respectively. The same colored lowercase indicates differences of means among the six water treatments in each treated time by Duncan's post-hoc method. The significance level was 0.05.
Fig. 4D–F shows the changes of AP's andrographolides contents on ‘per plant’ basis. AGP showed no statistical differences among water stresses in all treated times (Fig. 4D). DDAGP showed no statistical changes in time1 and time2 but significantly increased by SD in time3 (Fig. 4E). AGPs showed similar change patterns to DDAGP (Fig. 4F). Similarly, in time3, the increase of DDAGP by SD and MD compared to CK was 25.39%, 2.88% and the decrease by W1 and W2 was 13.34%, 27.88%. And the respective increase of AGPs by SD and MD was 20.28%, 2.20% and the decrease by W1 and W2 was 2.71%, 28.20%. To sum up, when AP was water stressed for 17 days (time3), severe drought stress (SD) resulted in a 1.53/1.25 fold increase in DDAGC/ DDAGP and a 1.44/1.20 fold increase in AGCs/AGPs (with 25.35% total biomass loss), and moderate drought stress (MD) resulted in a 1.24/ 1.29 fold increase in DDAGC/DDAGP and a 1.24/1.22 fold increase in AGCs/AGPs (with 16.58% total biomass loss). 3.5. Relationships between plant growth parameters and andrographolides contents under water stressed conditions The plant growth parameters were relatively easy available and cheaper than the data of the andrographolides contents, so we explored their relationships with andrographolides contents under water stressed conditions, trying to find the ones which could represent the andrographolides variations (Table 2). Two plant growth parameters, leaf area and root length, were not significantly correlated to any andrographolide content. And no significant correlations were found between AGC and any growth parameter.
Table 1 Significance (P-value) of univariate tests of within-subjects effects. Source
Leaf water potential
Leaf area
Plant height
Stem diameter
Root length
Shoot DW
Root DW
DWs
Time Time ⁎ treatment
0.026 0.046⁎
0.141 0.141
0.000⁎⁎ 0.612
0.000⁎⁎ 0.655
0.136 0.000⁎⁎
0.000⁎⁎ 0.687
0.090 0.078
0.001⁎⁎ 0.609
Source Time Time ⁎ treatment
Root-shoot ratio 0.001⁎⁎ 0.008⁎⁎
AGC 0.004⁎⁎ 0.642
DDAGC 0.000⁎⁎ 0.572
AGCs 0.000⁎⁎ 0.008⁎⁎
AGP 0.034⁎ 0.887
DDAGP 0.003⁎⁎ 0.239
AGPs 0.001⁎⁎ 0.371
DW: dry weight (g); AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/plant’) basis.
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Fig. 2. Changes of AP's leaf area, stem diameter, plant height, and root length under different water treatments. Other legends are the same as in Fig. 1.
The correlation coefficients between other plant growth parameters with DDAGC/AGCs were very low (b0.30), even if they were significant. Lower correlation coefficients were also found in plant height and stem diameter with DDAGP/AGPs (b0.40). These lower correlations could be ignored because of not much use in practice. However, relatively higher positive correlation coefficients were found in shoot DW, root DW and DWs with AGP/DDAGP/AGPs, especially with DDAGP/AGPs (close to 0.50). These results demonstrated that a close relationship existed between plant dry weight and bioactive contents when expressed on ‘per plant’ basis.
143.7 mM salinity stress applied before AP flowering led to the maximum increases of AG and DDAG. In summary, our study suggested that water stress duration time should be considered when applying different degrees of drought/flooding to AP, and in another respect this also reflected the well adaptability of AP to water stress. Therefore, in the following part, to clarify the water stress effects on AP, we would discuss its responses in the longest treated time (time3), rather than the other two times (time1, time2).
4. Discussion
The dynamics of AP's leaf potential during water stress treatments implied its adapted processes (Fig. 1). In drought stress, the cellular dehydration causes osmotic stress, plant responds to accumulate osmoregulation substances (betaine, proline, soluble sugar, etc.), which decreases the value of leaf potential [13]. And accompanied with the increase of tissue water content, the value of leaf potential also increased. The responses of AP's leaf area, stem diameter, plant height and root length to different water stress treatments revealed that these plant growth and morphological characteristics were not significantly affected except by severe drought/flooding stress (Fig. 2A–D). This growth stability implied the good tolerance of AP to water stress. During the experimental period, AP was still in its fast growing stage, so the water stress could be compensated rapidly and serious damage could be avoided. But when in severe drought stress conditions, the scarcity of water finally changed AP's growth physiology. Besides preventing excessive water deficits by reducing plant water transpiration through stomatal closure, and absorbing much more water from underground by elongating root length, AP adjusted its photosynthetic assimilate distribution strategies and more assimilation were allocated to roots than the
4.1. Water stress effects related to the treated time In our study, repeated measures ANOVA showed that water stress treated time was statistically effective while time and treatment interactions were not, except for a few parameters (Table 1). The time effects of water stress were further proved by plant morphological, dry weight, and bioactive component changes (Figs. 2-4). For example, water stress decreased shoot DW and DWs compared to CK in every treated time, while these decreases were non-significant when treated with short time (7 days, time1) even by severe drought stress (SD), and became significant by SD when stress time prolonged (12 days, time2; 17 days, time3). In the case of bioactive contents, drought (SD, MD) induced increases of AGC, DDAGC, and AGCs were non-significant in time1 and time2, but the increases of DDAGC and AGCs by SD were significant in time3. Similar change patterns were found in DDAGP and AGPs as affected by SD. The needs of relatively longer stress time and higher stress intensity to trigger productive effects in AP were also revealed in salinity stress. For instance, Talei et al. [13] found a 30-day,
4.2. Water stress changed the biomass allocation of AP
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Fig. 3. Changes of AP's shoot DW (shoot dry weight), root DW (root dry weight), DWs (total dry weight), and root-shoot ratio under different water treatments. Other legends are the same as in Fig. 1.
above-ground. The significant increases of root-shoot ratio in the severe drought stress also indicated the allocation pattern changed compared to control. Consistent with this, AP's shoot, root, and total DW was also decreased variously when confronted with water stress (Fig. 3A– C). Farooq et al., [14] thought the plant growth reduction caused by drought stress was a result of impaired mitosis, cell elongation and expansion. To be noticed, waterlogging stress significantly decreased the root DW but not the shoot DW, indicating the influence of flooding stress was more severe on the below-ground rather than the above-ground. Oxygen-depletion is one of the most serious symptoms when plants meet with flooding, roots undergone anaerobic respirations are hard to survive. The smaller root biomass was also found in Islam's research [15]; their study proved that under waterlogging stress the metabolism and carbon budget was more restricted in root than shoot. 4.3. Drought stress enhanced the level of DDAG in AP Researchers found the proper harvest time for this annual herb AP is between the budding stage and early blooming stage, i.e., the transformation of vegetative stage to reproductive stage, which is about 100–120 days after seeding stage [6,16–18]. Studies also proved that during this time, both the dry matter accumulation and total andrographolides production were gradually reached the peak and maintained relative stability [17,18]. Here we applied water stress prior to the final harvest time to get the higher andrographolides production. Our results showed that drought stress (SD, MD) induced considerable increases of DDAG and AGs while flooding stress (W1, W2) resulted in their decreases in AP. Similar results were reported in water
stressed amur corktree (Phellodendron amurense), a pharmaceutical resource plant of traditional Chinese medicine cortex phellodendri [19]. Drought and waterlogging stress inhibited the growth of amur corktree, its height, diameter and biomass were significantly lower than those of the control, but drought (soil water potential was −80 – −60 KPa) increased the contents of three major alkaloids (berberine, jatrorrhizine, palmatine) while waterlogging stress (soil water potential was −20–0 KPa) decreased these alkaloids contents. In hawthorn (Crataegus laevigata and C. monogyna), it was found that water-deficit stress caused the increase of the desired polyphenolics levels while flooding stress led to the decrease of them [20]. We speculated that the main reason was the cell hypoxia environment caused by flooding stress, which restricted the biosynthesis of plant bioactive compounds. The phenomena of drought-stress induced bioactive secondary metabolites accumulation were also observed in other medicinal plants. For example, it was found that drought stress increased the major secondary metabolites, hyperforin concentration in St. John's wort (Hypericum perforatum) leaves [21], total indole alkaloid content in Catharanthus roseus shoots and roots [22,23], artemisinin in annual wormwood (Artemisia annua L., Asteraceae) leaves and whole plants [24], etc. Reasons about this increase were discussed a lot. In summary, this drought-stress induced bioactive components accumulation in medicinal plants is thought to be a response and adaptation to environmental stress and there are several hypotheses explaining it. Kleinwächter and Selmar [25–27] had summarized the phenomena of drought stress-related concentration increases of natural products and proposed the following theory: Reduction of CO2 fixation from water shortage results in the oversupply of reduction equivalents (NADPH + H+), and thus the metabolic processes are shifted to
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Fig. 4. Changes of AP's andrographolides contents on ‘%’ and ‘per plant’ basis under different water treatments. AG: andrographolide; DDAG: 14-deoxy-11,12-didehydroandrographolide; AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/ plant’) basis. Other legends are the same as in Fig. 1.
Table 2 Relationships between plant growth parameters and andrographolides contents under water stressed conditions.
2
Leaf area (cm ) Plant height (cm) Stem diameter (cm) Root length (cm) Shoot DW (g) Root DW (g) DWs (g) Root-shoot ratio
AGC (%)
DDAGC (%)
AGCs (%)
AGP (mg/plant)
DDAGP (mg/plant)
AGPs (mg/plant)
−0.264 −0.102 −0.156 0.033 −0.102 0.042 −0.066 0.178
−0.130 −0.249⁎ −0.186 0.162 −0.285⁎⁎
−0.230 −0.236⁎ −0.214⁎
−0.253 0.161 0.189 −0.032 0.368⁎⁎ 0.423⁎⁎ 0.408⁎⁎
−0.139 0.214⁎ 0.366⁎⁎
−0.219 0.231⁎ 0.360⁎⁎
0.000 0.481⁎⁎ 0.499⁎⁎ 0.515⁎⁎
−0.005 0.504⁎⁎ 0.509⁎⁎ 0.538⁎⁎
0.050
0.014
−0.001
−0.134 −0.259⁎ 0.228⁎
0.157 −0.280⁎⁎ −0.132 −0.253⁎ 0.215⁎
DW: dry weight (g); AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/plant’) basis. ⁎ Correlation is significant at the 0.05 level (2-tailed). ⁎⁎ Correlation is significant at the 0.01 level (2-tailed).
Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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stress [36].These highly heritable traits could be used for yielding drought tolerant AP with high bioactive contents as well. By the way, several studies suggested that the problem of drought-stress caused growth reduction could be alleviated by adding fertilization [23,45,48,49] or supplying elevated carbon dioxide during the stress [40,50]. This needs further research. Contrary to other researches, in our study, the amount of DDAG in AP was about at least 4-fold higher than AG. This may be explained by the instability of AG. Studies showed that fresh AP had higher AG content but AG was easily converted into DDAG during storage [7]. The other reason was our material handling method before the HPLC determination of the two diterpenoids. We dried AP in an oven at 60 °C for 96 h, but other researches oven-dried the material at relatively low temperature (such as 35–45 °C or shade dried) [51]. Studies also showed that thermal treatment made it easier for AG to transform into DDAG [52]. We also found that shoot DW, root DW and DWs were relatively highly correlated with bioactive contents when expressed on ‘per plant’ basis (Table 2). The higher plant dry weight meant the higher andrographolides contents in AP. This gave us good indicators representing medicinal quality when applying water treatments. The plant weight could be weighed rapidly at low cost, and it could be used for prompt and large-scale selection for high medicinal quality AP after stress.
pathways that consume the surplus reduction equivalents, such as the secondary metabolite biosynthesis of phenols, alkaloids and isoprenoids [25–31]. Their theories were kind of similar to other reducing equivalents accumulation circumstances such as dissipation system of excess light energy. For example, concomitant with chloroplast overreduction by excess light, plants re-oxidizes reduction equivalents through antioxidant system, photorespiration, and violaxanthine cycle to reduce damages [32,33]. The second type of hypothesis believes that drought stress causes the production of reactive oxygen species (ROS), besides antioxidant system, plant develops the secondary metabolites accumulation defense mechanism to protect plants from oxidative stress [21–23,34–36]. However, some scientists consider drought-stress induced ROS as a trigger of signal transduction pathway that promotes the gene expressions of bioactive components in medicinal plants [37,38]. These hypotheses were the most easily accepted interpretations and proved by many researches. The third type of hypothesis relates to the carbon/nutrient balance and growth differentiation balance hypotheses which predict a trade-off between plant growth and defense (though this kind of hypothesis lays emphasis on plant chemical defenses against pathogens and herbivores). When plant growth and development are inhibited by water deficit, the carbon fixed during photosynthesis tends to be allocated to the defense formation of secondary compounds [34,39–42]. However, these hypotheses were often specifically used when plants confronted with biotic stress. In short, reasonable drought stress (proper applying time, duration and intensity) seems to be beneficial for the biosynthesis and accumulation of bioactive secondary metabolites in medicinal plants [24,43–45]. Nonetheless, our results also demonstrated the inevitable biomass loss of AP accompanied with the severe and moderate drought induced increase of DDAG/AGs. And part of this andrographolides increase attributed to the reduction of biomass, as researched by Talei et al. [13] when discussing the salinity induced increase of AG. Therefore, the balance between stress-induced bioactive component enhancement and biomass reduction should be evaluated according to the different harvesting purposes in modern agronomical cultivation of AP [13,23,46]. Furthermore, as revealed by Taeil et al. [47], AG and DDAG exhibited moderate and high intraspecific hybridization heterosis and their high heritability was beneficial for producing higher andrographolide varieties. AG (and proline, net photosynthetic rate) also showed high broad-sense heritability in AP encountered salinity
5. Conclusion Water stress effects on AP related to the treated time, and most of the plant growth and andrographolides changes were significantly affected by severe drought/flooding stress in prolonged treated time. Severe and moderate drought induced increase of DDAG/AGs with inevitable biomass loss. Plant dry weight were relatively highly correlated with bioactive contents when expressed on ‘per plant’ basis. Declaration of Competing Interest None. Acknowledgements This work was supported by China's National Natural Science Foundation [31200305] and Special program for guidig local science and technology development by the central government [Guangxi ZY1949023].
Appendix A. Appendix Table A.1 Mauchly's test of sphericitya. Within subjects effect
Mauchly's W
Approx. chi-square
df
Sig.
Time
0.396 0.979 0.630 0.993 0.925 0.767 0.863 0.774 0.782 0.745 0.910 0.886 0.711 0.831 0.807
10.182 0.236 13.404 0.218 2.269 7.674 4.273 7.427 7.136 5.894 1.880 2.416 5.798 3.140 3.645
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
0.006 0.889 0.001 0.897 0.322 0.022 0.118 0.024 0.028 0.053 0.391 0.299 0.055 0.208 0.162
Leaf water potential Leaf area Plant height Stem diameter Root length Shoot DW Root DW DWs Root-shoot ratio AGC DDAGC AGCs AGP DDAGP AGPs
Epsilonb Greenhouse-Geisser
Huynh-Feldt
Lower-bound
0.624 0.979 0.730 0.993 0.930 0.811 0.880 0.816 0.821 0.797 0.918 0.898 0.776 0.856 0.838
0.951 1.000 0.886 1.000 1.000 0.994 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000
0.396 0.979 0.630 0.993 0.925 0.767 0.863 0.774 0.782 0.745 0.910 0.886 0.500 0.500 0.500
Tests the null hypothesis that the error covariance matrix of the orthonormalized transformed dependent variables is proportional to an identity matrix. DW: dry weight (g); AGC, DDAGC, and AGCs: AG, DDAG and AGs expressed on percent (‘%’, converted from ‘mg/g dry weight’) basis; AGP, DDAGP, and AGPs: AG, DDAG and AGs expressed on ‘per plant’ (‘mg/plant’) basis. a Design: Intercept + treatment; Within Subjects Design: time. b May be used to adjust the degrees of freedom for the averaged tests of significance. Corrected tests are displayed in the Tests of Within-Subjects Effects table.
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Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003
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Please cite this article as: X. Chen, Y. Xie, K. Wei, et al., Drought stress enhanced andrographolides contents in Andrographis paniculata, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2020.02.003