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Article

Manipulating the Difference between the Day and Night Temperatures Can Enhance the Quality of Astragalus membranaceus and Codonopsis lanceolata Plug Seedlings

by
Ya Liu
1,
Xiuxia Ren
1 and
Byoung Ryong Jeong
1,2,3,*
1
Department of Horticulture, Division of Applied Life Science (BK21 Plus Program), Graduate School of Gyeongsang National University, Jinju 52828, Korea
2
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Korea
3
Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea
*
Author to whom correspondence should be addressed.
Agronomy 2019, 9(10), 654; https://doi.org/10.3390/agronomy9100654
Submission received: 9 September 2019 / Revised: 15 October 2019 / Accepted: 17 October 2019 / Published: 18 October 2019
Browse Figures
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Abstract

:
Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f. are two famous medical species in Korea, China, and Japan, mainly used for treating diseases including cancer, obesity, and inflammation. Manipulation of the difference between the day and night temperatures (DIF) is an efficient horticultural practice to regulate the growth and development of vegetables in a glasshouse. However, little research has focused on how the DIF influences the plug seedling quality of medicinal plants. In this study, uniform plug seedlings were cultivated in three environmentally controlled chambers under an average daily temperature of 20 °C with negative (−10 °C), zero, or positive (+10 °C) DIFs, and the same relative humidity (75%), photoperiod (12 h), and light intensity (150 μmol·m−2·s−1 photosynthetic photon flux density with white LEDs). The results showed that the DIF had a noticeable effect on the growth, development, and morphology of A. membranaceus and C. lanceolata plug seedlings. The positive DIF (+10 °C) significantly increased the biomass (shoot, root, and leaf), stem diameter, and Dickson’s quality index, indicating an enhanced plug seedling quality. Moreover, the contents of primary and secondary metabolites, including soluble sugar, starch, total phenols and flavonoids, were higher with higher DIFs, where the maximum values were found at 0 °C or +10 °C DIF. Furthermore, the increases in the chlorophyll content and stomatal conductance were obtained in a positive DIF, indicating that a positive DIF was favorable to photosynthesis. An analysis of the gene expression showed that a positive DIF (+10 °C) up-regulated the expression of photosynthetic genes, including GBSS, RBCL, and FDX. In conclusion, the results of this study recommend a positive DIF (+10 °C) for enhancing the quality of A. membranaceus and C. lanceolata plug seedlings.

1. Introduction

Astragalus membranaceus Bunge and Codonopsis lanceolata Benth. et Hook. f. are two medicinal species that are widely distributed in East Asian countries, particularly Korea, China, and Japan. These two species have a long history of being used as medicine to treat many diseases including cancer, obesity, and inflammation in Asian countries [1,2]. In addition to having its medicinal values, C. lanceolata has been consumed as a high-class vegetable in Asian countries, especially in ancient Korea [3,4]. In recent years, more and more researchers have shown an increased interest in these two species, since more and more phytochemicals have been identified and applied in medicine for treating diseases [5,6]. However, most of those works focused on how to separate, extract, and characterize the medicinal compounds in A. membranaceus and C. lanceolata. Not much information is available on the cultivation of those two species in a glasshouse by manipulating the temperature regime.
Temperature is a dominant and controllable component in protected plant production, which strongly influences the crop growth, yield, and quality [7,8,9]. Manipulation of the difference between the day and night temperature (DIF) is an efficient horticultural practice to control plant morphology [10,11,12]. DIF is defined as the day temperature minus the night temperature, which means that if the day temperature is higher than the night temperature, it results in a positive DIF, and the opposite for a negative DIF [13]. Previous studies have reported that negative DIFs could be used for inhibiting stem elongation and increasing yield [11,14,15]. However, the opposite results have also been reported. For instance, Lund, et al. [16] found that a highly positive DIF does not seem to increase the elongation growth of Hibiscus rosa-sinensis L. Similarly, Lyngved, et al. [17] reported that the optimal values for biomass growth of Cyclamen persicum Mill. were under a positive DIF (+10 °C). It was reported that a negative DIF inhibited the growth and development of young cucumber plants [18]. Moreover, the mechanism of how the DIF affects the growth and morphology of plants is not well known [19]. Studies on the effects of the DIF on medicinal plants are limited especially in A. membranaceus and C. lanceolata plug seedlings.
Photosynthesis is the most important photochemical reaction that produces food for all organisms, using sunlight, water, and CO2. Many genes and proteins are involved in this process. In the photosynthetic chain, ferredoxin (FDX) codes one kind of iron–sulfur proteins, named ferredoxin [20]. In the non-cyclic photophosphorylation, protein is the last electron acceptor and transports them to NADP+ [21]. Ribulose bisphosphate carboxylase large chain (RBCL) is the key gene for the CO2 fixture in the CO2 assimilation [22]. Ribulose bisphosphate carboxylase (RuBisCO) catalyzes the reaction of ribulose bisphosphate and CO2 at the initial step of photosynthesis, which determines the photosynthetic rate [23]. Granule-bound starch synthase (GBSS) is an obbligato gene for the synthesis of amylose in starch, which plays a role in building the final structure of amylopectin [24,25,26]. Previous studies have reported the responses of those gene expressions to the temperature [27,28,29]. However, few studies have focused on how the DIF affects the expression of those genes in medical plant plug seedlings.
The objectives of this study were to demonstrate how the DIF influences the plug seedling quality of A. membranaceus and C. lanceolata and to identify an ideal temperature regime for production of high quality plug seedlings. Uniform plug seedlings were cultured under positive (10 °C), zero, and negative (−10 °C) DIFs for four weeks. The growth and morphological responses of plug seedlings to the different DIFs were investigated. The contents of the primary and secondary metabolites were also determined, such as soluble sugar, starch, total phenols and flavonoids. Additionally, the chlorophyll content and stomatal conductance were evaluated. The expression of photosynthesis-related genes including FDX, RBCL, and GBSS were assayed. The data in this study could provide a theoretical and practical basis for enhancing the quality of plug seedlings by manipulating the DIF, and useful information for growers and managers to regulate other medicinal plants.

2. Materials and Methods

2.1. Plant Materials and Treatments

Seeds of A. membranaceus and C. lanceolata were sown in 200-cell plug trays filled with the Bio Medium (FarmHannong Co. Ltd., Seoul, South Korea). The components of Bio Medium are 20% coir, 60% peat moss, and 20% pearlite. After germination, uniform plug seedlings were selected and cultivated in three different environmentally controlled chambers. The average daily temperature in the chambers was 20 °C, with negative (−10 °C), zero, and positive (+10 °C) DIFs: the day/night temperatures were 15/25 °C, 20/20 °C, and 25/15 °C, respectively. Each treatment was set up with the same relative humidity (75%), photoperiod (12 h), and light intensity (150 μmol·m−2·s−1 photosynthetic photon flux density with white LEDs). Before the experiment, the light intensity was adjusted and calibrated using a photo-radiometer (HD2102.1, Delta OHM, Padova, Italy). The experimental design was completely random design with three replications. Each replication included 20 plugs seedlings (total 60 seedlings per treatment). After four weeks of cultivation, some plug seedlings were harvested for measurements of the growth parameters and others were frozen immediately in liquid nitrogen for further analysis. The Dickson’s quality index was calculated according to a previous formula [30], which is
Dickson’s quality index = Total DW/((shoot length/stem diameter)+(shoot DW/root DW))
where “DW” represents the dry weight.

2.2. Contents of Soluble Sugar and Starch

The contents of soluble sugar and starch were determined by the anthrone colorimetric method [31]. In brief, leaf samples (0.2 g) were ground and then extracted in distilled water (14 mL) for 30 min at 100 °C. After a 15-min centrifugation at 3000 rpm, the supernatant was transferred into a new tube for assay of soluble sugar. For measurement of starch, the residue was re-extracted in distilled water mixed with perchloric acid (2 mL, 52%).
The supernatant (0.5 mL) was added to distilled water (1.9 mL) combined with anthrone (0.5 mL, 2%), and concentrated sulfuric acid (5 mL, 98%), followed by 15 min of incubation at 100 °C. The absorbance at 630 nm and 485 nm was recorded using a UV-spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). The calibration curves of soluble sugar and starch were made with standard solutions.

2.3. Contents of Total Phenols and Flavonoids

Total phenols and flavonoids in leaves were extracted with 80% methanol. The contents of total phenols and flavonoids were measured according to previous methods described by Manivannan et al. [32].

2.4. Hydrogen Peroxide Content

To determine the hydrogen peroxide content, leaves (100 mg) were ground and mixed with a TCA extracting solution (0.1%, 1.0 mL). The mixture was centrifuged for 15 min at 12,000 rpm, and then the supernatant was collected. The supernatant (0.3 mL), sodium phosphate buffer (50 mM, 0.5 mL), and potassium iodide (1 M, 0.5 mL) were mixed and then incubated for 30 min in the dark. The absorbance of the mixture was measured and recorded at 395 nm using a UV spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK) [31].

2.5. Assessments of the Chlorophyll Content and Stomatal Conductance

The chlorophyll content was measured by using the Plus Chlorophyll Meter (SPAD 502, Konica Minolta Sensing Inc., Osaka, Japan). The stomatal conductance was assessed at 10 am using the Decagon Leaf Porometer SC-1 (Decagon Device Inc., Pullman, WA, USA).

2.6. Gene Expression Analysis

Leaves of A. membranaceus and C. lanceolata (mixed sample, three biological replications) were ground into powder in liquid nitrogen and homogenized in a lysis buffer under a RNase-free condition. The total RNA was extracted using a commercial extraction kit (iNtRON Biotechnology, Seoul, Republic of Korea), and cDNA was converted from total RNA using the GoScript Reverse Transcription System (Promega, Madison, WI, USA). The gene expression was determined by employing a Rotor-Gene Q detection system (Qiagen, Hilden, Germany), and the value was calculated by using the 2−∆∆Ct method [33]. All primers were designed using Premier 5.0 (Premier Biosoft Inc., Palo Alto, CA, USA) and the primer sequences are presented in Table 1.

2.7. Data Collection and Analysis

The data were collected with three individual biological repeats, and are presented as the mean ± standard error. A one-way analysis of variance (one-way ANOVA) was performed to estimate the differences among treatments, followed by a Duncan multiple range test (p < 0.05) using SPSS (Statistical Package for the Social Sciences, version 21; International Business Machine Corp., Armonk, NY, USA). All figures in this study were prepared using the OriginPro software (version 9.0; OriginLab Corp, Northampton, MA, USA.)

3. Results

3.1. Growth, Development, and Morphology

In this study, the DIF had a significant influence on the growth, development, and morphology of A. membranaceus and C. lanceolata plug seedlings (Figure 1, Table 2 and Table 3). As shown in Figure 1, a morphological distinction of plug seedlings was observed with the increase from −10 °C DIF to +10 °C DIF. At the negative DIF (−10 °C), plug seedlings were inferior with a low number of roots, curved shoots and few leaves. In comparison, plug seedlings were stronger and more compact with straight shoots and well-developed roots at the positive DIF (+10 °C). As shown in Table 2, the dry weights of the shoot and root in A. membranaceus plug seedlings were 34.8 ± 2.7 and 4.3 ± 0.4 mg at +10 °C DIF, significantly higher than that at the other two DIFs. Similarly, the maximum stem diameter of 0.90 ± 0.02 mm was observed at +10 °C DIF. Importantly, the +10 °C DIF also increased the Dickson’s quality index (DQI), which was remarkably greater than that in −10 °C and 0 °C DIF. Similarly in C. lanceolata plug seedlings, the +10 °C DIF remarkably increased the growth parameters, such as shoot and root dry weights (98.9 ± 10.3 and 20.5 ± 2.2 mg), leaf area (8.5 ± 0.5 cm−2), stem diameter (2.06 ± 0.07 mm), and DQI (14.2 ± 0.8 × 10−4), in comparison to those at the −10 °C DIF (Table 3).

3.2. Contents of Soluble Sugar and Starch

As shown in Figure 2A,B, increases in the soluble sugar and starch contents were found in A. membranaceus and C. lanceolata seedlings with higher DIFs. The maximum soluble sugar and starch contents were found at +10 °C DIF, where they were 10.8 and 24.6 mg·g−1 FW in A. membranaceus, and 10.9 and 25.6 mg·g−1 FW in C. lanceolata, respectively—significantly higher than those in −10 °C DIF.

3.3. Contents of Total Phenols and Flavonoids

In A. membranaceus plug seedlings, the content of total phenols gradually increased with the increase in the DIF, whereas the content of total flavonoids went up first and then declined, as shown in Figure 2C. The highest contents of total phenols and flavonoids were 0.65 ± 0.1 mg·g−1 FW (10 °C DIF) and 0.34 ± 0.1 mg·g−1 FW (0 °C DIF), respectively. In C. lanceolata, the DIF had the same effects on the contents of total phenols and flavonoids. The maximum values of total phenols and flavonoids were 2.15 ± 0.03 and 0.78 ± 0.02 mg·g−1 FW in 0 °C DIF, followed by those in +10 °C DIF (Figure 2D).

3.4. Hydrogen Peroxide Content

As shown in Figure 3, the H2O2 content in both species decreased significantly with the increase in DIF. The lowest H2O2 content was 0.119 ± 0.001 mg·g−1 for A. membranaceus and 0.251 ± 0.004 mg·g−1 FW for C. lanceolata, both under a positive DIF.

3.5. Chlorophyll Content and Stomatal Conductance

The chlorophyll content in A. membranaceus and C. lanceolata plug seedlings was affected by the DIF. As shown in Figure 4A,B, a positive DIF resulted in much higher chlorophyll content than zero or negative DIF did in both species. The highest value was 33.9 ± 0.4 SPAD for A. membranaceus and 33.2 ± 0.6 SPAD for C. lanceolata. The data showed that the stomatal conductance increased with the increase in DIF, and the maximum was 594.9 ± 7.8 and 611.2 ± 71.1 mmol·m−2·s−1 in A. membranaceus and C. lanceolata plug seedlings, respectively (Figure 4C,D).

3.6. Gene Expression Analysis

The DIF had a significant influence on the expression of photosynthetic genes in both A. membranaceus and C. lanceolata (Figure 5). In A. membranaceus, expressions of the GBSS and RBCL genes were greatly upregulated in the 10 °C DIF than in the two other treatments, which were 5.7- and 1.8-fold higher than that in −10 °C DIF, respectively (Figure 5A). In C. lanceolata, the expressions of RBCL and FDX were significant higher in 10 °C DIF than those in −10 °C and 0 °C DIF, about 2.2 and 1.6 times larger than that in 0 °C DIF (Figure 5B).

4. Discussion

This study shows that DIFs significantly affect the growth of A. membranaceus and C. lanceolate plug seedlings. Morphologically, compact plug seedlings with straight shoots and well-developed roots were observed when grown in a positive DIF (+10 °C). The data also proved that +10 °C DIF upgraded the quality of plug seedlings with significantly increased biomass (shoot and root) and stem diameter. Importantly, the DQI was greater in seedlings grown with the +10 °C DIF than those in the other two treatments, implying the ability of the +10 °C DIF to yield high-quality plug seedlings. The biomass, stem diameter, and DQI are important parameters used to evaluate the quality of seedlings [34,35,36]. These results were in accordance with previous studies in tomato [19,37] and sweet pepper [38]. However, Bachman and McMahon [39] found that a negative DIF increased the stem diameter in petunia plants which differed from results in this study. The probable reason is the difference in the photoperiod. In their study, an 18-h photoperiod was employed—6 h longer than ours—which meant a lower consumption of carbohydrates during the night. Another reason may be the difference in species, which may exhibit different responses to different temperature regimes.
The DIF had a remarkable effect on the primary and secondary metabolism in both A. membranaceus and C. lanceolata. The highest contents of soluble sugar and starch observed at the 10 °C DIF, suggests that a positive DIF significantly promotes the accumulation of carbohydrates. As the main photosynthetic products, soluble sugar and starch were synthesized in leaves and then transported to accumulate where they were used for consumption in new tissues and organs. Hence, sufficient soluble sugar and starch produced by photosynthesis are of importance for the growth and development of plug seedlings. The positive DIF (10 °C) increased the contents of soluble sugar and starch, which benefited the growth and development of plug seedlings. Similar results were reported in tomato [19] and cucumber [7]. Moreover, William, et al. [40] reported that negative DIF regimes decreased the total soluble carbohydrates by 39% and 46% in the leaf and stem, respectively, compared to those in positive DIF regimes. The reason is the higher day temperature increases the activities of photosynthetic enzymes, including RBCL and FDX, while a lower night temperature inhibits the activities of respiration-related enzymes [41,42,43]. The data also proved that secondary metabolites were influenced by the DIF. Compared to 0 °C or +10 °C DIF, the −10 °C DIF inhibited the accumulation of total phenols and flavonoids. As photosynthetic products, carbohydrates are important substances for synthesizing secondary metabolites [44,45]. A lower concentration of soluble sugar and starch in −10 °C DIF is a negative factor for biosynthesis and accumulation of total phenols and flavonoids. As non-enzymatic antioxidants in nature, total phenols and flavonoids have the capacity to scavenge reactive oxygen species and maintain the homeostasis in plants [46]. The higher contents of total phenols and flavonoids observed in 0 °C or +10 °C DIF were more helpful to plug seedlings in coping with abiotic stresses [47], compared to the lower concentrations in −10 °C DIF. As important secondary metabolites, the higher contents of total phenols and flavonoids also suggested a higher medicinal value to humans [48].
Photosynthesis is a fundamental and important physiological process, greatly influencing the quality and yield of crops. This process is regulated by a series of genes, and affected by the day and night temperatures. In this study, an up-regulation of expressions of GBSS, RBCL, and FDX was found in the 10 °C DIF, compared to those in the −10 °C and 0 °C DIFs (Figure 5). The FDX gene codes the ferredoxin protein and is an important gene in the electron transport chain in photosynthesis. RBCL plays a key role in the CO2 fixture in dark and influences the photosynthetic rate. GBSS codes the granule-bound starch synthase and determines the presence of amylose in reserve starches [49]. A high expression of those genes in the 10 °C DIF suggested that a positive DIF had a beneficial effect on photosynthesis and carbohydrate accumulation. This partly explains the higher contents of soluble sugar and starch in plug seedlings grown with the 10 °C DIF. Moreover, the H2O2 content was significantly lower in the positive DIF than in the negative and 0 °C DIF. As one kind of reactive oxygen species, H2O2 is generated when plants fall into an unfavorable condition with abiotic or biotic stresses. Consequently, a suppressive expression of photosynthetic genes and degradation of proteins occur since H2O2 destroys the structure of nucleic acids and proteins and inhibits their function [50]. The data implies that the 10 °C DIF exposed the plug seedlings to a lower level of stresses, contributing to the higher expression of photosynthetic genes, such as RBCL, FDX, and GBSS. This partly explains the high quality of plug seedlings grown with the 10 °C DIF, compared to that with the other two treatments.
In photosynthesis, chlorophyll is a core component of the light-harvesting complex, playing a key role in capturing and transferring photons to the reaction center of the photosystem in the primary reaction. The data showed that the 10 °C DIF increased the chlorophyll content more than the −10 °C and 0 °C DIF did in both species, implying that a positive DIF enhanced photosynthesis by elevating chlorophyll contents. The results coincided with those of previous studies. Xiao, et al. [51] found that chlorophyll a and chlorophyll b contents in Solanum lycopersicum L. increased gradually with increasing DIF. Yuan [52] also found that chlorophyll a and b contents were significantly elevated under a positive DIF, and were lower with a negative DIF. Similarly, Bachman and McMahon [39] reported that chlorophyll a content was lower in a negative DIF (−6 °C) compared to the control. Additionally, Agrawal, et al. [53] found that the photosynthetic rate and chlorophyll content were lower at a negative DIF (−10 °C) than at a positive DIF (10 °C).
Stomata is the main channel for the air exchange between plants and the ambient conditions [54]. Carbon dioxide goes into the cell through the stomata and takes part in photosynthesis. Thus, stomatal conductance is an indicator of the stomata opening, which greatly influences the photosynthetic rate by controlling the carbon dioxide concentration in cells. The maximum stomatal conductance was observed at 10 °C DIF, suggesting that a positive DIF was beneficial for photosynthesis in A. membranaceus and C. lanceolata by promoting the stomata opening and carbon dioxide exchange. This was in accordance with the increased expression of the RBCL, FDX, and GBSS genes, and the chlorophyll content. These results are supported by previous studies. For instance, Xiao et al. [51] also reported that a high or low DIF decreased the stomatal conductance and the maximum was observed in 6 °C DIF at a daily mean temperature of either 18 °C or 25 °C. Yuan [52] showed that the stomatal conductance in Solanum lycopersicum significantly increased under a positive DIF, and decreased with a negative DIF. Moreover, the maximum stomatal conductance observed with the positive DIF (10 °C) was also in agreement with the highest content of carbohydrates, since photosynthesis was enhanced by an elevated stomatal conductance [55,56,57].

5. Conclusions

As an important horticultural practice, the DIF had a great influence on the growth, yield, and quality of plug seedlings. In this study, the effects of the DIF on the growth, morphology, and physiology of medicinal plant plug seedlings were demonstrated. The data showed that a positive DIF (+10 °C) had enhanced the quality of A. membranaceus and C. lanceolata plug seedlings by increasing the biomass (shoot, root, and leaf), stem diameter, and Dickson’s quality index. Additionally, the contents of soluble sugar, starch, total phenols and flavonoids were significantly enhanced with an increased DIF and their respective maximum values were found in elevated DIFs (0 °C or +10 °C). Furthermore, an increase in the chlorophyll content and stomatal conductance was obtained at +10 °C DIF, indicating that the positive DIF was favorable to photosynthesis. An analysis of the gene expression showed that photosynthesis-related genes, such as GBSS, RBCL, and FDX, were up-regulated by the positive DIF (+10 °C). In conclusion, the results of this study suggest that a positive DIF (10 °C) is a suitable method for enhancing the quality of A. membranaceus and C. lanceolata plug seedlings.

Author Contributions

Conceptualization, Y.L., X.R. and B.R.J.; funding acquisition, B.R.J.; investigation, Y.L. and X.R.; project administration, X.R. and B.R.J.; supervision, B.R.J.; writing—original draft, Y.L.; writing—review and editing, Y.L., X.R. and B.R.J.

Funding

This research was funded by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (Project No. 116057-03). Y.L. and X.R. were supported by a scholarship from the BK21 Plus Program, Ministry of Education, Republic of Korea.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Morphological characteristics of A. membranaceus (A) and C. lanceolata (B) plug seedlings affected by the difference between the day and night temperatures (DIF).
Figure 1. Morphological characteristics of A. membranaceus (A) and C. lanceolata (B) plug seedlings affected by the difference between the day and night temperatures (DIF).
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Figure 2. Contents of soluble sugar, starch, total phenols, and flavonoids in A. membranaceus (A,C) and C. lanceolata (B,D) plug seedlings affected by the DIF. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05.
Figure 2. Contents of soluble sugar, starch, total phenols, and flavonoids in A. membranaceus (A,C) and C. lanceolata (B,D) plug seedlings affected by the DIF. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05.
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Figure 3. Hydrogen peroxide content in A. membranaceus (A) and C. lanceolata (B) plug seedlings affected by the DIF. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
Figure 3. Hydrogen peroxide content in A. membranaceus (A) and C. lanceolata (B) plug seedlings affected by the DIF. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
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Figure 4. The effects of the DIF on the chlorophyll content and stomatal conductance in A. membranaceus (A,C) and C. lanceolata (B,D) plug seedlings. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
Figure 4. The effects of the DIF on the chlorophyll content and stomatal conductance in A. membranaceus (A,C) and C. lanceolata (B,D) plug seedlings. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
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Figure 5. The effects of the DIF on the gene expression in A. membranaceus (A) and C. lanceolata (B) plug seedlings. GBSS, granule-bound starch synthase; RBCL, ribulose bisphosphate carboxylase large chain; and FDX, ferredoxin. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
Figure 5. The effects of the DIF on the gene expression in A. membranaceus (A) and C. lanceolata (B) plug seedlings. GBSS, granule-bound starch synthase; RBCL, ribulose bisphosphate carboxylase large chain; and FDX, ferredoxin. Different letters (a, b, and c) indicate significant differences among treatments by Duncan’s multiple range test at a 0.05 level.
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Table
Table 1. The primer sequence for gene expression assay using quantitative real-time polymerase chain reaction.
Table 1. The primer sequence for gene expression assay using quantitative real-time polymerase chain reaction.
GeneForward PrimerReverse Primer
18S5′-CTCAACCATAAA CGATGCCGACC-3′5′-AGTTTCAGCCTTGCGACCATACTCC-3′
AmGBSS5′-ATAACATAGCGTATCAGGG-3′5′-CTCGGTCAGATTCTAACACT-3′
AmRBCL5′-TGGCTGTTCCTATCGTCA-3′5′-AAGTAATCTCCCTTTCTCCT-3′
β-Actin5′-CGAGAAGAGCTACGAGCTACCCGATGG-3′5′-CTCGGTGCTAGGGCAGTGATCTCTTTGCT-3′
ClFDX5′-CTTCGGCGTTTCTTCGT-3′5′-CTGCCAAACCCTTGATAACT-3′
ClRBCL5′-GCTTACCCATTAGACCTTT-3′5′-GGGACGACCATACTTGTT-3′
Am, Astragalus membranaceus; GBSS, granule-bound starch synthase; RBCL, ribulose bisphosphate carboxylase large chain; Cl, Codonopsis lanceolata; and FDX, ferredoxin. 18S and β-Actin were used as the housekeeping gene in A. membranaceus and C. lanceolata, respectively.
Table
Table 2. The effects of the day and night temperatures (DIF) on the growth parameters of A. membranaceus plug seedlings.
Table 2. The effects of the day and night temperatures (DIF) on the growth parameters of A. membranaceus plug seedlings.
DIF z (°C)Length (cm)Dry Weight (mg)Leaf Area (cm−2)No. of LeavesStem Diameter (mm)Root DW: Shoot DW RatioShoot Dry Weight Per Shoot Length (g·m−1)Root Dry Weight Per Root Length (g·m−1)Dickson’s Quality Index (×10−4)
ShootRootShootRootLeaf
−109.2 ± 0.3 b y3.3 ± 0.218.9 ± 1.4 c2.0 ± 0.1 c4.2 ± 0.3 c3.5 ± 0.3 c3.0 ± 0.0 c0.62 ± 0.03 c0.10 ± 0.010.21 ± 0.01 b0.06 ± 0.00 b1.3 ± 0.1 c
08.8 ± 0.3 b4.3 ± 0.228.0 ± 0.5 b2.8 ± 0.3 b6.4 ± 0.4 b4.6 ± 0.3 b3.5 ± 0.2 b0.78 ± 0.03 b0.10 ± 0.010.32 ± 0.01 a0.07 ± 0.01 b2.5 ± 0.2 b
1010.2 ± 0.2 a4.4 ± 0.534.8 ± 2.7 a4.3 ± 0.4 a7.5 ± 0.4 a6.2 ± 0.3 a3.8 ± 0.2 a0.90 ± 0.02 a0.13 ± 0.020.34 ± 0.02 a0.10 ± 0.02 a3.2 ± 0.2 a
F-test**NS*****************NS*******
z DIF, difference between day and night temperatures. y Different letters indicate significant separation within columns by Duncan’s multiple range test at a 0.05 level. NS, *, **, and ***, represent no significant difference, significant difference at p = 0.05, 0.01, or 0.001, respectively.
Table
Table 3. The effects of the DIF on the growth parameters of C. lanceolata plug seedlings.
Table 3. The effects of the DIF on the growth parameters of C. lanceolata plug seedlings.
DIF z (°C)Length (cm)Dry Weight (mg)Leaf Area (cm−2)Specific Leaf Weight (g·m−2)Stem Diameter (mm)Root DW: Shoot DW RatioShoot Dry Weight Per Shoot Length (g·m-1)Root Dry Weight Per Root Length (g·m−1)Dickson’s Quality Index (×10−4)
ShootRootShootRootLeaf
−1016.5 ± 0.43.9 ± 0.456.6 ± 3.5 b y11.1 ± 1.8 b8.3 ± 0.8 b6.0 ± 0.5 b13.8 ± 0.5 b1.51 ± 0.07 c0.19 ± 0.030.34 ± 0.02 b0.28 ± 0.04 b 5.9 ± 0.4 c
017.0 ± 0.53.9 ± 0.377.7 ± 8.1 ab 17.3 ± 2.7 ab10.5 ± 1.2 b7.4 ± 0.8 b14.1 ± 0.7 b1.82 ± 0.09 b0.22 ± 0.020.45 ± 0.04 a0.44 ± 0.05 a 9.8 ± 1.6 b
1017.9 ± 0.54.9 ± 0.498.9 ± 10.3 a20.5 ± 2.2 a14.4 ± 1.3 a8.5 ± 0.5 a16.8 ± 0.8 a2.06 ± 0.07 a0.21 ± 0.020.55 ± 0.05 a0.47 ± 0.03 a14.2 ± 0.8 a
F-testNSNS**********NS******
z DIF, difference between day and night temperatures. y Different letters indicate significant separation within columns by Duncan’s multiple range test at a 0.05 level. NS, *, **, and ***, represent no significant difference, significant difference at p = 0.05, 0.01, or 0.001, respectively.

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Liu, Y.; Ren, X.; Jeong, B.R. Manipulating the Difference between the Day and Night Temperatures Can Enhance the Quality of Astragalus membranaceus and Codonopsis lanceolata Plug Seedlings. Agronomy 2019, 9, 654. https://doi.org/10.3390/agronomy9100654

AMA Style

Liu Y, Ren X, Jeong BR. Manipulating the Difference between the Day and Night Temperatures Can Enhance the Quality of Astragalus membranaceus and Codonopsis lanceolata Plug Seedlings. Agronomy. 2019; 9(10):654. https://doi.org/10.3390/agronomy9100654

Chicago/Turabian Style

Liu, Ya, Xiuxia Ren, and Byoung Ryong Jeong. 2019. "Manipulating the Difference between the Day and Night Temperatures Can Enhance the Quality of Astragalus membranaceus and Codonopsis lanceolata Plug Seedlings" Agronomy 9, no. 10: 654. https://doi.org/10.3390/agronomy9100654

APA Style

Liu, Y., Ren, X., & Jeong, B. R. (2019). Manipulating the Difference between the Day and Night Temperatures Can Enhance the Quality of Astragalus membranaceus and Codonopsis lanceolata Plug Seedlings. Agronomy, 9(10), 654. https://doi.org/10.3390/agronomy9100654

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