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. 2017 Jun 29;7(3):167. doi: 10.1007/s13205-017-0795-5

Evaluating water deficit and glyphosate treatment on the accumulation of phenolic compounds and photosynthesis rate in transgenic Codonopsis lanceolata (Siebold & Zucc.) Trautv. over-expressing γ-tocopherol methyltransferase (γ-tmt) gene

Bimal Kumar Ghimire 1, Na-Young Son 1, Seung-Hyun Kim 1, Chang Yeon Yu 2, Ill-Min Chung 1,✉
PMCID: PMC5489442  PMID: 28660450

Abstract

The effect of water stress and herbicide treatment on the phenolic compound concentration and photosynthesis rate in transgenic Codonopsis lanceolata plants over-expressing the γ-tmt gene was investigated and compared to that in control non-transgenic C. lanceolata plants. The total phenolic compound content was investigated using high-performance liquid chromatography combined with diode array detection in C. lanceolata seedlings 3 weeks after water stress and treatment with glyphosate. Changes in the composition of phenolic compounds were observed in leaf and root extracts from transformed C. lanceolata plants following water stress and treatment with glyphosate. The total concentration of phenolic compounds in the leaf extracts of transgenic samples after water stress ranged from 3455.13 ± 40.48 to 8695.00 ± 45.44 µg g−1 dry weight (DW), whereas the total concentration phenolic compound in the leaf extracts of non-transgenic control samples was 5630.83 ± 45.91 µg g−1 DW. The predominant phenolic compounds that increased after the water stress in the transgenic leaf were (+) catechin, benzoic acid, chlorogenic acid, ferulic acid, gallic acid, rutin, vanillic acid, and veratric acid. The total concentration of phenolic compounds in the leaf extracts of transgenic samples after glyphosate treatment ranged from 4744.37 ± 81.81 to 12,051.02 ± 75.00 µg g−1 DW, whereas the total concentration of the leaf extracts of non-transgenic control samples after glyphosate treatment was 3778.28 ± 59.73 µg g−1 DW. Major phenolic compounds that increased in the transgenic C. lanceolata plants after glyphosate treatment included kaempherol, gallic acid, myricetin, p-hydroxybenzjoic acid, quercetin, salicylic acid, t-cinnamic acid, catechin, benzoicacid, ferulic acid, protocatechuic acid, veratric acid, and vanillic acid. Among these, vanillic acid showed the greatest increase in both leaf and root extracts from transgenic plants relative to those from control C. lanceolata plants following treatment with glyphosate, which could affect the 5-enol-pyruvyl shikimate-3-phosphate (EPSP) synthase, an enzyme in the shikimate pathway. We observed enhanced stomatal conductance (gs) and photosynthesis rate (A) in the transgenic plants treated with water stress and glyphosate treatment. The results of this study demonstrated large variations in the functioning of secondary metabolites pathway in response glyphosate and water stress in transgenic C. lanceolata.

Keywords: Codonopsis lanceolata, Glyphosate, Phenolic compound, Photosynthesis, Transgenic

Introduction

Plants constantly fight against biotic and abiotic environmental factors, including chemicals, water stress, and pathogen attack, which affect growth and yield (Bahrani et al. 2010), accumulation of compatible organic solutes (Sanchez-Diaz et al. 2008), and changes in endogenous phytohormones (Perales et al. 2005). Water availability is the most important factor that limits the productive potential of higher plants (Rodriguez et al. 2006; Bray et al. 2000). To survive under conditions of environmental biotic and abiotic stress, plants respond and adapt to these stresses by means of biochemical and physiological processes as well as morphological and developmental changes (Munns 2002). Maintenance of a high antioxidant capacity to scavenge toxic reactive oxygen species (ROS) has been correlated with increased tolerance of plants to a wide variety of environmental stresses (Zaefyzadeh et al. 2009; Chen et al. 2010); phenolic compounds (Usenik et al. 2004). Therefore, globally, the use of resistant genetically engineered crops has increased by >80% (James 2012), contributing immensely to yield, sustainable crop farming, and phytoremediation.

Weeds are a major problem for farmers during crop cultivation, and to control weeds, farmers use a variety of herbicides and weedicides every year. The increased use of herbicides has led to the appearance of tolerant or resistant weeds (Service 2007). Moreover, excessive use of herbicides becomes a major ecological risk when genetically modified organisms are released (GMOs) in the environment (Altieri 2005; Barton and Dracup 2000). Herbicides not only inhibit the growth of weeds, but they also inhibit the growth of useful crops in the field. A number of studies have revealed the influence of herbicide treatment on the phenolic compound composition of the plant, thereby influencing the disease resistance potential of plants (Donnini et al. 2016).

Phytochemicals are found at different levels in many aromatic medicinal plants (Perumalla and Hettiarachchy 2011; Negi 2012; Cowan 1999). In plants, these compounds are related to various functions such as defense against pathogens (Kutchan 2001; Housti et al. 2002). The phenolic profiles of plants may change in response to stress conditions such as those induced by the soil-condition, season, climate, plant component, and other parameters (Marian and Fereidoon 2004). Commercial herbicides exhibit that many different mechanisms of action and several enzymes involved in the biosynthesis of amino acids are the sites of action of herbicides (Duke 1990). Glyphosate (N-phosphonomethylglycine) is a systemic post-emergence broad spectrum herbicide, which inhibits EPSP synthase that is involved in the synthesis of aromatic amino acids in the shikimate pathway (Montgomery 1997; Duke et al. 2003). These amino acids are essential for the growth and survival of the plants (Hatcher and Kruger 1997). The physiological effects of glyphosate on plants include reduced protein synthesis, reduced stomatal conductance (Munoz-Rueda et al. 1986), inhibition of photosynthesis and carbon allocation (Geiger et al. 1986), production of ethylene and CO2 (Abu-Irmaileh et al. 1979), and phenolic compound metabolism (Hernandez et al. 1999). Glyphosate blocks the shikimate pathway, thereby reducing the synthesis of aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) and causes the accumulation of shikimate in affected plant tissues (Lydon and Duke 1988). Glyphosate has been shown to increase phenylalanine ammonia-lyase (PAL) synthesis in plant and synthesize more flavonoids as a possibly protective reaction in response to oxidative damage (Duke and Hoagland 1978).

Codonopsis lanceolata (Siebold & Zucc.) Trautv. of the family Campanulaceae are mostly found in moist and low mountains and hills of Korea, Japan, and China (Jung et al. 2006). The roots of these plants are known for their various nutritional and medicinal properties and consumed as vegetable in Korea (Kim et al. 2003). In addition, the root of this plant plays a part in the traditional Chinese medicine due to their antioxidant (Oh and Kim 2006), antimicrobial, anti-inflammatory (Li et al. 2007), and immunomodulatory properties (Byeon et al. 2009). It is known to contain many important bioactive chemicals including alkaloids, saponins, vitamins, essential oils, tannins, polyphenolics, and steroids (Ichikawa et al. 2009) which are supposed to be responsible for the various biological activities including caspase-dependent apoptosis activity in humans (Lee et al. 2005), regulatory effects (Lee et al. 2007), lipid peroxidation and anti-aging properties (Batista et al. 2007), and anti-cancer activities, α-glucosidase inhibition, and tyrosinase inhibition (Kim et al. 2009). Previously, we generated transgenic C. lanceolata lines (Ghimire et al. 2008), which over-express the γ-tmt gene, using Agrobacterium mediated transformation. Comparative analyses of the effects of glyphosate and water stress on accumulation of phenolic compounds and photosynthetic rate in transgenic plants in response to over-expression of the γ-tmt gene have not been documented. Therefore, in this experiment, we selected transgenic lines to evaluate the glyphosate effects in terms of their total phenolic compounds and photosynthetic rate.

Materials and methods

Chemicals

All solvents used were of analytical grade. Methanol was obtained from Baker (Phillipsburg, NJ).

Transgenic plant generation and selection

Transgenic C. lanceolata were generated by transforming the γ-tocopherol methyltransferase (γ-tmt) gene, which encodes key enzymes involved in the final step of tocopherol biosynthesis, according to the Agrobacterium mediated method. The vector (pYBI130) was introduced into Agrobacterium tumefaciens (LBA 4404). A vector was inserted into leaf explants of C. lanceolata plants that contain the neomycin phosphotransferase gene (npt-II) under the control of the nopaline synthase (NOS) promoter as a selectable marker. A construct consisting of γ-tmt cDNAs from Arabidopsis thaliana under the control of cauliflower mosaic virus (CaMV) 35S promoter and NOS terminator was inserted into the genome of the C. lanceolata plants. The obtained regenerants were prescreened using the polymerase chain reaction (PCR) method with specific primers for the neomycin phosphotransferase npt-II gene, which were N-1 (5′GAA-GCT-ATT-CGG-CGG-CTA-TGA-CTG-3′) as the sense primer and N-2 (5′ATC-GGG-AGC-GGC-GGC-GAT-ACC-CTA-3′) as the anti-sense primer. Plants that exhibited a 700-bp (npt-II) DNA fragment were used for further selection by means of southern blot analysis. The details of plant transformation, preselection, selection, and transgenic plant analysis have been previously described (Ghimire et al. 2008). The reference specimens were collected from the Plant Biotechnology Department, Kangwon National University, South Korea. The transgenic line (T1, T2, and T3) that showed the highest level of mRNA for the introduced cDNAs was used to investigate the metabolic effects caused by the water stress and glyphosate treatment on the transgenic C. lanceolata (Fig. 1).

Fig. 1.

Fig. 1

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a PCR analysis from leaves of transgenic C. lanceolata using specific primers of amplification of 700 bp npt-II gene. Lane 1 molecular marker, lane 2 the plasmid pYBI130 as a positive control, lane 3 the genomic DNA from untransformed plants, lanes 4 and 5 the genomic DNA of T2 transformants. b PCR analysis from leaves of transgenic C. lanceolata using specific primers of amplification of the 1070 bp fragment of γ-tmt gene. Lane 1 molecular marker, lane 2 the genomic DNA from untransformed plants, lane 3 the plasmid pYBI130 as a positive control, lanes 4–9 the genomic DNA of T2 transformants

Measurement of photosynthetic rate after water stress and glyphosate treatment

To investigate the physiological significance of water stress and glyphosate treatment, the photosynthetic rate of three C. lanceolata transgenic lines was determined and compared to that of non-transgenic control plants. Seeds of wild-type C. lanceolata and transgenic plants were obtained from the Bio herb Research Center, Kangwon National University, South Korea and transgenic plants over-expressing the γ-tmt gene were previously generated in the Plant Biotechnology Laboratory, Kangwon National University, Korea. The seeds of both control and transgenic C. lanceolata were germinated on vermiculite and perlite mixture (3:1) in a plant growth chamber at 25 °C and 80% relative humidity, providing a photoperiod of 16 h light and 8 h dark. After 2 weeks, seedlings were transferred to bigger pots containing vermiculite and soil (3:1) and were maintained in a glass house at 28/18 ± 2 °C with 40 ± 5% humidity and at a PPFD of 400 μmol m−2 s−1 in a green house. When the plantlets contained five leaves, they were individually transplanted in pots (190 × 195 mm) filled with a soil mixture (garden soil, perlite, vermiculite, and sand) and grown in a greenhouse under natural light at day/night temperatures of 28/18 °C and 40 ± 5% humidity and at a PPFD of 400 μmol m−2 s−1. Water stress applied to both control and transgenic C. lanceolata plants. Water stress treatment was initiated by the withdrawal of the water supply (reduced by 60%) compared to control plants. All the plantlets were watered after every 2 days.

Glyphosate were sprayed outside the greenhouse using a backpack sprayer under constant pressure of 2.2 kg f cm−2 of CO2. The plants were grown at 28/18 ± 2 °C during glyphosate treatment with humidity of 40 ± 5% and wind speed of 5–7 km/h. Glyphosate solution was sprayed uniformly on the plants which were in the five-to-seven-leaf stage in the morning at 10:30 using Kwang Sung sprayers, Model no. KS-10-1, South Korea. Care was taken to avoid glyphosate run-off from leaves during plant treatment. The pots were placed in the controlled greenhouse, with temperature ranging from 28 ± 2 °C (day) to 25 ± 2 °C (night) with relative humidity of 70% during s 16/8 photoperiod and the plantlets were irrigated every day. The absence of glyphosate treatment treated as control plant. The experiments were repeated in a completely randomized manner with four pots per replication and were repeated three times at a concentration of 1.3 mg/mL. Data were collected over 2 weeks of spraying transgenic plants with glyphosate. Stomatal conductance (gs), net CO2 assimilation rate (A), and the ratio of internal CO2 concentration (C i) were measured according to the methods described by Ivan et al. (2001) using a portable photosynthesis system (LCA-4; Analytical Development Co., Hoddesdon, Herts, UK) at an air temperature 28 ± 1 °C. All measurements were performed three times during August and September on sunny days between 10:00 and 14:00, on the surface of leaves under a saturating PPFD of 1500 µmol m−1 s−1. Measurements were taken at the center of the leaf surface immediately after the decrease in CO2 concentration was stable. Each leaf was allowed to stabilize for 4–6 min before measurements of gs, C i, and A were made. After the completion of physiological analysis, plant sample from all the treatments was harvested for analyzing the biochemical parameters.

Phenolic compound analysis after water stress and glyphosate treatment

The leaves and roots (10 g) from each transgenic line and non-transformed control plants were harvested, dried at room temperature (28 °C), and ground to crude powder. About 2 g of dried crude powder was soaked in 20 ml of 80% methanol for 48 h. The mixture was filtered through No. 1 Whatman filter paper (Schleicher & Schuell, Whatman International Lts., Maidstone, England) to remove debris. The filtrate was concentrated to dryness under vacuum rotary evaporator at 40 °C. The dried samples were redissolved in 10 mL of 80% HPLC grade methanol solution. An aliquot of each sample was filtered through a 0.45-μm filter unit (TITAN syringe filter nylon membrane) and then analyzed by high-performance liquid chromatography (HPLC). HPLC analysis was carried out on a product from Shimadzu company (Shimadzu Instruments CO., LTD, Japan) with a pump model LC-10AD VP and a detector model SPD-M10A VP (Photodiode Array detector). YMC-Pack ODS-AM-303 (5 μm, 250 × 4.6 mm I.D) analytical HPLC column was employed for quantitative analyses. The substances being measured during the analytic procedure were monitored and determined at a UV wavelength of 280 nm. Solvent gradient HPLC analysis was applied using the modified method described by Thiruvengadam and Chung (2015). The mobile phase consisted of solvents A and B. Solvent A consisted of 0.1% glacial acetic acid in distilled water and solvent B was 0.1% glacial acetic acid in acetonitrile. The solvent flow rate was 1 mL min−1, and the wavelength of the photodiode array detector (PDA) was 280 nm. The injection volume was 20 µL of the sample, and the linear gradient of the HPLC solvent was as follows: B was increased from 8 to 10% for 2 min, then from 10 to 30% for 25 min, from 30 to 90% for 24 min, and from 90 to 100% for 2 min, and was then maintained at 100% of B for 5 min before returning to the initiation stage. The next sample was injected after 15 min. Genuine standards of 28 phenolic compounds were purchased from Sigma (USA) and Extrasynthese (France) and were dissolved in dimethylsulfoxide (DMSO) and then used to generate calibration curves. The standard concentrations were obtained at three concentrations, 10, 50, and 100 ppm, and a high linearity of r 2 > 0.996 was obtained from each curve. Benzoic acid, caffeic acid, chlorogenic acid, t-cinnamic acid, m-coumaric, o-coumaric acid, p-coumaric acid, ferulic acid, gallic acid, gentisic acid, homogentisic acid, p-hydroxybenzoic acid, protocatechuic acid, pyrogallol, resveratrol, rutin, salicylic acid, 5-sulfosalicylic acid, syringic acid, vanillic acid, vanillin, biochanin A, (+) catechin, formononetin, hesperetin, kaempferol, myricetin, naringenin, naringin, and quercetin were each identified by their retention times or by chromatography with other authentic examples, and concentrations were calculated by comparing the peak areas of samples with those of the standards (Table 1).

Table 1.

Calibration curves of equation of 28 phenolic compound standards

Sl. no. Phytochemicals Abbreviation Concentration Linearity (r 2) Equation
(µg/ml)1
1 Benzoic acid BE 0.5 0.9995 y = 7335.1x − 5972.7
2 Chlorogenic acid CH 0.5 0.9954 y = 7335.1x − 5972.7
3 t-Cinnamic acid t-C 0.5 0.9994 y = 169586x − 221,863
4 m-Coumaric acid m-C 0.5 0.999 y = 99520x + 366,589
5 o-Coumaric acid o-C 0.5 0.999 y = 30,379.0x + 146.33
6 p-Coumaric acid p-C 0.5 0.985 y = 10,243.0x − 5998.60
7 Ferulic acid FA 0.5 0.9992 y = 63695x − 165,433
8 Gallic acid GA 0.5 0.9994 y = 35535x − 79,194
9 Gentisic acid GE 0.5 0.999 y = 3183.7x + 496.00
10 Homogentisic acid HO 0.5 0.9977 y = 15919x − 81,841
11 p-Hydroxybenzoic acid p-HY 0.5 0.9999 y = 27919x + 43,418
12 Protocatechuic acid PR 0.5 0.9971 y = 29859x − 174,443
13 Pyrogallol PY 0.5 0.999 y = 3320x + 2150
14 Resverotrol RE 0.5 0.999 y = 31,260.0x − 58.97
15 Salicylic acid SA 0.5 1 y = 15377x + 7783.7
16 5-Sulfosalicylic acid SSA 0.5 0.9988 y = 7115.8x + 21,563
17 Syringic acid SY 0.5 0.9997 y = 62213x + 69,563
18 Vanillic acid VA 0.5 1 y = 34743x + 2786.6
19 Vanillin VN 0.5 0.999 y = 39,731.0x − 3975.80
20 Veratric acid VE 0.5 1 y = 34957x + 15,741
21 Biochanin A BA 0.5 0.9979 y = 42516x + 5293
22 Catechin CN 0.5 0.9979 y = 6810.3x + 29,358
23 Kaempferol KA 0.5 0.9994 y = 32211x − 57,175
24 Myricetin MY 0.5 0.9955 y = 22104x − 195,936
25 Naringenin NA 0.5 0.999 y = 23,238.0x − 787.89
26 Rutin RN 0.5 0.999 y = 34957x + 15,741
27 Quercetin QN 0.5 0.9944 y = 22509x − 213,591
28 Formononetin FN 0.5 0.9992 y = 274000x + 21,300
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Statistical analysis

All experiments were repeated at least three times. The data shown represent mean ± standard deviation. The data were statistically analyzed using analysis of variance (ANOVA), and differences were assessed to be significant at a 5% level of probability (P < 0.05).

Results

Accumulation of phenolic compounds after glyphosate treatment

In this study, we treated transgenic C. lanceolata with glyphosate and ascertained the phenolic compound concentration in the resistant transgenic lines and control plants. We found quantitative differences in the content of total phenolic compounds between leaf and root samples from transgenic and control plants (Table 2). A significant increase in the total phenolic compound concentration was revealed in leaf extracts from transgenic plants treated with glyphosate, and ranged from 4744.37 ± 81.81 to 12,051.02 ± 75.00 µg/g DW relative to the lower phenolic compound concentration in control plants (3778.28 ± 59.73 µg/g DW). The major phenolic compounds that were increased in leaf extracts from C. lanceolata treated with glyphosate were kaempferol, catechin, benzoic acid, ferulic acid, protocatechuic acid, veratric acid, gallic acid, myricetin, p-hydroxybenzjoic acid, quercetin, t-cinnamic acid, and vanillic acid. The most pronounced enhancement was observed in the concentration of quercetin, which ranged from the equivalent of 42.03 to 51.34% of the total phenolic compounds. Biochanin A and p-coumaric acid, which were not detected in control plants, were observed in T2 and T3 transgenic lines, respectively. A considerable amount of phenolic acids were observed to decrease in transgenic plants, and included salicylic acid. The total phenolic compound concentration decreased in glyphosate treated root extracts of transgenic lines ranging from 1437.96 ± 36.43 to 1748.73 ± 46.40 µg/g DW, as compared with control plants (3020.13 ± 46.86 µg/g DW). Similarly, the concentration of myricetin decreased in transgenic roots following treatment with glyphosate as compared to control plants. Significantly increased levels of vanillic acid were revealed in the T2 and T3 transgenic root extracts at 222.00 ± 2.65 and 172.67 ± 6.43 µg/g DW, respectively, after treatment with glyphosate as compared to control plants at 6.67 ± 1.53 (µg/g DW). Chlorogenic acid and veratric acid, which were not detected in the root of control plants, were observed in root extracts from the T2 transgenic line. Homogenic acid, p-coumeric acid, and p-hydroxybenzoic acid were not detected in transgenic roots but were present in transgenic leaf extracts of C. lanceolata plants.

Table 2.

Quantities of phenolic compounds from control and transgenic C. lanceolata after treating with glyphosate

Plant lines Phenolic compounds (µg/g)c
BE CH t-CI p-C FE GA HO p-HY PR SA
AC1 307.89 ± 6.54d 182.00 ± 7.45b 71.67 ± 2.08e 0.00a 64.33 ± 3.51c 115.33 ± 3.05c 125.00 ± 4.58b 41.67 ± 1.53c 176.00 ± 2.65d 74.67 ± 3.05f
AT1 370.88 ± 6.17e 0.00a 76.33 ± 1.53f 0.00a 163.33 ± 9.45e 89.00 ± 9.0b 120.33 ± 5.51b 327.00 ± 14.53d 142.33 ± 2.52bc 33.00 ± 2.00c
AT2 561.63 ± 5.74f 1175.00 ± 16.00d 87.33 ± 2.08g 0.00a 173.67 ± 7.09f 293.67 ± 6.81e 377.67 ± 3.21c 24.67 ± 2.52b 382.33 ± 3.86f 0.00a
AT3 688.00 ± 10.53g 0.00a 91.00 ± 1.00h 44.35 ± 4.66b 146.33 ± 5.13d 187.00 ± 10.14d 0.00a 784.00 ± 4.58e 326.67 ± 6.03e 77.33 ± 2.08e
BC1 184.67 ± 6.51b 0.00a 36.33 ± 1.53c 0.00a 0.00a 61.67 ± 2.08a 0.00a 0.00a 151.00 ± 5.57b 3.50 ± 0.17b
BT1 130.33 ± 10.41a 0.00a 41.33 ± 1.55d 0.00a 0.00a 55.67 ± 4.04a 0.00a 0.00a 146.00 ± 3.00c 3.24 ± 0.14b
BT2 225.67 ± 12.50c 237.67 ± 6.43c 32.33 ± 2.52b 0.00a 54.33 ± 4.04b 63.00 ± 2.00a 0.00a 0.00a 126.00 ± 2.52a 52.96 ± 2.63d
BT3 337.80 ± 2.00c 0.00a 26.33 ± 3.06a 0.00a 0.00a 65.33 ± 4.04a 0.00a 0.00a 130.33 ± 4.51a 4.33 ± 1.53b
Plant lines Phenolic compounds (µg/g)c
SSA SY VA VE BCA CA KA MY RU QU Total
AC1 10.58 ± 0.59b 0.00a 13.00 ± 2.65b 55.00 ± 3.61c 0.00a 58.81 ± 2.17a 445.33 ± 3.51e 194.00 ± 5.19a 33.00 ± 2.00e 1810.00 ± 5.57d 3778.28 ± 59.73e
AT1 0.00a 0.00a 22.00 ± 2.00c 3.50 ± 0.50b 0.00a 0.00a 749.67 ± 13.79f 183.33 ± 7.23a 28.00 ± 2.65d 2435.67 ± 4.93e 4744.37 ± 81.81f
AT2 14.68 ± 14.67c 0.00a 158.00 ± 4.00e 118.67 ± 1.53e 22.66 ± 2.52d 1315.55 ± 4.10d 885.67 ± 3.05 g 773.33 ± 14.01d 22.400 ± 2.65c 4632.00 ± 6.56f 11,018.93 ± 100.40 g
AT3 0.00a 0.00a 34.67 ± 1.53d 108.67 ± 2.52d 0.00a 2952.00 ± 7.21f 159.67 ± 2.08d 529.00 ± 1.00c 91.33 ± 6.51f 5831.00 ± 10.00 g 12,051.02 ± 75.00 h
BC1 10.83 ± 0.04b 0.00a 6.67 ± 1.53a 0.00a 12.33 ± 2.52c 2060.67 ± 9.50e 44.00 ± 200b 224.67 ± 14.22b 0.79 ± 0.19a 223.00 ± 3.00b 3020.13 ± 246.86d
BT1 0.00a 0.00a 5.83 ± 1.04a 0.00a 8.00 ± 2.00b 927.66 ± 9.07c 0.00a 218.67 ± 12.50b 0.00a 212.00 ± 2.65a 1748.73 ± 46.40c
BT2 0.00a 0.00a 222.00 ± 2.65 g 5.59 ± 0.37b 7.67 ± 1.53b 0.00a 44.00 ± 2.00b 189.50 ± 9.50a 6.14 ± 0.06b 243.00 ± 6.56c 1509.86 ± 55.31b
BT3 0.00a 0.00a 172.67 ± 6.43f 0.00a 6.07 ± 0.89b 211.09 ± 1.80b 96.67 ± 2.08c 183.67 ± 7.57a 0.00a 203.67 ± 2.52a 1437.96 ± 36.43a
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BE benzoic acid, CH chlorogenic acid, t-CI t-cinnamic acid, p-C p-coumaric acid, FE ferulic acid, GA gallic acid, HO homogentisic acid, p-HY p-hydroxybenzoic acid, PR protocatechuic acid, SA salicylic acid, SSA 5-sulfosalicylic acid, SY syringic acid, VA vanillic acid, VE veratric acid, BCA biochanin A, CA (+) catechin, KA kaempferol, MY myricetin, RU rutin, QU quercetin

Data in each column with the same letter were not significantly different according to Duncan’s multiple comparison test (P < 0.05)

AC1—Leaf of control plant; AT1, AT2, and AT3—leaf of transgenic plant treated with glyphosate

BC1—Root of control plant; BT1, BT2, and BT3—root of transgenic plant treated with glyphosate

Accumulation of phenolic compounds after water stress

The effect of water stress on the phenolic compound composition of transgenic C. lanceolata and control plants is summarized in Table 3. The results from the present studies revealed that water stress of transgenic and non-transgenic C. lanceolata plants caused significant variation in the phenolic compound composition in the root and leaf extracts of transgenic lines as compared to the control plants. The total concentration of phenolic compounds in the leaf extracts of transgenic samples after water stress ranged from 3455.13 ± 40.48 to 8695.00 ± 45.44 µg g−1 DW, whereas the total concentration of the leaf extracts of non-transgenic control samples was 5630.45.91 µg g−1 DW. The predominant phenolic compounds that increased after the water stress in the transgenic leaf were (+) catechin, benzoic acid, chlorogenic acid, ferulic acid, gallic acid, rutin, vanillic acid, and veratric acid. The most pronounced enhancement was observed in the concentration of quercetin (54.20% of the total phenolic compounds). p-Hydroxybenzoic acid, which were not detected in control plants, were observed in the leaf extracts of T1, T2, and T3 transgenic lines, respectively (Table 3). Similarly, kaempferol, which were not detected in control plants, were observed in the root extracts of T1, T2, and T3 transgenic lines, respectively. A considerable amount of phenolic acids were observed to decrease in transgenic plants, and included homogentisic acid and syringic acid. The total phenolic compound concentration increased in water stress leaf and root extracts of transgenic lines compared to control plants.

Table 3.

Quantities of phenolic compounds from control and transgenic C. lanceolata plants after water stress

Plant lines Phenolic compounds (µg/g)
BE CH t-CI p-C FE GA HO p-HY PR SA
AC1 159.33 ± 3.05c 691.67 ± 3.05e 37.67 ± 1.53c 36.33 ± 2.52b 75.67 ± 1.53c 56.33 ± 2.08c 134.67 ± 1.53c 0.00a 150.67 ± 5.13bc 555.00 ± 5.55g
AT1 315.00 ± 3.61d 1005.00 ± 3.61f 681.33 ± 6.66f 0.00a 163.33 ± 1.55e 82.00 ± 2.65e 115.00 ± 2.64b 23.00 ± 2.00b 0.00a 211.67 ± 2.08c
AT2 312.00 ± 2.65d 3066.33 ± 1.53h 31.00 ± 1.00b 495.00 ± 3.61c 96.00 ± 2.69d 67.33 ± 2.08d 0.00a 134.33 ± 1.53d 151.00 ± 1.00b 1293.00 ± 2.64h
AT3 454.00 ± 2.65f 1548.67 ± 6.43g 44.00 ± 2.65d 0.00a 72.00 ± 2.00c 80.00 ± 2.00e 114.33 ± 2.08b 74.33 ± 2.52c 155.00 ± 1.00c 383.00 ± 2.64f
BC1 43.33 ± 3.22a 250.00 ± 5.57c 24.67 ± 3.21a 0.00a 255.00 ± 1.00f 51.33 ± 1.53b 0.00a 0.00a 0.00a 173.00 ± 2.67b
BT1 323.00 ± 2.65e 455.67 ± 5.451d 53.33 ± 2.08e 0.00a 94.33 ± 3.51d 115.00 ± 2.65f 190.67 ± 5.03d 25.00 ± 2.64b 212.00 ± 2.65e 258.67 ± 11.84e
BT2 62.33 ± 1.53b 0.00a 27.00 ± 2.00ab 0.00a 54.00 ± 2.61a 57.12 ± 2.08c 0.00a 0.00a 0.00a 4.00 ± 0.50a
BT3 65.33 ± 4.04b 223.67 ± 2.52b 36.67 ± 2.81c 0.00a 65.33 ± 3.51b 0.00a 0.00a 0.00a 165.00 ± 3.00d 233.67 ± 1.52d
Plant lines Phenolic compounds (µg/g)
SSA SY VA VE BCA CA KA MY RU QU Total
AC1 0.00a 26.00 ± 1.80c 16.00 ± 1.50b 4.00 ± 1.00b 0.00a 28.83 ± 0.76a 462.33 ± 7.77f 214.00 ± 1.00b 8.00 ± 1.00c 2974.33 ± 5.11g 5630.83 ± 45.91f
AT1 0.00a 23.66 ± 2.52b 35.00 ± 2.50c 47.33 ± 1.52e 0.00a 48.14 ± 1.01b 105.00 ± 3.61e 280.67 ± 2.52e 64.00 ± 1.00e 255.00 ± 1.00c 3455.13 ± 40.48e
AT2 0.00a 21.33 ± 1.53b 251.67 ± 2.08e 0.00a 0.00a 160.33 ± 2.52d 96.00 ± 2.65d 252.00 ± 6.08d 22.00 ± 1.50d 426.67 ± 1.52f 6875.99 ± 36.61g
AT3 0.00a 2.00 ± 0.22a 39.00 ± 1.80c 44.67 ± 1.53d 0.00a 32.66 ± 2.08a 623.67 ± 9.02g 307.00 ± 3.61f 7.67 ± 0.57c 4713.00 ± 2.64h 8695.00 ± 45.44h
BC1 0.00a 167.33 ± 2.08d 7.00 ± 1.10a 43.33 ± 1.52d 0.00a 384.67 ± 4.04f 0.00a 213.67 ± 3.21b 4.00 ± 1.00b 234.67 ± 3.51b 1852.00 ± 33.66b
BT1 0.00a 0.00a 385.33 ± 6.43g 0.00a 0.00a 106.67 ± 3.79c 103.00 ± 3.00de 324.00 ± 2.65g 7.00 ± 1.12c 363.00 ± 2.65e 3016.67 ± 58.14d
BT2 0.00a 0.00a 180.67 ± 4.55d 0.00a 0.00a 204.67 ± 4.04e 64.67 ± 3.51 0.00a 0.00a 223.33 ± 2.08a 877.79 ± 22.90a
BT3 0.00a 0.00a 294.67 ± 3.21f 18.67 ± 1.55 0.00a 592.33 ± 6.51g 42.33 ± 1.53b 236.00 ± 2.00 0.00a 263.33 ± 2.08d 2237.00 ± 34.29
Open in a new tab

BE benzoic acid, CH chlorogenic acid, t-CI t-cinnamic acid, p-C p-coumaric acid, FE ferulic acid, GA gallic acid, HO homogentisic acid, p-HY p-hydroxybenzoic acid, PR protocatechuic acid, SA salicylic acid, SSA 5-sulfosalicylic acid, SY syringic acid, VA vanillic acid, VE veratric acid, BCA biochanin A, CA (+) catechin, KA kaempferol, MY myricetin, RU rutin, QU quercetin

Data in each column with the same letter were not significantly different according to Duncan’s multiple comparison test (P < 0.05)

AC1—Leaf of control plant; AT1, AT2, and AT3—leaf of transgenic plant treated with water deficit

BC1—Root of control plant; BT1, BT2, and BT3—root of transgenic plant treated with water deficit

Measurement of photosynthesis rate after water stress and glyphosate treatment

The physiological effects of water stress and glyphosate treatment on the transgenic C. lanceolata investigated by measuring the photosynthesis rate (A), stomatal conductance (gs), and CO2 concentration (C i) and compared them to those of the non-transgenic control plants (Table 4). In the glyphosate treatment experiment, the aerial parts of transgenic plants sprayed with glyphosate revealed higher A (3.91 ± 0.950 to 5.44 ± 0.31 µmol m−2 s−1) and gs (0.5 ± 0.004 to 0.06 ± 0.004 cm s−1) relative to the glyphosate treated control plants 2.28 µmol m−2 s−1 and 0.05 cm s−1, respectively). In this study, we observed gs and A in the transgenic plants treated with water stress after 21 days relative to those observed in the non-transgenic control plants (Table 5). In the water stress experiment, transgenic plants grown under greenhouse conditions showed higher A (3.58 ± 0.66 to 4.67 ± 0.91 µmol m−2 s−1) and gs (0.5 ± 0.001 to 0.06 ± 0.001 cm s−1) relative to water stressed control plants (2.09 ± 0.69 µmol m−2 s−1 and 0.05 ± 0.004 cm s−1, respectively).

Table 4.

Photosynthetic performance of transgenic and non-transgenic (control) C. lanceolata plants treated with glyphosate after 21 days

Plant lines A (µmol m−2 s−1) gs (cm s−1) C i (µmol mol−1)
C1 2.28 ± 0.12d 0.05 ± 0.001b 377.15 ± 4.29a
T1 5.07 ± 0.11b 0.05 ± 0.005b 308.50 ± 4.94c
T2 5.44 ± 0.31a 0.06 ± 0.004a 277.83 ± 2.06d
T3 3.91 ± 0.950c 0.05 ± 0.004b 348.30 ± 2.301b
Open in a new tab

A photosynthetic rate, gs stomatal conductance, C i CO2 concentration

Plant lines: C control plant, T1, T2, T3 transgenic plants

The data represent the mean of 8–10 independent determinations ± SD. Data in each column with the same letter were not significantly different according to Duncan’s multiple comparison test (P < 0.05)

Table 5.

Photosynthetic performance of transgenic and non-transgenic (control) C. lanceolata plants under water stress after 21 days

Plant lines A (µmol m−2 s−1) gs (cm s−1) C i (µmol mol−1)
C1 2.09 ± 0.69d 0.05 ± 0.004b 384.97 ± 3.92a
T1 4.67 ± 0.91a 0.05 ± 0.001b 286.90 ± 4.31d
T2 4.07 ± 0.31b 0.05 ± 0.002b 321.60 ± 3.83c
T3 3.58 ± 0.66c 0.06 ± 0.001a 342.80 ± 6.65b
Open in a new tab

A Photosynthetic rate, gs stomatal conductance, C i CO2 concentration

Plant lines: C Control plant, T1, T2, and T3 transgenic plants

The data represent the mean of 8–10 independent determinations ± SD

Data in each column with the same letter were not significantly different according to Duncan’s multiple comparison test (P < 0.05)

Discussion

This study investigated the physiological effects of glyphosate on the phenolic compound composition and photosynthesis rate of transgenic C. lanceolata plants. We found that transgenic lines of C. lanceolata had a higher photosynthesis rate (A), stomatal conductance (gs), and elevated phenolic concentration following glyphosate treatment. A high level of phenolic acid suggests that alternative biosynthesis of phenolic compounds was activated by glyphosate treatment due to inhibition of 5-enolpyruvylshikimate-3-p-synthase. Glyphosate has been shown to reduce/inhibit the production of phenolics and other phytochemical responses for disease resistance (Greaves and Sargent 1986). However, in this study, we observed increased levels of total phenolic compounds in transgenic leaves treated with glyphosate. After glyphosate treatment, the levels of gallic acid, t-cinnamic acid, and quercetin increased in the transgenic leaf extracts, whereas vanillic acid increased in the root extracts of transgenic C. lanceolata plants treated with glyphosate. Some of the phenolic compounds that were not present initially in the transgenic C. lanceolata plants were observed in transgenic C. lanceolata plants treated with glyphosate. This result indicates that some of these phenolic compounds are produced only after glyphosate treatment. However, in transgenic roots treated with glyphosate, we observed a decrease in the total phenolic compound concentration relative to that observed in the control plants. Several phenolic compounds (chlorogenic acid, ferulic acid, and veratric acid) that were not detected in control plants were found in transgenic plants (T2) sprayed with glyphosate. The presence of these compounds in transgenic lines indicates that genetic or chemical disruption of the metabolic pathway of phenolic compounds can lead to an increase in the phenolic compound concentration and in glyphosate resistance. In a similar study, an increase in phenolic compounds such as hydrobenzoic acid after glyphosate treatment was reported by Hernandez et al. (1999). However, a number of studies reported decreased phenolic compound concentration after glyphosate treatment in plants such as soybean, lupine, ryegrass, and pigweed (Lydon and Duke 1988; Marchiosi et al. 2009). In this study, the concentration of gallic acid increased in leaf extracts of transgenic plants following treatment with glyphosate. Since glyphosate is known to block EPSP synthase (Duke 1988), which is an enzyme in the shikimate pathway, the increase in gallic acid concentration observed in the presence of glyphosate indicates that an alternative pathway might exist for the synthesis of gallic acid. Ossipov et al. (2003) indicated that the most possible alternative biosynthesis pathway for gallic acid during glyphosate treatment is from dehydroshikimic acid. Consistent with our result, Becerra-Moreno et al. (2012) in their study reported increased shikimic acid (SA) and chlorogenic acid content following treatment with glyphosate.

We evaluated the metabolic response to water stress in transgenic C. lanceolata plants. In the present study, introduction of the γ-tmt gene in C. lanceolata plants resulted in an increased photosynthesis rate following water stress. In a previous study, we generated transgenic C. lanceolata plants by over-expressing the γ-tmt gene, which had increased levels of α-tocopherol and antioxidant property (Ghimire et al. 2008, 2011). It is believed that α-tocopherol protects pigments and proteins in the photosynthetic pigments (Kanwischer et al. 2005; Maeda et al. 2008; Matrige et al. 2008; Semchuk et al. 2009). It is likely that the higher α-tocopherol concentration in transgenic C. lanceolata plants infers increased protection of these organelles from the effect of water stress. Another possible reason for the higher photosynthesis rate could be decreased levels of reactive oxygen species (ROS) in transgenic C. lanceolata plants due to the higher antioxidant property of α-tocopherol (Ghimire et al. 2011). Moreover, in the previous study, we found that ROS scavenging enzymes were up-regulated in transgenic C. lanceolata plants indicating that the γ-tmt gene is potentially involved in oxidative stress in transgenic C. lanceolata plants (Seong et al. 2009). It seems likely that the higher ROS scavenging activity of α-tocopherol can withstand the water stress. Moreover, it has been argued that tocopherol protects the cellular machinery, and can thus improve plant defense responses (Espinozaa et al. 2013). The author reported a delay in leaf senescence and reduced oxidative leaf damage in tobacco plants due to increased α-tocopherol levels as a result of drought-induced over-expression of a Homogentisate phytyltransferase (VTE2.1) gene. Gajewska and Sklodowska (2007), and Collin et al. (2008) reported that increased levels of tocopherol confer enhanced tolerance to plants against drought. The tocopherol content correlates positively with tolerance to water deficit in different species (Guo et al. 2006; Munne-Bosch et al. 1999) and genes in the tocopherol pathway might respond differently to different environmental stresses (Kanwischer et al. 2005; Abbasi et al. 2007). Moreover, transgenic plants with higher levels of tocopherol outperform the wild-type plants in the prevention of oxidative damage to the membrane and photosynthetic apparatus during drought treatment (Liu et al. 2008). It has been shown that α-tocopherol contributes to carbohydrate metabolism and photoassimilate transport (Maeda et al. 2006), provides stability and protection of phenylpropanoid compounds in plants (Yao et al. 2015), and plays an important role in adaptive mechanisms to environmental conditions (Loyola et al. 2012). On the other hand, the level of α-tocopherol is increased in response to a variety of abiotic stresses in photosynthetic plant tissues (Noctor 2006). Therefore, it is likely that an increased concentration of α-tocopherol and phenolic compounds has a direct influence on the increased photosynthetic rate in transgenic C. lanceolata plants during water stress. Some of phenolic compounds are efficient endogenous antioxidants (Larson 1988), can scavenge ROS, and can prevent chloroplasts from being damaged (Ye et al. 2000). In particular, we observed increased in the catechin, benzoic acid, chlorogenic acid, ferulic acid, gallic acid, rutin, vanillic acid, and veratric acid in water stressed transgenic plant. In the similar studies, higher level of phenolic compound was reported under water stressed condition (Hameed et al. 2013; Ayaz et al. 2000; Abreu and Mazzafera 2005; Hura et al. 2009; Kirakosyan et al. 2004). It has been shown that water stress increase the levels of ROS in plant cells (Smirnoff 1993). Hura et al. (2009) indicated that increase in ferulic acid act as photoprotector/or as a ROS scavenger, thus a reliable indicator of the resistance to drought stress. Thus, in this study, it is possible that increased phenolic compounds in the transgenic plant with water stressed containing higher concentration of ferulic acid could gain more water stress tolerance by protecting photosynthetic apparatus relative to control plants. However, Cheruiyot et al. (2007), Sánchez-Rodríguez et al. (2011), Yildiz-Aktas et al. (2009) observed degradation of phenolic compound concentration during water stress relative to control plants. The result indicates that water deficit affected the physiological and biochemical properties of transgenic C. lanceolata by regulating the synthesis of secondary metabolites. Hence, it is possible that a higher concentration of phenolic compounds in the transgenic plants contributed to the higher photosynthetic activity observed in water stress transgenic plants. However, the decline in photosynthetic rate and stomatal closure has been reported during water stress (Flexas et al. 2009; Ghannoum 2009). Therefore, it remains to be determined whether increased levels of phenolic compounds and α-tocopherol have a direct effect on increased water stress resistance in transgenic C. lanceolata plants.

Acknowledgements

The authors express their gratitude to the 2015 KU Brain Pool of Konkuk University for supporting this work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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