Regulation of the Phenylpropanoid Pathway: A Mechanism of

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Regulation of the Phenylpropanoid Pathway: A Mechanism of Selenium Tolerance in Peanut (Arachis hypogaea L.) Seedlings Guang Wang, Liying Wu, Hong Zhang, Wenjia Wu, Mengmeng Zhang, Xiao-Feng Li, and Hui Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01054 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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Journal of Agricultural and Food Chemistry

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Regulation of the Phenylpropanoid Pathway: A Mechanism of Selenium Tolerance in

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Peanut (Arachis hypogaea L.) Seedlings

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Guang Wang, Liying Wu, Hong Zhang, Wenjia Wu, Mengmeng Zhang, Xiaofeng Li, Hui Wu *

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College of Food Science and Engineering, South China University of Technology, Guangzhou

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510640, China.

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Corresponding author:

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*Hui Wu, College of Food Science and Engineering, South China University of Technology,

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Wushan Road 381, Guangzhou, Guangdong, China.

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E-mail: [email protected], Phone/Fax: (+86)20-87112853.

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ACS Paragon Plus Environment


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Abstract: To clarify the mechanisms of selenium (Se) tolerance in peanut seedlings, we grown

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peanut seedlings with sodium selenite (0, 3, and 6 mg/L), and investigated the phenylpropanoids

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metabolism in seedling roots. The results showed that selenite up-regulated the expression of

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genes and related enzyme activities responding for the phenylpropanoids biosynthesis cascade,

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such as phenylalanine ammonia-lyase, trans-cinnamate-4-hydroxylase, chalcone synthase,

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chalcone isomerase, and cinnamyl-alcohol dehydrogenase. Selenite significantly increased

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phenolic acids and flavonoids which contributed to the alleviation of selenite-induced stress.

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Moreover, selenite enhanced the formation of endodermis in roots, which may attributed to the

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up-regulation of lignin biosynthesis mediated by the selenite-induced changes of H2O2 and NO,

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probably regulated the selenite uptake from external medium. Accumulation of polyphenolic

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compounds via the phenylpropanoid pathway may be one of the mechanisms of the increasing

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selenite tolerance in plant, by which peanut seedlings survived in seleniferous soil and

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accompanied with accumulation of Se.

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Keywords: peanut; selenium; phenylpropanoid pathway; phenolic; lignin; flavonoid.

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Introduction

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Selenium (Se) is an essential micronutrient in human diets which is vital for human

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antioxidation and hormone balance, deficiency of Se may result health problems.1 There are

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several effective ways to increase Se level in crops, one of the main approaches is the

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application of Se fertilizers to plants, and selenite has been used as a Se fertilizer.2 Studies have

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demonstrated that adding selenite to soil increased Se content in the peanut kernel,3, 4 hence

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peanuts (Arachis hypogaea L.) can be used as one of the Se accumulators. Se is beneficial for

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some plants by increasing resistance and antioxidant capacity of plants subjected to stressful

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environment at a low concentration.5 However, there is no research shows that Se is necessary

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for high plants, and what’s more, a certain high concentration of Se may become toxic to plants

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like heavy metals.6 Therefore, further research on the mechanisms of Se tolerance is extremely

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important for the Se accumulation of crops. Our previous research has demonstrated that low

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concentrations of selenite enhanced the antioxidant capacity of peanut seedlings, which included

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the increase of antioxidant enzymes and non-enzymatic compounds.7 These responses

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contributed to the alleviation of selenite-induced oxidant stress. Studies reported that Se alerted

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the polyphenolic compounds in tomato,8 buckwheat,9 and purple potatoes.10 We hypothesized

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that the increase of polyphenolic compounds induced by Se may also be one of the mechanisms

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of increasing Se tolerance or detoxification of excessive Se, but this is needed to be confirmed.

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Phenylpropanoids are a large group of polyphenolic compounds including phenolic acids,

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flavonoids, and lignin synthesized in plants via the phenylpropanoid pathway,11 which is one of

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the most important and best understood plant secondary metabolism pathways.12 The pathway

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starts with the general phenylpropanoid metabolism and then leads to several main branches,

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such as lignin and flavonoids biosynthesis pathways.13 There are three key enzymes involved in

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the general phenylpropanoid metabolism: l-phenylalanine ammonia lyase (PAL; EC 4.3.1.24),

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cinnamate 4-hydroxylase (C4H; EC 1.14.13.11), and 4-coumarate: coenzyme A ligase (4CL;

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EC 6.2.1.12).14 In dicotyledonous plants, the first step of the pathway is the deamination of

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L-phenylalanine by PAL to yield cinnamic acid, which is then hydroxylated to form p-coumaric

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acid by C4H. The resultant p-coumaric acid is a key phenylpropanoid intermediate which is

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either activated its coenzyme A (CoA) thioester by 4CL, or further modified to different

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phenolic acids. Derivatives of these phenolic acids can then be modified to lignin. The

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p-coumarate CoA also can undergo polymerization or modification to yield stilbenoid and

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flavonoids finally.15,

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phenylpropanoid pathway in response to biotic or abiotic stresses.17 As mentioned above, Se

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may act as a trigger for the synthesization of phenolic compouds in crops. However, the

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mechanisms of selenite on the regulation of the phenylpropanoid pathway in peanut seedlings

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have not been investigated to date. Research on the metabolism of how selenite affects

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phenylpropanoid will be useful to improve our understanding on how plant resist Se and how Se

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is accumulated in plants, and it can also be useful to help develop Se accumulated products

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which is of more nutrition and better quality. Therefore, the purpose of this study was to

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elucidate the regulation mechanisms of the phenylpropanoid pathway and the main direction of

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carbon flow in peanut seedling roots in response to low concentration of sodium selenite during

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development.

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Phenylpropanoid compounds are induced in plants via the

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Materials and Methods

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Chemicals

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Sodium selenite (Na2SeO3), p-coumaric acid, caffeic acid, and ferulic acid standards were

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purchased from Sigma Co. (St. Louis, MO, USA). Phenylalanine, and cinnamic acid were

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purchased from Aladdin biological technology Co. (Shanghai, China). Hydrogen peroxide

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(H2O2) and nitric oxide (NO) kits were purchased from NanJing JianCheng Bioengineering

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Institute (Jiangsu, China). Plant RNA isolation kit was purchased from Aidlab Company

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(Beijing, China). RNase-free DNase I and the First-strand cDNA Synthesis Kits were purchased

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from Takara (Dalian, China). FastStart Essential DNA Green Master was purchased from Roche

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(Mannheim, Germany). Ultrapure water was obtained from a Milli-Q water purification system

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(Millipore, Bedford, MA, USA). Other chemicals were obtained from local sources and were of

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analytical grade.

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Plant materials and selenite treatments

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Peanut seedlings were cultivated using the method as our previous study.7 Briefly, selected

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healthy peanuts seeds were sterilized with 75% ethanol first, and then washed with distilled

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water followed by being soaked in distilled water for 8 h at 25°C. After the soaking, the seeds

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were pre-germinated in the dark with temperature at 25°C and the relative humidity (RH) as

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90%. Twenty four hours later, the pre-germinated seeds were sowed into a sprouter with the size

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of 370 × 270 × 60 mm and the quartz sand as the culture medium. The pre-germinated seeds

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were then developed in the sprouter for 10 days at 25°C and 80% RH in the dark with different

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concentrations (0, 3, and 6 mg/L) sodium selenite irrigated to it. Samples were collected every

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24 hours from the second day to the tenth day after sowing. The roots were cut from peanut

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seedlings of each treatment and then respectively grounded into fine powder in the presence of

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liquid nitrogen. The powder samples were stored at -80°C for further analysis.

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H2O2 and NO measurement

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For measuring H2O2 and NO levels, 1.0 g sample powder of each treatment was

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homogenized in 3 mL of 50 mM K-P buffer (pH 6.5) and then centrifuged at 12,000 × g for 20

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min at 4°C. The supernatant was collected for the measurements of H2O2 and NO. All indices

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were measured according to the instructions of the test kits.

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Enzymes extraction and determination

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Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was extracted and analyzed by the

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method of Dickerson et al. (1984)18 with slightly modification. Briefly, sample powder (1.0 g

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for each treatment) was homogenized in 5 mL of 50 mM ice-cold sodium borate (pH 7.0)

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containing 2% insoluble polyvinylpolypyrrolidone (PVP), 1 mM EDTA, and 3 mM

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β-mercaptoethanol. The homogenate was filtered through four layers of cheesecloth and

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centrifuged at 12,000 × g for 20 min at 4°C, the supernatant was collected. The reaction mixture

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contained 15 M L-phenylalanine, 30 mM sodium borate buffer (pH 8.8), and 0.1 mL

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supernatant with a total volume of 3.0 mL, which were then incubated at 37°C for 1 h and

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stopped by the addition of 0.2 mL 6 N HCl. PAL activity was determined directly by

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spectrophotometric measurement of the conversion of L-phenylalanine to trans-cinnamic acid at

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290 nm. The activity was expressed on a fresh weight basis (U / g FW). 6


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The cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11) activity was assayed by the method

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described by C. J. LAMB and P. H. RUBERY (1975).19 Briefly, sample powder (1.0 g for each

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treatment) was homogenized in 5 mL of 50 mM ice-cold sodium phosphate buffer (pH 7.6)

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containing 1 mM EDTA, 3 mM β-mercaptoethanol, and 2% (w/v) polyvinylpyrrolidone (PVP).

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The homogenates were centrifuged at 12,000 ×g for 20 min at 4°C, and the supernatants were

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used as the enzyme extraction for determination of enzymes activities. The assay mixture

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contained 2 µM trans-cinnamic acid, 50 mM sodium phosphate buffer pH 7.6, 5 µM glucose

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6-phosphate (disodium salt, pmoles), 2 µM NADP (sodium salt), and 0.2 mL enzyme extraction.

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The assay mixtures were preincubated at 30°C for 5 min before the reaction started by the

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addition of the enzyme extraction. The reaction mixtures were incubated for 30 min at 30°C

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with vigorously shaking, which were then stopped by the addition of 0.2 mL 6 N HCl. Any

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resultant precipitate was removed by centrifugation at 12000 ×g for 10 min and 2.5 mL of the

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supernatant was then adjusted to pH 11 by addition of a calculated amount of 5 M NaOH.

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4-Hydroxy-trans-cinnamic acid production was measured by the absorption of this solution at

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340 nm against a reference sample lacking of trans-cinnamic acid which had been through the

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same procedure.

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The 4-Coumarate: CoA ligases (4CL, EC 6.2. 1.12) activity was assayed using a method as

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Dickerson et al. (1984).18 Briefly, sample powder (1.0 g for each treatment) was homogenized

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with 5 mL of of 50 mM ice-cold Tris-Hcl buffer (pH 7.8) containing 15 mmol/L

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mercaptoethanol, 25% glycerol (v/ v), and 0.2 g of PVP. The homogenates were centrifuged at

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12,000 × g for 20 min. The supernatant served as an enzyme source. The 4CL reaction mixtures

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contained 10 mM p-coumaric acid, 5 mM ATP, 5 mM MgCl2, 1 mM COA, 0.2 mL protein

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extracts, and assay buffer to make the volume 3 mL. The blank (reference) mixtures contained

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the same components but without COA. Enzyme activity was measured as the increase in

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absorbance at 333 nm. The extinction coefficient for 4-coumarate CoA, 23 × l03 M-1 cm-l was

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used to calculate the amount of product synthesized per minute. All samples were assayed at

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least three times and the average velocity was calculated. Activity was expressed on a fresh

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weight basis (U / g FW).

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Measurement of the total flavonoids

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The sample powder (4.0 g for each treatment) was extracted with 20 mL 80% methanol for 3

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hours at 55°C in the dark. After being centrifuged at 12,000 × g for 10 min at room temperature

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(25°C), the supernatant was collected. The residue was extracted three times at the same

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condition, and the combined supernatants were used for the measurement of total flavonoids.

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The total content of flavonoids was determined according to Zhishen et al.20 with some

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modifications. Briefly, an aliquot (2 mL) of extracts or rutin standard solution was added to 10

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mL volumetric flask containing 3 mL distilled water.0.4 mL of a 5% NaNO2 solution was added

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and mixed well. After 6 min, 0.6 mL of a 10% AlCl3 solution was added and the mixture was

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allowed to stand for 6 min. Then 4 mL of 1 M NaOH was added and mixed well again. After 10

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min, the mixture was centrifuged at 1, 2000 × g for 5 min. Absorbance of the supernatant was

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measured at 510 nm against the blank consisting of extraction solvent instead of a sample. The

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flavonoid content was calculated by the following calibration curve: A = 2.6593C - 0.0042, R² =

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0.9992, where A is the absorbance and C is the concentration of rutin standard solution. The

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total flavonoid content was expressed as milligram (mg) of rutin equivalent per gram (g) of

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fresh weight (FW).

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Analysis of bound phenolic acids by HPLC

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Bound phenolic acid preparation procedures were according to the methods described by

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Krygier and Sosulski,21 and modified in our laboratory. Briefly, after three times of extraction with

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80% methanol as described above, the pellet was dried to remove organic solvent. The residue

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which contained bound phenolic acids was initially dispersed in 4 M NaOH for 4 h in the 37°C

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shaking water bath, acidified to pH 2 with 6 M HCl, and centrifuged at 12,000 × g for 10 min. The

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supernatant was extracted three times with equal volumes of hexane to remove free fatty acids and

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other lipid contaminants. The bound phenolic acids were then extracted three times with diethyl

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ether-ethyl acetate (1:1) at a solvent to water phase ratio of 1:1. The ether-ethyl acetate extracts

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were evaporated to dryness under vacuum at 37°C, which were then redissolved in methanol. The

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samples were filtered through a 0.45-µm fluoroethylene filter for further analysis. The phenolic

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acids were analyzed using a high-performance liquid chromatography (HPLC) system with a 2996

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photodiode array detector (DAD) from Waters (Milford, MA, USA) and a column of Agilent zorbax

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300sb-C18 column (4.6 × 250 mm). The mobile phase consisted of 28% acetonitrile, 1% acetic acid,

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and 71% water. Flow rate was kept at 1 mL/min. The compounds were detected between 200 and

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400 nm with DAD. The phenolic acids were initially identified by a direct comparison of their

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retention times and absorption characteristics with the standards. The data were analyzed and

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processed using the installed Empower 4 software. 9



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Determination of lignin content

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The lignin was extracted and assayed according to the method of Fukuda, H. and A.

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Komamine (1982)22 with some modification. Briefly, sample powder (1.0 g for each treatment)

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was homogenized in distilled water with an ultrasonic wave generator. After centrifugation at

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1,000 × g for 5 min, the supernatant was discarded and the pellet was washed three times with

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95% ethanol and twice with ethanol-hexane (1:1, v/v). The washed pellet was dried to constant

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weight at 50°C. The dry samples (10 mg for each treatment) were incubated with 1 mL of the

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freshly made acetyl bromide solution (25% v/v acetyl bromide in glacial acetic acid) at 70°C for

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30 min. Then, 0.9 mL of 2 M NaOH, 5 mL of glacial acetic acid and 0.1 mL of 7.5 M

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hydroxylamine hydrochloride were added and mixed well, the total volume made up to 10 mL

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with glacial acetic acid. After centrifugation at 1,000 × g for 5 min, the absorbance of the

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supernatant was measured at 280 nm with a spectrophotometer. The lignin content was

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expressed as A280 per gram (g) of fresh weight (FW).

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Observation of root anatomical structure

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After cutting from seedlings, the peanut roots were fixed with 2.5% glutaraldehyde for 24

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hours at 4°C. The glutaraldehyde fixing solution was then removed and the samples were rinsed

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with PBS (pH 7.0) three times. After that, the samples were dehydrated successively with 20, 40,

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60, 80 and 100% alcohol (2 hours for each), and soaked in tertiary butanol at 4°C. The samples

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were freeze dried and then coated with gold using a sputter coater (ETD-2000, Beijing

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Elaborate Technology Development Ltd., China). A scanning electron microscope (SEM,

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Hitachi 3700N, Tokyo, Japan) was performed to observe the root anatomical structure and

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photograph images.

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Relative quantification of mRNA levels

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Total RNA was isolated from root using a plant RNA isolation kit (Aidlab Company,

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Beijing, China) and then treated with a RNase-free DNase I (Takara, Dalian, China) to eliminate

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the residual genomic DNA. Total RNA was converted into cDNA using the First-strand cDNA

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Synthesis Kit (Takara, Dalian, China), and the cDNA solution was taken as the template for

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quantitative real-time PCR (qPCR) analysis.

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Primer pairs for qPCR were designed using primer premier 5 software. The studied gene

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sequences of peanuts (Arachis hypogaea L.) were found in GenBank of NCBI (see Table 1 for

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details). Gene-specific primers were selected so that all the resulting PCR products had

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approximately the same size of 200 bp. The quality of PCR products were visually inspected by

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2% agarose gel electrophoresis (see Figure 1), and the generation of only one single band of the

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expected size was taken as a criterion for specificity.

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The qPCR was performed as described in our previous study.7 The relative quantification of

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gene expressions were analyzed by the 2–∆∆CT method and the results were expressed as extent

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of change with respect to control values.23 Each sample was tested in triplicate to obtain an

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average Ct value.

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Statistical Analysis

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All the experiments were carried out in triplicate, and all data were expressed as means ±

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standard deviation (SD). Statistical analysis was performed by SPSS 19 (SPSS Inc., Chicago, IL,

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USA). One-Way ANOVA with Tukey multiple comparisons test were used to determine

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differences between selenite treatment groups and control groups. The probabilities of less than

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5% (p < 0.05) were considered significant.

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Results

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Selenite up-regulated PAL and C4H gene expressions and enzyme activities

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The expression levels of phenylpropanoid genes were analyzed by qPCR. As shown in

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Figure 2a, the phenylalanine ammonialyase (PAL) gene expression levels of all treatments

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raised mildly from days 2 to 5 but raised rapidly from days 6 to 10. On day 10, the PAL

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expression levels of 3 and 6 mg/L selenite treatments were 2 folds and 2.5 folds higher than the

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control, respectively. Accordingly, the PAL activities were enhanced by selenite treatments

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when compared to the control, the trends of which were all raised mildly during peanut

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seedlings development (see Figure 2b).

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In the case of cinnamate-4-hydroxylase (C4H) gene, no significant changes of expression

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were detected earlier than 5 days for both selenite treatments and control, but the expression

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trend of selenite treatments were enhanced significantly by day 6. On the day 9, the C4H

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expression levels of 3 and 6 mg/L selenite treatments reached 2.7 and 3.5 folds of the control,

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respectively. We also found that the effect of selenite on the expression levels of C4H were

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dose-dependent, what perfectly matched with respective metabolite levels (see Figure 2c-d);

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The C4H activities in selenite treatments were significant greater than the control by day 6.

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Selenite enhanced the bound phenolic acids and 4CL activity

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Identification and evaluation of bound phenolic acids were performed by the HPLC method.

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As shown in Figure 3a-b, by comparing their retention time and absorption characteristics with

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the corresponding phenolic acid standards using a photodiode array detector (DAD), the

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separated components of the bound phenolic acids were identified to be caffeic acid, p-coumaric

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acid, and ferulic acid, respectively. The levels of these phenolic acids were presented in Figure

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3c-e. These phenolic acids were all significantly enhanced by selenite in a dose-dependent

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manner, especially, p-coumaric acid and caffeic acid biosynthesis in peanut seedling roots were

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significantly promoted by selenite treatments. As it can be seen in the Figure 3c-e, the highest

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amount of phenolic acid during development was p-coumaric acid (100-370 µg/g FW), followed

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by caffeic acid (15-40 µg/g FW), and the least was ferulic acid (3.5-14 µg/g FW). We also

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observed that selenite treatments dramatically enhanced the 4CL activities in a

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concentration-dependent manner when compared to the control (Figure 3f), which was known

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as a key enzyme catalyzing phenolic acids to their respective CoA esters.

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The effects of selenite on CAD gene expression and lignin biosynthesis

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We have noticed a considerable increase in the expression level of CAD gene in peanut

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seedlings with 3 and 6 mg/L selenite treatments, the most significant increase was observed in 6

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mg/L selenite treatment from days 5 to 10 (see Figure 4a). Accordingly, the lignin contents were

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enhanced by selenite treatments, as shown in Figure 4b, the change trends of lignin in all

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treatments and control were first decreased and then increased. Selenite treatments significantly

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enhanced the lignin formation when compared to the control during seedlings development.

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Selenite treatments caused a dose-dependent increase in lignin content from days 5 to 10.

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Associated with the results of phenolic acids and 4CL activities described above, we found that

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selenite treatments induced accumulation of lignin content with a concomitant increase of

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phenolic acids and CAD and 4CL activities.

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Selenite induced H2O2 generation and decreased NO level

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Selenite could induce reactive oxygen species (ROS) formation during peanut seedlings

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development, as shown in Figure 4c, selenite treatments (3 and 6 mg/L) caused a

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dose-dependent increase in hydrogen peroxide (H2O2) content from days 2 to 5, but the H2O2

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contents were reduced to nearly normal levels by day 6. This result was in accordance with the

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H2O2 content of peanut seedling hypocotyl in our previous research.7 We found that the high

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H2O2 levels on selenite treatments were not obviously connected to cell death in young root

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meristems, since there was no difference on the root development of peanut seedlings.

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Dramatically, the selenite-induced changes of NO levels in peanut seedlings were negatively

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correlated to the lignin contents during development. As shown in Figure 4d, Selenite treatments

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decreased NO level in a concentration-dependent manner. On day 10, the NO content of the 3

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and 6 mg/L selenite treatments were 2 and 4 folds lower than the control, respectively.

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Selenite enhanced the formation of endodermis in root

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We studied the anatomical structure changes of the roots of peanut seedlings with SEM. As

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can be seen from Figure 5, although there were no differences of external morphology between

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the normal control and selenite treatments, the anatomical structure have significant variations.

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Especially, the cells density in endodermis were different between treatments. The cells of

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endodermis in control were relative loosened, while they were arranged densely in treatment of

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3 mg/L, and higher-density in treatment of 6 mg/L (see the area of red arrows in Figure 5a-c).

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The endodermis clearly deposited as a ring in the transverse section of the selenite treatments,

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and the endodermis area of selenite treatments were much thicker than the control.

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Selenite up-regulated CHS and CHI genes and promoted the biosynthesis of flavonoids

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As shown in Figure 6a, the total flavonoids of peanut seedling roots were enhanced by 3 and

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6 mg/L selenite treatments when compared to the normal control during development, especially

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after a development of 5 days. The highest increase of total flavonoids content was found in 6

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mg/L selenite treatment on day 10. The expression level of CHS and CHI genes were presented

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in Figure 6b-c, we have observed only slight level of expression for CHS and CHI when the

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peanut seedlings were grown on normal medium supplemented with water, but relative high

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level of expression when selenite treatments were applied. CHS and CHI expression levels

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showed a faster and more significant uptrend by day 5 and reached the optimum expression

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level on day 10. The expression levels of CHS and CHI in 6 mg/L selenite treatment from days

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5 to 10 were 2-10 folds and 1.5-9 folds of the control, respectively. The effects of selenite on

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these two genes were all dose-dependentand had a similar tendency during development. These 15



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results indicated that CHS and CHI were co-regulation in response to selenite treatment.

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Discussion

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Selenite up-regulated relative gene expressions and enzyme activities in the general

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phenylpropanoid metabolism

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In plants, excessive Se may induced toxic to plants like many heavy metals do. 6 Evidence

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showed that low concentrations of toxic metals induced hormetic effects by activating plant

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stress defense mechanisms. This mechanism involve the metal sensing systems in plant cells

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and the second messengers such as Ca2+ and reactive oxygen species (ROS) that regulate gene

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expression via cellular signaling transduction cascades.

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demonstrated that selenite treatments increased ROS generation in the early stage of

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development. 7 The mechanisms of Se tolerance in peanut seedlings may be similar to the metal

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tolerance of plant involving complex cellular signaling transduction cascades. Research has

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confirmed that phenylpropanoids contribute to various aspects of plant responses against biotic

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and abiotic stimuli.17 There was evidence shows that phenolic accumulation was connected with

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metals tolerance in plants. For example, phenolic compounds were implicated as additional

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physiological mechanism of aluminum tolerance in soybean plants.

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mechanisms of Se tolerance in peanut seedlings, phenolic compounds induced by selenite

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treatments and the mechanisms were investigated in the present work. As described above, all

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flavonoids, lignins, and an immense diversity of phenolic compounds are derived from

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phenylalanine. PAL and C4H are the upstream genes belonging to the general phenylpropanoid

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metabolism. PAL is the first rate-limiting enzyme which controls the synthesis of cinnamic acid

24

In our previous study, we have

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To elucidate the

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from L-phenylalanine.26 And then the cytochrome P450 superfamily’s C4H hydroxylates

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cinnamic acid into p-coumaric acid which is finally transformed to lignin and flavonoids.14, 27 It

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has been suggested that these key enzymes play a significant role in regulating the accumulation

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of phenolic compounds in response to biotic and abiotic stresses. The increase of PAL activity

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strengthens the lignification process in soybean seedling roots.28 The enhanced flavonoid

340

production on Glycyrrhiza uralensis Fisch was associated with the elevated mRNA levels and

341

enzyme activities of PAL and C4H. 12 The increased transcription and the coordinated activation

342

of PAL and C4H in selenite treatments suggested that the downstream branches of

343

phenylpropanoid pathway would also be regulated by selenite in peanut seedlings. Therefore,

344

mechanisms of the effects of selenite on phenolic acids, lignin, and total flavonoids need to be

345

studied further in more details.

346

347

Bound phenolic acids were enhanced by selenite

348

Phenolic acids are the intermediate products of the phenylpropanoid pathway, in the

349

pathway, the further hydroxylation of p-coumaric acid leads to formation of caffeic acid, which

350

is then transferred into ferulic acid or chlorogenic acid.29 It is well known that the phenolic acids

351

exhibit strong antioxidant capacity and play important role in resistance of biotic and abiotic

352

stress.30 The increase of phenolic acids may exert the ability to scavenge the excessive amounts

353

of ROS in peanut seedlings, thus contributing to the reduction of the selenite toxicity. Bound

354

phenolic acids are also the important molecules which are involved in cell wall structure by

355

cross-linking with lignin components, they have a profound effect on the growth of the cell wall

356

and its mechanical properties,31 hence the increase in phenolic acids may also contribute to the 17



357

reinforcement of cell wall. The results of phenolic acids enhanced by selenite treatments are in

358

agreement with the previous report showing that selenite regulated the phenolics accumulation

359

on purple potatoes.10 The accumulative effects of phenolic acids in peanut seedlings induced by

360

selenite were similar to that of some metal ions. Cultivation of Lentil seedlings in the presence

361

of Cu2+ resulted in an increase in ferulic acid (156–140% of the control) and p-coumaric acid

362

(106–124% of the control). 32

363

There are three shared steps of the synthesis of lignin and flavonoids in the

364

phenylpropanoid pathway. The first two shared steps are catalyzed by PAL and C4H, the last

365

shared step is with 4-coumarate: CoA ligase (4CL) as a catalyst which helped the generation of

366

p-coumaroyl CoA, caffeoyl CoA and feruloyl CoA from their respective substrate acid.33 These

367

hydroxycinammoyl-CoA esters can be used as the important intermediates in the

368

phenylpropanoid compounds metabolism. The increase of 4CL activity suggested that selenite

369

treatments lead the flow of carbon into the branched pathways of phenylpropanoid metabolism

370

in peanut seedling roots, such as lignin synthesis pathway.

371 372 373

The enhanced formation of endodermis in roots, which regulated the selenite uptake, may attributed to the up-regulation of lignin biosynthesis mediated by H2O and NO.

374

Lignin, a complex polymeric product which is composed of differing amounts of

375

p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) subunits,34 is produced by using

376

hydroxycinammoyl-CoA esters as intermediates.11 Cinnamyl alcohol dehydrogenase (CAD) is a

377

key enzyme involved in the step of monolignol biosynthesis, which catalyzes the reduction of

18


Page 18 of 41

Page 19 of 41


378

hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols (p-coumaryl,

379

coniferyl and sinapyl alcohol, respectively).35, 36 In the last step of the lignin synthesis pathway,

380

POD catalyzes the hydroxycinnamyl alcohols polymerization that leads to corresponding lignin

381

subunits synthesis. H2O2 is a necessary substrate for the cell wall’s lignifying process catalyzed

382

by POD.26, 37 Our previous research has shown that the peroxidase (POD) activities of peanut

383

seedlings were enhanced by selenite treatments during development,

384

synthesize lignin in peanut seedling roots. This explained the decrease of H2O2 content and the

385

increase of lignin content in peanut seedling roots of selenite treatments in the later stage of

386

development (Figure 4b-c). These results also demonstrated that the lignin formation was

387

closely corresponded with the metabolism of the phenolic acids, and the activities of 4CL and

388

CAD.

7

which used H2O2 to

389

NO is a highly diffusible gas act as a synchronizing chemical messenger involved in the

390

regulation of different physiological processes in plants. At a low concentration, sodium

391

nitroprusside (a NO donor) induced soybean seedlings root growth.38 But at a high

392

concentration, inhibition of hypocotyl and internode elongation can be triggered by sodium

393

nitroprusside in the condition of darkness.39 Research also demonstrated that NO inhibited

394

activities of POD and PAL and thus regulating the synthesis of lignin and cellulose.40 These

395

results suggested that NO plays a multiple functional role in plant development. Our research

396

showed that the selenite-induced decrease of NO generation in peanut seedlings was

397

accompanied with an increase of lignin level (Figure 4b-d). We hypothesize that the NO

398

production decreases inversely regulated the lignin levels. Our finding is supported by a striking

19



399

fact revealed by Gabaldo´n et al. (2005)41 : the process of producing NO and cell wall lignifying

400

in the xylem differentiation of Zinnia elegans are two negative related metabolism. Associated

401

with the results of 4CL and CAD activities described above, we hypothesized that the increase

402

of lignin induced by selenite was regulated by the changes of NO which may act as a chemical

403

messenger mediated the 4CL and CAD activities in the phenylpropanoid pathway.

404

Plants have evolved multiple defense strategies to combating invaders, cell walls provide an

405

effective barrier to any invaders that are able to gain access to interior spaces.29 Lignin is an

406

embedding material for the cellulosic polymers of the secondary cell walls. It is also the major

407

polymer in the middle lamellae between adjacent cell walls.42 Besides, in the root of higher

408

plants, lignin functioned as a structures deposited material with which the casparian strips are

409

formed in the transverse section of endodermal cells encircling the vascular system.43

410

Endodermal Casparian strips are generally understood to be useful in the prevention of the

411

apoplastic passage (through the cell wall) of ions from the cortex to the stele.44 Because of the

412

selective function of the endodermis, plant roots are able to selectively take up both essential

413

nutrients and water from the soil, and block the passive flow of solutes in the apoplast.45 Lignin

414

is one of the most important components deposition in endodermal cells of plant root, sealing of

415

cell walls have been postulated to be one of the mechanisms explaining the role of lignins in

416

resistance.46 Therefore, lignin is thought to play an important role in the defense mechanism

417

contributing to against environmental stress. In the present work, selenite treatments

418

significantly enhanced the formation of endodermis, which may be attributed to the

419

up-regulation of lignin biosynthesis via the lignin branch of the phenylpropanoid pathway

20


Page 20 of 41

Page 21 of 41


420

mediated by the H2O2 and NO. These responses contributed to the prevention of excessive

421

selenite intake from external environment. There is evidence showed that Se concentration in

422

the xylem exudate of root was always lower than that of the external medium when tomato grew

423

in selenite medium.47 There is also evidence showed that at least part of selenite uptake by plant

424

is associated with the root metabolism.48 We hypothesize that the increase of lignin and the

425

enhanced formation of endodermis in roots may also be one of the mechanisms of regulating the

426

selenite uptake from external medium.

427 428

Selenite significantly up-regulated CHS and CHI genes and thus increasing the total

429

flavonoids by the flavonoid branch of the phenylpropanoid pathway

430

Flavonoids are one of the largest and the most diverse classes of phenolic compounds

431

exhibiting central functions in plant growth and development.49 Flavonoids are also known to

432

enhance tolerance to different environmental factors and stress conditions.50 All flavonoids and

433

other phenolic compounds are derived from phenylalanine, by the way of general

434

phenylpropanoid metabolism that produces hydroxycinnamic acids (p-coumaric acid) and their

435

activated CoA esters. Chalcone was produced from p-coumaryl- CoA and malonyl-CoAs with

436

the catalyzation of chalcone synthase (CHS) which is the flavonoid biosynthesis entry point

437

enzyme after p-coumaroyl-CoA being formed. And then the chalcone isomerase (CHI) catalyzes

438

the formation of naringenin, which serves as the common precursor for a large number of

439

downstream flavonoids.51 Therefore, CHS and CHI are the key genes encoding the early,

440

un-branched segment of the flavonoids biosynthesis pathway. The increase of total flavonoids

21



441

on peanut seedling roots in the present work were attributed to the upregulative effect of selenite

442

on these two genes, which also be closely connected with the upstream genes (PAL, C4H, and

443

4CL) of the general phenylpropanoid metabolism. These results were in accordance with the

444

former reaserch on the effects of metal on plants:

445

(Lupinus luteus L.) with Lead (Pb) increased plant roots flavonoid contents by 54%.52 As

446

flavonoids possess antioxidant properties, the protective role of flavonoids against

447

environmental stress might be associated with their ability of scavenging reactive oxygen

448

species. They counteracted the effect of oxidative burst occurring during growth with selenite,

449

thus contributing to the reduction of the selenite-induced toxicity on peanut seedlings.

evidences showed that yellow lupine

450

In summary, our study gives the first insight into the phenylpropanoid metabolism in peanut

451

seedlings during development with low concentrations of selenite treatment, which shows

452

evidences for how the peanut seedlings survived in response to selenite treatments. Selenite

453

up-regulated the relative genes expression and enzyme activities, and last enhanced the

454

phenylpropanoids including bound phenolic acids, lignin, and total flavonoids during peanut

455

seedling development. The increase generations of these phenylpropanoid compunds enhanced

456

the antioxidant capacity and stabilized and strengthened the cell structure, thus contributing to

457

the alleviation of selenite-induced toxicity. Furthermore, selenite enhanced the formation of

458

endodermis in roots, which may attributed to the up-regulation of lignin biosynthesis mediated

459

by H2O and NO, probably regulated the selenite uptake from external medium. Via regulating

460

the phenylpropanoid pathway, peanut seedlings survived in 3 and 6 mg/L selenite treatments

461

accompanied with biosynthesis of antioxidant compounds implied in plant development and

22


Page 22 of 41

Page 23 of 41


462

responded to stresses. We concluded that the phenylpropanoid pathway could be mediated by

463

selenite, which contributed to the increase of Se tolerance and Se accumulation in peanut

464

seedlings. The results founded in this study provided important evidences for the further

465

research on Se tolerance and Se accumulation in plants.

466

23



467

Acknowledgments

468

This work was financially supported by National Natural Science Foundation of China (Grant

469

No. 31270636), National Natural Science Foundation of China (Grant No. 31201330), Key

470

Science & Technology Brainstorm Project of Guangzhou (20130000202), and Program for New

471

Century Excellent Talents in University (NCET-12-0192).

472 473

Notes

474

The authors declare no competing financial interest.

475 476

24


Page 24 of 41

Page 25 of 41


477

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Applied Microbiology and Biotechnology 2011, 91, 949-956.

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52. Izbiańska, K.; Arasimowicz-Jelonek, M.; Deckert, J., Phenylpropanoid pathway metabolites promote

607

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61-67.

609

31



Page 32 of 41

610

Table 1. Quantitative Real-Time PCR Primers Used for Evaluation of Steady-State mRNA

611

Levels of PAL, C4H, CAD, CHS, CHI, and Actin genes. primers

mRNA protein

GenBank

amplicon name

sequence

accession no.

(bp)

PAL-F PAL

C4H

CAD

CHS

CHI

actin

GU477587.1

170 PAL-R

5’-GCAACTTCTTGGCGGCTTTA-3’

C4H-F

5’-ATGGTGGCTCGCAAACAA-3’

EF371920.1

193 C4H-R

5’-CAAACGCCCCAAAGTGATT-3’

CAD-F

5’-ACACCCCTCTTCAATTCGTT-3’

EU331152.1

196 CAD-R

5’-CTCACATCGTTCTTCTCCAGTC-3’

CHS -F

5’-CGGCAACGAAAGCCATAA-3’

AY192572.1

217 CHS -R

5’-TTGTTCTCCGCCAAGTCC-3’

CHI -F

5’-GGACCCTGAAATAGTGAACCA-3’

JN412735.1

176 CHI -R

5’-CAGCAGTCTCAATCTGAACCC-3’

actin-F

5’-ATTGTCTTGAGTGGTGGTTCC-3’

DQ873525.1

121 actin-R

612

5’-CTTGTCGGAGGTTCTTTCGG-3’

F：Forward primer;

5’-ACTTCCTCTCTGGTGGTGCTA-3’

R：Reverse primer. 32


Page 33 of 41


613

FIGURE LEGENDS

614

Figure 1. The PCR products from gene-specific primers confirmed by 2% agarose gel

615

electrophoresis.

616 617

Figure 2. Enzymes and relative gene expression levels of peanut seedlings with different

618

selenite treatments (0, 3, 6 mg/L) in the general phenylpropanoid metabolism. (a) mRNA

619

expression level of PAL. (b) enzyme activity of PAL. (c) mRNA expression level of C4H. (d)

620

enzyme activity of C4H. * indicates P < 0.05, ** indicates P < 0.01 compared with control at a

621

given time.

622

623

Figure 3. Effects of sodium selenite on phenolic acids and the key enzyme of the

624

phenylpropanoid pathway in peanut seedling roots. HPLC/UV trace for phenolic acid standards

625

(a) and bound phenolic acids extract of peanut seedling roots (b) which were monitored with

626

photodiode array detector from 200 nm to 400 nm, the photograps were taken at 310nm. The

627

effect of selenite treatments on p-coumaric acid (c), caffeic acid (d), and ferulic acid (e) levels in

628

peanut seedling roots. The effect of selenite treatments on the 4CL enzyme activity (f). *

629

indicates P < 0.05, ** indicates P < 0.01 compared with control at a given time.

630

631

Figure 4. Effects of selenite on lignin synthesis. Selenite induced changes in CAD mRNA

632

expression level (a), lignin content (b), H2O2 content (c), and NO content (d) in peanut seedling

633

roots during development. * indicates P < 0.05, ** indicates P < 0.01 compared with control at a

634

given time. 33



635 636

Figure 5. Anatomical structure of peanut seedling root of different selenite treatments (0, 3, 6

637

mg/L) on day 5 of development. The root anatomical structure was observed in a scanning

638

electron microscopy (Hitachi 3700N, Tokyo, Japan) at an accelerating voltage 20.0 kV.

639 640

Figure 6. Effects of selenite on the total flavonoids and mRNA expression levels of relative

641

genes on the flavonoids synthesis branch pathway. (a) Total flavonoids of peanut seedling roots.

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(b) mRNA expression level of CHS. (c) mRNA expression level of CHI. * indicates P < 0.05, **

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indicates P < 0.01 compared with control at a given time.

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Figure 1. The PCR products from gene-specific primers confirmed by 2% agarose gel electrophoresis. 298x160mm (300 x 300 DPI)



Figure 2. Enzymes and relative gene expression levels of peanut seedlings with different selenite treatments (0, 3, 6 mg/L) in the general phenylpropanoid metabolism. (a) mRNA expression level of PAL. (b) enzyme activity of PAL. (c) mRNA expression level of C4H. (d) enzyme activity of C4H. * indicates P < 0.05, ** indicates P < 0.01 compared with control at a given time. 609x422mm (300 x 300 DPI)


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Figure 3. Effects of sodium selenite on phenolic acids and the key enzyme of the phenylpropanoid pathway in peanut seedling roots. HPLC/UV trace for phenolic acid standards (a) and bound phenolic acids extract of peanut seedling roots (b) which were monitored with photodiode array detector from 200 nm to 400 nm, the photograps were taken at 310nm. The effect of selenite treatments on p-coumaric acid (c), caffeic acid (d), and ferulic acid (e) levels in peanut seedling roots. The effect of selenite treatments on the 4CL enzyme activity (f). * indicates P < 0.05, ** indicates P < 0.01 compared with control at a given time. 419x442mm (300 x 300 DPI)



Figure 4. Effects of selenite on lignin synthesis. Selenite induced changes in CAD mRNA expression level (a), lignin content (b), H2O2 content (c), and NO content (d) in peanut seedling roots during development. * indicates P < 0.05, ** indicates P < 0.01 compared with control at a given time. 604x427mm (300 x 300 DPI)


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Figure 5. Anatomical structure of peanut seedling root of different selenite treatments (0, 3, 6 mg/L) on day 5 of development. The root anatomical structure was observed in a scanning electron microscopy (Hitachi 3700N, Tokyo, Japan) at an accelerating voltage 20.0 kV. 450x450mm (300 x 300 DPI)



Figure 6. Effects of selenite on the total flavonoids and mRNA expression levels of relative genes on the flavonoids synthesis branch pathway. (a) Total flavonoids of peanut seedling roots. (b) mRNA expression level of CHS. (c) mRNA expression level of CHI. * indicates P < 0.05, ** indicates P < 0.01 compared with control at a given time. 969x219mm (300 x 300 DPI)


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Table of Contents Graphic 45x30mm (600 x 600 DPI)


Regulation of the Phenylpropanoid Pathway: A Mechanism of

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