2019-12-14 12:48:32
Jasna Milanovića, Jana Oklestkovab, Anamari Majdandžićc, Ondřej Novákb, Snježana Mihaljevićc,
Institute for Plant Protection, Croatian Centre for Agriculture, Food and Rural Affairs, Gorice 68b, 10000, Zagreb, Croatia b Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University & Institute of Experimental Botany ASCR, Šlechtitelů 27, 783 71, Olomouc, Czech Republic c Ruđer Bošković Institute, Bijenička cesta 54, 10000, Zagreb, Croatia
Introduction
Viroids are subviral pathogens characterized by small (246–401 nt), circular, single-stranded RNA molecules (Diener, 1971). Despite their lack of protein-coding capacity, viroids are capable of causing serious
diseases in numerous species worldwide. Potato spindle tuber viroid (PSTVd) is a type member of the family Pospiviroidae that provokes disease in potato, tomato and some other economically important herbaceous and woody plants (Flores et al., 2011). At the early stage of PSTVd infection in potato, growth reduction or any other symptoms may be barely visible. At the later stage, the infection culminates in systemic spread of the viroid, concomitant with the appearance of symptoms such as stunted plant growth with upright and pointed leaflets. The most prominent symptoms develop on the below-ground
part of plant, i.e. tubers that become small and spindly. Unlike plant resistance to most other pathogens, no known naturally occurring resistance to PSTVd in cultivars of potato has been established thus far (Kovalskaya and Hammond, 2014).
Although potato spindle tuber disease and its causal agent PSTVd have been well characterized (Owens and Hammond, 2009), molecular aspects related to symptom expression in potato are not fully understood. Using tomato-PSTVd interaction as a model system, it has been shown that PSTVd infection alters the expression of host genes that encode products involved in defense/stress response, cell wall structure, chloroplast function, protein metabolism, and other diverse functions (Itaya et al., 2002). An important part of the transcriptional changes found to be affected in response to PSTVd infection deals with genes involved in biosynthesis and signaling of phytohormones (Owens et al., 2012). Moreover, it has been presumed that disease symptoms are the results of viroid-induced disruptions of phytohormone homeostasis in host plants.
Phytohormones that play a central role in the activation of plant defense responses are salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid (ABA) (Alazem and Lin, 2015). Other phytohormones,
which are known mostly for their roles in plant growth and development, such as auxins, cytokinins, gibberellins, and brassinosteroids (BR), are also implicated in plant defense signaling pathways but their
role in plant defense and symptom development is less well studied.
During defense responses, phytohormone signaling pathways are not isolated, but rather interconnected with each other as well as with a complex regulatory network involving various developmental processes and defense-signaling pathways (Robert-Seilaniantz et al., 2011). Recently, it is becoming increasingly apparent that the reactive oxygen species (ROS) as second messengers participate in phytohormone signaling that coordinately regulate plant development and stress tolerance (Xia et al., 2015).
To date, only one study on potato-PSTVd interaction dealing with the detection of changes in the plant transcriptome has been reported, indicating alternations in phytohormone biosynthesis and metabolism of infected potato plants (Katsarou et al., 2016). However, no studies focusing on changes in endogenous hormone content in potato during PSTVd infection have been conducted thus far. The level of phytohormones in plant cells and tissues has been determined by the joint action of biosynthesis, conjugation, oxidation, and transport of phytohormones (Tanaka et al., 2005). Therefore, quantification of endogenous phytohormone levels in host plant tissue could be a valuable contribution toward better understanding of the role of phytohormones in the regulation of plant-viroid interactions.
In this study, we investigated changes in phytohormone and antioxidative responses that accompany systemic spread of PSTVd infection and the onset of early symptoms in susceptible potato cv. Désirée.
Changes in the endogenous content of SA, JA, cis-OPDA, ABA, IAA, and CS in leaves and tubers of potato plants were compared with the expression analysis of selected phytohormone-related genes. In addition, we estimated changes in the antioxidative status of infected plants by measuring cellular levels of hydrogen peroxide, ascorbate and glutathione, as well as the activity of antioxidant enzymes such as ascorbate peroxidases (APX, EC 1.11.1.11), guaiacol peroxidase (POX, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1), and catalases (CAT, EC 1.11.1.6). Possible effects of these changes on the development of disease-associated symptoms in potato are discussed.
Materials and methods
Plant material
Potato plants (Solanum tuberosum L. cv. Désirée) were propagated from single-eye tuber cuttings, and young plants were grown in a growth chamber at light intensity of 160 μmol m−2 s−1 for a 16-h photoperiod, a relative humidity of 70 ± 2%, and day/night temperature cycle of 20/18 ± 2 °C. Before being mechanically inoculated, the vegetatively propagated plants were tested for the absence of pospiviroids by one-step RT-PCR assay (Qiagen Hilden, Germany) using universal primers for pospiviroids (Verhoeven et al., 2004).
Mock- and PSTVd-inoculated plants were grown in separate growth chambers, at light intensity of 160 μmol m−2 s−1 for a 16-h photoperiod, a relative humidity of 70 ± 2%, and day/night temperature of 28/24 ± 2 °C, in conditions that enhance viroid replication and symptom development (Owens et al., 2012). Leaf samples (second and third from the top) were taken to confirm systemic spread of PSTVd at 8
weeks post-inoculation (wpi). Immediately after the infection was proven, samples of three to four leaves placed just above the inoculated leaves were taken for endogenous hormone, gene expression and anti-oxidant analysis. The top two leaves were omitted to minimize the developmental-related effects on phytohormone content. Tuber samples were taken at the same time. The harvested material was immediately frozen in liquid nitrogen and stored at −80 °C until further analysis.
Viroid strain and inoculation procedure
Potato plants were inoculated with the well-characterized PSTVd isolate (GeneBank accession number KF418768) (Milanović et al., 2014). For inoculum preparation, approximately 4 mg of frozen plant material infected with PSTVd was ground with a mortar and pestle in 1 mL of 20 mM sodium phosphate buffer (pH 7.4) containing 2% polyvinylpyrrolidone (Mw 10,000). Plants were inoculated when they had 3–4 fully developed leaves.
The second and third leaves of at least 5 plants per experiment were dusted with carborundum powder (silicon carbide, 400 mesh) and inoculated mechanically with the PSTVd-containing buffer suspension. Plants that were dusted with carborundum and mock-inoculated only with the buffer, served as healthy controls (at least 3 per experiment).
RNA and gene expression analysis
Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Plants infected by PSTVd were identified by one-step RT-PCR using specific primers for PSTVd (Di Serio, 2007) following a
procedure described by Faggioli et al. (2005). A PSTVd-positive control PV-0064 obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) was used to assess test validity.
To examine the expression of potato genes, real time PCR analysis was performed. DNase-treated (Thermo Fisher Scientific, Waltham, MA, USA) total RNA (5 μg) was reverse transcribed using SuperScript II Plus RNase H− Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The Primer3web (v 0.4.0, http://primer3.ut.ee/) was used to design primers
for selected transcripts, which are shown in Supplementary Table S1. Real-time PCR assays were performed on an ABI 7300 Sequence Detection System (Applied Biosystems, Carlsbad, CA) using 100 ng template cDNA, Power SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and universal cycling conditions. The potato elongation factor 1 (EFα1) gene (PGSC0003DMG400023270), which is not influenced by different stress conditions (ExpósitoRodríguez et al., 2008), was used as a reference gene. The amplification efficiency for all genes was determined by measuring the quantification cycle value (Cq) using a serial dilution of the pool of all cDNA samples.
Normalized relative quantity of the target gene was calculated based on the gene specific amplification efficiency (Hellemans et al., 2007). The ratio of the target gene and the geometric mean of the relative quantities of the reference genes were normalized against the control sample (mock-inoculated plant). The PCR amplicons of the studied genes were confirmed by sequencing (data not shown). All real-time PCR assays were repeated at least twice in three replicates.
Endogenous phytohormone analysis
The endogenous content of SA, JA, cis-OPDA, ABA, and IAA were simultaneously quantified in MeOH extracts pre-cleaned by microscale solid-phase extraction on silica-based aminopropyl matrix (Varian, Darmstadt, Germany) by ultra-high pressure liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) using an ACQUITY UPLC® I–Class System (Waters, Milford, MA, USA) and a triple quadrupole mass spectrometer XevoTM TQ–S MS (Waters MS Technologies, Manchester, UK), as previously described (Floková et al., 2014). In a separate set of samples, the extraction and purification of
endogenous brassinosteroid castasterone (CS) followed by its quantifi- cation by UHPLC-MS/MS were performed as described by Oklestkova et al. (2017). The endogenous contents of phytohormones quoted are mean values from three extracts of two experiments.
Hydrogen peroxide, ascorbate and glutathione analysis
The hydrogen peroxide (H2O2) content in leaves was determined according to the method described by Mukherjee and Choudhuri (1983), using a BioSpec-1601 spectrophotometer (Shimadzu, Kyoto,
Japan). The ascorbate content was assayed using the method described by Gillespie and Ainsworth (2007). The reaction mixture was measured at 525 nm, and the amount of total and reduced ascorbate was calculated from the calibration curve prepared with the standard L-ascorbic acid (Merck, Munich, Germany). Oxidized ascorbate (DHA) is calculated as the difference between total (AA + DHA) and reduced (AA) ascorbate. The glutathione content was assayed following the method described by Smith (1985). The reaction mixture was measured at 412 nm, and the content of total and reduced glutathione in the samples were calculated from the calibration curve prepared with the standard GSSG (Merck, Munich, Germany). Concentration of the oxidized glutathione (GSSG) was calculated as the difference between total (GSH + GSSG) and reduced (GSH) glutathione. The assays were carried out in triplicate.
Antioxidant enzyme activity assays
For total soluble protein extraction, approximately 500 mg of frozen tissue samples was ground to a fine powder in liquid nitrogen and homogenized in 1 ml of extraction buffer solution (50 mM phosphate buffer containing 10 mM EDTA and 2% (w/v) polyvinyl polypyrrolidone). The samples were centrifuged for 30 min at 16,000 x g at 4 °C, and the supernatant was stored at −80 °C for further analysis.
Total soluble protein concentration for all samples was determined according to Bradford (1976). The activity of soluble SOD was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium following the method of Giannopolitis and Reis (1977). POX activity was determined according to the method of Chance and Maehly (1955). APX activity was measured according to
Nakano and Asada (1981). CAT activity was determined by the stable complex formed by ammonium molybdate and H2O2 (Luhová et al., 2003), using a correction factor to exclude the interference that arises from the presence of amino acids and proteins in the sample that contains catalase enzyme (Hadwan and Abed, 2015). All enzyme activity assays were carried out in triplicate.
Photosynthetic pigment quantification
Total chlorophylls and carotenoids were extracted from lyophilized leaf tissues in 80% acetone and the absorbance of acetone extract was measured at 647, 664, and 453 nm. The concentration of chlorophyll a and b, total chlorophyll, and carotenoids (mg g−1 dry weight) was calculated using formulae given by Lichtenthaler (1987). The concentrations of photosynthetic pigments quoted are mean values from four extracts of two experiments.
Statistical analysis
Each experiment had at least three mock-inoculated and 5 PSTVd infected plants, and was replicated twice. The values are presented as mean ± standard error. The Student’s t-test was used to calculate the significant differences between mock- and PSTVd-inoculated plants. A probability of p ≤ 0.05 was considered significant.
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1-(3-chloro-2-fluorophenyl)-1H-1,2,3-triazole-4-carboxylic acidCatalog No.:AA01A4PU CAS No.:1039876-00-0 MDL No.:MFCD11192456 MF:C9H5ClFN3O2 MW:241.6063 |
2-Amino-4-chloro-3-fluorobenzoic acidCatalog No.:AA003A5S CAS No.:1039878-71-1 MDL No.:MFCD11193652 MF:C7H5ClFNO2 MW:189.5715 |
4-[(4-iodophenyl)amino]butanoic acidCatalog No.:AA01AJSL CAS No.:1039879-15-6 MDL No.:MFCD11194074 MF:C10H12INO2 MW:305.1123 |
3-(2-Ethylphenyl)-2-propenoic acidCatalog No.:AA008RNJ CAS No.:103988-23-4 MDL No.:MFCD06205460 MF:C11H12O2 MW:176.2118 |
2,3-dimethyl-2,3-dihydro-1-benzofuran-5-carboxylic acid, Mixture of diastereomersCatalog No.:AA01EL0H CAS No.:103988-32-5 MDL No.:MFCD31616054 MF:C11H12O3 MW:192.2112 |
4,7-Dimethyl-1H-indole-2-carboxylic acidCatalog No.:AA007F54 CAS No.:103988-96-1 MDL No.:MFCD02664423 MF:C11H11NO2 MW:189.2105 |
7-chloro-8-fluoro-1,2,3,4-tetrahydroquinolineCatalog No.:AA01C1W5 CAS No.:1039881-41-8 MDL No.:MFCD11192412 MF:C9H9ClFN MW:185.6259 |
[4-(dimethylamino)-3-nitrophenyl]methanolCatalog No.:AA01A6O4 CAS No.:1039882-00-2 MDL No.:MFCD11191404 MF:C9H12N2O3 MW:196.2032 |
3-(chloromethyl)-5-(2,3-dichlorophenyl)-1,2,4-oxadiazoleCatalog No.:AA01BHUP CAS No.:1039885-60-3 MDL No.:MFCD11189767 MF:C9H5Cl3N2O MW:263.5078 |
4-(2-methoxy-2-oxoethoxy)-3,5-dimethylbenzoic acidCatalog No.:AA019YPP CAS No.:1039886-43-5 MDL No.:MFCD11188423 MF:C12H14O5 MW:238.2366 |
6-Ethoxy-1h-indole-2-carboxylic acidCatalog No.:AA008RNH CAS No.:103989-09-9 MDL No.:MFCD02664460 MF:C11H11NO3 MW:205.2099 |
2-Oxazolidinone, 3-(4-acetylphenyl)-Catalog No.:AA007WS3 CAS No.:103989-12-4 MDL No.:MFCD02946492 MF:C11H11NO3 MW:205.2099 |
(2-methylnaphthalen-1-yl)boronic acidCatalog No.:AA003HN1 CAS No.:103989-84-0 MDL No.:MFCD03452758 MF:C11H11BO2 MW:186.0148 |
3-(2-chlorophenoxymethyl)-1-benzofuran-2-carboxylic acidCatalog No.:AA01A9F5 CAS No.:1039891-11-6 MDL No.:MFCD13381158 MF:C16H11ClO4 MW:302.7091 |
3-[(thiophen-2-ylsulfanyl)methyl]-1-benzofuran-2-carboxylic acidCatalog No.:AA019Y3H CAS No.:1039891-26-3 MDL No.:MFCD12469026 MF:C14H10O3S2 MW:290.3574 |
(3-Fluoro-4-[4-(pyrimidin-2-yl)piperazin-1-yl]phenyl)methanamineCatalog No.:AA019WRN CAS No.:1039891-92-3 MDL No.:MFCD12913219 MF:C15H18FN5 MW:287.3353 |
1-[4-(Aminomethyl)-2-fluorophenyl]piperidin-4-olCatalog No.:AA019VDV CAS No.:1039892-60-8 MDL No.:MFCD11191685 MF:C12H17FN2O MW:224.2746 |
[4-(4-ethylpiperazin-1-yl)-3-fluorophenyl]methanamineCatalog No.:AA01ABEN CAS No.:1039892-84-6 MDL No.:MFCD11191697 MF:C13H20FN3 MW:237.3164 |
4-(tert-butoxy)pyridin-3-amineCatalog No.:AA01A1KV CAS No.:1039893-67-8 MDL No.:MFCD11196185 MF:C9H14N2O MW:166.2203 |
[4-(2,3-dichlorophenoxy)phenyl]methanamineCatalog No.:AA01AKOB CAS No.:1039894-09-1 MDL No.:MFCD11191753 MF:C13H11Cl2NO MW:268.1385 |
