Dynamics of total proteins during stress alleviation in tomato
infected with cucumber mosaic virus
Rana Muhammad Sabir M Tariq Corresp.,
Dildar Gogi 3
1
2
3
1
, Nighat Sarwar
2
, Imran Ul Haq
1
, Shahbaz Talib Sahi
1
, Muhammad
Department of Plant Pathology, University of Agriculture Faisalabad, Faisalabad, Pakistan
Plant protection division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan
Department of Entomology, University of Agriculture Faisalabad, Faisalabad, Pakistan
Corresponding Author: Rana Muhammad Sabir M Tariq
Email address: [email protected]
Background: Phytoalexins are the secondary metabolites produced in crop plants after the
attack of necrotizing pathogens. They are aimed to support homeostasis in stressed
plants. Proteins are also part of this regime that is manipulated according to the type of
infection or environmental stress. Chemical elicitors substitute the traditional fungicides
and safe to use. They also modify protein levels due to their direct or indirect influence on
the defense pathways. Methodology: An in vitro study was conducted on tomato seedlings
inoculated with cucumber mosaic virus (CMV). Plants were treated with salicylic acid and
BTH at different intervals to find out the alterations in the total soluble proteins. Purpose of
study was to investigate the variation in total proteins during the viral attack and role of
elicitors in this case. Results: Results indicated that protein level was initially reduced
(133.3 mg) in the inoculated plants prior to the application of elicitors. However, BTH alone
or combined with SA significantly enhanced total soluble proteins (200.3 mg) in tomato
leaves when applied on weekly intervals. Single application of Salicylic acid gave similar
response as in case of non-inoculated plants, however weekly applied SA reduced the total
proteins to much lower extent (83.6 mg) under the CMV stress. Discussion: Modification of
total proteins in a single tomato cultivar along with homogenous environmental factors
indicates the active role of chemical elicitors in the defense pathways. BTH is a structural
homologue to SA but expressed a remarkable functional contrast under viral attack
particularly in solanaceous crops. This novelty in plant science is worthy of discussion.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2105v1 | CC BY 4.0 Open Access | rec: 6 Jun 2016, publ: 7 Jun 2016
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Title: Dynamics of total Proteins during stress alleviation in tomato infected with cucumber
2
mosaic virus; escorted by application of safe chemicals.
3
Short title: Protein variation in CMV infected Tomato
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5
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7
8
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*1Rana M. Sabir Tariq, 2Nighat Sarwar, 1Imran ul Haq, 1Shahbaz Talib Sahi, 3Muhammad Dildar Gogi.
1Department
of Plant Pathology, University of Agriculture, Faisalabad.
Institute for Agriculture and Biology, Faisalabad.
3Department of Entomology, University of Agriculture, Faisalabad.
2Nuclear
*Corresponding Author’s Email: [email protected]
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ABSTRACT
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Background: Phytoalexins are the secondary metabolites produced in crop plants after the
12
attack of necrotizing pathogens. They are aimed to support homeostasis in stressed plants.
13
Proteins are also part of this regime that is manipulated according to the type of infection
14
or environmental stress. Chemical elicitors substitute the traditional fungicides and safe to
15
use. They also modify protein levels due to their direct or indirect influence on the defense
16
pathways.
17
Methodology: An in vitro study was conducted on tomato seedlings inoculated with
18
cucumber mosaic virus (CMV). Plants were treated with salicylic acid and BTH at
19
different intervals to find out the alterations in the total soluble proteins. Purpose of study
20
was to investigate the variation in total proteins during the viral attack and role of elicitors
21
in this case.
22
Results: Results indicated that protein level was initially reduced (133.3 mg) in the
23
inoculated plants prior to the application of elicitors. However, BTH alone or combined
24
with SA significantly enhanced total soluble proteins (200.3 mg) in tomato leaves when
25
applied on weekly intervals. Single application of Salicylic acid gave similar response as in
26
case of non-inoculated plants, however weekly applied SA reduced the total proteins to
27
much lower extent (83.6 mg) under the CMV stress.
28
Discussion: Modification of total proteins in a single tomato cultivar along with
29
homogenous environmental factors indicates the active role of chemical elicitors in the
30
defense pathways. BTH is a structural homologue to SA but expressed a remarkable
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2105v1 | CC BY 4.0 Open Access | rec: 6 Jun 2016, publ: 7 Jun 2016
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functional contrast under viral attack particularly in solanaceous crops. This novelty in
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plant science is worthy of discussion.
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Introduction:
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Tomato (Solanum lycopersicum) belongs to family Solanaceae. It is the 2nd important
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vegetables grown all over the world (Akhtar et. al., 2010). It has increased the interest for the
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fresh market due to its frequent use in a variety of processed foods (Georgé et. al., 2011).
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Phenolic, ascorbic acid and Carotenoids of tomato contain antioxidants properties. Its regular
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consumption can reduce the risk of prostate cancer (Giovannucci, 1999). Among the tomato
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viruses CMV is most destructive plant virus. Among the plant viruses it has broadest host range
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(Stevenson, 2004) and is serious pathogens for tomato cultivation in temperate climates
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(Kyriakopoulou et. al., 2000).
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Among the chemical elicitors, BTH can activate SAR response against various plant viruses
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including CMV in tomato, and cantaloupe (Anfoka, 2000; Smith-Becker et. al., 2003), Turnip
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crinkle virus and Tobacco mosaic virus (Lawton et. al., 1996). SA has key role in the plant
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kingdom as being important part of signal transduction. In case of plant viruses, SA can induce
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the metabolic pathways that directly or indirectly interference with two general processes of
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replication and systemic movement of in the host plant (Murphy and Carr, 2002).
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Total soluble proteins under biotic and abiotic stress indicate an active part of defense related
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compounds. They are reliable indicator of stress response in crop plants. However their
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quantification was studies during application of elicitors in the plants under stress.
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MATERIALS & METHODS:
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Tomato variety Nagina without special resistance against CMV was sown under insect free
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environment within the glass house. Eight treatments including positive and negative control
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were maintained in the pot experiment. One month old plant nursery was sown after 1 hour root
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dip into the solutions of the treatment formulations. While control plants were dip into the
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distilled water for same time. Treatment combinations were maintained as follows,
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T1= Salicylic acid weekly foliar application, T2= Salicylic acid one foliar spray post inoculation
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T3= BTH (Benzothiadiazole-7-carbothioic acid S-methyl ester) foliar application on weekly
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basis, T4= BTH single foliar spray post inoculation, T5= BTH+ Salicylic acid weekly treatment
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T6= BTH+ Salicylic acid single spray post inoculation, T7= Healthy control, T8= Inoculated
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control.
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Mechanical inoculation:
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Mechanical inoculation of plants was done after 1st foliar spray of chemical elicitors. Inoculum
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source was the infected tomato leaves with typical shoestring disease ground in 0.02M phosphate
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buffer, pH 7.4 (1 g/ml); in a pestle and mortar. Later this homogenate was squeezed through a
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very fine muslin cloth. Young leaves of 4-5 week-old healthy tomato plants were dusted with
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500-mesh carborandum powder and mechanically inoculated with the freshly extracted sap using
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cotton swabs. Later on, the inoculated Plants were rinsed gently in a stream of water to remove
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extra inoculum and kept under insect-free environment (Akhtar et. al., 2010).
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Virus Testing:
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Samples from tomato leaves were collected after the appearance of symptoms on the leaves.
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Virus testing was done through DAS-ELISA and protocols were followed as described by
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Hussain et. al. (2004) with commercial polyclonal antibodies to CMV (BIOREBA AG
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Switzerland).
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
Wells of ELISA plate were coated with CMV antibodies diluted in coating buffer at
1:200.
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
The coated plate was incubated at 40C for overnight.
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
After incubation the plate was washed with PBS-Tween 3-4 times at 5-minute intervals.
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
Then wells were filled with the sap of CMV- infected tissue extracted in extraction buffer
and two wells were filled with each of buffer, CMV negative and positive sample.
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
The plate was incubated for overnight at 4oC and washed 3 times with PBST.
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
200µl of enzyme conjugate diluted at 1:200 was added and plates incubated for overnight
at 40C followed by washing as in step 3.
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
200µl freshly prepared substrate buffer containing p- nitro phenyl phosphate (75ug/ml)
was added to each well.
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
visually for the development of yellow colour and read in reader at 405 nm.
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Incubation was done at room temperature for 30 minutes and reaction was observed

The reaction was stopped by adding 50µl 3M NaOH to each well.
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Sample Extraction and biochemical Analysis:
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For extraction and estimation of enzymes and other biochemical parameters leaf and fruit
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samples from glass house were ground in extraction buffer specific for different
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enzymes/biomolecules and centrifuged at 15,000×g for 20 min at 4°C. The supernatant was
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separated and used for assay.
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Total soluble proteins:
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For protein estimation in different parts of maize, 5 µL of supernatant and 95 µL 150 mM NaCl
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were mixed with 1.0 mL of dye reagent (100 mg). Coomassie Brilliant Blue G-250 dye was
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dissolved in 50mL of 95% ethanol and 100 mL 58% (w/v) phosphoric acid and dilute to one
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litter and the mixture was left for 5 min to form a protein dye complex. Then, the absorbance was
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measured at 595 nm (Bradford, 1976).
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Statistical analysis:
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All experiments were conducted in triplicates using completely randomized design (CRD). The
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significance was ascertained by analysis of variance and Tukey (HSD) test at p<0.05 and where
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applicable at P<0.01 using XL-STAT software.
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RESULTS:
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ELISA:
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Presence or absence of virus was confirmed by typical viral symptoms and ELISA. ELISA plate
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was assessed visually. Positive reaction (yellow colour) was observed with CMV-infected
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(symptomatic) plants but negative reaction indicated the absence of virus as in case of healthy (-
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ve) control. However, negative reaction was rare due to unchecked aphid feedings in tunnel
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throughout the cropping season. Mechanical inoculation was also successful and all treated
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samples positively indicated the presence of CMV. Colour development was due to conversion
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of p-Nitro phenyl phosphate into p-Nitro phenol phosphate. Positive and negative control
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samples were also loaded as standards available in the ELISA kit.
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PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2105v1 | CC BY 4.0 Open Access | rec: 6 Jun 2016, publ: 7 Jun 2016
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Total soluble Proteins (mg g-1 fresh weight):
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Proteins are assumed to play key role in plant metabolic system and defense activity against the
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infections. They include pathogenesis related proteins as well. According to the results protein
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level was slightly decreased after mechanical inoculation of CMV. Among the treatments,
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(B+SA)W (200.33 mg) and BW (200.33 mg) induced higher level of total proteins. While SA1
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(169 mg) demonstrated similar level of proteins; as in case of non-inoculated plants. But B1
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(113.67 mg) and SA-w (83.67 mg) reduced the total protein concentration in tomato leaves to
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much lower extent.
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DC= positive control, HC= negative control, SA= salicylic acid, B= BTH, BSA= BTH+Salicylic
acid, W= weekly sprays, 1= single spray
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Discussions:
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In the present studies we have discussed the dynamic progress of protein contents while
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alleviating the CMV infection in tomato with application of chemical elicitors. It is generally
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observed that protein contents show a great diversity while crops experience the biotic or abiotic
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stress. Our results indicated the minor decrease in total proteins during initial challenge by
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cucumber mosaic virus (CMV). Previous studies on tomato support our findings where protein
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level was reduced after salt stress artificially induced by NaCl (Doganlar et. al., 2010). Tomato
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also responds with increased level of heat shock proteins and other secondary metabolites under
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heat stress. However, increase or decrease in the protein contents is not limited to the stress but
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the genetic variations (susceptible or resistant varieties) are also an important factor that
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indicates the level of proteins in tomato (Vishwanath et. al., 2011; Amini and Ehsanpour, 2005).
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While, our conclusions were drawn from tomato var. Nagina. It had no special resistant against
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CMV.
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Moreover, our experiment was based on the application of safe chemicals under biotic stress.
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Protein levels were significantly increased in our treatments particularly BTH and BTH+SA
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when applied on weekly basis. Increased expression of proteins in BTH treated tomato plants
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under CMV infection has been previously documented that was accompanied with reduced
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vegetative characters (Kalogirou, 2012). Similarly it was observed that combined treatment of
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these elicitors also expressed higher level of proteins in tomato. Our conclusion is supported by
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the work of Thakur and Sohal (2014); investigated different concentrations of SA and BTH on
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physico-chemical parameters of Brassica juncea and B. napus up to four consecutive weeks.
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Total soluble protein increased among all the treatments particularly in combine application of
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SA and BTH. It was also suggested that the combinations of elicitors promoted growth and
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metabolic activities. Similar conclusion was drawn from the rice cultivars treated with foliar
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application of BTH and SA to combat sheath blight disease. It induced a large amount of defense
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related proteins and stress tolerance while applied in combination.
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Another significant observation from the results is minimum amount of proteins in the host
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plants treated with SA on weekly basis. Excessive application of SA might alleviate or inhibit the
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stress response imposed by the mechanical injuries and CMV or both. Previous studies on
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tomato leaves artificially wounded and treated with SA and acetylsalicylic acid (ASA) have
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shown to inhibit proteinase inhibitor synthesis (Doherty et. al., 1988; Peda-Cortés et. al., 1993).
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These chemicals were shown to be potent inhibitors of systemin-induced and jasmonic acid (JA)-
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induced synthesis of proteinase inhibitor mRNAs and proteins in tomato leaves. Such inhibitions
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are not restricted to wounding but SA also inhibits the Synthesis of Proteinase inhibitors induced
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by Systemin and Jasmonic Acid in Tomato Leaves (Steven et. al., 1995).
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Conclusion: Total proteins show significant alterations in the infected plants. However,
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chemical elicitors alter their quantitative production by interacting with defense pathways.
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Moreover, BTH show an altered response versus salicylic acid, though considered its structural
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analogue. Further investigations may help to find out the mechanisms involved in the BTH
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induced defense pathways and Molecular mechanism involved in vegetative stress imparted by
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it.
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Acknowledgment: The authors thank to Mr. Kahlid Pervaiz Akhtar (Pincipal Scientist, NIAB)
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and Dr. Amjad Hameed (Pincipal Scientist, NIAB) for coordination in the field and lab
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experiments.
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