Proteomics Related With Pathogenesis of Tomato and Maize Plants Treated With Bio Products and Salicylic Acid

8/11/2019 Proteomics Related With Pathogenesis of Tomato and Maize Plants Treated With Bio Products and Salicylic Acid

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PROTEOMICS RELATED WITH PATHOGENESIS OF TOMATO AND MAIZEPLANTS TREATED WITH BIO PRODUCTS AND SALICYLIC ACID

Sarah Boyd Lade

University of Lleida

tutor

Vicente Medina


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This project is approved by Vicente Medina, University of Lleida 2011

Vicente Medina


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INDEX

Summary and key words 4Special Thanks 5Table Key 6

Figure Key 7Acronyms and Abbreviations Key 9

I. Project Outline 10

1. Goals 102. Contents 103. Overall Objectives 104. Work Plan 11

II. Introduction 12

1. Background Information 122. The Process of Pathogenesis: Bacteria, Fungus and Virus 123. Resistance Pathways 134. Bio products 145. Salicylic Acid 156. Summary of Recognized families of PRs and Related proteins 157. Carbon and Oxygen Isotopes 16

III. Study 1 17

1. Study Design 172. Material and Methods 183. Results 224. Discussion 375. Conclusions 41

IV. Study 2 42

1. Study Design 422. Material and Methods 433. Results 49

4. Discussion and Conclusions 64

V. Bibliography 67

VII. Annexes 72


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SUMMARY AND KEY WORDS

This project explores resistant protein (PR) expression, the plant defense system and induction pathwaysof these PRs by bio products and salicylic acid. Tomato and maize plants were used as objects ofexperimentation and were first treated with three different bio stimulators and analyzed via western blot

and proteomics to deduce PR-2 and PR-3 protein expression and the significance of their apparition. Itwas found that all three bio-stimulators augment PR expression in tomato plants. Bio stimulator 2induces a high PR-2 level and bio stimulator 3 induces a high PR-3 level.

Next, plants were subject to three different concentrations of salicylic acid (SA) application, 0.2g/L,0.5g/L and 0.8g/L and both PR and physiological parameter data was collected. The PR portion of the

project is still unfinished, as time was a constraint in completion of the proteomic and western blot procedures. The physiological tests showed us that there is a change in photosynthetic activity in all plants one week after treatment, but there is not a significant difference between treatment groups per se.It was clear that the control group had the lowest stomatal conductance of all, and 0.8g/L, the highest.

Conclusions remain in concrete as plants treated with 0.8g/L SA are harmed foliar-ly and undergo seriousshock at day 2, but fare the best in terms of stomatal conductance after one week. In general, thesignificant difference of overall stomatal conductance of 0.2g/L or 0.5g/L when compared with thecontrol, was the same.

Key words: Salicylic acid, proteomics, western blot, bio stimulators, induction pathways, PRexpression, PR-2, PR-3, tomato, maize, physiological parameters.


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SPECIAL THANKS

Dr. Vicente Medina from the ETSEA Department of Vegetable Production and Forest Science (DPVCF)designing the following experiment. Without your knowledge and help, this experiment would not have

been a success or even have existed.

Dr. Tania Falcioni for expertise in the related fields and for endless advice on the experimental design.Also, for always assisting when called on.

Isabel Snchez Lpez from Faculty of Medicine in the Proteomic and Genomic Service of the Universityof Lleida, for offering her previous studies for analysis in this project. Also, for advising us in the proper

protocol for protein extraction.

Dr. Juan Pedro Ferrio from the ETSEA Department of Silviculture for spending the time to conductnumerous physiological measurements and then help to analyze the results.

Clara Nierga Parra who worked diligently in the laboratory under a grant to share her extensiveknowledge base of western blot procedure. I could not have done this project without her lessons.

The Univeristy of Lleida for facilitating the infrastructure necessary to successfully run and complete thisexperiment in the greenhouses and the laboratory.


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TABLE KEY

Table 1. Protein Identification of Tomato at T=1 29

Table 2. Protein identification for maize at one weeks time, T=1 30

Table 3. Protein Quantification 33

Table 4. Experimental design for salicylic acid treatments to tomato and maize plants. 42

Table 5. IRGA photosynthetic readings for Tomato (Mean Standard Error) 50

Table 6. IRGA photosynthetic readings for maize. (Mean +/- Standard Error) 58

Table 7. Western blot resolving and stacking mixtures 75

Table 8. Protein quantities and sample codes 77

Table 9. Sample preparation 83

Table 10. Stock solution measurements for 3 SDS-page gels 84

Table 11. Protein extraction data - Tomato, Time = 0 days 91

Table 12. Protein extraction data - Tomato, Time = 48h 92

Table 13. Protein extraction data - Tomato, Time = 7days 92

Table 14. Tomato SPAD readings 95

Table 15. Maize SPAD readings 96


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FIGURE KEYFigure 1. Overall Project Design 11

Figure 2. Project 1 Design 18

Figure 3. Tomato Time = 0 days 23

Figure 4. "Spot" Table Tomato Time = 0 days 23

Figure 5. Tomato Time = 1 week 24

Figure 6. "Spot" Table Tomato Time = 1 week 25

Figure 7. Maize Time = 0 days 26

Figure 8. "Spot" Table Maize Time = 0 days 26

Figure 9. Maize Time = 1 week 26

Figure 10. "Spot" Table Maize T=1 27

Figure 11. Western blot results Membrane 1 32

Figure 12. Western blot results membrane 2 32

Figure 13. Comparative analysis of PR expression in each sample (value of BE3T1 is not considered because wasvery far out of the range of over expression) 34

Figure 14. Comparative analysis of PR expression in each sample - value of BE3T1 is considered. 35

Figure 15. Western blot of the proteome PR-2 36

Figure 16. Western blot of the proteome PR-3 36

Figure 17. Proteome of PT215 (BE3T1-3) 36

Figure 18. Analyzing molecular weight of PR-2 and PR-3 spots via proteomic and western blot gels 40

Figure 19. Tomato plant time and treatment groups 45

Figure 20. Maize plant time and treatment groups 45

Figure 21. 90% LSD Scatterplot analysis of gs variance, where Time = 0 days 51

Figure 22. Time = 0 days, control plants (L) and 0.2 g/L salicylic acid treatment plants (R) 52

Figure 23. 90% LSD scatterplot analysis of gs variance, where Time = 2 days 52

Figure 24. Time= 2 days. Comparing control plant (Left) with treatment group 0.8 g/L salicylic acid (Right) 53

Figure 25. Time =2 days. Top: Control (Left), 0.2g/L salicylic acid (SA) (Right). Bottom: 0.5g/L SA (Left),0.8g/L SA (Right) 54
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Figure 26. 90% LSD scatterplot analysis of gs variance, where Time = 7 days 55

Figure 27. Comparing salicylic acid treatments to tomato at 2 and 7 days, considering standard error 56

Figure 28. Tomato treated with salicylic acid, mean gs by day 57

Figure 29. 90% LSD scatterplot analysis of variance for gs, where Time = 0 days 59

Figure 30. Maize, T= 2 days. Al treatment groups: Control (Left), 0.2 g/L, 0.5 g/L and 0.8 g/L 60

Figure 31. Comparing maize treatments at 2 and 7 days, considering standard error 60

Figure 32. Maize treated with salicylic acid, stomatal conductance by day 61

Figure 33. Maize plants at day 7, control (Left), 0.2g/L salicylic acid (SA), 0.5g/L SA, 0.8g/L SA (Right) 62

Figure 34. Tomato SPAD chlorophyll readings, comparing different salicylic acid treatment groups on day 2 62

Figure 35. Comparative analysis of average SPAD readings by day, tomato treated with salicylic acid 63

Figure 36. Tomato plants (var Boludo) in the growing chamber. The 4 leaf development stage 72

Figure 37. Transplanted tomato plants at the time of bio-product treatment 72

Figure 38. Example of "matching" proteins on proteomic gel 73

Figure 39. PR proteins induced by Samsun tobacco by TMV infection (Bol et al. 1990) 85

Figure 40. Example of tomato light chamber lighting inconsistency 88

Figure 41. IRGA Model -LCA 4 photosynthetic activity reader 88

Figure 42. SPAD chlorophyll meter model 88

Figure 43. Salicylic acid method of application 89

Figure 44. Tomato plant harvest and flash freeze in liquid nitrogen 89

Figure 45. Ball mill grinding method 90

Figure 46. Ball mill powder to be used for isotope analysis 90

Figure 47. Plants on day 0 (Left) and on day 7 (Right) 94

Figure 48. Day 2 (Left) and Day 7 (Right) 94

Figure 50. Control (Left) and 0.2g/L (Right) 95

Figure 52. Control (Left) and 0.8g/L-Plant 1 (Right) 95

Figure 49. Control (Left) and 0.5g/L (Right) 95

Figure 51. Control (Left) and 0.8g/L-Plant 2 (Right) 95
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ACRONYMS AND ABBREVATIONS KEY

APX1 ascorbic peroxidase

ATP Adenosine triphosphateDPVCF Departament de Producci Vegetal i Cincia ForestalETSEA Escola Tcnica Superior dEnginyeria Agrri aGADPDH Gliceraldehyde 3-phosphate dehydrogenaseHPLC High performance liquid chromatographyISR Induced systemic resistancekDa kilodaltonLAR Local acquired resistanceLC-MS/MS Liquid chromatography-mass/mass spectrometryMALDI-TOF (MS) Matrix-Assisted Laser Desorption/Ionization - Time-Of-FlightMAMPs/PAMPs - microbe associated (or pathogen associated) molecular patternsMDMV Maize dwarf mosaic virus NADPH Nicotinamide adenine dinucleotide phosphate

NF - Nod factor PAL phenylalanine ammonia-lyasePSI Photosystem IPRs Proteins related with pathogenesisPRR - pattern recognition receptorsPTI - PAMP triggered immunityPVCF - Produccin vegetal y ciencias forestalesPVX Potato virus X RuBisCO Ribulose-1,5-bisphosphate carboxyl oxigenaseSA Salicylic acidSAR Systemic acquired resistanceSR Systemic resistanceSGP Servicio de Genmica y Protemica de la UdL

TMV Tobacco mosaic virusTPI Triosephosphate isomeraseUdL Universidad de Lleida


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I. PROJECT OUTLINE

1. Goals

To analyze and replicate two anterior studies conducted in the Department of PVCF at UdL in agreement

with a company. The two experiments are distinct but may have common conclusions and discussionitems that can be extracted from the findings of both experiments.

This study also served to extend the anterior studies by reproducing and testing all methods used in theWestern Blot procedure. Proteomic methodology was not reproduced in this study due to the costlynature of the procedure.

A final goal of this project was to compile and perform a succinct series of procedure for physiologicalanalysis of tomato and maize plants treated with salicylic acid.

2. Contents

This project contains background information and research related to resistant protein (PR) expression,the plant defense system and induction pathways of these PRs by bio products and salicylic acid. Goals,

procedures (materials and methods) and results from anterior proteomic and western blot projects are presented and joined. In the case of the western blot experiment, all methodologies were reproduced byour team to wholly understand the procedure and so that any modifications to the protocols could beelaborated. Conclusions and discussion unite the studies and their findings.

In the second part of the project, all methodologies regarding plant production, salicylic acid applicationand physiological parameter studies were created or revised. This part of the project is unfinished, as

time was not allotted to complete the proteomic and western blot procedures; however, conclusions could be drawn from results obtained from physiological tests.

Final conclusions unite all completed parts of the two projects thus far. Closing remarks project futurestudies related with the project's conclusions.

3. Overall Objectives

1. Connect studies and conclusions about treated plants' protein contents and physiological reactions.2. Comment on the effect that the application of certain bio products and salicylic acid to tomato and

maize plants has upon the PR-2 and PR-3 expression in the proteome.3. Establish an ideal concentration of salicylic acid application to tomato and maize plants.4. Analyze the effects of salicylic acid and bio products on protein contents (and defense pathways) intomato and maize plants and compare the metabolic processes that are altered.


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4. Work Plan and Project Design

The following graphic depicts the evolution of the project platform. The preliminary project (1), was the proteomic study of tomato and maize plants treated with 3 different bio products. The same bio product

treated plants were used in a Western Blot analysis to detect the quantity of proteins expressed in eachtime block.

Figure 1. Overall Project Design

STUDY 1(Previous)

Proteomic study of plants treated with3 Bio products

(A company agreement)

STUDY 2

Proteomic and PR study of plantstreated with different concentrations of

Salicylic Acid

PR study of Bio product treated plantsutilizing Western Blot Procedure

Physiological Parameters: photosyntheticactivity, chlorophyll content and total

organic matter

STUDY 3

Proteomic and PR study of pathogeninfected plants treated with Salicylic

Acid and taking into consideration all

Physiological parameters

Task:

Verify PR study methodology and results

Conclude Study 1 by discussing Proteomic and PR study results

Design experiment utilizing Salicylic Acid, but leave all other parameters equal

ADD Physiological Parameters

Leave Study 2 ready to conclude in the

next phase - all samples are ready for Proteomic and analyze PR results


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II. INTRODUCTION

1. Background Information

A plant has both biotic and a biotic defense mechanisms. When a plant is confronted with a pathogen, or

a threat to its health, these mechanisms are induced to defend the plant from harm. Unlike theconstitutive defense, induced defense mechanisms are only activated by a pathogen or related attack(Collinge et al., 2001). These mechanisms of defense many times act together in order to detain theadvance of a provoked pathogen. Examples are cellular death caused by the hypersensitive reaction, theaccumulation of secondary metabolites with antimicrobial activity, such as phytoalexins, the productionof reactive oxygen species, the accumulation of hydrolytic enzymes and the synthesis of pathogenesisrelated proteins (PRs). An increase in enzyme activity during infection has also been commonlyobserved. These enzymes are responsible for the synthesis of phenylpropenoid compounds such as

phenylalanine ammonia-lyase (PAL) or intermediated flavanoids known for their antifungal andantibacterial capacities (Gomez-Vasquez, et al., 2004).

2. The Process of Pathogenesis: Bacteria, Fungus and Virus

A number of biochemical and physiological changes are associated with pathogenic infection (Low andMerida, 1996). Infection relies on the interaction between the gene products of the plant and the pathogenon the cellular and molecular levels. Successful evasion of a hosts surveillance system and subsequentactivities of metabolites of the pathogen (enzymes and toxins) encoded by pathogen genes characterizethe initial infection and then counteract the effects of various defense-related antimicrobial compounds

present already or produced by the host plants (Narayanasamy, 2008). The process of pathogenesis for bacterial, viral and fungal infections are similar, yet distinct.

Plant viruses enter the plant via a cell entering the plant or via a vector carrier that acts as a vehicle oftransmission. The virus particles multiply at the site of infection (Soto, et al., 2009). They are visiblyapparent by means of localized symptoms such as necrotic spots on the leaves or mottling. The virus thenusually spreads systemically throughout the plant, either in the vascular system, or directly from cell tocell. The plant's response system to this type of infection is referred to as the 'hypersensitive response'which is manifested as: the synthesis of new proteins (PRs), the increase of production of cell wall

phenolics, the release of active oxygen species, the production of phytoalexins and the accumulation ofsalicylic acid. The most challenging obstacle in viral spread within a plant is crossing of the plant cellwalls (Cann, 2009).

Studies performed in bacteria related plant pathogen interactions indicate that the rst -line of plantdefense is triggered upon the recognition of general elicitors, known as microbe associated (or pathogen-associated) molecular patterns (MAMPs/PAMPs), by plant trans membrane pattern recognition receptors(PRR). This recognition results in PAMP-triggered immunity (PTI) or basal defense, that negativelyaffects the progress of bacterial infection before the microbe gains a hold in the plant. In general,

bacterial infection in plants is two-sided. It can be detrimental or beneficial, depending upon the type of bacteria which is the cause of infection. Phyto pathogenic bacteria, or detrimental bacteria, enter plant


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tissue either by wounds or natural openings, such as stomata, and occupy the apoplast of plant tissues orthe xylem where they multiply and spread. This process induces the activation of hydrolytic enzymes andtoxins in the plant. Rhizobial infection (PGPR), on the other hand, causes the formation of nitrogen-fixing nodules on the roots of the plant. The process of infection by this type of bacteria involves mutualsecretion and correct recognition of several signal molecules by the plant and the bacteria alike.

Flavanoids excreted by the plant induce the bacterium to produce a lipo-chito-oligosaccharide nodulationsignal known as the Nod factor (NF) (Britannica).

Fungus, or Saprophytes, also affect plants via entrance through plant openings or damaged plant material.If this option does not exist, then they have to ability to colonize plants by excreting a cocktail ofhydrolytic enzymes, including cutinases, cellulases, pectinases and proteases. These enzymes weaken thecuticle and epidermal cell walls of the plants (Knogge, 1996). Fungal infection generally causes wilting,

browning and dropping of plant material. Upon colonization of the plant cells, the fungus excretes toxinsor plant hormone-like compounds into the plant which manipulate the plants physiology to the benefit ofthe pathogen. These toxins are usually plant specific, and can simply kill plant cells for the purpose offungal nutrient uptake, or they can redirect cellular machinery through the production of specific

phytotoxins (Knogge, 1996).

3. Resistance Pathways

An infected plant usually develops resistance dominated by local acquired resistance (LAR). Thisresistance manifests 2 or 3 days after the primary infection and is restricted in the zone which surroundsthe initial sites of infection. LAR leads to the activation of defense systems throughout all of the plant(similar to a vaccination) and after some days, the resistance is able to manifest is non-inoculated parts ofthe plant. This resistance is also known as Systemic Acquired Response (SAR). This system ofresistance could be related to the expression of resistance genes and with the accumulation of PRs

(Pieterse and van Loon, 1999) , in this case then, it can be said that Systemic Acquired Response (SAR) isdifferent than the immune response in mammals, specific in most cases, and independent of the inductororganism (Ryals et al., 1996).

Systemic Acquired Resistance (SAR)

The first SAR studies were conducted by measuring the plant/virus interaction in tobacco andArabadopsis plants infected with Tobacco Mosaic Virus (TMV) (Glazebrook et al., 1997). Theinvestigation conducted on the model plants indicated that salicylic acid (SA) is the molecule with themost evidence of participation in the SAR pathways (Mauch-Mani and Metraux, 1998) and this"protection" is correlated with its increased levels in both local and system acquired response pathways(Lawton et al., 1995) . From these studies, it was proven that SAR was not only able to stimulate plantresistance to pathogens, but also to different molecular inductors that are called "bio-stimulators".

System Acquired Response (SAR) and the proteins associated in the process have been identified to actindependently from other plant resistance responses. There is evidence to prove that the proteins encoded

by the SAR marker genes are directly related to pathogen resistance in plants, though not all genes


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expressed during the response to pathogen resistance are involved in SAR (Ryals et al ., 1996). SARgenes are distinct and are expressed to a stronger degree when resistance within the plant is maintained.What more, SAR marker genes have been identified to be induced even in unaffected plant tissue. Due tothis, expression in the pathogen-less environment of this experiment will lead to clear gene and proteinexpression. Monocots (like maize) and dicots (such as tomato) have different gene expressions when

affected with pathogens, but many genes homologous with SAR genes expressed in dicots are apparent inmonocot species as well. It had not been determined whether the induction of these genes can be directlycorrelated to the onset of SAR in the species tested. (Nasser, et al., 1988; White et al ., 1987).

Induced Systemic Resistance (ISR)

Another type of SR develops from the colonization of the plant's roots by rhizophere- dwellingorganisms, particularly plant growth promoting rhizobacteria (PGPR), and is known more specifically asInduced Systemic Response (ISR). Mainly, its metabolic route is different (Madriz-Ordenana, 2002).The use of these combined with arbuscular mycorrhizae (AMF) in the induction of SR is the most recent.The genus Rhizobium spp. and Azospirillum spp. and the mychorrizal fungus of the Glomus spp. genusare the most utilized (Bashan, 1998) and some have been efficiently isolated and multiplied, thus

permitting the formulation of inoculants for their application at the production scale. PGPR are not only biologic control agents that facilitate nutrient uptake but they also produce phyto hormones (Montesinoset al., 2002), inducing an increase in enzymatic activities such as PAL, peroxidases polyphenoloxidase,B-1,3-glucanase and chitanase (van Loon and Bakker, 2005, 2006).

According to Ruz et al. (2004), understanding the natural mechanisms by which plants have the capacityto defend themselves can eventually lead to the production of plants with higher levels of resistance.With this as the principle objective, we thus plant this project, from the time when the plant receives thestimulation signal until the time that proteins are synthesized in response to the stress. This takes place in

different processes and like existing knowledge of the stress factor, synthesis of secondary metabolitesthat act as transduction signals, gene expression and PR synthesis, it needs to be elucidated.

ISR is independent of salicylic acid but involves jasmonic acid and ethylene signaling. On the molecularlevel, ISR induces a set of genes distinct from the PR genes, while SAR induces a set of pathogenesisrelated proteins (Siddiqui)

4. Bio-Products

To better understand the action of three bio-products created by the company, the first part of thisexperiment has been designed to determine the possible use of three bio products to control phyto

pathogenic agents. The study examines proteins elicited by the application of these products to twodifferent plant species: tomato ( var. Boludo ) and maize ( var. B-73 ). Protein contents in both species wereanalyzed by separating them via bi-dimensional gel electrophoresis and then identifying them with massspectrometry MALDI-TOF. To complement this study, foliar proteins were extracted as well andanalyzed via the Western Blot.


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The original experiment analyzed the effects of repeated application of the products over the course of amonth, though this paper will only examine the effects of the products over a two week time frame.

5. Salicylic Acid

Salicylic acid is synthesized naturally in plants in response to mechanical damage, necrosis and oxidativestress. Compounds resulting from the degradation of cells or cell walls might be involved in eliciting thesystemic signal and ISR can thereby be induced by different types of pathogens or antagonistic invaders(Heil et al., 2002). As stated above, salicylic acid (SA) must accumulate to some degree endogenously orexogenously in order for SAR to take place. The exact mechanisms, however, by which SA induces SARis unknown but it has been hypothesized that SA induced enzyme inhibition activities have an effect inSAR signaling (Ryals, 1996). It is known that SA is considered a key component in defense signaltransduction and induces a full set of systemic acquired resistance genes (Halim, et al. , 2006; Maleck, etal. , 2000). The exogenous application of SA may influence physiological plant processes such astranspiration rate (E) (Larque-Saavedra, 1979), stomatal closure (Rai, et al., 1986) membrane

permeability (Barosky and Einhellig, 1993), growth and photosynthesis (Arfan, et al. , 2007; El-Tayeb,2005; Gunes, et al., 2007) and antioxidant capacity (Ananievaa, et al., 2004).

The activation of various PRs proteins in plant tissues is a major biochemical and molecular event when plants are subjected to pathogen exposure. Induction is achieved through many signaling pathwayelements, including different receptor components or chemical elicitors such as salicylic acid (SA),ethylene, jasmonic acid and systemin (Ward, et al. , 1991; Xu, et al., 1994; Maleck et al., 2000; Campos etal., 2002).

6. Summary of relevant recognized families of PRs and related proteins

'Pathogenesis-related proteins' is a collective term relating to all microbe-induced proteins and theirhomologues, including enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase and

polyphenoloxidase which are generally present constitutively and only increase during pathogenicinfection. There exist a number of enzyme activities that increase in response to pathogen attack andwhich also may play a role in plant defense, but are not considered PRs, so the term 'inducible defense-related proteins' is used to classify them. These proteins are not detectable in healthy tissues thoughinduction at the protein level is demonstrated after infection by one or more pathogens. Thus, inducibledefense related proteins include both PRs and those defined in the anterior. Note that the term 'defenserelated' refers to the fact that these proteins are induced in association with resistance responses but doesnot by itself imply functional role in defense. Due to the fact that some of these proteins have potentialanti-microbial activities, a role in resistance to pathogens is plausible, but not currently accepted (VanLoon, et al., 2006).

Another manner of expression of PR genes is in 'primed' plants. Priming refers to the latent state which a plant will maintain despite being exposed to an inducing stimulus. This situation leads to an earlier andstronger expression once the plant actually responds to an antagonistic pathogen invasion; in comparisonto non-induced plants (Heil et al. , 2002; Verhagen et al., 2004).


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PRs are defined based upon their most prominent biological and biochemical properties. The PR-2 familyare -1,3-endoglucanases and are characterized as limiting pathogen activity, growth, and spread. PR-3,-4,-8 and -11 are endochitinases and can therefore act as fungi. PR-1 are perhaps the most perplexing

proteins, as they do not fit any specific classification.

7. Carbon and Oxygen Isotopes

The abundance of isotopes in plants reveals many physiological processes in plants because their tissuesare constructed from the smallest molecules. Some examples of these molecules are CO 2, NO 3

-, NH 4+ and

H20. They are so small that the presence of even one extra neuron is enough to alter the behavior of theentire molecule.

Carbon isotope ratios in plant tissues are used infer photosynthetic water-use efficiency (WUE). WUE isalso defined as the ratio of net photosynthesis to transpiration (A/E). Carbon isotope levels and WUE arecorrelated because during photosynthesis, CO 2 enters the leaf and the CO 2 molecules diffuse down aconcentration gradient into the leaf. The CO 2 diffuses against a countervailing diffusive flux of watervapor out of the leaf due to transpiration. Subsequent partial closure of the leaf stomata reduces stomataconductance, which reduces both gas fluxes. The decline in net photosynthesis is less than the decline intranspiration, and water-use efficiency increases.

Oxygen isotopes in plant tissues come from water, thus that processes that affect the isotopic ratio ofwater will affect the oxygen isotope ratios in plants. Hydrogen isotopes can be considered in a similarway to oxygen isotopes, though we are only interested in analyzing one of the two since the results would

be very similar. The main source of isotopic variation in this case would be transpiration activity withinthe plant reflected via levels of evapo transpiration from the leaf surface.

With this information we create the following hypothesis; physiological data taken from photosyntheticactivity results will lead us to conclusions regarding the circumstances that affect evapo transpiration ratesof the plants. The evapo transpiration levels taken by the LI-COR could vary from that of the isotope test

because the isotopes better reveal a summary of the transpiration of the plant over the course of the plant'shistory. The LI-COR measures more the evapo transpiration of the plant in the moment that the test wastaken. The higher the evapo transpiration rate of a plant, the more stomata conductance the plant isundergoing and therefore the less stress the plant has endured. After various salicylic acid applications tothe plant, less stress will indicate that the plant has responded better to the concentration level.

Measuring WUE from carbon isotope levels has more efficacy for measuring A/E than looking at closedsystem gas exchange results. WUE measures isotopes with a long integration time, while closed-systemgas exchange techniques measure A/E on an instantaneous basis and only on a few leaves. Carbonisotope analysis offers a measure of WUE integrated over the time during which carbon in the plant wasfixed (Schulze and Caldwell).


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III. STUDY 1

1. Experimental Design

1.1. Objectives

Original Objectives:

1. Analyze the protein content of tomato and maize plants before and after their treatment with three bio products.2. Establish the proteins which are affected by each one of the bio products and define the metabolic

processes that are altered.3. Seat the physiological knowledge base for future efficacy tests of these products in the control ofdifferent pathogenic agents.4. Complete the experiment that was started in a previous agreement with the company.

Revised Objectives:

The objectives created for this project were:

1. Analysis of the protein content (proteome) of tomato and maize plants at the time of treatment withthree bio products and one week later .2. Via proteome analysis, establish the proteins which are affected by each one of the bio products anddefine the metabolic processes that are altered.3. Comparative analysis of the quantity of PR-2 and PR-3 expressed in tomato and maize plants at thetime of treatment with bio products and one week later, compared with the control.

4. Determine which protein sample contains the most PR-2 and PR-3 based on western blot experimentsand map where these proteins fall on the proteome.5. Attempt to identify unknown protein spots that were found on the original tomato and maize proteomemaps (T0 and T1) as PR-2 and PR-3.

1.2. Design

According to parameters set forth in the agreement with a company, tomato plants var. Boludo and maize plants B-73 were maintained in a growth chamber with controlled temperature conditions, 14h light at 25

C and 10h darkness at 18

C. The maize plants were grown in controlled greenhouse conditions.

The tomato and maize plants were treated with three different bio-products a week after beingtransplanted and with a phenology of 4 developed leaves. 5 plants were used per bio product percollection day. Thus a total of 40 tomatoes ( var. Boludo) and 40 maize plants ( B-73) were needed. Theexperimental design was random blocks.


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Samples were collected and analyzed at 0 and 1 weeks, post-application of the bio-products andimmediately conserved at -80C. Vegetative development was compared for the plants with distincttreatments, so as to detect the possible incidence of other illnesses or plagues. The analysis andidentification of proteins, according to the agreement, was conducted in the Proteomic and GenomicService (SPG) of UdL.

The protein extractions performed for each plant sample were separated and identified via bi dimensionalelectrophoresis and mass spectrometry MALDI-TOF. These procedures were carried out according to

pre-fixed protocols by the SPG and UdL and have established standards according to the instructions presented by the commercial houses in each case (see annexes).

Plants which were never treated were compared with the others as controls.

2. Material and Methods

Material and methods are divided into three distinct sections. The first is the proteomic experiment (2.1),the second the western blot experiment (2.2) and the third (2.3) combines material and methods from thetwo experiments to identify PR-2 and PR-3 on the proteome.

2.1. Proteomic experiment

All seed and planting methodology set forth in this experiment was replicated and elaborated in Part 2 ofthe Project. See Annex I for pictures of the transplanted plants and moment of application.

2.1.2. Bio-products and treatment

There were three bio-products used. All products were imported by the company of the agreement andquantities used were delegated by them as well.Bio-product 1: DA 3 cc/lBio-product 2: SK 2 cc/l

40 Plants 40 Plants

Control Treatments TreatmentsControl

5 Nontreated

5 Nontreated

5 Bio product 15 Bio product 25 Bio product 3

5 Bio product 15 Bio product 25 Bio product 3

Weekly collection of samples (0 and 1 week = 2 collections)

Figure 2. Project 1 Design


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Bio-product 3: JV 4 cc/lAll products were used in their powder form, and then diluted in water for application to the plants. Plantleaves were completely dampened with the bio-product solutions. The control was treated only withwater.

2.1.3. Bi-dimensional gel electrophoresis

The tomato proteins were extracted according to the TCA/Acetone protocol, which can be found AnnexII . Extracts were analyzed in 2 dimensional gels and it was determined that the proteomic spots weresuitable for proper analysis (figures 3 and 5, see below).

The first dimension, or iso-electronic focusing, was performed as it is explained in Annex XIII .

The gels were stained with Flamingo Gel flourochrome (Bio -Rad) according to the companyinstructions. The image was acquired with the Versadoc MP4000 system (Bio-Rad) 1. Spot detection andgel analysis was conducted first with the PDQUEST program (Bio-Rad) and the second time, manually. 2

Normalization was realized with the regression model LOESS. Only the spots present on all of the gelswere utilized in the statistical analysis. The experimental design had three biological repetitions.

The identification of the selected proteins was carried out according to the methodology outlined below(2.1.4.).

2.1.4. Gel digestion with trypsine and mass spectrophotometry: Realization Stage

Protein spots were manually extracted from the prepared gels and digestion cases in situ with trypsine (20ng/ul, Promega) according to the factory instructions including the reduction and the alquilation.

This way, the separated peptides could be in the presence of -ciano-4-hydroxicinamico (LaserBio Labs)and thus transferred to a MALDI plate in the form of a matrix.

The spectrums were obtained via the mass spectrophotometer Voyager DE PRO MALDI-TOF (AppliedBiosystems) operated in a positive reflection mode. The acquired spectrums were processed using theData Explorer software (Informatica Corp.) until their complete analysis using a mixture of known

peptides (ProteoMix 4, LaserBio Labs) in the external calibration and trypsine auto digestion peptides inthe internal calibration.

The proteins were identified based on their peptide footprint compared with the SwissProt databasethrough the MASCOT program (Matrix Science, Boston, MA, USA). In establishing the parameters, itcan be assumed that the peptides are mono isotopic, oxidized from the methionine remains (variables) andmethyl carbamide in cistene residue (fixed) with a maximum peptide mass tolerance of 100 ppm.

1 Initially silver nitrate was used, but it presented problems and we had to change the stain. 2 On account of irregularities in the assessment, this analysis has been conducted two times.


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The figures that summarize the proteins altered by each bio product are presented in the results section.The figures are the accumulated results of studying the gels for each sample and comparing them with therespective control. An example of a matching can be observed in Annex III .

2.2. Western Blot (WB) experiment

The Western Blot experiment was conducted utilizing the same samples as in the proteomic experiment.The goal was to see which samples contained the most abundant amounts of PR-2 and PR-3, which weremarked with antibodies. Unlike the Proteomic experiment, WBs were only run for tomato plant samples(see Annexes IV to VIII ).

2.2.1. Gel Preparation

Gels were made to measure 9cm x 7cm. Table A-1 can be found in Annex V , and contains " Formulas formixing the resolving and stacking gels" . All quantities should be doubled, as two gel were always madein this case. All " Buffer formulas" , for buffers needed in the Western Blot procedure can be found inAnnex VI.

The gels were prepared and each injected into the 2 glass plate apparatus' and allowed to set. First theresolving gel, and once it had set, the stacking gel on top of it. The apparatus checked for any leaks andthe 15 forked comb was placed in the top of the gels.

A running buffer of 1x concentration was mixed and poured over both gels. The gels were allowed to setover night.

2.2.2. Electrophoresis

Proteins were extracted from the tomato leaves according to methods in the " Western Blot proteinextraction protocol ", which can be found in Annex IV .

To run the electrophoresis, the gel comb is removed from the set gels. Gel wells are cleaned and preparedfor filling with protein mixtures. Each well can be cleaned with a small syringe and the running buffer toeliminate any bubbles. All wells need to be cleaned and left in the same condition.

Protein mixtures are made according to the quantity of each protein present per extraction. The finalvolume had to be 12 l because a 15 well gel can only hold this volume. The table of protein codes and

protein quantities used for the experiment can be found in Annex VII . Since the proteins are all found indifferent concentrations, they had to be diluted so that they were placed at a 'normalized' quantity of 10 l

per mixture/well. The protein mixture for 'normalization' can also be found in Annex VII.

The wells were loaded with the molecular marker in the middle and to fit the electrophoresis conditions of20 mA/ gel for 1 hour, the measurement was adjusted accordingly; 20 * 2 gels = 40 mA/ 2 gels.


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2.2.3. Transfer

Gels were then transferred to PVDF (polyviniliden), membranes for easier analysis. Two transfers aremade, one for PR-2 (glucanase) and another for PR-3 (chitinase). In the transfer machine, a sandwich is

created: paper, gel, PVDF membrane, paper. The electric current runs from top to bottom negative to positive, catoide to anoide. The 2 PVDF (0.5) membranes must always be hydrated with MetOH transfer buffer (20 % methanol). Transfer conditions were set to be: 60mA/ membrane/ 1h.

2.2.4. Incubation and Revelation

The complete protocol " Incubation and Revelation" can be found in Annex VIII. During this part of theexperiment, the gels are incubated in antibodies for PR-2 and PR-3. Commercial house antibodyinformation for PR-2 and PR-3 can be found in Annexes IX and X , respectively. They are also incubatedin a secondary antibody called IgG rabbit. Information from the commercial house regarding thisantibody can be found in Annex XI.

Finally, the membranes were washed and treated with transcription factor EF-1. This transcription factoris very stable protein that appears the same under any conditions presented in the experiment. For this, itis considered a factor of normalization and will serve as the correction for the PR-2 and PR-3 results thatappear on the gel. Commercial house information about EF-1 can be found in Annex XII.

2.2.5. PVDF Membrane Stain

Colorant solution = 50% methanol, 10% acetic acid and 0.1% Coomassie BlueDecolorant solution = 50% methanol, 10% acetic acid.

The membranes were submerged in the colorant solution and agitated at 115 RPM overnight or until the protein bands could be seen. The colorant was poured off and the de-colorant poured over the membraneand allowed to sit still for 5 minutes or until clean. The de-colorant was then poured off and themembrane was left to dry.

2.3. PR-2 and PR-3 Identification on the Proteome

This portion of the experiment combined the above experiences of analyzing the 2-dimensional proteomegel and the WB membrane transfer. The WB experiment revealed which bio product treatments elicitedthe greatest increase in the presence of PR-2 and PR-3 in the tomato plants.

With these results, 2 dimensional gels were created to map the proteomes of sample PT215 (whichgrossly over expressed PR-2 and PR-3). The proteome of this sample was transferred to PVDFmembrane and the membrane immunoblotted with PR-2 and PR-3 antibodies, according to the WB

procedure outlined above and in Annexes IV - XI . This procedure allowed us to see where these PRsappear on the proteome.


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2.3.1. Isoelectron Focusing and Immunoblotting

First, sample PT215 with protein weight 1.89 g/l underwent iso electron focusing (IEF). The extracted

sample was prepared for IEF under conditions that can be found in Annex XIII . Also in the annex is thegel preparation protocol and IEF settings that were used. The following day, two gels were transferred toPVDF membranes and immunoblotted for PR-2 (glucanase) and PR-3 (chitinase) according to theWestern Blot protocol which can be found in Annex XIII.

3. Results

3.1. Proteomic Experiment

Featured here are photographs taken of the gels and their corresponding charts which interrelate the"spots" (or proteins) which have appeared on the gel. The spots indicated are those of interest and thevalues of proteins of plants subject to different treatments are given from the protein densitometer at thedifferent Times 0 and 1. The last column of these charts is indicated with colors to match the precedingfigure. These colors indicate if the alteration is caused by one or more of the bio products and which ofthose are active. Rise or decline in protein content is indicated with ( ) or ( ), in relation to the control. All images are of a bi dimensional gel of extracts from the tomato plant ( var. Boludo) or maize plant ( B73) stained with Flamingo Gel fluorochro me. It sums the different treatments in the moment of bio

product application (Time 0, T0 or Time 1, T1). The molecular weight (kDa) and the iso electric point(pl) are indicated in the figures. The different colors show the proteins that have been altered individually

by each bio product (B1, B2 or B3) or those altered by variations of them (B1 and B2, B1 and B3 or B1,B2 and B3).


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Tomato plant extracts (var. Boludo)

Figure 3. Tomato Time = 0 days

* These spots are equal to 6706 (they are not identified for redundancy, as they are all the same protein).

Figure 4. "Spot" Table Tomato Time = 0 days


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Figure 5. Tomato Time = 1 week


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In the first column of the following table, the identified processed spots (dark blue cells) are indicated,along with spots that were processed, but not identified (green cells). The last column is indicated withcolors that match the preceding figure. These colors indicate if the alteration is caused by one or more ofthe bio products and which of those are active

.*These spots were not identified because their quantity in the prepared gels was not sufficient to assure their accurate

identification via mass spectrometry MALDI-TOF .

Figure 6. "Spot" Table Tomato Time = 1 week


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Maize plant extracts (var. B73)

Figure 7. Maize Time = 0 days

Figure 8. "Spot" Table Maize Time = 0 days

Figure 9. Maize Time = 1 week


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*These spots were not identified because their quantity in the prepared gels was not sufficient to assure their accurateidentification via mass spectrometry MALDI-TOF.

Figure 10. "Spot" Table Maize T=1

3.1.1. Observation and Symptom Registration

Analysis of the total protein content of the extracts has allowed us to see that all three bio products have a

significant effect on the stress levels of treated plants versus non-treated plants. In tomato, bio-product 3induces variation for the largest quantity of proteins (28). Bio products 1 and 2 are less potent, as theyonly induce variation in 11 and 21 proteins, respectively. In contrast, maize plants displayed very similar

protein variation for all three bio products: 13 for bio product 1, 14 for 2 and 10 for 3. The overalleffects of bio products 2 and 3, was more limited in maize plants than in tomato plants. Bio product 1exhibited similar variation in both species, though was slightly higher in maize.


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The three bio products interfere in the Calvin Cycle. It can be said that the three bio products induce adecrease in the protein contents of RuBisCO in tomato; both for long and short chain proteins. Howeverin maize plants, this is not the case and the bio products hardly have any effect at all. The plants treatedwith bio product 2 significantly increased their RuBisCO content during the two weeks. These changescan be translated to mean that bio products 2 and 3, especially 2, stress the plant less - the stress being

much lower still in maize.

Of the proteins that form the complete light harvesting complex (LHC), it can be highlighted that therewas only an increase in protein binding with chlorophyll in the tomato plants treated with bio products 2and 3; this didn't vary in maize. In the case of b io product 1, no change was observed in this respect.Ferredoxin levels slightly increased in tomato plants treated with bio product 2 and in maize plants treatedwith B3. This indicates an improvement in the photosynthetic activity, diminished via the degradation ofRuBisCO in the bio products. From what we can deduce, bio products 2 and 3 stimulate chloroplastactivity.

Enzymes which are implicated in glycolysis, such as adolase, drop in all cases - both for tomato andmaize. In change, GADPDH, related to a number of metabolic processes such as transcription andcellular apoptosis, increased - especially in the case of maize.

APX1 (ascorbic peroxidase) indicates the activation of Systemic Adquired Resistance (SAR). The dropin levels of APX1 in maize plants treated with bio product 2 leads us to conclude that there is little futureinterest in treating maize plants with bio product 2.

Control plants, without bio product treatment, maintained similar identified protein concentrationsthroughout the entire development of the plant. Slight increases in photosynthesis-related proteins and inmembrane traffic were detected.

3.1.2. Analysis of Proteome

In the trials completed with the tomato plants, a significant variation in 71 "proteomic spots" could beobserved. The above tables clearly outline the proteins that have shown a rise or decrease in density foreach bio-product applied to the tomato and maize plants. In the maize plants, a significant variation wasobserved in a total of 38 "proteomic spots" of interest.

The "spots" variation at Time 0 is not considered, so their variation could be related with the product. Inthe end, a total of 64 samples: 38 of tomato and 26 of maize, were selected for identification by massspectrometry MALDI-TOF/MS via their peptide footprints.


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3.1.4. Protein Identification

Tomato

Table 1. Protein Identification of Tomato at T=1

"Spot" Protein identified Function Location B1 B2 B31505 RuBisCO 1 (long chain) Calvin-Benson Cycle

(via C3 photosynthesis)

Chloroplast

3711 " " " 5706 " " " 6605 " " "

3101 RuBisCO (small chain)3A/3C, 3B, 2A

" "

4303 RuBisCO (small chain)3A/3C, 3B

" "

2506 RuBisCO activase " " 4202 Chlorophyll a/b binding

proteinPhotosythesis (lightreception and energydistribution)

Chloroplastthylakoid

5201 " " " 7102 Photosystem I FE-S

Center, ferro sulfate protein (PSI)

Oxide-reduction (e-transport)

Mitochondriamembrane

4304 Unidentified 2 4401 " 4405 " 5202 " 5502 " 6203 " 6204 " 6293 " 6303 " 6901 " 6902 " 1 Ribulose-1, 5-biphosphate carboxilase oxygenase. 2 The peptide map is insufficient and does not show anysignificant identification


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Maize

Table 2. Protein identification for maize at one weeks time, T=1

"Spot" Protein identified Function Location B1 B2 B3

5606 RuBisCO1

(long chain) Calvin-Benson Cycle(photosynthesis viaC3)

Chloroplast

5204 RuBisCO 3-epimerases " " 1607 Malic enzyme dependent

on NADP" Cytosol

3303 Ferredoxin - NAPDreductase

Photosynthesis (e-transport)

ChloroplastThylakoid

3702 Transketolase " Chloroplast 2607 " " " 3607 " " " 3613 " " " 4611 " " " 4101 Subunit IV A of

Photosystem I (complexof cytochrome b6f)

e- transport "

6302 NAD-dependentepimerase/ dehydratase

" Cytosol

3305 Adolase 2 Glucolosis Mitochondrialcytosol

7409 GADPDH 1 3 " Cytosol 8401 GADPDH 2 " " 8402 " " "

7301 GBLP beta subunit of protein regulated byguanine-nucleotide

Regulation of proteinsythesis

Membranes

4202 APX1 (ascorbate peroxidase) PR9

Detoxification of peroxides (antioxidant)

Cytosol

4503 Potential maize protein Unknown function 6202 " " 7303 " " 2603 Unidentified 4 4202 " 5201 "

7103 " 7204 " 1 Ribulose-1, 5-biphosphate carboxilase oxygenase 2 Fructose-1, 6-biphosphate adolase 3 Glyceraldehyde 3-phosphatedehydrogenase 4 The peptide map is insufficient and does not carry and significant identification


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3.2. Western Blot Experiment

Two membranes were prepared; one to reveal quantities of PR-2 and the other to reveal quantities of PR-3. Protein/ elongation factor EF-1 was used on both gels as a control, as it is a normalization factor thatinsures consistent results despite variable conditions.

The following figure is a chart of the selected proteomes and their representation in the western blot gelfor PR2 analysis. The above numbers represent the protein code according to bio product treatment whilethe first set of lower numbers represents the treatment given to the sample and the second line of lowernumbers represents the sample number within each treatment group. KT0 is control at time 0, KT1 iscontrol at time 1. BE1T1 is "bio elicitor" or bio product treatment 1, at time 1. BE2T1 is bio product 2 attime 1, and BE3T1 is bio product 3 at time 1.

Membrane 1:

Anti PR-2 82 83 84 85 86 87 91 92 93 163 166 169 209 212 215 Protein code

KT0 KT0 KT0 KT1 KT1 KT1 BE1T1 BE1T1 BE1T1 BE2T1 BE2T1 BE2T1 BE3T1 BE3T1 BE3T1 treatment1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 plant number (trial)

Anti EF- 1 82 83 84 85 86 87 91 92 93 163 166 169 209 212 215 Protein code

KT0 KT0 KT0 KT1 KT1 KT1 BE1T1 BE1T1 BE1T1 BE2T1 BE2T1 BE2T1 BE3T1 BE3T1 BE3T1 treatment1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 plant number (trial)

Below, the entire membrane is depicted. The darker lines (below) are those of EF-1 and the lighter lines(above) are of PR-2. Each vertical strip was created by a different protein. The abbreviations, or proteincodes, can be found in the above depictions and range from 82 - 215. These numbers have significance inthe SPG laboratory but are merely ways to organize the protein samples taken from the three plant trialssubject to the three bio stimulators.


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Blau Coomassie MEMBRANE

Membrane 2:

Antibody PR-382 83 84 85 86 87 91 92 93 163 166 169 209 212 210 Protein code

Antibody EF-1

Blau Coomassie MEMBRANE

If there would be any problem in the buffer mixing, or in any other part of the experiment, it would beapparent by means of viewing the success of failure of the EF- 1 . For example, in PR -3 gel well 169,the result is a very dark color. This most likely indicates that the well was 'overcharged' with too much

protein, or there existed some error in the pipette when diluting the protein mixture. We can therefore

Figure 11. Western blot results Membrane 1

Figure 12. Western blot results membrane 2


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correct such an error by creating a ratio with the more stable results obtained from the EF-1 proteinmixtures.

Below are the results from the quantification experiment in which all PR-2 and PR-3 values are correctedvia a 1/100 ratio PR-2 or 3/EF-1.

3.2.1. Quantification

This process takes the ratio of PR-2 or PR-3 to the EF- 1 to correct any errors which may have occurredat any point in the experiment with running the PR protein samples in the WB gel.

Table 3. Protein Quantification

Protein Code Sample PR-2/ EF- 1 ratio* andaverages

PR-3/ EF- 1 ratio* and

averagesPT082 KT0-1 100

37.33

100

45.69PT083 KT0-2 10.340187 32.1311753PT084 KT0-3 1.68038778 4.93075417PT085 KT1-1 34.0139046

65.09

26.2353242

82.43PT086 KT1-2 85.2536784 87.4569223PT087 KT1-3 76.0186862 133.601654PT091 BE1T1-1 60.9863834

175.90

125.369519

171.27PT092 BE1T1-2 129.067426 191.33699PT093 BE1T1-3 337.654855 197.08768PT163 BE2T1-1 178.827237

483.98

215.849941

318.23PT166 BE2T1-2 531.603261 267.88831PT169 BE2T1-3 741.505504 470.941152

PT209 BE3T1-1 212.403629239.57 or5797.61

222.757206

611.70PT212 BE3T1-2 266.729729 391.943111PT215 BE3T1-3 16913.71 1220.38972

* Ratio refers to 100. Value not included in the graph because it was very far out of the range of over expression.

A statistical data analysis of Table 3. has been considered imperative to determining theefficacy of the results obtained. An analysis of variance was conducted on the two sets ofdata to look at the least significant difference (90% LSD). When considering a 90%confidence interval, results yield insignificance between group means. Per se, we cannotdraw conclusions when comparing the PR-2 data set including the outlier with the PR-2data set sans.


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Figure 13. Comparative analysis of PR expression in each sample (value of BE3T1 is not considered because was very farout of the range of over expression)


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Figure 14. Comparative analysis of PR expression in each sample - value of BE3T1 is considered.

These results allow us to see that the samples treated with bio product 2 (BE2T1) demonstrate a high PR-2 level and that samples treated with bio product 3 (BE3T1) demonstrate both high PR-2 and PR-3 levels.In the case of the PR-2 figure 12, the results of the sample 3 treated with bio product 3 (BE3T1) are notincluded in the sample's average and are not depicted on the graph. This was because PR-2 was sodrastically over expressed by bio product 3 in sample 3 that it drastically alters the aspect of the figure.This sample corresponds to the well filled with protein sample code PT215. In figure 13, the value of thePR-2 over expression is included in the average. Since the composition of the bio stimulators has not

been released by the company, accurate conclusions cannot be drawn for this result.

In order to confirm the results from this first project, we tried to copy all procedures of the original projectand obtain the same results. Two trials were conducted and unfortunately for different reasons, we didnot come to any clear results.

3.3. PR-2 and PR-3 Identification on the proteome

By transferring proteomic results for sample code PT215, also referred to as protein sample 3 treated with bio product 3 (the sample which most drastically over expressed PR-2 and PR-3 in the WB experiment) toPVDF membranes and immunoblotting for PR-2 and PR-3, results regarding the general location of these

proteins on the proteome were revealed. Below, three membranes are depicted. The first is of the PVDFmembrane of sample PT215 immunoblotted with PR-2, the second is of a PVDF membrane


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immunoblotted with PR-3, and the third is of a bi dimensional gel stained with Oriole, so that the strengthof PR-2 and PR-3 expression can be observed in the context of the gel overall.

Membrane 1:Anti PR-2

Membrane 2Anti PR-3

Membrane 3GEL stained with ORIOLE (total protein)

pI3 10 NL

PR 3

PR2

Mw 33

Figure 15. Western blot of the proteome PR-2

Figure 16. Western blot of the proteome PR-3

Figure 17. Proteome of PT215 (BE3T1-3)


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Based on the above three gels, we can observe that PR-2 and PR-3 do appear on an immunoblotted bidimensional proteome gel analysis. When looking at the proteome sans PR-2 and PR-3 antibodies, it isclear that the proteins are not expressed in a perceivable quantity. Their location on the proteome is

equivalent to 33 KD, and according to a table found in the article by Aglika Edreva et al. , "Pathogenesis-related proteins: research progress in the last 15 years", this molecular weight corresponds exactly withthat of Gluc b (33 kD), which functions as a -1,3- glucanase, and Ch. 32 (32 kD) and Ch. 34 (34 kD),which function as chitinases. This table can be found in Annex XIV.

4. Discussion

Analysis of the protein content (proteome) of tomato and maize plants at the time of treatment with threebio products and one week later.

In tomato, everything indicates that bio product 3 (B3) causes variation in the greatest number of proteins(16). Bio product variation is in a lower number of proteins, 6 and 15, respectively. In maize, however,the number of proteins that vary is quite similar for the three bio products. Bio product one varies asimilar number of proteins in both tomato and maize, though slightly more in maize. It seems that theeffect of bio products 2 and 3 is more limited in maize than in tomato and that bio product 1 has the mostefficacy in maize.

In terms of time, it was clear that the tomato plant proteins varied greatly in this first week followingapplication of the bio products (T1). This is the case in many similar proteomic projects. In any case, itis a discovery which leads to the conclusion that it would be of interest to follow protein variation inresponse to different products of abiotic and biotic agents at 48 and at 72 hours after treatment, or at least

the time which allows observation of gradual protein disappearance. This is a conclusion which tiesdirectly into Study 2, as we study the effects of different concentration of salicylic acid at different time

periods throughout a week.

Via proteome analysis, establish the proteins which are affected by each one of the bio products anddefine the metabolic processes that are altered.

The control plants, or those without bio products, maintained similar concentrations of identified proteinsthroughout their development. They demonstrate slight spikes in proteins related with photosynthesis andin membrane traffic.

In the case of tomato, there are only two proteins for bio product 1 that were unidentifiable. Of these two,only one augmented. This is such a low number that when all is said and done, it can be considered thatthe variation caused by bio product 1 is not quantifiable.

Treatments with bio products 2 and 3 to tomato demonstrated an increase in concentration of manyunidentifiable proteins: in bio product 2 there were nine and in bio product 3 there were also nine.


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In the case of maize, there were not many proteins to identify. Only two for bio products 1 and 2 and fourfor bio product 3; of them, two are shared with bio products 1 and 2. For this, the study can be consideredalmost "complete".

From the identified proteins, it has become apparent that the three bio products interfere in the CalvinCycle. More accurately, the three bio products induce a general decrease in the quantity of proteins of theRuBisCO complex in tomato; long chain more than short chain, and the effect is greater in tomato than inmaize, where it is practically nil. In tomato, it seems that bio products 2 and 3 seem to activate shortchain RuBisCO. It seems that bio products 2 and 3 stress the plant less so than bio product 1.

RuBisCO activase does no vary in maize but does so in tomato, though in a variable form. It increaseswith bio product 1, lowers with bio product 3 and does not vary with bio product 2. Its importance in thefunction of joining RuBisCO was highlighted by (Spreitzer and Salrucci, 2003) who demonstrated that itis necessary for the maximum catalytic activity of the enzyme and in many occasions contributes to thespecificity of CO 2/O2. In conditions of stress, such as high temperatures, it disassociates and anchors tothe thylakoid membrane to act as a chaperone - associating itself with newborn polypeptide complexes.Thus, in that which concerns this study, its low quantity in the chloroplast could make it lose bothfunctions of diminishing transcriptions and protein levels.

For all the considerations that have been presented regarding RuBisCO proteins, it is important toremember that the degradation of the proteins in the RuBisCO family is usual in situations of a biotic(Feller et al., 2008) and of biotic stress (Orcutt and Nilsen, 2000).

Of the proteins that form part of the LHC complex, it can be emphasized that the chlorophyll union protein in tomato plants treated with bio products 2 and 3 augments, but does not change in maize. This

increase indicates a-normal enzymatic activity in the chloroplasts (Sindelarova et al. , 2005), which isnormally associated with situations of abiotic (such as excess or lack of light) and biotic stress (like

pathogens). In the case of bio product 1, no change was registered in this respect. Ferredoxin levels, the protein associated in electron transport and which is also in chloroplasts, increased slightly in tomato plants treated with bio product 2 and in maize plants treated with bio product 3. This could indicate animprovement in the photosynthetic activity hindered by the degradation of RuBisCO in these bio

products. It also indicates higher presence of transketolase in the case of treated maize plants. From allthis, it can be deduced that bio products 2 and 3, differ from bio product 1 and seem to better the activityof chloroplasts. In maize, bio product 2 slightly decreased the ferrosulfur protein (Fe-S Center) in Photosystem I (PSI).

Glucolysis or glycolysis is the metabolic route in charge of oxidizing glucose with the final result ofobtaining energy for the cell. In the tests conducted, the enzymes implied in glucolysis or glycolysis (TP1and adolase) decrease in all cases - as much tomato as maize. In change, the GADPDH, which iscatalyzed in the sixth step of glucolysis, releases fructose and energy, and was a protein that was


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increased by bio product 1 in maize. GADPDH has been related with various metabolic processes such asthe activation of transcription and the initiation of cellular apoptosis 3 (Tarze et al ., 2007).

It is important to stress the decrease of APX1 (ascorbate peroxidase) in the case of maize treated with bio product 2. The peroxidases are known as indicators of Systemic Acquired Response (SAR) for plants

against pathogens (Graskova et al. , 2001). For this, their increase is related with the defense of the plantagainst stress. This decrease, can be confirmed because B2 has less interest in maize, thus lowering itscontent.

It is here that we come to the discussion regarding the solicitation of pathogenesis related proteins withthe salicylic route in what is called Systemic Acquired Resistance (SAR). In Study 2, we will WesternBlot protein extraction from tomato samples treated with salicylic acid for PR-2 and PR-3 in order toobserve the solicitation of these proteins. These PRs do not appear in Induced Systemic Response (ISR)that is promoted by rhizo bacteria and other growth promoting agents that could be case of some or of allthe bio products.

Comparative analysis of the quantity of PR-2 and PR-3 expressed in tomato and maize plants at the timeof treatment with bio products and one week later, compared with the control.

Since the WB procedure was only conducted on tomato plant samples, we cannot comment on theexpression of PR-2 and PR-3 in maize. This is a subject for future study.

In tomato, it was concluded that bio product 3 induced the greatest expression of PR-2 and PR-3 at time1, one week after product application. Bio product 2 was close behind in inducing the tomato plants toexpress these resistance proteins.

Determine which sample contains the most PR-2 and PR-3 based on western blot experiments and mapwhere these proteins fall on the proteome.

Via the western blot analysis, it was observed that tomato plants treated with bio products 2 and 3 containthe highest quantities of PR-2 and also contain high quantities of PR-3. Of all the samples, tomato plant

protein sample PT215 had the highest expression of both PR-2 and PR-3. This sample was therefore usedto immunoblot the location of PR-2 and PR-3 on the proteome.

Attempt to identify unknown protein spots that were found on the original tomato and maize proteomemaps (T0 and T1) as PR-2 and PR-3.

3 Cellular plant death, similar to cellular death in animals, is the mechanism by which plants regulate many physiological processes such as germination, differentiation, growth, reproduction and seed production. Cellular plant death also plays a role inother important processes; such as resistance in unfavorable environmental conditions.


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There were 11 unidentified proteins noticeable on the tomato proteome and 5 on the maize proteome 7days after the bio product treatment to the plants. The unidentified proteins that were expressed in tomato

plants were "spot" numbers 4304, 4401, 4405, 5202, 5502, 6302, 6204, 6923, 6303, 6901 and 6902.Unidentified "spot" numbers in maize plants were 2603, 4202, 5201, 7103 and 7204. Since maize plant

protein samples were not analyzed according to the western blot procedure, commentary cannot be made

regarding the identification of these unknown protein "spots".

From the western blot results (see below) of the tomato "spots", however, it seems that unidentified"spots" 6293, 6302, 6204 and 6303 on Tomato proteome T=1 are in the same general vicinity as PR-2 andPR-3 on the WB of the proteome PT215. This, however, is an approximation. The molecular weight ofthe PR-2 and PR-3 spots detected on the proteome via WB was determined to be about 33 (kD). Thisweight was correlated by an anterior study, to be tandem to the molecular weights of b-1,3-Glucanase (33kD) and to chitinase (32, 34 kD).

One week after bio product application, the quantities of all these unknown proteins had increased."Spots" 6302, 6204 and 6293 were exclusively affected by bio products 2 and 3, while "spot" 6303 wasaffected by all three bio products; 1 caused the protein level to drop, while bio products 2 and 3 caused itto increase.

Figure 18. Analyzing molecular weight of PR-2 and PR-3 spots via proteomic andwestern blot gels


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5. Conclusions

1. The three bio products induce situations of stress in the tomato and maize plants whichis principally reflected in the reduction of the levels of long chain RuBisCO, and in theincrease of enzymes in the chlorophyll union.

2. Bio products 2 and 3 have a stronger effect over tomato plants and alter a large numberof foliar proteins in this species.

3. Bio product 3 alters the greatest number of tomato leaf proteins.

4. Bio product 1 has the greatest effect on maize.

5. Bio product 2 stresses the plants less, but in the case of maize, reduces the ascorbate peroxidase (PR9) levels which could lower defenses against pathogens.

6. The detected increase in the concentration of RuBisCO reductase upon the applicationof bio product 1 could be the calming effect of the degradation of RuBisCO.

7. The increase of GADPDH that is caused by all three bio products is in harmony with the predisposition that it defends against environmental stresses.

8. All three bio-stimulators augment PR expression in tomato plants.

9. Bio stimulator 2 induces a high PR-2 level

10. Bio stimulator 3 induces a high PR-3 level. When the grossly over expressed sample istaken into consideration, bio stimulator 3 causes a very high PR-2 level in the tomato plants.

11. The molecular weight of the PR-2 and PR-3 spots detected on the proteome via WBapproximately correlates to the molecular weights of b-1,3-Glucanase (33 kD) and to chitinase(32, 34 kD).


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III. STUDY 2

1. Experimental Design

1.1 Objectives

1. Determine an ideal concentration for salicylic acid application to tomato and maize plants based upon proteomic, PR and physiological response.

2. Evaluate the effects that different concentrations of salicylic acid has on PR proteinexpression.

3. Use physiological measurement data results to conclude effects that application of differentconcentrations of SA to tomato and maize plants has upon biological processes influenced by

photosynthetic rate and chlorophyll levels, over one weeks time.

4. Conclude effects that SA application has on tomato and maize plants based on carbon andoxygen isotope results.

1.2 Design

The experiment was organized into four treatment groups, as follows; control 0.2g/L application ofsalicylic acid, 0.5g and 0.8g. The treatment groups were then divided into three application times; 0, 2days (48h) and 7 days. For each treatment group, five plants of tomato and five of maize were planted.In the actual experiment, only three plants of each plant type were used for salicylic acid application andsample extraction, but extra plants were grown to accommodate for the possibility of unpredicted plant

death.

Table 4. Experimental design for salicylic acid treatments to tomato and maize plants.

Treatment Time Blocks: T = Number of PlantsTomato Maize

Control 0 5 548h 5 57 days 5 5

0.2g SA 0 5 548h 5 57 days 5 5

0.5g SA 0 5 548h 5 57 days 5 5

0.8g SA 0 5 548h 5 57 days 5 5

The total number of plants seeded were 60 tomato and 60 maize.


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Physiological Parameters

Physiological parameters are broken into three distinct parts. Plant were analyzed for 1) photosyntheticactivity, 2) chlorophyll and 3) total organic matter (TOM). In the future, there will be four distinct parts,

as soluble organic matter (SOM) will be added to the project. All testing was completed for photosynthetic activity and chlorophyll, and thus conclusions extracted from results, but TOM isunfinished.

1.3 Calendar of events

2.5 months: Time from planting specimens to maturity of 4 leaves, minimum (experimentation inETSEA cultivation chambers and greenhouses).2 days: Photosynthesis and chlorophyll physiological tests conducted1 week: SOM extraction preparation.2 weeks: WB protein extraction.6 weeks: TOM extraction sent to Davis, CA to be analyzed.

1.4 Outstanding Tasks

- Proteomic analysis and interpretation.- Western blot analysis and interpretation.- Proteins which are unable to be identified in Lleida to be analyzed in the Service in Barcelona.- Analyze results TOM extraction, draw conclusions.- Background research and protocol creation for and conduct SOM extractions 4. .

2. Material and Methods

2.1 Planting and Pre-harvest Treatment: Tomato and Maize

Planting

The planting date of the Tomato was February 9, 2011. The tomatoes were planted at a ratio of 4 seeds per pot, alternating varieties; 2 old and 2 new varieties were planted for each pot. The new varietywas LOTE 67728267 Tomato Boludo and the old variety was LOTE 728627 Tomato Boludo 04-05. Allseeds came from Gelboplant , Malgrat de Mar, Spain. The seeds were planted at a depth of 5 10 cm. in asphagnum peat media called Traysubtrat . This media has 8 10kg organic matter and a pH of 5.5 6.5.Cooked clay pots measuring 12.5cm x 12.5cm were used. All seeds were watered with about 50 mL. tapwater upon planting. Large pots were used so that the plants would not need to be transplanted later.

4 These extracts will need to be sent to laboratories in Davis, CA to be analyzed. Conclusions need to be drawn from the results.As of today, samples are milled and ready for use in the UdL Silviculture laboratory


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Planting and growing conditions for corn were distinct from those of the tomato. The corn was of variety Panis 73B . 2 seeds were placed per pot. In the case of the maize, instead of using cooked clay pots, aswas used in the planting of the tomato, plastic trays with pots measuring 15cm x 15cm were utilized.The same planting media was used as for the tomato plants. Greenhouse conditions were maintainedconstant from Project 1 and the maize plants were grown in controlled greenhouse conditions.

Pre-harvest

All 60 pots of tomato seeds were immediately transported to the growing space, which was a growingchamber with the following temperature and light settings: 24 degrees night time temperature, at 10 hoursnight; 18 degrees daytime temperature, at 14 hours day time. The plants were watered three times perweek for the following weeks every Monday, Wednesday and Friday in the morning; usually beforenoon.

Watering of tomato plants was done with a watering can until salicylic acid treatments were put on the plants. Then the method was changed to a 50mL. graduated cylinder. The water was carefully applied tothe root base of the plants.

The growing chamber used for the tomato plants is the ING climas grow chamber with humidityReference: GROW 1300/HR and equipped with florescent lights positioned vertically on the doors on oneside of the chamber. The chamber had two shelves only, which together fit all 60 terra cotta pots. Oneshelf was placed at a height of 1m and the other was the ground floor of the chamber 5. One time duringweek 2, and another time at T = 0, all of the plants were rotated so that the plants on the bottom couldhave an equal opportunity at receiving full potential light radiation. The rotation involved plants on the

bottom shelf being moved to the top, and the plants which had been at the back of the grow chambermoved to the front (and thus closer to the lights).

Watering methodology for the corn was distinct from that of the tomato plants, though wateringfrequency was identical; three times weekly, specifically every Monday, Wednesday and Friday in themornings. Water was applied with an in-house hose, freely to the plants, from above. Upon applicationof salicylic acid, all plants were watered at the root base, so as not to disturb the salicylic acid on the plantleaves.

5 The florescent lights do not reach to the very bottom of the chamber, and so it was clear that the pots placed on the bottom werenot in a position to receive the same amount of light as those on the higher shelf. This is depicted in Annex XVI (picture 1).


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Figure 19. Tomato plant time and treatment groups

T = 7d

T = 48h

T = 0

Control 0.2 0.5 0.8

Documents

Proteomics Related With Pathogenesis of Tomato and Maize Plants Treated With Bio Products and Salicylic Acid