PROTEOMICS RELATED WITH PATHOGENESIS OF TOMATO AND MAIZE

PLANTS TREATED WITH BIO PRODUCTS AND SALICYLIC ACID

Sarah Boyd Lade
University of Lleida






tutor
Vicente Medina
2

















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






Vicente Medina


3

INDEX
Summary and key words 4
Special Thanks 5
Table Key 6
Figure Key 7
Acronyms and Abbreviations Key 9

I. Project Outline 10
1. Goals 10
2. Contents 10
3. Overall Objectives 10
4. Work Plan 11

II. Introduction 12
1. Background Information 12
2. The Process of Pathogenesis: Bacteria, Fungus and Virus 12
3. Resistance Pathways 13
4. Bio products 14
5. Salicylic Acid 15
6. Summary of Recognized families of PRs and Related proteins 15
7. Carbon and Oxygen Isotopes 16

III. Study 1 17
1. Study Design 17
2. Material and Methods 18
3. Results 22
4. Discussion 37
5. Conclusions 41

IV. Study 2 42
1. Study Design 42
2. Material and Methods 43
3. Results 49
4. Discussion and Conclusions 64

V. Bibliography 67
VII. Annexes 72
4

SUMMARY AND KEY WORDS

This project explores resistant protein (PR) expression, the plant defense system and induction pathways
of these PRs by bio products and salicylic acid. Tomato and maize plants were used as objects of
experimentation 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. It
was found that all three bio-stimulators augment PR expression in tomato plants. Bio stimulator 2
induces 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 serious
shock at day 2, but fare the best in terms of stomatal conductance after one week. In general, the
significant difference of overall stomatal conductance of 0.2g/L or 0.5g/L when compared with the
control, was the same.

Key words: Salicylic acid, proteomics, western blot, bio stimulators, induction pathways, PR
expression, PR-2, PR-3, tomato, maize, physiological parameters.
5

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 University
of 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 conduct
numerous physiological measurements and then help to analyze the results.

Clara Nierga Parra who worked diligently in the laboratory under a grant to share her extensive
knowledge 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 this
experiment 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 KEY
Figure 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 was
very 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 triphosphate
DPVCF Departament de Producci Vegetal i Cincia Forestal
ETSEA Escola Tcnica Superior dEnginyeria Agrria
GADPDH Gliceraldehyde 3-phosphate dehydrogenase
HPLC High performance liquid chromatography
ISR Induced systemic resistance
kDa kilodalton
LAR Local acquired resistance
LC-MS/MS Liquid chromatography-mass/mass spectrometry
MALDI-TOF (MS) Matrix-Assisted Laser Desorption/Ionization - Time-Of-Flight
MAMPs/PAMPs - microbe associated (or pathogen associated) molecular patterns
MDMV Maize dwarf mosaic virus
NADPH Nicotinamide adenine dinucleotide phosphate
NF - Nod factor
PAL phenylalanine ammonia-lyase
PSI Photosystem I
PRs Proteins related with pathogenesis
PRR - pattern recognition receptors
PTI - PAMP triggered immunity
PVCF - Produccin vegetal y ciencias forestales
PVX Potato virus X
RuBisCO Ribulose-1,5-bisphosphate carboxyl oxigenase
SA Salicylic acid
SAR Systemic acquired resistance
SR Systemic resistance
SGP Servicio de Genmica y Protemica de la UdL
TMV Tobacco mosaic virus
TPI Triosephosphate isomerase
UdL 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 discussion
items 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 the
Western Blot procedure. Proteomic methodology was not reproduced in this study due to the costly
nature of the procedure.

A final goal of this project was to compile and perform a succinct series of procedure for physiological
analysis 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 by
our team to wholly understand the procedure and so that any modifications to the protocols could be
elaborated. Conclusions and discussion unite the studies and their findings.

In the second part of the project, all methodologies regarding plant production, salicylic acid application
and 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 future
studies 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) in
tomato 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 each
time block.






Figure 1. Overall Project Design
STUDY 1
(Previous)
Proteomic study of plants treated with
3 Bio products
(A company agreement)

STUDY 2
Proteomic and PR study of plants
treated with different concentrations of
Salicylic Acid


PR study of Bio product treated plants
utilizing Western Blot Procedure
Physiological Parameters: photosynthetic
activity, chlorophyll content and total
organic matter
STUDY 3
Proteomic and PR study of pathogen
infected 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 the
constitutive 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 the
advance of a provoked pathogen. Examples are cellular death caused by the hypersensitive reaction, the
accumulation of secondary metabolites with antimicrobial activity, such as phytoalexins, the production
of reactive oxygen species, the accumulation of hydrolytic enzymes and the synthesis of pathogenesis
related proteins (PRs). An increase in enzyme activity during infection has also been commonly
observed. These enzymes are responsible for the synthesis of phenylpropenoid compounds such as
phenylalanine ammonia-lyase (PAL) or intermediated flavanoids known for their antifungal and
antibacterial 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 and
Merida, 1996). Infection relies on the interaction between the gene products of the plant and the pathogen
on the cellular and molecular levels. Successful evasion of a hosts surveillance system and subsequent
activities of metabolites of the pathogen (enzymes and toxins) encoded by pathogen genes characterize
the 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 of
transmission. The virus particles multiply at the site of infection (Soto, et al., 2009). They are visibly
apparent by means of localized symptoms such as necrotic spots on the leaves or mottling. The virus then
usually spreads systemically throughout the plant, either in the vascular system, or directly from cell to
cell. 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 of
salicylic acid. The most challenging obstacle in viral spread within a plant is crossing of the plant cell
walls (Cann, 2009).

Studies performed in bacteria related plantpathogen interactions indicate that the rst-line of plant
defense 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 negatively
affects 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
13

tissue either by wounds or natural openings, such as stomata, and occupy the apoplast of plant tissues or
the xylem where they multiply and spread. This process induces the activation of hydrolytic enzymes and
toxins 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 mutual
secretion 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 nodulation
signal 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 of
hydrolytic enzymes, including cutinases, cellulases, pectinases and proteases. These enzymes weaken the
cuticle 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 toxins
or plant hormone-like compounds into the plant which manipulate the plants physiology to the benefit of
the pathogen. These toxins are usually plant specific, and can simply kill plant cells for the purpose of
fungal 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). This
resistance manifests 2 or 3 days after the primary infection and is restricted in the zone which surrounds
the 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 of
the plant. This resistance is also known as Systemic Acquired Response (SAR). This system of
resistance 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) is
different than the immune response in mammals, specific in most cases, and independent of the inductor
organism (Ryals et al., 1996).

Systemic Acquired Resistance (SAR)

The first SAR studies were conducted by measuring the plant/virus interaction in tobacco and
Arabadopsis plants infected with Tobacco Mosaic Virus (TMV) (Glazebrook et al., 1997). The
investigation conducted on the model plants indicated that salicylic acid (SA) is the molecule with the
most 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 plant
resistance 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 act
independently 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
14

expressed during the response to pathogen resistance are involved in SAR (Ryals et al., 1996). SAR
genes 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 to
this, expression in the pathogen-less environment of this experiment will lead to clear gene and protein
expression. 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 in
monocot species as well. It had not been determined whether the induction of these genes can be directly
correlated 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- dwelling
organisms, particularly plant growth promoting rhizobacteria (PGPR), and is known more specifically as
Induced 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. genus
are 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 (Montesinos
et 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 capacity
to 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 the
stimulation 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 metabolites
that 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 molecular
level, ISR induces a set of genes distinct from the PR genes, while SAR induces a set of pathogenesis
related proteins (Siddiqui)

4. Bio-Products

To better understand the action of three bio-products created by the company, the first part of this
experiment 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 two
different plant species: tomato (var. Boludo) and maize (var. B-73). Protein contents in both species were
analyzed by separating them via bi-dimensional gel electrophoresis and then identifying them with mass
spectrometry MALDI-TOF. To complement this study, foliar proteins were extracted as well and
analyzed via the Western Blot.

15

The original experiment analyzed the effects of repeated application of the products over the course of a
month, 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 oxidative
stress. Compounds resulting from the degradation of cells or cell walls might be involved in eliciting the
systemic 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 or
exogenously in order for SAR to take place. The exact mechanisms, however, by which SA induces SAR
is unknown but it has been hypothesized that SA induced enzyme inhibition activities have an effect in
SAR signaling (Ryals, 1996). It is known that SA is considered a key component in defense signal
transduction and induces a full set of systemic acquired resistance genes (Halim, et al., 2006; Maleck, et
al., 2000). The exogenous application of SA may influence physiological plant processes such as
transpiration 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 pathway
elements, 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 et
al., 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 their
homologues, including enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase and
polyphenoloxidase which are generally present constitutively and only increase during pathogenic
infection. There exist a number of enzyme activities that increase in response to pathogen attack and
which 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 though
induction at the protein level is demonstrated after infection by one or more pathogens. Thus, inducible
defense related proteins include both PRs and those defined in the anterior. Note that the term 'defense
related' refers to the fact that these proteins are induced in association with resistance responses but does
not by itself imply functional role in defense. Due to the fact that some of these proteins have potential
anti-microbial activities, a role in resistance to pathogens is plausible, but not currently accepted (Van
Loon, 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 and
stronger expression once the plant actually responds to an antagonistic pathogen invasion; in comparison
to non-induced plants (Heil et al., 2002; Verhagen et al., 2004).
16


PRs are defined based upon their most prominent biological and biochemical properties. The PR-2 family
are -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 tissues
are constructed from the smallest molecules. Some examples of these molecules are CO
2
, NO
3
-
, NH
4
+
and
H
2
0. They are so small that the presence of even one extra neuron is enough to alter the behavior of the
entire molecule.

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

Oxygen isotopes in plant tissues come from water, thus that processes that affect the isotopic ratio of
water will affect the oxygen isotope ratios in plants. Hydrogen isotopes can be considered in a similar
way 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 within
the plant reflected via levels of evapo transpiration from the leaf surface.

With this information we create the following hypothesis; physiological data taken from photosynthetic
activity results will lead us to conclusions regarding the circumstances that affect evapo transpiration rates
of 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's
history. The LI-COR measures more the evapo transpiration of the plant in the moment that the test was
taken. The higher the evapo transpiration rate of a plant, the more stomata conductance the plant is
undergoing and therefore the less stress the plant has endured. After various salicylic acid applications to
the 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 closed
system gas exchange results. WUE measures isotopes with a long integration time, while closed-system
gas exchange techniques measure A/E on an instantaneous basis and only on a few leaves. Carbon
isotope analysis offers a measure of WUE integrated over the time during which carbon in the plant was
fixed (Schulze and Caldwell).
17

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 of
different 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 with
three bio products and one week later.
2. Via proteome analysis, establish the proteins which are affected by each one of the bio products and
define 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 the
time 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 experiments
and map where these proteins fall on the proteome.
5. Attempt to identify unknown protein spots that were found on the original tomato and maize proteome
maps (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 being
transplanted and with a phenology of 4 developed leaves. 5 plants were used per bio product per
collection day. Thus a total of 40 tomatoes (var. Boludo) and 40 maize plants (B-73) were needed. The
experimental design was random blocks.

18

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

The protein extractions performed for each plant sample were separated and identified via bi dimensional
electrophoresis 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 the
two 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 of
the 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 and
quantities used were delegated by them as well.
Bio-product 1: DA 3 cc/l
Bio-product 2: SK 2 cc/l
40 Plants 40 Plants
Control Treatments Treatments Control
5 Non
treated
5 Non
treated
5 Bio product 1
5 Bio product 2
5 Bio product 3
5 Bio product 1
5 Bio product 2
5 Bio product 3
Weekly collection of samples (0 and 1 week = 2 collections)
Figure 2. Project 1 Design
19

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

2.1.3. Bi-dimensional gel electrophoresis

The tomato proteins were extracted according to the TCA/Acetone protocol, which can be found Annex
II. Extracts were analyzed in 2 dimensional gels and it was determined that the proteomic spots were
suitable 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 company
instructions. The image was acquired with the Versadoc MP4000 system (Bio-Rad)
1
. Spot detection and
gel 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 gels
were 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 (20
ng/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 (Applied
Biosystems) operated in a positive reflection mode. The acquired spectrums were processed using the
Data 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 in
the internal calibration.

The proteins were identified based on their peptide footprint compared with the SwissProt database
through the MASCOT program (Matrix Science, Boston, MA, USA). In establishing the parameters, it
can be assumed that the peptides are mono isotopic, oxidized from the methionine remains (variables) and
methyl 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.
20

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 the
respective 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 were
marked 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 for
mixing the resolving and stacking gels". All quantities should be doubled, as two gel were always made
in this case. All "Buffer formulas", for buffers needed in the Western Blot procedure can be found in
Annex VI.

The gels were prepared and each injected into the 2 glass plate apparatus' and allowed to set. First the
resolving gel, and once it had set, the stacking gel on top of it. The apparatus checked for any leaks and
the 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 set
over night.

2.2.2. Electrophoresis

Proteins were extracted from the tomato leaves according to methods in the "Western Blot protein
extraction 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 prepared
for filling with protein mixtures. Each well can be cleaned with a small syringe and the running buffer to
eliminate 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 final
volume 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 in
different 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 of
20 mA/ gel for 1 hour, the measurement was adjusted accordingly; 20 * 2 gels = 40 mA/ 2 gels.

21


2.2.3. Transfer

Gels were then transferred to PVDF (polyviniliden), membranes for easier analysis. Two transfers are
made, 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 the
experiment, the gels are incubated in antibodies for PR-2 and PR-3. Commercial house antibody
information for PR-2 and PR-3 can be found in Annexes IX and X, respectively. They are also incubated
in a secondary antibody called IgG rabbit. Information from the commercial house regarding this
antibody can be found in Annex XI.

Finally, the membranes were washed and treated with transcription factor EF-1. This transcription factor
is very stable protein that appears the same under any conditions presented in the experiment. For this, it
is considered a factor of normalization and will serve as the correction for the PR-2 and PR-3 results that
appear 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 Blue
Decolorant 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 membrane
and allowed to sit still for 5 minutes or until clean. The de-colorant was then poured off and the
membrane 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 proteome
gel and the WB membrane transfer. The WB experiment revealed which bio product treatments elicited
the 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 (which
grossly over expressed PR-2 and PR-3). The proteome of this sample was transferred to PVDF
membrane 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 PRs
appear on the proteome.
22



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 the
gel preparation protocol and IEF settings that were used. The following day, two gels were transferred to
PVDF membranes and immunoblotted for PR-2 (glucanase) and PR-3 (chitinase) according to the
Western 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 the
values of proteins of plants subject to different treatments are given from the protein densitometer at the
different Times 0 and 1. The last column of these charts is indicated with colors to match the preceding
figure. These colors indicate if the alteration is caused by one or more of the bio products and which of
those 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 (B
73) stained with Flamingo Gel fluorochrome. 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).

23

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
24


Figure 5. Tomato Time = 1 week

25

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 with
colors that match the preceding figure. These colors indicate if the alteration is caused by one or more of
the 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
26

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
27


*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 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 3
induces variation for the largest quantity of proteins (28). Bio products 1 and 2 are less potent, as they
only 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 overall
effects of bio products 2 and 3, was more limited in maize plants than in tomato plants. Bio product 1
exhibited similar variation in both species, though was slightly higher in maize.
28

The three bio products interfere in the Calvin Cycle. It can be said that the three bio products induce a
decrease in the protein contents of RuBisCO in tomato; both for long and short chain proteins. However
in maize plants, this is not the case and the bio products hardly have any effect at all. The plants treated
with bio product 2 significantly increased their RuBisCO content during the two weeks. These changes
can 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 there
was only an increase in protein binding with chlorophyll in the tomato plants treated with bio products 2
and 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 treated
with B3. This indicates an improvement in the photosynthetic activity, diminished via the degradation of
RuBisCO in the bio products. From what we can deduce, bio products 2 and 3 stimulate chloroplast
activity.

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

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

Control plants, without bio product treatment, maintained similar identified protein concentrations
throughout the entire development of the plant. Slight increases in photosynthesis-related proteins and in
membrane 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 be
observed. The above tables clearly outline the proteins that have shown a rise or decrease in density for
each bio-product applied to the tomato and maize plants. In the maize plants, a significant variation was
observed 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. In
the end, a total of 64 samples: 38 of tomato and 26 of maize, were selected for identification by mass
spectrometry MALDI-TOF/MS via their peptide footprints.






29




3.1.4. Protein Identification

Tomato

Table 1. Protein Identification of Tomato at T=1

"Spot" Protein identified Function Location B1 B2 B3
1505 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
protein
Photosythesis (light
reception and energy
distribution)
Chloroplast
thylakoid

5201 " " "
7102 Photosystem I FE-S
Center, ferro sulfate
protein (PSI)
Oxide-reduction (e-
transport)
Mitochondria
membrane

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 any
significant identification




30

Maize

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

"Spot" Protein identified Function Location B1 B2 B3
5606 RuBisCO
1
(long chain) Calvin-Benson Cycle
(photosynthesis via
C3)
Chloroplast
5204 RuBisCO 3-epimerases " "
1607 Malic enzyme dependent
on NADP
" Cytosol
3303 Ferredoxin - NAPD
reductase
Photosynthesis (e-
transport)
Chloroplast
Thylakoid

3702 Transketolase " Chloroplast
2607 " " "
3607 " " "
3613 " " "
4611 " " "
4101 Subunit IV A of
Photosystem I (complex
of cytochrome b6f)
e- transport "
6302 NAD-dependent
epimerase/ dehydratase
" Cytosol
3305 Adolase
2
Glucolosis Mitochondrial
cytosol

7409 GADPDH 1
3
" Cytosol
8401 GADPDH 2 " "
8402 " " "
7301 GBLP beta subunit of
protein regulated by
guanine-nucleotide
Regulation of protein
sythesis
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-phosphate
dehydrogenase
4
The peptide map is insufficient and does not carry and significant identification


31

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 that
insures consistent results despite variable conditions.

The following figure is a chart of the selected proteomes and their representation in the western blot gel
for PR2 analysis. The above numbers represent the protein code according to bio product treatment while
the first set of lower numbers represents the treatment given to the sample and the second line of lower
numbers represents the sample number within each treatment group. KT0 is control at time 0, KT1 is
control at time 1. BE1T1 is "bio elicitor" or bio product treatment 1, at time 1. BE2T1 is bio product 2 at
time 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 treatment
1 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 treatment
1 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 protein
codes, can be found in the above depictions and range from 82 - 215. These numbers have significance in
the SPG laboratory but are merely ways to organize the protein samples taken from the three plant trials
subject to the three bio stimulators.











32

Blau Coomassie MEMBRANE











Membrane 2:

Antibody PR-3
82 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 be
apparent 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
33

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

Below are the results from the quantification experiment in which all PR-2 and PR-3 values are corrected
via 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 occurred
at 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* and
averages
PR-3/ EF-1 ratio* and
averages
PT082 KT0-1 100
37.33
100
45.69
PT083 KT0-2 10.340187 32.1311753
PT084 KT0-3 1.68038778 4.93075417
PT085 KT1-1 34.0139046
65.09
26.2353242
82.43
PT086 KT1-2 85.2536784 87.4569223
PT087 KT1-3 76.0186862 133.601654
PT091 BE1T1-1 60.9863834

175.90
125.369519
171.27
PT092 BE1T1-2 129.067426 191.33699
PT093 BE1T1-3 337.654855 197.08768
PT163 BE2T1-1 178.827237
483.98
215.849941
318.23
PT166 BE2T1-2 531.603261 267.88831
PT169 BE2T1-3 741.505504 470.941152
PT209 BE3T1-1 212.403629
239.57 or
5797.61
222.757206
611.70
PT212 BE3T1-2 266.729729 391.943111
PT215 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 the
efficacy of the results obtained. An analysis of variance was conducted on the two sets of
data to look at the least significant difference (90% LSD). When considering a 90%
confidence interval, results yield insignificance between group means. Per se, we cannot
draw conclusions when comparing the PR-2 data set including the outlier with the PR-2
data set sans.
34



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

35



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 not
included in the sample's average and are not depicted on the graph. This was because PR-2 was so
drastically 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 the
PR-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 project
and obtain the same results. Two trials were conducted and unfortunately for different reasons, we did
not 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) to
PVDF 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 PVDF
membrane of sample PT215 immunoblotted with PR-2, the second is of a PVDF membrane
36

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

Membrane 1:
Anti PR-2










Membrane 2
Anti PR-3











Membrane 3
GEL stained with ORIOLE (total protein)
pI3 10 NL












PR3
PR2
Mw 33
KD
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)
37



Based on the above three gels, we can observe that PR-2 and PR-3 do appear on an immunoblotted bi
dimensional proteome gel analysis. When looking at the proteome sans PR-2 and PR-3 antibodies, it is
clear 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 with
that 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 three
bio 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 a
similar number of proteins in both tomato and maize, though slightly more in maize. It seems that the
effect of bio products 2 and 3 is more limited in maize than in tomato and that bio product 1 has the most
efficacy in maize.

In terms of time, it was clear that the tomato plant proteins varied greatly in this first week following
application of the bio products (T1). This is the case in many similar proteomic projects. In any case, it
is a discovery which leads to the conclusion that it would be of interest to follow protein variation in
response 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 ties
directly 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 and
define the metabolic processes that are altered.

The control plants, or those without bio products, maintained similar concentrations of identified proteins
throughout their development. They demonstrate slight spikes in proteins related with photosynthesis and
in 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 that
the variation caused by bio product 1 is not quantifiable.

Treatments with bio products 2 and 3 to tomato demonstrated an increase in concentration of many
unidentifiable proteins: in bio product 2 there were nine and in bio product 3 there were also nine.
38


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

From the identified proteins, it has become apparent that the three bio products interfere in the Calvin
Cycle. More accurately, the three bio products induce a general decrease in the quantity of proteins of the
RuBisCO complex in tomato; long chain more than short chain, and the effect is greater in tomato than in
maize, where it is practically nil. In tomato, it seems that bio products 2 and 3 seem to activate short
chain 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 increases
with bio product 1, lowers with bio product 3 and does not vary with bio product 2. Its importance in the
function of joining RuBisCO was highlighted by (Spreitzer and Salrucci, 2003) who demonstrated that it
is necessary for the maximum catalytic activity of the enzyme and in many occasions contributes to the
specificity of CO
2
/O
2
. In conditions of stress, such as high temperatures, it disassociates and anchors to
the 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 both
functions of diminishing transcriptions and protein levels.

For all the considerations that have been presented regarding RuBisCO proteins, it is important to
remember 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 is
normally 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 an
improvement 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 all
this, it can be deduced that bio products 2 and 3, differ from bio product 1 and seem to better the activity
of chloroplasts. In maize, bio product 2 slightly decreased the ferrosulfur protein (Fe-S Center) in Photo
system I (PSI).

Glucolysis or glycolysis is the metabolic route in charge of oxidizing glucose with the final result of
obtaining energy for the cell. In the tests conducted, the enzymes implied in glucolysis or glycolysis (TP1
and adolase) decrease in all cases - as much tomato as maize. In change, the GADPDH, which is
catalyzed in the sixth step of glucolysis, releases fructose and energy, and was a protein that was
39

increased by bio product 1 in maize. GADPDH has been related with various metabolic processes such as
the 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 plant
against stress. This decrease, can be confirmed because B2 has less interest in maize, thus lowering its
content.

It is here that we come to the discussion regarding the solicitation of pathogenesis related proteins with
the salicylic route in what is called Systemic Acquired Resistance (SAR). In Study 2, we will Western
Blot protein extraction from tomato samples treated with salicylic acid for PR-2 and PR-3 in order to
observe 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 all
the bio products.


Comparative analysis of the quantity of PR-2 and PR-3 expressed in tomato and maize plants at the time
of 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 the
expression 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 time
1, one week after product application. Bio product 2 was close behind in inducing the tomato plants to
express these resistance proteins.


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

Via the western blot analysis, it was observed that tomato plants treated with bio products 2 and 3 contain
the 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 used
to 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 proteome
maps (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 in
other important processes; such as resistance in unfavorable environmental conditions.
40

There were 11 unidentified proteins noticeable on the tomato proteome and 5 on the maize proteome 7
days 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 and
PR-3 on the WB of the proteome PT215. This, however, is an approximation. The molecular weight of
the PR-2 and PR-3 spots detected on the proteome via WB was determined to be about 33 (kD). This
weight was correlated by an anterior study, to be tandem to the molecular weights of b-1,3-Glucanase (33
kD) 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 was
affected by all three bio products; 1 caused the protein level to drop, while bio products 2 and 3 caused it
to increase.




Figure 18. Analyzing molecular weight of PR-2 and PR-3 spots via proteomic and
western blot gels
41

5. Conclusions

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

2. Bio products 2 and 3 have a stronger effect over tomato plants and alter a large number
of 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 application
of 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 is
taken 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 WB
approximately correlates to the molecular weights of b-1,3-Glucanase (33 kD) and to chitinase
(32, 34 kD).
42

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 protein
expression.

3. Use physiological measurement data results to conclude effects that application of different
concentrations 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 and
oxygen isotope results.

1.2 Design

The experiment was organized into four treatment groups, as follows; control 0.2g/L application of
salicylic acid, 0.5g and 0.8g. The treatment groups were then divided into three application times; 0, 2
days (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 and
sample 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 Plants
Tomato Maize
Control 0 5 5
48h 5 5
7 days 5 5
0.2g SA 0 5 5
48h 5 5
7 days 5 5
0.5g SA 0 5 5
48h 5 5
7 days 5 5
0.8g SA 0 5 5
48h 5 5
7 days 5 5

The total number of plants seeded were 60 tomato and 60 maize.
43


Physiological Parameters

Physiological parameters are broken into three distinct parts. Plant were analyzed for 1) photosynthetic
activity, 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 is
unfinished.

1.3 Calendar of events

2.5 months: Time from planting specimens to maturity of 4 leaves, minimum (experimentation in
ETSEA cultivation chambers and greenhouses).
2 days: Photosynthesis and chlorophyll physiological tests conducted
1 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 variety
was LOTE 67728267 Tomato Boludo and the old variety was LOTE 728627 Tomato Boludo 04-05. All
seeds came from Gelboplant, Malgrat de Mar, Spain. The seeds were planted at a depth of 5 10 cm. in a
sphagnum 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. tap
water 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
44

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, as
was 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 maintained
constant 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 growing
chamber with the following temperature and light settings: 24 degrees night time temperature, at 10 hours
night; 18 degrees daytime temperature, at 14 hours day time. The plants were watered three times per
week for the following weeks every Monday, Wednesday and Friday in the morning; usually before
noon.

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 to
the root base of the plants.

The growing chamber used for the tomato plants is the ING climas grow chamber with humidity
Reference: GROW 1300/HR and equipped with florescent lights positioned vertically on the doors on one
side of the chamber. The chamber had two shelves only, which together fit all 60 terra cotta pots. One
shelf was placed at a height of 1m and the other was the ground floor of the chamber
5
. One time during
week 2, and another time at T = 0, all of the plants were rotated so that the plants on the bottom could
have 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 chamber
moved to the front (and thus closer to the lights).

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

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 were
not in a position to receive the same amount of light as those on the higher shelf. This is depicted in Annex XVI (picture 1).
45


Figure 19. Tomato plant time and treatment groups






















T = 7d
T = 48h
T = 0
Control 0.2 0.5 0.8
Tomato
treatment
groups
and time
blocks
Maize
treatment
groups
and time
blocks
Control 0.2 0.5 0.8
T = 0
T = 7d
T = 48h
Figure 20. Maize plant time and treatment groups
46


2.2. Physiological Measurements

2.2.1. Photosynthetic Activity

IRGA is also called the LI-COR plant analyzer. It measures the photosynthetic activity in the plant;
stomata conductance, photosynthesis and transpiration. The plant's leaf photosynthetic activity causes the
air exiting the measurement chamber, where the leaf is, to have a lower CO
2
concentration and a higher
H
2
O concentration than the "reference" air entering the chamber. The "reference" air entrance is at the
end of a tube placed at a distance from the locale of the plants. The tube was placed 3m from the plants,
and allowed to hang over a balcony so as to be exposed to passing air, which would not be too laden with
CO
2
. This instrument was used to take samples for all plants at every stage of the experiment. Five
samples per plant were taken, and the average measurements were calculated. See Annex XV for the
instrument's protocol and Annex XVI (2) for a photograph of the instrument. All measurements were
taken at, or just before, midday in order to optimize the ambient light parameter and to allow the plants to
adjust to the day time photoperiod, which began at the same time every day. All stomatal conductance
(gs) readings are recorded with units of mol CO
2
m
-2
s
-1
where m is meters and s is seconds.

In the case of small plant size, a parameter which our experiment encountered, the leaf can be placed
inside the viewing window of the apparatus in as 'half'. Then, all calculations can be multiplied by two at
the time of calculation. This method was applied for both the tomato and maize plants.

2.2.2. Chlorophyll measurements

Chlorophyll content of the plant leaves was measured using SPAD. In order to do so, plants were taken
from the growth chamber and measured three times each.

The protocol for using SPAD is quite short so it is explained here. First the SPAD meter was calibrated
by pushing the meter closed until the apparatus beeped, confirming its calibration.

The SPAD meter pincher is placed on the leaf so that it is filled completely by the leaf area. It was
important to ensure that the leaf did not double or fold, for this could alter the final reading. The pincher
was closed on the leaf until a reading was registered and the apparatus beeped to confirm success. Each
plant was measured three times, and the average of the three measurements was used for data analysis. A
photograph of the SPAD meter can be seen in picture 3 in Annex XVI.

2.3. Salicylic Acid Application

The salicylic acid solution mixtures were made my diluting a salicylic powder, from Commercial House
Rhodia Operations (see Annex XVI, picture 4) and with molecular weight 138.12g/mole, in 100mL
distilled H
2
0. The solution was applied to the leaves of the tomato and maize plants via the spray method.
Using a spray nozzle, the solution was sprayed individually on each plant until the plants leaves were
47

thoroughly covered and clearly wet on the upper surfaces; about 10 sprays per plant. The solution was
allowed to dry for 20 minutes on the T = 0 samples before the plants were taken to the lab for sampling.

It was important to make sure that the plants which were sprayed with different concentrations of the
solution were not allowed to touch leaves between them as to avoid contamination between trials.

First, both tomato and the maize plants were treated with salicylic acid which was soon followed by the
first plant material harvest. The second harvest was 48 hours (2 days) after the first SA treatment and 7
days after the first SA treatment.

We randomly selected and labeled the plants according to the treatment groups and to the time breaks. It
was important that the plants utilized for the experiment were physiologically fit for harvest and had a
minimum of 4 leaves.

2.4. Harvest

pproximately thirty minutes after salicylic acid treatment, 3 of each treatment group for the T=0 plants,
were taken to the lab to be harvested. It was assumed that thirty minutes was about enough time for the
plants to sufficiently dry and for the SA to seep into the foliar material.

In a sterile environment, each plant was mechanically stripped of its leaves using scissors, until 0.5g. of
plant material could be tallied on a scale. The plant material was cut into pieces the size of a penny.
Samples were stored in previously labeled test tubes and flash frozen in liquid nitrogen. After a few
minutes, they were placed in the freezer at -80 C. See picture 5 in Annex XVI for methodology pictures.
The plants of the T = 2 days and those of T = 7 days were harvested utilizing the same methodology.

2.5. Post Harvest Physiological Tests

At the harvest of T = 0, extra leaves of the tomato and maize plants were stored in small paper bags and
allowed to dry so that the dry material could be powder milled and used to analyze for total and soluble
organic matter. In the ETSEA Silviculture Lab, the dry plant material was taken from the paper bags,
crushed with tweezers, and appropriate sized test tubes were filled with this crumble. Methodology
pictures can be seen in picture 1, Annex XVI.

It was made sure that a minimum of .1g plant material was placed in the test tubes. Three samples were
used per treatment group and test tubes were filled and saved for both tomato and maize.

2.5.1. TOM: Total Organic Matter - Carbon and Oxygen Isotopes

Refer to Annex XVI to see pictures describing the process of sample storage, treatment and grinding.
The samples were ground to a fine powder in a ball mill (Retsch MM301, Haan Germany) for isotope
analysis. The fine powder was then capsulated so that it could be analyzed.

48

All samples were used for the two analysis processes. The same samples would be placed in different
capsules for carbon isotope analysis and for oxygen isotope analysis. The two processes are nearly
identical, though the sample quantity and capsules used for each are distinct. This capsulation process
involved the use of a very fine scale which measured accurately to the mg. Mettler Toledo MX5 scale
was used to measure 1.0 mg +/- 0.01mg sample for the carbon isotope analysis and 0.4mg +/- 0.05mg for
the oxygen isotope analysis.

Using a small spatula/spoon, samples were measured and placed in "Ludiswiss" 0.03mL Sn 98 (tin) -
LOT: 602721capsules for Carbon isotope analysis and in 0.03 ml Ag 99.99 (silver) - LOT: 602889
capsules for oxygen isotope analysis. The weight of the sample within the capsules was recorded (by
means of first 'taring' the scale with only the capsule) and all sample weights were meticulously recorded.
Once an accurate weight was reached for each sample, the capsules were folded closed with tweezers.
This methodology involved the pinching of the open top of the capsule, doubling the capsule with an 's'
motion and then manipulating the tweezers to fold the capsule into a neat square.

Samples were placed in a plastic plate with dividers and numbered like a grid to keep the capsules
organized. This plate was accompanied by a data table with corresponding numbers to record sample
weights.
6


2.6. Protein Extraction

Protein extractions of the tomato plants were conducted on April 29, May 3 and May 5 for T = 0, T = 48
hours and T = 7 days, respectively according to the TCA/Acetone protein extraction protocol which can
be found in Annex IV. Plant material was taken from the freezer -80 C and stored in a foam box full of
ice.

Eppendorf test tubes had been labeled beforehand so that there was a quantity of 3 tubes per sample. 3
extracts per 3 leaf samples from the same treatment group and time is a total of 9 required test tubes per
time and treatment group.

With mortar and pestle, the samples were ground in liquid nitrogen until they were a fine powder. It was
made sure that the plant material was always kept frozen, or partially submerged in liquid nitrogen.

The scale was "tared" with an Eppendorf test tube, which was subsequently filled with 0.1-0.2g. of the
plant powder. All weights were recorded into data tables which can be found in Annex XVII.

Test tubes were stored, open, in a small foam 'boat' created by our lab for specifically holding test tubes.
These boats were suspended in liquid nitrogen until all 9 samples (3 extracts per 3 leaf samples from the
same treatment group and time) were completed. The foam piece was extracted from the liquid nitrogen

6
Capsulated samples were sent May 30, 2011 to the "UC Davis Stable Isotope Facility", Department of Plant and Soil Sciences.
One Shields Ave, Mail Stop 1, Davis, CA 95616, USA.

49

and allowed to sit on ice for 5 minutes or until all nitrogen had percievably evaporated from the test tubes.
The test tubes were promptly closed and stored in the freezer at -80C. This last technique was developed
to evade the problem of 'popping'. It was found that if we closed the test tubes promptly after filling them
with the crushed samples, any remaining liquid nitrogen would cause the test tubes to pop and explode
from the pressure created by the gas.

Maize plant samples have not yet undergone the process of protein extraction.

3. Results

3.1. Proteomic and Western Blot Experiments

Thus far, these parts of the experiment are unfinished. Next steps include:

1. Extract maize proteins from leaf samples was taken over the course of two days. All tomato
protein extractions are complete and stored at -80 C. The protocol for WB protein extraction
can be found in Annex IV.
2. Conduct Proteomic analysis of protein extractions according to the protocol presented in
Study 1, found in Annexes II (Proteomic protein extraction) and XIII (Proteomic Protocol).
3. Conduct Western Blot analysis (Annex V and VIII) for all protein extractions of tomato and
maize.
4. Immunoblot the proteome of the sample whose PR-2 and PR-3 levels were most
drastically affected by salicylic acid treatments and attempt to identify any unidentified "spots"
on the proteome which are induced by salicylic acid after 2 days and 7 days.
5. Compare salicylic acid results with bio product results and discuss any new conclusions.

3.2. Physiological Measurements
Physiological measurements are critical in seeing how the metabolic functions of the plant change as the
plants adjust to become more 'resistant' upon treatment with salicylic acid. Measurements were taken at
time 0, before treatment with salicylic acid, after 2 days, and after 7 days. The three physiological
parameters studied for tomato and maize were photosynthetic activity, chlorophyll content and total
organic matter (TOM).
3.2.1. Gas exchange parameters (IRGA)
Leaf response in tomato and maize plants was determined based on three principle factors; photosynthesis
(A), transpiration (E) and stomatal conductance (gs). Generally, photosynthesis did not vary within the
plants. This parameter is extremely light sensitive; if there was cloud coverage, then the reading could
change. Likewise, transpiration readings are very sensitive to humidity conditions. For this reason, these
two parameters are not very reliable to look at on their own. Stomatal conductance, on the other hand, is
less sensitive to short-term environmental changes and is in a direct relationship to the plant's
transpiration rate; per se, it is completely dependent upon stomatal conductance. The lower the reading of
50

stomatal conductance in a plant, the more stress the plant is undergoing. Indeed, it is one of the most
reliable and sensitive indicators of plant stress (Medrano et al. 2002).

3.2.2. Tomato photosynthetic activity

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

Time
(days)
Treatment
(dosis)
# of
Readings
mol CO
2
m
-2

s
-1
A mean
mmol H
2
O m
-2

s
-1
gs mean
mmol H
2
O m
-2

s
-1
E mean
0 0 3 2.3 0.66 31 5.9 0.5 0.08
0 0.2 3 1.5 0.29 151 25.0 1.9 .027
0 0.5 3 1.8 0.54 88 8.5 1.3 0.10
0 0.8 3 2.2 0.36 76 9.6 1.2 0.10
2 0 3 2.1 0.39 72 5.9 1.0 0.03
2 0.2 3 1.8 0.32 63 4.1 0.9 0.06
2 0.5 3 1.6 0.32 67 14.9 1.0 0.19
2 0.8 3 1.3 0.06 34 5.6 0.6 0.07
7 0 3 1.5 0.23 84 15.9 1.1 0.13
7 0.2 3 2.3 0.31 107 14.9 1.6 0.15
7 0.5 3 1.5 0.56 163 14.4 2.2 0.42
7 0.8 3 1.8 0.43 124 13.5 1.9 0.18

The table shows the actual mean readings for three different leaves (of three different plants) for each
time group and each treatment. In the table alone, it can be observed that stomatal conductance (gs)
readings are lowest at 0 days and highest at 7 days. As predicted, mean E and A readings are difficult to
comprehend and analyze because there is little pattern to their flux. The gas exchange analysis thus
concentrates on gs readings.
Comparing treatment groups as a function of the day

On day 0, the weather was cloudy and so the stomatal conductance readings cannot be used. This is
unfortunate because day 0 was to be used as a control for all other days. It was predicted that all plants
51

would have non- statistically significant stomatal conductance readings. The opposite was true. There
was a 5% significance between control plants and treatment group 0.2g/L and a 2% difference between
both control plants and treatment group 0.5g/L and control and 0.8g/L. It can thus be concluded that
readings for day 0 will not be utilized in the analysis.

A graphic depicting the statistical significance between plant treatment groups can be seen below.


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

In the graph, we see that treatment group 0 (control) has an overall stomatal conductance that is
considerably lower than the other groups. This may be on account of time that the plants were left out of
the growing chamber before measurements were recorded. It is also possible that the measurements of
the 0 (control) group were taken before those of the other groups, while the plants were still in shock to
'out-of-grow-chamber' conditions.

Below are pictures of the control plants and the 0.2g/L treatment plants, which had a difference of 5%
significance between them. The readings for the control plants were the lowest of all and those for
0.2g/L, the highest. If anything, it appears that the plants from the control group in fact have more leaves
are more vigorous that those of treatment group 0.2g/L.

Scatterplot analysis of gs variance T=0
Plant treatment groups
52


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


Next, we look at the variance between tomato plant treatment groups on day 2. The only statistically
significant difference existed between the control group (0) and treatment group 0.8 g/L SA. There was a
2% difference. The 0.8 g/L treatment group's lower stomatal conductance indicates that these plants are
under more stress than the plants from the other treatment groups.

Depicted below is a scatterplot showing the differences between plants and treatment groups at day 2.
treat2
Scatterplot for gs
g
s
0 0.2 0.5 0.8
0
0.03
0.06
0.09
0.12
0.15

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


Scatterplot analysis of gs variance T=2 days
Plant treatment groups
53



Figure 24. Time= 2 days. Comparing control plant (Left) with treatment group 0.8 g/L salicylic acid (Right)
54


The above graphic depicts the progression of toxicity to the foliar areas of the tomato plants. The control
plant is very healthy, while the plant treated with 0.2g/L SA demonstrates slight wilting at the leaf edges.
The plant treated with 0.5g/L (bottom left) demonstrates wilting on all foliar surfaces and clear damage to
the newest growth. Finally, the tomato plant treated with 0.8g/L SA is damaged. The two lowest leaves
ready to fall from the plant. The new growth is in a similar, ready-to-fall state.
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)
55

Last but not least, we look at the differences between plants in different treatment groups on day 7.
Unfortunately, there is not any statistical significance between treatment groups to draw conclusions
from. Treatment group 0.5g/L has 2% significance, but only because there is one plant which is an outlier.
The following scatterplot visually depicts these findings.

treat2
Scatterplot for gs
g
s
0 0 .2 0 .5 0 .8
0
0 .0 3
0 .0 6
0 .0 9
0 .1 2
0 .1 5

Figure 26. 90% LSD scatterplot analysis of gs variance, where Time = 7 days

The changes in average stomatal conductance for all plants from day 2 to day 7, (not taking treatments
into consideration) had a statistically significant increase of 6%. Notice that day 0 data was discarded
because the results were erratic.
The bar graph below is a visual representation of the increase in stomatal conductance for all treatment
groups between days 2 and 7. Though statistical significance tells us little, comparing data from days 2
and 7 is the still interesting because the plants have 'recovered' from the shock of SA application and the
true effects of SA on the plants can be observed accordingly.
Both treatment groups 0.5g/L and 0.8g/L appear to have the most drastic increase when looking at the
change in bar sizes from day 2 to day 7. We know that the change in the 0.5g/L treatment group is due to
an outlier. The results of the 0.8g/L plants, however, is a bit surprising and indicates that when tomato
plants treated with 0.8g/L of salicylic acid recover from the initial toxicity caused by the treatment, then
they have the ability to succeed and have a healthy stomatal conductance.
The bar graph also allows us to see that after 7 days, control plants exhibited the lowest stomatal
conductance as a group. This result is a surprising because at day 2, the control plants exhibited the
highest stomatal conductance as a group. From this, we can draw the conclusion that plants treated with
salicylic acid indeed require one week to physiologically recover from the initial effects of treatment, or
"shock", and use the treatment to their benefit.

Scatterplot analysis of gs variance T=7 days
Plant treatment groups
56


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

Looking at stomatal conductance as a function of treatment group
If the treatment group stomatal conductance (gs) averages are compared with the control group average,
there is a statistically significant increase of 4% gs in both plants treated with 0.2g/L and with 0.5g/L
salicylic acid. This result is very interesting because it indicates that treating plants with 0.2g/L salicylic
acid has the same effect on the stomatal conductance and transpiration of the plant as treating it with
0.5g/L salicylic acid.
In this case, it could be argued that treating a plant with 0.2g/L salicylic acid is better since this
concentration does not ever cause visible foliar damage to the plant.

Looking at stomatal conductance as a function of time
This result is similar to section 2.1, but different in the sense that it looks at the difference between
stomatal conductance averages as a function of the day. Observing the changes in stomatal conductance
between days allows us to further draw on the times when the plants were overall the most active, and
when they were the most shocked, or photo synthetically inactive.
There is statistically significant drop of 2% between days 0 and 2, a 3% increase between days 0 and 7,
and a 6% increase between days 2 and 7. Once again, results commenting on day 0 should be taken
lightly due to the wild nature of the data on account of the cloudy weather. In any case, this result
confirms that the change in stomatal conductance on all plants between 2 and 7 days is statistically
significant.
The graph below depicts this. In the graphic it also is apparent how the overall stomatal conductance of
the plants drops from day 0 to day 2, and then rises dramatically from day 2 to day 7.

Bar graph of treatment gs at days 2 and 7
57

0 2 7
Means and 95.0 Percent LSD Intervals
time2
44
64
84
104
124
144
(X 0.001)
g
s
c
o
r
r


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

Tomato plants visual observations
Observed visual differences among treatments in tomato plants were notable. Annex XVIII displays a
series of pictures, highlighting the most important observations to the study. First, it was interesting to
look at the difference in healthy growth of control tomato plants. The control tomato plants are the main
source of comparison for all treatment groups. Picture1 in the annex compares the group of control
tomato plants on day 0 and on day 7.
We were also concerned with qualifying the extent of foliar damage that the different concentrations of
SA caused to the tomato plants. The most drastic damage was caused on plants in the 0.8g/L treatment
group. Surprisingly enough, the plants managed to recover their vigor (though still showing signs of
damage) between days 2 and 7. This progression is documented and can also be seen in the Annex XVIII,
pictures 2.
Finally, it was interesting to compare the final, day 7, state of different treatment groups' foliage to the
control plant. In Annex XVIII, 3, there is a series of 4 pictures comparing first 0.2g/L, then 0.5g/L and
finally two pictures of 0.8g/L treatment to a control plant. It is clear that the 0.2g/L plant is the most
vigorous, perhaps even more vigorous than the control. 0.5g/L shows significant damage, and finally,
0.8g/L shows the most damage; but in two different ways. The first figure of 0.8g/L depicts the tomato
plant as browned and necrotic as a result of salicylic acid contact with the foliar tissue. The second photo,
however, differs in that it depicts the 0.8g/L treated plant as smaller, yellowed, and a bit wilted.

3.2.3. Maize gas exchange results

Like the tomato, all maize plants exhibited quite distinct initial stomatal conductance levels. Maize plants
were measured in their growing environment and were not extracted from the greenhouse to take
Overall gs Means and 95% LSD intervals by
day
Plant treatment groups
58

readings, so environmental shock cannot be taken into consideration when looking at the differences in
initial gs readings. Similar to the tomato plants, the control plants had the lowest readings at 0 days, and
the 0.8g/L treatment group plants, the highest.
After 2 days, the control group as well as treatment groups 0.2g/L and 0.5g/L all rose in their stomatal
conductance levels. Treatment group 0.8g/L dropped considerably to be lower than all other groups.
At 7 days, all plants had stabilized to have very similar stomatal conductance readings. On average, these
readings are the same as those taken at day 0. In order from lowest stomatal conductance to highest;
treatment group 0.5g/L, 0.8g/L, control and finally, 0.2g/L. It can be said that treatment group 0.2g/L was
under the least shock and 0.5g/L, the most.
Table 6. IRGA photosynthetic readings for maize. (Mean +/- Standard Error)
Time
(days)
Treatment
(dosis) N
mol CO
2
m
-2

s
-1
A mean
mmol H
2
O m
-2

s
-1
gs mean
mmol H
2
O m
-
2
s
-1
E mean
0 0 3 1.9 0.16 37 4.1 0.7 0.05
0 0.2 3 2.3 0.46 79 16.0 1.2 0.21
0 0.5 3 2.0 0.15 47 5.6 0.8 0.04
0 0.8 3 2.3 0.13 97 18.4 1.5 0.23
2 0 3 14.6 1.01 108 12.9 3.1 0.30
2 0.2 3 14.5 0.82 89 7.4 2.9 0.20
2 0.5 3 14.1 0.51 101 16.6 3.3 0.37
2 0.8 3 13.3 0.66 85 5.2 3.1 0.11
7 0 3 7.6 2.03 69 13.0 1.4 0.27
7 0.2 3 7.5 2.08 72 17.9 1.6 0.28
7 0.5 3 6.1 1.34 56 8.9 1.5 0.20
7 0.8 3 4.7 0.63 62 12.1 1.7 0.23

The table shows the actual mean readings for three different maize leaves (of three different plants) for
each time group and each treatment. All data has been included as a reference for further studies, if
needed.
Comparing stomatal conductance of the treatment groups as a function of the day
59

On day 0, the weather was cloudy and so the stomatal conductance readings cannot be used because they
are very irregular. This is unfortunate because like in tomato, maize day 0 was to be used as a control for
all other days. It was predicted that all plants would have non- statistically significant stomatal
conductance readings. The opposite was true. There was a 4% significance between control plants and
treatment group 0.2g/L and a 6% difference between control plants and treatment group 0.8g/L. There
was no calculable significant difference between the control group and 0.5g/L. It can thus be concluded
that readings for day 0 will not be utilized in the analysis because there is too much significant difference
between treatment groups at this time. A graphic depicting the statistical significance between plant
treatment groups at time/day 0 can be seen below.



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

In the graph, we see that treatment group 0 (control) has an overall stomatal conductance that is
considerably lower than the other groups. Tomato results were similar. In tomato, the difference was
attributed to shock from 'out-of-grow-chamber' conditions, but maize plants were measured in the same
greenhouse environment in which they were growing. Perhaps there was more cloud coverage at the time
of measurement than with the latter groups.
There was not any statistically significant difference between treatment groups on day 2, nor on day 7.
Depicted below is a photograph taken on day 2 of all maize treatment groups. The control is on the left,
followed by 0.2g/L, 0.5g/L and finally, 0.8g/L.
60



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

There were not any signs of toxicity on the plant leaves. The changes in average stomatal conductance
for all plants from day 2 to day 7, (not taking treatments into consideration) had a statistically significant
decrease of 3% .

The bar graph below is a visual representation of the increase in stomatal conductance for all treatment
groups between days 2 and 7. Though statistical significance tells us little, comparing data from days 2
and 7 is the still interesting because the plants have 'recovered' from the shock of SA application and the
true effects of SA on the plants can be observed accordingly.




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

Bar graph of treatment gs at days 2 and 7
61

The graph is surprising. We can see that the control group remained virtually the same, while all other
treatment groups decreased in their stomatal conductance.
The bar graph also allows us to see that after 7 days, plants in treatment group 0.2g/L exhibited the lowest
stomatal conductance as a group. This result is a surprising because at day 2, the 0.2g/L plants exhibited
the same stomatal conductance as the other two salicylic acid treatment groups.
On average, there was a statistically significant difference between treatment group averages of 0.2g/L
and 0.5g/L, of 2%, and there was also a difference between groups 0.5g/L and 0.8g/L of 3%.
Treatment group 0.8g/L treatment group had the highest average stomatal conductance throughout the
week and the 0.5g/L treatment group, the lowest.
Looking at stomatal conductance as a function of time
Observing the changes in stomatal conductance between days would allow us to draw on the times when
the plants were overall the most active, and when they were the most shocked, or photo synthetically
inactive. There was an overall 3% significance in the decrease in stomatal conductance between days 2
and 7. Tomato plants had statistically significant results between these two days as well, though in the
case of tomato, the stomatal conductance between days 2 and 7 rose, while that of maize declined.


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

Maize plants visual observations
Based on results that have been summarized above, it was interesting to visually compare the control
treatment group with 0.8g/L treatment group after 7 days. Although the control group's stomatal
conductance was numerically higher than that of 0.8g/L treatment group's at days 2 and 7, the overall
stomatal conductance of treatment group 0.8g/L was higher. Below is a picture comparing the four
treatment groups on day 7. The difference in overall aspect between treatment group plants is notable. We
see the control group plants (far left) considerably weaker looking and more wilted. The 0.5g/L treatment
group plants are the largest and vigorous and the 0.8g/L treatment group plants slightly smaller, but
equally, if not more, 'perky'.
62



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

Foliar damage was not an issue in the case of maize plants.

3.2.4. Chlorophyll Content

The chlorophyll content of the plants is another indicator of photosynthetic activity. In the case of tomato,
tests rendered significant results only on day 2. There was notable difference between the control and
distinct treatment groups, but never between treatment groups themselves. The reason for such
insignificant results in general could have been a mechanical error, due to folded leaves at the time of
measurement, or a number of other factors.


Figure 34. Tomato SPAD chlorophyll readings, comparing different salicylic acid treatment groups on day 2
63



It is curious that the SPAD reading for the control was so low on day 2, because at time 0, the reading was
considerably higher. Once again, this could be due to a mechanical error, or different leaves were used in
taking the average of 5 measurements.
In the graph, we can observe that chlorophyll content is highest of all in 0.5g/L treatment group plants.
This is a visual observation, as the reading is not statistically significant over the 0.2 and 0.8g/L treatment
groups. Tables with the SPAD reading for tomato and maize can be found in Annex XIX.
Though the results between days were not statistically significant to speak of, it is interesting to see a
graph of these results, to observe the slight change in plant chlorophyll content between tomato plant
treatment groups and between days. This graph is depicted below.


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

Results for chlorophyll content in maize plants was not significant neither by day nor by treatment nor the
interaction of the two.

64

4. Discussion and Conclusions

Thus far, we do not have enough results to discuss and draw conclusion regarding the premier objective:
Determine an ideal concentration for salicylic acid application to tomato and maize plants based upon
proteomic, PR and physiological response.

So to respond and draw conclusions for each objective which has proven relevant in this study, we begin
with the second objective: Use physiological measurement data results to conclude effects that
application of different concentrations of SA to tomato and maize plants has upon biological processes
influenced by photosynthetic rate and chlorophyll levels, over one week time.

This study has brought us to one important conclusion above others, and that is that analyzing maize
plants for physiological parameters is very difficult. Maize results regarding photosynthetic activity have
some, but little significance; while those regarding chlorophyll content are completely insignificant.
Also, photosynthetic activity results were heavily influenced by the light and cloud cover during the days
and times of data gathering. This is the principle reason why all results from time 0 had to be thrown
away.

The results lead us to conclude that the photosynthetic activity of maize plants is negatively affected by
time because the day with the lowest overall stomatal conductance, for all treatment groups, was day 7.
From lowest gs to highest at day 7: 0.2g/L, 0.5g/L, 0.8g/L and finally ,the control. Treatment group
0.2g/L was under the most shock. There was not significant interaction to speak of between treatment
groups and days. Perhaps the results require more time to see a significance difference in comparison
with time 0. If the gs decreases it means the plants are undergoing prolonged stress opening the
possibility that they could be producing more PRs. At day 7, all plants stabilized and had similar gs
readings.
In looking at the tomato plants, we come to a very different discussion. Plants treated with 0.8g/L were
most significantly stressed at day 2. Visual observations confirm this. Not only this, but all treated plants
were foliar-ly stressed 2 days after salicylic acid application. This is why the control group had the
highest stomatal conductance on this day.

There is no significant difference between treatment groups one week after salicylic acid application. It
was clear that the control group had the lowest stomatal conductance of all, and 0.8g/L, the highest. It
can be said that plants treated with 0.8g/L SA are harmed foliar-ly and undergo serious shock at day 2,
but fare the best in terms of stomatal conductance after one week. In general, the significant difference of
overall stomatal conductance of 0.2g/L or 0.5g/L when compared with the control, was the same. In other
words, the two treatments have the same physiological affect on the plant groups. The foliar damage
however, is a factor which weighed more heavily in the 0.5g/L treatment group and lets us conclude that
0.2g/L is the better dilution of the two (based on physiological response results).

Foliar damage is a heavy topic of discussion because perhaps it would have been different, and less
severe, if the plants had been older.

65

The 0.2g/L was the only treatment group that presented only slight foliar damage, but damage
nonetheless. This damage disappeared with time and was hardly visible at day 7. The damage caused to
0.5 and 0.8g/L treatment groups however was irreparable.

There is a significance difference in the stomatal conductance for all plants between days 2 and 7.
Comparing data from days 2 and 7 is the interesting because by day 7, the plants have 'recovered' from
the shock of salicylic acid application and the true effects of salicylic acid on the plants can be observed
accordingly.

Evaluate the effects that different concentrations of salicylic acid has on PR protein expression

Uniting the studies would involve analyzing the proteomes of the protein samples which contain the
highest quantities of PR-2 and PR-3. Based on the physiological tests, it can be assumed that the samples
with the highest level will be the 0.8g/L treatment at day 7. The 0.2g/L treatment at day 7 runs close
behind in stomatal conductance levels, and so may have very high PR levels too; what more, is a better
treatment overall due to foliar implications. We ask: does initial shock and then later stabilization lead to
bigger and better PR protein expression (as in 0.8g/L tomato) OR does less damage, shock and recovery
lead to better PR protein expression (as in 0.2g/L)? Per se, are the damages worth the final results?

When the proteome of 0.2g/L for 7 days is mapped, then it should be immunoblotted for PR-2 and PR-3,
to see where these proteins land on the proteome and to aid in the identification of any unknown defense
proteins in the tomato.

When analyzing the proteome, it will be interesting to identify all known proteins, as was done in Study
1. The rise and fall of certain proteins can be analyzed to draw connections and conclusions regarding the
biological pathways and systems involved in SAR and how these interrelate with the solicitation of
specific PRs.

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

When analyzing these results, it will be interesting to compare isotope values to the stomata conductance
and evapo-transpiration values extracted from the photosynthesis tests. Will the results confirm each
other? If not, then it will imply that the physiological tests are unstable to rely on. The isotopes better
reveal a summary of the transpiration of the plant over the course of the plant's history. We will be able
to extract conclusions based on the knowledge that the higher the evapo transpiration rate of a plant, the
less stress the plant has endured and the more effective the corresponding salicylic acid application.
66

Future study

To continue this work, Study 3 will conclude Study 2 and will go further by examining the effects that
salicylic acid has on the physiological parameters of the plants, PR-2 and PR-3 apparition and
concentration, while taking into consideration the introduction of pathogens to the plants.

To take the investigation further, an added parameter may be microscopy. The current study design
allows us to look at changes in the plants on the molecular and the physiological levels, but it would be
interesting to see cellular changes too.












67

IV. BIBLIOGRAPHY

Ananievaa, E.A., K.N. Christov and L.P. Popova. "Exogenous treatment with salicylic acid leads to
increased antioxidant capacity in leaves of barley plants exposed to Parquat." J Plant Physiol 161(3)
(2004): 319-28.

Arfan, M., H.R. Athar, and M. Ashraf. "Does exogenous application of salicylic acid through the rooting
medium modulate growth and photosynthetic capacity in two differently adapted spring wheat cultivars
under salt stress?" J Plant Physiol 164 (2007): 685-94.

Barosky, R.R. and F.A. Einhellig. "Effects of salicylic acid on plant water relationship." J Chem Ecol 19
(1993): 237-47.

Bashan, Y. "Innoculants of plant growth-promoting bacteria for use in agriculture." Biotechnol. Adv. 16
(1998): 729-770.

Benhamou, N., S. Gagne, D. Le Quere and L. Dehbi. "Bacterial-mediated induced resistance in
cucumber: Beneficial effect the endophytic bacterium Serratia plymuthica on the protection against
infection by Pythium ultimum." Phytopathology 90 (2000): 45-56.

. "Induction of defense related ultra structural modifications in pea root tissues inoculated with
endophytic bacteria." Plant Physiol 112 (1996c): 919-929.

. "Induction of differential host responses by Pseudomonas florescens in Ri T-DNA transformed Pea
roots after challenge with Fusarium oxysporum f. sp. pisi and Pythium ultimum." Phytopathology 86
(1996b): 1174-1185.

. "Pre-inoculation of Ri T-DNA transformed Pea roots with Pseudomonas florescens inhibits
colonization by Pythium ultimum Trow: an ultra structural and cytochemical study." Planta 1999 (1996a):
105-117.

Britannica, Encyclopedia. Nature and importance of plant diseases. 22 February 2011
<http://www.britannica.com/EBchecked/topic/463327/plant-disease/63295/General-considerations>.

Campos, M.A., S.G. Ribeiro, D.J. Rigden, D.C. Monte and M.F. Grossi de Sa. "Putative pathogenesis-
related genes within Solanum nigrum L. var. americanum genome: isolation of two genes coding for PR5-
like proteins, phylogenic and sequence analysis." Physiological and Molecular Plant Pathology 61 (2002):
205-216.

Cann, Dr. Alan.MicrobiologyBytes: Plant Viruses. 8 April 2009. 23 February 2011
<http://www.microbiologybytes.com/virology/Plant.html>.

68

Collinge, D.B., J. Borch, K. Madriz-Ordenana and M-A.Newman. "The responses of plants to
pathogens." Hawkesford, P. and MJ Buchner. Molecular analysis of plant adaptation to the environment.
Kluwer Academic Publishers, 2001.

El-Tayeb, M.A. "Response of barley grains to the interactive effect of salinity and salicylic acid." Paltn
Growth Regul 45 (2005): 215-24.

Feller, U., I. Anders and K. Demirevska. "Degradation of RuBisCO and other chloroplast proteins under
abiotic stress." Gen Appl. Plant Physiology, Special Issue 34 (1-2) (2008): 5-18.

Glazebrook, J., E.E. Rogers and F.M. Ausubel. "Use of Arabadopsis for genetic dissection of plant
defense responses." Annual Review of Genetics 3 (1997): 547-569.
Gomez-Vasquez, R., et al. "Phenylpropanoids, Phenylaline ammonia lyase and peroxidases in elicitor-
challenged Cassava (Manihot esculenta) suspension cells and leaves." Annals of Botany 94 (2004): 87-97.

Graskova, I.A., S.V. Vladimirova and E.G. Rikhvanov. "The mechanism of peroxidase activation in
bacterial pathogenesis is different in the cells of potato cultivars resistant and nonresistant to the
pathogen." Doklady Biological Sciences Volume 379 (1-6) (2001): 347-349

Gunes, A., A. Inal, M. Alpaslam, F. Erslan, E.G. Bagsi and N. Cicek. "Salicylic acid induced changes on
some physiological parameters symptomatic for oxidative stress and mineral nutrition in maize (Zea mays
L.) grown under salinity." J Plant Physiol 164 (2007): 728-36.

Halim, V.A., A. Vess, D. Scheel and S Rosahl. "The role of salicylic acid and jasmonic acid in pathogen
defense." Plant Biol 8(3) (2006): 307-313.

Heil, Martin and Richard M. Bostock. "Induced Systemic Resistance (ISR) Against Pathogens in the
Context of Induced Plant Defenses." Annals of Botany 89 (2002): 503-512.

Hunt, M. and J. Ryals. "Systemic acquired resistance signal transduction." Crit. Rev. Plant Sci. 15 (1996):
583-606.

Knogge, W. "Fungal infection of Plants." The Plant Cell 8 (1996): 1711-1722.

Larque-Saavedra, A. "Stomatal closure in response to acetylsalicylic acid treatment." Z Pflanzenphysiol
93 (1979): 371-5.

Lausanne, Ecole Polytechnique Federale de. Electron Microscopy. 2010. 20 February 2011
<http://come.epfl.ch/electron-microscropy>.

Lawton, K., K. Weymann, L. Friedrich, B. Vernooij, S. Uknes and J. Ryals. "Systemic acquired resistance
in Arabadopsis requires salicylic acid but not ethylene." Molecular Plant-Microbe Interactions 8 (1995):
863-870.
69


Low, P.S. and J.R. Merida. "The oxidative burst in plant defense: Function and signal transduction."
Physiol. Plant 96 (1996): 533-542.

Madriz-Ordenana, K. "Mecanismos de defensa en las interacciones planta-patogeno." Manejo Integrado
de Plagas (Costa Rica) 63 (2002): 22-32.

Maleck, K., A. Levine, T. Euglem, A. Morgan, J. Schmid, K.A. Lawton, J.L. Dangle and R.A. Dietrich.
"The transcriptome of Arabadopsis thaliana during systemic acquired resistance." Nature Genetics 26
(2000): 403-410.

Mauch-Mani, B. and A.J. Slusarenko. "Systemic acquired resistance in Arabadopsis thaliana induced by
predisposing infection with pathogenic isolate of Fusarium oxysporum." Mol. Plant Microbe Interact. 7
(1996): 203-212.

Mauch-Mani, B. and J.P. Metrauz. "Salicylic acid and system acquired resistance to pathogen attack."
Annals of Botany 82 (1998): 535-540.

Medrano H., J.A. Escalona, J. Bota, J. Gulias and J. Flexas. (2002) Regulation of photosynthesis of C
3
plants in response to progressive drought: stomatal conductance as a reference parameter. Annals of
Botany 89, 895 - 905.
Montesinos, E., A. Bonterra, E. Badosa, J. Frances, J. Alemany, J. Llorente, I. Moragrega . "Plant-
microbe interactions and the new biotechnological methods of plant disease control." Int. Microbiol. 5
(2002): 169-175.
Narayanasamy, P. Molecular Biology in Plant Pathogenesis and Disease Management: Disease
Development Volume 2. Springer, 2008.

Nasser, W., M. de Tapia, S. Kauffmann, S. Montasser-Kouhsari and G. Burkard. "Identification and
characterization of maize pathogenesis-related proteins. Four maize PR proteins are chitinases." Plant
Mol. Biol. 11 (1988): 529-538.

Neuenschwander, U. "Systemic acquired resistance." Stacey, G. and .T. Keen. In Plant-Microbe
Interactions Volume 1. New York: Chapman and Hall, 1996. 81-106.

Orcutt, S.M. and E.T. Nilsen. The physiology of plants under stress: soil and biotic factors. John Wiley &
Sons, 2000.

Park, K.S. and J.W. Kloepper. "Activation of PR-1a promoter by rhizobacteria that induce systemic
resistance against Pseudomonas syringae pv. tabaci." Biological Control 18 (2000): 2-9.

Pieterse, CMJ and L.C. van Loon. "Salicylic acid- independent plant defense pathways." Trends in Plant
Science 4 (1999): 52-58.
70


Pierterse, C.M.J., J.A. Van Pelt, B.W.M. Verhagen, J. Ton, S.C.M. Van Wees, K.M. Leon-Kloosterziel
and L.C. Van Loon. "Induced systemic resistance by plant-growth promoting rhizobacteria." Symbiosis
35 (2003): 39-54.
Proteomics. 14 December 2010. 23 February 2011
<http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Proteomics.html>.

Rai, V.K., S.S. Sharma and S. Sharma. "Reversal of ABA-induced stomatal closure by phenolic
compounds." J exp Bot 37 (1986): 129-34.

Ramamoorthy, V., T. Raguchander and R. Samiyappan. "Induction of defense-realted proteins in tomato
roots treated with Pseudomonas flourescens Pf1 and Fusarium oxysporum f.sp. lycopersici." Plant and
Soil 239 (2002): 55-68.

Ruz, L., E. Badosa and E. Montesinos. "Las proteinas de estres en plantas." Phytoma Espana 159 (2004):
49-51.

Ryals, John A. , U.H. Neuenschwander, M.G. Willits, A. Molina, H.Y. Steiner and M.D. Hunt. "Systemic
acquired resistance." The Plant Cell 8 (1996): 1809-1819.

Samiyappan, R., S. Harish, R. Radjacommare and D. Saravanakumar. "Exploitation of Plant Growth
Promoting Rhizobacteria (PGPR) in horticultural crops." Keshavachandran, R. and et al. Recent Trends in
Horticultural Biotechnology Vol. I and II. Vellanikkara, Kerala, India, 2005. 59-83.

Schulze, Ernst-Detlef and Martyn M. Caldwell. Ecophysiology of photosynthesis. Berlin: Springer-
Verlag, 1994.

Siddiqui, Zaki A. PGPR: Biocontrol and Biofertilization. Dordrecht, The Netherlands: Springer, 2006.
Sindelarova, M., L. Sindelar, N. Wilhelmova and D. Prochazkova. "Changes in key enzymes of viral-
RNA biosynthesis in chloroplasts from PVY and TMV infected tobacco plants." Biologia Planetarium 49
(3) (2005): 471-474.

Soto, Maria J. A. Dominguez-Ferreras, D. Perez-Mendoza, J. Sanjuan, J. Oliveras. "Mutualism versus
Pathogenesis: the give and take in plant-bacteria interactions." Cellular Microbiology (11)3 (2009): 381-
388.

Spreitzer, R.J. and M.E. Salrucci. "Structure, regulatory interactions, and possibilities for a better
enzyme." Annual Review of Plant Biology 53 (2003): 449-475.

Tarze, A., M. Deniaud and E. Le Bras. "GAPDH, a novel regulator of the pro-apoptotic mitochondrial
membrane permeabilization." Oncogene 26 (18) (2007): 2602-2620.
Uknes, S., et al. "Acquired resistance in Arabadopsis." Plant Cell 4 (1992): 645-656.
71

Van Loon, L.C. and E.A. Van Strien. "The families of pathogenesis-related proteins, their activities, and
comparative analysis of PR-1 type proteins." Physiological and Molecular Plant Pathology 55 (1999): 85-
97.

Van Loon, L.C., M. Rep and C.M.J. Pieterse. "Significance of Inducible defense-related Proteins in
Infected Plants." Annu. Rev. Phytopathol. 44 (2006): 135-62.

Van Loon, L.C and P.A.H.M. Bakker. "Root-associated bacteria inducing systemic resistance."
Gnanamanickam, S. Plant-associated bacteria. Dordrecht, The Netherlands: Springer, 2006. 269-316.

Verhagen, B.W.M., J. Glazebrook, T. Zhu, H-S. Chang, L.C. van Loon and C.M.J. Pieterse. "The
transcriptome of the rhizobacteria-induced systemic resistance in Arabadopsis." Molecular Plant-Microbe
Interactions 17 (2004): 895-908.

Viswanathan, R., R. Nandakumar and R. Samiyappan. "Role of pathogenesis-related proteins in
rhizobacteria-mediated induced resistance against Colletotrichum falcatum in sugarcane." Journal of Plant
Disease Protection 110 (2003): 524-534.

Ward, E.R., S.J. Uknes, S.C. Williams, S.S. Dichner, D.L. Wiederhold, D.C. Alexander, P. Ahl-Goy, J.P.
Metraux and J.A. Ryals. "Coordinate gene activity in response to agents that induce systemic acquired
resistance." Plant Cell 3 (1991): 49-59.

White, R.F., E.P. Rybicki, M.B. Von Wechmar, J.L. Dekker, J.F. Antoniw. "Detection of PR-1 type
proteins in Amaranthanceae, Chenopodaciaceae, Gramineae and Solanaceae by immunoblotting." J. Gen.
Virol. 68 (1987): 2043-2048.

Xu, Y., P.L.F. Chang, D, Liu, M.L. Narasimhan, K.G. Raghothama, P.M. Hasegawa and R.A. Bressan.
"Plant defense genes are synergistically induced by ethylene and methyl jasmonate." Plant Cell 6 (1994):
1077-1085
72

VI. ANNEXES
Annex I
IMAGES OF PLANT DEVELOPMENT IN PROJECT 1


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


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




73

Annex II
PROTEIN EXTRACTION PROTOCOL

Procedure
- Grind with a mortar and pestle 1g. tomato leaves in liquid N
2

- Divide 0.1g/tube the frozen powder in cold microtubes with a +/- 0.02g. margin of error.
- Submerge in liquid Nitrogen and conserve at -80 C until moved to the SPG.

TCA/Acetone method
0. Fill the tubes with the cold solution of 10% TCA/ acetone/ 0.07% DTT. Mix well with vortex.
1. Incubate the tubes at -20 C (protein precipitation) for 5h.
2. Centrifuge at maximum velocity for 30 min and at 4 C. Eliminate the supernatant.
3. Clean the "pellet" by filling the tube with acetone and re suspend the pellet with a vortex or with a
pipette point.
4. Centrifuge at maximum velocity for 5min.
5. Repeat steps 3 and 4 two more times, or until the pellet is a white color.

Annex III
MATCHING EXAMPLE


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

74


Example of "matching". The gels that were compared by the PDQuest program, for only one replica.
Total number of "matchings" = 64
Annex IV
WESTERN BLOT: PROTOCOL FOR PROTEIN EXTRACTION OF TOMATO LEAF

1. Grind 0.5g of tomato leaf with a mortar and pestle with liquid N
2

2. Separate the powder in 3 cold micro tubes. Put 0.10.2 g/ tube +/- 2mL of reason
3. Submerge the tubes in liquid N
2
and conserve at 80 C.
PROTEIN EXTRACTION

1. Add 2ml of 10% TCA Acetone + 20mM DTT (in glass and 20C) + protease inhibitor.
2. Incubate at 20C for at least 2h. White scales should be apparent suspended in the liquid.
3. Centrifuge 14.000rpm/4C/30min (eliminate the supernatant with a blue point).
4. Wash the pellet 3 times with: 21,5mL Acetone + 20mM DTT. Break apart the pellet. Re-
suspend the pellet with the vortex or with a blue point. A white pellet should remain.
5. Centrifuge 14.000 rpm/4C/15min.
6. Dry on ice (1030min).
7. Add 100250L Lysis Buffer (PER 4, Sigma). Room temperature (RT). Be careful to not create
foam, as it does easily when mixed with the detergent.
8. Termomix or rotator. Mix at (800850rpm) Over Night/ 22 C.
9. Centrifuge 14.000rpm/10min.
10. Take off the supernatant.
11. Protein quantification via the Bradford method.
a. Dilute the unknown concentration of extracted protein to 1/100/L.
b. With this known dilution, create 3 more samples of different concentrations. An
additional dilution/ test tube will be the blank or constant. These different dilutions
will allow us to fit the Abs readings to the extracted formula (created in the trials -
graphs) and whereupon predict the molecular weight of the proteins.
c. Run the program and record all readings.
12. Make portions (1WB x 25 mg, 2 x 125g, or that which remains) and freeze everything at -20 C.
REAGENTS

100% trichloro acetic acid (TCA) (Sigma, #91228)
DTT (GEHealthcare, #15379) Stock 1M
Protease inhibitor (Sigma, #P9599)
Acetone
Lysis Buffer PER4 (Sigma, #C0356) Portions of 1ml at -20 C. Completely unfreeze at room temperature.
(Do not heat above 30 C).
Bradford Reagent (Bio-Rad Protein Assay Dye Reagent Concentrate, 450 ml, #500 - 0006.)

Equipment
75


Refrigerated centrifuge for micro tubes.
Spectrophotometer (Bradford)
Termomix, or spinner.

Annex V
WESTERN BLOT: GEL PREPARATION

Table 7. Western blot resolving and stacking mixtures
Resolving (one gel) Two gels R Stacking (one gel) Two Gels S
6mL 12mL 6mL 12mL
9% 9% 4% 4%
30% acrylamide/bis (mL) 1.8 3.6 0,8 1.6
4x TRIS-HCl-SDS pH
8,8 (mL)
1.5 3.0 - -
4x TRIS-HCl-SDS pH
6,8 (mL)
- - 1.5 3.0
H2O (mL) 2.7 5.4 3.7 7.4
10% APS (L) 30 L 60 30 60
TEMED (L) 3 L 6 3 6

Annex VI

WESTERN BLOT: BUFFERS

Tris = Trizma base (Sigma)

Protein Extraction Buffer: prepared freshly, for 100 ml, add:
- 0.2M Tris-HCl pH = 7.5 20ml Tris-HCl 1M (pH = 7.5)
- 5mM EDTA 1ml EDTA 0.5M pH = 8
- 0.1% Tween-20 100l Tween-20
- Protease inhibitors

76

4x Tris-Cl/SDS pH 6.8: stored in fridge, 100 ml
- 6.05g Tris-base in 40ml H
2
0
- adjust pH = 6.8
- add 2ml of 20% SDS and H
2
0 until 100ml

4x Tris-Cl/SDS pH 8.8: stored in fridge, 500ml
- 91g Tris base in 300ml H
2
0
- adjust pH = 8.8
- add 10ml of 20% SDS and H
2
0 500ml

Loading Buffer 6x: stored at RT, 10 ml
- 1.25ml Tris-HCl 1M pH = 8
- 2ml 20% SDS
- 6ml glycerol
- ~ 50mg bromophenol blue
10ml H
2
0

Polyacrylamide-bis Polyacrilamide from Sigma (A3574)

Running Buffer 5x: stored at RT, 1L
- 15.14g Tris
- 72.7g glycine
- 25ml 20% SDS
1L H
2
0, do not adjust the pH

Transfer Buffer = Running Buffer 1x concentration that contains 20% methanol, 1L
- 200 ml methanol
- 200 ml Running Buffer 6x
- 600 ml H20

Blocking Buffer: 0.5 L
- 3% Bovine Serum Album (Fraction V)
- Make up in PBS 1x and sterile filter, then add 0.05% Tween-20, Keep at 4 C to prevent bacterial
contamination

PBS 10X: stored at RT, 1L
- 2g KH
2
PO
4

- 29g NA
2
HPO
4
* 12 H
2
O
- 2g KCl
- 80g NaCl

TBS 1X: 1L
It can be used instead of PBS
77

- 150mM: Tris-HCl 1M pH = 7.5 50ml
- 50mM: 8.76g NaCl

PBS-T 10x: same as PBS, but adding 0.1% Tween-20

Annex VII
PROTEIN QUANTITIES AND SAMPLE CODES

Experiment conducted by the SPG
Table 8. Protein quantities and sample codes
Well # Protein code Sample Quantity of Protein
(g/l)
1 PT082 KT0-1.1 1.860
2 PT083 KT0-2.1 4.040
3 PT084 KT0-3.1 4.078
4 PT085 KT1-1.1 4.088
5 PT086 KT1-2.1 4.210
6 PT087 KT1-3.1 2.150
7 PT091 BE1T1-1.1 4.455
8 PT092 BE1T1-2.1 2.185
9 PT093 BE1T1-3.1 3.541
10 PT163 BE2T1-1.1 3.00
11 PT166 BE2T1-2.1 3.90
12 PT169 BE2T1-3.1 4.01
13 PT209 BE3T1-1.1 1.33
14 PT212 BE3T1-2.1 2.25
15 PT215 BE3T1-3.1 1.89
Protein Mixture:
10l sample (protein) 2l me (Beta mercaptanol ethanol)
3.5l LB (loading buffer) 6x fill up to 12l water
78

Annex VIII
WESTERN BLOT: INCUBATION AND REVELATION

1. Membrane transfer from gel to 2 PVDF membranes (transfer conditions: 60mA/ membrane/ 1h)
2. Blocking of the membranes: I-block 0.3% in PBS (without Ca++) + 0.1% Tween-20 (1h minimum)
3. Incubation of the first antibody Ab: TBS + 0.1% Tween-20 + 5% I-block. (1h, room temperature - RT)
MB1_Ab anti PR-2: 1/2500
MB2_Ab anti PR-3: 1/2500
4. Wash the first antibody; TBS + 0.1% Tween-20 + 5% I-block (3 washes for 5min each).
5. Incubation of the secondary antibody Ab: TBS + 0.1% Tween-20 + 5% I-block.
MB1/MB2_Ab anti IgG rabbit: 1/100000
6. Wash second antibody: TBS + 0.1% Tween-20 (5 washes for 5min each).
7. Wash with Immobilion Western Chemiluminescent HRP Substrate, 5'/mb.
8. Expose the membrane at 300" for 60sec x 3 exposures.
a. Wash the membranes with AzidaNa at 0.02% (solution stock at 2%) minimum 1h/ RT.
b. Incubation of the primary antibody Ab: TBS + 0.1% Tween-20 + 5% I-block (1h, RT).
- MB1_Ab anti EF-1: 1/1000
- MB2_Ab anti EF-1: 1/1000
c. Wash the first antibody Ab: TBS + 0.1% Tween-20 + 5% I-block. 3 washes for 5' each.
d. Incubate the secondary antibody Ab: TBS + 0.1% Tween-20 + 5% I-block
- MB1/MB2_Ab anti IgG rabbit: 1/100000
e. Wash the secondary antibody Ab: TBS + 0.1% Tween-20 (5 washes at 5min each).
f. Develop with Immobilon Western Chemiluminescent HRP Substrate, 5'/mb.
- luminol (dark) 750l
- peroxide (light) 750l
Mix the two chemicals, and put them over the membranes, allowing 5min for reaction.
Put the membranes in small plastic folios for protection.
g. Expose 300" for 60sec x 3 exposures.
- BIORAD Quantity One Machine
i. Program: Chemi Doc
ii. place one membrane in the center of the machine drawer and
center with a ruler
iii. Press "Epi White" on the machine: to see the focus and to
center, then turn off
iv. On the computer: "Freeze", "Live Acquire" (Exposure time
300.00; Starting exposure: 60.00 sec.; Number exposures: 3)
v. Rightclick and "Transform" to make the image brighter
vi. "Autoscale"
vii. Open images and resave: Export image to TIFF and to save
and view elsewhere. Export view excluding overlays.


79

Annex IX
PR-2 PRODUCT INFORMATION


80

Annex X
PR-3 PRODUCT INFORMATION


81

Annex XI
RABBIT IGG PRODUCT INFORMATION


82

Annex XII
EF-1 PRODUCT INFORMATION


83


Annex XIII

ISOELECTRIC FOCUS

Experiment conducted by the SPG

Protocol: A total of 3 strips per sample were needed so the rehydration volume of the strip was 125l x 3
= 375 (400). The weight of protein for one strip is 20g so for 3= 60 (64g).
- Strips: 7cm IPG; pH 3-10 NL (Bio-Rad)
- Re-hydration volume of the strip: 125l
- Weight of the protein on the strip: 20g
- Apply the sample: during the re hydration.
- Rehydration: activate at 50v/20 C/12h. Place the strip with its acidic side (+) oriented facing the
anode (red/+).
- Focus conditions: 20C; final voltage: 4.000v; total v-h: 8.000-10.000; ramp type: fast.

Focus program: 7 cm

S1: fast, 50v12h
S2: fast, 150v1h
S3: fast, 300v1 h
S4: fast, 1.000v3 h
S5: fast, 4.000v (9.000vh)
S6: fast, 500v10 h

- Freeze the strips at -80 C

SAMPLE PREPARATION

Table 9. Sample preparation
Sample
(protein
code)
(g/l) Sample (l) RHB (l) DTT 1M (l) Ampholytes pH 3-10
(l)
PT215 1.89 34 342 20 4

The extracted sample was prepared for IEF under the following conditions:

1. All samples were unfrozen and precipitated in an RHB vial.
2. The sample was prepared according to the recipe table below, then samples were left to agitate
at 22 C/ hours.
3. Place strips with the acidic side (+) oriented facing the anode (+).
4. Cover strips with mineral oil.
84

5. According to the protocol, IEF was run for the 7cm. strip. The number of gels was
introduced for focusing (3). The total focus was 13.053vh.
6. Balance the strips: they need 2.5 ml/strip. Incubate with each solution for 10-15
min.
Solution I:
6M urea 4.32g
0.375M Tris-HCl 3ml 1.5M Tris-HCl, pH = 8.8
2% SDS 24ml 10% SDS
20% glycerol 2.4ml
2% DTT 200mg
For 10ml, weigh 200 mg DDT.

Solution II:
6M urea 4.32g
0.375M Tris-HCl 3ml 1.5M Tris-HCl, pH = 8.8
2% SDS 24ml 10% SDS
20% glicerol 2.4ml
2.5% IAA 250mg

7. Place the strip on the electrophoresis gel with the extreme acid (+) on the left and the gel
facing you, touching the plastic to the posterior glass. Push the strip until it is in contact with the
face of the gel (it may be necessary to lubricate the strip with a bit of Running Buffer). Eliminate
all the bubbles between the gel and the strip and the strip and the glass. Cover the tempered
agarose.
8. Mount the gels in the electrophoresis box. In the upper tank (catode) put the Running Buffer
2x.
9. Run the gels: 5 mA/gel for 30 min. Then, 20 mA/gel-final.

SDS-PAGE GEL PREPARATION

3 gels were needed: 2 transfer gels and 1 gel to stain with Oriole. Three SDS/PAGE gels at 9% R were
prepared. 50% isopropanol was added and then the gel was left to polymerize at room temperature (RT).
The following solution is for a gel volume at 9%, small R (7cm strip).
Table 10. Stock solution measurements for 3 SDS-page gels
Stock solution 3
gels (ml)
H
2
O MilliQ 6.45
Acrilamide/Bisacrilamide 30% 4.5
Tris-HCl 1.5 M pH 8.9 3.75
SDS 10% 0.15
PSA 10% 0.12
TEMED 0.015
85

Annex XIV

PATHOGENESIS-RELATED PROTEINS: RESEARCH PROGRESS IN THE LAST 15 YEARS


Figure 39. PR proteins induced by Samsun tobacco by TMV infection (Bol et al. 1990)
86

Annex XV
BASIC STEPS FOR PHOTOSYTHETIC MEASUREMENTS - IRGA

Stage 1: Equipment Settings

1. Mount the lower part of the measurement chamber (pincher A)
2. Put the spring in place. Mount the upper part of the measurement chamber (pincher B) above pincher A
and connected to the spring.
3. Above pincher B, mount the acrylic sheet, though first positioning the radiation sensor.
4. Connect the cables (red, white, black) of the pincher to the equipment according to the color scheme
(red to red, white to white, and black to black).
5. Place the memory card in the corresponding slot.
6. Turn on the equipment using the I key. From now on, the I key will be used to go to elected
screens and menu options.
7. Wait until the equipment is warmed up. "Warming, realizing bip).

Stage 2: Aspects to check before making measurements

8. Before measuring, first check that the equipment configuration corresponds to the type of
measurement chamber that you are using. The chamber that is used for the measurement of graminae
leaves is Narrow and the parameters that correspond to this type of chamber are:
area = 5.80.

The parameters correspondent to the chamber configuration detailed on the screen indicate the following;
Log = off Cfg = narrow uset = 200
area = 5.80 T1 mtd = rb set = 0.30
Hfact = ... Qset = ... Trw = ...

The chamber configuration is item Cfg. To change the type of chamber, position the cursor on Cfg, then
push the I key and using the up key, change the configuration by moving between distinct options.

9. Before measuring, the next step is to calibrate the equipment. In order to do so, move with the I key
to the screen where there appears the menu option Calibrate. Select with the up buttons in Calibrate. The
calibration should be performed each day before taking measurements and corresponds to the option CO2
flow check.

To perform the calibration, it is required to take the closed chamber (without leaf) to an open air area (for
example next to the plants). Pressing DoCal will perform the calibration; it is required to press DoCal
another time in order to finalize the process.

10. The last step to perform before taking measurements is generating the file where the measurement
data points will be stored. In order to do so, press the I key until the screen takes you to the Output
87

option menu. Select Output, Logging, Set file, Set log, New file and write the name of the advanced file
with letters using the keys above.

Step 3: Measuring

11. To take the measurements of the Cref and C' an values, they should more or less be the same (these
values refer to the concentration of CO2 externally and in the chamber). These values can be observed in
the first screen of the menu.
12. Position the leaf inside the chamber and maintain the chamber so that the solar sensor is
perpendicular to the sun and so that the radiation that the sensor receives is uniform with that which the
rest of the chamber receives. The radiation value is referenced with the letter Q.
13. Once the leaf is placed inside the chamber, the registration of gas exchange begins. The
photosynthesis level is correspondent to the letter A. To register the Amax value, there are two
methodologies from which to choose:
- register the Amax value for a fixed amount of measurement time (for example, register the
photosynthetic value for 50 seconds after the leaf has been placed in the measurement
chamber).
- periodically measure the Amax value (for example, every 15 seconds for one minute and
using time 0 as the first time that you place the leaf inside the chamber). With this
information, it will be clear that the initial A value is low, but increases incrementally for 50 - 60
seconds and from then on oscillates at an average value.
To store the values every 10 - 15 seconds, push the black button that is located in the upper
handle of the chamber.
IMPORTANT: When measuring, it is recommended to observe the A and Ci values, to ensure
that the measurement is being taken correctly. The values which immediately appear on the screen of A
are detailed on the screen and are presented in the following format:
A: 6.04 E: 0.03 Ci: 381
gs: 0.19 T1c: 25.1 u: 201
uset: 200 Power Record 1
(numeric values are only examples...)
The number of data registered in the selected archive in step 10 is that which is recorded as the
record.
14. To finish the measurement for one leaf, remove it from the chamber. To continue measurements with
other leaves, repeat all passes from Step 3.
15. When all measurements are completed, turn off the equipment by pressing the I key for 3 seconds.

NOTE:
- To measure under conditions of low radiation the radiation value (Q) should be at least 1000mol m
-2

seg
-1
with measurements taken between 10:30h and 14:00h at the latest.
- As a reference, it should be considered that the maximum A values are around 20-30mmol m
-2
seg
-1
.


88

Annex XVI
REFERENCE PICTURES

1. The grow chamber light inconsistency:






2. IRGA Model - LCA 4

3. SPAD Chlorophyll Meter Model







Figure 40. Example of tomato light chamber lighting inconsistency
Figure 41. IRGA Model -LCA 4 photosynthetic activity reader
Figure 42. SPAD chlorophyll meter model
89



4. Salicylic Acid method of application







5. Plant harvest and flash freeze leaf in liquid Nitrogen.


Figure 43. Salicylic acid method of application
Figure 44. Tomato plant harvest and flash freeze in liquid nitrogen
90

Annex XVI
ISOTOPE EXTRACTIONS FOR CARBON AND OXYGEN

1. Mechanically grinding dry plant materials with tweezers. The samples were ground to a fine powder in
a ball mill

Figure 45. Ball mill grinding method

2. Powder to be used for carbon and oxygen isotope analysis

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



91

Annex XVII
PROTEIN EXTRACTION DATA - TOMATO

Table 11. Protein extraction data - Tomato, Time = 0 days
Date 4/29/2011
Wt /g
Date 4/29/2011
Wt /g
Date 4/29/2011
Wt /g
Tomato Sample TC0 1 Sample TC0 2 Sample TC0 3
control Replica TC0 1.1 0.08 Replica TC0 2.1 0.16 Replica TC0 3.1 0.15
T=0 TC0 1.2 0.11 TC0 2.2 0.11 TC0 3.2 0.11
TC0 1.3 0.08 TC0 2.3 0.12 TC0 3.3 0.08

Date 4/29/2011
Wt /g
Date 4/29/2011
Wt /g
Date 4/29/2011
Wt /g
Tomato Sample T0,2.0 1 Sample T0,2.0 2.1 Sample T0,2.0 3
A.S: 0,2g/L Replica T0,2.0 1.1 0.08 Replica T0,2.0 2.1 0.12 Replica T0,2.0 3.1 0.1
T=0 T0,2.0 1.2 0.13 T0,2.0 2.2 0.09 T0,2.0 3.2 0.12
T0,2.0 1.3 0.1 T0,2.0 2.3 0.16 T0,2.0 3.3 0.12

Date 5/3/2011
Wt /g
Date 5/3/2011
Wt /g
Date 5/3/2011
Wt /g
Tomato Sample T0,5.0 1 Sample T0,5.0 2 Sample T0,5.0 3
A.S: 0,5g/L Replica T0,5.0 1.1 0.09 Replica T0,5.0 2.1 0.12 Replica T0,5.0 3.1 0.2
T=0 T0,5.0 1.2 0.11 T0,5.0 2.2 0.09 T0,5.0 3.2 0.08
T0,5.0 1.3 0.14 T0,5.0 2.3 0.09 T0,5.0 3.3 0.05

Date 5/3/2011
Wt /g/
Date 5/3/2011
Wt /g
Date 5/3/2011
Wt /g
Tomato Sample T0,8.0 1 Sample T0,8.0 2 Sample T0,8.0 3
A.S: 0,8g/L Replica T0,8.0 1.1 0.12 Replica T0,8.0 2.1 0.1 Replica T0,8.0 3.1 0.12
T=0 T0,8.0 1.2 0.1 T0,8.0 2.2 0.14 T0,8.0 3.2 0.15
T0,8.0 1.3 0.07 T0,8.0 2.3 0.2 T0,8.0 3.3 0.12







92


Table 12. Protein extraction data - Tomato, Time = 48h
Date 3/5/2011
Wt /g/
Date 3/5/2011
Wt /g/
Date 3/5/2011
Wt /g/
Tomato Sample TC48 1 Sample TC48 2 Sample TC48 3
control Replica TC48 1.1 0.1 Replica TC48 2.1 0.12 Replica TC48 3.1 0.12
T=48h TC48 1.2 0.11 TC48 2.2 0.12 TC48 3.2 0.1
TC48 1.3 0.13 TC48 2.3 0.16 TC48 3.3 0.14

Date 3/5/2011
Wt /g/
Date 3/5/2011
Wt /g/
Date 3/5/2011
Wt /g/
Tomato Sample T0,2.48 1 Sample T0,2.48 2 Sample T0,2.48 3
A.S:
0,2g/L Replica
T0,2.48
1.1 0.09 Replica T0,2.48 2.1 0.15 Replica T0,2.48 3.1 0.09
T= 48h
T0,2.48
1.2 0.09 T0,2.48 2.2 0.13 T0,2.48 3.2 0.11

T0,2.48
1.3 T0,2.48 2.3 0.17 T0,2.48 3.3 0.11

Date 3/5/2011
Wt /g/
Date 3/5/2011
Wt /g/
Date 3/5/2011
Wt /g/
Tomato Sample T0,5.48 1 Sample T0,5.48 2 Sample T0,5.48 3
A.S:
0,5g/L Replica
T0,5.48
1.1 0.13 Replica T0,5.48 2.1 0.13 Replica T0,5.48 3.1 0.1
T=48 h
T0,5.48
1.2 0.11 T0,5.48 2.2 0.12 T0,5.48 3.2 0.08

T0,5.48
1.3 0.07 T0,5.48 2.3 0.1 T0,5.48 3.3







Tomato
A.S:
0,8g/L
There were not any samples for this treatment group since all plants died before
leaf samples could be taken.
T=48 h


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

Date 5/3/2011
Wt /g/
Date 5/3/2011
Wt /g/
Date 5/3/2011
Wt /g/
Tomato Sample TC7 1 Sample TC7 2 Sample TC7 3
Control Replica TC7 1.1 0.19 Replica TC7 2.1 0.11 Replica TC7 3.1 0.12
T=7days TC7 1.2 0.18 TC7 2.2 0.15 TC7 3.2 0.09
TC7 1.3 0.13 TC7 2.3 0.12 TC7 3.3 0.01
93












Date 5/3/2011
Wt /g/
Date 5/3/2011
Wt /g/
Date 5/3/2011
Wt /g/
Tomato Sample T0,2.7 1 Sample T0,2.7 2 Sample T0,2.7 3
A.S:
0,2g/L Replica T0,2.7 1.1 0.11 Replica T0,2.7 2.1 0.12 Replica T0,2.7 3.1 0.22
T=7days T0,2.7 1.2 0.1 T0,2.7 2.2 0.1 T0,2.7 3.2 0.12
T0,2.7 1.3 0.05 T0,2.7 2.3 0.15 T0,2.7 3.3 0.12

Date 5/4/2011
Wt /g/
Date 5/4/2011
Wt /g/
Date 5/4/2011
Wt /g/
Tomato Sample T0,5.7 1 Sample T0,5.0 2 Sample T0,5.0 3
A.S:
0,5g/L Replica T0,5.7 1.1 0.13 Replica T0,5.7 2.1 0.12 Replica T0,5.7 3.1 0.11
T=7days T0,5.7 1.2 0.13 T0,5.7 2.2 0.1 T0,5.7 3.2 0.12
T0,5.7 1.3 0.09 T0,5.7 2.3 0.11 T0,5.7 3.3 0.09

Date 5/4/2011
Wt /g/
Date 5/4/2011
Wt /g/
Date 5/4/2011
Wt /g/
Tomato Sample T0,8.7 1 Sample T0,8.7 2 Sample T0,8.7 3
A.S:
0,8g/L Replica T0,8.7 1.1 0.14 Replica T0,8.7 2.1 0.12 Replica T0,8.7 3.1 0.11
T=7days T0,8.7 1.2 0.15 T0,8.7 2.2 0.09 T0,8.7 3.2 0.11
T0,8.7 1.3 0.1 T0,8.7 2.3 0.15 T0,8.7 3.3 0.13


Fell in N
2
liq., the lost weight is unknown
Used in the first extraction
94

Annex XVIII
TOMATO VISUAL OBSERVATIONS

1. Control growth. Day 0 vs. day 7.

Figure 47. Control plants on day 0 (Left) and on day 7 (Right)



2. 0.8 g/L plant 1 - recovery observation. Day 2 vs. day 7.

Figure 48. Day 2 (Left) and Day 7 (Right)
95

3. Control compared with each treatment group on day 7.

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



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

Annex XIX
SPAD READINGS

Tomato
Table 14. Tomato SPAD readings
Time Treatment Plant # SPAD: ave.
0 0 1
31.4
36.4
25.7
31.16

0 0 2
0 0 3
0 0.2 1
24.4
25.7
27.8
25.96

0 0.2 2
0 0.2 3
0 0.5 1 38.6 32.16
Figure 49. Control (Left) and 0.5g/L (Right)
Figure 51. Control (Left) and 0.8g/L-Plant 2 (Right)
96

0 0.5 2 25.9
32

0 0.5 3
0 0.8 1
31.1
24.3
38.8

31.4

0 0.8 2
0 0.8 3
2 0 1
23.5
23.7
24
23.73

2 0 2
2 0 3
2 0.2 1
26.4
24.5
26.4
25.76

2 0.2 2
2 0.2 3
2 0.5 1
26.2
28
27.1
27.1

2 0.5 2
2 0.5 3
2 0.8 1
28.2
27.2
25.5
26.96

2 0.8 2
2 0.8 3
7 0 1
27.6
23.8
25.8
25.73

7 0 2
7 0 3
7 0.2 1
27.3
24.9
28.7
26.96

7 0.2 2
7 0.2 3
7 0.5 1
23.7
27.5
32.7
27.96

7 0.5 2
7 0.5 3
7 0.8 1
32.3
25.6
29.2
29.03

7 0.8 2
7 0.8 3

Maize
Table 15. Maize SPAD readings
Time Treatment Plant # SPAD
0 0 1 31.4
0 0 2 30.9
0 0 3 34
0 0.2 1 32.8
0 0.2 2 26.5
0 0.2 3 32.9
0 0.5 1 34.2
0 0.5 2 36
0 0.5 3 34.5
97

0 0.8 1 27.1
0 0.8 2 34.6
0 0.8 3 30.7
2 0 1 31.3
2 0 2 35.8
2 0 3 33.8
2 0.2 1 31.3
2 0.2 2 30.1
2 0.2 3 34.5
2 0.5 1 33.8
2 0.5 2 31
2 0.5 3 35.6
2 0.8 1 35.8
2 0.8 2 34.3
2 0.8 3 34.4
7 0 1 27.4
7 0 2 31.4
7 0 3 34.3
7 0.2 1 40.8
7 0.2 2 34.7
7 0.2 3 37.6
7 0.5 1 38.1
7 0.5 2 36.4
7 0.5 3 34.6
7 0.8 1 35.3
7 0.8 2 39.3
7 0.8 3 34.3