1

G PROTEINS CORRELATIONS WITH GLYCOMICS, PROTEOMICS AND

METABOLOMICS IN PLANT NANOFEMTOPHYSIOLOGY FOR PROTECTED

AGRICULTURE

Luis Alberto Lightbourn Rojas

1*, Josefa Adriana Sañudo Barajas

1,2, Josefina León Félix

1,2,

José Basilio Heredia1,2

, Rosabel Vélez de la Rocha1,2

, Rubén Gerardo León Chan1, Luis

Alfonso Amarillas Bueno1, Talia Fernanda Martínez Bastidas

1, Gisela Jareth Lino López

1.

1División de Generación, Excogitación y Transferencia de Conocimiento. Bioteksa S.A de C.V.

(Bionanofemtotecnología en Sistemas Agrológicos). www.bioteksa.com. Carretera Las Pampas Km. 2.5, Col.

Industrial, CP 33981. Jiménez, Chihuahua México. lalr@bioteksa.com. 2Centro de Investigación en Alimentación y Desarrollo, A.C., Unidad Culiacán Carret. a Eldorado Km. 5.5

Campo El Diez, Culiacán, Sin., 80129 México. Tel: (667) 7605536.

Key words: Bionanofemtophysiology, genomatic-epigenetic nutrition, Lightbourn Biochemical Model, In

cerebrum.

INTRODUCTION

The main features of plant metabolism are the ability to adapt and respond to changing

environments, likewise temperature, salinity, light levels, nutrient deficiency and drought

(Lloyd and Zakhleniuk, 2004). Plant growth and development is mediated by a great

diversity of signaling pathways, coordinated by exogenous factors that regulate all

physiological processes as well as cell division and differentiation, photosynthesis and

respiration. In this regard, the heterotrimeric G proteins are an important factor as signal

mediators in the transduction of diver´s external signals (Fujisawa et al., 2001).

The G proteins are constituted by three subunits, α, β and γ, organized in a highly

conserved structure and typically bound to a specific G protein-coupled receptors, which

are a primary component of its signaling pathway (Trusov et al., 2009). This receptor

recognizes a huge range of ligands, including biogenic agents, pigments, peptides, insect

pheromones, fungal and environmental changes, which allows the plant to respond to a

wide arrange of stimulus. Furthermore, the G proteins are involved in the germination,

oxidative stress, and opening of ion channels and stomata (Millner, 2001; Nilson and

Assmann, 2010). However, it is still unclear how G proteins perform that diverse

functionality in plants despite of the multiple related studies.

Plant nutrition in protected agriculture involves the consideration of variables of vital

importance, which is about the soil-plant-water-atmosphere equilibrium and its biological,

physical and chemical approach under limiting conditions. Therefore, light intensity,

temperature and relative humidity must be considered as factors that define the

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thermodynamic of the processes associated directly to the metabolome and both delimited

by the photosynthetic and respiratory phenomena.

Biomass production, seen as the rate of tissue formation, is directly proportional to the

work exerted by the plant to survive and produce. For this reason, nutrition in protected

environments requires specifically designed molecules, based on the architecture of cells

and in synergy with the delicate and precise metabolic processes that would allow the

genome can be expressed in proteome. Also, that the, metabolome and the secretome work

in sync with changes, flows and rhythms of the various own phases of metabolic

oscillations and the molecular diffusion of genomatic-epigenetic nutrition.

The tautochrone of the light beam towards the leaf surface under coverage conditions is of

fundamental importance not only for photosynthesis but also for respiration. The transverse

vibrations of the light path through the filter material used in protected agriculture would

create, constriction spaces in specialized organelles to capture the luminous intensity,

directly affecting all processes of energy management, both generative and vegetative. This

would generate a different approach for handling nutrition in protected agriculture.

According to the Lightbourn Biochemical Model (LBM), the efficiency and effectiveness

in the assimilation of nutrients from the molecules needed to feed plants, would directly be

connected to their femtologic architecture, which is determined and determinant by the

nanological architecture of the cellular membrane.

The basic steps for an architectural nano-femto design are: 1. Topological studies of the cell

membrane, 2. Thermodynamic and stereochemical considerations, 3. Metabolic

engineering, 4. Molecule design, 5. Molecule production, 6. Exo-endogenous nutrient

traceability, 7. A new way to interpret the analysis of soil, water and plant, 8. Correlational

glycobiology, 9. Ad hoc nutrition program, 10. Projectable and precise results, 11. Reliably

quantifiable results, 12. Economy: energy resources and low environmental impact

equivalent to greater sustainability.

The depthness of analysis is in accordance with practical needs and specific purposes, being

advisable the valuation of more associated and related parameters. This research work

presents the proteomic and glycomic analysis, and correlates them with G proteins in plant

nanofemtophysiology produced under protected agriculture.

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METHODOLOGY

This research includes an evaluation of analysis in vivo, in vitro and in cerebrum.

In vivo

This stage was developed at the Paralelo 38 Farm located in the Culiacan Sinaloa valley:

East-North 24°35´23" latitude, 107°30´54" length, East-south 24°34´53" latitude,

107°31´01" longitude, West-north 24°35´23" latitude, 107°31´24" length, West-south

24°34´53" latitude, 107°31´24" length.

In vitro

The in vitro part was developed by the BIOTEKSA RESEARCH TEAM performing the

following activities:

Protein extraction. Protein was extracted from petiole according to the Mechin et al.

(2007) method. Tissue was macerated in mortar, and the protein was precipitated with

trichloracetic acid and 2-mercaptoethanol using cold acetone. Protein concentration was

determined by Bradford method (1976) using BSA as standard.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein

electrophoresis was carried out 12 % SDS-PAGE gels according to Laemmli method

(1970) in Miniprotean chamber (BIO-RAD) with Tris-HCl 25 mM, pH 8.3, at 70 V for 3 h.

Gels were stained with Coomassie Brilliant Blue R-250 [Coomassie Brilliant Blue R-250 at

0.04% (w/v): methanol 40% (v/v), acetic acid 10% (v/v), agua 50% (v/v)] and distained

with the same solution without Coomassie Brilliant Blue (Garfin, 1990).

Two dimension electrophoresis. The 2-DE analysis of proteins was carried out using IPG

strips (Immobilized pH gradient, BIORAD) of 3 to 10 and 4 to 7 of pH. The strip was

hydrated with 300 µg of protein samples in 125 µL of hydration buffer [4% (w/v) (3-[3-

cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 8 M urea, 50 mM

dithiothreitol (DTT), 0.2% (v/v) ampholytes, 0.002% (w/v) bromophenol blue]. Strips were

rehydrated for 16 h at 20 °C. Isoelectric focusing (IEF) of proteins was conducted using a

Protean IEF Cell (Bio-Rad) during 7.5 h at 20 °C. After IEF, IPG strips were equilibrated in

reducing equilibration buffer [50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 2%

(w/v) SDS, 0.002% (w/v) bromophenol blue, 1% (w/v) DTT] and then placed in the same

equilibration buffer without DTT but containing 2.5% (w/v) iodoacetamide for 10 min

each. Subsequently the proteins Isoelectric focusing onto strips were transferred to a

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vertical 12% SDS-PAGE gel and the second dimension was performed as above.

Immunodetection of proteins by Western Blotting. Proteins were separated in SDS-

PAGE gel either 1-DE or 2-DE electrophoresis. The proteins were transferred to a PVDF

membrane (Bio-Rad) using a wet electroblotting chamber (Mini Trans-Blot Electrophoretic

Transfer Cell, Bio-Rad) with transfer buffer (0.025 M Tris-HCl, pH 8.3, 0.192 M glycine,

and 7.5% (v/v) isopropanol) at 100 V for 75 min (Villanueva, 2008). Membrane was

incubated in blocking solution [5% skim milk in Tris-buffered saline (TBS, 0.020 M Tris-

HCl, pH 7.5, 0.5 M NaCl)] for 2 h at 20 C. Membrane was washed twice with 0.05% (v/v)

Tween-20 in Tris-buffered saline (TTBS) and was then shaken for 12 h at 4 °C in TTBS

containing antibody against Anti-Gα-Subunit Internal (1:5,000). Immediately the

membrane was incubated 2 h at 20 ºC with anti-rabbit antibody conjugated (Bio-Rad)

(1:30,000 on TTBS). Finally, the PVDF membrane was transferred in Tris buffer (0.1 M

Tris, pH 9.5, 0.0005 M MgCl2) containing p-nitroblue tetrazolium chloride (NBT) (Bio-

Rad) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Bio-Rad) for 1 h. The reaction

was stopped by washing with distilled water.

Analysis of Carbohydrates

Phloem sap extraction. The sap was obtained by centrifugation, were removed the ends of

the leaf petiole and placed into mesh Miracloth and then in Falcon tube filter (without

membrane) was centrifuged at 10.000 rpm for 10 min at 4 ° C.

Analysis of Carbohydrates. The sap was obtained and analyzed for total and neutral sugar

by the anthrone (Yemm and Willis, 1954) and alditol acetates methods, respectively. The

alditol acetates method consisting in an hydrolysis with trifluoroacetic acid (TFA) 2 N for 1

at 120 °C, reduction with sodium borohydride (20 mg / mL) and subsequent acetylation

with acetic anhydride and imidazole (10:1), finally were injected onto gas chromatograph

for analysis [equipped with a FID detector (250 ° C ), a DB-23 capillary column (30 m X

0.25 mm) (210 ° C), helium as carrier gas at constant flow (3 mL / min)], myo-inositol (100

mg /mL) was used as internal standard (Albersheim et al., 1967; Blakeney et al., 1983).

Also, soluble (free sugars, soluble in acetone), insoluble (sugars polysaccharide, insoluble

in alcohol) and neutral sugars were obtained. The insoluble sugars were obtained adding to

the sap four volumes (v) of ethanol, and incubating for 12 h at 4 ° C. Soluble sugars were

obtained by addition of cold acetone (4:1 v / v) and incubating at -20 ° C for 12 h. Insoluble

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sugars were obtained by centrifugation at 3000 rpm for 5 min, whereas the soluble were

centrifuged at 10,000 rpm for 15 min at 4 ° C. The insoluble sugars were determined by the

reduction group method (Gross, 1982) and glucose, sucrose and fructose by enzymatic

method that involves taking three absorbance readings (Abs) at 340 nm: Abs1, the sample

(100 L) was incubated with invertase 10 min, followed by the addition of NADP + enzyme

plus ATP and imidazole. Abs2, incubation with hexokinase plus glucose 6-phosphate

dehydrogenase (G6P) for 10 min. Abs 3, incubation with PGI enzyme (phosphoglucose

isomerase) for 10 min. All incubations were performed at 30 ° C.

In cerebrum

This part consisted in the integration of all results and observations obtained, and the

application of The Lightbourn Biochemical Model.

RESULTS

Figures 1A, B and C, show the protein profiles of bell pepper from three phenological

stages, showing variation in protein content. Figure 1D shows the profile of recognition by

immunoblotting with antibody against to the alpha subunit of G protein (anti-Gα-Subunit

Internal), finding the detection of bands with molecular weights of 57, 46 and 37 kDa,

molecular weights reported for G-proteins in other plant species.

A B C

D

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Figure 1. Protein electrophoretic profile from bell peper in flowering (A) fructification (B)

and production (C) stage and Western blot (D). MPM, Molecular weight marker (kDa).

Figure 2 shows the proteins profile of 2-DE gels from bell pepper and immunodetection by

Western blot, showing the recognition of a protein 57 kDa with an isoelectric point of 5.9.

We also analyzed the phloem sap proteins from bell pepper by SDS-PAGE (Figure 3A) and

Western blot (Figure 3B). Recognition was found in greater intensity of a band of 67 kDa

in the lanes 1, 3 and 4 corresponding to transplantation, fructification and

fructification/production stages. In flowering stage were detected 2 proteins of 28 kDa and

24 kDa. Furthermore, in the production stage (lane 5) protein recognition was found at 67

and 28 kDa in addition to others bands.

A B

Figure 2. Protein electrophoretic profile from bell peper on 2-DE gel (A) and

immunodetection by Western blot of G-protein (Anti-Gα-Subunit Internal). MPM,

molecular weight marker in kDa.

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Figure 3. Protein electrophoretic profile from phloem sap from bell peper in different

stages (A) and its immunodetection by Western blot (B). MPM, molecular weight marker

in kDa. 1, Transplantation in protected agriculture; 2, flowering; 3, fructification; 4,

fructification /Production; 5, Production.

Figure 4A, shows the concentrations of glucose, fructose and sucrose from bell pepper in

different stages on unprotected agriculture. In the flowering stage showed the least amount

of these sugars with 0.36, 0.05, 0.06 and 0.66 mg/mL of phloem sap, respectively. Whereas

the highest content was found at transplantation of plants.

From the soluble non-polymerized neutral sugars results (Figure 4B), it was found that

glucose was the predominant sugar, followed by galactose, mannose, rhamnose, arabinose,

xylose and fucose. The neutral sugars of polysaccharides obtained from the precipitation

with alcohol showed, the following order from highest to lowest concentration, galactose,

arabinose, xylose, glucose, mannose, rhamnose and fucose (Figure 4C).

A B

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Figure 4. Sugar content of sap from peppers cultivated under unprotected agriculture and harvested at different developmental stages.

C

A B

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In cerebrum

LIGHTBOURN

BIOCHEMICAL MODEL

BIONANOTECNOLOGY

BIODYNAMIC NUTRITION FOR

HIGH COMPETITIVENESS

MATHEMATICAL

ANALYSIS

CONVERGENCE

LIMITS

MATCHING

DEVELOPMENT OF

SPECIFIC

TECHNOLOGIES FOR

NUTRITION EDAPHIC

AND LEAF

AGROLOGICAL

ENVIRONMENTAL

MODEL

AGRICULTURAL

CROPS

HYPERPRODUCTIVITY

OBTAIN

+

SUPPORT

+

SUSTAIN

BASED ON

THEMATCHING

MATHEMATICS

GENOMATIC NUTRITION

EXISTENCE

RECURRENCE

TRASSCIENCE

CONTINUUM

AGRICULTURAL

CROPS

HYPERPRODUCTIVITY

PHASICAL

SYNCHRONIZATION

BIOLOGY

PHYSICAL

CHEMISTRY

AGROLOGY

HOMEODYNAMIC

OPERATINGOPERATING

OPERATING

INTENSIVE

EXTENSIVELY

IN VIVO

IN VIVO

IN SILICO

COHOMOLOGY

CALCULATION OF MULTIPLE

IN

EUCLIDEAN SPACE

AND

NON-EUCLIDEAN SPACE

HOMOLOGY

GENERATING

KNOWLEDGE AND

TECHNOLOGICAL

INNOVATION

CECREATE SYSTEM

TOPOLOGICAL

BIONANOTECHNOLOGY

SOIL

PLANT

WATER

ATMOSPHERE

ESTABLISHING

CONTROLS

MATHEMATICAL

MODELS FOLLOWING

RIEMANN

FINSLER

GAUSS

HERMITE

LEBESGUE

BESSEL

MARKOV

ABEL

JACOBI

HAMILTON

NOSE

HOOVER

ISSUING SOLUTIONSCALCULATION OF VARIATIONS

CYCLOID CURVETHE PROBLEMS AND CHALLENGES

=NUTRITION PROGRAMS

MUST BE:

ECOLOGICALEFFICIENT

EFFECTIVE

PROFITABLEINSTRUMENT HAVING AS FOCAL

COLLECTION AND PROCESSING OF FIELD

AND LABORATORY

IDENTIFICATIONDEFINITIONACCURACY

FUNCTIONAL VARIABLES

GENERATING MATHEMATICAL AND

TOPOLOGICAL MODELS FOR THE FUNCTIONAL

ANALYSIS

DEVELOPMENT AND CONSTRUCTION OF MATHEMATICAL MODELS

FOR SIMULATION HOMODYNAMICS PROCESSES

BIONANOTECHNOLOGYCAL IN PLANT NUTRITION

GENETATED MODELS MATHEMATICAL INDUCTION

NONLINEAL

CHAOTICSTOCHASTIC

FRACTAL

CONTROL: MATHEMATICAL ANALYSIS TO ENSURE

CALCULATION OF MULTIPLE

IN

EUCLIDEAN SPACE

AND

NON-EUCLIDEAN SPACE

MATHEMATICAL ANALYSIS

SYSTEMIC DESCRIPTION

OF THE ENVIRONMENTAL

MODEL

GENERAL OBJETIVE

Figure 5. Lightbourn Biochemical Model for application in bionanotechnological colloidal

nutrition (Lightbourn, 2011).

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DIFFUSION

ELASTICITY

PERMIABILITY

BIOPHYSIC CHEMICAL

CÉLULAS Y

DIFUSIÓN

STRUCTURE

PHOTOCHEMICAL AND

PHOTOSYNTHESIS

WATERLIGHT

TEMPERATURE AND ENERGY

QUANTUM FLUXBIOPHYSICAL-CHEMISTRY

DEEXITEMENT

TAUTOCHRONIE RAY OF LIGHT

TRANSPORT

QUANTUM FLOWS

SOLUTES

CONTINUUM

BIOINFORMATIC

BIOENERGETIC

PLANT AND FLOWS

BIOMASS FORMATION CONTROL

SURFACE TENSION

CAPILLARITYCIRCULAR

DICHROISM

POTENTIAL PHYSICOCHEMICAL

PHOTOISOMERATION

COMPLEXITY

G PROTEIN

ANTOCIANINICAPREDICTIVA

GLICÓMICAPROTEÓMICAFUNCIONALES

QUANTUMCHEMISTRY

SOILPLANTWATER

ATMOSPHERE

MOLECULARBIOLOGY

QUANTUM PHYSICALPIGMENTS

RADIATION

CONDUCTANCES

REDOX

CELLULAR ARCHITECTUREMOLECULAR ARCHITECTURE

TRANSCRIPTOME

PROTEOME

GENOME

METABOLOME

SECRETOME

TOPOLOGYTRIBOLOGY

TERMODYNAMIC

EXISTENCERECURRENCE TRANSCIENCE

CONDUCTION AND

CONVECTION

PHOTOPHOSFORYLATION

BALANCES

LATENTHEAT

EFFICIENCIE

RESISTANCES

TRANSPIRATION

EDDY DIFFUSION COEFFICIENT (TURBULENT

DIFFUSION)

RELATIONSHIP SYSTEMS

WATER

SOIL

PLANT

ATMOSPHERE

NADP

ATP

CHEMIOOSMOSIS

GOLGI + ER

GIBBS ENERGY

MITOCHONDRIACHOROPLAST

FLUJOS CUÁNTICOS

BIOMETRICSHOMOLOGICAL NANONOLOGY

BNF/MBL

FEMTOLOGY

SYMMETRICALCO-

HOMOLOGICAL

SIENCE DRIVEN:LIGHTBOURN INSTITUTE FOR PLA NT DISRUPTIVE

BIONANOFEMTOPHYSIOLOGY

QUANTUM FLOWS

QUANTUM FLOWS

Figure 6. General diagram of the "Science Driven" Lightbourn Institute.

PERSPECTIVES

This work represents the beginning of a comprehensive integrative research involving the

proteomic, glycomic and metabolomics disciplines, which aims to the analysis of predictive

molecules of the physiological state of plants, with the purpose of establishing a biometric

homological system that work in practice for the formation of biomass and thereby, to

achieve a more consistent production, better quality and higher yield per unit in extended

periods of harvest. Therefore, the initial objective of this research project is to understand

and implement techniques to identify and characterize the G proteins, as major regulatory

molecules in plant metabolism.

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sugars in plant cell-wall polysaccharides by gas-liquid chromatography.

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Blakeney A.B., Harris P.J., Henry R.J., Stone B.A. 1983. A simple and rapid preparation of

alditol acetates for monosaccharide analysis. Carbohydrate Research 113: 291-299.

Fujisawa Y., Kato H., Iwasaki Y. 2001. Structure and function of heterotrimérica G

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Garfin D.E. 1990. One-dimensional gel electrophoresis. In: Methods in Enzymology.

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Gross K.C. 1982. A rapid and sensitive spectrophotometric method for assaying

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Hooley R. 1999. A role for G proteins in plant hormone signaling?. Plant Physiology

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Trusov Y., Sewelam N., Rookes J., Kunkel M., Nowak E., Schenk M., Botella J. 2009.

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Villanueva M. 2008. Electrotransfer of proteins in an environmentally friendly methanol-

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