This study analyzed G proteins, proteomics, glycomics and metabolomics in plants grown under protected agriculture. Protein profiles showed variation between phenological stages, and western blot detected G protein subunits of 37, 46, and 57 kDa. Two-dimensional electrophoresis identified a 57 kDa protein with a pI of 5.9. Phloem sap proteins also detected G protein subunits of 28, 67 kDa between stages. Sugar analysis found glucose, fructose and sucrose varied between stages, with neutral sugars dominated by glucose, galactose and mannose. The Lightbourn Biochemical Model was applied to integrate results and propose bionanotechnology and biodynamic nutrition for high agricultural competitiveness and sustainability

1. G PROTEINS CORRELATIONS WITH GLYCOMICS, PROTEOMICS AND
METABOLOMICS IN PLANT NANOFEMTOPHYSIOLOGY FOR PROTECTED
AGRICULTURE
Luis Alberto Lightbourn Rojas1*, Josefa Adriana Sañudo Barajas1,2, Josefina León Félix1,2,
José Basilio Heredia1,2, Rosabel Vélez de la Rocha1,2, Rubén Gerardo León Chan1, Luis
Alfonso Amarillas Bueno1, Talia Fernanda Martínez Bastidas1, Gisela Jareth Lino López1.
1
División 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.
2
Centro 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
1

2. 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.
2

3. 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
3

4. 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
4

5. 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
5

6. 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.
6

7. A B
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).
7

8. A B
C
Figure 4. Sugar content of sap from peppers cultivated under unprotected agriculture and harvested at different developmental stages.
8

9. In cerebrum
LIGHTBOURN
BIOCHEMICAL MODEL
OBTAIN BIONANOTECNOLOGY
+ GENERAL OBJETIVE BIODYNAMIC NUTRITION FOR
SUPPORT
+ HIGH COMPETITIVENESS
SUSTAIN AGRICULTURAL
CROPS
HYPERPRODUCTIVITY
MATHEMATICAL
MUST BE: ANALYSIS
ECOLOGICAL AGROLOGICAL  CONVERGENCE
EFFICIENT
COLLECTION AND ENVIRONMENTAL  LIMITS
PROCESSING OF FIELD EFFECTIVE
PROFITABLE MODEL  MATCHING
AND LABORATORY
INSTRUMENT HAVING AS FOCAL
GENOMATIC NUTRITION DEVELOPMENT OF
ISSUING SOLUTIONS SPECIFIC
SYSTEMIC DESCRIPTION TECHNOLOGIES FOR
BASED ON CALCULATION OF VARIATIONS
OF THE ENVIRONMENTAL CYCLOID CURVE NUTRITION EDAPHIC
MODEL THEMATCHING
MATHEMATICS THE PROBLEMS AND CHALLENGES AND LEAF
=
NUTRITION PROGRAMS
EXISTENCE
IDENTIFICATION RECURRENCE AGRICULTURAL DEVELOPMENT AND CONSTRUCTION
DEFINITION CONTROL: MATHEMATICAL TRASSCIENCE CROPS
OF MATHEMATICAL MODELS
FOR SIMULATION HOMODYNAMICS
ACCURACY ANALYSIS TO ENSURE HYPERPRODUCTIVITY PROCESSES
FUNCTIONAL CONTINUUM BIONANOTECHNOLOGYCAL IN
VARIABLES PLANT NUTRITION
CALCULATION OF MULTIPLE PHASICAL CALCULATION OF MULTIPLE
IN SYNCHRONIZATION IN MATHEMATICAL
EUCLIDEAN SPACE EUCLIDEAN SPACE COHOMOLOGY
HOMOLOGY AND
ANALYSIS
AND
NON-EUCLIDEAN SPACE NON-EUCLIDEAN SPACE
HOMEODYNAMIC
BIOLOGY
GENERATING PHYSICAL
IN VIVO GENETATED MODELS
MATHEMATICAL AND OPERATING OPERATING
TOPOLOGICAL MODELS CHEMISTRY MATHEMATICAL INDUCTION
FOR THE FUNCTIONAL AGROLOGY NONLINEAL
ANALYSIS EXTENSIVELY CHAOTIC
IN VIVO STOCHASTIC
FRACTAL
INTENSIVE GENERATING
OPERATING KNOWLEDGE AND
TECHNOLOGICAL
INNOVATION
IN SILICO
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
Figure 5. Lightbourn Biochemical Model for application in bionanotechnological colloidal
nutrition (Lightbourn, 2011).
9

10. CIRCULAR
CAPILLARITY DEEXITEMENT DICHROISM
STRUCTURE WATER QUANTUM FLUX
QUANTUM BIOPHYSICAL-CHEMISTRY LIGHT
FLOWS
DIFFUSION SURFACE
RELATIONSHIP SYSTEMS PHOTOISOMERATION
TAUTOCHRONIE
TENSION RAY OF LIGHT
ELASTICITY CÉLULAS BIOPHYSIC
Y
DIFUSIÓN
CHEMICAL TRANSPORT
SOIL QUANTUM
MOLECULAR PLANT CHEMISTRY COMPLEXITY
BIOMASS FORMATION BIOLOGY WATER
PERMIABILITY CONTROL GLICÓMICA POTENTIAL
ATMOSPHERE
PROTEÓMICA PHYSICOCHEMICAL
G PROTEIN
QUANTUM FUNCIONALES
PIGMENTS PHYSICAL
ANTOCIANINICA SOLUTES
BIOMETRICS SIENCE DRIVEN: PREDICTIVA
HOMOLOGICAL NANONOLOGY LIGHTBOURN INSTITUTE SYMMETRICAL
PHOTOCHEMICAL FOR PLA NT DISRUPTIVE CO-
AND BIONANOFEMTOPHYSIOLOGY HOMOLOGICAL FLUJOS
FEMTOLOGY CUÁNTICOS
PHOTOSYNTHESIS
GENOME TRANSCRIPTOME
WATER
PHOTOPHOSFORYLATION PLANT
QUANTUM METABOLOME BNF/MBL PROTEOME
FLOWS CONTINUUM
SECRETOME BIOINFORMATIC
RADIATION BALANCES
SOIL
ATMOSPHERE
LATENT CELLULAR ARCHITECTURE
TEMPERATURE MOLECULAR ARCHITECTURE
AND ENERGY HEAT
TRIBOLOGY TOPOLOGY NADP REDOX
EFFICIENCIE GOLGI +
CONDUCTION
AND TERMODYNAMIC ATP ER
CONDUCTANCES
CONVECTION RESISTANCES
CHEMIOOSMOSIS BIOENERGETIC
EXISTENCE GIBBS
TRANSPIRATION RECURRENCE
PLANT AND ENERGY
TRANSCIENCE
FLOWS
MITOCHONDRIA
QUANTUM CHOROPLAST
EDDY DIFFUSION FLOWS
COEFFICIENT (TURBULENT
DIFFUSION)
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.
10

11. REFERENCES
Albersheim P., Nevins D. J., English P. D., Karr A. 1967. A method for the analysis of
sugars in plant cell-wall polysaccharides by gas-liquid chromatography.
Carbohydrate Research 5: 340-345.
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
proteins in plants. Plant Cell Physiology: 42; 789-794.
Garfin D.E. 1990. One-dimensional gel electrophoresis. In: Methods in Enzymology.
Deutscher M.P. (Editor). Academic Press, San Diego, California, pp. 425-488.
Gross K.C. 1982. A rapid and sensitive spectrophotometric method for assaying
polygalacturonase using 2-cyano-acetamide. HortScience 17: 933-934.
Hooley R. 1999. A role for G proteins in plant hormone signaling?. Plant Physiology
Biochemical: 37; 393-402.
Trusov Y., Sewelam N., Rookes J., Kunkel M., Nowak E., Schenk M., Botella J. 2009.
Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes
mechanisms independent of salicylic acid, jasmonic acid/ethylene and abscisic acid-
mediated defense signaling. The Plant Journal: 58; 69-81.
Lightbourn Rojas L.A. 2011. Arquitectura Celular y Arquitectura Molecular. In: MODELO
DE GESTIÓN DE TECNOLOGÍA BIOTEKSA I+D+i = 2i. Fabro Editores. ISBN
978-0-9833321-7-6.
Lloyd C., Zakhleniuk V. 2004. Responses of primary and secondary metabolism to sugar
accumulation revealed by microarray expression analysis of the Arabidopsis mutant,
pho3. Journal of Experimental Botany: 55; 1221-1230.
Nilson E., Assmann M. 2010. Heterotrimeric G proteins regulate reproductive trait
plasticity in response to water availability. New Phytologist: 185; 734-746.
Méchin V., Damerval C., Zivy M. 2007. Total Protein Extraction with TCA-Acetone. In:
Plant Proteomics: Methods and Protocols. Thiellement H., Zivy M., DamervaL C.,
Méchin V. (Editors). Springer Protocols. Pp. 1-8.
Millner P. 2001. Heterotrimeric G-proteins in plant cell signaling. New Physiology: 151;
165-174.
Villanueva M. 2008. Electrotransfer of proteins in an environmentally friendly methanol-
free transfer buffer. Analytical Biochemistry 373:377-379.
Xue C., Hsueh Y., Heitman J. 2008. Magnificent seven: roles of G protein-coupled
receptors in extracellular sensing in fungi. FEMS Microabiology: 32; 1010-1032.
Yemm E.W., Willis A.J. 1954. The estimation of carbohydrates in plant extracts by
anthrone. Biochemical Journal. 57: 508-514.
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