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  • 1. G PROTEINS CORRELATIONS WITH GLYCOMICS, PROTEOMICS AND METABOLOMICS IN PLANT NANOFEMTOPHYSIOLOGY FOR PROTECTED AGRICULTURELuis 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, LuisAlfonso 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. 5.5Campo El Diez, Culiacán, Sin., 80129 México. Tel: (667) 7605536.Key words: Bionanofemtophysiology, genomatic-epigenetic nutrition, Lightbourn Biochemical Model, Incerebrum. INTRODUCTIONThe main features of plant metabolism are the ability to adapt and respond to changingenvironments, likewise temperature, salinity, light levels, nutrient deficiency and drought(Lloyd and Zakhleniuk, 2004). Plant growth and development is mediated by a greatdiversity of signaling pathways, coordinated by exogenous factors that regulate allphysiological processes as well as cell division and differentiation, photosynthesis andrespiration. In this regard, the heterotrimeric G proteins are an important factor as signalmediators 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 highlyconserved structure and typically bound to a specific G protein-coupled receptors, whichare a primary component of its signaling pathway (Trusov et al., 2009). This receptorrecognizes a huge range of ligands, including biogenic agents, pigments, peptides, insectpheromones, fungal and environmental changes, which allows the plant to respond to awide arrange of stimulus. Furthermore, the G proteins are involved in the germination,oxidative stress, and opening of ion channels and stomata (Millner, 2001; Nilson andAssmann, 2010). However, it is still unclear how G proteins perform that diversefunctionality in plants despite of the multiple related studies.Plant nutrition in protected agriculture involves the consideration of variables of vitalimportance, 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 delimitedby the photosynthetic and respiratory phenomena.Biomass production, seen as the rate of tissue formation, is directly proportional to thework exerted by the plant to survive and produce. For this reason, nutrition in protectedenvironments requires specifically designed molecules, based on the architecture of cellsand in synergy with the delicate and precise metabolic processes that would allow thegenome can be expressed in proteome. Also, that the, metabolome and the secretome workin sync with changes, flows and rhythms of the various own phases of metabolicoscillations and the molecular diffusion of genomatic-epigenetic nutrition.The tautochrone of the light beam towards the leaf surface under coverage conditions is offundamental importance not only for photosynthesis but also for respiration. The transversevibrations of the light path through the filter material used in protected agriculture wouldcreate, constriction spaces in specialized organelles to capture the luminous intensity,directly affecting all processes of energy management, both generative and vegetative. Thiswould generate a different approach for handling nutrition in protected agriculture.According to the Lightbourn Biochemical Model (LBM), the efficiency and effectivenessin the assimilation of nutrients from the molecules needed to feed plants, would directly beconnected to their femtologic architecture, which is determined and determinant by thenanological architecture of the cellular membrane.The basic steps for an architectural nano-femto design are: 1. Topological studies of the cellmembrane, 2. Thermodynamic and stereochemical considerations, 3. Metabolicengineering, 4. Molecule design, 5. Molecule production, 6. Exo-endogenous nutrienttraceability, 7. A new way to interpret the analysis of soil, water and plant, 8. Correlationalglycobiology, 9. Ad hoc nutrition program, 10. Projectable and precise results, 11. Reliablyquantifiable results, 12. Economy: energy resources and low environmental impactequivalent to greater sustainability.The depthness of analysis is in accordance with practical needs and specific purposes, beingadvisable the valuation of more associated and related parameters. This research workpresents the proteomic and glycomic analysis, and correlates them with G proteins in plantnanofemtophysiology produced under protected agriculture. 2
  • 3. METHODOLOGYThis research includes an evaluation of analysis in vivo, in vitro and in cerebrum.In vivoThis 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-south24°34´53" latitude, 107°31´24" length.In vitroThe in vitro part was developed by the BIOTEKSA RESEARCH TEAM performing thefollowing 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 withtrichloracetic acid and 2-mercaptoethanol using cold acetone. Protein concentration wasdetermined by Bradford method (1976) using BSA as standard.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteinelectrophoresis 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 at0.04% (w/v): methanol 40% (v/v), acetic acid 10% (v/v), agua 50% (v/v)] and distainedwith the same solution without Coomassie Brilliant Blue (Garfin, 1990).Two dimension electrophoresis. The 2-DE analysis of proteins was carried out using IPGstrips (Immobilized pH gradient, BIORAD) of 3 to 10 and 4 to 7 of pH. The strip washydrated 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 mMdithiothreitol (DTT), 0.2% (v/v) ampholytes, 0.002% (w/v) bromophenol blue]. Strips wererehydrated for 16 h at 20 °C. Isoelectric focusing (IEF) of proteins was conducted using aProtean IEF Cell (Bio-Rad) during 7.5 h at 20 °C. After IEF, IPG strips were equilibrated inreducing 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 sameequilibration buffer without DTT but containing 2.5% (w/v) iodoacetamide for 10 mineach. 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 PVDFmembrane (Bio-Rad) using a wet electroblotting chamber (Mini Trans-Blot ElectrophoreticTransfer 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 wasincubated 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 TTBScontaining antibody against Anti-Gα-Subunit Internal (1:5,000). Immediately themembrane 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 MTris, 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 reactionwas stopped by washing with distilled water.Analysis of CarbohydratesPhloem sap extraction. The sap was obtained by centrifugation, were removed the ends ofthe leaf petiole and placed into mesh Miracloth and then in Falcon tube filter (withoutmembrane) 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 sugarby the anthrone (Yemm and Willis, 1954) and alditol acetates methods, respectively. Thealditol acetates method consisting in an hydrolysis with trifluoroacetic acid (TFA) 2 N for 1at 120 °C, reduction with sodium borohydride (20 mg / mL) and subsequent acetylationwith acetic anhydride and imidazole (10:1), finally were injected onto gas chromatographfor analysis [equipped with a FID detector (250 ° C ), a DB-23 capillary column (30 m X0.25 mm) (210 ° C), helium as carrier gas at constant flow (3 mL / min)], myo-inositol (100mg /mL) was used as internal standard (Albersheim et al., 1967; Blakeney et al., 1983).Also, soluble (free sugars, soluble in acetone), insoluble (sugars polysaccharide, insolublein alcohol) and neutral sugars were obtained. The insoluble sugars were obtained adding tothe sap four volumes (v) of ethanol, and incubating for 12 h at 4 ° C. Soluble sugars wereobtained 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 werecentrifuged at 10,000 rpm for 15 min at 4 ° C. The insoluble sugars were determined by thereduction group method (Gross, 1982) and glucose, sucrose and fructose by enzymaticmethod 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 + enzymeplus ATP and imidazole. Abs2, incubation with hexokinase plus glucose 6-phosphatedehydrogenase (G6P) for 10 min. Abs 3, incubation with PGI enzyme (phosphoglucoseisomerase) for 10 min. All incubations were performed at 30 ° C.In cerebrumThis part consisted in the integration of all results and observations obtained, and theapplication of The Lightbourn Biochemical Model. RESULTSFigures 1A, B and C, show the protein profiles of bell pepper from three phenologicalstages, showing variation in protein content. Figure 1D shows the profile of recognition byimmunoblotting with antibody against to the alpha subunit of G protein (anti-Gα-SubunitInternal), 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 byWestern 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) andWestern blot (Figure 3B). Recognition was found in greater intensity of a band of 67 kDain the lanes 1, 3 and 4 corresponding to transplantation, fructification andfructification/production stages. In flowering stage were detected 2 proteins of 28 kDa and24 kDa. Furthermore, in the production stage (lane 5) protein recognition was found at 67and 28 kDa in addition to others bands. A BFigure 2. Protein electrophoretic profile from bell peper on 2-DE gel (A) andimmunodetection by Western blot of G-protein (Anti-Gα-Subunit Internal). MPM,molecular weight marker in kDa. 6
  • 7. A BFigure 3. Protein electrophoretic profile from phloem sap from bell peper in differentstages (A) and its immunodetection by Western blot (B). MPM, molecular weight markerin 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 indifferent stages on unprotected agriculture. In the flowering stage showed the least amountof these sugars with 0.36, 0.05, 0.06 and 0.66 mg/mL of phloem sap, respectively. Whereasthe highest content was found at transplantation of plants.From the soluble non-polymerized neutral sugars results (Figure 4B), it was found thatglucose was the predominant sugar, followed by galactose, mannose, rhamnose, arabinose,xylose and fucose. The neutral sugars of polysaccharides obtained from the precipitationwith alcohol showed, the following order from highest to lowest concentration, galactose,arabinose, xylose, glucose, mannose, rhamnose and fucose (Figure 4C). 7
  • 8. A B CFigure 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 HOOVERFigure 5. Lightbourn Biochemical Model for application in bionanotechnological colloidalnutrition (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. PERSPECTIVESThis work represents the beginning of a comprehensive integrative research involving theproteomic, glycomic and metabolomics disciplines, which aims to the analysis of predictivemolecules of the physiological state of plants, with the purpose of establishing a biometrichomological system that work in practice for the formation of biomass and thereby, toachieve a more consistent production, better quality and higher yield per unit in extendedperiods of harvest. Therefore, the initial objective of this research project is to understandand implement techniques to identify and characterize the G proteins, as major regulatorymolecules in plant metabolism. 10
  • 11. REFERENCESAlbersheim 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. 11