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Determinación del contenido de prolina en plantas de fríjol (Phaseolus vulgaris)

Authors:
Determinación del
contenido de prolina en
plantas de fríjol como
respuesta al estrés por la
aplicación de oxifluorfen
Universidad Nacional de Colombia
Sede Bogotá
Facultad de Agronomía
Correo electrónico: dfrinconga@unal.edu.co
Daniel Rincón y Verónica Hoyos
INTRODUCCIÓN
Determinación del contenido de prolina en plantas de fríjol como respuesta al estrés por la aplicación de oxifluorfen
de los aa bajo condiciones
ambientales normales .
del total de aa bajo condiciones
de estrés (Kumar et al., 2010).
Prolina en
plantas <5%
80%
~Eubacteria
~Protozoos
~Invertebrados marinos
~Plantas después de varios tipos de estrés
(Verbruggen y Hermans, 2008)
Acumulación de
prolina
en plantas de tomate con PEG (25%),
con respecto al testigo no tratado
(Rhodes et al., 1986).
fue la [prolina] reportada
319 veces mayor
¿Para qué se acumula prolina?
Contribuye a presión osmótica (cerca a 0.1 MPa) en
células de halófitas.
Actividad de prolina deshidrogenasa (PDH) comparada
con su planta cercana Arabidopsis thaliana.
Thellungiella halophila
Halófitas ↑[prolina]
~Estabilización de la estructura terciaria de las proteínas
(chaperona)
(Munns y Tester, 2008).
~Buffer en el citosol.
~ Fuente de N y C.
~ Señalización de vías de estrés para adaptación (Verbruggen y
Hermans, 2008).
~ Estabiliza membranas celulares por la interacción con
fosfolípidos.
~ Elimina radicales hidoxilo (Claussen, 2005).
~ Incrementa la actividad de la super óxido dismutasa (Hua y
Guo, 2002).
Prolina: posiblemente contribuyen con
Estudios con reportes
de incremento en la
Especie
Proporción
[prolina]
Control
Condición de
estrés
Referencia
Arabidopsis
thaliana L.
3
1 C (5 días)
Wanner y Junttila
(1999)
Beta vulgaris L.
10
-2,1 MPa
Gzik (1996)
Beta vulgaris L.
4
NaCl: 4 M
Gzik (1996)
Solanum
lycopersicum
(L.) H. Karst. cv.
Counter
3-4
Solución nutritiva
11 veces mayor a
la [ ] estándar
Claussen (2005)
Zea mays L.
21
10 dS m-1 NaCl
+ PEG 6000
Köşkeroğlu y
Tuna (2010)
CONCENTRACIÓN DE PROLINA
Dando lugar a la producción de especies reactivas de oxígeno (ROS) (Iannone et al., 2012),
las cuales son inhibidas por prolina (Chen y Dickman, 2005) ¿cómo ocurre esto?
Tipos de estrés que dan lugar al incremento
en el contenido de prolina en plantas
Osmótico (Claussen, 2005) Salino (Gzik, 1996)
Metales pesados (Theriappan et al., 2011)
Alta intensidad lumínica (Mansour, 2000)
Frío (Wanner y Junttila 1999)
Aplicación de
herbicidas Paraquat (Chen y Dickman, 2005)
Bentazon (Galhano et al., 2011)
Oxifluorfen
Nombre IUPAC: 2-cloro-4-trifluorometilfenil-3-etoxy-4-nitrofenil eter Difeniléter
Familia química (HRAC)
Propiedades
físicas y químicas
Estructura
Peso molecular
361,7
Presión de vapor
1 x 10-6 mm Hg (media-baja)
Solubilidad
Compuesto puro: 0,1 ppm (baja)
pKa
No presenta disociación
log Kow
>2,77 (lipofílico)
Usos
Pre y posemergencia
Control de malezas de hojas anchas
y gramíneas anuales (Ahrens, 1994).
Oxifluorfen en
plantas
Glutamato tRNAGlu
Glutamil-tRNAGlu
Glutamato-1-semialdehido
Glutamil-tRNA transferasa
Hidrometilbilano
Porfobilinógeno
Uropofirinógeno III
Glutamil-tRNA sintetasa
5-aminolevulinato deshidratasa
Hidrometilbilano sintasa
Uropofirinógeno III sintasa
Uropofirinógeno III carboxilasa
Coproporfirinógeno III
Coproporfirinógeno III oxidasa
Protoporfirinógeno IX
Protoporfirinógeno IX oxidasa
Protoporfirina IX
Quelatasa de
magnesio Ferroquelatasa
Mg-protoporfirina IX Protohemo
Biliverdina IX
Fitocromobilina
Ficobilinas
Hemo
Mg-protoporfirina IX monometilester
Mg-protoporfirina IX metiltransferasa
Divinil protoclorofilido
Ciclasa
Protoclorofilido
NADPH: Protoclorofilido oxidoreductasa
Clorofila a sintasa
Clorofilido a + Fitil-PP
Vinil reductasa
Clorofila a
Clorofila b
Sitio de acción de oxifluorfen
Vía del tetrapirol en plantas y punto de
inhibición por parte de oxyfluorfen.
Modificado de (Böger et al, 2002).
Interactúa con oxígeno
Su
acumulación causa O2
O2 peroxidación de la membrana
(Grossman et al., 2010)
Oxidación no enzimática
Luz
Reactive
Oxygen
Species
(ROS)
Si ROS superan el sistema
defensivo antioxidante celular
estrés oxidativo a daño de
ácidos nucleicos, proteínas y
lípidos.
(Ray et al., 2012)
Especies de oxígeno radicales o
no radicales formadas por la
reducción parcial de oxígeno.
Generación endógena por
fosforilación oxidativa
mitocondrial, en el PSI o PSII o
por interacciones con fuentes
exógenas como compuestos
xenobióticos.
Superóxido (O2), peróxido de
hidrógeno (H2O2), radicales hidroxilo
(HO•), oxígeno molecular (O2)
Sistemas de defensa
antioxidante en plantas
Enzimático
Superóxido dismutasa, catalasa, ascorbato peroxidasa,
glutationa reductasa, monodeshidroascorbato
reductasa, glutationa peroxidasa, guaicol peroxidasa,
glutationa-S-transferasa.
No enzimático
Ácido ascórbico, glutationa, compuestos fenólicos,
alcaloides, tocoferoles y aminoácidos no proteicos.
↑ [ROS] → desbalance en el funcionamiento del
sistema antioxidante (Radyukina et al., 2008).
Inhibición
de
fotosíntesis
Formación
de etileno Formación
de etano
Blanquea-
miento de
cloroplas-
tos
Inhibición
de
crecimiento
Muerte
celular
DIFENILÉTERES
Efectos adicionales por la aplicación de
(Grossman et al., 2010)
Determinación del
contenido de prolina en
plantas de fríjol como
respuesta al estrés por la
aplicación de oxifluorfen
OBJETIVO
Cuantificar el contenido de prolina
biosintetizada por efecto de la aplicación
oxifluorfen en plantas de fríjol común
(Phaseolus vulgaris) cv. ICA Cerinza.
METODOLOGÍA
MATERIAL VEGETAL
Plantas de frijol común (Phaseolus vulgaris)
cv. ICA Cerinza.
15 días después de emergencia.
APLICACIÓN DE HERBICIDA
Cámara de aplicación de herbicida
Presión: 28 PSI (constante)
Boquilla: Tee Jeet 8002
Volumen de aplicación:250 L ha-1
TRATAMIENTOS
T1: 480 g i.a. ha -1 (dosis comercial)
T2: 960 g i.a. ha -1
Testigo sin aplicación.
4 repeticiones por tratamiento.
METODOLOGÍA
METODOLOGÍA
Tomar 0,3 g de material vegetal
Homogenización: 10 mL de solución acuosa de ácido sulfosalicílico (3%)
Filtrar en papel Whatman No. 2
2 Tomar en un tubo de ensayo 2 mL del filtrado
Agregar 2 mL de ácido de ninhidrina
Agregar 2 mL de ácido acético glacial Tiempo: 1 h
Temp.: 100 C
Detener la
reacción en
hielo
1
CUANTIFICACIÓN DE PROLINA (Bates, 1973)
24 h después de aplicación, importante!!: efecto de oxifluorfen ocurre en
presencia de luz
Extraer la mezcla de la reacción con 4 mL de tolueno
Mezclar vigorosamente durante 15 20 seg.
Leer la absorbancia a 520 nm.
Usar tolueno como blanco.
METODOLOGÍA
3
4
5
CUANTIFICACIÓN DE PROLINA
Cálculo del contenido de prolina
Incremento promedio
en los niveles de prolina
de 99% (T1) y 528%
(T2) con respecto al
testigo.
0
10
20
30
40
50
60
70
80
90
100
Testigo
T1
T2
Prolina (μmol g-1 PF)
RESULTADOS
, confirmando así a prolina como variable
determinante en la medida de la respuesta al
estrés causado por la aplicación de
en plantas de fríjol.
La cuantificación del contenido de prolina
permitió establecer el estrés ocasionado por la
aplicación de oxifluorfen.
El contenido de prolina en plantas de frijol se
incrementó al doble con una dosis comercial de
oxifluorfen y 5,28 veces por efecto de una
aplicación al doble de la dosis comercial.
CONCLUSIONES
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America.
Claussen, W. 2005. Proline as a measure of stress in tomato plants. Plant
Science, 168: 241248.
Bates, L. 1973. Rapid determination of free proline for water-stress
studies. Plant and Soil, 39: 205-207.
Chen, C. y M. Dickman. 2005. Proline suppresses apoptosis in the fungal
pathogen Colletotrichum trifolii. Proc Natl Acad Sci USA. 102 (9): 3459
3464.
Delauney, A. y D. Verma. 1993. Proline biosynthesis and osmoregulation in
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Deuschle, K., D. Funck, G. Forlani, H. Stransky, A. Biehl y D. Leister. 2004.
The role of Δ1-pyrroline-5-carboxylate in proline degradation. The Plant
Cell, 16: 34133425
Galhano, V., H. Santos, M. Oliveira, J. Gomes-Laranjo, F. Peixoto. 2011.
Changes in fatty acid profile and antioxidant systems in a Nostoc muscorum
strain exposed to the herbicide bentazon. Process Biochemistry, 46: 2152
2162.
BIBLIOGRAFÍA
Gzik, A. 1996. Accumulation of proline and pattern of α-amino acids in
sugar beet plants in response to osmotic, wáter and salt stress.
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BIBLIOGRAFÍA
Determinación del contenido de
prolina en plantas de fríjol como
respuesta al estrés por la aplicación
de oxifluorfen
Universidad Nacional de Colombia
Sede Bogotá
Facultad de Agronomía
Correo electrónico: dfrinconga@unal.edu.co
Gracias.
Daniel Rincón y Verónica Hoyos
ResearchGate has not been able to resolve any citations for this publication.
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