Download - apoplasto redox cebolla.PDF
-
8/12/2019 apoplasto redox cebolla.PDF
1/10
-
8/12/2019 apoplasto redox cebolla.PDF
2/10
-
8/12/2019 apoplasto redox cebolla.PDF
3/10
enzymatic activities were assayed for AF and SSF obtained from eachroot zone.
Guaiacol peroxidase (GPX) was determined according to Zhengand Van Huystee (1992). The reaction mixture contained 10 mMsodium phosphate (pH 6), 0.1 ml of 0.3% (v/v) H2O2, and 0.1 ml of1% (v/v) guaiacol. Reaction was followed at 470 nm (extinctioncoefficient = 26.6 mM
1 cm1) for 5 min at 30 8C.
Glucose-6-phosphate dehydrogenase (G6PDH) assay was devel-oped in 100 mM TRIS-HCl, pH 8, containing 1 mM MgCl2, 0.2 mM
NADP+, and 1 mM glucose-6-phosphate. Measurements were at340 nm (Weimar and Rothe, 1986). Enzyme activities were expressedas nmol of substrate metabolized min
1 g1 FW. The presence of this
activity in AF was used as an estimation of cytosolic contamination.It should be noted that all values given in this work concerningapoplastic constituents have been corrected for cytosolic contamina-tion according to the values of G6PDH activity in AF and thecorresponding SSF.
In vivo detection of total peroxidase activity
The method of De Pinto and Ros-Barcelo (1997) was used. Someonions growing under normal conditions and after treatment for 48 hwith 1 mM ASC or 2 mM GalL were transferred to media consistingof 0.1 M TRIS-acetate, 0.1 mM 4-chloro-naphthol, and 0.9 mM
H2O2, at pH 5. After several minutes of incubation a dark reactionbegan to be appreciable in the roots, and pictures were immediatelyobtained.
Protein determination
Protein was determined in both AF and SSF by the dye-bindingmethod of Bradford (1976), using c-globulin as a standard.
Statistical analysis
The effects of ASC and GalL on root sprouting and elongation weretested by both analysis of variance (ANOVA) and the KruskalWallis
test. In all the other experiments mean values were compared usingStudents t-test. Significance levels of 95% (P
-
8/12/2019 apoplasto redox cebolla.PDF
4/10
-
8/12/2019 apoplasto redox cebolla.PDF
5/10
-
8/12/2019 apoplasto redox cebolla.PDF
6/10
-
8/12/2019 apoplasto redox cebolla.PDF
7/10
-
8/12/2019 apoplasto redox cebolla.PDF
8/10
-
8/12/2019 apoplasto redox cebolla.PDF
9/10
adding a known concentration of ascorbate to the GPX assay
cuvette (Cordoba-Pedregosaet al., 1996).
The local effect of ASC on peroxidase activity can
explain the results ofin vivo staining in roots treated with
ASC or GalL, and supports the idea of a direct effect of
ASC treatment on cell wall metabolism. Moreover, continu-
ous inhibition of apoplastic peroxidase by treatments with
ASC but not with GalL could result in an increase inperoxidase protein synthesis and secretion in a typical
feedback mechanism. Thus, when the corresponding AF
are obtained and GPX assayed, a higher activity can be
found, as indicated in Fig. 4C, but only after the lag-phase
has finished. This fact can explain the increase in GPX
activity detected in apoplasts after ASC treatment and the
lack of staining in intact roots using 4-chloro-naphthol.
Recently, it has been proposed that ASC is also involvedin cell wall loosening via its participation in non-enzymatic
scission of cell wall polysaccharides (Fry, 1998; Miller
and Fry, 2001). This mechanism includes the formation of
hydroxyl radicals, which can be produced in the cell wall
from hydrogen peroxide formed by a reaction involvingASC, Cu2+, and oxygen. In this regard, it has been proposed
that there is participation of a peroxidase in the formation
of the peroxide (Liszkay etal., 2003). This hypothesis, which
has been partially tested in vitro and in vivo (Schopfer,
2001; Schopferet al., 2002), provides an additional explan-
ation of the role of ASC as a regulator of cell expansion.
In the present experiments, the highest ASC accumulations
have been found in those zones with the higher elonga-
tion rates. As stated above, treatment with ASC did not have
the same effect as GalL, and most probably the immersion
of the roots directly in ASC could accelerate the reaction
and the trigger of mechanisms involved in the stimulation
of root growth.
Conclusions
ASC and GalL treatments induce stimulation of rootsprouting and elongation probably by increasing metab-
olism in the symplastic compartment and through a more
direct effect on the apoplast. In this compartment, ASC oxi-
dation seems to be important and, therefore, direct incu-
bation with ASC was more effective than GalL. Consistent
with its effect on root growth, increased ASC content modi-
fies peroxidase activity in the different zones of the root.
Investigations are currently being developed to ascertain
the role of the enzymes involved in ascorbate metabolism inplants and in its maintenance in an appropriate redox status
in each compartment in roots with increased elonga-
tion rates.
Acknowledgements
This work was supported by the Spanish Ministerio de Educacion yCultura (grant BMC2002-01078) and by the Junta de Andaluca(grant CVI-276). MdCC-P was supported by grant CVI-276).
References
Andrews J, Adams SR, Burton KS, Edmondson RN. 2002. Partialpurification of tomato fruit peroxidase and its effect on the
mechanical properties of tomato fruit skin.Journal of Experimen-
tal Botany 53, 23932399.Andrews J, Malone M, Thompson DS, Ho LC, Burton KS. 2000.
Peroxidase isozyme patterns in the skin of maturing tomato fruit.
Plant, Cell and Environment23, 415422.
Arrigoni O. 1994. Ascorbate system in plant development. Journalof Bioenergetics and Biomembranes26, 407419.
Arrigoni O, Calabrese G, De Gara L, Bitonti MB, Liso R.1997. Correlation between changes in cell ascorbate and growth
of Lupinus albus seedling. Journal of Plant Physiology 150,302308.
Arrigoni O, De Tullio MC. 2002. Ascorbic acid: much more thanjust an antioxidant.Biochimica et Biophysica Acta 1569,19.
Beyer RE. 1994. The role of ascorbate in antioxidant protection ofbiomembranes: interaction with vitamin E and coenzyme Q.
Journal of Bioenergetics and Biomembranes26, 350358.Bradford MM. 1976. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle of
protein-dye binding.Analytical Biochemistry72, 248254.Buettner GR. 1993. The pecking order of free radicals and
antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate.Archives of Biochemistry and Biophysics300,535543.
Citterio S, Sgobarti S, Scippa S, Sparvoli E. 1994. Ascorbic acideffect on the onset of cell proliferation in pea root. Physiologia
Plantarum92, 601607.Cordoba F, Gonzalez-Reyes JA. 1994. Ascorbate and plant
cell growth. Journal of Bioenergetics and Biomembranes 26,399405.
Cordoba-Pedregosa MC, Cordoba F, Villalba JM, Gonzalez-Reyes JA. 2003a. Zonal changes in ascorbate and hydrogenperoxide contents, peroxidase, and ascorbate-related enzyme
activities in onion roots.Plant Physiology 131,697706.Cordoba-Pedregosa MC, Cordoba F, Villalba JM, Gonzalez-
Reyes JA. 2003b. Differential distribution of ascorbic acid,peroxidase activity, and hydrogen peroxide along the root axis in
Allium cepa L. and its possible relationship with cell growth anddifferentiation.Protoplasma 221,5765.
Cordoba-Pedregosa MC, Gonzalez-Reyes JA, Canadillas M,Navas P, Cordoba F. 1996. Role of apoplastic and cell-wallperoxidases on the stimulation of root elongation by ascorbate.Plant Physiology 112,11191125.
Cordoba-Pedregosa MC, Villalba JM, Cordoba F, Navas P,Gonzalez-Reyes JA.2000. The key role of cell wall peroxidasesand apoplastic ascorbate in the regulation of cell expansion in
onion roots. Poster. Fifth International Conference on Plasma
Membrane Redox Systems and their role in Biological Stress and
Disease. Hamburg, Germany.De Cabo RC, Gonzalez-Reyes JA, Cordoba F, Navas P. 1996.
Rooting hastened in onions by ascorbate and ascorbate free radical.Journal of Plant Growth Regulation15, 5356.
De Cabo RC, Gonzalez-Reyes JA, Navas P.1993. The onset of cellproliferation is stimulated by ascorbate free radical in onion root
primordia.Biology of the Cell 77, 231233.De Pinto MC, Ros-Barcelo A. 1997. Cytochemical localization of
pheno-oxiding enzymes in lignifying Coleus blumei stems.Euro-
pean Journal of Histochemistry41, 1722.De Tullio MC, Paciola C, Dalla Vechia F, Rascio N, DEmerico S,
De Gara L, Liso R, Arrigoni O. 1999. Changes in onion rootdevelopment induced by the inhibition of peptidyl-prolyl hydrox-
ylase and influence of ascorbate system on cell division and
elongation.Planta109, 424434.
Enhanced ascorbate and onion root growth 693
-
8/12/2019 apoplasto redox cebolla.PDF
10/10