apoplasto redox cebolla.pdf

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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


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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).

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