Plant Science 150 2000 163 – 170
A physiological characterization of Mn-tolerant tobacco plants selected by in vitro culture
Geraldina Santandrea, Tiziana Pandolfini, Andrea Bennici
Department of Plant Biology, Uni6ersity of Florence, P. le Cascine
28
,
50144
Florence, Italy Received 4 June 1999; received in revised form 13 September 1999; accepted 13 September 1999
Abstract
In previous research, an in vitro stepwise procedure permitted us to obtain Nicotiana tabacum regenerated plant lines able to grow in the presence of Mn at 2 and 5 mM Mn-tolerant plants. These plants showed several morpho-physiological and
cytological differences in comparison to the Mn-sensitive regenerated plants. In particular, the number of xylem cells and the degree of lignification appeared to be influenced differently by these Mn concentrations. In the present work these Mn-tolerant
and Mn-sensitive N. tabacum plants, maintained in the presence of Mn 2 and 5 mM, have been characterized with regards to the uptake of Mn and Fe, the activity of extracellular peroxidases in the stems, and the activity of superoxide dismutase, ascorbate
peroxidase, and glutathione reductase in the leaves. The leaf response to an increasing Mn concentration in the medium, corresponded a parallel decrease of Fe content. Plants tolerant of 5 mM Mn showed almost a doubling Mn content over that of
the 5 mM Mn-sensitive plants. In the stem, 2 and 5 mM Mn inhibited the extracellular free peroxidases guaiacol peroxidases either in the Mn-tolerant plants or in the Mn-sensitive plants. In the Mn-sensitive plants treated with 2 mM Mn the activity of
the peroxidases of the ionically and covalently bound wall peroxidases was also depressed. In 5 mM Mn-tolerant plants, an enhanced activity of the covalently bound wall peroxidases was observed. The effect of Mn on the covalently bound wall
syringaldazine peroxidases was identical to that observed in the guaiacol peroxidases; the activity was significantly higher in the Mn-tolerant plants grown in the presence of 5 mM Mn. In the leaf, the increase of Mn content inhibited the activity of guaiacol
peroxidase, ascorbate peroxidase and superoxide dismutase in the Mn-tolerant as well as in the Mn-sensitive plants. However, the effect was greater in the Mn-sensitive plants. Only glutathione reductase did not show significant variation except for the 2 mM
Mn-sensitive plants, where an increased activity was detected. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Mn-tolerance; Nicotiana tabacum; Plant regeneration; Peroxidases; Glutathione reductase; Superoxide dismutase www.elsevier.comlocateplantsci
1. Introduction
Manganese is an essential trace element for plants where it is involved in redox reactions as a
cofactor for many enzymes. In particular it is the metal component of superoxide dismutase SOD
[1,2]. The most important function of Mn is its role as a constitutive element in the water-splitting
system of PSII [3,4].
An excess of Mn can lead to toxicity conditions in natural and agricultural sites and especially in
acid soils [5,6]. Manganese toxicity symptoms are observed in
shoots as growth retardation, brown lesions, foliar chlorosis and crinkled leaves [7,8]. The response of
plants to excess Mn at physiological level consists principally in a decline in photosynthetic rate and
also in a reduction of respiration [9,10]. The Mn critical toxicity content varies widely depending on
the plant species, age, temperature, light intensity and soil nutrient balance [4].
Toxic Mn effects are found also in different in in vitro developmental processes such as germina-
tion, callus induction and growth, and shoot re- generation from callus [3,8,11,12]. Moreover, high
Mn level has direct cytotoxic effects such as exten- sive cytoplasmic injures, mitochondria modifica-
Corresponding author. Tel.: + 39-55-328-8270; fax: + 39-55-360- 137.
E-mail address
:
abennicicesit1.unifi.it A. Bennici 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 8 0 - 6
tion and plasma membrane ruptures in the outer root cap and meristematic cells [13].
The physiological mechanisms of Mn toxicity and tolerance are still not well known. Many
reports suggest that Mn excess may cause the induction of oxidative stress [4]. In this case, as a
consequence of the uptake of toxic concentrations of the metal, several defense enzymes such as
SOD, ascorbate peroxidase ASPX, glutathione reductase GR, guaiacol peroxidase GPX, are
induced in the chloroplasts and cytosol as protec- tion against oxidative stress [14 – 17].
Peroxidases POD are oxido-reductases which occur practically in all plants. They are present as
several isoenzymes which are found in soluble, ionically bound and covalently bound forms [18].
Their form and distribution is, probably, related to different physiological functions [19,20] An in-
crease of POD activity has been considered as a metabolic response under various stress conditions
including heavy metal stress [21]. In the cell wall- located bound POD activity plays a key role in
controlling the production and deposition of lignin in vascular tissue [22,23]. In non-lignified primary
cell walls, POD may be involved in the cross-link- ing of cinnamic acids such as ferulic acid, the
production of which is implicated in the enhance- ment of cell adhesion in parenchyma tissues [24 –
26]
POD may also prevent oxidative damage to plasmamembranes, acting as peroxide radical
scavengers [14]. A previous research established an in vitro pro-
cedure for the selection of Nicotiana tabacum var. BEL W3 callus lines and regenerated plants toler-
ant to high doses of Mn 2 and 5 mM. The performance of the Mn-tolerant plants with regard
to several morphological, anatomical and cytologi- cal characteristics, in comparison to the Mn-sus-
ceptible regenerated plants, was described [12]. In particular, Mn was also shown to affect the num-
ber of xylem elements and the degree of lignifica- tion, which differed from plants grown in the
presence of 2 and 5 mM Mn compared to the control. Moreover, in these plants, damaged
chloroplasts and a reduction in their number were also observed, especially for those treated with 5
mM Mn.
With an aim of better explaining these previous results and to characterize Mn-tolerant plants
from the physiological point of view, the present study was planned to determine in selected plants
growing in the presence of Mn at 2 and 5 mM, 1 the activity of extracellular POD in the stems
using two electron donors such as guaiacol and syringaldazine, 2 the activity of SOD, ASPX and
GR in the leaves.
2. Materials and methods
