=Paper=
{{Paper
|id=Vol-1498/HAICTA_2015_paper7
|storemode=property
|title=Effect of Auxins on Mechanical Injury Induced Oxidative Stress in Prunus sp. Rootstocks
|pdfUrl=https://ceur-ws.org/Vol-1498/HAICTA_2015_paper7.pdf
|volume=Vol-1498
|dblpUrl=https://dblp.org/rec/conf/haicta/PopovicMPKPD15
}}
==Effect of Auxins on Mechanical Injury Induced Oxidative Stress in Prunus sp. Rootstocks==
https://ceur-ws.org/Vol-1498/HAICTA_2015_paper7.pdf
Effect of Auxins on Mechanical Injury Induced
Oxidative Stress in Prunus sp. Rootstocks
Milan Popović1, Đorđe Malenčić1, Dejan Prvulović1, Biljana Kiprovski1, Aleksandra
Popović1, Dušica Dorić1
1
Faculty of Agriculture, University of Novi Sad, Trg D. Obradovića 8, 21000 Novi Sad,
Serbia, e-mail: dusicab@polj.uns.ac.rs
Abstract. The effect of exogenously applied auxins on oxidative stress was
studied in six rootstock selections of Prunus cerasus, P. mahaleb and P.
fruticosa aiming at the effect of phytohormones on the level of the lipid
peroxidation (LP). Standard rootstock PHL-A was used as a control. Leaves
were collected on 0, 1st, 3rd and 7th day of inserting cuttings into the rooting
substrate. Auxin mixture consisted of 0.8% α-naphthylacetic acid and 0.5%
indolebutyric acid. Differences in intensity of the LP between auxin-treated
and -untreated cuttings were examined. Almost all of investigated selections
had lower LP intensity after auxin application (11.4-47.2%). The most
prominent change was in leaves of PHL-A (49.2%). LP-lowering effect were
recorded in leaves of P. fruticosa (56.9%) on 7th day and in P. mahaleb on 1st
and 3rd day (5.9, 5.8%). Intensity of LP could be used as one of the
biochemical parameters in further rootstock selection and production.
Keywords: Prunus sp. rootstocks, softwood cuttings, auxins, oxidative stress,
lipid peroxidation
1 Introduction
Rootstocks should be adapted to environmental conditions, expected to be easily
propagated, to result in uniform fruit tree behavior in the orchard. Selection success
in breeding of vegetative rootstocks for sweet and sour cherry is dependent not only
on genetic variability, but also on expediting propagation processes as a precondition
for investigation of special combining abilities between rootstock and scion
(Bošnjaković et al., 2012; Bošnjaković et al., 2013). To expedite propagation of
rootstock selections, propagation by softwood cuttings is preferable.
Oxidative metabolism of normal cells and different stress situations generate
highly reactive oxygen species (ROS). The ROS, such as, superoxide radical (O2·-),
hydrogen peroxide (H2O2), hydroxyl radical (·OH), and singlet oxygen (1O2) have
been implicated in a number of physiological disorders in plants (Malenčić et al.,
2012; Blomster et al., 2011), which in turn leads to a decrease in plant productivity.
To prevent oxidative stress, plants have evolved a complex antioxidant system
composed of non-enzymatic and enzymatic mechanisms that scavenge ROS (Casano
et al., 2004; Malenčić et al., 2010). The formation of auxins conjugates may serve as
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a protection against oxidative degradation (Taiz and Zeiger, 2006). Antioxidant
defense systems have co-evolved with aerobic metabolism to counteract oxidative
damage from ROS. A ROS signal can be generated in a group of cells in the plant in
response to wounding, pathogen attack or a local abiotic stress, and be transferred to
the entire plant. The integration of ROS with auxin signaling networks, triggered by
environmental factors, is known as the stress-induced morphogenic response. In this
response, ROS and auxin metabolism interfere and lead to morphological changes
that help avoid deleterious effects of environmental stress (Mittler et al., 2011).
Lipid peroxidation (LP) is a natural metabolic process occurring in aerobic
conditions and presents the most investigated effect of ROS on structure and function
of cell membrane. Thus, the aim of this study was to measure the intensity of LP in
the leaves of six cherry rootstocks selections in order to investigate the effect of
exogenously applied auxins on the oxidative stress induced by mechanical injury
during rooting of softwood cuttings.
2 Material and Methods
The effect of exogenously applied auxins on mechanical injury induced oxidative
stress was studied in six rootstock selections: Prunus cerasus L., (OV21, OV22), P.
mahaleb L., (M4, M6) and P. fruticosa Pall. (SV2st, SV4). Standard vegetative
rootstock-PHL-A was used as a control, due to successful rooting. Softwood cuttings
of investigated selections were collected from ex situ mother trees from the
experimental field of the Faculty of Agriculture at Rimski Šančevi, near Novi Sad.
The experiment was carried out in a plastic house under a fogging system with 95-
98% relative humidity in average. The rooting substrate was a blend of white
sphagnum and perlite. The 15-20 cm long terminal cuttings were treated with
exogenously applied auxin mixture consisted of 0.8% α-naphthylacetic acid (NAA)
and 0.5% indolebutyric acid (IBA). Leaves of investigated rootstocks were collected
on 0, 1st, 3rd and 7th day after cutting and inserting softwood cuttings into the rooting
substrate. Experiment was carried out with a total of 60 softwood cuttings per
selection (30 auxin-treated and 30 auxin-untreated cuttings).
As a measure of LP intensity, the amount of malondialdehyde (MDA) was
determined spectrophotometrically by the thiobarbituric acid-reactive-substances
(TBARS) assay (Hodges, 1999). Leaves of softwood cuttings were first
homogenized and then extracted in 10% trichloroacetic acid (TCA) in ratio 1:5 (w/v)
and centrifuged at 12000 x g for 30 min at 4 °C. One cm3 of supernatant was
incubated with 4 cm3 20% TCA containing 0.5% TBA for 30 min at 95 °C. The
reaction was stopped by cooling on ice for 10 min and the product was centrifuged at
10000 x g for 15 min. The absorbance of the TBARS was measured at 532 nm and
600 nm and their concentration was determined using the MDA extinction
coefficient of 155 mM cm-1 and expressed as nmol MDA g-1 fresh weight.
54
3 Results and Discussion
The present investigation showed that the oxidative stress induced by mechanical
injury in the leaves of rootstock selection of sweet and sour cherry, characterized by
an accumulation of MDA, may be attributed to the genotypic differences in
wounding tolerance. Results of Malenčić et al. (2012) showed a positive effect of
exogenously applied NAA on LP-lowering effect in standard vegetative rootstock
Gisela 5, as well as in four investigated sweet and sour cherry rootstocks selections.
Similar was recorded for standard rootstock PHL-A in our experiment where a
positive auxin effect on lowering LP intensity was noticed between 1st and 3rd
collecting day, but on 7th day, an increase of LP was 65% compared to control (Fig.
1). In P. mahaleb, selection M4, MDA production was significantly lower in auxin-
treated cuttings on 1st and 3rd collecting day. On 7th day LP intensity was similar in
auxin-treated cuttings of both P. mahaleb selections, M4 and M6 (24.2% and 2.5%,
respectively), comparing to untreated cuttings (23.9%, 5.5%) (Fig. 2 and Fig. 3). The
enhancement of MDA accumulation, a cytotoxic product of lipid peroxidation, was
recorded in P. fruticosa selection SV2 on 3rd collecting day in both treated and
untreated cuttings, contrary to 7th collecting day where auxin application showed
lowering effect on LP intensity (Fig. 4). Lowering effect on LP intensity was noticed
on 1st and 7th day in SV4 selection, also. Higher degree of membrane damage was
noticed in untreated cuttings on 7th collecting day (154.5% of control) (Fig. 5).
Fig. 1. LP intensity in auxin treated and untreated standard vegetative rootstock PHL-A
55
Fig. 2. LP intensity in auxin treated and untreated rootstock selection P. mahaleb - M4
Fig. 3. LP intensity in auxin treated and untreated rootstock selection P. mahaleb - M6
56
Fig. 4. LP intensity in auxin treated and untreated rootstock selection P. fruticosa - SV2
Fig. 5. LP intensity in auxin treated and untreated rootstock selection P. fruticosa - SV4
According to Cheong et al. (2002), wounding negatively regulates IAA responsive
genes, revealing a new level of crosstalk between wounding and auxin response in
plants. Studies of expression patterns of genes regulated by wounding provided new
information on the interactions between wounding and other signals, such as
pathogen attack, abiotic stress factors, and plant hormones.
A great genetic variability in response of rootstocks of Prunus cerasus L., P.
mahaleb L. and P. fruticosa Pall. toward wounding and oxidative stress was
established. Despite the fact that auxin has a positive effect on rooting and LP-
lowering effect in most of treated selections, its application seems to has no universal
LP-mitigating effect for treated rootstocks. Among all investigated selections, the
57
best auxin LP-lowering effect was in P. mahaleb, M6 selection during the entire
experiment. Also, the results gained from LP assay proved to be invaluable marker
for resistance to the oxidative stress induced by mechanical injury and impact of
propagation in sweet and sour rootstock selection.
Acknowledgement. This research was a part of the project 'Selection of sweet and
sour cherry dwarfing rootstocks and development of intensive cultivation technology
based on sustainable agricultural principles', evidence number TR-31038 financed by
the Ministry of Education, Science and Technological development of the Republic
of Serbia, as well as a part of the Hungary-Serbia IPA Cross-border programme:
"Joint development of higher education and training programmes in plant biology in
support of knowledge-based society“ (Planttrain), project id: husrb/1203/221/173,
2015-2016.
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