Theor. Exp. Plant Physiol.

(2015) 27:215226
DOI 10.1007/s40626-015-0046-2

Cytosolic ascorbate peroxidase and Cu, Zn-superoxide

dismutase improve seed germination, plant growth,
nutrient uptake and drought tolerance in tobacco
M. Faize . E. Nicolas . L. Faize .
P. Daz-Vivancos . L. Burgos . J. A. Hernandez

Received: 21 July 2015 / Accepted: 24 October 2015 / Published online: 9 November 2015
Brazilian Society of Plant Physiology 2015

Abstract The effects of over-expression of two better growth performance that correlated with an
cytosolic antioxidant enzymes (Cu, Zn-SOD and/or improved photosynthetic capacity and nutrient uptake.
APX) on plant nutrition, gas exchange, chlorophyll Moreover, cytsod or cytapx genes promoted seed
fluorescence, seed viability and germination in trans- germination, and enhanced tolerance to mild water
genic tobacco (Nicotiana tabacum cv. Xanthi) under stress. In addition, this enhanced antioxidant capacity
deficit irrigation or salinity conditions were investi- protected seeds from ageing during prolonged storage,
gated. Three transgenic lines of tobacco were used in and stimulated germination under salt stress condi-
this study: line 17, harboring 2 copies of the cytosolic tions. These results suggest that cytosolic antioxidant
CuZn-SOD (cytsod) gene; line 51, with 2 copies of the transgenes are useful tools to improve drought toler-
cytosolic APX (cytapx) gene and line 39, harboring ance, nutrient uptake and seed germination under
one copy of each gene. Over-expression of cytosolic stressful conditions.
antioxidants enzymes in tobacco plants resulted in a
Keywords Germination
Plant growth
Mineral
nutrition
Salinity
Seed aging
Seed physiology
Electronic supplementary material The online version of
this article (doi:10.1007/s40626-015-0046-2) contains supple-
mentary material, which is available to authorized users. 1 Introduction
M. Faize
Group of Biotechnology and Plant Physiology, Faculty of Environmental stresses exert adverse effects on plant
Sciences, University Chouaib Doukkali, 24000 El Jadida, growth and development because of the reactive
Morocco oxygen species (ROS) that are overproduced under
E. Nicolas these stressful conditions. Among them, anion super-
Irrigation Department, CEBAS-CSIC, Campus oxide (O2
-), hydrogen peroxide (H2O2) and hydroxyl
Universitario de Espinardo, P.O. Box 164, 3010 Murcia, radicals (.OH) are the most damaging ROS in plants
Spain (Perl-Treves and Perl 2002).
L. Faize
P. Daz-Vivancos
L. Burgos
Water and salt stress are serious stress factors that
J. A. Hernandez (&) limit agricultural production in many regions worldwide
Group of Fruit Trees Biotechnology, Department of Plant (Alscher et al. 1997; Hernandez et al. 2001). Both, water
Breeding, Centro de Edafologa y Biologa Aplicada del deficit and salinity primarily affect photosynthetic CO2
Segura (CEBAS-CSIC), Campus Universitario de
Espinardo, P.O. Box 164, 30100 Murcia, Spain assimilation leading to a restricted plant production
e-mail: jahernan@cebas.csic.es (Flexas et al. 2002). This lower CO2 availability favors

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216 Theor. Exp. Plant Physiol. (2015) 27:215226

the ROS generation in the chloroplast inducing an plants (Asada 1999). SODs are metaloenzymes that
oxidative stress at cellular level (Asada 1999; Faize et al. catalyzes the dismutation of O2
- to O2 and H2O2
2011). In addition, under drought and salt stress (Fridovich 1975). APXs, the main enzyme of the ASC-
conditions ROS production also increased in other cell GSH cycle, are one of the most important key enzymes
compartments such as mitochondria or peroxisomes that scavenge potentially harmful H2O2 in different
(Hernandez et al. 1993; Alscher et al. 1997; Mittova cell compartments (Noctor and Foyer 1998; Asada
et al. 2003; Bartoli et al. 2004). 1999; Jimenez et al. 1997; Diaz-Vivancos et al. 2006).
Germination can also induce ROS production Besides their detrimental effect, ROS can also play
(Hendry 1993; McDonald 1999; Kranner et al. a key role in the completion of germination and should
2010), being traditionally considered as a negative be considered as a messenger or transmitters of
effect. Seeds rapidly increase oxygen uptake and environmental cues during seed germination (Bailly
oxidative phosphorylation in order to support the et al. 2008, Barba-Espin et al. 2010, 2011; Causin et al.
energy for the germination process (Tommasi et al. 2012; Diaz-Vivancos et al. 2013). In recent works, it
2001). Production of H2O2 has been demonstrated has been reported that the imbibition of pea seeds with
during the early imbibition stage of seeds of soybean low H2O2 levels increased the germination rate as well
(Puntarulo et al. 1991), tomato (Morohashi 2002), as the growth of the seedlings in a concentration-
radish (Schopfer et al. 2001), wheat (Caliskan and dependent manner, linked to the induction of some
Cuming 1998), and sunflower (Bailly et al. 2002). If proteins related to plant signaling and development,
ROS over-generation is not tightly controlled, they can cell elongation and division and cell cycle control, as
cause detrimental effects during the initial phases of well as to a strong decrease in ABA contents (Barba-
growth and development of embryos and seedlings Espin et al. 2010; 2011; Diaz-Vivancos et al. 2013). In
(Cakmak et al. 1993). Arabidopsis thaliana, germination was associated
Seeds can also be subjected to the detrimental effect with an accumulation of superoxide and hydrogen
of ROS production during extreme desiccation and peroxide in the radicle (Leymarie et al. 2012). In
storage, and they can lose germination ability and radish, germination was also accompanied with
viability during prolonged storage periods (McDonald increase in ROS originated from seed coat and embryo
1999). The excess of ROS accumulation induces a (Oracz et al. 2009).
reduced germination rate, which is considered as an Previously, we transformed tobacco (Nicotiana
indication of seed ageing. Aged seeds loss plasma tabacum cv. Xanthi) plants with cytosolic Cu-ZnSOD
membrane integrity (Priestley et al. 1985) which leads and/or APX genes. These transgenic lines displayed
to an inability of cells to maintain osmotic turgor, and greater tolerance to mild water stress, by stimulating the
therefore to seed death (Parrish et al. 1982). For these antioxidant capacity in both the cytosol and the
reasons, antioxidative mechanisms have been chloroplast (Faize et al. 2011). We extended this work
regarded as being of particular importance for the to see if this higher antioxidative capacity can also be
success of germination (de Gara et al. 1997; Tommasi important to increase the germination of the derived T1
et al. 2001). The antioxidant mechanisms are catego- seeds and vigor of the tobacco seedlings. We also
rized into enzymatic and non-enzymatic antioxidants. attempted to study the effect of these cytosolic trans-
Non-enzymatic antioxidants include ascorbate (ASC), genes in plant growth, mineral content, photosynthesis
glutathione (GSH), tocopherol, flavonoids, and car- (Vcmax and Jmax parameters) and seed physiology under
otenoids. The antioxidant enzymes are important to unfavorable environmental conditions.
cope with abiotic stress disorders in adult plants as
well as for the success of germination (Bailly et al.
2008; Barba-Espin et al. 2010). They include super- 2 Materials and methods
oxide dismutase (SOD), the ASC-GSH cycle
enzymes, catalase and peroxidases that are involved 2.1 Plant material, seed germination, plant vigor
in the scavenging of ROS in plant cells (Noctor and and biomass
Foyer 1998; Asada 1999). Among the antioxidative
enzymes, SODs and ascorbate peroxidases (APXs) Three transgenic lines of tobacco (Nicotiana tabacum
play a pivotal role in ROS detoxification in higher cv. Xanthi) (T0 plants) were used in this study: line 17

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Theor. Exp. Plant Physiol. (2015) 27:215226 217

harboring 2 copies of the cytosolic Cu-ZnSOD (cyt- the 3 days duration of the experiment (Faize et al.
sod) gene; line 51 with 2 copies of the cytosolic APX 2011).
(cytapx) gene and line 39 harboring one copy of each Photosynthetic capacity of leaves was estimated
gene. All of these cytosolic genes are under the control from the analysis of A-Ci response curves. Measure-
of the constitutive duplicated CaMV35S promoter ments were made using an open gas exchange system
(Faize et al. 2011). Transgenic and non-transgenic (Li6400, Li-Cor Inc., Nebraska, USA) with an inte-
plants were grown in pots with peat substrate in the grated leaf chamber fluorometer (LI-6400-40, Li-Cor
greenhouse under a 16/8 h day/night photoperiod at Inc., Nebraska, USA). The curves were performed
25 C. under saturating irradiance (1800 lmol m-2 s-1) and
T1 seeds were collected from T0 plants 60 days under constant leaf temperature (25 C) by controlling
after self-pollination at anthesis and used for further the CO2 concentration of inlet air in 11 steps from 50 to
experiments. They were used immediately (new 1400 lmol mol-1. Diffusion leaks when performing
seeds) or stored for up to 4 years at 8 C (aged seeds). the curves were taking into account by applying the
New seeds were germinated on Petri dish on three manufacturers equation to determine the diffusion
layers of filter paper disks soaked with 5 ml of sterile coefficient. Six leaves per line and water treatment were
distilled water and incubated at 25 C up to 10 days. measured. Photosynthetic parameters maximum rate of
In order to investigate the effect of ectopic expres- carboxylation by ribulose 1,5-biphosphate carboxylase/
sion of cytsod and cytapx genes on seed germination oxygenase (Rubisco) (Vcmax) and the maximum rate of
and plant growth vigor, seed germination rates were electron transport (Jmax) were determined according to
tested in non-transformed and transgenic T1 lines. the Farquhar model of leaf photosynthesis (Farquhar
Seeds were surface sterilized with diluted sodium et al. 1980). The temperature dependencies of photo-
hypochlorite solution (2 %) for 5 min followed by synthetic parameters were calculated according to
three washes with sterilized distilled water. Seeds Bernacchi et al. (2002) modified taking into account
were then germinated on three layers of filter paper the effect of mesophyll conductance (gm). ACi curves
disks soaked with 5 ml of sterile distilled water or with were fitted with a non-rectangular hyperbola version of
5 ml of 100 mM NaCl solution. Seeds were incubated the biochemical model of leaf photosynthesis following
at 25 C with a 16/8 h photoperiod. The rate of Ethier and Livingston (2004). The Ci cut-off point was
germination was determined every 2 days by counting determined based on the method proposed by Ethier
seeds with radicle emergence of at least 2 mm in et al. (2006). From this analysis Vcmax, Jmax and gm
length. The time course (measured in days) taken to were determined.
reach 50 % of germinated seeds was also determined The fluorescence of chlorophyll was measured with
(T50). All assays were replicated four times using 50 a chlorophyll fluorometer (IMAGIM-PAM M-series,
seeds per replicate. Heinz Walz, Effeltrich, Germany) in detached leaves
Growth parameters, such as roots and shoots length from well-irrigated and water stressed tobacco lines
and shoots fresh weight from germinated seeds were after 3 days. After dark-incubation of plants (15 min),
measured 14 days after seed imbibition, and seedlings the minimum and the maximal fluorescence yields
were transferred to pots with peat substrate and were monitored. Kinetic analyses were carried out
acclimatized in an environmental-controlled green- with actinic light (81 lmol quanta m-2 s-1 PAR) and
house. The height and the weight of shoots were repeated pulses of saturating light at 2700 lmol
measured again at 4 and 8 weeks after germination. quanta m-2 s-1 PAR for 0.8 s at intervals of 20 s.
The effective PSII quantum yield (Y(II)), the non-
2.2 Water stress assay, gas exchange photochemical quenching (NPQ) and the coefficients
measurements and chlorophyll fluorescence of non-photochemical quenching (qN), and the pho-
analysis tochemical quenching (qP) were analyzed.

Three week-old control (non-transformed) and trans- 2.3 Mineral content

genic plants (T0 plants) grown in a greenhouse were
deprived from water during 3 days. Control plants Mineral content was analyzed on the leaves of non-
were irrigated with 50 ml of water every day during transformed and transgenic tobacco T0 plants grown

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in normal conditions or exposed to 3 days mild water 3 Results

stress. Leaves were washed with distilled water, dried
at 65 C, ground and stored at room temperature. 3.1 Effect of cytsod and cytapx on maximum rate
Inorganic solute analysis was performed by the of carboxylation by Rubisco (Vcmax)
Ionomic Services at CEBAS-CSIC (Murcia, Spain). and maximum rate of electron transfer (Jmax)
Briefly, samples were digested using a high-perfor-
mance microwave reaction (Ultraclave; Milestone, The effect of cytsod and cytapx on Vcmax and Jmax was
Shelton, CT, USA) with HNO3:H2O2 (4:1, v/v) and investigated in plants well irrigated and in plants
then used for macro and micronutrient determination subjected to irrigation deficit (Table 1). Vcmax data
with inductively coupled plasma-optical omission were affected by both the Line and Irrigation
spectrometry (ICP-OES), in a Thermo ICP-ICAP Treatment factors and a significant interaction was
6500 DUO (Thermo Scientific, England). observed. However, Jmax and the ratio Jmax/Vcmax were
affected only by the Irrigation Treatment factor.
2.4 Statistical analysis Under well irrigated conditions, line 39 displayed a
significantly higher Vcmax than the non-transformed
The effects of the overexpression of cytsod and/or control, but the transgenic lines 17 and 51 presented a
cytapx on leaf mineral nutrition and gas-exchange significant lower Vcmax value than non-transformed
parameters measured in non-transformed and trans- line. Line 39 also exhibited the highest Jmax values
genic lines under well-irrigated and deficit irrigation (Table 1). However, under irrigation deficit condi-
conditions were tested by a two-way ANOVA, tions, Vcmax and Jmax decreased in all cases and values
whereas those related to seedling growth parameters were quite similar in all tobacco lines. Finally, the
in T1 plants were tested by one-way ANOVA. Within ratio Jmax/Vcmax significantly differed between irriga-
each irrigation treatment, lines were compared with tion treatments but not among lines, and data suggest
non-transformed controls by a Dunnetts test. PCA that, on average, the ratio is higher under irrigation
analysis was also performed for mineral content using deficit being line 39 the one that showed the highest
STATISTICA software. values (Table 1).

Table 1 Maximum rate of carboxylation by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Vcmax; lmol CO2
m-2 s-1)
Treatment Line Vcmax Jmax Jmax/Vcmax

Irrigated Control 91.8 18.2 101.5 21.1 1.11 0.23

17 71.9 1.6* 100.4 5.1 1.40 0.07
39 148.4 8.6* 140.7 12.6 0.95 0.12
51 79.2 11.6* 113.06 7.5 1.43 0.09
Irrigation deficit Control 62.1 2.2 92.5 6.8 1.49 0.08
17 55.4 9.1 78.1 2.1 1.41 0.23
39 50.2 2.8 82.3 5.5 1.64 0.15
51 56.3 4.4 78.1 7.2 1.39 0.07
F valuesa
Source of variation Vcmax Jmax Jmax/Vcmax
Line (A) 5.06** 1.06 1.05
Irrigation treatment (B) 50.04*** 17.49*** 9.08**
A9B 9.00*** 1.49 1.71
-1 -2 -1
Maximum rate of electron transport (Jmax lmol e m s ) and Jmax:Vc.max ratio in the different genotypes after exposure to the
irrigation (I) or irrigation deficit (ID) treatments. Data are the mean SE from four replicates
F values significant at 99.9 % (***), 99 % (**) or 95 % (*) levels of probability
a
F values from two-way ANOVA for Vcmax, Jmax and Jmax/Vcmax.

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Theor. Exp. Plant Physiol. (2015) 27:215226 219

The images of chlorophyll fluorescence parameters PC (which accounted for 58 % for the total variability
showed that under well irrigated conditions lines 39 of the basic set of variables). The second PC accounted
and 51 displayed the highest qP and Y(II) values, for 15.8 % of the variability and was mostly due to Na
whereas non-transformed plants showed the highest and Mn concentration) (Online Resource 2).
values for the non-photochemical quenching param-
eters (qN and NPQ) (Online Resource 1). Under 3.3 Effect of cytsod and cytapx on seed
irrigation deficit conditions a decrease in the photo- germination under various conditions
chemical quenching parameters [qP and Y(II)] as well
as the qN parameter was observed in non-transformed The effect of ectopic expression of cytsod and cytapx
plants (Online Resource 1). A decrease in qP also on seed germination was investigated in new and aged
occurred in line 51 that was accompanied by an seeds as well as on seeds soaked in 100 mM NaCl
increase in the NPQ parameter, related to the safe (Fig. 1). For this experiment, we used seeds produced
dissipation of the excess light energy. However, under by self-pollinating the T0 lines and therefore around
deficit irrigation conditions, line 17 did not change the 6 % of the seeds were non transgenic (corresponding
qP parameter and even an increase in the non- to a segregation 1:16 of two independently inherited
photochemical parameters (qN and NPQ) was copies of the nptII gene).
observed. Finally, in line 39 deficit of irrigation also When new seeds were sown in the presence of
decreased qP, but did not significantly alter Y(II) and 100 mM NaCl germination rate was severely affected
NPQ as compared to the irrigated control (Online in the non-transgenic line (only 2 % of seeds germi-
Resource 1). nated after 10 days, Fig. 1a). The percentage of
germination was also affected in line 39 (around
3.2 Effect of cytsod and cytapx and water stress 20 % of seeds germinated after 10 days). This line
on mineral macro- and micronutrients showed significant differences in the germination rate
with the non-transformed control at 5 days. However,
Leaf mineral content (macro- and micronutrients) was germination rate was less affected in lines 17 and 51
determined in both non-transformed and transformed (T50 at 5 and 7 days, respectively), which reached
tobacco lines under deficit of irrigation and under well values of about 67 % for line 17, and 58 % for line 51
irrigated conditions. When analyzed separately, (Fig. 1a).
although the differences were not statistically signif- Germination rate was also determined in aged seeds
icant, the levels of most of leaf macro and micronu- that were stored for 4 years at 8 C (Fig. 1b). Germi-
trients under well-watered conditions were higher in nation was severely affected in the non-transformed
the transformed lines than in the non-transformed seeds (less than 20 % of seeds germinated after
control. Under well irrigated conditions all the trans- 10 days), while it was barely affected in line 39
formed lines showed slightly lower C contents than (T50 of 3.5 days) and not affected in the lines 17 and
non-transformed plants (Table 2). Line 39 had signif- 51 (T50 of 2 days) (Fig. 1b). According to Dunnetts
icantly higher Na and B contents than non-trans- test the three transgenic lines exhibited significantly
formed plants, whereas line 51 showed higher Mg, B, higher germination ability than the non-transformed
Mn and Zn levels than the non-transformed plants control (P \ 0.05).
(Tables 2, 3). Under deficit of irrigation the most
important changes were observed in line 51, which 3.4 Effect of cytsod and cytapx on plant vigor
showed the highest Na, B and Mn contents, whereas and biomass
line 17 also displayed lower Mn levels than non-
transformed plants (Tables 2, 3). Transgenic lines (T1 plants) exhibited elevated vigor,
When mineral elements contents were evaluated by showing significantly higher shoot and root length than
a principal component analysis (PCA) they showed a the non-transformed plants (Table 4). After 2 weeks,
significant correlation. PCA yielded two PC which root growth was significantly higher in transgenic
explained 73.8 % of the total data variance. N, P, K, tobacco lines than in non-transformed plants. Root
Mg, S, Fe, B, Cu and Zn contributed mostly to the first length was 2.7-fold and 1.8-fold higher in lines 17 and

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Table 2 Macronutrient content in leaves from non-transformed and transformed tobacco lines (control, line 17, line 39, and line 51) under irrigated (I) and irrigation deficit (ID)
conditions (3 days of withholding water)
Treatment Line C (%) N (%) P (%) K (%) Na (%) Ca (%) Mg (%) S (%)

I Control 41.8 0.19 1.74 0.14 0.308 0.02 3.22 0.14 0.045 0.01 1.65 0.10 0.52 0.015 0.26 0.014
Line 17 41.1 0.23* 2.06 0.28 0.355 0.01 3.50 0.48 0.047 0.01 1.92 0.28 0.55 0.011 0.28 0.025
Line 39 41.1 0.20* 2,48 0.13 0.399 0.04 4.20 0.32 0.094 0.03* 1.93 0.17 0.54 0.01 0.32 0.01
Line 51 41.1 0.10* 2.78 0.27 0.416 0.04 4.17 0.4 0.057 0.01 2.09 0.21 0.62 0.03* 0.35 0.03
ID Control 41.3 0.19 2 0.41 0.338 0.01 3.60 0.43 0.090 0.01 1.85 0.15 0.54 0.024 0.28 0.032
Line 17 41.8 0.28 1.86 0.24 0.324 0.04 2.96 0.39 0.089 0.01 1.72 0.14 0.48 0.037 0.25 0.032
Line 39 41.6 0.21 2.84 0.28 0.385 0.01 4.41 0.23 0.078 0.01 1.88 0.09 0.53 0.007 0.32 0.01
Line 51 40.6 0.57 2.51 0.07 0.434 0.02 4.66 0.35 0.175 0.01* 1.75 0.24 0.58 0.03 0.36 0.01
Source of variation F valuesa
Line (A) 6.42** 18.07*** 13.67*** 17.26*** 13.95*** 0.76 13.78*** 19.96***
Irrigation Treatment (B) 0.11 0.11 0.02 0.89 56.0*** 1.15 5.96* 0.12
A9B 5.57** 2.41 1.44 2.53 20.33*** 3.43* 4.42* 1.10
Data are the mean SE from 3 replicates. Asterisks represent significant differences (P \ 0.05) between each transgenic line and the wild type control, within each irrigation
treatment, according to Dunnetts test (results in bold)
F values significant at 99.9 % (***), 99 % (**) or 95 % (*) levels of probability
a
F values from two-way ANOVA for the different macronutrient analysed
Theor. Exp. Plant Physiol. (2015) 27:215226
Theor. Exp. Plant Physiol. (2015) 27:215226 221

Table 3 Micronutrient content in leaves from non-transformed and transformed tobacco lines (control, line 17, line 39, and line 51)
under irrigated (I) and irrigation deficit (ID) conditions (3 days of withholding water)
Treatment Line Fe (ppm) B (ppm) Cu (ppm) Mn (ppm) Zn (ppm)

I Control 45.08 5.61 28.94 0.64 3.99 0.71 0.52 0.015 21.54 1.42
Line 17 48.98 4.72 31.52 2.12 4.51 0.97 0.55 0.011 24.88 2.55
Line 39 57.39 7.03 33.54 2.51* 5.85 0.2* 0.54 0.01 23.51 0.12
Line 51 71.24 9.02 41.9 1.03* 6.6 0.9* 0.62 0.03* 30.78 2.78*
ID Control 38.26 12.45 34.44 2.15 4.4 1.56 0.54 0.024 24.87 1.74
Line 17 45.19 5.94 31.08 1.18 4.78 1.36 0.48 0.00* 23.11 3.32
Line 39 61.79 5.94 34.11 1.17 5.35 0.79 0.53 0.007 28.35 3.68
Line 51 58.08 6.88 40.87 1.98* 6.04 0.16 0.58 0.03* 28.23 0.32
Source of variation F valuesa
Line (A) 15.21*** 44.78*** 7.84** 6.05** 8.62**
Irrigation Treatment (B) 3.12 2.96 0.24 112.06*** 1.02
A9B 2.54 4.49* 0.72 7.54** 3.68*
Data are the mean SE from 3 replicates. Asterisks represent significant differences (P \ 0.05) between each transgenic line and the
wild type control, within each irrigation treatment, according to Dunnetts test (results in bold)
F-values significant at 99.9 % (***), 99 % (**) or 95 % (*) levels of probability
a
F values from two-way ANOVA for the micronutrient analysed.

39, respectively, whereas in line 51 the effect was even enhanced tolerance to mild water stress that correlated
more evident, and root reached a length fourfold higher with higher water use efficiency and better photosyn-
than non-transformed plants (Table 4). thesis rates, as well as with increased antioxidant
When compared to the non-transformed plants, capacity in both soluble and chloroplast fractions
shoot length was significantly higher in the transgenic (Faize et al. 2011). In this work we showed that these
lines, being 1.7-fold in line 17 and about twofold in transgenic lines exhibited modified photosynthetic
lines 39 and 51, after 2 weeks of germination. Four capacity, enhanced mineral content and seed germi-
weeks after germination all the transgenic lines nation as well as plant growth.
showed higher vigor than the non-transformed plants,
although values were only statistically significant for 4.1 Photosynthetic capacity
lines 17 and 51. Two months after germination, shoot
length was about 2.5-fold higher in lines 17 and 39 A direct effect of water stress is disruption of
whereas line 51 reached a shoot length 4.3-fold higher photosynthesis. The maximum Rubisco activity
than non-transformed plants (Table 4). (Vcmax) and electron transport capacity (Jmax) are the
The transgenic lines showed also higher biomass when key parameters determining photosynthetic capacity
compared to the non-transformed plants at 2 weeks after (Dickson and Tomlinson 1996). Vcmax is a measure-
germination, being 2.4-fold higher in the line 17, 1.6-fold ment of the process by which Rubisco catalyzes the
in line 39 and about 4-fold in line 51 when compared with reaction of ribulose 1, 5-biphosphate (RuBP) with CO2
non-transformed plants. However, differences were sig- to produce the carbon compounds that become triose
nificant only for lines 17 and 51 (Table 4). phosphates, whereas Jmax reflects electron transport
through the thylakoid membrane, which is critical to
produce NADPH and ATP, and then provide the
4 Discussion metabolic energy necessary to produce triose phos-
phates. In this study we showed that under well-
We generated transgenic tobacco transformed with watered conditions, lines 39 and 51 exhibited the
cytsod (line 17), cytapx (line 51) or containing both highest Jmax values that correlated with high net
transgenes (line 39). These transgenic lines showed photosynthesis (Faize et al. 2011) and the qP

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and increases in NPQ in non-transformed plants and in

line 51. However, lines 17 and 39 maintained Y(II)
and also increased NPQ as a safe mechanism of excess
energy dissipation. Some authors, point out that the
reduction in Vcmax can be due to an inactivation or loss
of Rubisco, reducing the carboxylation efficiency,
while the reduction in Jmax seems to be associated with
a diminution of sedoheptulose-1,7-bisphosphatase, a
key regulatory enzyme in the Calvin cycle (Allen et al.
1997; Nogues and Baker 2000; O lcer et al. 2001). In
fact, the decrease in CO2 assimilation would reduce
the demand for ATP and NADPH, resulting in
decreased electron transport (Zhou et al. 2004). The
ratio Jmax/Vcmax can be considered as a parameter
indicating light absorbed/light used for CO2 fixation.
For this reason, values close to one could indicate a
balanced photosynthetic process, as observed in line
39, or even in non-transformed plants under well
irrigated conditions. Under deficit of irrigation line 39
performed as non-transgenic plants in relation to the
Jmax/Vcmax parameter. However, line 39 showed
higher qP and Y(II) values than non-transgenic plants.
Lines 17 and 51 (harboring only one transgene)
showed constitutively high Jmax/Vcmax values under
Fig. 1 a Germination percentage from new tobacco seeds both irrigation treatments. Values greater than one, as
imbibed in 100 mM NaCl. b Germination percentage from aged occurred under irrigation deficit conditions in non-
tobacco seeds. Aged seeds derived from non-transformed
control (NT) and transformed tobacco lines (line 17, line 39
transformed plants and line 39, could indicate an
and line 51) were harvested 2 months after anthesis and stocked excess of light energy that can lead to the photoinhi-
for 4 years at 8 C. Data are mean and standard errors from 4 bition of photosynthesis as well as ROS over-gener-
independent replicates. 50 seeds were used for each replicate ation. In fact, these tobacco lines suffered a higher
parameter. Under irrigation deficit conditions, decrease in photosynthetic rate (AN) (near 50 %
decreases in Vcmax and Jmax were observed in all decrease) than lines 17 and 51 (about 25 % decrease)
cases that correlated with decreases in qP and Y(II) (Faize et al. 2011). However, these transgenic lines

Table 4 Seedling growth parameters at various days after seed imbibition (DAI) in non-transformed and in transgenic tobacco lines
Line Root length (mm) Shoot length (mm) Shoot fresh weight (mg)
14 DAI 14 DAI 28 DAI 60 DAI 14 DAI

Control 2.9 0.4 7.6 1.8 71 14 78 6 308 88

17 7.8 0.9* 12.7 2.1* 129 33* 179 13 733 186*
39 5.1 0.8* 15.7 1.8* 94 10 196 42 508 51
51 11.6 1.1* 17.2 2* 129 29* 340 123* 1192 191*
F valuesa
96.77*** 18.09*** 5.23** 10.99*** 27.22***
Data are means and standard error from 5 to 15 replicates. Asterisks represent significant differences (P \ 0.05) between each
transgenic line and the wild type control, within each irrigation treatment, according to a Dunnetts test
F values significant at 99.9 % (***), 99 % (**) or 95 % (*) levels of probability
a
F values from one-way ANOVA for the growth parameters analysed.

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Theor. Exp. Plant Physiol. (2015) 27:215226 223

showed higher APX and SOD activity in the chloro- TBARS content and electrolyte leakage values (Faize
plasts (Faize et al. 2011), which may help the et al. 2011).
dissipation of excess excitation energy under environ-
mental stress conditions (Asada 1999). 4.3 Seed physiology

4.2 Leaf mineral nutrition and plant growth Our transgenic tobacco plants exhibited a tight
control of ROS production via an enhanced antiox-
Micronutrients such as Fe, Cu, Mn and Zn are idant capacity (Faize et al. 2011). ROS can act as
cofactors of different metalloenzymes, including signaling molecules regulating plant growth and
APX, catalase, peroxidases (POXs) and SOD isoen- development (Gechev et al. 2006), being also
zymes (Fe-, Mn- and CuZn-SOD), along with their involved in seed germination and seedling growth
role in other important processes including protein (Kranner et al. 2010; Barba-Espin et al. 2010).
synthesis, enzyme activation or carbohydrate metabo- Increased growth by over-expression of antioxidants
lism (Marschner 1997). The content in these micronu- enzymes has also been reported in transgenic
trients was somehow higher in transgenic lines than in tobacco expressing pepper ascorbate peroxidase-like
non-transformed plants. This could be related with the gene (Sarowar et al. 2005).
enhanced activity of those metalloenzymes described The reactivation of metabolism following seed
previously (Faize et al. 2011). Moreover, line 51 imbibition may provide an important source of ROS
presented higher B content under well irrigated and (Bailly 2004; Kranner et al. 2010). For this reason,
irrigation deficit conditions that may contribute to a anti-oxidative mechanisms have been regarded as
better membrane integrity as reported by Faize et al. being of particular importance for the success of
(2011) for this transgenic line. Boron is an essential germination, and most of them increase during
micronutrient for plant growth and development, and germination (De Gara et al. 1997; Tommasi et al.
it is involved in a number of metabolic pathways and 2001; Bailly 2004; Barba-Espin et al. 2010). Uncon-
functions such as cell wall synthesis and structure trolled ROS accumulation during germination may
lignification, carbohydrate metabolism, phenol meta- lead to seed deterioration. For this reason ROS
bolism and plasma membrane integrity (Marschner accumulation must be tightly regulated by the scav-
1997). enging mechanisms during seed germination (Bailly
Several factors may contribute to the changes in 2004). These deleterious effects of ROS, with the
element contents of the leaves. A large size of the root concomitant decrease in the antioxidative defenses,
system will help to access nutrient with limited are responsible for decreased structural integrity and
diffusion such as phosphates (Marschner 1997). increased seed mortality (Priestley et al. 1985;
Increased leaf mineral nutrient may help to maintain Smirnoff 1993). Therefore, reinforcement of antiox-
efficient photosynthesis and thus biomass. A correla- idative system can lead to improved seed germination
tion between vigor and tolerance to water stress has and plant vigor. In this sense, our results showed that
been reported in several plants such as Arabidopsis overexpression of cytapx or cytsod increased seed
(Narang et al. 2000) and rice (Price et al. 2002). Under germination, vigor and plant biomass under non-stress
our experimental conditions, overexpression of cytsod conditions in T1 plants.
and/or cytapx genes stimulated the growth of T1 Salt stress can affect different physiological pro-
tobacco plants. This was especially noticeable in line cesses, including seed germination (Darra et al. 1973;
51, which exhibited better growth parameters than the Heikal et al. 1982). Salinity is detrimental to both seed
non-transformed control. Under normal conditions, as viability and vigor during germination (Ungar 1978).
well as under drought stress, line 51 exhibited the In salt-sensitive species, germination under saline
highest maximum net photosynthesis (AN) that conditions produced osmotic potential increases
showed a good relationship with leaf mass area (Faize which can inhibit the imbibition process by restricting
et al. 2011). In addition, the elevated qP and Y(II) water uptake (Brandford 1990). In the present work,
values observed for this line are indicative of its better we showed that salt stress drastically reduced the rate
photosynthetic capacity. Moreover, line 51 showed a of germination in non-transformed tobacco, being
better membrane protection as observed by its lower transgenic lines less affected. Surprisingly, line 39,

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which overexpressed both transgenes, exhibited an accumulation during storage. Viability losses during
intermediate phenotype. This differential behavior storage is a result of ROS accumulation as well as a
could be related to the activity level of ROS scaveng- decrease in antioxidant mechanisms, which ultimately
ing enzymes. Although we have not analyzed the lead to oxidative damage to essential macromolecules
activity of antioxidant enzymes in germinated tobacco during seed imbibition (Bailly et al. 2008; Rajjou et al.
seeds at 10 DAI, transgenic T1 young tobacco plants 2008). Therefore, the regulation of ROS generation and
(4 weeks old) showed a higher constitutive antioxi- antioxidant defenses is of vital importance to maintain
dant capacity than non-transgenic plants (Faize et al. seed viability. In our work, aged seeds from trans-
2012). However, line 39 displayed lower APX, formed lines showed no loss of viability after 10 days
monodehydroascorbate reductase (MDHAR), POX post-imbibition, contrary to the control seeds, which
and SOD activity levels than lines 17 and 51 (Faize showed a very low germination rate, indicating serious
et al. 2012). This fact could in part explain the seed deterioration during storage. Our results are in
intermediate response to salinity observed during the agreement with those of Lee et al. (2010) who showed
germination of line 39 seeds. It has been extensively that overexpression of SOD and APX in tobacco
reported that salt stress induced an oxidative stress in plastids resulted in enhanced seed viability of aged
plants (Hernandez et al. 2001). Hence, the overex- seeds. Their transgenic lines exhibited low electrolyte
pression of antioxidants enzymes (APX and/or Cu, leakage and maintained their membrane integrity
Zn-SOD) can lead to a better control of ROS level during imbibition, due to higher antioxidant ability,
during the germination process under salinity condi- which results in a lower accumulation of ROS. Our
tions. This hypothesis is supported by the fact that results demonstrate that overexpression of cytsod and
addition of antioxidants such as reduced ascorbate or cytapx in the cytosol can also increase seed longevity
glutathione improved the germination rate of Ara- and germination rates under unfavorable environments.
bidopsis seeds under NaCl stress (Borsani et al. 2001). Taken together, these results suggest that cytosolic
Probably, the overexpression of cytsod or cytapx can antioxidants mechanisms can be useful tools to improve
minimize the salt-induced oxidative stress during the several physiological processes such as seed germina-
germination of tobacco seeds, improving their germi- tion, nutrient uptake, plant growth and a better photo-
nation rates. Moreover, different works reported that synthetic response under abiotic stress conditions.
ROS, when tightly regulated, can be beneficial for the
germination process (Bailly et al. 2008; Kranner et al. Acknowledgments PDV acknowledges the CSIC and the
Spanish Ministry of Economy and Competitiveness for his
2010; Barba-Espin et al. 2010; 2011; Diaz-Vivancos
Ramon y Cajal research contract, co-financed by FEDER
et al. 2013). In previous work, we reported that the funds. This work was supported by the Spanish Ministry of
imbibition of pea seeds with low H2O2 levels Economy and Competitiveness (Project CICYT BFU2009-
increased the germination rate and the early seedling 07443) co-financed by FEDER funds, and the Spanish Ministry
of Economy and Competitiveness (Project INIA, RTA2013-
growth, and this response was correlated with the 00026-C03-00).
induction of cytosolic and stromatic apx transcripts
and their corresponding activity as well as with the
increase of other antioxidant enzymes such as
MDHAR and POX (Barba-Espin et al. 2010). References
Improvement of seed germination using tylakoidal
apx or plastidial sod and apx has been already reported Allen DJ, McKee IF, Farage PK, Baker NR (1997) Analysis of
in tobacco (Sun et al. 2010; Lee et al. 2010). However, the limitation to CO2 assimilation on exposure of leaves of
this is the first report of the involvement of cytosolic two Brassica napus cultivars to UV-B. Plant Cell Environ
20:633640
ROS-scavenging enzymes during germination. Alscher RG, Donahue JL, Cramer CL (1997) Reactive oxygen
Seed germination is reported to be either suppressed species and antioxidants: relationships in green cells.
or slowed down during aging (McDonald 1999). In our Physiol Plant 100:223224
conditions, seed storage for 4 years severely affected Asada K (1999) The waterwater cycle in chloroplasts: scav-
enging of active oxygens and dissipation of excess photons.
germination in non-transformed tobacco, while it had Annu Rev Plant Physiol Plant Mol Biol 50:601639
no detrimental effect on germination of transgenic Bailly C (2004) Active oxygen species and antioxidants in seed
seeds, probably due to the lack of detrimental ROS biology. Seed Sci Res 14:93107

123
Theor. Exp. Plant Physiol. (2015) 27:215226 225

Bailly C, Bogatek-Leszczynska R, Come D, Corbineau F (2002) von Caemmerer-Berry leaf photosynthesis model. Plant
Changes in activities of antioxidant enzymes and lipoxy- Cell Environ 27:137153
genase during growth of sunflower seedlings from seeds of Ethier GJ, Livingston NJ, Harrison DL, Black TA, Moran JA
different vigour. Seed Sci Res 12:4755 (2006) Low stomatal and internal conductance to CO2
Bailly C, El-Maarouf-Bouteau H, Corbineau F (2008) From versus Rubisco deactivation as determinants of the photo-
intracellular signaling networks to cell death: the dual role synthetic decline of ageing evergreen leaves. Plant Cell
of reactive oxygen species in seed physiology. Comptes Environ 29:1682184
Rendus Biol 331:806814 Faize M, Burgos L, Faize L, Piqueras A, Nicolas E, Barba-Espin
Barba-Espin G, Diaz-Vivancos P, Clemente-Moreno MJ, G, Clemente-Moreno MJ, Alcobendas R, Artlip T, Her-
Albacete A, Faize L, Faize M, Perez-Alfocea F, Hernandez nandez JA (2011) Involvement of cytosolic ascorbate
JA (2010) Interaction between hydrogen peroxide and peroxidase and Cu/Zn-superoxide dismutase for improved
plant hormones during germination and the early growth of tolerance against drought stress. J Exp Bot 62:25992613
pea seedlings. Plant Cell Environ 33:981994 Faize M, Burgos L, Faize L, Petri C, Barba-Espin G, Daz-
Barba-Espin G, Diaz-Vivancos P, Job D, Belghazi M, Job C, Vivancos P, Clemente-Moreno MJ, Alburquerque N,
Hernandez JA (2011) Understanding the role of H2O2 Hernandez JA (2012) Modulation of tobacco bacterial
during pea seed germination: a combined proteomic and disease resistance using cytosolic ascorbate peroxidase and
hormone profiling approach. Plant Cell Environ Cu, Zn-superoxide dismutase. Plant Pathol 61:858866
34:19071919 Farquhar GD, von Caemmerer S, Berry JA (1980) A biochem-
Bartoli CG, Gomez F, Martinez DE, Guiamet JJ (2004) Mito- ical-model of photosynthetic CO2 assimilation in leaves of
chondria are the main target for oxidative damage in leaves C-3 species. Planta 149:7890
of wheat (Triticum aestivum L.). J Exp Bot 55:16631669 Flexas J, Bota J, Escalona JM, Sampol B, Medrano H (2002)
Bernacchi CJ, Portis AR, Nakano H, von Caemmerer S, Long SP Effects of drought on photosynthesis in grapevines under
(2002) Temperature response of mesophyll conductance. field conditions: an evaluation of stomatal and mesophyll
Implications for the determination of Rubisco enzyme limitations. Funct Plant Biol 29:461471
kinetics and for limitations to photosynthesis in vivo. Plant Fridovich I (1975) Superoxide dismutases. Ann Rev. Biochem
Physiol 130:19921998 44:147149
Borsani O, Valpuesta V, Botella MA (2001) Evidence for a role Gechev TS, Van Breusegem F, Stone JM, Denev I, Laloi C
of salicylic acid in the oxidative damage generated by NaCl (2006) Reactive oxygen species as signals that modulate
and osmotic stress in Arabidopsis seedlings. Plant Physiol plant stress responses and programmed cell death. BioEs-
126:10241030 says 28:10911101
Brandford KJ (1990) A water relations analysis of seed germi- Heikal MM, Shaddad MA, Ahmed MM (1982) Effect of water
nation rates. Plant Physiol 94:840849 stress and gibberellic acid on germination of flax, sesame
Cakmak I, Strbac D, Marschner H (1993) Activities of hydrogen and onion seeds. Biol Plant 24:124129
peroxide-scavenging enzymes in germinating wheat seeds. Hendry GAF (1993) Oxygen, free radical processes and seed
J Exp Bot 44:127132 longevity. Seed Sci Res 3:141153
Caliskan M, Cuming AC (1998) Spatial specificity of H2O2- Hernandez JA, Corpas FJ, Gomez M, del Ro LA, Sevilla F
generating oxalate oxidase gene expression during wheat (1993) Salt-induced oxidative stress mediated by activated
embryo germination. Plant J 15:165171 oxygen species in pea leaf mitochondria. Physiol Plant
Causin HF, Roqueiro G, Petrillo E, Lainez V, Pena LB, 89:103110
Marchetti CF, Gallego SM, Maldonado SB (2012) The Hernandez JA, Ferrer MA, Jimenez A, Ros-Barcelo A, Sevilla F
control of root growth by reactive oxygen species in Salix (2001) Antioxidant systems and O.- 2 /H2O2 production in
nigra Marsh. seedlings. Plant Sci 183:197205 the apoplast of Pisum sativum L. leaves: its relation with
Darra BL, Seth SP, Singh H, Mendriatta RS (1973) Effect of NaCl-induced necrotic lesions in minor veins. Plant
hormone directed presoaking on emergence and growth of Physiol 127:817831
osmotically stressed wheat (Triticum sativum L) seeds. Jimenez A, Hernandez JA, del Ro LA, Sevilla F (1997) Evi-
Agron J 65:292295 dence for the presence of the ascorbate-glutathione cycle in
De Gara L, de Pinto MC, Arrigoni O (1997) Ascorbate synthesis mitochondria and peroxisomes of pea (Pisum sativum L.)
and ascorbate peroxidase activity during the early stage of leaves. Plant Physiol 114:275284
wheat germination. Physiol Plant 100:894900 Kranner I, Roach T, Beckett RP, Whitaker C, Minibayeva FV
Diaz-Vivancos P, Rubio M, Mesonero V, Periago PM, Ros (2010) Extracellular production of reactive oxygen species
Barcelo A, Martnez-Gomez P, Hernandez JA (2006) The during seed germination and early growth in Pisum sati-
apoplastic antioxidant system in Prunus: response to plum vum. J Plant Physiol 167:805811
pox virus. J Exp Bot 57:38133824 Lee YP, Baek KH, Lee HS, Kwak SS, Bang JW, Kwon SY
Diaz-Vivancos P, Barba-Espn G, Hernandez JA (2013) Eluci- (2010) Tobacco seeds simultaneously over-expressing Cu/
dating hormonal/ROS networks during seed germination: Zn superoxide dismutase and ascorbate peroxidase display
insights and perspectives. Plant Cell Rep 32:14911502 enhanced seed longevity and germination rates under stress
Dickson R, Tomlinson P (1996) Oak growth, development and conditions. J Exp Bot 61:24992506
carbon metabolism in response to water stress. Ann For Sci Leymarie J, Vitkauskaite V, Hoang H, Gendreau E, Chazoule V,
53:181196 Meimoun F, Corbineau F, El-Maarouf-Bouteau H, Bailly C
Ethier GJ, Livingston NJ (2004) On the need to incorporate (2012) Role of reactive oxygen species in the regulation of
sensitivity to CO2 transfer conductance into the Farquhar Arabidopsis seed dormancy. Plant Cell Physiol 53:96106

123
226 Theor. Exp. Plant Physiol. (2015) 27:215226

Marschner H (1997) Mineral nutrient of higher plants, 2nd edn. Puntarulo S, Galleano M, Sanchez RA, Boveris A (1991)
London Academic Press Inc, London, pp 313396 Superoxide anion and hydrogen peroxide metabolism in
McDonald MB (1999) Seed deterioration: physiology, repair soybean embryonic axes during germination. Biochim
and assessment. Seed Sci Technol 27:177237 Biophys Acta 1074:277283
Mittova V, Tal M, Volokita M, Guy M (2003) Up-regulation of Rajjou L, Lovigny Y, Groot SPC, Belghazi M, Job C, Job D
the leaf mitochondrial and peroxisomal antioxidative sys- (2008) Proteome-wide characterization of seed aging in
tems in response to salt-induced oxidative stress in the wild Arabidopsis: a comparison between artificial and natural
salt-tolerant tomato species Lycopersicon pennellii. Plant aging protocols. Plant Physiol 148:620641
Cell Environ 26:845856 Sarowar S, Kim EN, Kim YJ, Ok SH, Kim KD, Hwang BK, Shin
Morohashi Y (2002) Peroxidase activity develops in the JS (2005) Overexpression of a pepper ascorbate peroxi-
micropylar endosperm of tomato seeds prior to radicle dase-like 1 gene in tobacco plants enhances tolerance to
protrusion. J Exp Bot 53:16431650 oxidative stress and pathogens. Plant Sci 169:5563
Narang RA, Bruene A, Altmann T (2000) Analysis of phosphate Schopfer P, Plachy C, Frahry G (2001) Release of reactive
acquisition efficiency in different Arabidopsis accessions. oxygen intermediates (superoxide radicals, hydrogen per-
Plant Physiol 124:17861799 oxide, and hydroxyl radicals) and peroxidase in germi-
Noctor G, Foyer C (1998) Ascorbate and glutathione: keeping nating radish seeds controlled by light, gibberellin, and
active oxygen under control. Ann Rev Plant Physiol Plant abscisic acid. Plant Physiol 125:15911602
Mol Biol 49:249279 Smirnoff N (1993) The role of active oxygen in the response of
Nogues S, Baker NR (2000) Effects of drought on photosyn- plants to water deficit and desiccation. New Phytol
thesis in Mediterranean plants grown under enhanced UV- 125:2758
B radiation. J Exp Bot 51:13091317 Sun WH, Duan M, Shu DF, Yang S, Meng QW (2010) Over-
Oracz K, El-Maarouf-Bouteau H, Kranner I, Bogatek R, Cor- expression of StAPX in tobacco improves seed germination
bineau F, Bailly C (2009) The mechanisms involved in and increase early seedling tolerance to salinity and
seed dormancy alleviation by hydrogen cyanide unravel osmotic stresses. Plant Cell Rep 29:917926
the role of reactive oxygen species as key factors of cellular Tommasi F, Paciolla C, de Pinto MC, De Gara L (2001) A
signaling during germination. Plant Physiol 150:494505 comparative study of glutathione and ascorbate metabo-
Parrish DJ, Leopold AC, Hanna MA (1982) Turgor changes lism during germination of Pinus pinea L. seeds. J Exp Bot
with accelerated ageing of soybeans. Crop Sci 22:666669 52:16471654
Perl-Treves R, Perl A (2002) Oxidative stress: an introduction. Ungar IA (1978) Halophyte seed germination. Bot Rev
In: Inze D, Van Montagu M (eds) Oxidative stress in plants. 44:233264
Taylor and Francis, London, pp 132 Zhou YH, Yu JQ, Huang LF, Nogues S (2004) The relationship
Price AH, Cairns JE, Horton P, Jones HG, Griffiths H (2002) between CO2 assimilation, photosynthetic electron trans-
Linking drought-resistance mechanisms to drought avoid- port and waterwater cycle in chill-exposed cucumber
ance in upland rice using a QTL approach: progress and leaves under low light and subsequent recovery. Plant Cell
new opportunities to integrate stomatal and mesophyll Environ 27:15031514
responses. J Exp Bot 53:9891004
Priestley DA, Warner BG, Leopold AC, McBride MB (1985)
Organic free radical levels in seeds and pollen: the effects
of hydration and ageing. Physiol Plant 70:8894

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