Bulletin UASVM Horticulture, 70(1)/2013, 172-179

Print ISSN 1843-5254; Electronic ISSN 1843-5394

Effects of some Abiotic Factors on Brassica Oleracea Var. Capitata Sprouts

Antoanela PATRAS, Camelia Elena LUCHIAN, Marius NICULAUA,

Vasile STOLERU Faculty of Horticulture, University of Agricultural Sciences and Veterinary Medicine of Iaşi; 3, Mihail

Sadoveanu Alley, 700490, Iaşi, Romania; elapatras@yahoo.fr

Abstract. Nowadays there is a continuous increase of sprouts consumption. The white cabbage is

known as a resistant plant at different abiotic factors. It has the capacity to accumulate heavy metals

without visible phytotoxicity symptoms, when the concentration is not excessively high. The

morphological and biochemical modifications induced in cabbage sprouts by two heavy metals (Cu

and Mn) were analysed comparing with the effects of salt stress. The metal accumulation in sprouts

was measured, because over a certain level, it may become dangerous for the consumers health. The

sprouts were grown in a germinator in specific conditions for 10 days, in the presence of CuCl2,

MnCl2, NaCl or water respectively. For each treatment, we used three concentrations. It was analyzed

the accumulation of each metal in the cabbage sprouts (by AAS method), the influence of each metal

concentration on the seeds germination and on the sprouts growth (by biological determination) and

photosynthetic pigments concentration (by spectrophotometric method). The treatment with Mn and

Na at the studied concentrations is not inhibiting the sprouts growth (only Cu does) and is not inducing

phytotoxicity symptoms (contrary, is stimulating the growth in some cases). The accumulation of the

heavy metals in sprouts is significant and may become dangerous for the consumer health, if the

quantity of the ingested sprouts is important.

Keywords: salt stress, heavy metals, sprouts, white cabbage.

INTRODUCTION

In many areas of the globe, because of the increasing necessity of food, the

vegetables are grown under unfavorable conditions, like the presence of heavy metals or high

salinity.

Salt-affected soils have been identified in practically all climatic regions: about

95 million hectares of land all over the world are affected by high salinity (Mittal et al, 2012).

Sodium at normal level in plants is important for the osmotic equilibrium and the membrane

permeability. At high levels, appears salt stress phenomena, which are manifesting by osmotic

disorders associated with physiological responses similar to those induced by drought stress

(Chaves et al., 2009). The cell growth and photosynthesis are processes affected by high

salinity (Munns et al., 2006). It has been demonstrated also, that plants under salt stress are

affected by high illumination resulting photoinhibition (Ohnishi and Murata, 2006). High

light stimulates the oxygen reduction and the generation of reactive oxygen species (ROS),

responsible for many damages of chlorophyll, proteins, DNA, lipids and other important

biomacromolecules, thus irremediable affecting plant metabolism, growth and yield (Mittal et

al., 2012).

The contamination of the environment with heavy metals is mainly due to the

industry, agricultural practices or domestic activities. However, copper and manganese are

two essential microbioelements (Reeves and Baker, 2000), thanks to their role in redox

reactions and to the participation to enzymatic systems. Both Cu and Mn are important in

photosynthesis: Cu is a component of primary electron donor in photosystem I and of various

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proteins like plastocyanin of photosynthetic system and Mn is need for water splitting at

photosystem II. Also, they participate in the antioxidant protection of plant, being involved in

the elimination of superoxide radicals, because of their implication in the functioning of

superoxide dismutases (SOD). SOD constitute a family of metaloenzymes, including Mn-

SOD and Cu/Zn-SOD, the metal component (like Mn, Cu + Zn or other), being different,

depending of the isoenzyme (Bannister et al., 1987). We can find Mn-SOD in mitochondria

and bacteria and Cu/Zn-SOD in higher plants, mushrooms and animals (Pelmont, 1995).

Nevertheless, Cu and Mn uptake in excess to the plant requirements cause serious

phytotoxic effects. Cu provokes plant growth retardation and leaf chlorosis. It generates

reactive oxygen species and oxidative stress and, as consequence, causes disturbance of

metabolic pathways and damage to biomacromolecules (Nagajyoti et al., 2010). Mn provokes

leaf chlorosis and necrotic brown spotting. It reduces the photosynthetic rate and inhibits

synthesis of chlorophyll (Clarimont et al., 1986).

Many researchers studied the correlation between the metal concentration in soil and

its accumulation in different plants (Fergusson, 1990; Fytianos et al., 2001) and different

plant organs (Rascio and Navari-Izzo, 2011) and also the way that each metal affects the

morpho-physiological and biochemical characteristics of plants (Moustakas et al., 1997;

Nagajyoti et al., 2010; Oancea et al., 2005; Pandey et al., 2009; Pop, 2010). There are huge

differences between plants concerning the capacity to absorb different metals. Also, it was

demonstrated that certain plants that absorb a specific quantity of metal manifest phytotoxic

effects and other plants, accumulating similar quantities of metal have no morpho-

physiological changes (Rascio and Navari-Izzo, 2011). In this last case, when the plant

accumulates metals at high concentrations, without manifesting any visible changes, the

consumption of plant-based food may cause a serious risk to human health (Wenzel and

Jackwer, 1999).

Nowadays there is a continuous increase of sprouts consumption, because of their

properties and benefits for health. We can find on the market a great variety of different types

of sprouts in which Brassicaceae family is well represented. The Brassica oleraceae var.

capitata (white cabbage) is known as a resistant plant at different abiotic factors. It was

demonstrated that it has the capacity to accumulate some heavy metals without visible

phytotoxicity symptoms, when the concentration is not excessively high (Anjum et al., 2012).

Our study analyses the influence of sodium, copper and manganese stress, at low

concentrations (that can be commonly found on the cultivated soil), on Brassica oleraceae

var. capitata germination, sprouts development and chlorophyll content. The metals

accumulation in sprouts was measured, because over a certain level, it may become dangerous

for the consumers health.

MATERIALS AND METHODS

Seeds germination

The analysed sprouts were obtained from seeds of Brassica oleraceae var. capitata,

the cultivar Copenhagen Market, commercialised by Agrosel. The seeds were equally

distributed in Petri dishes of 10 cm diameter containing filter paper moistened in distillated

water for the blank and in NaCl, CuCl2 and respectively MnCl2 solutions for the treated

samples. Each metal solution was prepared in three concentrations: 0.01 mM, 0.20 mM and

respectively 0.40 mM. The Petri dishes were introduced in a Germinator MLR-351 in specific

conditions for 10 days: constant humidity (70%); the temperature between 5 am – 1 pm was

20°C, between 1 pm – 9 pm was 27°C and between 9 pm – 5 am was 20°C; light 16h/day and

dark 8h/day.

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General measurements

The influence of each metal concentration on the seeds germination was appreciated

by the number of germinated seeds from 100 seeds. The sprouts growth was visual estimated

by biological determination.

Determination of metal content in sprouts

The accumulation of each metal in the cabbage sprouts was measured by atomic

absorption spectrometry (AAS), with the Atomic Absorption Spectrophotometer - Shimadzu

AA 6300. In order to prepare the samples for AAS, the sprouts were washed (for the removal

of the heavy metal ions from the external surface), oven dried in an electric oven at 105°C for

4h30min. After dry weight determination, the oven-dried samples were calcinated at 550°C

and digested with HCl.

The metal concentration was expressed in mg/g dry weight. We calculated the

relative metal accumulation in sprouts as:

[Me] (%) = ([Me]t / [Me]b) x 100,

[Me] - relative metal accumulation,

[Me]t - metal concentration in treated sprouts,

[Me]b - metal concentration in blank.

Measurement of photosynthetic pigments

The content of photosynthetic pigments of the sprouts was measured using the

spectrophotometric method (UV-Vis Spectrophotometer 200, Analytic Jena), after trituration

and extraction with acetone 90% (Ikan, 1991). The absorbance was measured at: 662 nm -

chlorophyll a, 644 nm - chlorophyll b - and 440.5 nm - carotenoids.

Statistical analysis

All measurements were made on triplicate and the data were statistically analysed

using Student’s t test (Snedecor and Cochran, 1984).

RESULTS AND DISCUSSIONS

Germination rate

The 10 days old sprouts were examined and we established the germination rate and

the morphological characteristics. Mn induces a very small stimulation of the sprouts

development and Na has no visible effects, at the studied concentrations, but the growth was

inhibited in the case of Cu treatments after 8 days.

The explanation concerning the Mn action may be that it activates some seed’s

antioxidant systems, like Mn-SOD. At low Mn concentrations, the activation of the enzymatic

systems does not take place any longer, according to the obtained results. In the case of the

action of manganese ions on the plantlet, this mechanism is no longer valuable (the sprout has

not a protection tegument, like the seed) so that the higher Mn ions concentrations may act

aggressively and inhibit the growth.

The germination rate was slightly influenced by all treatments: Na is stimulating the

germination in the order: Na 0.01 mM > Na 0.2 mM > Na 0.4 mM. Mn is stimulative only at

0.2mM (89%) and 0.4 mM (86%), compared to the blank (84%) and has no influence at

0.01mM. Cu is inhibiting the germination in all concentrations and the inhibition is stronger

when the CuCl2 concentration increases (Fig. 1). Similar results were obtained by Radoviciu

et al. (2009), in the case of germination of corn seeds in the presence of copper and

manganese. Also, Rîşca et al. (2008) observed the stimulation of the wheat seeds germination,

induced by MnCl2.

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Fig. 1. The germination rate of white cabbage seeds in the presence of different concentrations of Na,

Mn and Cu ions

Metals accumulation in sprouts

In the case of all three studied metals, we observe that the accumulation in sprouts

was more important when the treatment solution was more concentrated (Fig. 2, Fig. 3,

Fig. 4). But, this increase is not linear, and one explanation is that heavy metal uptake in plant

is not linear either, in response to the increasing concentration, as Nagajyoti also observed

(2010). For Mn and especially for Cu (in case of high concentration treatments), we noticed

an important heavy metal accumulation in cabbage sprouts, compared to the blank. In the case

of Na, the accumulation is insignificant: for 0.4 mM treatment we observe the higher

accumulation, which is only 1.17 times increased compared to the blank.

Fig. 2. The concentration of Na in sprouts (mg/g

dry weight) for the three NaCl treatments,

compared to the blank

Fig. 3. The concentration of Mn in sprouts (mg/g

dry weight) for the three MnCl2 treatments,

compared to the blank

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Fig. 4. The concentration of Cu in sprouts (mg/g dry weight) for the three CuCl2 treatments, compared

to the blank

The graphical representation of the relative metal accumulation in sprouts (Fig. 5)

reveals that in the case of small concentration treatment (0.01 mM), Mn and Cu are

accumulated in small concentrations and Na accumulation is insignificant. When metal

concentration in the treatment solution is 0.2 mM, Mn accumulation in sprouts increases

approximately 10 times than in the blank and Cu accumulation, about 14 times, while Na

accumulation is still insignificant. For the 0.4 mM treatment, Na concentration in sprouts is

still very small (117%), but Mn and especially Cu are strongly accumulated (about 15,

respectively 22 times more than their concentrations in the untreated sprouts). This confirms

that sodium is slower absorbed in plants, compared to other cations (Mn, Cu) and its level in

10 days old sprouts has small values. But, the important Cu and Mn bioaccumulations and

eventual biomagnifications in the food chain (Anastasio, 2006, Imran 2008), can be extremely

dangerous to human health.

Fig. 5. The relative metal accumulation in sprouts (%) compared to the blank (100%)

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Content of photosynthetic pigments

Na, Mn and Cu treatments induce small modifications concerning the photosynthetic

pigments concentration in sprouts (Fig. 6 and Tab. 1). Na 0.01 mM weakly stimulates the

chlorophyll a and inhibits chlorophyll b and carotenes. The medium Na concentration

stimulates chlorophyll a and b concentrations and inhibits carotenes concentration, reported to

blank. Na 0.4 mM has the stronger inhibition effect upon all photosynthetic pigments. Bartha

(2012) also observed the fluctuations of chlorophyll a and b content for different varieties, in

the case of Na treatment applied to the lettuce: for some varieties, he observed the increasing

of pigments concentration, compared to blank, for others, a decrease.

In the case of Mn treatments, the global effect is the inhibition of photosynthetic

pigments. The exception is for the medium concentration (0.2 mM) treatment, when the

chlorophyll a content is slightly increasing. The other two Mn treatments seriously decrease

the sprouts content in photosynthetic pigments. An explanation may be the fact that the excess

of Mn inhibits the synthesis of chlorophyll, by blocking a Fe-concerning process, as also

Clarimont reported (1986).

Cu decreases the content of all photosynthetic pigments applied in 0.01 mM and

0.4mM concentrations and increases it at 0.2 mM. The carotenoids content registers the most

important increase compared to the blank, reflected also in the color of the sprouts.

We notice that for all 3 metals, the concentrations of photosynthetic pigments have

the highest values in the case of the middle treatment concentration (0.2 mM), compared to

the other two concentrations.

Fig. 6. The chlorophyll a, chlorophyll b and carotenes concentrations in white cabbage sprouts after

Na, Mn and Cu treatments

The optimal ratio chl a / chl b is 3 / 1 and it was obtained in the case of Na 0,2 mM

treatment. Close results were obtained for Cu 0.2 mM and Na 0.01 mM. The total chlorophyll

content has the highest values in case of treatments with 0.2 mM solutions and the order is:

0.2 mM Na > 0.2 mM Cu > 0.2 mM Mn > blank. The smallest total chlorophyll content is

reached for the treatment with the 2 heavy metals (Mn and Cu) in solutions 0.01 mM; then the

total chlorophyll increases at 0.2 mM and decreases again at bigger concentration (0.4 mM).

Pandey (2009) explained that the decreased content of the pigments may be the result

of reduced synthesis and/or enhanced oxidative degradation of these pigments by the

oxidative stress produced by heavy metals. The carotenoids are known to be potent quenchers

of reactive oxygen species. As the carotenoids protect chlorophyll from photo-oxidative

destruction, a reduction in carotenoids under excess of heavy metals might be a reason for the

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decrease in chlorophyll. Also, excess of divalent heavy metal ions compete with Fe for uptake

by binding with biomolecules of which iron is a constituent.

Tab. 1

The total chlorophyll content, chlorophyll a /chlorophyll b ratio and carotenoids / total chlorophyll

ratio of white cabbage sprouts treated with Na, Mn and Cu

Treatment Total chl (chl a + chl b)

(mg/100g fresh weight)

chl a / chl b car / (chl a + chl b)

blank 101.42±1.43a 2.599±0.05

a 0.293±0.01

a

Na 0.01 mM 98.66±1.41a 2.838±0.04

a 0.290±0.01

a

Na 0.2 mM 136±2.03b 3.037±0.13

b 0.208±0.00

b

Na 0.4 mM 92.43±1.04a 2.594±0.03

a 0.283±0.01

a

Mn 0.01 mM 78.6±0.78a 2.795±0.01

a 0.293±0.01

a

Mn 0.2 mM 102.5±1.54a 2.674±0.08

a 0.284±0.01

a

Mn 0.4 mM 87.37±0.97b 2.798±0.01

b 0.308±0.02

b

Cu 0.01 mM 97.05±1.33a 2.768±0.02

a 0.290±0.01

a

Cu 0.2 mM 112.92±1.85b 2.858±0.11

b 0.359±0.02

b

Cu 0.4 mM 97.34±1.57a 2.787±0.01

a 0.291±0.01

a

a indicates statistically significant at p<0.001

b indicates statistically significant at p<0.02

CONCLUSION

The influence on the germination process by the three studied metals is reduced: Na

and Mn stimulate the germination rate in all analysed concentrations (Na by osmotic

mechanism and Mn stimulating enzymatic systems); Cu inhibits the germination (inducing

oxidative stress). Differences concerning the growth rate or the morphological parameters

were not very significant in the case of Mn and Na treatments, comparing to the blank, but the

growth was inhibited in the case of Cu treatments. This correlates perfectly with the metal

accumulation in sprouts, as Na is slowly accumulating and has a very small increase

compared with Mn and especially Cu. The metals concentration in sprouts is more important

when the treatment solution is more concentrated.

The photosynthetic pigments were slightly influenced both by the nature of the metal

applied and by its concentration. Mn inhibits the photosynthetic pigments, by blocking a

process involving Fe. In all treatments, the 0.2 mM concentration is more beneficial for

photosynthetic pigments. Bigger concentrations may interfere with their synthesis or provoke

oxidative stress which affects the pigments.

Generally, white cabbage sprouts are resistant at metal accumulation. Only for

copper are visible phytotoxic changes.

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