Early seedling growth affected by CuSO4 and its combination with PEG 6000 in maize

Several abiotic stress factors are faced by the plants in nature, including metal exposure and water deficit condition. The present study was undertaken to investigate the effects of copper and its combination with water deficit, on growth and anatomical characteristics of Zea mays L. (maize) cv. Ganga safed-2 seedlings. Seeds were treated with CuSO4 (0-1000µM) for inducing Cu stress, PEG 6000 (0-10%) for inducing water deficit stress and their combination for combined stress for 5 days. Germination %, growth parameters, % phytotoxicity, and root anatomical characteristics were analyzed. Treatment of maize seeds with 0-1000µM CuSO4 significantly reduced almost all the growth parameters, except germination %. Root growth was inhibited significantly at 100µM and higher concentrations of CuSO4, however, for shoot growth, ?300µM are inhibitory. Germination percentage was not affected by the supplementation of Cu, indicating the tolerant nature of Ganga safed-2 maize genotype at germination stage. Treatment with Cu (?300µM) and PEG 6000 (10%), decreased the growth of maize seedlings with prominent effect on root by Cu and on the shoot by 10% PEG. Anatomical modifications in root were noticed with both the stresses, individually and in combination.


Introduction
Heavy metal contamination is the pervasive threat that makes a significant contribution to environmental pollution.Copper (Cu) is a naturally occurring element of earth crust, a transitional metal and usually present in soils within the range of 0-250 µg/g (1).However, its concentration in soils often varies due to its release into the environment by anthropogenic activities, such as, by the use of bactericides, fungicides and industrial wastes from manufacturing companies dealing with the production of metal, electric appliances, etc. (2,3).It is an essential trace element for plants required for normal growth and development.It may act as a structural element in regulatory proteins, like, plastocyanin and a constituent of many enzymes, such as, cytochrome C oxidase, Cu/zinc superoxide dismutase, ascorbate peroxidase, diamine oxidase, polyphenol oxidase etc. Being a component/cofactor for enzymes, it participates in various physiological processes, such as, photosynthesis, respiration, antioxidant mechanism and hormone metabolism (4)(5)(6)(7).Apart from its essential role in plants, above threshold concentrations Cu becomes extremely toxic and causes inhibition of seed germination rate, root length and shoot elongation, root/shoot ratio and dry biomass as well as chlorosis and necrosis, that leads to inhibition of plant growth or even cell death (8,9).Excessive Cu alters the pigment and protein components (e.g., chlorophyll content and PSII), thylakoid membrane structure and CO2fixation reactions of photosynthesis.Further, it induces oxidative stress that leads to overproduction of reactive oxygen species that can severely damage membrane lipids, proteins and DNA (10,11).Furthermore, Cu toxicity can induce anatomical changes in plant roots, such as, decrease in thickening of endodermis, increase in thickness of exodermis and reduction of the diameter of vascular bundles (12).Structural modifications in the form of thickening of epiblema, endodermis, cell wall of xylem and cortical parenchyma, increase in the number of protoxylem and metaxylem elements, increased proportion of pith have been observed in several reports due to heavy metal elements, such as, copper, cadmium, chromium and lead (13)(14)(15)(16).
Water deficit stress is an important abiotic factor that causes dehydration and osmotic imbalance in the cell, consequently water potential of the plant tissues is decreased that affects crop yields (17).Polyethylene glycol (PEG) compounds have been frequently used to stimulate water deficit effects in plants since, PEG of high molecular weight range (6000 or above) cannot enter the pores of plant cells (18,19).It reduces seed germination percentage, root length, seedling water content, decreases chlorophyll content and inhibits enzymes of chlorophyll biosynthesis, damages the thylakoid membrane structure thereby decreases photosynthesis and increases the activity of antioxidative enzymes (20)(21)(22)(23).Water deficit and heavy metal toxicity are often co-occurring abiotic stresses in the natural environment.Recent studies showed that concurrent exposure of heavy metals Ni, Cu, Co, and Cr with drought alters xylem structure and reduces conduit density, hydraulic conductance and root and shoots growth that resulted in smaller plants and increases early mortality of Acer rubrum L. (Red maple) (24).Lead treatment in combination with drought has been found to significantly affect the dry mass production, photosynthetic activity and quantum yield of PSII and cellular ultrastructure of Populus cathayana L. (25).Combined application of excess Cu and water deficit on tobacco leaves showed increased proline and abscisic acid content with decreased leaf relative water content (26).
Zea mays L. is a cereal crop used as human food, animal feed and raw material for manufacture of many industrial products, so, it is crucial to understand its performance under various stresses and their combinations.Responses of plants under combination of stresses are limited to a small number of reports.Thus, present study was designed to understand the changes in growth characteristics and anatomical behavior in maize seedlings in relation to Cu stress and its combination with water deficit stress.

Materials and Methods
Uniform and healthy seeds of Zea mays L. cv.Ganga safed-2 (obtained from Ganga Kaveri seeds Pvt. Ltd., Hyderabad) were selected and surface-sterilized by soaking in 0.1% HgCl2 for 1-2 min and then washed thoroughly with tap water followed by distilled water.Seeds were placed in 20 cm diameter Petri plates (10 seeds per plate) lined with filter paper discs and treated with different concentrations of CuSO4.5H2Oviz.0, 10, 50, 100, 300, 500 and 1000 µM and allowed to grow in a light growth chamber in continuous light of intensity about 40W/m 2 at 25±3ºC for five days.Distilled water was used as control.On the basis of observations of pilot experiments with 0-1000µM concentrations of Cu on growth parameters, 10 and 100µM concentrations were selected for combination studies with water deficit.Water deficit was induced by polyethylene glycol (PEG) 6000 (5% and 10% solutions).These concentrations of PEG were decided after performing germination and growth studies with 0-30% PEG solutions (data not shown).The combinations of PEG and Cu used were 5% PEG + 10µM CuSO4, 5% PEG + 100µM CuSO4, 10% PEG + 10µM CuSO4 and 10% PEG + 100µM CuSO4.After five days seedlings were examined for various growth parameters.Copper sulphate (CuSO4.5H2O) of SQ grade-Qualigens Fine Chemicals and PEG 6000 from HiMedia procured from a local dealer were used in the study.
Germination percentage was noted on 3 rd day after emergence of 2 mm radicle by formula of Close and Wilson (27): x 100 Total number of seeds grown Growth parameters, such as, root length, shoot length, root shoot ratio, seed vigor, root and shoot fresh and dry weights, tolerance index and percentage phytotoxicity in root and shoot as well as root anatomical features of maize seedling were determined on completion of 5 th day of growth.Root and shoot length was measured with the help of thread and centimeter scale.Root shoot ratio was obtained by dividing lengths of root and shoot tissues.Seed vigor was calculated using formula of Baki and Anderson ( 28     prepared in 0.1M sodium acetate buffer (pH 4.4).Anatomical changes were then observed under Olympus microscope mounted with digital camera.
Each observation is a mean of at least 5 replicate experiments and data are presented as mean ± standard error.All data were analyzed by Student's 't' test for mean comparison and to test the significance of mean difference obtained for various treatments.

Results
Germination and growth parameters of Cu treated maize seeds are presented in Table 1.When seeds were treated with 0-1000µM concentrations of CuSO4, there was no significant change in germination percentage.However, a concentration dependent decrease in root length was observed with increasing CuSO4 concentrations, that was non-significant up to 50µM and significant at 100µM (p<0.05) and higher concentrations, ≥300 µM (p<0.001).Shoot length was marginally increased at 100µM Cu concentration.Further, at higher concentrations of Cu (300-1000µM), a significant reduction in shoot length was noted.Root-shoot ratio was decreased with increasing concentration of Cu.Seed vigor was reduced gradually and significantly at 300-1000µM concentrations with only 29% value of seed vigor at 1000µM Cu, while at lower Cu levels (10-100µM), the change in seed vigor was non-significant (Table 1).
Table 2 presents the data for germination percentage and seedling growth parameters of maize treated with PEG 6000 (5% and 10%) treatment alone and its combination with 10µM and 100µM CuSO4.Germination percentage was significantly reduced with the treatment of both 5% and 10% PEG, however, when supplied along with 10µM and 100µM Cu, no significant change was observed (Table 2).Seedling root length, shoot length and seed vigor were significantly (p<0.001)decreased in both individual PEG and its combined treatments with Cu (Table 2).Root-shoot ratio was significantly reduced at 5% PEG treatment individually and in combination with Cu.However, the ratio was found to be significantly higher at 10% PEG treatment alone and in combination with metal (Table 2).
Effects of supplementation of Cu on fresh and dry weights of maize tissues are shown in Fig. 1A and 1B.At 300-1000µM concentrations, both root and shoot fresh and dry weights were decreased gradually and significantly (p<0.001),however, at low Cu levels i.e. 0-50µM, no significant change was noticed (Fig. 1A and 1B).At 100µM Cu concentration, fresh weight of root was reduced significantly, while, shoot fresh weight was increased (Fig. 1A).Similar pattern of change was observed with dry weights of root and shoot tissues (Fig. 1B).Treatment with only PEG and PEG+Cu, reduced the root and shoot fresh weights significantly (Fig. 1C).Root dry weight was decreased with individual PEG and combined treatments (Fig. 1D).However, shoot dry weight was slightly increased at individual 5% PEG treatment and decreased in combination; nevertheless, both the changes were nonsignificant.With the supply of 10% PEG both individually and in presence of Cu, shoot dry weight was reduced significantly (Fig. 1D).
When maize seeds were supplied with 300-1000µM concentrations of CuSO4, percentage phytotoxicity increased with maximum toxicity of 79% in roots and 53% in shoots at highest concentration, however, at low levels (0-100µM), it was increased in roots and decreased in shoots (Fig. 2A).Percentage phytotoxicity due to PEG and combined treatments (Cu+PEG) increased in both root and shoot tissues (Fig. 2B).At 5% PEG and its combined treatment with Cu, % phytotoxicity was higher in roots as compared to shoots, while at 10% PEG and its combination with Cu, it was more in shoots than in roots (Fig. 2B).Due to increase in % phytotoxicity, tolerance index was consequently decreased in all treatments (Fig. 2A and 2B).In root anatomical sections of maize seedlings, appropriate number of unicellular root hairs was seen in well defined epiblema cell lining when treated with 0-100µM Cu.
Large sized metaxylem vessels and smaller pith in stellar region was identified (Fig. 3A and 3B).With increasing Cu concentration (≥300µM), a gradual increase in parenchymatous cell layers in cortex region, increased pith size with overall increase in area of stellar region and comparative decrease in size of metaxylem vessels was noticed (Fig. 3B).Due to increased cortex and pith size in stellar region, an overall increase in the width and thickening of root was noted, especially at highest dose of metal (1000µM).Further, irregularity in distribution and arrangement of cells of epidermis and damage of root hairs was resulted, with the effect being more prominent at 1000µM CuSO4.Due to damage of epidermal cells, an additional modification i.e. enlargement of cells in outermost lining of cortex was seen that causes thickening of overall exodermis of cortex (Fig. 3B).Appropriate number of root hairs was visualized when 10 and 100µM Cu combined with 5% PEG 6000 were supplied to the maize seedlings (Fig. 3C).However, in combination of 10µM Cu with 10% PEG increased number of root hairs with irregularities in shape and arrangement of epiblema cell lining was noticed.Further, thinning of roots and smaller cortex region was another structural change (Fig. 3C).No distinctive changes in stellar region were noticed in any of the combinations studied, however, treatment with combination of 100µM Cu and 10% PEG caused damage of epiblema with less number of root hairs (Fig. 3C).

Discussion
Copper being an essential heavy metal is required for normal plant growth and development.However, at elevated concentrations in soil it becomes toxic.Non-significant difference in % germination of maize seeds treated with varying concentrations of CuSO4 in the present study indicates that probably Ganga safed-2 maize variety is resistant towards Cu toxicity even up to 1000 concentration at germination stage.It might be due to utilization of its own reserve by the seeds for germination and hence the process is not affected by presence of metal ions in the medium (31).Mahmood et al. (32) has reported a similar non-significant change in germination in maize at 3-12ppm Cu treatment, however, Radoviciu and coworkers (33) noticed slight improvement in germination % at 3ppm Cu in maize, that decreased on higher Cu doses (30 and 300ppm).Significant reduction in germination percentage obtained by treatment with PEG 6000, could be due to inhibition of seed imbibition process caused by reduced water potential gradient between seed and medium.This process delayed seed metabolism activation and hence germination (34).Supply of 100µM Cu significantly reduced root growth of maize seedling, while, shoot length was increased with the same treatment.This might be due to some protective mechanism for reduced translocation of Cu from root to shoot (35) or the amount of Cu that transfers from root to shoot fulfills shoot Cu requirement in trace amounts to enhance its growth (36).An increased shoot length over control at 10ppm Cu has been reported in wheat (37).At concentration >100µM Cu, both root and shoot length were significantly reduced with the effect being more pronounced for the earlier one (Table 1).Similar to the observation of the present study, inhibitory effects of Cu on root and shoot has also been reported in wheat (37) and maize seedling (38).Heavy metal induced root inhibition may involve metal interference with cell division and hence abnormal mitosis (39).Water deficit induced by PEG treatment reduces the rate of cell division and elongation which results into decreased root and shoot length (40).Further, in water deficit condition, cell wall elasticity also decreases because of hormonal and hydraulic signals which in turn may reduce the final size of the cells and the growth (41).Significant reduction in root length by combined treatment of 10% PEG and 100µM CuSO4 indicates synergistic effect of both the stresses as PEG induced water deficit condition becomes more severe in presence of Cu (Fig. 1C and 1D).For shoot length, significant reduction during combined treatment of 10% PEG and CuSO4 seems to be due to prominent effect of water deficit stress only (Table 2).
Decreased fresh weight of root and shoot tissues might be related with reduced growth of these tissues under stress condition.Significant reduction in weights among combination of 10% PEG and CuSO4 is probably due to more pronounced effect of water deficit condition only (Table 2).In maize, reduced root fresh weight and dry weight with PEG 6000 has also been observed by Li et al. (42).Lower activities of photosynthetic enzymes under water stress condition might be a possible cause of reduced dry matter (43).In the present study, 79% phytotoxicity was noticed in the roots at 1000µM Cu level, while in shoots the toxicity was 53% (Fig. 2A).Gang et al. (36) reported a percentage phytotoxicity of 95 and 91% at 500ppm Cu concentration in wheat root and shoot tissues, respectively.However, 100% phytotoxicity at Cu concentrations ≥300ppm has been reported in both root and shoots of tomato seedlings (44).Increase in percentage phytotoxicity in both root and shoot by the supplementation of PEG and its combination with Cu could be due to lesser water availability in 10% PEG containing medium which might be inadequate for growth.Anatomical changes in the cortical region due to Cu supplementation indicate root adaptation for survival under stress conditions.Increased thickness of the root as a result of greater number of cells in cortex and pith region is one of the morphological traits that contribute to heavy metal tolerance of maize (45).Thickening of exodermis under Cu stress in wheat has been suggested as a means of protection against penetration of excess toxic agents from the medium (12,14).Greater root system mainly of fine roots and large number of root hairs could be an adaptive strategy towards water deficit stress when PEG (10%) was supplemented along with 10µM Cu.

Conclusion
It seems that Cu concentration higher than 100µM is toxic for root environment, and doses of ≤100µM fulfils Cu requirement in shoot tissue of Zea mays L. cv.Ganga safed-2 cultivar.Unaffected germination percentage due to Cu supply demonstrates that probably this variety of maize is tolerant towards Cu toxicity at germination stage.Higher concentrations of Cu (≥300µM) and PEG 6000 (10%), negatively influence growth of maize seedlings with effects being substantial for root growth in Cu treated seedlings and for shoot growth in 10% PEG treated seedlings.Further, increase in the severity of stresses, individual and in combination causes more reduction in growth attributes.Furthermore, combination of both the water deficit and metal stress affects the root characteristics in a synergistic manner.Anatomical modifications in root architecture due to both the stresses represent an adaptive trait towards tolerance of maize.
): Seed vigor = Seedling length x Germination percentage Electrical weighing balance was used to determine fresh and dry weights of roots and shoots.Dry weight was obtained after keeping the tissues in a hot air oven at 80ºC for a period of 4 days.Tolerance index was calculated by formula given by Iqbal and Rahmati (29): Tolerance index = Mean root length in treatment applied x 100 Mean root length in distilled water (control) For calculation of percentage phytotoxicity in root and shoot, following equation was used (30): % Phytotoxicity = Root/Shoot length of control -Root/ Shoot length of treatment x 100 Root/Shoot length of control Histological sections of maize roots were prepared by free hand sectioning.Thin sections were stained with 0.05% toluidine blue solution,

Fig. 1A :
Fig. 1A: Effect of CuSO4 on root and shoot fresh weights.Fig. 1B: Effect of CuSO4 on root and shoot dry weights.Fig. 1C: Effect of combination of PEG 6000 and Cu on root and shoot fresh weights.Fig. 1D: Effect of combination of PEG 6000 and Cu on root and shoot dry weights.

Fig. 2 .
Fig. 2. A: Effect of CuSO4 on % Phytotoxicity and Tolerance index.B: Effect of combination of PEG 6000 and CuSO4 on % Phytotoxicity and Tolerance index.

Table 1 :
Effect of CuSO4 on germination percentage and growth parameters in maize seedlings ns = not significant, * , ** and *** = significant at p<0.05, p<0.01 and p<0.001 respectively.Values are mean ± SE and in parentheses relative values are given.

Table 2 :
Effect of combination of PEG 6000 and CuSO4 on germination percentage and growth parameters in maize seedlings.