Legume Research, 38 (2) 2015 : 185-193

AGRICULTURAL RESEARCH COMMUNICATION CENTRE

Print ISSN:0250-5371 / Online ISSN:0976-0571

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Improving vitamin content and nutritional value of legume yield through water and hormonal seed priming A. Janeczko1*, M. Dziurka1, A. Ostrowska1, J. Biesaga-Koscielniak1 and J. Koscielniak Department of Plant Physiology, University of Agriculture in Krakow, Podluzna 3, 30-239 Krakow, Poland. Received: 21-10-2014 Accepted: 30-12-2014

DOI: 10.5958/0976-0571.2015.00072.7

ABSTRACT Seed priming is one of the methods to improve plant vigour, overcome difficult habitat conditions, and consequently obtain a higher yield. Far less is known about effect of parental seed priming on quality of the seeds produced. The aim of our study was to evaluate the effect of water and hormonal (brassinosteroid) seed priming on the chemical content of the seed yield of pea and lupine, grown in a pot experiment and in the field. Pre-sowing water soaking of parental plant seeds resulted in significant increased protein, lipid, sugar, vitamin E, C and provitamin A content in seeds collected from these plants compared to the content in seeds collected from the control plants (without pre-sowing seed soaking). Priming with brassinosteroid enhanced these effects. The results showed that seed priming may be effectively used as a simple method for improving the quality of legume seeds. Key words: 24-Epibrassinolide, Lupine, Nutritional value, Pea, Seed priming, Vitamins. INTRODUCTION Legumes are recommended as an important component of the human diet as a rich source of protein, vitamins, and fats. In addition, cultivation of legumes increases N levels in soil, which makes these species an important part of the rotation system. For these reasons, the legume crop occupies a significant place in world agriculture. At the same time, many species of legumes are sensitive to environmental stresses which limits the range of their cultivation. In many European countries low temperatures at the beginning of the growing season, greatly limits the range of crops, such as soybean. In other regions of the world (Asia and Africa), cultivation of legumes is in turn associated with the occurrence of extreme weather conditions and also with poor or saline soils. Therefore, agriculture in these regions uses the simple method of pre-sowing seed soaking in water (water priming or hydropriming), which has beneficial effects on plant growth and development. This results in a faster and more uniform emergence, less need to re-sow, more vigorous plants, better stress tolerance, earlier flowering and harvest, and higher grain yield (Rashid et al. 2006 and literature cited there). Seeds may also be primed with inorganic salts or hormonal substances such as auxins, which may positively influence plant stress resistance (Farooq et al. 2006; Iqbal and Ashraf, 2007; Ella et al. 2011). There are also beneficial

effects from priming with brassinosteroids (BR) (Fariduddin et al. 2008; Hayat and Ahmad, 2003; Hasan et al. 2008). BR are plant steroid hormones involved in the regulation of growth and developmental processes of plants (Cheon et al. 2010). BR also increases the yield of crops (Fariduddin et al. 2008; Janeczko et al. 2010; Ramraj et al. 1997) as well as improves the quality of the yield (Vardhini and Rao, 1998, 2002). Janeczko et al. (2009) reported that pre-sowing seed soaking with brassinosteroid changed the composition of rape oilseed and soybean mainly by increasing the content of tocopherols (vitamin E) and -carotene (provitamin A). These and other vitamins, such as ascorbic acid (vitamin C), are important substances in the human diet affecting the value of plant products. Due to the significant role they play, there have been efforts to increase the content of these compounds in fruits and seeds of edible plants by scientists all over the world. Thus, these compounds are objects of interest of metabolomic engineering (Ajiawi and Shintani, 2004; Paine et al. 2005). The direction of improving the nutritional value of plants through genetic manipulation will probably grow, but this does not preclude the parallel promotion of other methods, especially those based on the use of simple and environmental friendly techniques. Moreover, the use of different methods not only to improve the content of essential nutrients but also to regulate metabolism in food products is an integral part in obtaining functional foods.

*Corresponding author’s e-mail: [email protected]. 1The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Krakow, Poland.

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The aim of this study was to evaluate the effect of water and hormonal (brassinosteroid) seed priming on the chemical content of seed yield of Pisum sativum L. and Lupinus luteus L. Within the scope of our interest was the content of basic nutritional components (soluble proteins, total lipids, and soluble sugars) as well as components with regulatory properties for human (vitamin E, vitamin C, and provitamin A). MATERIALS AND METHODS Plant material, growth and treatments: This work presents the results of experiments conducted in a pot culture in a foil tunnel house in 2010, and in natural field conditions in 2011. The weather conditions of the 2011 vegetation period are given in Table 1. The experiment was performed on two cultivars of pea Pisum sativum L. (Roch and Wiato) and two cultivars of yellow lupine Lupinus luteus L. (Mister and Talar). Seeds were obtained from the Poznañ Plant Breeding Ltd. (Poland). In the pot experiment, plants were grown in pots (diameter: 22 cm, height: 20 cm) filled with a mixture of soil:peat:sand in a 2:2:1 ratio (five plants per pot). Pots were kept in a foil tunnel house. The seeds were sown in early April and were watered once a week with Hoagland nutrient solution (Hoagland and Arnon1950) containing macro- and microelements. During growth, plants had natural light conditions typical for the spring/summer season in Poland (latitude: 50°03' N, longitude: 19°55' E). In the field experiment (2011 growing season), plants were grown on experimental plots at the Agricultural University in Krakow. Seeds were sown in early April (latitude: 50°03' N, longitude: 19°55' E) in brown soil of class II wheat complex. Plants in the field were cultured in rows placed in two belts (one belt was 2 m wide), separated by a 1 m path. Space between rows was 20 cm. Within the belts there were randomly placed blocks dedicated to particular species and treatments to minimalize possible effect of soil diversity on growing plants. After plant germination, seedlings were thinned to give all plants the same growth conditions and obtain the same distance between plants in row (20 cm). During vegetative growth, plants were manually weeded several times. In the seedling stage, fertilizer containing macro- and microelements was applied (Azofoska, Inco

Veritas SA, Poland; 40 g m-2). No other agrochemicals were used during vegetative growth. In both pot and field experiments, three treatments were applied to each species. The three treatments were: nontreated seeds, seeds soaked before sowing for 24 hours in water (water seed priming), seeds soaked before sowing for 24 hours in a solution of brassinosteroid: 24-epibrassinolide (24-epibrassinolide seed priming). Concentrations of 24epibrassin olide were chosen based on preliminary experiments (seedling growth response) as well as data from the literature (Janeczko et al. 2010 and Ramraj et al. 1997). For lupine seeds, a solution of 0.5 mg dm-3 was used; for pea seeds, the concentration was 0.25 mg dm -3. The 24epibrassinolide (BR27 - symbol proposed by Zullo and Kohout (2004)) used in the experiments was purchased from SigmaAldrich (Poznan, Poland). The stock solution contained 4.1 mM BR27 in 50% ethanol (dissolved in ultrasonic bath). Working solutions used for the experiments were prepared by diluting the stock solution with distilled water. Analysis of the chemical composition of seeds: Seeds collected at the same time, at the end of the season (beginning of September, latitude: 50°03' North, longitude: 19°55' East) were milled in Unidrive1000 laboratory homogenizer (CAT, Germany). Fine powder was used for further analyses. Chemicals needed were bought from Sigma-Aldrich (Poznañ, Poland). Soluble protein content was measured spectrophotometrically in 0.005 g samples, according to the Bradford (1976) method. Absorbance was read at 595 nm using microplate reader Synergy II (BioTek, USA). The calibration curve was calculated using bovine serum albumin. Total lipid content was measured in 0.2 g samples, according to Bligh and Dyer (1959), after extraction of material, chloroform phase was collected, evaporated and the mass of total lipids was estimated. Soluble (reducing and non-reducing) sugars were analysed spectrophotometrically according to the modified method of Dubois et al. (1951). Samples (0.005 g) were agitated on rotatory shaker (RL-2002, JWE, Poland) in 1 cm3 of deionized water (15 min). Next, samples were centrifuged at 2100 x g (15 min) and 0.04 cm3 of supernatant was

TABLE 1: Rainfall and temperature in the 2011 growing season. Month

Sum of rainfall per month (mm) Average temperature per month (°C)

III

IV

V

VI

VII

VIII

Total rainfall and average temperature during the growing season

15.2

77.7

48.0

33.0

186.4

73.1

433.4

3.7

10.3

13.6

18.2

17.6

19.1

13.8

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transferred to tubes containing 0.4 cm3 of deionized water. Next, 0.4 cm3 of 5% phenol and 2 cm3 of concentrated sulphuric acid were applied. Samples (0.02 cm 3) were transferred to 96-well plates after 20 min of incubation. Absorbance was read at 490 nm with the use of a microplate reader (Synergy II). The calibration curve was made using glucose solutions. Vitamin E(α-, γ-, δ- tocopherol) and provitamin A (-carotene) were measured in extracts from 0.5 g of seed samples. The extraction procedure was done as described in the work of Janeczko et al. (2009). High-performance liquid chromatography measurements were done as described in Biesaga-Koscielniak et al. (2014) on HPLC Agilent 1200 system (USA). Vitamin C (ascorbic acid) was determined spectrophotometrically in 0.01g samples by modified CUPRAC method (Ozyürek et al. 2007) using a microplate reader. The amount of ascorbic acid was calculated with the use of calibration curve made for ascorbic acid standard solutions.

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Statistical approach: Statistical significance was estimated based on Duncan’s test (P  0.05) using program Statistica 10 (StatSoft Inc., USA). Chemical analyses for each treatment (no priming, water priming, BR27 priming) were conducted in three replicates. One replicate consisted of ground seeds collected from 10 plants. Mean values, presented on figures and in tables, marked with the same letters (separately for pot and field experiment and separately for each cultivar) do not differ significantly according to Duncan’s test; P  0.05. RESULTS AND DISCUSSION Water seed priming and brassinosteroid priming modified the composition of newly formed pea and lupine seeds but the effect was partly dependent on the species and cultivar. Also observed was the influence of growth conditions (field and pot experiments) on quality composition of the collected seeds. The effect of seed priming on the content of proteins, soluble sugars, and total lipids in harvested seed: Soluble protein content in pea (cv. Roch and Wiato) and lupine seed yield (cv. Mister and Talar), obtained from plants of which

TABLE 2: Content of soluble proteins, sugars and total lipids in the seeds (mg g-1) of pea and lupine born on plants grown from seeds not primed or primed before sowing in water and BR27 solution. Mean values marked with the same letters do not differ significantly according to Duncan’s test; P  0.05. POT EXPERIMENT Pea cv. Roch

Lupine cv. Wiato

cv. Mister

cv. Talar

Pre-sowing seed treatment Untreated seeds Water priming BR27 priming

271.1c 302.2b 346.3a

Untreated seeds Water priming BR27 priming

60.1b 65.1b 99.9a

Untreated seeds Water priming BR27 priming

87.9b 99.9a 96.4a

Untreated seeds Water priming BR27 priming

271.1c 361.9b 372.2a

Untreated seeds Water priming BR27 priming

67.7b 68.6b 78.7a

Untreated seeds Water priming BR27 priming

121.9c 130.0b 137.1a

Description of figures

Soluble proteins 253.4b 231.6b a 312.7 257.3a 307.4a 244.4a Soluble sugars 90.7c 100.1b b 101.1 102.1b 122.8a 180.9a Total lipids 80.9b 243.8b 83.2b 257.1a 96.4a 260.2a FIELD EXPERIMENT Soluble proteins 190.8b 150.2b b 194.9 153.1b a 224.4 160.9a Soluble sugars 90.9b 101.2b a 101.5 111.6a 100.1a 112.7a Total lipids 89.9c 100.1c 110.1b 119.1b 120.9a 177.2a

200.1c 226.8b 263.1a 100.4c 136.1b 167.7a 100.9b 105.8b 122.5a

200.9c 252.3b 261.5a 60.6b 68.4a 69.9a 139.0b 146.1a 145.9a

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the seeds were not primed, ranged from 200.1 mg g-1 to 271.1 mg g-1 (pot experiment) and from 150.2 mg g-1 to 271.1 mg g1 (field experiment) (Table 2). Pre-sowing soaking in water, resulted in increased protein content in the seeds collected from these plants compared to the content in the seeds from the control plants (without pre-sowing seed soaking). In the pot experiment, this effect was independent of species and cultivar; the increase in protein content was at a level ranging from 11.1% to 23.4%. In the case of the field experiment for both pea and lupine, statistically significant effect was observed only in one of the two cultivars tested (cv. Roch 33.5%, cv. Talar 25.6%).

On the other hand, statistically significant increase in the protein content of seeds (calculated relative to ‘water priming’) obtained after the application of hormonal priming ranged between 2.8%–16%. This effect was statistically proven for all cultivars in the field experiment and for two cultivars (cv. Roch, cv. Talar) in the pot experiment.

it averaged 13.3%). Assuming amounts of sugars obtained with ‘water priming’ as 100%, the effect of the hormone remained high in the pot experiment (21.5%–77.2%), but was not detectable in the field experiment, except for cv. Roch. Total lipid content in pea seeds (cv. Roch and cv. Wiato) and lupine seeds (cv. Mister and cv. Talar) ranged from 80.9 mg g-1 to 243.8 mg g-1 (pot experiment) and from 89.9 mg g-1 to 139.0 mg g-1 (field experiment) (Table 2). Pre-sowing soaking in water resulted in an increased lipid content in the seeds collected from these plants compared to the content in the seeds from the control plants (without pre-sowing seed soaking). An increase in the lipid content after application of pre-sowing water soaking in newly formed seeds was observed in cv. Roch and cv. Mister (pot experiment) and in all cultivars in the field experiment. The highest increase in lipid content (22.5%) was observed in pea plants of cv. Wiato in the field experiment. The application of priming with 24epibrassinolide further increased the total lipid content in the seeds. Compared to the seeds collected from the controls without pre-sowing seed soaking, a statistically significant increase in the lipid content was observed regardless of the species, cultivar, and plant growth conditions. Again, assuming that 100% lipid content was obtained in waterprimed objects, an effect of the hormone was not present in cv. Roch (pot experiment) and cv. Talar (field experiment). For the other cultivars, the statistically significant increase in total lipids was about 16% in the pot experiment and from 5.5% to 48.8% in the field experiment.

Soluble sugar content in pea seeds (cv. Roch and Wiato) and lupine (cv. Mister and Talar) ranged from 60.1 mg g-1 to 100.4 mg g-1 (pot experiment) and from 60.6 mg g-1 to 101.2 mg g-1 (field experiment) (Table 2). Pre-sowing soaking in water resulted in increased sugar content in the seeds collected from these plants compared to the content in the seeds from the control plants (without pre-sowing seed soaking). An increase in the sugar content after application of pre-sowing water soaking in newly formed seeds was observed in cv. Wiato and cv. Talar in the pot experiment and in cv. Wiato, cv. Mister, and cv. Talar in the field experiment. The highest increase in sugar content was observed in cv. Talar by 35.6% and 12.9% in, respectively, the pot and field experiment. In the cv. Roch there was no difference in sugar content in seeds either in the pot or field experiment, however priming with 24-epibrassinolide did increase the content of soluble sugars. Compared to the seeds collected from the control (without pre-sowing seed soaking), a significant increase in sugar content was observed regardless of the species, cultivar, and plant growth conditions. There was a stronger effect observed in the pot experiment, where the increase in sugars ranged from 35.4% to 80.7% (in the field

The effect of seed priming of parental plants on the content of vitamins E, C and provitamin A in seed yield: Tocopherols (vitamin E) content in pea (cv. Roch and Wiato) and lupine seeds (cv. Mister and Talar) in the pot experiment ranged from 0.50 g g -1 to 0.82 g-1 (-tocopherol), from 40.4 g-1 to 59.9 g-1 (-tocopherol) and 0.64 g to 1.57 g (-tocopherol) (Figs. 1–2). The content of tocopherols in the pea and lupine seeds obtained in the field culture ranged from 0.40 g g -1 to 1.01 g g -1 -tocopherol), 13.9-41.0 g (-tocopherol), and 4.10-7.44 g (-tocopherol) (Figs. 1–2). From a practical point of view, the most important changes are in the content of γ -tocopherol, as the percentage of this compound is the highest in the entire pool of tocopherols. Pre-sowing soaking of seeds of parental plants in water resulted in the collection of seeds from these plants with elevated γ -tocopherol content (cv. Roch and Talar in pot and field experiments). The highest percentage of γ-tocopherol increase (20.8%) was found in pea seeds in the pot experiment and in lupine seeds (20.0%) in the field experiment. The application of priming with 24epibrassinolide further increased -tocopherol content in the

The application of hormone priming (24epibrassinolide) further increased the content of soluble protein. As compared to the seeds collected from the control without pre-sowing seed soaking, a statistically significant increase in the protein content was observed regardless of the species, cultivar, and plant growth conditions (field and pot experiments). The largest increase in protein content (37.3%) occurred in the seeds of cv. Roch in the field experiment.

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seeds. In the pot experiment, a significant increase of the content of -tocopherol (22.5%–44.8%) was detected for three cultivars tested. In the pea cv. Wiato, only one trend occurred that was statistically insignificant. In the field experiment, an increase in the content of this compound was present in all tested cultivars (37.2%–48.3%). Assuming the -tocopherol content obtained with ‘water priming’ as 100%, the effect of the hormone is still present in all varieties in the field experiment and in three varieties in the pot experiment. Both water priming and hormonal priming also increased the content of - and -tocopherols in the developed seeds. Of particular note is the twofold increase of -tocopherol in lupine cv. Mister and cv. Talar in the field experiment and -tocopherol in lupine cv. Mister in the pot experiment after BR administration.

FIG 1: Content of vitamin E (tocopherols) in the seeds of pea born on plants grown from seeds not primed or primed before sowing in water and BR27 solution. Mean values marked with the same letters (separately for pot and field experiment and separately for each cultivar) do not differ significantly according to Duncan’s test; P  0.05.

FIG 2: Content of vitamin E (tocopherols) in the seeds of lupine born on plants grown from seeds not primed or primed before sowing in water and BR27 solution. Mean values marked with the same letters (separately for pot and field experiment and separately for each cultivar) do not differ significantly according to Duncan’s test; P  0.05.

Vitamin C content in pea seeds (cv. Roch and Wiato) ranged from 69.6 g g-1 to 76.7 g g-1 (pot experiment) and from 80.1 g g-1 to 110.1 g g-1 (field experiment) (Fig. 3). Vitamin C content in lupine seeds (cv. Mister and Talar) ranged from 10.2 g g-1 to 12.3 g g-1 (pot experiment) and from 5.1 g g-1 to 8.1 g g-1 (field experiment) (Fig. 4). Presowing soaking in water resulted in an increased vitamin C content in the seeds collected from these plants compared to the content in the seeds from the control plants (without presowing seed soaking). In the pea cv. Wiato (field experiment) and both cultivars from the pot experiment, a statistically significant increase in the vitamin C content that ranged from 5.7% to 17.5% was observed. Hormonal (brassinosteroid) priming resulted in a significant increase of the vitamin C in the seeds of both cultivars of pea tested, but in case of cv. Wiato, the content of vitamin C obtained in ‘hormonal priming’ object (pot experiment) did not differ from that

FIG 3: Vitamin C and provitamin A levels in seeds of pea born on parental plants grown from seeds not primed or primed before sowing in water and BR27. Mean values marked with the same letters (separately for pot and field experiment and separately for each cultivar) do not differ significantly according to Duncan’s test; P  0.05.

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acquired in ‘water priming’. Neither water priming or hormonal priming of seeds had any effect on the vitamin C content in newly formed seeds of lupine. Provitamin A content in pea seeds (cv. Roch and Wiato) ranged from 0.24 g-1 to 0.25 g-1 (pot experiment) and from 0.06 g g-1 to 3.33 g g-1 (field experiment) (Fig. 3). Provitamin A content in lupine seeds (cv. Mister and Talar) was higher and ranged from 1.69 g g-1 to 1.75 g g-1 (pot experiment) and from 0.04 g g-1 to 2.79 g g-1 (field experiment) (Fig. 4). In the case of pea seeds, no effect on the content of provitamin A was detected in newly formed seeds when either water priming or hormonal priming seeds of parental plants. Pre-sowing seed soaking in water of parental lupine plants resulted in an increased provitamin A content in the seeds collected from these plants compared to the content in the seeds from the control plants (without presowing seed soaking). However, there were differences between cultivars. Water priming gave a positive result only in lupine cv. Talar in the pot experiment (8.6% increase in provitamin A content). Hormonal (brassinosteroid) priming resulted in a significant increase of the provitamin A in the seeds of cv. Mister (field and pot experiments). In the case of plants of cv. Talar, provitamin A content in seeds obtained in ‘hormonal priming’ object was not significantly different than was obtained in the ‘water priming’ object. Many studies have shown the beneficial effects of pre-sowing seed soaking in various plant species including legumes (Harris et al. 1999; Khalil et al. 2001; Khan et al. 2008). A beneficial effect was typically found for water priming or priming with solutions of salts, hormones, other signalling substances, etc., on uniform emergence, seedling vigour, and yield structure. Causes of seed priming effects

FIG 4: Vitamin C and provitamin A levels in seeds of lupine born on parental plants grown from seeds not primed or seeds primed before sowing in water and BR27. Mean values marked with the same letters (separately for pot and field experiment and separately for each cultivar) do not differ significantly according to Duncan’s test; P  0.05.

on subsequent health of the plant, stress resistance, or productivity are probably due to metabolic changes in early stages of development of the treated seeds. In 2001, Gallardo et al. (2001) conducted proteomic analyse of Arabidopsis seed germination and priming. They showed that among approximately 1,300 total seed proteins resolved in twodimensional gels, changes in the abundance (up- and downregulation) of 74 proteins were observed during germination. The authors also described two low-molecular weight heat shock proteins specifically upregulated during osmopriming. Gao et al. (1999) characterized plasma and tonoplast membrane aquaporins appearing in primed seeds of Brassica napus. Job et al. (1997) reported an increase in 11-S globulins associated with the storage protein mobilization in primed sugar beet (Beta vulgaris) seeds. Water priming and KClpriming improved rice seedling establishment in flooded soil, enhanced the capacity to scavenge reactive oxygen species in seeds by increasing activity of antioxidant enzymes, and enhanced carbohydrate mobilization (Ella et al. 2011). Changes have also been found in the hormonal metabolism that occurs as a result of water seed priming (Janeczko and Swaczynová, 2010). All these and similar metabolic changes surely significantly contribute to better plant performance from the beginning of germination. Better germination and seedling vigour have in turn subsequent effects on growth and production. The effect of parental seed priming on the quality of obtained yield has been studied to a much lesser extent in comparison to the effects of priming on uniform emergence, seedling vigour, and yield structure (Farooq et al. 2006). Meanwhile, in addition to the descriptions of the effects of priming on the improvement of yield and its structure, one can also expect additional improvements to the quality of new seeds. In this study, water seed priming resulted in increased vitamin, soluble protein, soluble sugar, and lipid content in new generation seeds. Additional application of 24-epibrassinolide further enhanced this effect. The mechanism of enrichment of seed yield in proteins, sugars, and lipids as a result of water priming is probably connected with the generally better performance of plants obtained from primed seeds (Harris et al. 1999). As for brassinosteroids, the effect of BR on protein production may be a result of enhanced activity of RNA and DNA polymerases, which are engaged in physiological responses to hormone (Kalinich et al. 1986). It is known that these hormones stimulate protein synthesis in plant leaves (Nakajima et al. 1996; Sirhindi 2009; Anuradha and Rao, 2001) and seeds (Vardhini and Rao, 1998). Similarly, increased sugar production and sugar accumulation in seeds seem to be direct effects of well proved

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positive BR influence on many aspects of the photosynthesis process (Braun and Wild, 1984; Yu et al. 2004) and assimilate transport to generative organs (Fuji and Saka, 2001). As for explaining the stimulating effect of BR on lipid metabolism, further studies are required. In the literature, exogenous application of brassinolide and 24-epibrassinolide by foliar spray also resulted in an elevated lipid concentration in seeds of Arachis hypogaea L. (Vardhini and Rao, 1998). We can only suspect that it may be connected with anti-stress activity of this group of hormones (Bajguz and Hayat, 2009), especially since the level of antioxidative/protective compounds such as tocopherols and -carotene dissolved in lipid fraction were elevated as well. Tocopherols, together with ascorbate and glutathione, are associated with the response of plants to stress (Smirnoff 1996 and Szarka et al. 2012). Together, they form the so-called triad which counteracts the increase of reactive oxygen species (Smirnoff 1996 and Szarka et al. 2012). Increased tocopherols as a result of BR treatment were reported earlier only by our group in pea and lupine seeds after root and foliar application (Biesaga-Koscielniak et al. 2014) and in oilseed rape seeds after parental seed priming (Janeczko et al. 2009). We also demonstrated earlier that BR may increase the concentration of ascorbic acid in seeds after root and foliar application of this hormone to selected legume plants (Biesaga-Koscielniak et al. 2014). BR increased also ascorbic acid level in the leaves of chromium-stressed Raphanus sativus L. (Choudhary et al. 2011) while lowering it in tomato fruits (Ali et al. 2006 and Vardhini and Rao, 2002). Carotenoids (including -carotene) are important components for photosynthesis, photoprotection, the production of phytohormones and coloration of flowers (Bartley and Scolnik, 1995 and Cazzonelli 2011). Brassinosteroids increased levels of -carotene in tomato fruits after root application as well as in pea and lupine seeds

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after root and foliar application (Ali et al. 2006 and BiesagaKoscielniak et al. 2014). Seed priming with BR increased provitamin A concentration in soybean and oilseed rape seeds (Janeczko et al. 2009). Increases in the amount of antioxidants, as well as increased amounts of storage material, not only bring advantages in terms of human nutrition, but may also be useful in the case of unfavourable environmental conditions during germination and the early growth of the next generation of plants. CONCLUSIONS Seed priming is one of the methods used in many countries to improve plant vigour, overcome difficult habitat conditions, and consequently obtain higher yields. It is therefore important to know that the effects of water or hormonal priming are also associated with favourable changes in the composition of new generations of seeds. In the current experiment we showed an increase in vitamins, proteins, carbohydrates, and lipids in the seed yield obtained from legumes grown from seeds primed with water. This makes priming a simple “ecological” method useful for improving the quality of legume seed yield. This favourable trend was further enhanced by the use of hormonal priming with brassinosteroid. Water and/or hormonal seed priming provide a simple method for producing functional food with increased amounts of important regulatory components. ACKNOWLEDGMENTS Research conducted within project N310452238 was financed by the Polish Government. We would like to thank the employees of the Department of Agricultural Chemistry, Poznan University of Life Sciences, for providing rainfall and temperature data for the 2011 growing season. We would also like to thank Dr Magdalena Mirek (Institute of Plant Physiology, PAS) for her technical assistance.

REFERENCES Ajiawi, I. and Shintani, D. (2004). Engineered plants with elevated vitamin E: a nutraceutical success story. Trends Biotechnol. 22:104–107. Ali, B., Hayat, S., Hasan, A.S. and Ahmad, A. (2006). Effect of root applied 28-homobrassinolide on the performance of Lycopersicon esculentum. Sci. Hortic. 110:267–273. Anuradha, S. and Rao, S.S.R. (2001). Effect of brassinosteroids on salinity stress induced inhibition of seed germination and seedling growth of rice (Oryza sativa L.). Plant Growth Regul. 33:151–153. Bajguz, A. and Hayat, S. (2009). Effects of brassinosteroids on the plant responses to environmental stresses. Plant Physiol. Biochem. 47:1–8. Bartley, G.E. and Scolnik, P.A. (1995). Plant carotenoids: pigments for photoprotection, visual attraction, and human health. Plant Cell 7:1027–1038. Biesaga-Koscielniak, J., Dziurka, M., Ostrowska, A., Mirek, M., Koscielniak, J. and Janeczko, A. (2014). Brassinosteroid improves content of antioxidants in seeds of selected leguminous plants. Aust. J. Crop Sci. 8:378–388.

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