CC BY
1008
Journal of Stress Physiology & Biochemistry, Vol. 6 No. 3 2010, pp. 102-113 ISSN 1997-0838 Original Text Copyright © 2010 by Singh, Chaturvedi, Bose
ORIGINAL ARTICLE
EFFECTS OF SALICYLIC ACID ON SEEDLING GROWTH AND NITROGEN METABOLISM IN CUCUMBER (CUCUMIS SATIVUS L.)
Singh, Pramod Kumar A*, Chaturvedi, Varan Kumar A, Bose, Bandana B
APlant Physiology Lab, Department of Botany, Udai Pratap Autonomous College, M.G. Kashi Vidyapeeth University, Varanasi-221002, (U.P.), INDIA
BDepartment of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005, (U.P.), INDIA
*Fax: +91-0542-2281799; Phone: +91-9415388189
*Email- [email protected]
Received May 28, 2010
Salicylic acid is involved in the regulation of metabolic activity and defense mechanism in plants under various stress conditions. Present study was conducted to determine the effects of salicylic acid (10 to 500 ^M) on seedling growth, development and nitrogen use efficiency in cucumber (Cucumis sativus L.) plants with or without nitrogen nutrient. Salicylic acid increased contents of chlorophyll, total non-structural carbohydrate and total nitrogen, as well as nitrate assimilation through the induction of nitrate reductase (EC 1.6.6.1) activity in isolated cucumber cotyledons. Accumulation of salicylic acid was two-fold higher in cotyledons without nitrate supply in comparison to that with nitrate supply. Further 50 ^M of SA induced enhancement in seed germination and growth characteristics. However higher salicylic acid concentrations inhibited above physiological characteristics. Results show that, field application of salicylic acid need optimum physiological concentration (e.g., 50 ^M) to increase nitrogen use efficiency particularly during germination and seedling growth.
key words: Cucumber (Cucummis sativus L.), Cucumber cotyledons, Nitrate-nutrition response, Nitrate reductase activity, Salicylic acid.
ORIGINAL ARTICLE
EFFECTS OF SALICYLIC ACID ON SEEDLING GROWTH AND NITROGEN METABOLISM IN CUCUMBER (CUCUMIS SATIVUS L.)
Singh, Pramod Kumar A*, Chaturvedi, Varun Kumar A, Bose, Bandana B
APlant Physiology Lab, Department of Botany, Udai Pratap Autonomous College, M.G. Kashi Vidyapeeth University, Varanasi-221002, (U.P.), INDIA
BDepartment of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005, (U.P.), INDIA
*Fax: +91-0542-2281799; Phone: +91-9415388189
*Email- [email protected]
Received May 28, 2010
Salicylic acid is involved in the regulation of metabolic activity and defense mechanism in plants under various stress conditions. Present study was conducted to determine the effects of salicylic acid (10 to 500 ^M) on seedling growth, development and nitrogen use efficiency in cucumber (Cucumis sativus L.) plants with or without nitrogen nutrient. Salicylic acid increased contents of chlorophyll, total non-structural carbohydrate and total nitrogen, as well as nitrate assimilation through the induction of nitrate reductase (EC 1.6.6.1) activity in isolated cucumber cotyledons. Accumulation of salicylic acid was two-fold higher in cotyledons without nitrate supply in comparison to that with nitrate supply. Further 50 ^M of SA induced enhancement in seed germination and growth characteristics. However higher salicylic acid concentrations inhibited above physiological characteristics. Results show that, field application of salicylic acid need optimum physiological concentration (e.g., 50 ^M) to increase nitrogen use efficiency particularly during germination and seedling growth.
key words: Cucumber (Cucummis sativus L.), Cucumber cotyledons, Nitrate-nutrition response, Nitrate reductase activity, Salicylic acid.
Phenylpropanoids are increased or it may be de-novo synthesized in response to adverse environmental conditions, which play an important role in regulation of biochemical, physiological and molecular responses in plants (Singh et al. 2007). These include effects on
nitrate (NO3-) assimilation, ion uptake, enzyme regulation, membrane organization,
photosynthetic carbon dioxide assimilation and nutrient deficiency in plants (Barkosky and Einhellig 1993; Uzunova and Popova 2000; Mateo et al. 2006; Lattanzio et al. 2009). Levels of some
compounds related to secondary metabolism show a sensitive response to nutrient deficiency in plants (Chisaki and Horiguchi 1997; Kovacik et al. 2007). Accumulation of phenolic compounds is a symptom of nutrient-stress, while production of different classes of phenolics depends on the nature of stress (Weisskopf et al. 2006). Higher levels of phenolics explain diagnosis of nutrient disorders and the visual symptoms caused by nutrient deficiency in shoot culture of organo (Lattanzio et al. 2009). However, effect of secondary metabolites on growth and
development of plants under limited availability of nitrogen (N) nutrient is not clear.
To attain optimal growth and development plants tend to maintain constant levels of essential nutrients, despite their limited
availability in most soils. These limitations are usually due to low nutrient concentration or accessibility (Schachtman and Shin 2007). To cope with reduced nutrient availability, plants trigger physiological and developmental
responses aimed to increase nutrient acquisition that, in many cases, alter the whole plant morphology and metabolism (Lopez-Bucio et al. 2003). Plants use adaptive mechanisms to
stimulate growth in the organs that directly participate in nutrient acquisition (Hermans et al. 2006; Svistoonoff et al 2007). Relative availability of soil ammonium and nitrate to most plants will become increasingly important in determining their productivity as well as their quality as food (Bloom et al. 2010). This is the case of plants grown under low N-supply, which triggers proliferation of lateral roots, resulting in increased amount of surface availability for N-uptake (Lopez-Bucio et al. 2003). These responses to maintain N-supply for plants may be helpful at maturity but, during germination and seedling growth morphological alteration cannot be sufficient.
Therefore, phenolic acids (PAs) based regulation of N-metabolism due to environmental constraints requires more study to understand germination and seedling growth under N-deficiency. Recently, salicylic acid (SA) received attention after it was determined that it can induce resistance to pathogens as well as abiotic stress tolerance in plants (Gautam and Singh 2009; Pieterse et al. 2009; Ramirez et al. 2009). Analyzing the role of secondary compounds, such as PAs may provide a method for the diagnosis of nutrient disorder in plants. Therefore, effects of exogenous SA on growth, development and nutrient metabolism in cucumber is determined to understand physiological responses to N-nutrition.
The objectives of this study were to investigate role of SA in regulation of NR (nitrate reductase) activity, chlorophyll synthesis, carbohydrate content, total N-content, NO3- assimilation; percent seed germination, seedling development and dry mass of cucumber (Cucumis sativus L.) plants.
MATERIALS AND METHODS
Plant materials and culture conditions (Experiment 1):
Seed of cucumber (Cucumis sativus L.) cv. HY-0512 were obtained from Indian Institute of Vegetable Research (IIVR), Varanasi. Seeds were sterilized with 0.01% HgCl2 for about 10 min, washed thoroughly with tap water followed by distilled water. Seeds were placed on moist Whatman No. 1 filter paper in acid washed Petri dishes (15x15 cm) for germination in an incubator at 250C ± 20C for 48 h. After this period, cotyledons of uniform size were isolated and allowed to green and expand under constant illumination for 72 h in culture room, temperature maintained at 25 ± 20C. Cotyledons were transferred to Petri dishes containing SA treatment
(10, 50, 100 and 500 ^M) with or without NO3-. Treated tissues were exposed to continuous illumination with light intensity of 100 ^W m-1s-2 in the culture room for 48 h, after which cotyledons were subjected to biochemical analyses. Controls were incubated either in distilled water (without NO3-) or with 20 mM KNO3.
Analysis of growth parameters (Experiment 2):
Dynamics of growth analysis of Cucumis sativus L. cv HY-0512 were started from 7 days old seedlings raised in sterilized Petri-dishes after a 6 h treatment of pre-soaked seeds with different (10, 50, 100, 500) ^M SA in presence as well as absence of NO3- (20 mM KNO3). Percent germination was recorded for 7 days and seeds were considered germinated when the radical became visible. Analyses were at 7 and 14 days to determine root and shoot lengths. Dry weight of 14 days old seedlings was determined after they were placed in oven at 600C until a constant weight was obtained. Other seedlings were transferred to pots containing black soil as a growth medium. Pots were provided only tap water.
Estimation of chlorophyll content:
To determine chlorophyll content 72 h old fully expanded cotyledons weighing 100 mg (Precision Balance, Model No. CB-125) were collected after 24 h after start of treatment with SA, placed in 80% acetone and homogenized to extract the chlorophyll. The resulting solution was extracted through preweighted filter paper using a Buchnner funnel. The volume of the remaining acetone-chlorophyll solution was measured; solutions were kept in dark tubes in ice to minimize chlorophyll degradation. Absorbance of solutions was measured at 645 nm and 663 nm using a digital spectrophotometer (Perkin-Elmer) for chlorophyll a and b, respectively, and chlorophyll contents were calculated using Arnon's equation (1949).
Estimation of NR activity:
In vivo NR activity was determined by the method of Hageman and Hucklesby (1971) with slight modification. For determination of NR activity 100 mg of shredded cotyledons were placed directly into 10 ml of incubation medium (300 mM KNO3 as substrate in 1% isopropanol). The reaction was performed in the dark for 30 min in a water bath maintained at 300C with constant shaking. NR activity was calculated as the amount of enzyme, which produced micromoles of nitrite g-1 fresh weight in 1 h. The amount of nitrite was determined spectrophotometrically at 540 nm.
Determination of SA content using HPLC:
Content of SA in cucumber cotyledons was determined by Daayf et al. (1997). 1.0 gm of cucumber cotyledons from each treatment were macerated in pestle and mortar with 80% aqueous ethanol (80:20, 10 ml) and homogenate was centrifuged at 1500 rpm for 15 minutes. Supernatant was treated with light petroleum ether and filtered through Whatman paper no. 1. Clear supernatants were evaporated under vacuum at room temperature. The residue was dissolved in 1 ml HPLC grade methanol, filtered through membrane filter (Millipore, 0.45^) and stored at 40 C for HPLC analysis. Further analysis were performed using (Shimadzu Corporation, Kyoto Japan) comprising LC-20 ATVP reciprocating pumps, a variable SPD-20A UV-VIS detector at 280 nm, C-18 reverse HPLC column 250x4.6 mm I'd. Particle size 5^C-18, (Phenomenex USA) at 360 C. Concentration of SA was calculated by comparing peak areas of reference compounds with that in the sample.
Amount of SA Peak area of sample x Amount of standard x 20
„ , . = Peak area of standard
(mg of sample)
Analytical methods:
One hundred mg of dried cotyledons were used for N-analysis. The N-content was determined by a
modified micro-Kjeldahl method after digestion with concentrated H2SO4 (Lang 1958). Total non-structural carbohydrates (TNC) in cotyledons were assayed for total soluble sugars and starch. Total sugar content was analyzed with the method of Scheible et al.
(1997). The starch content was measured as glucose content, following an enzymatic hydrolysis of starch residues (McCready et al. 1950).
Statistical analysis:
The experiment was arranged in a complete block design with five replications. Tests of significance between treatments were done using analysis of variance (ANOVA) and Duncan’s multiple range tests (Little and Hills 1978).
RESULTS
Growth analysis:
SA induced several affects depending on the concentration applied and high doses were required to observe inhibitory action in cucumber plants. Percent seed germination was highest at 50 ^M of SA with or without NO3- and effect of SA was more significant in absence of NO3- than in the presence of NO3- (Table 1). However, 500 ^M SA caused reduction in germination by 30.2% in respect to the control. Studies were performed for 14 days to determine the influence of SA on seedlings growth. Results indicated that 20 ^M of NO3- in conjugation with 50 ^M of SA increased root and shoot length, while higher doses of SA were inhibitory with or without NO3- (Table 1). Growth parameters determining effect of SA in cucumber have been influenced by the specific concentration of treatment rather than the supply of external NO3-. To overcome this complication, all concentrations were plotted against total plant dry mass. 50 ^M of SA exhibited highest dry matter (g per plant) in 14 days old seedlings, while higher doses of treatment reduced plat dry matter even in presence of external NO3-(Table 1).
Chlorophyll content:
Total chlorophyll content was recorded as the sum of chlorophyll a and b. The 50 ^M SA produced the highest chlorophyll content, which gradually declined thereafter at higher concentrations (100-500 ^M) in the absence and presence of NO3- (Table 2). 50 ^M SA increased near about 5 times higher chlorophyll content in cotyledonary tissues in comparison to aqueous control whereas total chlorophyll content reduced significantly at 500 ^M SA treatment both in presence and absence of NO3- (20 mM KNO3).
Non- structural carbohydrates:
The content of soluble sugar increased at 50 ^M SA compared to the aqueous control as well as NO3-control and decreased at higher concentrations (Table 2). The effect of SA was more significant in absence of external NO3-. The 50 ^M of SA without NO3-produced 3 folds increases in the content of sugars, the least being in plants treated with 500 ^M SA. In the presence of external NO3-, only 50 ^M of SA produced increases in sugar content compared to the control, while 500 ^M SA reduced sugar levels. Similar trends were observed for starch content, except at the 100 and 500 ^M concentrations SA where, the starch content was sharply reduced comparison to the control (Table 2). TNC status in the cotyledons did not respond at higher supply of NO3- nutrition, indicates counter action of exogenous NO3- to SA whereas, in the absence of exogenous NO3-, increase in TNC was due to increasing concentration of soluble sugars at 50 ^M SA treatment.
Nitrogen content:
PA induced changes in the level of N-content were analyzed on dry weight basis in 7 days old cucumber cotyledons. N-content increased significantly by treating with 50 ^M SA in comparison to aqueous control, while the N-level declined sharply at higher concentrations (100-500 ^M SA) (Table 2). Concentration based SA response
of SA was more significant in absence of exogenous NO3- in comparison to with NO3-. In absence of exogenous nitrate, highest level of N was observed at 50 ^M SA (64.15 mg g-1 of dry weight) whereas, in the presence of exogenous nitrate it was 52.41 mg g-1 of dry weight (Table 2). External NO3- interactive properties with SA may be due to inhibition of NO3-uptake at higher concentrations of SA.
NR activity:
To see the effect of SA on possible correlation between the NO3- assimilation and NR activity, SA treated cucumber cotyledons were demonstrated for NR activity in absence as well as presence of NO3-nutrition (Table 3). In absence of exogenous NO3-, SA increased 5 fold of NR activity (^M NO2- h-1 g-1 fresh weight) at 50 ^M and then significantly reduced at higher concentrations of the SA (Table 3). While, in presence of exogenous NO3-, increase in NR activity was observed maximum at 50 ^M of SA with gradual reduction at higher doses of SA. In an
attempt to check the effect of SA on the rate of enzyme action, NR activity was calculated in terms of percent control and was found that 50 ^M of SA (without NO3-) increased NR activity by 371% however, higher concentration (500 ^M) reduced it by 16% of aqueous control.
SA content in absence and presence of NO3":
SA content was determined in 7 days old
cucumber cotyledons by a reverse phase HPLC to investigate the effect of exogenous NO3- (20 mM KNO3) supply on SA accumulation. N-deficiency showed significant accumulation of SA in
cotyledons. SA content was 2 fold high in 7 days old
cucumber cotyledons without NO3- in comparison to with NO3- in control (Figure 1). SA accumulation was reduced (19%) under the supply of exogenous NO3-at 50 ^M SA. Data presented by figure 1 showed a correlation between accumulation of SA and
exogenous supply of NO3-.
Table 1. Differential effect of pre-soaking seed treatment of SA on percentage seed germination, root length, shoot length and plant dry-weight (DW) of cucumber seedlings in absence and presence of 20mM
SA (^M) Germination % Root length (cm) Shoot length (cm) Plant DW -1 (gplant )
48 Hrs. 7 Days 14 Days 7 Days 14 Days 14 Days
Control (-Nitrate) e 67.3 ± 0.089 2.96 ± 0.009g c 6.0 ± 0.172 e 10.1 ± 0.008 f 15.5 ± 0.102 c 6.6 ± 0.089
(+Nitrate) d e b b e d
70.4 ± 0.092 3.00 ± 0.179 6.23 ± 0.013 14.6 ± 0.178 17.7 ± 0.092 6.8 ± 0.179
10 (-Nitrate) b 73.0 ± 1.409 c 4.2 ± 0.178 a 7.5 ± 0.172 c 13.5 ± 0.017 c 19.1 ± 0.089 7.0 ± 0.283g
(+Nitrate) c d c d d h
72.3 ± 0.172 3.76 ± 0.017 5.9 ± 0.102 11.4 ± 0.282 18.7 ± 0.179 6.9 ± 0.424
50 (-Nitrate) a 80.4 ± 0.214 a 7.2 ± 0.268 a 7.4 ± 0.014 a 15.3 ± 0.424 a 22.8 ± 0.018 7.8 ± 0.008i
(+Nitrate) a b b b b 7.9 ± 0.172j
78.8 ± 0.141 5.1 ± 0.141 6.4 ± 0.214 14.7 ± 0.102 21.7 ± 0.214
100 (-Nitrate) f 50.9 ± 0.172 f 2.5 ± 0.282 d 4.8 ± 0.017 f 9.25 ± 0.014 13.1 ± 0.283g f 5.0 ± 0.102
(+Nitrate) 30.5 ± 0.141* h 1.86 ± 0.014 e 4.6 ± 0.021 7.6 ± 0.042g h 12.8 ± 0.424 e 4.8 ± 0.092
500 (-Nitrate) 47.1 ± 0.102g 2.2 ± 0.424g f 3.25 ± 0.282 h 6.5 ± 0.141 11.2 ± 0.008i a 4.9 ± 0.018
(+Nitrate) h 43.3 ± 0.141 h 1.9 ± 0.282 2.9 ± 0.424g 5.3 ± 0.2681 10.9 ± 0.172j b 4.7 ± 0.214
*CD 1.889 0.216 0.179 0.17 0.106 0.206
Footnote:
Each value represented as mean ±SE (n=5), mean values followed by same letter (s) are not significantly different (P < 0.05)
CD: critical difference
Table 2. Biochemical changes in cotyledonary tissue-content of chlorophylls, carbohydrates and total nitrogen
in cucumber cotyledons in response to SA in absence and presence of nitrate 20 mM KNO3.
SA (^M) Total Chlorophyll Total Sugars Total Starch Nitrogen
-1 -1 -1 -1
(mg g F.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.)
b g e g
Control (-Nitrate) 0.028 ± 0.002 4.46 ± 0.577 17.50 ± 1.102 15.2 ± 0.051
ab f d f
(+Nitrate) 0.065 ± 0.005 7.00 ± 0.601 21.75 ± 0.603 30.02 ± 0.201
ab b b d
10 (-Nitrate) 0.132 ± 0.001 8.20 ± 0.071 31.25 ± 0.571 25.4 ± 0.057
ab c c b
(+Nitrate) 0.106 ± 0.005 9.00 ± 0.151 22.50 ± 2.801 48.0 ± 0.057
ab a a a
50 (-Nitrate) 0.157 ± 0.001 13.5 ± 0.501 36.25 ± 5.701 64.15 ± 0.011
ab c d b
(+Nitrate) 0.142 ± 0.005 9.50 ± 0.702 21.50 ± 0.502 52.41 ± 0.281
a d f c
100 (-Nitrate) 0.091 ± 0.001 8.00 ± 0.801 12.00 ± 0.201 36.2 ± 0.036
ab e g e
(+Nitrate) 0.077 ± 0.005 7.20 ± 0.151 11.75 ± 1.101 34.25 ± 0.005
ab h h h
500 (-Nitrate) 0.072 ± 0.005 2.20 ± 0.154 9.70 ± 1.502 9.09 ± 0.021
ab i i i
(+Nitrate) 0.070 ± 0.001 1.75 ± 0.571 2.45 ± 0.503 10.94 ± 0.051
CD* 0.0121 0.934 0.712 0.106
Footnote:
Each value represented as mean ±SE (n=5), mean values followed by same letter (s) are not significantly different (P < 0.05)
CD: critical difference
Table 3. Effect of SA on nitrate reductase activity in cucumber cotyledons grew with distilled water in absence and presence of nitrate (20 mM KNO3).
Enzyme activity (NR) in cotyledonary tissues (^M. NO2- h-1 g-1 fresh weight)
SA (^M) Without NO3- % of control With NO3- % of control
Control 122.00 + 0.5d 100 512.00 ±1.1c 100
10 275.00 ± 2.8b 225 655.00 + 1.0d 127
50 575.00 ± 2.8a 471 710.00 + 5.7a 138
100 260.00 ±3.0c 231 442.00 + 1.5d 86
500 103.00 + 2.8e 84 280.00 + 2.0e 54
Footnote:
Each value represented as mean ±SE (n=5), mean values followed by same letter (s) are not significantly different (P < 0.05)
CD: critical difference
Exogenous SA (uM)
Figure 1. Determination of SA content, by measuring fresh weight of 7 days old cucumber cotyledons through reverse phase HPLC approach under increasing exogenous application of SA (10 mM - 500 mM) in absence and presence of 20 mM KNO3.
Footnote: mean ± SE (n=5)
DISCUSSION
Plants have evolved adaptive responses to grow in soils with low amount of one or several nutrients. These responses implicate complex metabolic changes generated by nutrient deficiency. SA induced several affects depending on the concentration applied. Higher doses of SA were required to observe inhibitory action in cucumber plants. Percentage of seed germination was found significantly higher at 50 ^M of salicylic acid and sharply reduced at higher doses both in absence as well as presence of exogenous nitrate. Higher levels of SA may inhibit nitrate uptake system and cause retardation in growth and development. Glass (1974) observed concentration based inhibitory potency of PAs on
ion-uptake in barley (Hordeum vulgare L. cv. Karlsberg). Rajjou et al. (2006) have also been reported similar observations on seed germination and seedling establishment of Arabidopsis thaliana. SA might be involved in mobilization of internal tissue NO3- and chlorophyll biosynthesis to increase the functional state of the photosynthetic machinery in plants (Shi et al. 2006), or it may induce accumulation of a-amino levulinic acid (a-ALA) in cotyledons. Ananiev et al. (2004) reported increases in chlorophyll biosynthesis in excised cotyledons of Cucurbita pepo L. (zucchini), cv. Cocozelle in response to growth regulator. This induction may be due to the interaction of PAs with light (McClure 1997; Hemm et al. 2004) producing higher rates of
carbohydrate synthesis through photosynthetic activity. This is possibly due to changes in membrane organization at higher SA level or to chelation of some important elements of cellular and organeller membrane (Uzunova and Popova 2000). It is not clear why N-content increased, when NO3- was not applied. However; internal nitrate may provide an inductive concentration to NR activity at lower concentrations of SA and/or SA induced modulation of nitrogen use efficiency (NUE) in cucumber cotyledons (Singh and Singh 2008). It may be that increase in NO3- assimilation was dependent on the physiological concentration (e.g. 50 ^M) of SA when NO3- was absent.
The imbalance between demand and N-supply in crops can result in either sub-optimal yield or the addition of environmentally damaging excesses of fertilizer. The uptake and assimilation of N by roots is known to change with supply in a manner that suggests that the N status of plants is somehow sensed and can feedback to regulate these processes with interaction of phytohormones (Rubio et al. 2009). Limited N-availability reduces the growth and plant productivity and induces secondary metabolism (Lattenzio et al. 2009; Chisaki and Horiguchi 1997). The results from our HPLC analysis support the hypothesis that SA favored growth and development by increasing NUE in cucumber. In absence of NO3-, accumulation of SA in cucumber play protective role for nutritional disorder. Previous results support exogenous application of 50 ^M SA was beneficial for growth and development in comparison to high doses (500 ^M) of SA (Wang and Li 2003).
The possible explanation for the concentration-based effect of SA on NR activity is that NR activity was induced and/or prevention of enzyme degradation was prevented. Results indicated that concentrations of SA at 10 to 50 ^M might induce NR synthesis by mobilization of intracellular NO3-, and provide protection to in vivo NR degradation in
absence of NO3- (Singh et al. 1997). Fariduddin et al. (2003) reported increased NR activity due reduced concentrations of SA while higher concentrations were observed to be inhibitory to NR activity in Brassica juncea Czern & Coss cv. Varuna.
Effect of SA on carbon and N-metabolism:
In higher plants, NO3- assimilation is dependent on the supply of carbon skeletons, indicating a close interaction between carbon and N-metabolism. Increase in the level of PAs in plants under stress of N-nutrition has been reported (Dixon and Paiva 1995). NO3- assimilation proceeds at a low rate in plants with low carbohydrate levels (Stitt et al. 2002). Certain sugars increase N-assimilation rate and amino acid synthesis (Morcuende et al. 1998). Studies with mustard (Brassica juncea Czern & Coss cv. Varuna) and wheat (Triticum aestivum L.) reported direct relationships between photosynthetic CO2 assimilation and NO3- assimilating enzymes in response to SA (Fariduddin et al. 2003; Singh and Usha 2003). In these studies, plants were treated by foliar application of SA; however, in this work we tested pre-soaking seed with SA in absence and presence of NO3-. The rate of NO3- assimilation in cotyledons increased in response to 50 ^M SA, with increases in amounts of soluble sugars and starch at same SA concentrations (Table 2), though accumulation of starch content is low compared to that of total N in cucumber (Table 2). The effect of exogenous SA on physiological characteristics of plants may depend on its concentration as well as nutritional conditions of the plants.
Present study indicates a positive correlation between chlorophyll content and total N in cucumber cotyledons. Moreover, it seems that effect of SA was more significant in absence of NO3- than in presence of nitrate. Increases in N-content, and chlorophyll content at lower concentration of SA, indicates that the acid plays a regulatory role during the biosynthesis of active photosynthetic pigments.
Although the direct effect of SA on chlorophyll biosynthesis in plants is not clearly understood, a-ALA mediated enhancement in chlorophyll biosynthesis by benzyladenine (synthetic SA) (Ananiev et al. 2004). Reduction in level of total N and chlorophyll content at 500 ^M SA may be due to the breakdown/degradation of chlorophyll or inhibition of foliar proteins required for production of photosynthetic pigments.
Conclusions:
SA response against nutrient stress is a new study in the field of crop physiology. Excessive use of chemical fertilizers in agriculture industries has appeared as a threat to soil health and yield. Results indicated that seed imbibition with SA affected physiological processes related to growth and development in cucumber plants. At lower concentrations, SA significantly increase rate of seed germination and plant dry mass even if added NO3-was 20 ^M. Plants treated with 10 and 50 ^M SA had higher chlorophyll levels and NO3- assimilation through the induction of NR activity. However 100 and 500 ^M were detrimental to plant health. SA, a natural endogenous growth regulator, if used exogenously, may improve plant growth and yield of cucumber.
Ackn owledgment:
Authors thank to Dr. A.K. Singh, Head, Department of Botany, Udai Pratap Autonomous College, M. G. Kashi Vidyapeeth University, Varanasi, for providing facilities during the course of study.
REFERENCES
Ananiev, E.D., Ananieva, K., Todorov, I. (2004) Effect of methyl ester of jasmonic acid, abscisic acid and benzyladenine on chlorophyll synthesis in excised cotyledons of Cucurbita
pepo (Zucchini). Bulg. J. Plant Physiol. 30(1-2), 51-63.
Arnon, D.I. (1949) Copper enzymes in isolated chloroplast; poly- phenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15.
Barkosky, R.R. and Einhellig, F.A. (1993) Effect of salicylic acid plant-water relationships. J. Chem. Ecol. 19, 237-247.
Bloom, A.J., Burger, M., Asensio, J.S.R. and Cousins, A.B. (2010) Carbon Dioxide Enrichment Inhibits Nitrate Assimilation in Wheat and Arabidopsis. Science 328(5980): 899-903.
Chisaki, N. and Horiguchi, T. (1997) Responses of secondary metabolism in plants to nutrient deficiency. In Plant nutrition for sustainable food production and environment. Ando T, Fujita, K., Mae, T., Matsumoto, H., Mori, S. Sekija, J. (Eds.) Kluwer academic publishers, pp. 341-345.
Daayf, F., Schmitt, A. and Belanger, R.R. (1997) Evidence of Phvtoalexins in Cucumber Leaves lnfected with Powdery Mildew following Treatment with Leaf Extracts of Reynoutria sa chalinensis. Plant Physiol. 113, 719-727.
Dixon, R.A. and Paiva, N.L. (1995) Stress-induced phenylpropanoid metabolism. The Plant Cell 7(7), 1085-1097.
Fariduddin, Q., Hayat, S. and Ahmad, A. (2003) Salicylic acid influences net photosynthetic rate, carboxylation efficiency, nitrate reductase activity, and seed yield in Brassica juncea. Photosynthetica 41(2), 281-284.
Gautam, S., Singh, P.K. (2009) Salicylic acid-induced salinity tolerance in corn grown under NaCl stress. Acta Physiol. Plant. 31, 1185— 1190.
Glass, A.D.M. (1974) Influence of phenolic acids upon ion uptake. J. Exp. Bot. 25(6), 11041113.
Hageman, R.H. and Hucklesby, D.P. (1971) Nitrate reductase. In: San Pietro A. (Ed.), Vol. XXII, Part A, pp 491-503, Methods in enzymology. Academic Press, London.
Hemm, M.R., Rider, S.D., Ogas, J., Murry, D.J. and Chapple, C. (2004) Light induces phenylpropanoid metabolism in Arabidopsis roots. The Plant J. 38(5), 765-778.
Hermans, C., Hammond, J.P., White, P.J. and Verbruggen, N. (2006) How do plants respond to nutrient shortage by biomass allocation? Trends Plant Sci. 11, 610-617.
Kovacik, J., Klejdus. B., Backor, M. and Repcak, M. (2007) Phenylalanine ammonia-lyase activity and phenolic compounds accumulation in nitrogen-deficient Matricaria chamomilla leaf rosettes. Plant Science 172(2), 393-399.
Lang, C.A. (1958) Simple micro determination of Kjeldahl nitrogen in biological materials. Annal. Chem. 30(10), 1692-1694.
Lattanzio, V., Cardinali, A., Ruta, C., Fortunato, I.M., Lattanzio, V.M.T., Linsalata, V. and Cicco, N. (2009) Relationship of secondary metabolism to growth in oregano (Origanum vulgare L.) shoot cultures under nutritional stress. Env. & Exp. Botany 65, 54-62.
Little, T.M. and Hills, F.J. (1978) Agricultural experimentation. John Wiley & Sons, Berlin.
Lo'pez-Bucio, J., Cruz-Rami'rez, A. and Herrera-Estrella, L. (2003) The role of nutrient availability in regulating root architecture. Curr. Opin. Plant Biol. 6, 280-287.
Mateo, A., Funck, D., Muhlenbock, P., Kular, B., Mullineaux, P.M. and Karpinski, S. (2006) Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis. J. Exp. Bot. 57(8), 1795-1807.
McClure, J.M. (1997) The physiology of phenolic compounds. Recent Adv. Phytochem. 12, 525556.
McCready, R.M., Guggolz, J., Silviera, V. and Owens, H.S. (1950) Determination of starch and amylose in vegetables. Anal. Chem. 22(9), 1156-1158.
Morcuende, R., Krapp, A., Hurry, V. and Stitt, M.
(1998) Sucrose feeding leads to increased rates of nitrate assimilation, increased rates of a -oxogluatarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves. Planta 206(3), 394-409.
Pieterse, C.M.J., Reyes, A.L., Ent, S.V.D. and Wees, S.C.M.V. (2009) Networking by small-molecule hormones in plant immunity. Nature Chem. Bio. 5(5), 308-316.
Rajjou, L., Belghazi, M., Huguet, R., Robin, C., Moreau, A., Job, C., Job, D. (2006) Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms. Plant Physiol. 141, 910-923.
Ramirez, A.A. et al. (2009) Evidence for a role of gibberellins in salicylic acid-modulated early plant responses to abiotic stress in Arabidopsis seeds. Plant Physiology 150, 1335-1344.
Rubio, V., Bustos, R., Irigoyen, M.L., Cardona-Lo 'pez, X., Rojas-Triana, M. and Paz-Ares, J. (2009) Plant hormones and nutrient signalling. Plant Mol. Biol. 69, 361-373.
Schachtman, D.P. and Shin, R. (2007) Nutrient sensing and signaling: NPKS. Annu. Rev. Plant Biol. 58, 47-69.
Scheible, W.R., Fontes, A.G., Lauerer, M., Rober, B.M., Caboche, M. and Stitt, M. (1997) Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 9(5), 783-798.
Shi, Q., Bao, Z., Zhu, Z., Ying, Q., Qian, Q. (2006) Effects of different treatments of salicylic acid on heat tolerance, chlorophyll fluorescence and antioxidant enzyme activity in seedlings of
Cucumis sativa L. Plant Growth Reg. 48(2), 127-135.
Singh, A., Singh and P.K. (2008) Salicylic acid induced biochemical changes in cucumber cotyledons. I. J. Agri. Biochem. 21(1-2), 35-38.
Singh, B. and Usha, K. (2003) Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress. Plant Growth Reg. 39(2), 137-141.
Singh, P.K., Bose, B., Kumar, M. and Singh, A. (2007) Physiological and molecular actions of salicylate in plant. In: (Bose B, Ranjan H Eds), vol. 2 Advances in Physiology, Biochemistry and Molecular Biology in Plants. Nipa Pub, New Delhi, pp. 1-19.
Singh, P.K., Koul, K.K., Tiwari, S.B. and Kaul, R.K. (1997) Effect of cinnamate on nitrate reductase activity in isolated cucumber cotyledons. Plant Growth Reg. 21(3), 203-206.
Stitt, M., Muller, C., Matt, P., Gibon, Y., Carillo, P., Morcuende, R., Scheible, W.R. and Krapp, A. (2002) Steps towards an integrated view of
nitrogen metabolism. J. Exp. Bot. 53(370), 959-970.
Svistoonoff, S., Creff, A., Reymond, M., Sigoillot-Claude, C., Ricaud, L. and Blanchet, A. et al. (2007) Root tip contact with low-phosphate media reprograms plant root architecture. Nat. Genet. 39, 792-796.
Uzunova, A.N. and Popova, L.P. (2000) Effect of salicylic acid on leaf anatomy and chloroplast ultra structure of barley plants. Photosynthetica 38(2), 243-250.
Wang, M. and Li, Z. (2003) Nonideal gas flow and heat transfer in micro- and nanochannels using the direct simulation Monte Carlo method. Phys. Rev. E68(4), 046704 - 046710.
Weisskopf, L., Tomasi, N., Santelia, D., Martinoia, E., Langlade, N.B., Tabacchi, R. and Abou-Mansour, E. (2006) Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage. New Phytologist 171(3), 657-668.
