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Chilean journal of agricultural research

versão On-line ISSN 0718-5839

Chilean J. Agric. Res. vol.77 no.1 Chillán mar. 2017

http://dx.doi.org/10.4067/S0718-58392017000100010 

Research

Re-watering: An effective measure to recover growth and photosynthetic characteristics in salt-stressed Brassica napus L.

Qaiser Javed1  , Yanyou Wu2  *  , Deke Xing1  , Ahmad Azeem1  , Ikram Ullah1  , Muhammad Zaman3 

1Jiangsu University, Ministry of Education, Institute of Agricultural Engineering, 212013, Zhenjiang, Jiangsu, China.

2Chinese Academy of Sciences, Institute of Geochemistry, 550081, Guiyang, Guizhou, China.

3University of Agriculture, Department of Irrigation & Drainage, 38000, Faisalabad, Punjab, Pakistan

ABSTRACT

Salinity is one of major environmental problem which is limiting the agricultural production. This research was conducted to evaluate the effect of re-watering on Brassica napus L., and determination of an appropriate regime for dilution of salted water by studying photosynthetic and growth response of B. napus to salt stress and subsequent re-watering. Plants were treated with NaCl (Nc1: 2.5, Nc2: 5, Nc3: 10; g L-1); Na2SO4 (Ns1: 2.5, Ns2: 5, Ns3: 10; g L-1) and mixed salts treatments (M1: Nc1+ Ns3; M2: Nc3+ Ns1; M3: Nc2+ Ns2; g L-1) and 0 as control, followed by re-watering. In salt stress phase, maximum reduction in net photosynthetic rate (PN) was noted 79.54%, 80.72%, 84.54%, and 74.84% for Nc3, Ns3, M1 and M2, respectively, under high concentration levels. To maintain PN, carbonic anhydrase (CA) activity was stimulated and kept water status stable under low (Nc1 and Ns1) to medium concentration levels (Nc2, Ns2 and M3), and the decreases in PN under Nc2, Ns2 and M3 were 48.28%, 55.58% and 58.69%, respectively. However, during re-watering phase, growth and physiological parameters were recovered well due to regulation of CA activity under low to medium concentration levels. Relatively as compare to other stress levels more recovery in PN was found after re-watering under medium concentration levels, which were 44.94%, 53.45% and 63.04%, respectively. Though aimed at consideration of high production in B. napus, the best re-watering time was found to be when plants undergo medium concentration levels. Therefore, this study provides a new method for dilution of saline irrigation based on plant physiology.

Key words: Carbonic anhydrase activity; growth; re-watering; salt stress; photosynthetic traits.

INTRODUCTION

The stress due to salinity is a foremost environmental factor that severely affects the productivity of crop all over the world. Salinity is a major problem that affects 6% of the world's land and 20% of irrigated land (Chinnusamy et al., 2005). According to reports represented by United Nations, 50% of world crops lands are salt affected (Yokoi et al., 2002). Moreover, 40% of China which is equivalent to 8.1 Mha of the total cultivated land affected by salinity (Su et al., 2013) and water shortage (Wang et al., 2011). However, soil salinity affects about one third of the total irrigated crop land in North-West region of China (Chen et al., 2010). Overcoming stress due to water deficiency and salts accumulation is a foremost issue in these areas to ensure agricultural sustainability and continues production of food. Brassica napus L. is considered comparatively moderate salt tolerant crop grown under variant environmental conditions (Humaira and Rafiq, 2004) like temperature, salinity, and drought (Maggio et al., 2005; Hayat et al., 2007). Among all rapeseed crops, B. napus is one of the world's best and leading edible oil crops due to its improved oil content, high nutritious content, enrichment and stability in yield (Zum Felde et al., 2007). Furthermore, it contains less than 2% erucic acid and 5%-8% saturated fats which is lower than any other oil-seed crops (Raymer, 2002).

Salt accumulation in irrigated land from equally groundwater sources and irrigation increase the salinity to levels which creates physiological disturbances in plants, as a result, plant growth, plant quality, and plant yield are affected (Toorchi et al., 2011). The stress caused by salinity reduces overall photosynthetic capacity (Ashraf and Harris, 2013). From recent studies, it has been found that salt stress depresses the regulation of photosynthesis through limited stomatal opening (Chaves et al., 2009). On the other hand, the relative performance of stomatal conductivity and photosynthetic activity increases water-use efficiency (Vos and Groenwold, 1989) and leaf water potential. Leaf water potential, which is termed as energy status of water in leaves, tends to decrease as a result of decrease in relative water content (Arif et al., 2013). Consequently, some plants convert intracellular HCO3 - into CO2 and H2O by carbonic anhydrase (CA) activity to maintain leaf water status, when they suffer water deficiency. As a result, C and water source are provided for the photosynthesis process itself by plant (Hu et al., 2011; Xing and Wu, 2012). Hence, CA activity works for survival of plant under stressed condition (Wu et al., 2006).

The salinity and scarcity of fresh water limits sustainable agricultural production and development (Wan et al., 2007). Meanwhile, the quality of irrigation water is also becoming low. As a result, saline water irrigation and lower quality-water, such as saline groundwater have been used more readily in agriculture to overwhelmed drought and sustain crop production (Verma et al., 2012; Li et al., 2015). Reuse of diluted saline water for irrigation of plants becomes the readily available water when water resources are scare. It would be a reasonable approach to use saline water as substitute resource for fresh water to irrigate the moderately salt tolerant crops such as B. napus. To move forward in this research field, re-watering or dilution of saline water is a new index which could be helpful for regulation of saline water in order to sustain agricultural productivity and economical irrigation. An appropriate dilution of salt water will save the water resource. Thus, the aim of this study was to find out threshold value in B. napus through physiological traits and growth status in different salt stresses and subsequent re-watering conditions. Afterwards, an appropriate regime for dilution of salted water was observed by studying the growth and photosynthetic response of B. napus.

MATERIALS AND METHODS

Plant material

The experiment was carried out at the Institute of Agricultural Engineering, Jiangsu University, Zhenjiang (32.20° N, 119.45° E), Jiangsu, China. Intact seeds of B. napus, identical in size and color, homogeneous and free from wrinkles, were chosen for this experiment. Seeds were cultivated in 20-cell tray, containing equal quantities of vermiculite washed with distilled water. The seeds were left to grow inside the growth chamber under day/night temperature cycle of 25/20 °C, and 60% RH. Plants were daily irrigated with Hoagland solution (Hoagland and Arnon, 1950). After 21 d, plants were transferred into greenhouse under natural lighting with (25/18) ± 2 °C (day/night) temperature and 70% RH. Homogenous healthy seedlings were exposed to salt stress induced by NaCl, Na2SO4 and combination of both salts at four levels, in which one is control level. The treatments of salts were NaCl (Nc1: 2.5, Nc2: 5, Nc3: 10, and 0 as control) g L-1; Na2SO4 (Ns1: 2.5, Ns2: 5, Ns3: 10, and 0 as control) g L-1 and in mixed salts (M1: Nc1 + Ns3; M2: Nc3 + Ns1; M3: Nc2 + Ns2 and 0 as control) selected for treatment with Hoagland solution. The controlled treatment received full strength Hoagland solution.

Re-watering was done on day 21 from the onset of salt stress treatment for 15 d. The order for re-watering was that plants were suffering in high stress level (10 g L-1 in both NaCl and Na2SO4) irrigated with medium stress level Nc3^2, Ns3^2 (5 g L-1 in both NaCl and Na2SO4, respectively), medium stress level (5 g L-1 in both NaCl and Na2SO4) irrigated with low stress level Nc2^1, Ns2^1 (2.5 g L-1 in both NaCl and Na2SO4, respectively) and low stress level (2.5 g L-1 in both NaCl and Na2SO4) irrigated with control level Nc1^0, Ns1^0 (0 g L-1 in both NaCl and Na2SO4, respectively). In mixed treatments, all levels re-watered with control (M1^0, M2^0, M3^0). This experiment was designed in a randomized block and five replicates were chosen for each physiological measurement.

Determination of growth parameters

Growth parameters were measured after treatment application and re-watering 3-times per week in both cases, respectively. The five replicates were chosen for each treatment, and also used to analyze the mean of each measurement. The measurements taken for growth analysis were: Plant length (PH); stem diameter (SD) and leaf area (LA). The LA was measured by a leaf area meter (Handheld Laser Leaf Area Meter, CI-203, CID Bio-Science, Camas, Washington, USA).

Determination of physiological characteristics

Leaves in salt stress phase and subsequently in re-watering phase were used for the determination of photosynthesis characteristics. Net photosynthetic rate (PN), stomatal conductance (gs) and water potential () were measured at 09:00-11:00 h after every 3 d in both salt stress and re-watering phase, respectively. Five plants from each treatment group were selected for the measurement. The photosynthetic active radiation (PAR), temperature and CO2 concentration during the measurements were 800 (mol m-2 s-1, 28 °C and 500 umol mol-1, respectively. A portable photosynthesis measurement system (LI-6400XT, LI-COR, Lincoln, Nebraska, USA) was used. Water use efficiency (WUE) was calculated according to the following equation: WUE = PN/Tr , where PN is the net photosynthetic rate and Tr is the transpiration rate. Leaf water potential () was measured with dew point microvolt meter in a C-52-SF universal sample room (Psypro; Wescor, Logan, Utah, USA).

Determination of carbonic anhydrase activity

The carbonic anhydrase (CA) activity was determined by using the pH method described by Wilbur and Anderson (1948) with modifications (Wu et al., 2011). The CA activity was expressed in Wilbur and Anderson (WA) units as WA [WAU g-1 (FW)] = (t0/t) - 1, where t0 and t were the time(s) measured for the pH change (8.2 to 7.2), with buffer alone (t0) and with sample (t). Leaf tissues (weight select according to leaf size usually used 0.1 to 0.2 g) quickly freeze in liquid nitrogen and ground with 3 mL extraction buffer (0.01 M barbitone sodium with 0.05 M mercaptoethanol, pH 8.3). The homogenate centrifuged at 10 000 × g, 0 °C for 5 min and then placed on ice for 20 min. In brief, CA activity was examined at 0 °C to 2 °C in a mixture containing 4.5 mL 0.02 M barbitone buffer (5,5-diethylbarbituric acid pH 8.3), 0.4 mL of the sample and 3 mL CO2 saturated H2O.

Calculation of re-watering water use efficiency

Re-watering WUE was calculated by the increment of and PN in leaves of B. napus from salt stress to subsequent re-watering phase. In the experiment, four treatment levels, control, 2.5%, 5% and 10% were marked as level 0, 1, 2 and 3, respectively. In stress phase, PN and under level 0, 1, 2 and 3 were expressed as PN l ((mol m-2 s-1) and l (MPa) respectively (l was the osmotic stress level, l = 0, 1, 2, 3); while in re-watering phase, PN and of leaves in salt stress levels 1, 2 and 3 after re-watering were expressed as PN l ( l - 1) and l(l - 1) respectively l(l - 1) indicated that leaves were re-watered from salt stress level l to salt stress level l - 1. In other words, leaves re-watered to adjacent lower salt stress level l > 1, and l was positive integer).

Relationship between plant leaf water potential and cell sap solute concentration (Q) is:

(1)

where l is plant leaf water potential (MPa); i is dissociation coefficient (i = 1); Q is cell sap solute concentration; R is gas constant (R = 0.0083 L MPa mol-1 K-1); T is thermodynamic temperature (273 + t °C) K.

The relationship between proportion of solute quality in the total quality of leaf (P, %) and cell sap solute concentration (Q) was expressed as:

(2)

where M is the relative molecular mass of cell sap solute, sugar C12H22O11, M is 342 g mol-1.

According to Equations [1] and [2], P could be rewritten as:

(3)

Proportion of water content in the total quality of leaf is 1 - P; WC (%) expressed as:

(4)

The leaves in salt stress levels 2, 3 and 4 were re-watered to adjacent lower salt stress levels respectively. The increment of PN(PN) and () were calculated as:

(5)

(6)

where l is the salt stress level, l > 1, and l is positive integer.

According to Equations [4] and [6], the increment of WC ((WC) could be calculated as:

(7)

where l is the salt stress level, l > 1, and l is positive integer; M is the relative molecular mass of cell sap solute, sugar C12H22O11, M is 342; ( is plant leaf water potential (MPa); i is dissociation coefficient (i = 1); R is gas constant (R = 0.0083 L MPa mol-1 K-1); 7 is thermodynamic temperature (273 + t °C) K.

So, the increment of WC ((WC) could be calculated as:

(8)

where (WC* l(l - 1) is increment of leaf water content per leaf area and per second (mmol m-2 s-1), m (g) is leaf fresh weight and A (cm2) is the area of chamber (A = 6 × 104).

According to Equations [5] and [8], re-watering WUE (WUER, mmol CO2 mol-1 H2O) is calculated as:

(9)

Statistical analysis

All measurements were subjected to ANOVA to discriminate significant differences (defined as P (( 0.05) between group means. Data are shown as the mean ± standard error (SE) (n = 5). These mean data were statistically analyzed under factorial design by using SPSS software version 13.0 (IBM Corporation, Armonk, New York, USA) and mean results were compared through LSD at 5% significance level (P < 0.05).

RESULTS

Photosynthetic traits in salt stress vs salt stress subsequently re-watering

The net photosynthetic rate (PN) and stomatal conductance (gs) expressed in two phases, salt stress phase and re-watering phase. The PN and gs significantly decreased with increasing salt concentration under different salt stress levels (Tables 1 and 2). Maximum reduction in PN (79.54%, 80.72%, 84.54%, and 74.84%) and in gs (81.74%, 87.79%, 90.20%, and 83.90%) was noted under high concentration (Nc3, Ns3, M1 and M2) of NaCl, Na2SO4 and mixed salts, respectively as compared to control. By comparing with control, PN (100%), there was slight reduction in PN (17.19% and 19.91%) and in gs (17.76% and 22.46%) recorded under low (Nc1 and Ns1) concentration of NaCl and Na2SO4 while the highest reduction in PN (84.54%) and in gs (90.20%) was observed under M1 concentration of mixed salts. In medium (Nc2, Ns2 and M3) concentration of NaCl, Na2SO4 and mixed salts, the reduction in PN was found 48.28%, 55.58% and 58.69%, while in gs was noted 52.51%, 56.40% and 59.85%, respectively.

Tables 1 and 2 also show the response of re-watering in PN and gs. It was observed that B. napus exhibited better results from stress phase to re-watering phase. The PN and gs increased significantly under low (Nc1^0 and Ns1^0) concentrations. Relatively, the maximum recovery were found under medium concentration (Nc2^1, Ns2^1 and M3^0) which were 44.94%, 53.45% and 63.04% in PN and 50.28%, 42.92% and 49.06% in gs, respectively. However, salt stress at high concentration, affected Pn (18.93%, 18.54%, 14.37% and 18.57%) and gs (20.11%, 16.76%, 13.42% and 13.87%) adversely followed the order as Nc3^2, Ns3^2, M1^0 and M2^0, respectively. However, additions of mixed salts at M3^0 revealed the same effect both on PN and gs as compared to PN and gs under Nc2^1, Ns2^1 levels, respectively (Tables 1 and 2). It was also cleared from results that responses of PN and gs towards NaCl concentration was better than Na2SO4 and mixed salts concentrations during re-watering phase.

Table 1 Effect of salt stress and re-watering on net photosynthetic rate (Pn). 

The means ± SE (n = 5) in the table indicated the significant difference in net photosynthetic rate during salt stress phase and afterwards the recovery under re-watering phase at P 0.05, according to one way ANOVA and LSD.

Nc1: 2.5 g L-1 NaCl; Nc2: 5 g L-1 NaCl; Nc3:10 g L-1 NaCl; Ns1: 2.5 g L-1 Na2SO4; Ns2: 5 g L-1 Na2SO4; Ns3:10 g L-1 Na2SO4; M1: Nc1 + Ns3; M2: Nc3 + Ns1; M3: Nc2 + Ns2; Nc1^0: (2.5 0) g L-1 NaCl; Nc2^1: (5 2.5) g L-1 NaCl; Nc3^2: (10 5) g L-1 NaCl; Ns1^0: (2.5 0) g L-1 Na2SO4; Ns2^1: (5 2.5) g L-1 Na2SO4; Ns3^2: (10 5) g L-1 Na2SO4; M1^0: (12.5 0) g L-1 Hoagland solution; M2^0: (12.5 0) g L-1 Hoagland solution; M3^0: (10 0) g L-1 Hoagland solution.

Table 2 Effect salt stress and re-watering on stomatal conductance (gs). 

The means ± SE (n = 5) in the table indicated the significant difference in stomatal conductance during salt stress phase and afterwards the recovery under re-watering phase at P 0.05, according to one way ANOVA and LSD.

Nc1:2.5 g L-1 NaCl; Nc2: 5 g L-1 NaCl; Nc3:10 g L-1 NaCl; Nst: 2.5 g L-1 Na2SO4; Ns2: 5 g L-1 Na2SO4; Ns3:10 g L-1 Na2SO4; M1: Nc1 + Ns3; M2: Nc3 + Ns1; M3: Nc2 + Ns2; Nc1^0: (2.5 0) g L-1 NaCl; Nc2^1: (5 2.5) g L-1 NaCl; Nc3^2: (10 5) g L-1 NaCl; Ns1^0: (2.5 0) g L-1 Na2SO4; Ns2^1: (5 2.5) g L-1 Na2SO4; Ns3^2: (10 5) g L-1 Na2SO4; M1^0: (12.5 0) g L-1 Hoagland solution; M2^0: (12.5 0) g L-1 Hoagland solution; M3^0: (10 0) g L-1 Hoagland solution.

CA activity and water potential in salt stress and subsequently in re-watering

The CA activity of B. napus under salt stress condition showed its regulation which varied with stress level (Figure 1). It activated significantly in low (Nc1, Ns1) to medium concentration levels (Nc2, Ns2 and M3) as compared to control. It had maximum values under Nc3 and Ns3 concentration levels. But at M1 and M2 concentration levels, CA activity was not activated due to under high stress condition especially under M1 concentration level. CA activity in NaCl treatment was significantly activated than Na2SO4 treatments. Also, CA activity was significantly activated in both single NaCl and Na2SO4 concentrations than mixed salts treatments.

In re-watering phase, CA activity showed better performance. The CA activity was successfully activated under Nc2^1, Ns2^1 and M3^0, concentrations respectively. However, salt stress subsequent re-watering resulted in an adverse effect under M1^0 and M2^0 concentrations (Figure 1). The CA activity of B. napus was the lowest at M1^0 and M2^0 concentration levels and nearly undetectable even after re-watering.

According to our results, significantly decreased going towards more negative with increasing salt stress (Table 3). The minimum decrease in was noted in low concentration levels (Nc1, Ns1) as compared with the control. However, the maximum decrease in was noted at high concentration levels (Nc3, Ns3, M1 and M2), respectively.

The means ± SE (n = 5) in the figure indicate significant differences between different stress and re-watering levels at P 0.05, according to one-way ANOVA and LSD. WAU is Wilbur and Anderson Unit which expresses the CA activity in WA units as WA [WAU g-1 (FW)] = (t0/t) - 1, where t0 and t were the time(s) measured for the pH change (8.2 to 7.2), with buffer alone (t0) and with sample (t) and FW was the fresh weight of leaves.

Figure 1 Effect of salt stress and subsequent re-watering on the regulation of carbonic anhydrase (CA) activity. 

Table 3 Effect salt stress vs. salt stress subsequently re-watering on water potential (). 

The means ± SE (n = 5) in the table indicated the significant difference in water potential during salt stress phase and afterwards the increment under re-watering phase at P 0.05, according to one way ANOVA and LSD.

Nc1: 2.5 g L-1 NaCl; Nc2: 5 g L-1 NaCl; Nc3:10 g L-1 NaCl; Ns1: 2.5 g L-1 Na2SO4; Ns2: 5 g L-1 Na2SO4; Ns3:10 g L-1 Na2SO4; M1: Nc1 + Ns3; M2: Nc3 + Ns1; M3: Nc2 + Ns2; Nc1^0: (2.5 0) g L-1 NaCl; Nc2^1: (5 2.5) g L-NaCl; Nc3^2: (10 5) g L-1 NaCl; Ns1^0: (2.5 0) g L-1 Na2SO4; Ns2^1: (5 2.5) g L-1 Na2SO4; Ns3^2: (10 5) g L-1 Na2SO4; M1^0: (12.5 0) g L-1 Hoagland solution; M2^0: (12.5 0) g L-1 Hoagland solution; M3^0: (10 0) g L-1 Hoagland solution.

While in medium (Nc2, Ns2 and M3) concentration of NaCl, Na2SO4 and mixed salts, the decrease in W was found to almost same. As a comparison between single salts and mixture of salts, it was observed that W was significantly less affected in NaCl concentrations than Na2SO4 and mixed salts concentrations.

In re-watering phase, the outcome of the results showed that of B. napus was recovered. The increment in also increased under low (Nc1^0 and Ns1^0) to medium concentration levels (Nc2^1, Ns2^1 and M3^0) and it decreased at high concentration levels (Nc3^2, Ns3^2, M1^0 and M2^0), respectively. However, the degree of salts were still showed the adverse effect on increment of W even during re-watering under high levels (Nc3^2, Ns3^2, M1^0 and M2^0) (Table 3).

Effect of salt stress on plant growth

The application of stress significantly affected PH, SD and LA of B. napus. By following the results of PH, SD and LA under salt stress, it appeared that increase in salts concentration decreased the values of PH, SD and LA (Table 4). Comparing to control, the PH decreased by 70.58%, 78.76%, 96.73% and 87.91% for Nc3, Ns3, M1 and M2, respectively, under high concentration of NaCl, Na2SO4 and mixed salts. It was found that PH (14.15 and 12.40 cm) under low (Nc1 and Ns1) concentration levels of NaCl and Na2SO4 is slightly affected as compared to PH (15.30 cm) under control. Upon comparing with other salts levels, B. napus exhibited maximum decrease in PH (96.73%) under mixed salts concentration (M1). While in medium (Nc2, Ns2 and M3) concentration of NaCl, Na2SO4 and mixed salts, the decrease in PH were recorded 43.79%, 49.67% and 49%, respectively. Reduction in SD was observed continuously from control to high concentrations levels (Nc3, Ns3, M1 and M2) of NaCl, Na2SO4 and mixed salts (Table 4). The control treatment showed the maximum SD (0.195 cm) followed by low concentrations (0.145 and 0.135 cm) with decrease of 25.37% and 30.77%, medium concentrations (0.095, 0.085 and 0.085 cm) with percent decrease of 51.28%, 56.41% and 56.41% and high concentrations 0.035, 0.015, 0.005, and 0.010 cm with decrease of 82.06%, 92.31%, 97.44% and 94.76%, respectively. Similarly, control had the highest LA (19.07 cm2) followed by low concentrations with decrease of 21.82% and 33.28% and medium concentrations with decrease of 51.34%, 54.06% and 54.12%. But, salt stress at high concentrations (Nc3, Ns3, M1 and M2) exerted a severe influence on LA and reductions found were 81.91%, 89.52%, 97.91% and 94.76%, respectively.

Table 4 Effect of salts stress on plant height (PH), stem diameter (SD) and leaf area (LA) in Brassica napus

The means ± SE (n = 5) in the table indicated the significant difference in plant height, stem diameter and leaf area during salt stress phase at P 0.05, according to one way ANOVA and LSD.

Nc1: 2.5 g L-1 NaCl; Nc2: 5 g L-1 NaCl; Nc3:10 g L-1 NaCl; Ns1: 2.5 g L-1 Na2SO4; Ns2: 5 g L-1 Na2SO4; Ns3:10 g L-1 Na2SO4; M1: Nc1 + Ns3; M2: Nc3 + Ns1; M3: Nc2 + Ns2.

Table 5 Effect of re-watering on plant height (PH), stem diameter (SD) and leaf area (LA) in Brassica napus

The means ± SE (n = 5) in the table indicate significant difference in plant height, stem diameter and leaf area during re-watering phase at P G.G5, according to one way AN0VA and LSo.

Nc1^0: (2.5 0) g L-1 NaCl; Nc2^1: (5 2.5) g L-NaCl; Nc3^2: (10 5) g L-1 NaCl; Ns1^0: (2.5 0) g L-1 Na2SO4; Ns2^1: (5 2.5) g L-1 Na2SO4; Ns3^2: (10 5) g L-1 Na2SO4; M1^0: (12.5 0) g L-1 Hoagland solution; M2^0: (12.5 0) g L-1 Hoagland solution; M3^0: (10 0) g L-1 Hoagland solution.

Afterwards, Table 5 shows the effect of salt stress and subsequent re-watering on PH, SD and LA of B. napus. A statistical analysis specified that the increments observed during re-watering were significant, except for the concentration at high levels (Nc3^2, Ns3^2, M1^0 and M2^0). The recovery in Ph (80.98% and 64.79%), Sd (84.37% and 78.13) and La (87.47% and 76.88%) were significantly higher under low concentrations (Nc1^0 and Ns1^0), respectively. Moreover by comparing with other stress levels after re-watering, relatively, the recovery in Ph (54.22%, 48.24% and 50.35%), Sd (53.13%, 43.75% and 46.87%) and La (58.07%, 49.84% and 49.39%) found under medium concentration (Nc2^1, Ns2^1 and M3^0). However, at high concentration, the degree of salt levels exhibited the adverse effect of salt stress on Ph, Sd and La. However, additions of mixed salts at M3^0 revealed the same effect on Ph, Sd and La as compared to Ph, Sd and La under Nc2^1, Ns2^1 levels, respectively (Table 5).

Water use efficiency and re-watering water-use efficiency

The WUE showed non-significant reduction from control (100%) to low concentrations (92.26% and 87%) under Nc1 and Ns1 followed by medium concentrations (80.80%, 75.23% and 73.06%) under Nc2, Ns2, and M3). The maximum WUE was recorded under high concentration (108.66%, 117.03%, 126.93% and 118.57%) at Nc3, Ns3, M1 and M2. However, the stress-persuaded maximum increase was recorded at M1 levels (Table 6). While, re-watering of plants reduced the effect of salts stresses significantly, and showed the significant increase in WUE. The results showed that values increased significantly at control, Nc1^0, Nc2^1, Nc3^2, Ns1^0 and Ns2^1 and Ns3^2, respectively in single salt but the increment was reduced in comparison with WUE under salt stress phase. WUE was the same at moderate levels (Nc2^1, Ns2^1 and M3^0), during re-watering. WUE had the maximum values under high levels (Nc3^2, Ns3^2, M1^0 and M2^0).

Re-watering water-use efficiency (WUEr) of B. napus at each stress level is shown in Figure 2. The WUER3^2 of B. napus under Nc3^2 and Ns3^2 concentration levels in both single salt was lower than control. The WUER2^1 of B. napus had maximum values under medium concentration levels at Nc2^1 and Ns2^1 followed by low concentrations levels WUER1^0 at Nc1^0 and Ns1^0, respectively. Comparatively, the plants treated with Na2SO4 concentration at Ns3^2 level effected more and showed lower WUER than the plants treated with NaCl concentration at Nc3^2 level. Although, WUER under mixed treatment WUERM1^0, WUERM1^0 and WUERM1^0 at M1^0 and M2^0 concentration levels was decreased badly. Among all the concentrations levels, WUER showed its better effects on B. napus performance at medium concentrations (Nc2^1, Ns2^1 and M3^0) of NaCl, Na2SO4 and mixed salts.

Table 6 Effect salt stress vs. salt stress subsequently re-watering on water use efficiency (WUE). 

The means ± SE (n = 5) in the table indicate significant difference in water use efficiency during salt stress phase and afterwards under re-watering phase at P 0.05, according to one way ANOVA and LSD.

Nc1: 2.5 g L-1 NaCl; Nc2: 5 g L-1 NaCl; Nc3:10 g L-1 NaCl; Ns1: 2.5 g L-1 Na2SO4; Ns2: 5 g L-1 Na2SO4; Ns3:10 g L-1 Na2SO4; M1: Nc1 + Ns3; M2: Nc3 + Ns1; M3: Nc2 + Ns2; Nc1^0: (2.5 0) g L-1 NaCl; Nc2^1: (5 2.5) g L-NaCl; Nc3^2: (10 5) g L-1 NaCl; Ns1^0: (2.5 0) g L-1 Na2SO4; Ns2^1: (5 2.5) g L-1 Na2SO4; Ns3^2: (10 5) g L-1 Na2SO4; M1^0: (12.5 0) g L-1 Hoagland solution; M2^0: (12.5 0) g L-1 Hoagland solution; M3^0: (10 0) g L-1 Hoagland solution.

Figure 2 Effect of salt stress and subsequent re-watering on re-watering water use efficiency (WUE). 

Relationship between P N , g s , WUE,and CA activity

The Pearson correlation coefficients for the relationship between the different physiological properties B. napus are shown in Table 7. Correlations between PN and physiological parameters were observed and PN was found to be positively correlated with gs and , but had no correlation with WUE and CA activity, which revealed the opposite trend during stress condition. The negative relationship between PN and WUE and CA activity suggested that the presence of salts could inhibit the growth of B. napus.

Table 7 Pearson correlation coefficients among different physiological parameters of Brassica napus (n = 5). 

*Correlation is significant at the 0.05 level. 2-tailed significance is used.

gs: Stomatal conductance; WUE: water use efficiency; : water potential;

CA: carbonic anhydrase activity; PN: net photosynthetic rate.

DISCUSSION

Photosynthetic response traits and growth

Photosynthesis characteristics were different in their response to different salt stress levels (Table 1). The variations in PN were found to be dependent on water status of leaves. The deficiency of water limited PN occurred due to through stomatal closure (Hu et al., 2009). Stomatal opening and closing are considered as response of drought stress for short term duration (Rouhi et al., 2007). Brassica napus was found as different in their PN response to different salt stress levels. Salt stress severely affects the PN of B. napus under high concentration levels (Nc3, Ns3, M1, and M2) (Table 1). It might be because of the water status of leaves disturbed by increasing salt stress through stomatal limitations. Similar results were reported by (Qasim et al., 2003) in canola and Brassica juncea L. (Siddiqui et al., 2008). Consequently, B. napus showed photosynthetic tolerance under low (Nc1, Ns1) to medium (Nc2, Ns2 and M3) concentration of NaCl, Na2SO4 and mixed salts. Thus, this situation demonstrated the threshold photosynthetic adaptability and tolerance of B. napus under medium concentration levels.

Salt stresses exerted a toxic effect on the PN due variations in within the tissues (Ashraf and Foolad, 2005). As the salts within the plant tissues increase, decreases and affects the opening and closing of stomata. This is in return lastly causes imbalance in gas exchange and disturbs the photosynthetic activity (Chartzoulakis et al., 2002a) and affected the plant growth development. At that point, C and water source provide by CA which enhance the activity of photosynthetic process because CA in leaves convert intracellular HCO3 - into CO2 and H2O ( Xing and Wu, 2012). The CA activity in B. napus was activated under low concentration (Nc1, Ns1) and showed good regulatory under medium concentration of NaCl (Nc2, Ns2 and M3), Na2SO4 and mixed salts (Figure 1). Therefore, WUE of leaves was enhanced in B. napus through regulation of CA and by maintaining the variations in leaf .

High salts concentrations caused a clear reduction in growth of B. napus (Table 4). The reduction in growth was due to decrease in PN. It is well documented by Parida and Das (2005), salt stress distresses leaf , gs and growth rate. A considerable increase occurred even during stress conditions in growth attributes under moderate concentration levels of NaCl, Na2SO4 and mixed salts (Nc2, Ns2 and M3) (Table 4). It indicated the growth performance of B. napus respond to PN and threshold adoptability under medium salts concentrations. Therefore, B. napus was thought to be species with single and mixed salts tolerance adaptability under medium stress conditions.

Re-watering effects

The application of re-watering had better effect on plants growth development. Re-watering had a positive impression on PN of leaves in B. napus. After application of re-watering, PN rate was recovered and maintained its status successfully under low (Nc1^0 and Ns1^0) concentration levels followed by moderate concentration (Nc2^l, Ns2^l and M3^0) levels, as compared to high levels. But under high concentration (Nc3^2, Ns3^2, M1^0 and M2^0) levels even in the re-watering phase, photosynthetic activity was restricted due to gs inhibition (Tables 1 and 2). It reflected that CA activity was also inhibited under high concentration (Figure 1) and water regulation caused by CA could not work. The need of supply of H2O and CO2 for photosynthesis was not enough to replenish leaf water status which became the reason of reduction in . Consequently, plant growth attributes like PH, SD and LA showed the unhealthy status due to inhibited water uptake movements. It is also may be due to the toxic effect of salt on growth (Silveira et al., 2009).

Re-watering water-use efficiency (WUER) is an important index of B. napus to adapt different behaviors to different salts stresses subsequent re-watering. It meant that increment in water content directed to the increment of PN in leaves of B. napus. Better effect of re-watering found in plants due to higher WUER (Figure 2). Water regulation caused by CA in plant can change the variation of or gs to some extent concentration levels of salts and the water regulation effect is hysteretic. However, the WUE is an instantaneous value and cannot reflect the re-watering effect on plant. As a result, WUER is a new index tends to indicate the better of re-watering effect on plant. A considerable decrease was noticed even during re-watering conditions in WUER at high levels (Nc3^2, Ns3^2, M1^0 and M2^0). According to Yousfi et al. (2016), after application of re-watering, there is partially recovery found in some species of Medicago laciniata (L.) Mill., due to severe drought stress. Upon re-watering of plants which suffered from high water stress condition under Nc3A2, Ns3A2, M1A0 and M2A0, indicated that it was difficult for plants to be recovered from rapid increase in the assimilation rate. The basic mechanism of photosynthetic biochemistry adopted by plant under stress condition is not impaired due to deficiency of water. However, the decrease in net CO2 uptake is not only the reason of stomatal closure to decrease photosynthetic rate (Cornic, 2000). Therefore, plants suffering from high salt stress stayed stunted.

The variation in osmotic potential and water potential occurred because of lower water content. For that reason, it was necessary for plants to be re-watered prior to their wilting stages. The regime is considered very important in plant tolerance adaptation to drought stress environment in which net photosynthetic rate is maintained and recovered during periods of drought and water-stress (Chartzoulakis et al., 2002b). Accordingly, salt tolerance adaptability of B. napus was better under low to moderate salt stress conditions. Therefore, dilution of salted water or re-watering of salted water could be done at Ns2, Nc2 regime, by considering the best level for threshold tolerance and production of B. napus under saline condition. However, if salinity is caused by mixture of salts then M3 regime is better to consider for dilution of saline irrigation because the relative effect of mixture of salt at M1 was the same with single salts under Nc2 and Ns2, respectively. It reflected that single salt might be more toxic to plant growth than mixture of salts.

CONCLUSION

In conclusion, according to this work, Brassica napus is able to tolerate salt stress under low to medium concentration levels. In this aspect, at regime of medium concentration, WUER left positive effects on the growth and developments of plants and also show better restorability. Thus, the effect of salinity in B. napus could be reduced by diluting the saline irrigation water and also by mixing of salts in response to physiological behaviors. Application of dilution of saline irrigation could be helpful to maintain crops productivity, reduce irrigation cost and save water resources.

ACKNOWLEDGEMENTS

This study was supported by the project of the National Natural Science Foundation of China (nr 31301243); the national key basic research program (973) special projects (2013CB956701), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the research foundation for introduce talents of Jiangsu University (13JDG030).

REFERENCES

Arif, S.W., Aqil, A., Shamsul, H., and Qazi, F. 2013. Salt-induced modulation in growth, photosynthesis and antioxidant system in two varieties of Brassica juncea. Saudi Journal of Biological Sciences 20:183-193. [ Links ]

Ashraf, M., and Foolad, M.R. 2005. Roles of glycinebetaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59:206-216. [ Links ]

Ashraf, M., and Harris, P.J.C. 2013. Photosynthesis under stressful environments: An overview. Photosynthetica 51:163-190. [ Links ]

Chartzoulakis, K., Loupassaki, M., Bertaki, M., and Androulakis, I. 2002a. Effects of NaCl salinity on growth, ion content and CO2 assimilation rate of six olive cultivars. Scientia Horticulturae 96:235-247. [ Links ]

Chartzoulakis, K., Patakas, A., Kofidis, G., Bosabalidis, A., and Nastou, A. 2002b. Water stress affects leaf anatomy, gas exchange, water relations and growth of two avocado cultivars. Scientia Horticulturae 95:39-50. [ Links ]

Chaves, M.M., Flexas, J., and Pinheiro, C. 2009. Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Annals of Botany 103:551-560. [ Links ]

Chen, W., Hou, Z., Wu, L., Liang, Y., and Wei, C. 2010. Evaluating salinity distribution in soil irrigated with saline water in arid regions of northwest China. Agricultural Water Management 97:2001-2008. [ Links ]

Chinnusamy, V., Jagendorf, A., and Zhu, J.K. 2005. Understanding and improving salt tolerance in plants. Crop Science 45:437-448. [ Links ]

Cornic, G. 2000. Drought stress inhibits photosynthesis by decreasing stomatal aperture - not by affecting ATP synthesis. Trends in Plant Science 5:187-188. [ Links ]

Hayat, S., B. Ali, Hasan, S.A., and Ahmad, A. 2007. Effect of 28-homobrassinolide on salinity-induced changes in Brassica juncea. Turkish Journal of Biology 31:141-146. [ Links ]

Hoagland, D.R., and Arnon, D.I. 1950. The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular 347:1-32. [ Links ]

Humaira, G., and Rafiq, A. 2004. Effect of different irrigation intervals on growth of canola (Brassica napus L.) under different salinity levels. Pakistan Journal of Botany 36:359-372. [ Links ]

Hu, H., Boisson-Demier, A., Israelsson-Nordstrom, M., Bohmer, M., Xue, S., Ries, A., et al. 2011. Carbonic anhydrase are upstream regulators of CO2-controlled stomatal movements in guard cells. Nature Cell Biology 12:87-93. [ Links ]

Hu, L.X., Wang, Z.L., and Huang, B.R. 2009. Photosynthetic responses of Bermuda grass to drought stress associated with stomatal and metabolic limitations. Crop Science 49:1902-1909. [ Links ]

Li, C., Jiaqiang, L., Zhao, Y., Xu, X., and Li, S. 2015. Effect of saline water irrigation on soil development and plant growth in the Taklimakan Desert Highway shelterbelt. Soil and Tillage Research 146:99-107. [ Links ]

Maggio, A., Pascale, S.D., Ruggiero, C., and Barbieri, G. 2005. Physiological response of field-grown cabbage to salinity and drought stress. European Journal of Agronomy 23:57-67. [ Links ]

Parida, A.K., and Das, A.B. 2005. Salt tolerance and salinity effects on plants: a review. Ecotoxicology and Environmental Safety 60:324-349. [ Links ]

Qasim, M., Ashraf, M., Ashraf, M.Y., Rehman, S.U., and Rha, E.S. 2003. Salt-induced changes in two canola cultivars differing in salt tolerance. Biologia Plantarum 46:629-632. [ Links ]

Raymer, PL. 2002. Canola: An emerging oilseed crop. p. 122-126. In Janick, J. and Whipkey, A. (eds.) Trends in new crops and new uses. ASHS Press, Alexandria, Virginia, USA. [ Links ]

Rouhi, V., Samson, R., Lemeur, R., and Van Damme, P. 2007. Photosynthetic gas exchange characteristics in three different almond species during drought stress and subsequent recovery. Environmental and Experimental Botany 59:117-129. [ Links ]

Siddiqui, Z.S., Muhammad, A.K., Beom, G.K., Jeon, S.H., and Taek, R.K. 2008. Physiological responses of Brassica napus genotypes to combined drought and salt stress. Plant Stress 2:78-83. [ Links ]

Silveira, J.A.G., Araujo, S.A.M., Lima, J.P.M.S., and Viegas, R.A. 2009. Roots and leaves display contrasting osmotic adjustment mechanisms in response to NaCl salinity in Atriplex nummularia. Environmental and Experimental Botany 66:1-8. [ Links ]

Su, J.J., Wu, S., Xu, Z.J., Qiu, S., Luo, T.T., Yang, Y.M., et al. 2013. Comparison of salt tolerance in Brassicas and some related species. American Journal of Plant Sciences 4:1911-1917. [ Links ]

Toorchi, M., Rana, N., Adnan, K., and Mohammad, R.S. 2011. Response of spring canola cultivars to sodium chloride stress. Annals of Biological Research 2:312-322. [ Links ]

Verma, A.K., Gupta, S.K., and Isaac, R.K. 2012. Use of saline water for irrigation in monsoon climate and deep water table regions: simulation modeling with SWAP. Agricultural Water Management 115:186-193. [ Links ]

Vos, J., and Groenwold, J. 1989. Genetic differences in water-use efficiency, stomatal conductance and carbon isotope fractionation in potato. Potato Research 32:113-121. [ Links ]

Wan, S., Yaohu, K., Dan, W., Shi-Ping, L., and Li-Ping, F. 2007. Effect of drip irrigation with saline water on tomato (Lycopersicon esculentum Mill) yield and water use in semi humid area. Agricultural Water Management 90:63-74. [ Links ]

Wang, R.S., Kang, Y.H., Wan, S.Q., Hu, W., Liu, S.P, and Liu, S.H. 2011. Salt distribution and the growth of cotton under different drip irrigation regimes in a saline area. Agricultural Water Management 100:58-69. [ Links ]

Wilbur, K.M., and Anderson, N.G. 1948. Electrometric and colorimetric determination of carbonic anhydrase. Journal of Biological Chemistry 176:147-154. [ Links ]

Wu, Y.Y., Shi, Q.Q., Wang, K., Li, P.P., Xing, D.K., Zhu, Y.L., et al. 2011. An electrochemical approach coupled with Sb microelectrode to determine the activities of carbonic anhydrase in the plant leaves. In Zeng, D. (ed.) Future intelligent information systems. Vol. 1. Lecture Notes in Electrical Engineering. Vol. 86. p. 87-94. Springer-Verlag, Berlin, Heidelberg, Deutschland. [ Links ]

Wu, Y.Y., and Xing, D.K. 2012. Effect of bicarbonate treatment on photosynthetic assimilation of inorganic carbon in two plant species of Moraceae. Photosynthetica 50:587-594. [ Links ]

Wu, Y.Y., Zhao, X.Z., Li, P.P., and Liu, C.Q. 2006. A study on the activities of carbonic anhydrase of two species of bryophytes, Tortula sinensis (Mull. Hal.) Broth. and Barbula convoluta Hedw. Cryptogamie Bryologie 27:349-355. [ Links ]

Xing, D.K., and Wu, Y.Y. 2012. Photosynthetic response of three climber plant species to osmotic stress induced by polyethylene glycol (PEG) 6000. Acta Physiologiae Plantarum 34:1659-1668. [ Links ]

Yokoi, S., Bressan, R.A., and Hasegawa, P.M. 2002. Salt stress tolerance of plants. JIRCAS Working Report 2002:25-33. [ Links ]

Yousfi, N., Ncib, S., Amari, R., and Chedly, A. 2016. Growth, photosynthesis and water relations as affected by different drought regimes and subsequent recovery in Medicago laciniata (L.) populations. Journal of Plant Biology 59:33-43. [ Links ]

Zum Felde, T., Baumert, A., Strack, D., Becker, H.C., and Mollers, C. 2007. Genetic variation for sinapate ester content in winter rapeseed (Brassica napus L.) and development of NIRS calibration equation. Plant Breeding 126:291-296. [ Links ]

Received: August 23, 2016; Accepted: December 27, 2016

*Corresponding author (wuyanyou@mail.gyig.ac.cn).

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