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5-Aminolevulinic acid alleviates the salinity-induced changes in Brassica napus as revealed by the ultrastructural study of chloroplast
Plant Physiology and Biochemistry 57 (2012) 84e92
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Research article
5-Aminolevulinic acid alleviates the salinity-induced changes in Brassica napus as revealed by the ultrastructural study of chloroplast Muhammad S. Naeem a, b, Hasitha Warusawitharana a, Hongbo Liu a, Dan Liu a, Rashid Ahmad b, Ejaz Ahmad Waraich b, Ling Xu a, Weijun Zhou a, c, * a b c
Institute of Crop Science and Key Laboratory of Crop Germplasm Resource of Zhejiang Province, Zhejiang University, Hangzhou 310058, China Department of Crop Physiology, University of Agriculture, Faisalabad 38040, Pakistan Agricultural Experiment Station, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2012 Accepted 18 May 2012 Available online 29 May 2012
5-Aminolevulinic acid (ALA) is an important plant growth regulator which is derived from 5-carbon aliphatic amino acid. The present study investigates the interaction of increasing NaCl-salinity and ALA on plant growth, leaf pigment composition, leaf and root Naþ/Kþ ratio and chloroplast ultrastructure in mesophyll cells of oilseed rape (Brassica napus) leaves. The plants were treated hydroponically with three different salinity levels (0, 100, 200 mM) and foliar application of ALA (30 mg l1) simultaneously. Ten days after treatment, higher NaCl-salinity significantly reduced the plant biomass and height. However, ALA application restored the plant biomass and plant height under saline conditions. A concentration-dependent increase in Naþ uptake was observed in the aerial parts of B. napus plants. On the other hand, ALA reduced Naþ uptake, leading to a significant decrease in Naþ/Kþ ratio. Accumulation of Naþ augmented the oxidative stress, which was evident by electron microscopic images, highlighting several changes in cell shape and size, chloroplast swelling, increased number of plastogloubli, reduced starch granules and dilations of the thylakoids. Foliar application of ALA improved the energy supply and investment in mechanisms (higher chlorophyll and carotenoid contents, enhanced photosynthetic efficiency), reduced the oxidative stress as evident by the regular shaped chloroplasts with more intact thylakoids. On the basis of these results we can suggest that ALA is a promising plant growth regulator which can improve plant survival under salinity. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: 5-Aminolevulinic acid Chloroplast ultrastructure Naþ/Kþ ratio Oilseed rape Photosynthetic pigments Salinity
1. Introduction Salinity is recognized as one of the major environmental stresses which limit agricultural production and constitutes a severe agricultural problem in many parts of the world. Approximately 7% of the world’s total land area is affected by salinity [1]. There is also a dangerous trend of a 10% annual increase in this area throughout the world. The most common salt composition of saline soils is sodium chloride [2]. Salinity unleashes various types of stress conditions in plants. Resultantly, it initiates complex responses that interfere plant morphology, physiology and metabolism [3]. It is commonly accepted that growth inhibition by salt stress is associated with
* Corresponding author. Institute of Crop Science and Key Laboratory of Crop Germplasm Resource of Zhejiang Province, Zhejiang University, Hangzhou 310058, China. Tel.: þ86 571 8820 8496; fax: þ86 571 8898 1152. E-mail address: [email protected] (W. Zhou). 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2012.05.018
alterations in the water relationships within the plant, caused by osmotic effects with specific ionic consequences. Moreover, salinity interferes with a multiple physiological processes like photosynthetic rate by decreasing the chlorophyll content [4]. Bleaching of chlorophyll is also considered as the first sign of salt poisoning in some plants while other species show a browning of isolated parts of the leaves. Salt stress, in addition to the known components of osmotic stress and ion toxicity, is also manifested as an oxidative stress [5] which is responsible for the structural and ultrastructural damage to mitochondria, chloroplast membranes [6] and photosynthetic apparatus through cellular accumulation of reactive oxygen species. Hernández et al. [7] in pea plants have suggested that salinity induces the swelling of thylakoids, reduction in starch contents, disorganization of grana and an increase in the number and size of plastoglobuli in chloroplasts as well as destruction of chloroplast envelop. Plant growth regulators (PGRs) are widely used to enhance stress resistance in agricultural crops for crop improvement. One of
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The extract was centrifuged at 4000 rpm for 10 min; the supernatant was then taken and diluted by 85% aqueous acetone to the suitable concentration for spectrophotmetric measurements. The extinction was measured against a blank of a pure 85% aqueous acetone at three wavelengths of 452.5, 644 and 663 nm. Chlorophyll a, chlorophyll b and carotenoids concentrations were calculated by using the following equations: Chlorophyll a ¼ 10.3 E663 e 0.98 E644 ¼ mg ml1 Chlorophyll b ¼ 19.7 E644 e 3:87 E663 ¼ mg ml1 Total carotenoids ¼ 4.2 E452.5 e {(0.0264 Chl a) þ (0.426 Chl b)} ¼ mg ml1. Finally, these pigment fractions were calculated as mg g1 fresh weight.
these PGRs is 5-aminolevulinic acid (ALA), which is also known as an essential biosynthetic precursor for the biosynthesis of tetrapyrrols such as heme and chlorophyll. ALA at low concentration has been found to promote growth and yield of several crops and vegetables [8]. Wang et al. [9] showed that treatment of Brassica campestris L. with ALA exercised a positive effect on the growth of the seedlings. Exogenous application of ALA to plants exerts diverse physiological effects, such as promotion in net photosynthetic rate, chlorophyll content, photosynthetic gas exchange and antioxidant system level, suppress Naþ uptake [10] under saline conditions. Moreover, Zhang et al. [11] has reported that ALA also functions as a protectant against oxidative damages of membranes. Oilseed rape (Brassica napus L.) is one of the world’s major oilseed crops and the most important source of edible oil [12], and it is moderately tolerant to salinity [13]. In view of the growing importance of oilseed rape, it is utmost important to exploit it against various environmental stresses like salinity. Keeping in view the importance of oilseed rape and unavailability of information regarding the effect of ALA on salinity-induced ultrastructural changes in oilseed rape leaves, the present study was planned. The main objectives of present investigation were to explore the effect of ALA on salinity-induced changes in leaf mesophyll cells with focus on chloroplast ultrastructures, because thylakoids are the membranes which undergo severe changes during stress conditions like salinity [14]. Moreover, this study also examined the ameliorative role of ALA to avoid cytotoxic damage caused by Naþ uptake on plant morphology and physiology.
The ratio of variable fluorescence to maximal fluorescence (Fv/ Fm), which is an indicator of the efficiency of the photosynthetic apparatus, was measured with a portable fluorometer (model FMS2 Hansatech Instruments Ltd., England). Ten days after treatment, topmost fully expanded leaves of measured plants were first adapted to total darkness with a Hansatech clip for 15 min. The unquenchable portion of fluorescence (F0) was determined by measuring beam [<0.05 mmol/(m2,s)]. The maximal fluorescence (Fm) was determined using a saturating pulse [1200 mmol/(m2s)]. The variable fluorescence (Fv) was determined by the formula: Fv ¼ FmF0.
2. Materials and methods
2.4. Determination of Naþ and Kþ content
2.1. Plant material and treatment conditions
Ten days after treatment, plants were harvested in each treatment, washed with tap water and distilled water thrice respectively and then blotted to remove the excessive water. After that, topmost fully expanded leaf and root samples were dried at 80 C in an oven for 48 h, and then ground into powder. One gram of sample was dry-ashed, extracted with 1:1 HNO3 and finally filtered. Concentrations of Naþ and Kþ elements were determined by flame atomic absorption spectrometry (model AA6300, SHIMADZU, Kyoto, Japan).
Healthy seeds of B. napus L. cv. ZS 758 were obtained from the College of Agriculture and Biotechnology, Zhejiang University and were sown in plastic pots (170 mm 220 mm) filled with peat. Thirty days after sowing, morphologically uniform seedlings were selected and plugged in plate holes on plastic pots (five plants per pot) containing half-strength nutrient solution [15], aerated continuously with an air pump, in the greenhouse. The pH of the solution was adjusted to 6.0. The seedlings were grown under light intensity in the range of 250e350 mmol m2 s1. The temperature was in the range of 16e20 C, and the relative humidity was approximately 55e60%. Each treatment was replicated three times. The solution was renewed every five days. After a two-week acclimatization period, solutions were adjusted to the desired salinities (0, 100, 200 mM NaCl) and plants were simultaneously treated with an aqueous solution of 5aminolevulinic acid (ALA, provided by Cosmo Oil Co. Ltd., Japan) at a concentration 30 mg L1 by foliar spray. The concentrations of NaCl and ALA were determined based on our preliminary studies. Lower as well as upper leaf surface was sprayed until wetted with a hand-held atomizer, as it was reported that absorption by the lower leaf surface is rapid and effective [16]. A subsequent application was made five days after 1st spray. Plants sprayed with distilled water served as the control. Ten days after treatment, plants were harvested and data was recorded for different parameters as described below: 2.2. Determination of photosynthetic pigments Chlorophyll a (Chl a), chlorophyll b (Chl b) and total carotenoids were spectrophotometrically determined according to Pei et al. [17]. After ten days of treatment, topmost fully expanded leaves were taken to extract the pigments. A sample of 0.5 g of fresh leaves was homogenized in a known volume of 85% (v/v) aqueous acetone.
2.3. Photosynthetic efficiency measurement
2.5. Transmission electron microscopy Topmost fully expanded leaf fragments without veins of randomly selected plants were fixed overnight in 4% glutaraldehyde (v/v) in 0.1 M PBS (Sodium Phosphate Buffer, pH 7.4) and washed three times with same PBS. The samples were post fixed in 1% OsO4 (osmium (VIII) oxide) for 1 h, then washed three times in 0.1 M PBS (pH 7.4) with 10 min interval between each washing. Then with 15e20 min interval, they were dehydrated in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100%) and finally by absolute acetone for 20 min. The samples were then infiltrated and embedded in Spurr’s resin overnight. After heating the specimens at 70 C for 9 h, ultra-thin sections (80 nm) were prepared and mounted on copper grids for viewing by a transmission electron microscope (JEOL TEM-1230EX) at an accelerating voltage of 60.0 kV [18]. Data for the size (circumference) of whole mesophyll cells and chloroplasts were recorded using software JeDa 801D Morphology Image Analysis Systems. Data were recorded from at least 25 cell samples and were averaged. 2.6. Statistical analysis One-way analysis of variance (ANOVA) was performed for plant growth, biomass and photosynthesis related parameters. Data were analyzed using SAS v.9 software. All results were expressed as mean
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from four replications. Least significant difference (LSD) test was applied at 5% level of probability to separate the means with statistically significant difference.
Table 2 Effects of salinity and 5-aminolevulinic acid on photosynthetic pigments in Brassica napus L. leaves. Each value is the mean of four replicates. Means followed by same small letters are not significantly different at P 0.05.
3. Results
ALA level (mg l1)
NaCl level (mM)
Chlorophyll (mg g1 FW) Chl a
Chl b
Total
3.1. Plant growth indices
0
0 100 200 0 100 200
0.72 0.59 0.41 0.79 0.68 0.61
0.24 0.18 0.14 0.28 0.20 0.17
0.96 0.77 0.55 1.08 0.88 0.78
3.2. Photosynthetic pigments content Effect of different levels of NaCl and ALA, alone or in combination was investigated on the photosynthetic pigments (Chl a, Chl b, total Chl and carotenoids) in oilseed rape topmost fully expanded leaves (Table 2). Chlorophyll and carotenoids contents were significantly decreased with increasing NaCl concentration and this decrease was highest under 200 mM NaCl compared to the control plants. However, the plants treated with ALA in combination with NaCl (100 and 200 mM) enhanced all types of pigments contents significantly compared to the corresponding plants under NaCl stress. Moreover, plants treated with ALA alone also increased their pigments contents significantly compared to that of control plants. 3.3. Photosynthetic efficiency Photosynthetic efficiency changes were recorded in terms of Fv/ Fm, induced by NaCl and ALA alone or combined in oilseed rape topmost fully expanded leaves (Fig. 1). A gradual decline was observed in photosynthetic efficiency with increasing salinity. However, the declining effect (22.11%) at 200 mM NaCl was much pronounced and significant compared to the control plants. However, the foliar application of ALA helped the plants overcome salinity stress and recover their photosynthetic efficiency significantly compared to the control plants. Photosynthetic efficiency values of 100 mM NaCl-treated plants and ALA together were comparable to those of control plants. Moreover, ALA significantly enhanced the photosynthetic efficiency of the plants under 200 mM NaCl compared to the plants under 200 mM NaCl alone. In addition, the maximum photosynthetic efficiency values were observed in plants treated with ALA alone.
Table 1 Effects of salinity and 5-aminolevulinic acid on shoot length and biomass of Brassica napus L. plants. Each value is the mean of four replicates. Means followed by same small letters are not significantly different at P 0.05. ALA level (mg l1)
NaCl level (mM)
Shoot length (cm)
0
0 100 200 0 100 200
24.74 24.59 19.89 25.19 24.74 22.60
30
a a c a a b
Shoot biomass (g plant1) Fresh
Dry
16.67 b 15.02 ab 9.78 d 17.29 a 16.65 ab 13.62 c
1.40 1.33 1.10 1.70 1.40 1.32
30
b d e a c d
0.43 0.38 0.27 0.46 0.44 0.39
b c d a ab c
Topmost fully expanded leaves and roots of oilseed rape plants were analyzed for Naþ and Kþ accumulation (Fig. 2). In the absence of salt, the Naþ contents were negligible (nearly 1 mg g1 DW). The present investigation revealed that Naþ accumulation was NaCl concentration-dependent. The aerial parts accumulated Naþ in more quantity as compared to root of the plants and variations were quite significant (P < 0.05) at both NaCl treatments. Salinity did not change the accumulation of Kþ significantly compared to the control plants. Subsequently, Naþ/Kþ ratios were significantly increased as salinity increased. Application of ALA reduced significantly the accumulation of Kþ and Naþ under 100 mM as well as 200 mM salinity stress, leading to a concomitant reduction in Naþ/ Kþ ratio compared to the plants under salinity stress alone. 3.5. Morphometric changes in whole mesophyll cells and chloroplasts Transmission electron micrographs of whole mesophyll cells and chloroplasts revealed several changes in the size of cells and chloroplasts of the treated plants (Table 3). Increasing salinity alone or in combination with ALA did not induce any significant change in cell size as compared to that of control. The plants exposed to the highest concentration of NaCl (200 mM) alone, reduced significantly the cell diameter over other treatments but this decrease was recovered if the plants treated with ALA and NaCl together.
1.2 NaCl NaCl+ALA
1
a a
a b
0.8
c
d 0.6
0.4
0.2
0 0
b b c a b b
b d e a c d
3.4. Naþ and Kþ uptake and accumulation
Fv/Fm
The effect of different levels of salinity (NaCl) and 5aminolevulinic acid (ALA) on plant growth was recorded in terms of shoot length and biomass (fresh and dry weight) (Table 1). It was observed that increasing salinity level imposed inhibitory effects on shoot length, fresh and dry weight of shoot. However, this inhibitory effect was more prominent at 200 mM NaCl-salinity level and significantly different (P < 0.05) as compared to their respective controls. ALA treatment helped the plants counteract salinity stress conditions. The plants under 100 mM sodium chloride salinity were completely recovered by ALA application while 200 mM NaCltreated plants also showed substantial recovery.
b c d a b c
Carotenoids (mg g1 FW)
100
200
NaCl (m M) Fig. 1. Effects of salinity (NaCl, 0e200 mM) and 5-aminolevulinic acid (ALA, 0e30 mg l1) on photosynthetic efficiency (Fv/Fm) in Brassica napus L. leaves. Means of four replicates SE. Means followed by same small letters are not significantly different by the LSD test at P 0.05.
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Fig. 2. Effects of salinity (NaCl, 0e200 mM) and 5-aminolevulinic acid (ALA, 0e30 mg l1) on sodium (Naþ), potassium (Kþ) uptake and Naþ/Kþ ratio in Brassica napus L. Means of four replicates SE. Means followed by same small letters are not significantly different by the LSD test at P 0.05.
Chloroplast size and width were increased significantly with increasing salinity as compared to that of control plants and this increase was reversed by foliar application of ALA. However, the chloroplast length was decreased significantly only in the plants treated with 200 mM NaCl alone. Foliar application of ALA to NaCltreated plants increased the chloroplast length and the length values were at par with that of control plants. 3.6. Ultrastructural changes in whole mesophyll cells NaCl or ALA alone or together induced changes in whole mesophyll cells (Fig. 3). In NaCl-treated plants, irregular shaped cells with relatively less developed cell wall, containing nucleus without nucleolus, wide intercellular spaces and swollen chloroplasts, especially under 200 mM NaCl (Fig. 3C) were observed as compared to the control or ALA alone treated plant cells (Fig. 3AD). However, foliar application of ALA reversed the aforementioned maladies induced by NaCl (Fig. 3E and F). 3.7. Ultrastructural changes in chloroplasts Chloroplasts of control leaves were elongated with a typical arrangement of thylakoid membranes having intact stacks of grana and stroma (Figs. 4 and 5A). Low salinity level (100 mM NaCl)
treated plants showed slight changes in the ultrastructure of chloroplast (Figs. 4 and 5B). Under higher saline conditions (200 mM NaCl), drastic changes were observed, and the chloroplasts were swollen (Fig. 4C) and contained a large number of plastoglobuli. Their thylakoid membranes showed dilations, the spaces between the membranes looked swollen, and undulated thylakoid areas developed (Fig. 5C). In particular, the number of grana stacks and starch grains were considerably reduced. Plants treated with ALA alone, showed a typical chloroplast ultrastructure with no significant change (Figs. 4 and 5D). However, foliar application of ALA on NaCl-treated plants helped to alleviate the damage and thus the chloroplast ultrastructures were more stable (Fig. 4). Chloroplasts were less swollen, having organized thylakoid membrane system, clearly differentiated into grana stacks, relatively less plastoglobuli and more starch grains were observed (Fig. 5E and F). 4. Discussion Salinity is a complex trait which affects almost every aspect of the physiology and biochemistry of plants through osmotic, ionspecific and oxidative stress. It needs to be addressed and answered urgently by any feasible strategy. To understand how 5aminolevulinic acid (ALA) ameliorates the salinity-induced
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Table 3 Effects of salinity and 5-aminolevulinic acid on the size of whole mesophyll cell and chloroplast in Brassica napus L. leaves. Means followed by same small letters in the same row are not significantly different at P 0.05. Parameter (mm)
Control
100 mM NaCl
200 mM NaCl
30 mg l1 ALA
100 mM NaCl þ 30 mg l1 ALA
200 mM NaCl þ 30 mg l1 ALA
Cell size Cell diameter Chloroplast size Chloroplast length Chloroplast width
90.22 ab 28.42 a 9.71 b 3.72 a 1.22 b
94.12 a 27.15 a 10.32 ab 3.55 a 1.34 ab
87.10 b 19.92 c 12.40 a 2.65 b 1.63 a
88.13 b 26.36 a 9.52 b 3.56 a 1.11 b
87.98 b 25.28 ab 8.26 b 3.65 a 1.29 ab
91.27 ab 22.14 bc 10.13 3.05 ab 1.43 ab
morphological, physiological, ultrastructural changes in oilseed rape (B. napus L.) leaves and how these changes (especially in chloroplasts of mesophyll cells) are related to physiological functions, the present study was conducted on topmost fully expanded leaves of oilseed rape. To the best of our knowledge, this is the first research about ALA ameliorative effect on salinity-induced changes in the leaf ultrastructure of oilseed rape.
Plant growth is a function of photosynthesis, water conductivity, osmotic potential, and threshold turgor among other factors. When these agents are insufficient, growth does not occur. In the present investigation, we observed a decreasing trend in the length, fresh and dry weight of shoot (Table 1) with increasing salinity which reflects the increased metabolic energy cost and reduced carbon gain, which are associated with salt adaptation. These findings
Fig. 3. Electron micrographs of whole mesophyll cells of oilseed rape (Brassica napus L.) under (A) control; (B) 100 mM NaCl alone; (C) 200 mM NaCl alone; (D) 30 mg l1 ALA alone; (E) 100 mM NaCl þ 30 mg l1 ALA; (F) 200 mM NaCl þ 30 mg l1 ALA. NaCl-treated mesophyll cells are showing a relatively less developed cell wall especially under 200 mM NaCl alone, nucleus without nucleolus, wide intercellular spaces, swollen chloroplasts as compared to the control or ALA alone, treated mesophyll cells. However, ALA þ NaCl combinations reversed the above mentioned maladies induced by NaCl. CW, cell wall; CH, chloroplast; ICS, intercellular spaces; N, nucleus; Nue, nucleolus; Vac, vacuole; S, sugar grains. Bars AF ¼ 5 mm.
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Fig. 4. Relatively low magnified view of chloroplast in mesophyll cells of oilseed rape (Brassica napus L.) under (A) control; (B) 100 mM NaCl alone; (C) 200 mM NaCl alone; (D) 30 mg l1 ALA alone; (E) 100 mM NaCl þ 30 mg l1 ALA; (F) 200 mM NaCl þ 30 mg l1 ALA. Under saline conditions, chloroplasts swelling with dilations in thylakoid membranes, reduction of starch granules, relatively more plastoglobuli are evident especially under 200 mM NaCl compared to the control or ALA alone, treated mesophyll cells. However, ALA þ NaCl combination reduced the chloroplasts swelling with more organized thylakoid membranes, increased the starch granules, relatively less plastoglobuli are evident. CW, cell wall; CH, chloroplast; st, stroma thylakoids; Gt, grana thylakoids; S, sugar grains; Pg, plastoglobuli. Bars AF ¼ 0.2 mm.
agree with those of Yusuf et al. [19] in Brassica juncea L. under NaCl stress. However, foliar application of ALA increased the aforementioned growth indices by improving salt tolerance; as reported by Watanabe et al. [10] in cotton under NaCl stress. Salinity induces a serious decline in chlorophyll content, fluorescence, caroteniods and photosystem II (PSII) activities in several plants [20]. Reduction observed in the present study (Table 2) may be either due to a decrease in chlorophyll synthesis or an increase in chlorophyll degradation or both reasons. In fact, salinity declines the accumulation of ALA [21] which is a precursor of all tetrapyrroles like protochlorophyllide (that is converted in chlorophyll when exposed to light). In the present study, exogenous application of ALA (a key precursor of chlorophyll) might induce biosynthesis of chloroplasts which is reflected by the raised chlorophyll content (Fig. 1). It is well documented that carotenoids play a protective role against photo-oxidation by dissipating the excessive energy of
excitation [6]. Accordingly, the reason for increased amount of leaf carotenoids in ALA-treated plants in the present investigation may be an influence of such protective mechanisms that counteracts the deleterious effects of oxidative damage resultant from salt stress. Our results are consistent with those of Wang et al. [22]; they reported that ALA may regulate the biosynthesis of chlorophyll. The effectiveness of ALA in enhancing photosynthetic efficiency can therefore be attributed to the significant improvement of chlorophyll a content and subsequently boosting light-harvesting capabilities of the treated plants. It is well known that the plants under saline conditions, accumulate more Naþ, which disturbs the ionic balance, plant metabolism and induce oxidative damage as revealed by the increasing sodium accumulation in leaves of oilseed rape plants with increasing salinity compared to the control plants. Plant tolerance toward salinity depends on the Kþ status in root and leaves [23]. In
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Fig. 5. Magnified view of chloroplast and thylakoid membranes in mesophyll cells of oilseed rape (Brassica napus L.) under (A) control; (B) 100 mM NaCl alone; (C) 200 mM NaCl alone; (D) 30 mg l1 ALA alone; (E) 100 mM NaCl þ 30 mg l1 ALA; (F) 200 mM NaCl þ 30 mg l1 ALA. CW, cell wall; CH, chloroplast; st, stroma thylakoids; Gt, grana thylakoids; S, sugar grains; Pg, plastoglobuli. Bars AF ¼ 0.1 mm.
this study, selective Kþ uptake is evident in leaves of the plants under increasing salinity. Although salinity did not affect Kþ content, but increased Naþ content in leaves substantially raised Naþ/Kþ ratio (Fig. 2). Cytosolic Naþ/Kþ ratio depends on the ability of plants to extrude Naþ either outside the cell or into the cell vacuoles by Naþ/Hþ antiport. Similar physiochemical structure of Naþ and Kþ may also affect the Naþ/Kþ ratio. Naþ/Kþ ratios equal to or smaller than 0.6 are necessary for an optimal metabolic efficiency in non-halophyte plants. Consistent with the findings of Watanabe et al. [10] on cotton seedlings, ALA reduced the accumulation of Naþ and Kþ, leading to a reduction of Naþ/Kþ ratio compared to the respective salt-treated plants. Higher accumulation of sodium in the leaves than roots (data not shown) suggest that ALA does not affect Naþ transport from the roots to the shoots but it might suppress the inflow of Naþ. Reports have shown that chloroplast ultrastructures in both halophytes and glycophytes are considerably affected by salinity [24]. Chloroplast swelling in salt-treated plants may be induced by an
osmotic imbalance between stroma and cytoplasm (Figs. 4 and 5). Membranes like thylakoids are particularly susceptible to disturbance by salt [25]. The distortion of grana stacking and swelling of thylakoids caused by salinity in the present study is probably due to a change in the ionic composition of the stroma liquid. This argument is also supported by the Naþ accumulation in the leaves in the present investigation. An increase in the number of plastoglobuli, observed in this study is a typical indicator of leaf senescence and is connected with the degradation of thylakoids or cell membrane, and they disappear if the stress conditions are over. The accumulation of lipid droplets (plastoglobuli) is also considered as reserves of energy for metabolism required to tolerate salinity. Plastoglobuli are ubiquitous in chloroplasts and chromoplasts, and play their role in salt tolerance. However, the decrease of starch granules in the present study is associated with osmotic stress and advancing of senescence [26]. Reduction in starch granules can also be attributed to enhanced conversion of starch into soluble sugars which are considered as compatible solutes and play an important role in osmotic adjustment.
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In fact, salinity induces the overproduction of reactive oxygen species (ROS) which initiate the chloroplast damage. Under salt stress conditions, plants reduce transpiration by closing the stomata, which consequently decreases the CO2 concentration inside the chloroplasts. The lower concentration of CO2 inside the chloroplasts reduces NADPþ generation by the Calvin cycle [27] and allows electrons from ferredoxin to reduce O2 as an alternative acceptor, the formation of superoxide ðO 2 Þ initiates a chain reaction to produce toxic ROS like hydrogen peroxide (H2O2) and hydroxyl radical ð$ OHÞ and induce oxidative stress to damage the chloroplasts in plants exposed to salinity. The plants seemed to use the additional energy supply under foliar application of ALA for increasing the investment in salinity tolerance mechanisms, for example, for reducing oxidative stress and water loss. Elongated chloroplasts with no swelling and only minor dilations of the thylakoids in ALA and NaCl-treated plants are the concrete indication of less oxidative stress. Another difference in the chloroplasts of the plants grown under NaCl and ALA application together was that the number of starch grains increased. This indicates that the plants accumulate starch under favorable growth conditions and they metabolize a larger amount of starch under saline condition to have a suitable pool of osmolytes like soluble sugars [22] for osmotic adjustment. Relatively less number of lipid droplets (plastoglobuli) in chloroplasts of plants treated with NaCl and ALA together is another indication of lesser oxidative stress, in other words we can say that ALA reduced the lipid peroxidation [28] of thylakoids and cell membranes by inducing the antioxidant system. This assumption is in accordance with a study which has shown that ALA induced the activities of heme-based antioxidant enzymes like APX and CAT [29]. The increasing activity of APX catalyzed the breakdown of H2O2 in Halliwell-Asada pathway originally described in the chloroplast [30], in order to protect the thylakoids against the potentially cytotoxic species of activated oxygen under the condition of NaCl stress. This argument is also supported by Bhaya and Castelfranco [31] and Castelfranco and Jones [32] who reported that exogenous application of ALA boasts up the activity of heme-based biomolecules. Thus, we can assume that exogenous ALA application alone or in combination with salinity enhanced the activity of hemebased enzymatic antioxidants and scavenged the ROS efficiently produced under salinity conditions, leading to avoid the damage of chloroplast ultrastructures. 5. Conclusions No apparent damage was observed on the aerial parts of plants in response to 100 mM salinity, which confirms the tolerant nature of B. napus L. cv. ZS 758 toward low salinity. However, exogenous application of ALA recovered the plants treated with 200 mM NaCl by inducing the conformational changes in the ultrastrutures of chloroplasts of mesophyll cells. Higher accumulation of sodium in the leaves than roots suggests that ALA does not affect Naþ transport from the roots to the shoots but it might suppress the inflow of Naþ. NaCl induced senescence-like situation in the leaves of oilseed rape as revealed by an increase in number of plastoglobuli and is connected with the degradation of thylakoids. However, ALA treatment avoids this physiological senescence by developing the more organized granal stacks in chloroplasts and reduced the number of plastoglobuli. Our study is based on the plants grown in hydroponics growth conditions. In order to study the more practical ameliorative effects of ALA on oilseed rape plants grown under saline conditions in the real soil environment further research is required.
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Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2011AA10A206), the National Key Science & Technology Supporting Program of China (2010BAD01B01, 2010BAD01B04), the National Natural Science Foundation of China (31000678, 31071698), the Industry Technology System of Rapeseed in China (nycytx-005), and the Higher Education Commission of Pakistan. References [1] T.J. Flowers, Improving crop salt tolerance, J. Exp. Bot. 55 (2004) 307e319. [2] R.W. Cumming, J.L. Elliot, Soil chemical properties, in: P.E.V. Charman, B.W. Murphy (Eds.), Soil: Their Properties and Management, Sydney University Press, Melbourne, 1991. [3] J.M. Cheesman, Mechanisms of salinity tolerance in plants, J. Plant Physiol. 87 (1988) 547e550. [4] M. Soussi, A. Ocana, C. Lluch, Effect of salt stress on growth, photosynthesis and nitrogen fixation in chickpea (Cicer arietinum L.), J. Exp. Bot. 14 (1998) 1329e1337. [5] J.M. Gómez, J.A. Hernández, A. Jiménez, L.A. Del Río, F. Sevilla, Differential response of antioxidative enzymes of chloroplasts and mitochondria to longterm NaCl stress of pea plants, Free Radic. Res. 31 (1999) 11e18. [6] R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci. 7 (2002) 405e410. [7] J.A. Hernández, E. Olmos, F.J. Corpas, F. Sevilla, L.A. Del Río, Salt-induced oxidative stress in chloroplasts of pea plants, Plant Sci. 105 (1995) 151e167. [8] R.C. Bindu, M. Vivekanandan, Hormonal activities of 5-aminolevulinic acid in callus induction and micropropagation, Plant Growth Regul. 26 (1998) 15e18. [9] L.J. Wang, W.B. Jiang, H. Liu, W.Q. Liu, L. Kang, X.L. Hou, Promotion by 5aminolevulinic acid of germination of Pakchoi (Brassica camestris ssp. chinensis var. communis Tsen et Lee) seeds under salt stress, J. Integr. Plant Biol. 47 (2005) 1084e1091. [10] K. Watanabe, T. Tanaka, H. Kuramochi, Y. Takeuchi, Improving salt tolerance of cotton seedling with 5-aminolevulinic acid, Plant Growth Regul. 32 (2000) 97e101. [11] Z.J. Zhang, H.Z. Li, W.J. Zhou, Y. Takeuchi, K. Yoneyama, Effect of 5aminolevulinic acid on development and salt tolerance of potato (Solanum tuberosum L.) microtubers in vitro, Plant Growth Regul. 49 (2006) 27e34. [12] E.J.J. Momoh, W.J. Zhou, B. Kristiansson, Variation in the development of secondary dormancy in oilseed rape genotypes under conditions of stress, Weed Res. 42 (2002) 446e455. [13] M. Ashraf, T. McNeilly, Salinity tolerance in brassica oilseeds, Crit. Rev. Plant Sci. 23 (2004) 157e174. [14] N. Geissler, S. Hussin, H.W. Koyro, Elevated atmospheric CO2 concentration ameliorates effects of NaCl salinity on photosynthesis and leaf structure of Aster tripolium L, J. Exp. Bot. 60 (2009) 137e151. [15] D.I. Arnon, D.R. Hoagland, Crop production in artificial solution with special reference to factors affecting yield and absorption of inorganic nutrients, Soil Sci. 50 (1940) 463e485. [16] H.M. Hull, H.L. Morton, J.R. Wharrie, Environmental influence on cuticle development and resultant foliar penetration, Bot. Rev. 41 (1975) 421e451. [17] Z.F. Pei, D.F. Ming, D. Liu, G.L. Wan, X.X. Geng, H.J. Gong, W.J. Zhou, Silicon improves the tolerance to water deficit stress induced by polyethylene glycol in wheat seedlings, J. Plant Growth Regul. 29 (2010) 106e115. [18] U. Najeeb, L. Xu, S. Ali, G. Jilani, H.J. Gong, W.Q. Shen, W.J. Zhou, Citric acid enhances the phytoextraction of manganese and plant growth by alleviating the ultrastructural damages in Juncus effusus L, J. Hazard. Mater. 170 (2009) 1156e1163. [19] M. Yusuf, S.A. Hasan, B. Ali, S. Hayat, Q. Fariduddin, A. Ahmad, Effect of salicylic acid on salinity induced changes in Brassica juncea L. J. Integr. Plant Biol. 50 (2008) 1096e1102. [20] C. Santos, H. Azevedo, G. Caldeira, In situ and in vitro senescence induced by KCl stress: nutritional imbalance, lipid peroxidation and antioxidant metabolism, J. Exp. Bot. 52 (2001) 351e360. [21] A. Stobart, W. Griffiths, I. Ameen-Bukhari, R. Sherwood, The effect of Cd2þ on the biosynthesis of chlorophyll in leaves of barley, Physiol. Plant 63 (1985) 293e298. [22] L.J. Wang, W.B. Jiang, B.J. Huang, Promotion of 5-aminolevulinic acid on photosynthesis of melon (Cucumis melo) seedlings under low light and chilling stress conditions, Physiol. Plant 121 (2004) 258e264. [23] F.J.M. Maathuis, A. Amtmann, Kþ nutrition and Naþ toxicity: the basis of cellular Kþ/Naþ ratios, Ann. Bot. 84 (1999) 123e133. [24] N.V. Paramonova, N.I. Shevyakova, Kuznetsov VlV, Ultrastructure of chloroplasts and their storage inclusions in the primary leaves of Mesembryanthemum crystallinum affected by putrescine and NaCl, Russ. J. Plant Physiol. 51 (2004) 86e96. [25] N.R. Baker, A possible role for photosystem II in environmental perturbations of photosynthesis, Physiol. Plant 81 (1991) 563e570.
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[26] J. Kutik, D. Hola, A. Vicankova, M. Smidova, M. Kocova, M. Kornerova, L. Kubinova, The heterogeneity of structural and functional photosynthetic characteristics of mesophyll chloroplasts in various parts of mature or senescing leaf blade of two maize (Zea mays L.) genotypes, Photosynthetica 39 (2001) 497e506. [27] K. Biehler, H. Fock, Evidence for the contribution of the Mehler-peroxidase reaction in dissipating excess electrons in drought-stressed wheat, Plant Physiol. 112 (1996) 265e272. [28] W.F. Zhang, F. Zhang, R. Raziuddin, H.J. Gong, Z.M. Yang, L. Lu, Q.F. Ye, W.J. Zhou, Effects of 5-aminolevulinic acid on oilseed rape seedling growth under herbicide toxicity stress, J. Plant Growth Regul. 27 (2008) 159e169.
[29] E. Nishihara, K. Kondo, M.M. Parvez, K. Takahashi, K. Watanabe, K. Tanaka, Role of 5-aminolevulinic acid (ALA) on active oxygen-scavenging system in NaCl-treated spinach (Spinacia oleracea), J. Plant Physiol. 160 (2003) 1085e1091. [30] Y. Nakano, K. Asada, Hydrogen peroxide scavenged by ascorbate-specific peroxidase in spinach chloroplasts, Plant Cell Physiol. 22 (1981) 867e880. [31] D. Bhaya, P.A. Castelfranco, Chlorophyll biosynthesis and assembly into chlorophyll-protein complexes in isolated developing chloroplasts, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 5370e5374. [32] P.A. Castelfranco, O.T.G. Jones, Protoheme turnover and chlorophyll synthesis in greening barley tissue, Plant Physiol. 55 (1975) 485e490.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
5-Aminolevulinic acid alleviates the salinity-induced changes in Brassica napus as revealed by the ultrastructural study of chloroplast Muhammad S. Naeem a, b, Hasitha Warusawitharana a, Hongbo Liu a, Dan Liu a, Rashid Ahmad b, Ejaz Ahmad Waraich b, Ling Xu a, Weijun Zhou a, c, * a b c
Institute of Crop Science and Key Laboratory of Crop Germplasm Resource of Zhejiang Province, Zhejiang University, Hangzhou 310058, China Department of Crop Physiology, University of Agriculture, Faisalabad 38040, Pakistan Agricultural Experiment Station, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2012 Accepted 18 May 2012 Available online 29 May 2012
5-Aminolevulinic acid (ALA) is an important plant growth regulator which is derived from 5-carbon aliphatic amino acid. The present study investigates the interaction of increasing NaCl-salinity and ALA on plant growth, leaf pigment composition, leaf and root Naþ/Kþ ratio and chloroplast ultrastructure in mesophyll cells of oilseed rape (Brassica napus) leaves. The plants were treated hydroponically with three different salinity levels (0, 100, 200 mM) and foliar application of ALA (30 mg l1) simultaneously. Ten days after treatment, higher NaCl-salinity significantly reduced the plant biomass and height. However, ALA application restored the plant biomass and plant height under saline conditions. A concentration-dependent increase in Naþ uptake was observed in the aerial parts of B. napus plants. On the other hand, ALA reduced Naþ uptake, leading to a significant decrease in Naþ/Kþ ratio. Accumulation of Naþ augmented the oxidative stress, which was evident by electron microscopic images, highlighting several changes in cell shape and size, chloroplast swelling, increased number of plastogloubli, reduced starch granules and dilations of the thylakoids. Foliar application of ALA improved the energy supply and investment in mechanisms (higher chlorophyll and carotenoid contents, enhanced photosynthetic efficiency), reduced the oxidative stress as evident by the regular shaped chloroplasts with more intact thylakoids. On the basis of these results we can suggest that ALA is a promising plant growth regulator which can improve plant survival under salinity. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: 5-Aminolevulinic acid Chloroplast ultrastructure Naþ/Kþ ratio Oilseed rape Photosynthetic pigments Salinity
1. Introduction Salinity is recognized as one of the major environmental stresses which limit agricultural production and constitutes a severe agricultural problem in many parts of the world. Approximately 7% of the world’s total land area is affected by salinity [1]. There is also a dangerous trend of a 10% annual increase in this area throughout the world. The most common salt composition of saline soils is sodium chloride [2]. Salinity unleashes various types of stress conditions in plants. Resultantly, it initiates complex responses that interfere plant morphology, physiology and metabolism [3]. It is commonly accepted that growth inhibition by salt stress is associated with
* Corresponding author. Institute of Crop Science and Key Laboratory of Crop Germplasm Resource of Zhejiang Province, Zhejiang University, Hangzhou 310058, China. Tel.: þ86 571 8820 8496; fax: þ86 571 8898 1152. E-mail address: [email protected] (W. Zhou). 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2012.05.018
alterations in the water relationships within the plant, caused by osmotic effects with specific ionic consequences. Moreover, salinity interferes with a multiple physiological processes like photosynthetic rate by decreasing the chlorophyll content [4]. Bleaching of chlorophyll is also considered as the first sign of salt poisoning in some plants while other species show a browning of isolated parts of the leaves. Salt stress, in addition to the known components of osmotic stress and ion toxicity, is also manifested as an oxidative stress [5] which is responsible for the structural and ultrastructural damage to mitochondria, chloroplast membranes [6] and photosynthetic apparatus through cellular accumulation of reactive oxygen species. Hernández et al. [7] in pea plants have suggested that salinity induces the swelling of thylakoids, reduction in starch contents, disorganization of grana and an increase in the number and size of plastoglobuli in chloroplasts as well as destruction of chloroplast envelop. Plant growth regulators (PGRs) are widely used to enhance stress resistance in agricultural crops for crop improvement. One of
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The extract was centrifuged at 4000 rpm for 10 min; the supernatant was then taken and diluted by 85% aqueous acetone to the suitable concentration for spectrophotmetric measurements. The extinction was measured against a blank of a pure 85% aqueous acetone at three wavelengths of 452.5, 644 and 663 nm. Chlorophyll a, chlorophyll b and carotenoids concentrations were calculated by using the following equations: Chlorophyll a ¼ 10.3 E663 e 0.98 E644 ¼ mg ml1 Chlorophyll b ¼ 19.7 E644 e 3:87 E663 ¼ mg ml1 Total carotenoids ¼ 4.2 E452.5 e {(0.0264 Chl a) þ (0.426 Chl b)} ¼ mg ml1. Finally, these pigment fractions were calculated as mg g1 fresh weight.
these PGRs is 5-aminolevulinic acid (ALA), which is also known as an essential biosynthetic precursor for the biosynthesis of tetrapyrrols such as heme and chlorophyll. ALA at low concentration has been found to promote growth and yield of several crops and vegetables [8]. Wang et al. [9] showed that treatment of Brassica campestris L. with ALA exercised a positive effect on the growth of the seedlings. Exogenous application of ALA to plants exerts diverse physiological effects, such as promotion in net photosynthetic rate, chlorophyll content, photosynthetic gas exchange and antioxidant system level, suppress Naþ uptake [10] under saline conditions. Moreover, Zhang et al. [11] has reported that ALA also functions as a protectant against oxidative damages of membranes. Oilseed rape (Brassica napus L.) is one of the world’s major oilseed crops and the most important source of edible oil [12], and it is moderately tolerant to salinity [13]. In view of the growing importance of oilseed rape, it is utmost important to exploit it against various environmental stresses like salinity. Keeping in view the importance of oilseed rape and unavailability of information regarding the effect of ALA on salinity-induced ultrastructural changes in oilseed rape leaves, the present study was planned. The main objectives of present investigation were to explore the effect of ALA on salinity-induced changes in leaf mesophyll cells with focus on chloroplast ultrastructures, because thylakoids are the membranes which undergo severe changes during stress conditions like salinity [14]. Moreover, this study also examined the ameliorative role of ALA to avoid cytotoxic damage caused by Naþ uptake on plant morphology and physiology.
The ratio of variable fluorescence to maximal fluorescence (Fv/ Fm), which is an indicator of the efficiency of the photosynthetic apparatus, was measured with a portable fluorometer (model FMS2 Hansatech Instruments Ltd., England). Ten days after treatment, topmost fully expanded leaves of measured plants were first adapted to total darkness with a Hansatech clip for 15 min. The unquenchable portion of fluorescence (F0) was determined by measuring beam [<0.05 mmol/(m2,s)]. The maximal fluorescence (Fm) was determined using a saturating pulse [1200 mmol/(m2s)]. The variable fluorescence (Fv) was determined by the formula: Fv ¼ FmF0.
2. Materials and methods
2.4. Determination of Naþ and Kþ content
2.1. Plant material and treatment conditions
Ten days after treatment, plants were harvested in each treatment, washed with tap water and distilled water thrice respectively and then blotted to remove the excessive water. After that, topmost fully expanded leaf and root samples were dried at 80 C in an oven for 48 h, and then ground into powder. One gram of sample was dry-ashed, extracted with 1:1 HNO3 and finally filtered. Concentrations of Naþ and Kþ elements were determined by flame atomic absorption spectrometry (model AA6300, SHIMADZU, Kyoto, Japan).
Healthy seeds of B. napus L. cv. ZS 758 were obtained from the College of Agriculture and Biotechnology, Zhejiang University and were sown in plastic pots (170 mm 220 mm) filled with peat. Thirty days after sowing, morphologically uniform seedlings were selected and plugged in plate holes on plastic pots (five plants per pot) containing half-strength nutrient solution [15], aerated continuously with an air pump, in the greenhouse. The pH of the solution was adjusted to 6.0. The seedlings were grown under light intensity in the range of 250e350 mmol m2 s1. The temperature was in the range of 16e20 C, and the relative humidity was approximately 55e60%. Each treatment was replicated three times. The solution was renewed every five days. After a two-week acclimatization period, solutions were adjusted to the desired salinities (0, 100, 200 mM NaCl) and plants were simultaneously treated with an aqueous solution of 5aminolevulinic acid (ALA, provided by Cosmo Oil Co. Ltd., Japan) at a concentration 30 mg L1 by foliar spray. The concentrations of NaCl and ALA were determined based on our preliminary studies. Lower as well as upper leaf surface was sprayed until wetted with a hand-held atomizer, as it was reported that absorption by the lower leaf surface is rapid and effective [16]. A subsequent application was made five days after 1st spray. Plants sprayed with distilled water served as the control. Ten days after treatment, plants were harvested and data was recorded for different parameters as described below: 2.2. Determination of photosynthetic pigments Chlorophyll a (Chl a), chlorophyll b (Chl b) and total carotenoids were spectrophotometrically determined according to Pei et al. [17]. After ten days of treatment, topmost fully expanded leaves were taken to extract the pigments. A sample of 0.5 g of fresh leaves was homogenized in a known volume of 85% (v/v) aqueous acetone.
2.3. Photosynthetic efficiency measurement
2.5. Transmission electron microscopy Topmost fully expanded leaf fragments without veins of randomly selected plants were fixed overnight in 4% glutaraldehyde (v/v) in 0.1 M PBS (Sodium Phosphate Buffer, pH 7.4) and washed three times with same PBS. The samples were post fixed in 1% OsO4 (osmium (VIII) oxide) for 1 h, then washed three times in 0.1 M PBS (pH 7.4) with 10 min interval between each washing. Then with 15e20 min interval, they were dehydrated in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100%) and finally by absolute acetone for 20 min. The samples were then infiltrated and embedded in Spurr’s resin overnight. After heating the specimens at 70 C for 9 h, ultra-thin sections (80 nm) were prepared and mounted on copper grids for viewing by a transmission electron microscope (JEOL TEM-1230EX) at an accelerating voltage of 60.0 kV [18]. Data for the size (circumference) of whole mesophyll cells and chloroplasts were recorded using software JeDa 801D Morphology Image Analysis Systems. Data were recorded from at least 25 cell samples and were averaged. 2.6. Statistical analysis One-way analysis of variance (ANOVA) was performed for plant growth, biomass and photosynthesis related parameters. Data were analyzed using SAS v.9 software. All results were expressed as mean
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from four replications. Least significant difference (LSD) test was applied at 5% level of probability to separate the means with statistically significant difference.
Table 2 Effects of salinity and 5-aminolevulinic acid on photosynthetic pigments in Brassica napus L. leaves. Each value is the mean of four replicates. Means followed by same small letters are not significantly different at P 0.05.
3. Results
ALA level (mg l1)
NaCl level (mM)
Chlorophyll (mg g1 FW) Chl a
Chl b
Total
3.1. Plant growth indices
0
0 100 200 0 100 200
0.72 0.59 0.41 0.79 0.68 0.61
0.24 0.18 0.14 0.28 0.20 0.17
0.96 0.77 0.55 1.08 0.88 0.78
3.2. Photosynthetic pigments content Effect of different levels of NaCl and ALA, alone or in combination was investigated on the photosynthetic pigments (Chl a, Chl b, total Chl and carotenoids) in oilseed rape topmost fully expanded leaves (Table 2). Chlorophyll and carotenoids contents were significantly decreased with increasing NaCl concentration and this decrease was highest under 200 mM NaCl compared to the control plants. However, the plants treated with ALA in combination with NaCl (100 and 200 mM) enhanced all types of pigments contents significantly compared to the corresponding plants under NaCl stress. Moreover, plants treated with ALA alone also increased their pigments contents significantly compared to that of control plants. 3.3. Photosynthetic efficiency Photosynthetic efficiency changes were recorded in terms of Fv/ Fm, induced by NaCl and ALA alone or combined in oilseed rape topmost fully expanded leaves (Fig. 1). A gradual decline was observed in photosynthetic efficiency with increasing salinity. However, the declining effect (22.11%) at 200 mM NaCl was much pronounced and significant compared to the control plants. However, the foliar application of ALA helped the plants overcome salinity stress and recover their photosynthetic efficiency significantly compared to the control plants. Photosynthetic efficiency values of 100 mM NaCl-treated plants and ALA together were comparable to those of control plants. Moreover, ALA significantly enhanced the photosynthetic efficiency of the plants under 200 mM NaCl compared to the plants under 200 mM NaCl alone. In addition, the maximum photosynthetic efficiency values were observed in plants treated with ALA alone.
Table 1 Effects of salinity and 5-aminolevulinic acid on shoot length and biomass of Brassica napus L. plants. Each value is the mean of four replicates. Means followed by same small letters are not significantly different at P 0.05. ALA level (mg l1)
NaCl level (mM)
Shoot length (cm)
0
0 100 200 0 100 200
24.74 24.59 19.89 25.19 24.74 22.60
30
a a c a a b
Shoot biomass (g plant1) Fresh
Dry
16.67 b 15.02 ab 9.78 d 17.29 a 16.65 ab 13.62 c
1.40 1.33 1.10 1.70 1.40 1.32
30
b d e a c d
0.43 0.38 0.27 0.46 0.44 0.39
b c d a ab c
Topmost fully expanded leaves and roots of oilseed rape plants were analyzed for Naþ and Kþ accumulation (Fig. 2). In the absence of salt, the Naþ contents were negligible (nearly 1 mg g1 DW). The present investigation revealed that Naþ accumulation was NaCl concentration-dependent. The aerial parts accumulated Naþ in more quantity as compared to root of the plants and variations were quite significant (P < 0.05) at both NaCl treatments. Salinity did not change the accumulation of Kþ significantly compared to the control plants. Subsequently, Naþ/Kþ ratios were significantly increased as salinity increased. Application of ALA reduced significantly the accumulation of Kþ and Naþ under 100 mM as well as 200 mM salinity stress, leading to a concomitant reduction in Naþ/ Kþ ratio compared to the plants under salinity stress alone. 3.5. Morphometric changes in whole mesophyll cells and chloroplasts Transmission electron micrographs of whole mesophyll cells and chloroplasts revealed several changes in the size of cells and chloroplasts of the treated plants (Table 3). Increasing salinity alone or in combination with ALA did not induce any significant change in cell size as compared to that of control. The plants exposed to the highest concentration of NaCl (200 mM) alone, reduced significantly the cell diameter over other treatments but this decrease was recovered if the plants treated with ALA and NaCl together.
1.2 NaCl NaCl+ALA
1
a a
a b
0.8
c
d 0.6
0.4
0.2
0 0
b b c a b b
b d e a c d
3.4. Naþ and Kþ uptake and accumulation
Fv/Fm
The effect of different levels of salinity (NaCl) and 5aminolevulinic acid (ALA) on plant growth was recorded in terms of shoot length and biomass (fresh and dry weight) (Table 1). It was observed that increasing salinity level imposed inhibitory effects on shoot length, fresh and dry weight of shoot. However, this inhibitory effect was more prominent at 200 mM NaCl-salinity level and significantly different (P < 0.05) as compared to their respective controls. ALA treatment helped the plants counteract salinity stress conditions. The plants under 100 mM sodium chloride salinity were completely recovered by ALA application while 200 mM NaCltreated plants also showed substantial recovery.
b c d a b c
Carotenoids (mg g1 FW)
100
200
NaCl (m M) Fig. 1. Effects of salinity (NaCl, 0e200 mM) and 5-aminolevulinic acid (ALA, 0e30 mg l1) on photosynthetic efficiency (Fv/Fm) in Brassica napus L. leaves. Means of four replicates SE. Means followed by same small letters are not significantly different by the LSD test at P 0.05.
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Fig. 2. Effects of salinity (NaCl, 0e200 mM) and 5-aminolevulinic acid (ALA, 0e30 mg l1) on sodium (Naþ), potassium (Kþ) uptake and Naþ/Kþ ratio in Brassica napus L. Means of four replicates SE. Means followed by same small letters are not significantly different by the LSD test at P 0.05.
Chloroplast size and width were increased significantly with increasing salinity as compared to that of control plants and this increase was reversed by foliar application of ALA. However, the chloroplast length was decreased significantly only in the plants treated with 200 mM NaCl alone. Foliar application of ALA to NaCltreated plants increased the chloroplast length and the length values were at par with that of control plants. 3.6. Ultrastructural changes in whole mesophyll cells NaCl or ALA alone or together induced changes in whole mesophyll cells (Fig. 3). In NaCl-treated plants, irregular shaped cells with relatively less developed cell wall, containing nucleus without nucleolus, wide intercellular spaces and swollen chloroplasts, especially under 200 mM NaCl (Fig. 3C) were observed as compared to the control or ALA alone treated plant cells (Fig. 3AD). However, foliar application of ALA reversed the aforementioned maladies induced by NaCl (Fig. 3E and F). 3.7. Ultrastructural changes in chloroplasts Chloroplasts of control leaves were elongated with a typical arrangement of thylakoid membranes having intact stacks of grana and stroma (Figs. 4 and 5A). Low salinity level (100 mM NaCl)
treated plants showed slight changes in the ultrastructure of chloroplast (Figs. 4 and 5B). Under higher saline conditions (200 mM NaCl), drastic changes were observed, and the chloroplasts were swollen (Fig. 4C) and contained a large number of plastoglobuli. Their thylakoid membranes showed dilations, the spaces between the membranes looked swollen, and undulated thylakoid areas developed (Fig. 5C). In particular, the number of grana stacks and starch grains were considerably reduced. Plants treated with ALA alone, showed a typical chloroplast ultrastructure with no significant change (Figs. 4 and 5D). However, foliar application of ALA on NaCl-treated plants helped to alleviate the damage and thus the chloroplast ultrastructures were more stable (Fig. 4). Chloroplasts were less swollen, having organized thylakoid membrane system, clearly differentiated into grana stacks, relatively less plastoglobuli and more starch grains were observed (Fig. 5E and F). 4. Discussion Salinity is a complex trait which affects almost every aspect of the physiology and biochemistry of plants through osmotic, ionspecific and oxidative stress. It needs to be addressed and answered urgently by any feasible strategy. To understand how 5aminolevulinic acid (ALA) ameliorates the salinity-induced
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Table 3 Effects of salinity and 5-aminolevulinic acid on the size of whole mesophyll cell and chloroplast in Brassica napus L. leaves. Means followed by same small letters in the same row are not significantly different at P 0.05. Parameter (mm)
Control
100 mM NaCl
200 mM NaCl
30 mg l1 ALA
100 mM NaCl þ 30 mg l1 ALA
200 mM NaCl þ 30 mg l1 ALA
Cell size Cell diameter Chloroplast size Chloroplast length Chloroplast width
90.22 ab 28.42 a 9.71 b 3.72 a 1.22 b
94.12 a 27.15 a 10.32 ab 3.55 a 1.34 ab
87.10 b 19.92 c 12.40 a 2.65 b 1.63 a
88.13 b 26.36 a 9.52 b 3.56 a 1.11 b
87.98 b 25.28 ab 8.26 b 3.65 a 1.29 ab
91.27 ab 22.14 bc 10.13 3.05 ab 1.43 ab
morphological, physiological, ultrastructural changes in oilseed rape (B. napus L.) leaves and how these changes (especially in chloroplasts of mesophyll cells) are related to physiological functions, the present study was conducted on topmost fully expanded leaves of oilseed rape. To the best of our knowledge, this is the first research about ALA ameliorative effect on salinity-induced changes in the leaf ultrastructure of oilseed rape.
Plant growth is a function of photosynthesis, water conductivity, osmotic potential, and threshold turgor among other factors. When these agents are insufficient, growth does not occur. In the present investigation, we observed a decreasing trend in the length, fresh and dry weight of shoot (Table 1) with increasing salinity which reflects the increased metabolic energy cost and reduced carbon gain, which are associated with salt adaptation. These findings
Fig. 3. Electron micrographs of whole mesophyll cells of oilseed rape (Brassica napus L.) under (A) control; (B) 100 mM NaCl alone; (C) 200 mM NaCl alone; (D) 30 mg l1 ALA alone; (E) 100 mM NaCl þ 30 mg l1 ALA; (F) 200 mM NaCl þ 30 mg l1 ALA. NaCl-treated mesophyll cells are showing a relatively less developed cell wall especially under 200 mM NaCl alone, nucleus without nucleolus, wide intercellular spaces, swollen chloroplasts as compared to the control or ALA alone, treated mesophyll cells. However, ALA þ NaCl combinations reversed the above mentioned maladies induced by NaCl. CW, cell wall; CH, chloroplast; ICS, intercellular spaces; N, nucleus; Nue, nucleolus; Vac, vacuole; S, sugar grains. Bars AF ¼ 5 mm.
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Fig. 4. Relatively low magnified view of chloroplast in mesophyll cells of oilseed rape (Brassica napus L.) under (A) control; (B) 100 mM NaCl alone; (C) 200 mM NaCl alone; (D) 30 mg l1 ALA alone; (E) 100 mM NaCl þ 30 mg l1 ALA; (F) 200 mM NaCl þ 30 mg l1 ALA. Under saline conditions, chloroplasts swelling with dilations in thylakoid membranes, reduction of starch granules, relatively more plastoglobuli are evident especially under 200 mM NaCl compared to the control or ALA alone, treated mesophyll cells. However, ALA þ NaCl combination reduced the chloroplasts swelling with more organized thylakoid membranes, increased the starch granules, relatively less plastoglobuli are evident. CW, cell wall; CH, chloroplast; st, stroma thylakoids; Gt, grana thylakoids; S, sugar grains; Pg, plastoglobuli. Bars AF ¼ 0.2 mm.
agree with those of Yusuf et al. [19] in Brassica juncea L. under NaCl stress. However, foliar application of ALA increased the aforementioned growth indices by improving salt tolerance; as reported by Watanabe et al. [10] in cotton under NaCl stress. Salinity induces a serious decline in chlorophyll content, fluorescence, caroteniods and photosystem II (PSII) activities in several plants [20]. Reduction observed in the present study (Table 2) may be either due to a decrease in chlorophyll synthesis or an increase in chlorophyll degradation or both reasons. In fact, salinity declines the accumulation of ALA [21] which is a precursor of all tetrapyrroles like protochlorophyllide (that is converted in chlorophyll when exposed to light). In the present study, exogenous application of ALA (a key precursor of chlorophyll) might induce biosynthesis of chloroplasts which is reflected by the raised chlorophyll content (Fig. 1). It is well documented that carotenoids play a protective role against photo-oxidation by dissipating the excessive energy of
excitation [6]. Accordingly, the reason for increased amount of leaf carotenoids in ALA-treated plants in the present investigation may be an influence of such protective mechanisms that counteracts the deleterious effects of oxidative damage resultant from salt stress. Our results are consistent with those of Wang et al. [22]; they reported that ALA may regulate the biosynthesis of chlorophyll. The effectiveness of ALA in enhancing photosynthetic efficiency can therefore be attributed to the significant improvement of chlorophyll a content and subsequently boosting light-harvesting capabilities of the treated plants. It is well known that the plants under saline conditions, accumulate more Naþ, which disturbs the ionic balance, plant metabolism and induce oxidative damage as revealed by the increasing sodium accumulation in leaves of oilseed rape plants with increasing salinity compared to the control plants. Plant tolerance toward salinity depends on the Kþ status in root and leaves [23]. In
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Fig. 5. Magnified view of chloroplast and thylakoid membranes in mesophyll cells of oilseed rape (Brassica napus L.) under (A) control; (B) 100 mM NaCl alone; (C) 200 mM NaCl alone; (D) 30 mg l1 ALA alone; (E) 100 mM NaCl þ 30 mg l1 ALA; (F) 200 mM NaCl þ 30 mg l1 ALA. CW, cell wall; CH, chloroplast; st, stroma thylakoids; Gt, grana thylakoids; S, sugar grains; Pg, plastoglobuli. Bars AF ¼ 0.1 mm.
this study, selective Kþ uptake is evident in leaves of the plants under increasing salinity. Although salinity did not affect Kþ content, but increased Naþ content in leaves substantially raised Naþ/Kþ ratio (Fig. 2). Cytosolic Naþ/Kþ ratio depends on the ability of plants to extrude Naþ either outside the cell or into the cell vacuoles by Naþ/Hþ antiport. Similar physiochemical structure of Naþ and Kþ may also affect the Naþ/Kþ ratio. Naþ/Kþ ratios equal to or smaller than 0.6 are necessary for an optimal metabolic efficiency in non-halophyte plants. Consistent with the findings of Watanabe et al. [10] on cotton seedlings, ALA reduced the accumulation of Naþ and Kþ, leading to a reduction of Naþ/Kþ ratio compared to the respective salt-treated plants. Higher accumulation of sodium in the leaves than roots (data not shown) suggest that ALA does not affect Naþ transport from the roots to the shoots but it might suppress the inflow of Naþ. Reports have shown that chloroplast ultrastructures in both halophytes and glycophytes are considerably affected by salinity [24]. Chloroplast swelling in salt-treated plants may be induced by an
osmotic imbalance between stroma and cytoplasm (Figs. 4 and 5). Membranes like thylakoids are particularly susceptible to disturbance by salt [25]. The distortion of grana stacking and swelling of thylakoids caused by salinity in the present study is probably due to a change in the ionic composition of the stroma liquid. This argument is also supported by the Naþ accumulation in the leaves in the present investigation. An increase in the number of plastoglobuli, observed in this study is a typical indicator of leaf senescence and is connected with the degradation of thylakoids or cell membrane, and they disappear if the stress conditions are over. The accumulation of lipid droplets (plastoglobuli) is also considered as reserves of energy for metabolism required to tolerate salinity. Plastoglobuli are ubiquitous in chloroplasts and chromoplasts, and play their role in salt tolerance. However, the decrease of starch granules in the present study is associated with osmotic stress and advancing of senescence [26]. Reduction in starch granules can also be attributed to enhanced conversion of starch into soluble sugars which are considered as compatible solutes and play an important role in osmotic adjustment.
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In fact, salinity induces the overproduction of reactive oxygen species (ROS) which initiate the chloroplast damage. Under salt stress conditions, plants reduce transpiration by closing the stomata, which consequently decreases the CO2 concentration inside the chloroplasts. The lower concentration of CO2 inside the chloroplasts reduces NADPþ generation by the Calvin cycle [27] and allows electrons from ferredoxin to reduce O2 as an alternative acceptor, the formation of superoxide ðO 2 Þ initiates a chain reaction to produce toxic ROS like hydrogen peroxide (H2O2) and hydroxyl radical ð$ OHÞ and induce oxidative stress to damage the chloroplasts in plants exposed to salinity. The plants seemed to use the additional energy supply under foliar application of ALA for increasing the investment in salinity tolerance mechanisms, for example, for reducing oxidative stress and water loss. Elongated chloroplasts with no swelling and only minor dilations of the thylakoids in ALA and NaCl-treated plants are the concrete indication of less oxidative stress. Another difference in the chloroplasts of the plants grown under NaCl and ALA application together was that the number of starch grains increased. This indicates that the plants accumulate starch under favorable growth conditions and they metabolize a larger amount of starch under saline condition to have a suitable pool of osmolytes like soluble sugars [22] for osmotic adjustment. Relatively less number of lipid droplets (plastoglobuli) in chloroplasts of plants treated with NaCl and ALA together is another indication of lesser oxidative stress, in other words we can say that ALA reduced the lipid peroxidation [28] of thylakoids and cell membranes by inducing the antioxidant system. This assumption is in accordance with a study which has shown that ALA induced the activities of heme-based antioxidant enzymes like APX and CAT [29]. The increasing activity of APX catalyzed the breakdown of H2O2 in Halliwell-Asada pathway originally described in the chloroplast [30], in order to protect the thylakoids against the potentially cytotoxic species of activated oxygen under the condition of NaCl stress. This argument is also supported by Bhaya and Castelfranco [31] and Castelfranco and Jones [32] who reported that exogenous application of ALA boasts up the activity of heme-based biomolecules. Thus, we can assume that exogenous ALA application alone or in combination with salinity enhanced the activity of hemebased enzymatic antioxidants and scavenged the ROS efficiently produced under salinity conditions, leading to avoid the damage of chloroplast ultrastructures. 5. Conclusions No apparent damage was observed on the aerial parts of plants in response to 100 mM salinity, which confirms the tolerant nature of B. napus L. cv. ZS 758 toward low salinity. However, exogenous application of ALA recovered the plants treated with 200 mM NaCl by inducing the conformational changes in the ultrastrutures of chloroplasts of mesophyll cells. Higher accumulation of sodium in the leaves than roots suggests that ALA does not affect Naþ transport from the roots to the shoots but it might suppress the inflow of Naþ. NaCl induced senescence-like situation in the leaves of oilseed rape as revealed by an increase in number of plastoglobuli and is connected with the degradation of thylakoids. However, ALA treatment avoids this physiological senescence by developing the more organized granal stacks in chloroplasts and reduced the number of plastoglobuli. Our study is based on the plants grown in hydroponics growth conditions. In order to study the more practical ameliorative effects of ALA on oilseed rape plants grown under saline conditions in the real soil environment further research is required.
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Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2011AA10A206), the National Key Science & Technology Supporting Program of China (2010BAD01B01, 2010BAD01B04), the National Natural Science Foundation of China (31000678, 31071698), the Industry Technology System of Rapeseed in China (nycytx-005), and the Higher Education Commission of Pakistan. References [1] T.J. Flowers, Improving crop salt tolerance, J. Exp. Bot. 55 (2004) 307e319. [2] R.W. Cumming, J.L. Elliot, Soil chemical properties, in: P.E.V. Charman, B.W. Murphy (Eds.), Soil: Their Properties and Management, Sydney University Press, Melbourne, 1991. [3] J.M. Cheesman, Mechanisms of salinity tolerance in plants, J. Plant Physiol. 87 (1988) 547e550. [4] M. Soussi, A. Ocana, C. Lluch, Effect of salt stress on growth, photosynthesis and nitrogen fixation in chickpea (Cicer arietinum L.), J. Exp. Bot. 14 (1998) 1329e1337. [5] J.M. Gómez, J.A. Hernández, A. Jiménez, L.A. Del Río, F. Sevilla, Differential response of antioxidative enzymes of chloroplasts and mitochondria to longterm NaCl stress of pea plants, Free Radic. Res. 31 (1999) 11e18. [6] R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci. 7 (2002) 405e410. [7] J.A. Hernández, E. Olmos, F.J. Corpas, F. Sevilla, L.A. Del Río, Salt-induced oxidative stress in chloroplasts of pea plants, Plant Sci. 105 (1995) 151e167. [8] R.C. Bindu, M. Vivekanandan, Hormonal activities of 5-aminolevulinic acid in callus induction and micropropagation, Plant Growth Regul. 26 (1998) 15e18. [9] L.J. Wang, W.B. Jiang, H. Liu, W.Q. Liu, L. Kang, X.L. Hou, Promotion by 5aminolevulinic acid of germination of Pakchoi (Brassica camestris ssp. chinensis var. communis Tsen et Lee) seeds under salt stress, J. Integr. Plant Biol. 47 (2005) 1084e1091. [10] K. Watanabe, T. Tanaka, H. Kuramochi, Y. Takeuchi, Improving salt tolerance of cotton seedling with 5-aminolevulinic acid, Plant Growth Regul. 32 (2000) 97e101. [11] Z.J. Zhang, H.Z. Li, W.J. Zhou, Y. Takeuchi, K. Yoneyama, Effect of 5aminolevulinic acid on development and salt tolerance of potato (Solanum tuberosum L.) microtubers in vitro, Plant Growth Regul. 49 (2006) 27e34. [12] E.J.J. Momoh, W.J. Zhou, B. Kristiansson, Variation in the development of secondary dormancy in oilseed rape genotypes under conditions of stress, Weed Res. 42 (2002) 446e455. [13] M. Ashraf, T. McNeilly, Salinity tolerance in brassica oilseeds, Crit. Rev. Plant Sci. 23 (2004) 157e174. [14] N. Geissler, S. Hussin, H.W. Koyro, Elevated atmospheric CO2 concentration ameliorates effects of NaCl salinity on photosynthesis and leaf structure of Aster tripolium L, J. Exp. Bot. 60 (2009) 137e151. [15] D.I. Arnon, D.R. Hoagland, Crop production in artificial solution with special reference to factors affecting yield and absorption of inorganic nutrients, Soil Sci. 50 (1940) 463e485. [16] H.M. Hull, H.L. Morton, J.R. Wharrie, Environmental influence on cuticle development and resultant foliar penetration, Bot. Rev. 41 (1975) 421e451. [17] Z.F. Pei, D.F. Ming, D. Liu, G.L. Wan, X.X. Geng, H.J. Gong, W.J. Zhou, Silicon improves the tolerance to water deficit stress induced by polyethylene glycol in wheat seedlings, J. Plant Growth Regul. 29 (2010) 106e115. [18] U. Najeeb, L. Xu, S. Ali, G. Jilani, H.J. Gong, W.Q. Shen, W.J. Zhou, Citric acid enhances the phytoextraction of manganese and plant growth by alleviating the ultrastructural damages in Juncus effusus L, J. Hazard. Mater. 170 (2009) 1156e1163. [19] M. Yusuf, S.A. Hasan, B. Ali, S. Hayat, Q. Fariduddin, A. Ahmad, Effect of salicylic acid on salinity induced changes in Brassica juncea L. J. Integr. Plant Biol. 50 (2008) 1096e1102. [20] C. Santos, H. Azevedo, G. Caldeira, In situ and in vitro senescence induced by KCl stress: nutritional imbalance, lipid peroxidation and antioxidant metabolism, J. Exp. Bot. 52 (2001) 351e360. [21] A. Stobart, W. Griffiths, I. Ameen-Bukhari, R. Sherwood, The effect of Cd2þ on the biosynthesis of chlorophyll in leaves of barley, Physiol. Plant 63 (1985) 293e298. [22] L.J. Wang, W.B. Jiang, B.J. Huang, Promotion of 5-aminolevulinic acid on photosynthesis of melon (Cucumis melo) seedlings under low light and chilling stress conditions, Physiol. Plant 121 (2004) 258e264. [23] F.J.M. Maathuis, A. Amtmann, Kþ nutrition and Naþ toxicity: the basis of cellular Kþ/Naþ ratios, Ann. Bot. 84 (1999) 123e133. [24] N.V. Paramonova, N.I. Shevyakova, Kuznetsov VlV, Ultrastructure of chloroplasts and their storage inclusions in the primary leaves of Mesembryanthemum crystallinum affected by putrescine and NaCl, Russ. J. Plant Physiol. 51 (2004) 86e96. [25] N.R. Baker, A possible role for photosystem II in environmental perturbations of photosynthesis, Physiol. Plant 81 (1991) 563e570.
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