How Bruguiera gymnorhizza seedlings respond to climate change induced salinity rise?

A study was undertaken during August 2017 to evaluate the effect of salinity on chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid and proline contents of hydroponically grown seedlings of Bruguiera gymnorhizza. The primary aim was to observe its tolerance to changing salinity. The selected seedlings were exposed to fi ve different salinity levels (2,5,10,15 and 20psu) for a period of 30 days and observations were done at a regular interval of 7,14,21 and 30 days respectively. The concentrations of chlorophyll exhibited signifi cant positive correlations with salinity (p<0.01). The chlorophyll a:b ratio in the plant varied between 2.39 to 3.71 throughout the period of investigation. The salinity fl uctuation did not affect the carotenoid level and proline content in the leaves of the species as evidenced from the insignifi cant r values. The results show that Bruguiera gymnorhizza of Indian Sundarbans region can tolerate and adapt to high saline condition as witnessed in the central sector of the deltaic complex around the Matla River. Research Article How Bruguiera gymnorhizza seedlings respond to climate change induced salinity rise? Prosenjit Pramanick1, Subrata Trivedi2, Sufi a Zaman1 and Abhijit Mitra3* 1Department of Oceanography, Techno India University West Bengal, Salt Lake Campus, Kolkata 700091, India 2Department of Biology, Faculty of Science, University of Tabuk, Ministry of Higher Education, Kingdom of Saudi Arabia 3Department of Marine Science, University of Calcutta, 35 B.C. Road, Kolkata 700019, India *Address for Correspondence: Abhijit Mitra, Department of Marine Science, University of Calcutta, 35 B.C. Road, Kolkata 700019, India, Email: abhijit_mitra@hotmail.com Submitted: 15 September 2017 Approved: 20 September 2017 Published: 22 September 2017 Copyright: 2017 Pramanick P, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Pramanick P, Trivedi S, Zaman S, Mitra A. How Bruguiera gymnorhizza seedlings respond to climate change induced salinity rise? Ann Adv Chem. 2017; 1: 047-052. https://doi.org/10.29328/journal.aac.1001005 Introduction Bruguiera gymnorhizza belongs to the “true mangroves” and is very common in the Orient. The name gymnorhizza comes from two Greek words “gymno” naked and “rhizza” root, naked root which refers to the exposed knee roots of Bruguiera gymnorhizza emerging from the ground. The main difference from other Bruguiera species is that Bruguiera gymnorhizza has the largest leaves, lowers, propagules and lenticels of all Bruguiera species. The name Large-Leafed Orange Mangrove of Bruguiera gymnorhizza comes from the orange lowers and the large leaves that can reach up to 25 cm in length. Salinity plays a crucial role in the growth and survival of mangroves. However saline condition is not a pre-requisite for their development, rather mangroves choose saline condition to avoid the competition with the more vigorous terrestrial plants. Based on the physiological studie [1,2] concluded that mangroves are not salt lovers, rather salt tolerant. However, excessive saline conditions retard seed germination, impede growth and development of mangroves. Indian Sundarbans, the famous mangrove chunk of the tropics is gradually losing few mangroves species (like Heritiera fomes, Nypa fruticans etc.) owing to increase of salinity in the central sector of the deltaic complex around the Matla River. Reports on alteration of growth in mangroves due to difference in salinity between western and central sectors of Indian Sundarbans are available [3]. However no study has yet been carried out on the effect of salinity luctuation on the biochemical components of mangroves under culture conditions How Bruguiera gymnorhizza seedlings respond to climate change induced salinity rise? Published: September 22, 2017 48/52 from this part of the Indian subcontinent. The effects of salinity on mangroves have been studied in relation to antioxidative enzymes [4,5], leaf structure, rates of transpiration, stomatal conductance and rates of photosynthesis [6,7] and changes in chloroplast structure and function [8,9] reported that Na+/H+ anti-port catalyzed exchange of Na+ for H+ across the vacuolar membrane of the cells of Bruguiera sexangula offer tolerance to ionic stress imposed by NaCl and this mechanism is important for cellular salinity adjustments. Also, the mechanism of acclimation to salt in mangroves was suggested to be linked to the changes in the vacuolar size in B. sexangula [10]. Further, one of the biochemical mechanisms by which mangroves counter the high osmolarity of salt was accumulation of compatible solutes [4]. Proline has also been found to be an effective osmoregulating compounds that increase under high saline condition as a mechanism to combat salinity stress. In this paper, we present the effect of increasing salinity on pigments and proline content of hydroponically grown seedlings of Bruguiera gymnorhizza with an aim to obtain insights into the changes in these biochemical components with salt acclimation. Materials and Methods Plant materials and culture conditions Seedlings of Bruguiera gymnorhizza were collected from Sundarbans mangrove ecosystem of India (22°16′40.6′′ N latitude and 88°38′18.4′′ E longitude) during August, 2017. They were raised in the laboratory condition by diluting the source water with stored rain water. The source water was collected from high saline zones of Sundarbans (salinity=30 psu). Two month old healthy seedlings were subjected to hydroponic culture in Hoagland’s nutrient medium (pH=5.8-6.0) under photosynthetically active radiation (PAR) of 1220-1236 μmol m-2s-1 .The preliminary experiments were carried out in the selected species at ive different salinities (2psu,5psu,10psu,15psu and 20psu respectively) in order to determine the optimum range of salinities in relation to photosynthetic pigments, carotenoids and proline. The cultures were aerated continuously with an air bubbler. The hydroponic cultures were maintained in a culture room under a 14h photoperiod at PAR of 300 μmol m-2s-1, 26±3°C and 80% RH. The culture medium was changed every 7days. Leaves were harvested at 7,14,21 and 30 days intervals to measure the pigment and proline concentrations. Extraction and estimation of pigments Leaves (0.5g) were homogenized in chilled N, N-dimethylformamide (DMF) in a mortar and pestle in dark at 4°C and the homogenates were centrifuged at 8800×g for 10 min. The supernatants were collected and absorption spectra at 663.8 and 646.8nm were recorded using Jasco V-530 UV–vis spectrophotometer for estimation of chlorophyll a, chlorophyll b and total chlorophyll following the procedure of [11]. For estimation of total carotenoids, leaf tissues (0.5g) were homogenized in chilled 80% (v/v) acetone and the homogenates were centrifuged at 8800×g for 10 min at 4°C in the dark. The absorbance of the acetone extracts was measured at 663,645 and 470 nm. Total carotenoids were calculated according to [12]. Estimation of free proline Free proline content was measured from leaf using 3% sulphosalicylic acid following the method of [13], using L-proline (Sigma) as standard. Statistical Analysis Statistical analysis of the results was carried out according to Duncan’s multiple range tests. Data were also subjected to analysis of correlation coef icient (r) in order to evaluate the inter-relationship between salinity, selected pigments and proline content of the leaves of the selected species following the method of [14]. How Bruguiera gymnorhizza seedlings respond to climate change induced salinity rise? Published: September 22, 2017 49/52 Results All the collected seedlings of Bruguiera gymnorhizza could tolerate salinity up to 20psu and could be maintained for more than 30days. The concentrations of chlorophyll increased signi icantly with salinity (Table 2). The total chlorophyll expressed on unit fresh weight basis increased considerably (Table 1). The chlorophyll a:b ratio in the plant, however, remained almost constant for the species and varied only marginally during the period under observation. In our experiments with differential salinity exposure the chlorophyll a:b ratio yielded values ranging between 2.39 to 3.71 (Table 1). The increase of the photopigments with aquatic salinity is statistically signi icant (Table 2) and re lects the ef iciency of photosynthetic machinery of the species even in high saline condition. As the chlorophyll a:b ratio remained unaffected at high saline condition in the selected species, it appears that the light harvesting complex (LHCs) of thylakoid membranes are little altered by salt exposure. The species thus seems to have a higher tolerance to increased salinity that may occur during climate change induced sea level rise in vulnerable islands of Sundarbans [15]. Till date there have been few studies on the effect of salinity on photosynthetic gas exchange in mangroves. [16] stated in his communication that the rate of light saturated photosynthesis decreases with increasing salinity of ambient media, attributing this to co-limitation of assimilation rate by stomatal conductance and photosynthetic capacity in response to differences in water status induced by the various salinity treatments. Thus, on the evidences available so far it is most likely that salinity exerts its effect on photosynthesis mainly through changes in leaf water status and this study reveals that the photosynthetic process may be affected at high saline condition due to decrease in chlorophyll a and b concentrations in mangroves. The present study is different from several works as the salinity of water has been altered naturally (through dilution with rain water) keeping the all the constituent Table 1: Effects of different salinities on p


Introduction
Bruguiera gymnorhizza belongs to the "true mangroves" and is very common in the Orient. The name gymnorhizza comes from two Greek words "gymno" naked and "rhizza" root, naked root which refers to the exposed knee roots of Bruguiera gymnorhizza emerging from the ground. The main difference from other Bruguiera species is that Bruguiera gymnorhizza has the largest leaves, lowers, propagules and lenticels of all Bruguiera species. The name Large-Leafed Orange Mangrove of Bruguiera gymnorhizza comes from the orange lowers and the large leaves that can reach up to 25 cm in length.
Salinity plays a crucial role in the growth and survival of mangroves. However saline condition is not a pre-requisite for their development, rather mangroves choose saline condition to avoid the competition with the more vigorous terrestrial plants. Based on the physiological studie [1,2] concluded that mangroves are not salt lovers, rather salt tolerant. However, excessive saline conditions retard seed germination, impede growth and development of mangroves. Indian Sundarbans, the famous mangrove chunk of the tropics is gradually losing few mangroves species (like Heritiera fomes, Nypa fruticans etc.) owing to increase of salinity in the central sector of the deltaic complex around the Matla River. Reports on alteration of growth in mangroves due to difference in salinity between western and central sectors of Indian Sundarbans are available [3]. However no study has yet been carried out on the effect of salinity luctuation on the biochemical components of mangroves under culture conditions from this part of the Indian subcontinent. The effects of salinity on mangroves have been studied in relation to antioxidative enzymes [4,5], leaf structure, rates of transpiration, stomatal conductance and rates of photosynthesis [6,7] and changes in chloroplast structure and function [8,9] reported that Na + /H + anti-port catalyzed exchange of Na + for H + across the vacuolar membrane of the cells of Bruguiera sexangula offer tolerance to ionic stress imposed by NaCl and this mechanism is important for cellular salinity adjustments. Also, the mechanism of acclimation to salt in mangroves was suggested to be linked to the changes in the vacuolar size in B. sexangula [10]. Further, one of the biochemical mechanisms by which mangroves counter the high osmolarity of salt was accumulation of compatible solutes [4]. Proline has also been found to be an effective osmoregulating compounds that increase under high saline condition as a mechanism to combat salinity stress. In this paper, we present the effect of increasing salinity on pigments and proline content of hydroponically grown seedlings of Bruguiera gymnorhizza with an aim to obtain insights into the changes in these biochemical components with salt acclimation.

Plant materials and culture conditions
Seedlings of Bruguiera gymnorhizza were collected from Sundarbans mangrove ecosystem of India (22°16′40.6″ N latitude and 88°38′18.4″ E longitude) during August, 2017. They were raised in the laboratory condition by diluting the source water with stored rain water. The source water was collected from high saline zones of Sundarbans (salinity=30 psu). Two month old healthy seedlings were subjected to hydroponic culture in Hoagland's nutrient medium (pH=5.8-6.0) under photosynthetically active radiation (PAR) of 1220-1236 μmol m -2 s -1 .The preliminary experiments were carried out in the selected species at ive different salinities (2psu,5psu,10psu,15psu and 20psu respectively) in order to determine the optimum range of salinities in relation to photosynthetic pigments, carotenoids and proline. The cultures were aerated continuously with an air bubbler. The hydroponic cultures were maintained in a culture room under a 14h photoperiod at PAR of 300 μmol m -2 s -1 , 26±3°C and 80% RH. The culture medium was changed every 7days. Leaves were harvested at 7,14,21 and 30 days intervals to measure the pigment and proline concentrations.

Extraction and estimation of pigments
Leaves (0.5g) were homogenized in chilled N, N-dimethylformamide (DMF) in a mortar and pestle in dark at 4°C and the homogenates were centrifuged at 8800×g for 10 min. The supernatants were collected and absorption spectra at 663.8 and 646.8nm were recorded using Jasco V-530 UV-vis spectrophotometer for estimation of chlorophyll a, chlorophyll b and total chlorophyll following the procedure of [11]. For estimation of total carotenoids, leaf tissues (0.5g) were homogenized in chilled 80% (v/v) acetone and the homogenates were centrifuged at 8800×g for 10 min at 4°C in the dark. The absorbance of the acetone extracts was measured at 663,645 and 470 nm. Total carotenoids were calculated according to [12].

Estimation of free proline
Free proline content was measured from leaf using 3% sulphosalicylic acid following the method of [13], using L-proline (Sigma) as standard.

Statistical Analysis
Statistical analysis of the results was carried out according to Duncan's multiple range tests. Data were also subjected to analysis of correlation coef icient (r) in order to evaluate the inter-relationship between salinity, selected pigments and proline content of the leaves of the selected species following the method of [14].

Results
All the collected seedlings of Bruguiera gymnorhizza could tolerate salinity up to 20psu and could be maintained for more than 30days. The concentrations of chlorophyll increased signi icantly with salinity ( Table 2). The total chlorophyll expressed on unit fresh weight basis increased considerably ( Table 1). The chlorophyll a:b ratio in the plant, however, remained almost constant for the species and varied only marginally during the period under observation. In our experiments with differential salinity exposure the chlorophyll a:b ratio yielded values ranging between 2.39 to 3.71 ( Table  1). The increase of the photopigments with aquatic salinity is statistically signi icant ( Table 2) and re lects the ef iciency of photosynthetic machinery of the species even in high saline condition. As the chlorophyll a:b ratio remained unaffected at high saline condition in the selected species, it appears that the light harvesting complex (LHCs) of thylakoid membranes are little altered by salt exposure. The species thus seems to have a higher tolerance to increased salinity that may occur during climate change induced sea level rise in vulnerable islands of Sundarbans [15].
Till date there have been few studies on the effect of salinity on photosynthetic gas exchange in mangroves. [16] stated in his communication that the rate of light saturated photosynthesis decreases with increasing salinity of ambient media, attributing this to co-limitation of assimilation rate by stomatal conductance and photosynthetic capacity in response to differences in water status induced by the various salinity treatments. Thus, on the evidences available so far it is most likely that salinity exerts its effect on photosynthesis mainly through changes in leaf water status and this study reveals that the photosynthetic process may be affected at high saline condition due to decrease in chlorophyll a and b concentrations in mangroves. The present study is different from several works as the salinity of water has been altered naturally (through dilution with rain water) keeping the all the constituent Units of all pigments are mg/gm fresh weight; unit of proline is nmol/gm fresh weight. Different letters besides fi gures indicate statistically different means as at p≤0.01. salts of brackish water constant unlike several previous studies where the plants were exposed to different NaCl concentrations [17,18] that are not the real image of ambient seawater. Various studies have shown that a number of mangrove species grow best at salinities between 4psu and 15psu [19,14,[20][21][22] and for Heritiera fomes, the preferred salinity range is much lower [23]. In this context, Bruguiera gymnorhizza seems to be a be itted species for arid, high saline zone.
The carotenoid pigment of the species showed a mosaic nature on exposure to different salinity. At the end of 7 and 21 days there was no decrease, but after 14 days the carotenoid content in the leaf decreased by 5%. Again the value remained constant at the end of 30days. Overall trend however, indicates no effect of salinity on carotenoid level of the species ( Table 2). The result implies that the species does not alter the synthesis of carotenoid under stress condition and is ideal for high saline environment. Our results is contradictory to several reports of decrease content of chlorophyll and carotenoids by salinity as observed in a number of glycophytes [24,25].
Proline accumulation is a common phenomenon in halophytes. As Bruguiera gymnorhizza is a true halophyte and a salt excreting species, it is of interest to study proline accumulation in response to salinity in this plant. It is well known that proline content in leaves of many plants gets enhanced by several stresses including salt stress [26,27]. Thus, we monitored the proline levels in leaves of the species treated with 2,5,10,15,20psu saline water for 7,14,21 and 30 days. Our results exhibited almost uniform proline content in leaves of the species which is contrary to several reports of accumulation of proline as compatible osmolyte during NaCl exposure [26][27][28]. The constancy of proline value con irms the unique tolerance power of the species even under salinity stress. This uniformity of proline level may be attributed to the activity of proline dehydrogenase, a catabolic enzyme of proline [26]. It appears that the enzyme remain unaffected in Bruguiera gymnorhizza even under high saline condition which is a unique adaptive mechanism.
Our results show that Bruguiera gymnorhizza of Indian Sundarbans region can easily be propagated in saline zone around the Matla River. Even at 15 and 20 psu salinity the chlorophyll pigments showed an increase. The high salinity could not affect the carotenoid and proline content of the species that usually increase under stressful situation.
Indian Sundarbans and its adjacent estuaries situated in the lower Gangetic region at the apex of Bay of Bengal are one of the less studied regions of the world ocean in context to impact of rising salinity luctuation on mangrove loral community, although the region sustains the 5 th largest mangrove chunk in the world (2120 km 2 in the Indian part and 4500 km 2 in the Bangladesh part). The present study is extremely important from the point of view of rising salinity in the central sector of Indian Sundarbans over a period of two decades [15] due to complete obstruction of the freshwater supply of Ganga-Bhagirathi-Hooghly River as a result of heavy siltation since the late 15 th century [23] and rising sea level [29] at the rate of 3.14 mm/yr, which is higher than the global average sea level rise of 2.12 mm/yr and 2.50 mm/yr along the Indian coastline [30]. Increased salinity and lack of freshwater is likely to result in a decrease in mangrove productivity, growth and seedling survival, and may change species composition favoring more salt tolerant species [31,32].
In summary, the results of the present study show that the mangrove Bruguiera gymnorhizza can easily be propagated under high salinity conditions and may be a better suited species for central sector of Indian Sundarbans where the environment is hypersaline in nature. The present study is relevant from the point of adaptation of the species to sea level rise and subsequent saline water intrusion into the islands of Indian Sundarbans. This deltaic system in the lower Gangetic region is vulnerable to climate change related effects owing to its location below the mean sea level and experiencing a sea level rise of 3.14 mm/yr.