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Journal of Stress Physiology & Biochemistry, Vol. 20, No. 3, 2024, pp. 19-28 ISSN 1997-0838 Original Text Copyright © 2024 by Basel Saleh
ORIGINAL ARTICLE
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Physiological and Molecular Response of Padina pavonica (Phaeophyta) brown Alga Towards Cadmium Heavy Metal
1 Department of Molecular Biology and Biotechnology, AECS, P.O. Box 6091, Damascus, Syria *E-Mail: ascientific22@gec.org.sy
Physiological and molecular response of Padina pavonica (phaeophyta) marine alga exposed to different cadmium (0, 2.5, 5 and 10 mg/L) concentrations after 4 days of exposure has been investigated. Physiological data revealed decrease in specific growth rate (SGR%), pigments (Chlorophyll a & b, total chlorophyll and total carotendois) content, osmotic potential with increased electric conductivity (EC) under Cd applied concentrations in the studied alga species. Whereas, carotendois pigments content increased in P. pavonica alga as Cd applied concentrations increased. As for molecular test, RAPD marker has been applied, a decrease in polymorphic bands (PB) from 95 to 87 has been recorded when applied Cd concentration increased from 2.5 to 10 mg/L. Whereas, genomic template stability (GTS%) as a qualitative measurement reflect DNA changes induced by Cd treatment was displayed by RAPD marker. Molecular study revealed increased GTS% value from 30.7 to 42.7% when applied Cd concentration increased from 2.5 to 10 mg/L. Based upon observed physiological (significant increase in carotendois content) and molecular data (decrease in the new induced bands number and increase in disappeared bands as Cd concentration increased from 2.5 to 10 mg/L) in P. pavonica alga, the current investigation could be assumed that P. pavonica alga adopted certain mechanism to minimize Cd stress damages.
Key words: Padina pavonica, Cd heavy metal, Physiological parameters, RAPD, Genotox-
Basel Saleh
*
Received March 11, 2024
icity, DNA variation
From very long time up till now, environmental pollution by heavy metals (HM) was considered as a global public health concern and has been dramatically increased due to their exponentially increased used for various purposes like e.g. industrial, agricultural, domestic and technological applications. Heavy metals (HM) as an abiotic stress caused perturbation of biological activity in living organisms, reflecting in changes at anatomical, morphological, physiological, biochemical and molecular aspects. Cadmium (Cd) among HM was considered as one of the most toxicants in ecosystems; and that their harmful effects on plant could be manifested by membrane distortion, reactive oxygen species (ROS) production, photosynthesis impair, DNA and RNA damages and cell death (Farid et al., 2013).
Many studies reported HM effect in living organisms on physiological parameters (relative growth, pigments including total chlorophyll, chlorophyll a & b, carotenoids, total carotenoids, osmotic potential and electric conductivity); e.g. Cd & Pb in the green alga Cladophora fracta (Lami et al., 2005); Cd in Hypnea musciformis (Rhodophyta) alga (Bouzon et al., 2012); Cd with chelator addition in cyanobacterium Microcystis aeruginosa (Zhou et al., 2006); Cd in Ulva prolifera and Ulva linza green algae (Jiang et al., 2013); Cd in Agarophyte Gracilaria domingensis (Rhodophyta) alga (dos Santos et al., 2012); Cd in Chlorella sp. algae (Kaplan et al., 1995); Cd in the two strains (Iranian and Australian) of Dunaliella salina green algae (Shariati and Yahyaabadi, 2006); Cd in Thalassia hemprichi (Tuapattinaya et al., 2016) and in Brassica juncea species (Kapoor et al., 2014); Ni and Zn in bean (Phaseolus vulgaris L.) (Zengin 2013). Cu, Pb, Zn and Cd metals in Ulva lactuca green alga (Saleh, 2015); Cd in two lichen species of Parmotrema tinctorum and Usnea barbata (Santos et al., 2022). Recently, Nowicka (2022) reviewed heavy metals toxicity in algae and mechanisms involved in their detoxification. More recently, Ejaz et al. (2023) reviewed different strategies developed by plants for heavy metals detoxification.
PCR-based DNA marker systems have been extensively employed for monitoring different effects of HM in living organisms on genetic stability through genetic variations in DNA pattern including the appearance of new bands induced or/and disappearance of control bands ones. For example, random amplified polymorphic DNA (RAPD) analysis for monitoring genomic stability induced by Cd stress in barley (Hordium vulgare) (Liu et al., 2005); RAPD for Cd stress in maize (Zea mays L.) (Mohsenzadeh et al., 2010); RAPD for Cu and Zn stresses in Cucumber (Cucumis sativus L.) (Aydin et al., 2012); RAPD for Cd stress in Okra (A. esculantus L.) (Aydin et al., 2013); random amplified microsatellite polymorphism (RAMP) marker for heavy metals (Cu, Pb, Cd and Zn) stress in U. lactuca green alga (Saleh, 2016) and RAPD for Cd and Pb stresses in Ramalina farinaceae lichen (Hamutoglu, 2021).
Thereby, the current study has been designed for investigation of physiological and molecular response of P. pavonica (phaeophyta) marine alga exposed to different cadmium (0, 2.5, 5 and 10 mg/L) concentrations after 4 days of exposure.
MATERIALS AND METHODS
Sampling
Samples of P. pavonica alga species were collected along the Syrian coast of the Mediterranean Sea. Sampling was carried out from 34°37734"N longitude, 38°29766"E latitude at 4 km North Lattakia - Syria. Only individuals with the similar size were harvested by hand with disposable gloves, biomass was washed with seawater where the alga were collected and then transported within a flask with 5 L seawater. Cultivation of algae and cadmium stress
Algae were washed twice after their arrival to laboratory with autoclaved artificial seawater ASW (500 mM NaCl, 10 mM KCl, 30 mM MgSO4, 10 mM CaCl2 and 10 mM Tris-HCl at pH 7.8) medium as previously described by Unal et al. (2010). Then, they divided to a fresh flask with a fresh ASW previously described solution and placed under controlled laboratory conditions (Temperature of 20'C, photoperiod of 12/12 h dark/light and illumination of 3195 Lux (~ 52.7 ^mol
photons m-2s-1) for 3 days before Cd stress application. The P. pavonica alga species was evaluated under different Cd concentrations under Cd (NO3)2 forma salt [(Standard solution (1000 mg/L) from Fisher Scientific - UK)].
The mentioned ASW was considered as a control. Whereas, Cd stress was applied by adding Cd to achieved 0, 2.5, 5 and 10 mg/L as final concentration for each treatment with three replicates/treatment. Experiment was carried out in flask with 300 mL ASW with or without Cd metal. The same previous described controlled conditions were maintained during the experiment stress application. Four days later, algae were harvested for physiological and molecular studies. Physiological study
The experiment was conducted in triplicates for 4 days. Algal specific growth rate (SGR%) was calculated in both control and stressed conditions according to Nielsen et al. (2012). Whereas, chlorophyll (Chl) and carotendois (Car) pigments were extracted in 80% acetone solvent and estimated as previously described by Lichtenthaler and Wellburn (1985). While, the osmotic potential was measured using a micro-osmometer (Osmomette) apparatus. Moreover, the electric conductivity (EC) was determined by an electric conductivity (Hanna HI 99301, Romania) instrument. Statistical analysis
Statistical analyses for physiological study, were performed using Statview 4.5 (Abacus 1996) statistical package at the 5% significance level (P = 0.05). Data were subjected to analysis of variance (ANOVA) for the determination of differences in means among Cd applied concentrations. Differences between means were tested for significance by Fisher's least significant difference (PLSD) test. Data are expressed as mean of three replicates.
Molecular study
Genomic DNA extraction
Total genomic algal DNA was isolated from (bulk of 3 replicates/ treatment) tissues for both of the control and stressed alga by a CTAB (cetyltrimethylammonium bromide) protocol as previously described by Doyle and Doyle (1987) with minor modifications. DNA
concentration was quantified by DNA Fluorimeter at 260/280 nm and adjusted to final concentration of 10 ng/^L. DNA was stored at -80 °C until needed. RAPD marker and data analysis
To evaluate Cd-genotoxicity in the studied P. pavonica alga species, 22 RAPD primers were tested (Table 1). RAPD assay was performed as previously described by Williams et al. (1990). Then PCR products were separated on a 1.5% ethidium bromide- stained agarose gel (Bio-Rad) in 0.5X TBE buffer. Electrophoresis was performed for 1.5 h at 85 V and visualized with a UV transilluminator. A VC 100bp Plus DNA Ladder (Vivantis) ladder standard was used to estimate molecular weight of amplification products. RAPD data analysis was performed by comparing the PCR products patterns for control with treated Cd algae at the mentioned Cd applied concentrations. Estimation of genomic template stability (GTS%)
Genomic template stability value was estimated as previously described by Atienzar et al. (2002).
RESULTS AND DISCUSSION
Effect of different cadmium (0, 2.5, 5 and 10 mg/L) concentrations has been investigated into P. pavonica (phaeophyta) marine alga after 4 days of exposure to Cd stress at physiological and molecular levels.
Physiological test revealed that cadmium stress caused significant SGR% (p < 0.001) reduction into the studied alga species (Table 2). Moreover, estimated pigmentation content including Chla, Chlfo and total chlorophyll was reduced following Cd stress in different manners according to the applied Cd concentrations (Table 2). In this regards, total chlorophyll decreased with no significant decline in P. pavonica alga as Cd applied concentrations increased.
As for estimated carotene and total carotendois, data showed that Cd stress caused decline in total carotendois in P. pavonica alga as Cd applied concentrations increased (Table 2). Whereas, carotendois pigments content significantly (p < 0.001) increased in the studied alga as Cd applied concentrations increased (Table 2).
Osmotic potential as a direct response of living organism against given abiotic stress, was significantly
(p < 0.001) decreased as Cd applied concentrations increased in the studied alga species (Table 2). While, Electric conductivity (EC) value increased as Cd applied concentrations increased in the studied alga species (Table 2).
In plant cells, wall acts as barrier to prevent the transport of heavy metals into the cytoplasm under low ion concentration. While, at high concentration the cell wall become disable to capturing all the metal ions and thereby some enter the cell causing cell damages. In sensitive plants exposed to heavy metals, the cytoplasm became disorganized even heavy metals entry the cytoplasm (Jamers et al., 2013).
Physiological test revealed decline in different pigments content in different manner according to the tested Cd concentrations. This reduction increased as Cd applied concentrations increased. In this regards, at 10 mg/L Cd, Ch a content deceased by 21% in P. pavonica alga (Table 2). Whereas, for Ch b, this index was reduced by 48% in P. pavonica alga. While, for total chlorophyll this reduction found to be 30% below their respective control in P. pavonica alga, at 10 mg/L Cd.
Similarly, significant decline in relative growth and total chlorophyll values as applied Cd & Pb concentrations increased has been recorded in the green alga C. fracta (Lami et al., 2005); decline in growth rate and Chl a content in H. musciformis alga exposed to different Cd (9-55 mg/L) concentrations (Bouzon et al., 2012); decline in growth rate and Chl a in M. aeruginosa has been observed following exposure to 0.73 mg/L Cd with chelator addition (0.33 mg/L free Cd, MINEQL calculation) (Zhou et al., 2006); SGR% significantly decreased by 93% and 39% in U. prolifera alga; whereas, this reduction was recorded to be 53% and 75% in U. linza alga at 1.8 and 3.7 mg/L Cd, respectively; combined with decline in Chl content by 18, 25 and 45% at 3.7, 7.4 and 14.8 mg/L Cd in U. prolifera alga, respectively and by 16, 20 and 39% for U. linza alga, respectively under the above applied Cd concentrations, respectively. Indeed, reduction in carotenoids by 16, 29 and 54%, respectively in the case of U. prolifera alga and by 13, 16 and 44% below the control for U. linza alga, respectively under the above applied Cd concentrations, respectively (Jiang et al.,
2013); reduction in pigmentations e.g. Chl a and phycobiliproteins has been observed in Agarophyte G. domingensis (Rhodophyta, Gracilariales) alga exposed to different Cd (0, 0.1, 0.2 and 0.3 mg/L) concentrations (dos Santos et al., 2012). However, in the current study, this parameter it sharply increased in P. pavonica alga by 1 and 80% at 2.5 and 10 mg/L Cd, respectively.
Researches demonstrated how living organisms | (plants and algae) developed different mechanisms defense systems against adverse effects caused by heavy metals stress through induction of different antioxidants compounds types. In this regards, Collen et al. (2003) reported that, algae exposed to heavy metals tend to accumulate some antioxidant defenses e.g. flavonoids and carotenoids pigmentations as a defenses mechanism against ROS induced by heavy metal exposure. Whereas, dos Santos et al. (2012) reported that, algae act as Cd includer or excluder to minimize Cd toxicity and thereby, prevent cell damages after Cd exposure. In this respect, Bouzon et al. (2012) reported that G. domingensis alga tends to accumulate certain antioxidants compounds e.g. flavonoids, tocopherols and carotenoids as a strategy to prevent the negative effects of ROS formation by heavy metals. Whereas, Zengin (2013) reported that carotenoid content significantly increased in the leaves bean (P. vulgaris L.) seedlings at different concentrations of Ni, Co, Cr and Zn heavy metals. Moreover, Pazoki et al. (2014) reported that Pb (0, 300, 600 and 900 mg/kg of soil) caused Chl a and b reduction with carotene content increasing in wheat (T. aestivum L.) as Pb concentrations increased from 300 to 900 mg/kg. Previously, Kaplan et al. (1995) stated the induction of phytochelatins in Chlorella sp. algae exposed to Cd. Moreover, Shariati and Yahyaabadi (2006) reported that increasing Cd concentrations caused a decline in chlorophyll content and increase of beta-carotene pigmentations in the two strains (Iranian and Australian) of D. salina green algae after 5 days exposure to different Cd concentrations (0, 0.005, 0.05 and 0.5 mg/L).
Similarly, Zengin (2013) reported increase in carotenoids content by 8%, 13.5% and 17% at 1.5, 2.0 and 2.5 mM Zn & by 15%, 23.1% and 27.3% at 0.1, 0.3
and 0.5 mM Ni, respectively in bean (Phaseolus vulgaris L.). Our data were in coherent with previous reports; e.g. Increase in carotenoids content in Thalassia hemprichii-Cd treated (Tuapattinaya et al., 2016) and in Brassica juncea-Cd treated species (Kapoor et al., 2014); in bean (Phaseolus vulgaris L.)-Ni and Zn treated (Zengin 2013) and recently in two lichen species of Parmotrema tinctorum and Usnea barbata when Cd concentrations increased from 10 to 500 |M (Santos et al., 2022).
Heavy metals in particularly Pb and Cd generated an oxidative stress, so some of plants species developed some mechanisms defense systems to combat their adverse negative effect through enzymatic systems (CAT, SOD, POX....) or non- enzymatic systems by producing phenolics, carotenoids, proline and some vitamins (Michalak, 2006; Ahmad et al., 2020). In has been demonstrated that carotenoids pigments serve as antioxidants against free radicals and photochemical damage (Sengar et al., 2008).
Carotenoids could be considered as the first defense line against singlet oxygen. They have special chemical structure that is able to neutralize or extinguish the singlet oxygen reactivity and to provide energy to the entire carotenoid molecule (Ramel et al., 2012). To be able to extinguish the singlet oxygen, carotenoids must have at least 9 double bonds with single bonds between the double bonds. This chemical composition is called a double bond conjugation. The energy of singlet oxygen was transferred to the carotenoid and returned to its original energy level. At the time, singlet oxygen has been converted into normal oxygen. In addition to singlet oxygen, the other kinds of ROS molecules can be neutralized by the carotenoids via electron transfer reactions, the formation of radical cluster formation or by transferring hydrogen atom. Thus, the carotenoids will increase concurrently with the increase of the cadmium heavy metal to prevent the damaging effects that can be caused by metal cadmium (Ramel et al., 2012). Increased of carotenoids content in response to heavy metals could play a significant role in photosystem protection as an antioxidant agent (Alsherif et al., 2022). Otherwise, the current study revealed decline in osmotic potential following different Cd applied concentrations. Similarly, Saleh (2015) reported decline in osmotic
potential compared to their respective control in U. lactuca after 5 days exposure to 4 heavy metals (Cu, Pb, Zn and Cd ions). Indeed, physiological data revealed that the estimated EC values increased as Cd concentrations in the studied alga compared to its respective control. In this regards, this index was increased by 83% over the control for P. pavonica alga.
As for molecular study, cadmium stress caused alteration of DNA profile expressed as polymorphic bands (PB) including induction of new bands in comparison to the control (a) and disappearance of a normal control bands (b). DNA changes profiles induced by Cd stress as yielded by OPE15, OPK17 and RPi-C8 RAPD primers in P. pavonica alga have been illustrated in Figure 1.
In this regards, decrease in (a) value from from 51 to 34 bands, has been recorded in P. pavonica (Figure 1) alga when applied Cd concentrations increased from 2.5 to 10 mg/L. Whereas, decrease in (b) value increased from 44 to 53 bands when applied Cd concentrations increased from 2.5 to 10 mg/L. Overall, decline in total PB value from 95 to 87 bands has been recorded in the studied alga when applied Cd concentrations increased from 2.5 to 10 mg/L.
Similar data have been reported by Mohsenzadeh et al. (2010), who reported genotoxicity of Cd (40 and 80 mg/L) in maize (Z. mays L.) seedlings after 7 days Cd exposure, using 11 RAPD primers. The previous study revealed that disappear bands increased from 33 to 45 bands combined with increased new bands also from 3 to 5 compared to their respective control, as Cd applied concentrations increased from 40 to 80 mg/L. Moreover, Aydin et al. (2013) reported Cd genotoxicity in Okra (A. esculantus L.) after 21 days of Cd (30, 60 and 120 mg/L) exposure, using 9 RAPD primers. The previous study revealed that polymorphic bands number decreased from 25 to 22 as Cd applied concentrations increased from 30 and 120 mg/L Cd.
Moreover, GTS% value as a qualitative measurement reflect DNA changes induced by Cd treatment was displayed by RAPD marker (Figure 3). In this regards, it increased from 30.7 to 42.7% in the studied P. pavonica alga when applied Cd concentrations increased from 2.5 to 10 mg/L.
Our data were coherent with results supported by Hamutoglu (2021), who reported that GTS% value increased from 69.04 to 80.95% and from 67.85 to 71.42% when applied both of Cd and Pb concentrations increased from 30 to 120 ppm after 72 h exposure using RAPD marker in Ramalina farinaceae lichen. Our data were also coherent with Soydam Aydin et al. (2012) who reported increase in GTS% values from 15.98 to 38.69% as ions (Cu and Zn) concentrations increased from 40 to 640 mg/L in Cucumber (Cucumis sativus L.) using RAPD marker. Similarly, Aydin et al. (2013) reported increase in GTS% from 59 to 72.5% as Cd applied concentrations increased from 30 to 120 mg/L Cd in Okra (A. esculantus L.) using RAPD marker.
The current investigation proved that increased GTS % as Cd applied concentrations increased, may be act Table 1: Selected RAPD primers for assessment of Cd stress
as a protective mechanism leading to best and effective DNA repairing. This phenomenon could be attributed to the fact, that the DNA alterations in RAPD patterns (appearance of new or/and disappearance of normal control bands); tendency to balance each other (Liu et al., 2005, Aydin et al., 2013).
Based upon observed physiological (significant increase in carotenoids content) and molecular data (decrease in the new induced bands number and increase in disappeared bands as Cd concentrations increased from 2.5 to 10 mg/L) in the studied P. pavonica alga, the current investigation could be assumed that P. pavonica alga adopted certain mechanism to minimize DNA damages induced by Cd stress.
in P. pavonica alga.
Primer Sequence 5' - 3' Primer Sequence 5' - 3'
RPi-C2 AACGCGTCGG OPD20 GGTCTACACC
RPi-C3 AAGCGACCTG OPE15 ACGCACAACC
RPi-C4 AATCGCGCTG OPG05 CTGAGACGGA
RPi-C5 AATCGGGCTG OPK17 CCCAGCTGTG
RPi-C6 ACACACGCTG OPR09 TGAGCACGAG
RPi-C7 ACATCGCCCA OPT18 GATGCCAGAC
RPi-C8 ACCACCCACC OPV03 CTCCCTGCAA
RPi-C9 ACCGCCTATG OPYIO CAAACGTGGG
RPi-C10 ACGATGAGCG OPW17 GTCCTGGGTT
OPA02 TGCCGAGCTG UBC159 GAGCCCGTAG
OPB17 AGGGAACGAG UBC702 GGGAGAAGGG
Table 2: Physiological parameters investigated in P. pavonica alga exposed to cadmium stress.
Parameter C T1 T2 T3
SGR% 0.003 X -0.075 Y -0.086 Y -0.088 Y
Chl a 5.049 X 4.232 Y 3.824 Y 3.970 Y
Chl b 2.308 X 1.710 X 1.431 X 1.198 Y
Total Chl 8.352 X 6.726 Y 5.939 Y 5.813 Y
Carotenoids 1.589 Z 1.599 Z 2.301 Y 2.864 X
Total Carotenoids 94.933 X 79.067 Y 71.200 Y 73.267 Y
Potential osmotic 79.000 Z 82.000 Z 92.333 X 106.333 W
EC 0.573 Z 0.577 Z 0.733 Y 1.050 X
C: control; T1: 2.5 mg/L Cd; T2: 5 mg/L & T3: 10 mg/L.
Figures sharing the same case letter are not significantly different at p = 0.05 probability by Fisher's PLSD test. SGR%: LSDo.05 Cd treatment: 2.271. Chl a: LSD005 Cd treatment: 0.708. Chl b: LSD0.05 Cd treatment: 1.034. Total Chl: LSDa05Cd treatment:1.619. Car: LSD0.05 Cd treatment: 0.461. Total Car: LSD0.05 Cd treatment: 13.273. Potential osmotic : LSD0 05 Cd treatment: 9.549. EC: LSD0.05 Cd treatment: 0.122.
Figure 1. RAPD pattern yielded by OPE15, OPK17 and RPi-C8 RAPD primers into P. pavonica alga species revealed DNA changes induced by different Cd concentrations. M: A VC 100bp Plus DNA Ladder (Vivantis) ladder standard.
C: control; Tl: 2.5 mg/L Cd; T2: 5 mg/L & T3: 10 mg/L.
T2 T3
Cd treatment
Figure 2. Polymorphic bands (PB) including new induced bands number (a) and disappeared of control bands number (b) using RAPD marker, into P. pavonica alga species revealed DNA changes induced by different Cd concentrations.
T1: 2.5 mg/L Cd; T2: 5 mg/L & T3: 10 mg/L.
Figure 3. Genomic template stability (GTS%) value induced by different Cd concentrations yielded by RAPD marker into P. pavonica alga species. T1: 2.5 mg/L Cd; T2: 5 mg/L & T3: 10 mg/L.
CONCLUSION
Physiological and molecular response of P. pavonica marine alga has been evaluated under different Cd concentrations for 4 days. For physiological test, Cd stress caused a decline in investigated physiological parameters (SGR%, Chl a & b, total chlorophyll and total carotenoids content and osmotic potential). Whereas, molecular test revealed increased GTS% from 30.7 to 42.7% when applied Cd concentrations increased from 2.5 to 10 mg/L, in the studied P. pavonica alga. Overall, data presented herein based upon observed physiological (significant increase in carotenoids content) and molecular data (decrease in the new induced bands number and increase in disappeared bands as Cd concentrations increased from 2.5 to 10 mg/L) in the studied P. pavonica alga, the current investigation could be assumed that P. pavonica alga was Cd tolerant. This observation could be related to the fact that P. pavonica alga adopted certain mechanism to minimize Cd toxicity at physiological level (significant increase of carotenoids pigments content) as Cd applied concentrations increased; where, carotenoids pigment acts as antioxidant and thereby, serves as osmoprotector involved in heavy metal tolerance mechanism by minimize ROS induction under heavy metal stress. Overall, P. pavonica alga could serve as a promising bioindicator for Cd pollution in marine ecosystems.
ACKNOWLEDGEM ENTS
I thank Professor Othman Ibrahim (director general of AECS) and Professor MirAli Nizar (head of Molecular Biology and Biotechnology Department in AECS) for their support, and also the Plant Biotechnology Group for technical assistance.
CONFLICTS OF INTEREST
The authors declare that they have no potential conflicts of interest.
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