Effects Of Exogenous Nitric Oxide On Germination And Growth Of Alfalfa Seedlings Under Cadmium Stress

Cadmium?Cd?is a toxic and non-essential heavy metal to either animal or plant.In order to maintain ecological balance and protect efficiently arable land from heavy metal pollution,it is very important to explore the resistance mechanism of plant against different heavy metals stresses and propose efficient techniques to ameliorate their resistance against heavy metal toxicity.Nitric oxide?NO?is a signaling molecule having key functions in mediating various physiological responses to both abiotic and biotic stresses.The role of NO in physiological responses and gene expression processes in response to heavy metal toxicity and the role of NO in plant defense pathways and implication in soil reclamation as well as in agricultural activities have not elucidated yet.Alfalfa?Medicago sativa L.?is an important forage crop,widely distributed in temperate zones of the world with 27 o C optimum temperature for seedling growth.Its extensive root system make it to be drought tolerant and can enhance the activity of degrading bacteria,promoting the bio-degradation rate of several organic contaminants such as polychlorinated biphenyls?PCBs?,polycyclic aromatic hydrocarbon?PAH?and trinitrotoluene?TNT?,especially in soils with low organic matter content.Alfalfa has been revealed to have strong resistance and be nutritive due to its Vitamins A,C,E,and K,minerals,and trace elements are present within it.Alfalfa due to its properties of fast-growing,deep rooted rhizosphere and high biomass producing plant it is a promising plant for phytoremediation of toxic metals.Alfalfa has been reported to be extremely resistant to heavy metal contaminants as well as bio-accumulator with a high absorption coefficient and it is highly effective in environmental remediation for Cd-contaminated soils.To explore the effects of Sodium Nitroprusside?SNP,a donor of NO?on Cd toxicity during germination of alfalfa seedlings and to elucidate its better application in phytoremediation against Cd contaminated soil we investigated germination parameters,oxidative stress level,antioxidant enzymes activities,hydrolyzing enzymes,storage substances reserve,fresh weight,dry weight and Cd uptake.Each experiment parameter was carried out for five days and the data were recorded every day with exactly 24 hours interval time.The pre-treatment experiments on the different concentrations of CdCl₂ on seed germination and seedling growth showed a clear increase in the severity of the negative effect as CdCl₂ concentration was increased.The CdCl₂ concentration chosen for the majority of the experiments here was 30?M,as it produced a significant reduction in seedling growth,whilenot seriously affecting the germination rate.Higher concentrations resulted in severely reduced germination rates,reducing the usable sample of seedlings produced per experiment.To investigate effects of SNP applied exogenously to alfalfa seeds submitted to Cd stress,different concentrations of SNP were tested: 25,50,100,200,300,400 and 500?M where each test was combined with 30?M CdCl₂.For germination parameters we investigated the germination percentage,germination index,seedling vigor index,root and shoot length.Reactive oxygen species?ROS?mechanism was investigated by studying Proline?Pro?level,Relative electric conductivity?REC?,Lipid peroxidation [in terms of malondialdehyde acid?MDA?level)],reactive oxygen species?ROS?such as H2O2 and O2-and antioxidant defense mechanism by studying activities of some antioxidant enzymes like superoxide dismutase?SOD?,peroxidase?POD?,ascorbate peroxidase?APX?,catalase?CAT?and glutathione reductase?GR?.Hydrolyzing enzymes activity was assayed by determining the activity of ?-amylase,?-amylases and storage substances content was investigated by determining the content in soluble proteins,soluble starch,soluble sugars and reducing sugars.Moisture content was measured by determining fresh and dry weight.The results showed that:?I?30?M CdCl₂ induced accelerated breakdown of stored food materials by significantly impair the process of seed germination,seedling growth,GI and seedling vigor index as it has been reported in several studies.During our experiments,different concentrations of SNP were shown to be able to alleviate the negative impact of heavy Cd stress on germination and early stage of seedling growth.However below the optimum concentration its stress alleviation significance was linearly increasing while the concentrations above the optimum resulted in a reverse trend.This response is more likely to be the result of activation of plant defense or tolerance mechanism by effective exogenous NO.This corroborates with previous studies about protective role of NO shown in germinating mug bean seeds submitted to Arsenic stress.Importance of exogenous NO against Cd toxicity has been elucidated after the study on rice seed germination and its seedling growth in Cd stress conditions where it has been reported that SNP is significantly able to increase germination percentage,GIand seedling vigor index,similar results have been reported in sesame seedlings.In our present study,exogenous NO which successfully alleviated Cd damage is likely to operate in a similar way to other biotic or abiotic stresses.However,above optimum concentrations of SNP,plant or seedlings exhibit the same defense mechanism to protect against toxic effects of SNP.The same results have been reported in perennial ryegrass submitted to Cd stress.200?M SNP have been found to be the most effective to inhibit Cd negative effects bysignificantly increasing germination percentage,seedling vigor index,and germination index,root length and shoot length.?II?Diverse mechanisms such as physiological changes are involved to block or alleviate abiotic or abiotic stresses.In response to environmental stress Pro accumulation and the regulatory role of NO in Pro metabolism under abiotic stresses has been reported in many plant species.In the present study,level of Pro in seedlings exposed to Cd stress was higher than those of control seedlings and their level further dose-dependently reduced with application of SNP especially for the high concentrations above 200?M;but with a long-time exposure,the concentrations above 200?M exhibited a trend in increase of the level in Pro content.As an active redox metal,Cd is able to induce the overproduction of ROS such as O2-and H2O2 produced in the seedlings under various abiotic stresses which in turn lead to membrane peroxidation,electrolyte leakage and oxidative stress.For our experiments we investigated the membrane leakage by measuring REC and involvement molecules such as MDA,H2O2 and O2-to elucidate the plant injury due to oxidative stress.Our experiment results showed that in time-dependent manner Cd induced the over-accumulation of these molecules leading to the oxidative injury in alfalfa seeds and inhibiting seedling growth.Cdcaused oxidation damage was also reported in Elymus dahuricus seeds.However under Cd stress,the protective role of SNP was demonstrated by the ability to alleviate H2O2,O2-content and protect cell from leaking as well as from membrane peroxidation by decreasing REC and accumulation of MDA.This lipid peroxidation results in reduced fluidity of membranes;which cause the increase membrane leakiness,damage of membrane protein and inactivation of receptors,enzymes and ions channels.NO inhibit the plant from oxidation damage by regulating general mechanism for cellular redox homeostasis and promoting the transformation of O2-to H2O2 and O2,also by enhancing H2O2-scavenging enzyme activities.To cope and neutralize the toxicity of ROS,plants have evolved endogenous systems of anti-oxidative enzyme system such as CAT,POD,SOD,APX,GR,etc.The physiological stress caused by Cd in the present study resulted in the increase in level of Pro and decrease antioxidant system which has a negative effect on tolerance capacity of alfalfa seedlings to overcome deleterious effects of stress caused by Cd.Neutralization of ROS by Pro is the primary method of metal detoxification.To assess whether the efficiency of exogenous NO to reduce the Cd stress as it was demonstrated by decreased membrane lipid peroxidation,H2O2,O2-and REC was due to antioxidant enzymes activity,the SOD,CAT,POD,APX and GR activities were studied.SOD as the most effective enzyme for protecting cells against oxidative stress by promoting the conversion of superoxide radical was tremendously inhibited by treating alfalfa seeds with Cd.Its activity was revealed to be significantly lowerthan seeds sown in control?CK?conditions.Due to weak hydrogen bond and low solidity of Zn2+-SOD complex,it has been reported that Cd2+ can easily substitute Zn2+ in active site of SOD which has a tremendous effect of SOD efficiency against for both abiotic and biotic stresses.In our study,accumulation of Cd induced the significant production of H2O2 and O2-which decreased the activity of SOD.The same results have been found in perennial ryegrass simultaneously submitted to Cd and Cu stress.The increase in the level of SOD activity with application of exogenous NO in present study corroborates earlier reports on rice and tomato seedlings.Exogenous NO increased POD activity during all days of treatment with a significant higher activity compared with seeds sown in Cd conditions,which indicates the vital role of POD in H2O2 scavenging activity.APX as well as CAT whose role is to scavenger ROS effects were also enhanced due to exogenous NO.APX activity was increased especially in early two days of germination where the following days it was decreased.The higher activities were observed in seeds sown in SNP concentrations less than 200?M whereas those in above concentrations the APX was significantly lower.CAT activity was remarkably observed in first 4 days of treatment among seeds sown in SNP conditions.For GR,except on the 1st day where the activity in Cd-treated seedlings was higher than in CK,the following days Cd induced the significant decrease in GR activity.By treating these seeds with SNP lead to a significant increase in GR activity however there was no regular change observed which means the relation between NO-induced GR activities is complex and still needs further researches.?III?Germination process is characterized by reserves mobilization such as carbohydrates and proteins by hydrolytic enzymes where the newly formed products are used in formation of new structures.These reserve materials are made available by hydrolytic enzymes to deliver fuel for respiration and various anabolic reactions in the form of anabolites.Enzymes accountable for starch breakdown and mobilization of these reserves materials are?-amylases and ?-amylases,and Cd has been reported to cause a restriction and impairment in these reserve mobilization.Application of SNP to germinating alfalfa seedlings sown in Cd conditions stimulated storage reserve mobilization and related enzymes.In time and dosedependent manner SNP activates ? and ?-amylases regulate the mobilization of soluble proteins,reducing sugar and soluble sugar content during germination alfalfa seeds sowed in Cd stress conditions.This is in accordance with our study where ? and ?-amylase activities were significantly depressed by only 30?M Cd.In these conditions,the quantities of soluble proteins and starch also have reduced drastically after the 2nd day of treatment.This is the major factor in the depression of seed germination.Our study revealed that application of SNP had a strong stimulating effect on germination of alfalfa seedlings under 30?mol Cd stress.Activity of amylases,solubleproteins and starch were highly significant as compared with the one in seedlings treated with only Cd.Our findings corroborated recent studies made on impact of exogenous NO on activity of amylases in germinating pea seeds submitted to Cd stress.SNP concentrations less than 100?M showed a moderate regulatory effect in soluble sugars mobilization for the first three days of treatment while above concentrations had a reversing effect.Except for seedlings treated with SNP concentrations ranging between in 25 and 100?M where the content in reducing sugars was relatively high,no significant difference in reducing sugars content was observed during all days and for all treatment;the production was almost constant.?IV?Cd as one of non-essential and harmful elements is reported to interfere in plant homeostasis by competing with other micro-and macro-nutrients in plant such as Ca2+and K+even with water transport.Occurrence of this is associated with reduction in both fresh and dry weight matter where the water content is reported to be highly affected by the gradual concentration of Cd in plant.In our study,as long as seeds were exposed to Cd stress,the fresh and dry weight content within seeds were also significantly affected gradually with exposure time compared with seeds in CK.conditions.Similar results have been reported in sunflower and in germinating sorghum seeds.Exogenous NO is reported to hinder the entry of heavy metals into plant cell wall or regulate cellular distribution and accumulation of excess Cd within plant cell wall.During our experiments,Cd accumulation within germinating alfalfa seeds was significantly counteracted by 200?M and above concentrations of SNP which yielded in an increase of both fresh and dry weigh.Similar results were obtained in several other previous researches such as in rice seedlings,against Mercury uptake in rice leaves and against Copper uptake in rice?Oryza sativa L.?seedlings.This suggests that SNP could be one of suitable compounds to protect seeds against heavy metal toxicity.To sum up,the present work demonstrates that 30?mol of Cd didn't significantly reduce germination percentage,but germination index,SVI,root length and shoot length were drastically affected by Cd toxicity.30?M CdCl₂ was found to induce the increase in Pro level,REC,MDA as well as overproduction of ROS.Cd has a significant hindrance in defense mechanism of alfalfa seeds by its ability to induce the decrease the activity of antioxidant enzymes.Activities of ?-amylase,?-amylases are significantly impaired by Cd toxicity resulting in impairment of storage substances in terms of soluble proteins,soluble starch,soluble sugars and reducing sugars.Cd can affect the content in fresh and dry weight and its accumulation within seedlings is linearly increased along exposure time.Exogenous NO is revealed to be one of compounds reversing the negative impact of Cd on germination percentage,hypocotyl length,root length,GI and on seedling vigor index.This Cd toxicitywas significantly counteracted by NO applied exogenously in dose-dependent manner where200?M SNP was found to be the most effective against 30?M Cd toxicity.Exogenous NO was found to be effective in terms of counteracting the oxidative damage caused by overproduction of ROS by the fact that it can decrease MDA level,REC,ROS.Exogenous NO was also found to boost the activity of antioxidant enzymes.Exogenous NO plays a major role in counteracting the impairment of storage substance caused by Cd toxicity by regulating mobilization of soluble starch,reducing sugar and soluble sugars via activation of ? and ?-amylases.Due to effects of exogenous NO,soluble protein mobilization was normalized.Exogenous NO counteracts Cd uptake by plant and increases the dry and fresh weigh matter.Generally,addition of 200?M SNP is revealed to be the most significant to alleviate 30?M Cd toxicity.Alfalfa plays important role in remediation of heavy metals like Cd,Cu,Pb,etc.contaminated soils especially in Northwest parts of China.Photosynthesis as a basis of physiological metabolism development,effects of NO on photosynthetic rate,transpiration rate,stomatal conductance,intercellular CO2 concentration,total chlorophyll content and xanthophyll cycle in alfalfa under Cd stress should be investigated in future researches.Further researches are recommended to elucidate genetic and proteomic analyses and additional physiological approaches to understand the details of exogenous NO in metabolic functions in plant,furthermore it will be helpful for elucidating involved mechanisms for better application in phytoremediation of heavy metals contaminated soils.