Copper Stress on Photosynthesis of Black Mangle ( Avicennia germinans )

The effects of copper toxicity on the photosynthetic activities of Avicennia germinans was investigated using two CuSO4 concentrations (0.062 and 0.33 M) added in Hoagland’s solution in an aerated hydroponic system. Photosynthesis and chlorophyll fl uorescence were measured after 30 h of copper stress. Results obtained in this study show that increasing levels of Cu+2 of 0.062 and 0.33 M Cu+2 resulted in a general reduction of the stomatal conductance (28 and 18%, respectively) and 100% of inhibition of net photosynthesis. Additionally, at these concentrations of Cu+2, reductions of chlorophyll fl uorescence parameters were also observed. These changes suggested that the photosynthetic apparatus of Avicennia germinans was the primary target of the Cu+2 action. It is concluded that Cu+2 ions causes a drastic decline in photosynthetic gas exchange and Chlorophyll fl uorescence parameters in A. germinans leaves.


INTRODUCTION
Mangrove ecosystems are among the most important features of the coastal environment in many tropical and subtropical areas.Many mangrove ecosystems are located close to urban development areas, which may be impacted by effl uents from industrial sources and urban runoff that often contains toxic concentrations of heavy metals (Cuong et al. 2005, Defew et al. 2005).Despite this, mangroves possess a great tolerance to relatively high levels of heavy metal pollution (Peters et al. 1997, De Lacerda 1998).Avicennia species in particular, are considered to be especially robust to heavy metals and accumulate metals in greater quantities than other mangrove species, before any visible signs of toxicity are evident (MacFarlane et al. 2003).
Copper (Cu +2 ) is essential plant micronutrients, and often occurs in high concentrations in mangrove forest due to their prevalence in sediments, up to 900 mg/kg (Chen et al. 2007).The uptake of Cu +2 in excess to nutritional requirements by mangroves may initiate a disruption of many physiological functions that can cause damage at the cellular level (MacFarlane and Burchett 2001).Most of the in vitro studies using isolated chloroplasts or excised leaves have reported a direct effect of copper on the photosynthetic electron transport chain (Mallick and Mohn 2003).These disturbances are correlated [665][666][667][668][669][670] with lipid peroxidation of thylakoid membranes or with the alteration of lipid protein interactions in the chloroplast membrane, affecting the light reaction processes, especially those associated with PSII (Perales-Vela et al. 2007).In vivo, the fi rst contact between copper and the plant is not directly at the chloroplast level and therefore, the mechanism of photosynthesis inhibition probably differs from that observed in vitro.
In this context, the objective of this work is to research if plants were able to tolerate higher concentrations of metal.We investigated the effect of an additional supply of copper on the photosynthetic activity of Avicenia germinans.The net photosynthetic rate and the chlorophyll a fl uorescence were measured to determine the physiological modifi cations induced by copper excess.

FIELD COLLECTION AND GERMINATION OF PLANT MATERIAL
Avicennia germinans (L.) Stearn., black mangle, seeds were obtained from Chabihau Bay, (21° 20' 38" N and 89° 05' 08" W) Yucatan, Mexico.The seeds of black mangle were germinated between moistened fi lter paper at 25°C for 3 d.The disinfection process was achieved by sequential steps starting with 1% NaOCl (Clorox) for 5 min, followed by a rinse with deionized sterile water.The propagules were then peeled with a sterile scalpel and the pericarp discarded.The naked propagules were further disinfected with 0.1% NaOCl and fi nally rinsed with deionized sterile water six times.One hundred fi fty propagules were then planted in 13.5 cm-diameter plastic pots (one plant per pot) fi lled with sterilized soil and grown at 28-35°C /24-26°C (day/night temperature regime) under a 12-h light-dark photoperiods and 60% relative air humidity in a greenhouse.

COPPER EXPOSURE
Twenty individuals, three-months-old Avicennia germinans seedlings were randomly allocated (n=4) and transferred to different fl asks containing Hoagland's solution supplemented with different concentrations (0.062 and 0.33 M) of CuSO 4 .5H 2 O.
Control plants were transferred to plastic containers with 500 mL Hoagland's solution without CuSO 4 .5H 2 O. Treated and control plants were exposed during a period of 30 h under hydroponics conditions and these exposures were performed in quadruplicates.Treated started at 9 am and the measures taken at 16 h were made during the night period.

GAS EXCHANGE
The rate of net photosynthesis (P The rate of net photosynthesis (P The rate of net photosynthesis ( n ), and stomata conductance (g conductance (g conductance ( s g s g ) of young fully expanded leaves were measured at 4, 8, 16, 24 and 30 h after exposure to treatments with copper using a portable infrared gas analyzer (LI-Cor Model 6200, Lincoln, NE, USA).All the photosynthetic measurements were standardised at 23°C, 400 μmol.m −2 .s−1 photosynthetically active radiation and each treatment was measured four times.Treatments started at 9 am and the measurements taken at 16 h were made during the night period.

FLUORESCENCE MEASUREMENTS
The determination of chlorophyll fl uorescence was carried out using a portable fl uorometer (Plant Effi ciency Analyzer-MK2-9600-Hansatech, Norfolk, UK) on completely expanded leaves of appropriate phytosanitary condition.The data were recorded from 10 ms up to 1 s with a data acquisition of every 10 ms for the fi rst 300 ms, then every 100 ms up to 3 ms and later every 1 ms.The signal resolution was 12 bits (0-4,000).For each treatment, the chlorophyll (Chl) a fl uorescence transients of 4 individual leaves were measured.Leaves were maintained in darkness for 5 min before taking the data on chlorophyll fl uorescence.The maximal intensity of the light source, providing an irradiance saturating pulse of 3,000 mmol photons.m -2 .s - was 667 COPPER STRESS ON Avicennia germinans used.Different chlorophyll fl uorescence parameters like the F and F 0 using the 0 using the 0 software Biolyzer 2.5 (Maldonado-Rodriguez 2002).The ratio of variable fl uorescence to maximal fl uorescence (F fl uorescence (F fl uorescence ) is an indicator of the effi ciency of the photosynthetic apparatus, while the ratio of variable fl uorescence to unquenchable portion of fl uorescence ( ) is an indicator of the size and the number of active photosynthetic reaction centers and (F and (F and ) represent the effi ciency of the watersplitting apparatus (Kriedemann et al. 1985).

STATISTICS ANALYSIS
All data presented are the mean values.Statistical analysis was carried out by one-way ANOVA for repeated measures followed by posthoc analysis by the Fisher test of least signifi cant difference with signifi cance set at P ≤ 0.05.P ≤ 0.05.P

PHOTOSYNTHESIS PARAMETERS
The increase of Cu +2 dose decreases all leaf gas exchange values, having the strongest negative effect on stomatal conductance (g on stomatal conductance (g on stomatal conductance ( s g s g ), and net photosynthesis (P (P ( n P n P ) (Fig. 1a, b).The values of stomatal conductance were lower in treated plants compared to control plants (Fig 1a).The reduction of stomatal conductance was similar in plants treated with 0.332 and 0.062 M Cu +2 (83 and 72 %, respectively).The net photosynthesis was signifi cantly (100% of inhibition) and most similarly affected by both Cu +2 concentrations (Fig 1b).

CHLOROPHYLL FLUORESCENCE PARAMETERS
The measurement of fl uorescence parameters in control plants showed an dropped from 4 to 8 h followed by a progressive rise from 8 to 24 h.towards the end of the dark hours to fi nally dropped again the following day to values similar found the previous day (Fig 2a).
On the contrary Cu +2 On the contrary Cu +2 On the contrary Cu -treated plants with 0.332 M also showed dial fl uctuations during the fi rst 24 h, but after 30 h of exposure to the metal, signifi cantly higher The F v 0 values were followed during the course of exposure Cu +2 concentrations (Fig. 2b and 2c).During the experiment, the plants exposed to 0.062 and 0.332 M Cu +2 , no difference was found in these parameters during a large part of the experiment.Effectively, only at 30 h (end of the experiment) could a slightly difference be observed between the treatments.In the case of values for control plants, they rise slightly from 4 to 8 h followed by a progressive drop from 8 to 24 h, towards the end of the dark hours, to finally rise again the following day to values similar found the previous day (Fig 2b and 2c).

DISCUSSION
Numerous studies on the physiological responses to excess amounts of heavy metal ions indicate that mangrove have developed various mechanisms to cope with this environmental threat.Some of them mechanisms appear to involve the presence of exclusion and sequestering processes that can moderate the metal uptake by roots and induce a substantial metal accumulation on the root tissues (De Lacerda 1998).
In this sense, Gonzalez-Mendoza and Zapata-Perez ( 2008) suggest that the radical architecture present in Avicennia germinans plants might alter the uptake of Cd by an integrated network of multiple response processes such as production of organic acids,

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COPPER STRESS ON Avicennia germinans antioxidative response, cell-wall lignifi cations and suberization.On the other hand, Gonzalez-Mendoza et al. (2007) showed that minimal concentrations of cadmium in foliar tissues affect several targets of photosystem II.More specifi cally the main targets of cadmium can be listed as a decrease in the number of active reaction centers and damage to the activity of the water-splitting complex.On the other hand, numerous metals are essential for living organisms at very low concentrations, but at high concentrations most of them are toxic and have a direct and adverse infl uence on various physiological and biochemical processes.In this sense the copper is an essential metal that participates in growth, metabolism and enzyme activities (Yruela 2005).The copper can be accumulated in the leaf tissue in high concentrations of numerous mangrove species in the fi eld including Kandelia spp., Kandelia spp., Kandelia Rhizophora spp.and Rhizophora spp.and Rhizophora Avicennia spp., Avicennia spp., Avicennia without apparent impact on plant health (Peters et al. 1997, Macfarlane et al. 2003).Even though, it is known that higher amounts of Cu +2 is known that higher amounts of Cu +2 is known that higher amounts of Cu end up in the leaves to be accumulated (Defew et al. 2005), the physiological effects of copper stress in plants of mangrove like Avicennia germinans are basically not known.Therefore, investigations on the mechanism of copper action on the photosynthetic apparatus are of special interest.In this way, we provide important evidence that aerial tissues of A. germinans are affected by low and high Cu +2 affected by low and high Cu +2 affected by low and high Cu concentrations.Our data showed that the highest sensitivity to Cu +2 data showed that the highest sensitivity to Cu +2 data showed that the highest sensitivity to Cu was exhibited by stomatal conductance (g exhibited by stomatal conductance (g exhibited by stomatal conductance ( s g s g ), and net photosynthesis (P photosynthesis (P photosynthesis ( n P n P ).Our study showed that at the experimental design used chlorophyll fl uorescence are less sensitive parameters of Cu +2 are less sensitive parameters of Cu +2 are less sensitive parameters of Cu toxicity.
The inactivation of net photosynthesis could be explained by a possible inhibition of the enzymatic processes in the Calvin cycle of Avicennia plants.Specifi cally, because the Cu +2 could induced a reduction in the synthesis or activity of Calvin cycle enzymes reducing the demand for CO 2 , resulting in reductions in stomatal conductance in this plants.
Additionally, the Cu +2 ions may also exert its toxicity in subcellular organelles, interfering with mitochondrial electron transport, respiration, ATP production and photosynthesis in the chloroplasts and may eventually cause plant death (Yruela 2005).Similar results were found by Ouzounidou and Ilias (2005) where Helianthus annuus plants exposed to Cu +2 for short time showed a decrease in the rate of net photosynthesis due to stomata closure, Rubisco inactivation and chlorophyll degradation.
Chlorophyll fl uorescence induction parameters have been shown to detect even subtle biochemical damage within photosynthetic reaction centers in a wide variety of both terrestrial and marine plants.The present investigation shows that a signifi cant decline in the /F 0 /F ratio can be due to the modifi cations in 0 ratio can be due to the modifi cations in 0 the unquenchable fl uorescence (F the unquenchable fl uorescence (F the unquenchable fl uorescence ( 0 F 0 F ) that affected the energy transfer from the antenna complex to the reaction centres (DeEll et al. 1999).Additionally, the decline in the /F 0 /F ratio is indicative of either a decline 0 ratio is indicative of either a decline 0 in the rate of photochemistry as the primary electron acceptor pool (Q n Q n Q ) became increasingly oxidized, or a reduction of the pool size of the primary electron acceptors associated with PS II activity (Krause and Weiss 1991).Another possible explanation for the changes in the In conclusion, excess Cu +2 produces a variety of toxic effects on photosynthesis.Based on both high sensitivity of leaf gas exchange parameters (stomatal DANIEL GONZÁLEZ-MENDOZA et al. conductance and net photosynthesis) than chlorophyll fl uorescence parameters to excess Cu 2+ and fastness of their measurement, we propose these parameters as suitable parameters for plant test systems, which can be utilized for successful screening for higher Cu 2+ tolerance among mangrove plants.

ACKNOWLEDGMENTS
The authors appreciate Jose Francisco Rodriguez, Reyna Collı, Jose Luis Febles, Jorge Novelo and Dr. Eduardo Batllori for assistance in seedling cultivation and experimental analysis and CONACyT (160136).

Figure 1 -
Figure 1 -Time course response of stomatal conductance (a) and net photosynthesis (b)., from untreated plants or plants treated with 0.062 M and 0.33 M Cu +2 during an exposure period of 30 h.The black bar indicates the dark hours.Each point is the mean from 4 replications.

Figure 2 -
Figure 2 -Time course response of various fl uorescent parameters for untreated A germinans plants or plants after 30 h exposure to 0.062 and 0.33 M Cu +2 (a)water -splitting apparatus of PSII, F 0 F 0 F /F 0 /F 0 v /F v /F , (b) the maximal photochemical yield of PSII in dark-adapted
ratio of the copper-treated plants 0 ratio of the copper-treated plants