Responses Of Growth,Nutrient Uptake,Photosynthesis And Related Physiological Parameters To Copper-toxicity In Citrus Seedlings

In recent years,copper pollution in agricultural soils has become a major concern for sustainable agricultural development,especially in developing countries due to the arbitrary use of pesticides,fungicides,industrial wastewater and sewage irrigation.Copper（Cu）,an essential mirconutrent,plays a vital role in the normal growth and development of higher plants.However,with the continuous accumulation of soil Cu,it will inevitably cause certain toxic effects on plants and affect plant growth and development.Citrus belong to evergreen fruit trees.Citrus are grown in 135 countries in the world and 985 counties（cities and districts）in China.Therefore,Citrus have very high commercial and economic values.Evidence shows that Cu-toxicity has occurred in some Citrus orchards and is becoming more and more significant.So far,studies on Cu-toxic effects on Citrus physiology are very limited.Seedlings of‘Shatian pummelo’（Citrus grandis）and‘Xuegan’（Citrus sinensis）were supplied daily with nutrient solution at a concentration of 0.5（control）,100,200,300,400 or 500μM CuCl₂ for six months by sand culture method.Thereafter,seedling growth,leaf,root and stem levels of nutrients,leaf gas exchange,concentrations of pigments,chlorophyll a fluorescence（OJIP）transients and related parameters,and leaf and root relative water content（RWC）,concentrations of nonstructural carbohydrates,H₂O₂ production rate and electrolyte leakage were examined in order（a）to understand Cu-toxic effects on growth,photosynthesis and related physiological parameters in Citrus seedlings;and（b）to elucidate the physiological mechanisms for the Cu-toxicity-induced inhibition of growth and photosynthesis in Citrus seedlings.The main results are as follows:1.Most of growth and physiological parameters were greatly altered only at 300-500μM Cu-treated seedlings.300-500μM Cu is considered to be excessive（toxic）.2.Generally speaking,Cu concentration in the leaves,stems and roots and Cu uptake per plant increased with increasing Cu supply;Nitrogen（N）,phosphorus（P）,potassium（K）,calcium（Ca）,magnesium（Mg）,boron（B）and iron（Fe）concentrations in leaves,stems and roots,sulfur（S）and zinc（Zn）in leaves and roots,and manganese（Mn）concentration in stems,as well as N,P,K,Ca,Mg,B and Fe uptake per plant decreased with increasing Cu supply.Also,Cu-toxicity altered the distributions of elements in roots,stems and leaves3.With the increase of Cu concentration,RWC of roots and leaves decreased,and the production rate of hydrogen peroxide and leakage of electrolytes increased in roots and leaves.4.Cu-toxicity increased the accumulation of Cu in roots.Over 93%of Cu was accuulatued in the roots of Cu-toxic Citrus seedlings.The preferential accumulation of Cu in roots might be a key mechanism of tolerance of Citrus plants to Cu-toxicity.In 400-500μM Cu-treated Citrus seedlings,many of fibrous roots became rotten and died.Most of no rotten fibrous roots became abnormally dark brown.Principal component analysis（PCA）showed that Cu-toxicity affected roots more than shoots.Thus,it is reasonable to assume that Cu-toxicity might impair the Citrus root growth and function,thus affecting the uptake of water and nutrients,and hence,decreasing the growth of shoots.5.Iron chlorosis occurred in the Cu-oxicity-treated young leaves.The decreased pigments in response to Cu-toxicity might be caused by the decreased biosynthesis due to deficiencies of nutrients,and accumulation of nonstructural carbohydrates（glucose,fructose,sucrose,total soluble sugars,starch and total nonstructural carbohydrates）,and structural damages to chloroplasts.6.Cu-toxic leaves had decreased maximum quantum yield of primary photosystem II（PSII）photochemistry（F_v/F_m）and quantum yield for electron transport(φ_Eo=ET_o/ABS),increased energy dissipation per reaction center（DI_o/RC）and quantum yield for energy dissipation(φ_Do=DI_o/ABS),and altered OJIP transients.This demonstrated that PSII complexes in Cu-toxic leaves suffered from photoinhibitory damage.Photosystem I（PSI）was a less sensitive excess Cu-inhibited site than PSII.The donor side of PSII in the excess Cu-treated leaves was less impaired than the acceptor side of PSII.7.Cu-toxicity-induced decrease of CO₂ assimilation in Citrus leaves was caused mainly by non-stomatal factors,including structural damage to thylakoids,altered sink-source relationship due to increased accumulation of nonstructural carbohydrates,decreased uptake of water and nutrients,increased production of reactive oxygen species（ROS）and an impaired photosynthetic electron transport chain.In summary,the effect of Cu toxicity concentration on citrus was significant between 300 and 500μM.Cu toxicity mainly accumulated in the root system of citrus to reduce water and nutrient uptake and shoot growth.Copper toxicity can cause structural damage to chloroplasts,nutrient deficiency and accumulation of unstructured carbohydrates,resulting in reduced photosynthetic pigmentation and biosynthesis.