Excess Copper Effects On Proteome And Related Physiological Parameters In Citrus Leaves

Copper?Cu?,a micronutrient required for the normal growth and development of higher plants,plays an important role in photosynthesis,respiration,antioxidation,cell wall metabolism and hormone response.It may become highly toxic when used in excess.For a long time,Cu-containing fungicidesand bactericides are widely used in agriculture to control fungal and bacterial diseases in cropsincluding Citrus in order to improve crop production and quality.So far,Cu contamination in agriculturesoils is on the rise all over the world.In old Citrus orchards,the excess accumulation of Cu insoils is a common phenomenon because of the extensive and continued use of Cu-containingagricultural chemicals against fruit and foliar diseases such as anthracnose and canker.Cu level and availability in soils under continuous Citrus production orchards increase withincreasing production period.Recently,Cu-toxic effects on Citrus growth and physiology have been investigated in somedetails,little is known about Cu-toxicity-induced alterations of protein profiles in Citrus.This present study examined excess Cu effects on seedling growth,leaf Cu content,gas exchange,and protein profiles identified by a two-dimensional electrophoresis?2-DE?based mass spectrometry?MS?approach after‘Xuegan'?Citrus sinensis?and‘Santianyou'?Citrus grandis?seedlingswere treated for six months with 0.5?control?,200,300,or 400?M Cu Cl₂.The objectives of this study were to identify Cu responsive proteins in Citrus leaves,to screen the candidate proteins possibly responsible for Cu tolerance in Citrus,and finally improve the tolerance of Citrus to Cu,so as to provide theoretical support for the high-yield and high-quality cultivationof Citrus and the sustainable development of Citrus industry.The main findings were as follows:1.C.sinensis?C.grandis?biomass remained littlechanged as Cu concentration in the nutrient solution elevated from 0.5 to 300?200??M,thendeclined at 400?300-400??M Cu.Biomass was lower in C.sinensis seedlings than that in C.grandisseedlings at each given Cu supply.Leaf Cu content increased with Cu supply and did not differ between the two Citrusspecies with the exception that its content in leaves was higher in C.sinensis than that in C.grandis at 300?M.Leaf CO₂ concentration and stomatal conductance?g_s?kept unchanged or increased as Cuconcentration in the nutrient solution rose from 0.5 to 200?M,then declined with further rise in Cuconcentration.Cu supply had little influence on the ratio of intercellular to ambient CO₂concentration?C_i/C_a?except for that C_i/C_ain C.grandis leaves was slightly higher at 200?M Cu thanthat at 300-400?M Cu.No significant differences were observed in leaf CO₂ concentration,g_s and C_i/C_abetween the two Citrus species over the range of Cu supply.Based on these results,seedlings that received 300-400?M Cu were regarded as Cu excess.2.Using a 2-DE based MS approach,a total of41 and 37 differentially abundant protein?DAP?spots were identified in 200,300 and/or 400 ?MCu-treated C.grandis and C.sinensis leaves,respectively,including some novel DAPs[viz.ferredoxin-NADP reductase,leaf-type isozyme?LFNR2?,sedoheptulose-1,7-bisphosphatase?SBPase?,6-phosphogluconolactonase 4?PGL4?,ferritin,S-adenosyl-L-homocysteine hydrolase and abscisic stress-ripening protein1-like]that were not reported previously in leaves and/or roots.Over 50%of these DAPs were involved inphotosynthesis,carbohydrate,and energy metabolism,followed by antioxidation and detoxification,protein folding and assembly?viz.,chaperones and folding catalysts?,and signal transduction.Morethan 80%of these DAPs were identified only in C.grandis or C.sinensis leaves.More DAPs increased inabundances than DAPs decreased in abundances were observed in Cu-treated C.grandis leaves,butthe opposite was true in Cu-treated C.sinensis leaves.3.For total DAPs in C.grandis leaves,there were eight significantly enriched KEGGpathways-namely carbon fixation in photosynthetic organisms?ko00710?,exosome?ko04147?,glycolysis/gluconeogenesis?ko00010?,fructose and mannose metabolism?ko00051?,photosynthesis?ko00195?,chaperones and folding catalysts?ko03110?,photosynthesis proteins?ko00194?andinositol phosphate metabolism?ko00562?.For total DAPsin C.sinensis leaves,carbon fixation in photosynthetic organisms,photosynthesis proteins,exosome,tricarboxylic acid?TCA?cycle?ko00020?and photosynthesis were the significantly enriched KEGGpathways.Principal component analysis?PCA?indicated that the association patterns of DAPs were more obvious in C.sinensis leaves than those in C.grandis leaves.4.Cu-toxicity might affect the abundances of proteins related to photosynthetic electron transport chain and CO₂ assimilation,thus decreasing electron transport rate and photosynthesis.Cu-toxic effects on photosynthetic electron transport chain were more pronounced in C.sinensis leaves than those in C.grandis leaves.Cu-responsive proteins related to antioxidation and detoxification,protein fold and assembly?viz.,chaperones and folding catalysts?,and signal transductionmight also play a role in Citrus Cu-toxicity and Cu-tolerance.To conclude,this study revealed some novel mechanisms on Cu-toxicity and Cu-tolerance in plants?Citrus?.