Root Responses Of Hydroponically Grown Rice（Oryza Sativa L.）to High Concentration Of ZnCl₂

Abiotic stresses such as high salinity,drought,and temperature extremes are a wellknown problem that significantly affects crop productivity.More recently,heavy metals have become an important environmental issue.Zinc(Zn)is an essential microelement for all organisms,participating in various physiological processes.However,a high concentration of Zn presents a high risk for organisms.Although Zn toxicity in crops is far less widespread than Zn deficiency,Zn concentration in soils has progressively increased in various regions over the last decades due to the different anthropogenic inputs.In plants,excess Zn reduces the growth of all plant organs,causes leaf chlorosis,reduces photosynthesis and cellular respiration,accumulates reactive oxygen species(ROS)and cell death.Rice(Oryza sativa L.)is not only one of the most important food crops in the world,being a basic food for nearly half of the world’s population,but is also an important model plant for research in monocots.While the insights into the molecular mechanisms underlying the Zn tolerance have been obtained from transcriptome and genome analysis,little effort has been made to understand rice(Oryza sativa L.)root responses to the toxic concentration of zinc at the proteomic level.Understanding the molecular responses of plant roots to the challenging environment contributes to engineering plants with improved stress tolerance.In addition,crops are the most sensitive to environmental challenges during their early life stages,seed germination and seedling establishment.Therefore,studying stress-responses of younger seedlings offer important data for agricultural improvement.Thus,in the present study,we mainly focused on the physiological and molecular responses of rice roots at the early growing stage.At first,we examine growth parameters and metabolic alterations caused by the excessive concentration of Zn.Then,we analyzed oxidative stress parameters and gene expression of stress-responsive genes.Finally,we studied Zn-induced proteomic changes in roots of rice by using two-dimensional electrophoresis(2-DE)technology.The main results are presented as followed:1.In the first part,we made a preliminary study to observe the effects of excess Zn on Oryza sativa growth.The growth was significantly inhibited by excess Zn.In roots,excess Zn reduces the growth of both primary and lateral roots.The shoot of rice seedlings exhibited higher sensitivity than roots toward excess Zn.All photosynthetic pigments including chlorophyll a,chlorophyll b,and carotenoids were strongly decreased in Zn-exposed plants.Fourier-transform infrared spectroscopy(FTIR)analysis of roots revealed that excess Zn negatively affected the content of carbohydrates and altered their composition.Also,excess Zn application significantly changed the relative secondary structure of proteins.2.In the second part,we examined oxidative stress parameters in Zn-exposed rice roots,by measuring the activities of antioxidant enzymes including superoxide dismutase(SOD),and guaiacol peroxidase(POD).In addition,we evaluate superoxide(O2-)and hydrogen peroxide(H2O2)accumulation and the content of reduced glutathione(GSH).Stress markers such as lipid peroxidation and electrolyte leakage were also included.The results are suggesting that increased root biomass at the lower Zn excess(10 mg/L)might be attributed to the increased root content of H2O2.Meanwhile,the concentration of 50 mg/L did not result in the accumulation of ROS and consequent membrane damage in the roots of rice.The opposite,a decreased H2O2 might be one of the reasons for the decreased root growth(particularly lateral roots).To further examined the mechanism of Zn-induced root growth inhibition,we considered the expression of genes encoding mitochondrially located proteins,mitochondrial-specific manganese superoxide dismutase(mtMn-SOD),alternative oxidase(AOX1a),and voltage-dependent anion channel(VDAC)by using quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR).The qRT-PCR was also performed for four other stress-inducible genes,glyoxalase I(GLYI),heme oxygenase 1(HO-1),polyphenol oxidase(PPO),and glucan synthase-like 5(GSL5).Our results indicate that excess Zn significantly up-regulated transcript abundance of genes involved in mitochondrial protection and cellular defense rather than induced oxidative stress as shown by the decreased accumulation of O2-and H2O2,lipid peroxidation,and electrolyte leakage.3.Finally,to further understand the mechanism of Zn-induced root growth inhibition,we conducted 2-DE to investigate which parts of cellular metabolism are affected by high Zn.2-DE profiles of rice proteome from both control and Zn-exposed seedling were quantitatively compared using PDQuest software,and a total of 43 proteins with sufficient confidence were identified by liquid chromatography-mass spectrometry(LC-MALDI-TOF/TOF MS).Zn significantly perturbed cellular processes.Proteins involved in abiotic and biotic stress response,sulfur metabolism,cell wall modification,and proteolysis were increased in abundance.In contrast,those proteins involving in energy production,secondary metabolism,and growth were down-regulated.Our data also indicate that plants exposed to excessive Zn were undergoing cell death processes.Importantly,two proteins that have been previously reported to confer tolerance to different heavy metals were reduced,translationally controlled tumor protein homolog(TCTP)and abscisic acid,stress and ripening 5(ASR5).We conclude that excessive Zn at high concentration has a great impact on various metabolic pathways in rice roots and that root growth inhibition was not accompanied by excessive accumulation of ROS.Perturbation in the mitochondrial transport chain(mETC)and the resulting decrease in energy production are the primary reasons for excessive Zn-induced growth reduction.These results provide the first evidence of root response of rice to excessive concentration of Zn,at the proteomic level.