Studies On The Mechanism Of High Zn Efficiency In Rice (Oryza Sativa L.)

Zinc deficiency in rice(Oryza sativa L.)is one of the most widespread micronutrient constraints in plants and often causes a significant reduction in yield. Zinc efficient genotypes are those that can grow better and produce higher yields than a standard genotype when grown in Zn-deficient conditions.Using the technologies as chlorophyll fluerescence,77 K Ch1 fluorescence,TEM,bioinformatics analysis and RT-PCR,with Zn-efficient(IR8192)and -inefficient(Erjiufeng)rice genotypes as plant materials,the relationship of zinc(Zn)efficiency in rice to differential photosynthetic capacity,physiological and morphological response and the expression patterns of ZIP were studied.The main results were as following:1.Zinc deficiency caused extensive declines in leaf chlorophyll(ch1)content,ratios of ch1 a:b,Pn,Fv/Fm,and Fv/Fo,indicating that the intrinsic quantum efficiency of the PSâ…¡units was damaged.Greater decline was observed the inefficient genotype (Erjiufeng)than in the efficient genotype(IR8192).The 77 K Ch1 fluorescence emission spectrum revealed that Zn-deficiency blocked the energy spillover from PSâ…¡to PSâ… and more excitation energy was distributed to PSâ…¡in IR8192 than Erjiufeng. The spectrum of Zn-deficient Erjiufeng was completely disordered,implying that the photosynthetic center was seriously damaged.Electron microscopy showed that Zndeficiency caused a severe damage to the fine structure of chloroplast and IR8192 had better preserved chloroplast ultrastructure as compared with Erjiufeng.These results indicate that Zn deficiency decreases leaf photosynthetic capacity primarily by reducing the number of PSâ…¡units per unit leaf area,and also reduces the photochemical capacity of the remaining PSâ…¡units.Therefore the maintenance of higher efficient photochemical capacity under low Zn stress is a key factor for the high Zn efficiency in rice.2.The moderate Zn deficiency significantly induced the larger root surface,longer total root length,longer total fine root length and more total root tips in IR8192(the Zn-efficient genotype),but no significant increase was observed in these root parameters of Erjiufeng(the Zn-ineeficient genotype).And greater values of these roots parameters were observed in the Zn efficient genotype IR8192 than in the Zn inefficient genotype Erjiufengeven under severe Zn deficiency.Under moderately Zn deficient condition,many swollen mitochondria were observed in the root tip cells of Erjiufeng,whereas most root cells in IR8192 remained intact.Under severe Zn deficient condition(pZn²⁺＞11.5),critical damage of the cells was observed in both genotypes.The vacuolation phenomena were popular,plasmolysis were observed other than the swollen mitochondria.These results suggested the Zn efficiency in IR8192 is closely associated with its high root tolerance to Zn-deficiency by maintaining intact root tip cells and functions.3.The detectable low macular weighted organic acids in the exudates from two genotypes were oxalic acid and citric acid.Zinc deficiency increased the oxalic acid exudation from both genotypes;comparing to the control,the citric acid exudates from Zn-deficient IR8192 were increased,however,it was decreased in Zn-deficient Erjiufeng.Under the Zn deficient conditions,the total phenolic acids and total soluble sugar exudates were also higher in IR8192 than Erjiufeng,suggesting that IR8192 can exudate more small organic molecules to activate the Zn²⁺in the rhizoshpere.In contrast to control,the citric acid and malic acid contents in roots of Zn-deficient IR8192 were lower than those in Erjiufeng,however,the ratio was reversed in the shoots.The results imply that IR8192 could transport the chelated Zn²⁺more efficiently than Erjiufeng according to the Zn-malic-acid-transportation model,There was no significant difference between the IMDH and SDH in there two genotypes, however,the activity of malic dehydrogenase was higher in Zn-deficient IR8192 than in Erjiufeng,this may resulted in more production of malic acid in IR8192 roots and the efficient transportation of malic-acid-chelated Zn²⁺to the shoots.4.Exposure of seedling to the Zn deficiency caused a general increase of the lipid peroxidation in both genotypes,the increased extent were higher in Erjiufeng as comparing with IR8192.The electrolyte leakage,H₂O₂ content and free Pro content also showed great increase after the rice seedlings were subjected to Zn deficiency. However,the increased percentages were much lower in IR8192 than in Erjiufeng, suggesting the lower oxidative stress in IR8192 caused by Zn deficiency.The relatively lower oxidant stress in Zn-deficient IR8192 may result from its higher efficient ROS scavenging enzymes.In general,the activity of SOD,POD,APX and CAT were higher in IR8192 when the seedlings were subjected to Zn deficiency.It is concluded that the greater tolerance in IR8192 to Zn deficiency was due to better synergies between the antioxidative enzymes which protected membrane system and maintain the functions,it may be an important mechanism for the high Zn efficiency in IR8192.5.ZRT,IRT-like protein(ZIP)comprises a large family of transition metal transporters in plants that have diverse functions to transport zinc,iron,copper,etc. Here,we provided a complete overview of this gene family in rice.Based on hidden Markov models and BLAST analysis,a total of 17 ZIP coding genes were identified and further studied by semi-quantitative RT-PCR analysis.Sequence analysis revealed that the 17 putative genes distribute randomly in 8 chromosomes and vary a lot in their sequence similarity.Although most of the predicted proteins had typical characteristics of the ZIP protein family,and were predicted to located in the plasma membrane,some of which seemed quite different,such as OsZIP9 and OsZIP10.The expression pattern for each gene in the Zn-efficient rice genotype(IR8192)and the Zn-inefficient rice genotype(Erjiufeng)was compared by semi-quantitative RT-PCR in roots,shoots and panicle from plants grown under Zn deficiency and normal conditions.Several ZIP genes were suggested only tissue specific expressed,such as OsZIP1(expressed only in root),OsZIP5(panicle),OsZIP8(root and panicle), OsZIP9(root and shoot),OsZIP13(root and panicle),OsZIP14(shoot),OsZIP15 (root and panicle).Under Zn deficit condition,the expression of OsZIP2,OsZIP4, OsZIP6,OsZIP7 and OsZIP14 were up-regulated in one or both genotypes,which indicated that they may be the possible Zn transporters in rice.Furthermore,the expression of OsIRT1,OsZIP3,OsZIP4,OsZIP6,OsZIP11,OsZIP15 was found to be much stronger in the roots of IR8192 than that of Erjiufeng,which suggested that those genes may contribute to the high Zn efficiency in rice.However,in the shoots the trends were not very obvious or even reversed for OsZIP4 and OsZIP6,which necessitates further investigation to elucidate the functions of each ZIP transporter in rice.