The Role Of Iron In The Plant Response To Ammonium Stress And Its Underling Mechanism

Ammonium is one of the main nitrogen sources for plant growth.However,due to the excess application of nitrogen fertilizer,especially the excess application of ammonium,and improper fertilization manners as well as detrimental environment factors,would easily result in a high level of ammonium accumulated in local soils,which inhibits the crops growth.Clarifying the mechanisms of ammonium toxicity and tolerance in plants may favor molecular breeding of ammonium-tolerant crop.Owing to the high level of ammonium in submergenic soil is often accompanied with high level of soluble iron?Fe?,we used Arabidopsis and rice plants as test species to investigate the effect and mechanism of iron regulating root growth under ammonium stress by using physiologic and molecular biology method.The main findings are as follows:The Arabidopsis plants fed with 2 mM NH₄⁺ had shorter roots than those fed with 2 mM NO₃^-in the agar growth mediums with same Fe supply.Increase in Fe supply further reduced the growth of roots under ammonium stress.In addition,in the solution growth medium,we also found that high level of iron supply also inhibited the growth of roots and shoots in the Arabidopsis plants under ammonium stress.Although rice was a kind of ammonium-tolerant plant which grown in flooded and anaerobic soil,an increase in iron supply could also inhibit the growth of roots under ammonium stress.These results indicated that iron plays an important role in inhibiting root grwoth under ammonium stress in plants.Both the analysis of root cell morphology by interference microscope and split plate experiment showed that the inhibition of the root elongation by ammonium should be caused by the decreased number of meristem cells and the length of elongation zone.Therefore,the root tip should be considered as the key target of iron acting on root growth inhibition under ammonium stress.Further analysis showed that there was no significant difference in the length of root elongation between the wild type and iron uptake or storage mutants?irtl and fer?of Arabidopsis.Therefore,either the iron uptake or the iron storage was not responsible for the inhibition of root growth under ammonium stress.By measuring the total iron and the apoplastic iron concentration in the root,we found that although the ammonium stress decreased the total iron concentration in roots,it clearly increased the accumulation of Fe in root apoplast,indicating that the accumulation of apoplastic iron in the root might be the main reason for the inhibition of root elongation under ammonium stress.In addition,Perls/DAB and Tumbull/DAB staining also showed an increased distribution of apoplastic iron in the root under ammonium stress,providing a further support for that the accumulation of apoplastic iron probably is a key factor resulting in inhibition of root growth under ammonium stress.Based on the above results,the mechanism underling how the apoplastic iron inhibite root growth under ammonium stress was further studied.The results showed that the root length of the multi-copper oxidase mutant lpr1-lpr2 was significantly higher than that of the wild type Col-0 under the 2 mM NH₄⁺ and 50?M Fe treatment,indicating that LPR multi-copper oxidase was involved in inhibition of root growth under ammonium stress.The Perls/DAB and Turnbull/DAB staining showed that the accumulation of apoplastic iron in roots of multi-copper oxidase mutant lpr1-lpr2 was less than in roots of Col-0 plants,indicating that LPR multi-copper oxidase was involved in the accumulation of apoplastic iron in roots under ammonium stress.In addition,aniline blue and NBT staining showed that the roots of the multi-copper oxidase mutant had less accumulations of callose deposition and superoxide under the 2 mM NH₄⁺ and 50 ?M Fe treatment,compared with wild type Col-0 plants.Taken together,we concluded that LPR multi-copper oxidase mediated iron accumulation in root apoplast is a key factor resulting in the inhibition of root growth under ammonium stress.