Copper-induced Initiation Of Lateral Root Is Mediated By AtNOA1 In Arabidopsis

On account of sessile lifestyle, plants had constantly adapted to the changing environment to survive and reproduce offspring. Plant’s root system not only provides structural anchor for itself, but also is site for acquisition of water and nutrients and responses to abiotic and biotic signals. Copper plays an important role in plant growth and development, but excessive copper is toxic to plants. Previous studies showed that copper involved in root architecture, but the molecular mechanism hasn’t been discussed. We found different exogenous concentrations of Cu2+ can induce the lateral root growth and development of wild-type Arabidopsis, and the effect of 50 ?M Cu2+ is most obvious. However, the mutation of Atnoa1 significantly inhibited the lateral root growth in the presence of copper, and complement plants of Atnoa1 Com-1、Com-2 rescued the inhibiting action. These results indicated copper induced the growth of lateral root, and Atnoa1 involved in this progress. Studies have proved that Atnoa1 regulate the balance of NO in plant, and we speculated that Cu2+ induced lateral root development may be associated with NO synthesis and balance. Treating WT、Atnoa1 and Com-1with different concentrations SNP, we found 20 ?M exogenous SNP can induced the lateral root formation of all above plant material. While the mutation of AtNOA1 gene can’t change the lateral root formation induced by NO. That is to say, AtNOA1 regulated Cu2+-induced lateral root formation may not depend on NO concentration and balance, but by other means.Oxidative stress may be the biggest injury caused by Heavy metal, DAB staining results revealed that H2O2 increased significantly in mutant Atnoa1 treated by copper. What role does the large increased H2O2 in the Cu2+-induced lateral root formation plays? NADPH peroxidase AtrbohD/F catalyses the generation of ROS and protein CEO1 is the antioxidant of Arabidopsis. When AtrbohD/F and ceo1 were treated with 50 ?M Cu2+, their lateral root number increased. Statistical analysis results show that the lateral root number of AtrbohD/F was 1.5 times of WT, while the number of ceo1 was as much as WT. Results implied that the content of H2O2 had little influence on lateral root formation. As a second messenger, Ca2+ plays great roles in plant normal growth and development as well as in adaptive responses to environmental stresses. Ca2+ signal consists of specific parameters such as content、spatial orientation、oscillation period、frequency、amplitude. In order to investigate the changes of [Ca2+]cyt dealt with cooper, we transformed WT、Atnoa1、Com-1 plants with AEQ plasmid. The test results proved that 50 ?M Cu2+-triggered increase of [Ca2+]cyt in WT and Com-1, but the increase in Atnoa1 mutant was abolished. Observation of [Ca2+]cyt change which marked by YC3.60 produced the same findings. The application of La3+ and EGTA inhibit of the increasing of [Ca2+]cyt, and the result demonstrated that the increased Ca2+ resulting from [Ca2+]cyt release. These results indicated that as the downstream of AtNOA1, Ca2+was involved in the copper-induced initiation of lateral root, and the regulation of [Ca2+]cyt change by AtNOA1 relied on the activity of Ca2+ channel.Auxin not only promotes LR initiation but also necessary for all stages of LR development. In order to observe auxin changes directly, hereditary material noa1-DR5::GUS and noa1-DR5::GFP were construct by hybridization. We used 50?M Cu2+ treat the above hereditary material in time course, then observed the distribution of auxin though the GUS staining and GFP transformation. GUS staining showed that in wild type auxin transferred from primary root tip to position of lateral root formation induced by copper treatment, and the course was inhibited in Atnoa1 mutant. By using confocal microscopy we observed that the transfer of auxin mainly occurred in vascular bundle and cortex in wild type. As the same with GUS staining results, Atnoa1 mutant lacked auxin transport by observing GFP fluorescence. Auxin transport depended on both auxin influx and outfluxe transporters, but whose activity was prevented by AtNOA1 had not been known, so auxin carrier inhibitors NPA and 1-NOA were used to treated wild type and Atnoa1 mutant. Application of NPA can effectively restore the inhibitory action of lateral root growth which induced by copper in Atnoa1 mutant, but the 1-NOA promote the inhibitor action. The same concentration of 1-NOA and NPA treatment showed the same result. So, we can draw the conclusion that AtNOA1 regulated the auxin influx activity.To sum up, the regulation of copper-induced lateral root formation which is mediated by AtNOA1 affect the Ca2+channel activity, reduce calcium ion concentration, change auxin influx transporter activity and eventually inhibit the transfer of auxin in plant.