Effects Of Herbivory By Spodoptera Litura On Nitrogen Distribution In Rice Plants

Nitrogen?N?is one of the important nutritional element in organisms,which is also a basic component of important organic molecules such as proteins,nucleic acids and chlorophyll.Organic nitrogen is one key component of nitrogen nutrients in animals and plants,among which amino acids are the most basic form.Additionally,amino acids are also a key factor linking food webs.As a critical defense signal in plants,jasmonic acid?JA?and methyl jasmonate?MeJA?play an important role in plant defense against insect herbivory.Insect herbivory can activate the jasmonic acid signaling pathway and cause the accumulation of jasmonates.N is the most important nutrient for both plants and insects.Its abundance directly affects the growth and development of plants and insects.Previous studies have revealed that JA induces the transport of organic nitrogen from shoot to root in tomato.However,there is little known about whether insect herbivory influences nitrogen distribution and the trade off between growth and defense.In this study,we used Spodoptera litura and rice?Oryza sativa L.cv.Nipponbare?seedlings as a model to investigate the effect of herbivore infestation on nitrogen allocation and its consequence on rice ratoon regeneration at molecular and physiological levels.Our results showed that insect herbivory S.litura could induce the alteration of nitrogen alolcation,thereby influencing nutrient assignment to growth and defense.Additionally,the finding that insect herbivory influences rice ratoon regeneration is important for guiding actual agricultural production,and also lay a good theoretical foundation for increasing the yield of ratooning rice.The main results of this study are as follows:1.Elemental Analyzer-Isotope Ratio Mass Spectrometry?EA-IRMS?was used to detect total nitrogen content in different tissues of rice after 48hours feeding by S.litura.Total nitrogen of the feeding region and the region below feeding in the first leaf were significantly decreased by 16%and 44%,respectively.And that in old root and new root were increased by 33%and 44%,respectively.Additionally,¹⁵N labeled glutamine was applied to rice leaves.It was shown that ¹⁵N of the feeding region and the region below feeding in the first leaf were significanlty decreased by 32%and 47%,respectively.And that in the base stem,old root and new root were increased by 13%,61%and 44%,respectively.These results indicated that feeding by S.litura on rice leaves could induce nitrogen migration from the feeding region to root in rice,and this transfer might be due to the swith of the relationship between source and sink.2.After S.litura invasion for 48 h,the pests on the rice leaves were removed,and after the rice was allowed to grow back for 48 hours,the changes in the nitrogen content in the foraged leaves and new roots were detected.Nitrogen was accumulated in the foraged leaves,while the total nitrogen content in the new roots of the treatment group was significantly lower than that of the control group,and decreased by 20%compared with the control group.In addition,100?M MeJA was used to simulate herbivore invasion and the rice was treated for 48 h.The total nitrogen in the feeding leaf was continuously decreased compared with that in other groups.These results further indicated that herbivore could induce nitrogen from shoot root,and the nitrogen could move back when the alarm went off.This migration might be regulated by JA signaling and other unknown signaling.3.Rice plants was cultured in nutrient solution with stable isotope labeled ¹⁵N(¹⁵NH₄¹⁵NO₃),and the plants were inoculated with the larvae of S.litura for 48 h and then the larvaer were removed from the plants.The total nitrogen content in new rice roots increased significantly 48 h after removal of insect larvae,and the total nitrogen content in old roots of rice increased significantly 96 h after lareval removal.At the same time,the content of ¹⁵N in the new roots of rice increased significantly 48 h after lareval removal,and the content of ¹⁵N in the new and old roots increased significantly 96 h after lareval removal.These results indicated that nitrogen uptake capacity was enhanced in the recovery time after insect herbivory.4.After S.litura invasion for 48 h,the content of total chlorophyll,chlorophyll a and chlorophyll b in rice leaves were significantly decreased compared with that in control.Further analysis showed that RuBisCo protein was degraded after herbivore invasion at that time,and RubisCo content was still decreased in recovery times compared with that in control.After 48 h of insect herbivory the feeding leaves were treated with 100?M MeJA for other 48 h,the content of RuBisCo protein in MeJA-treated leaves was decreased compared with that in control and recovery group.These results indicated that the migrated nitrogen was probably derived from RuBisCo degradation.5.The change of free amino acids in rice after feeding by S.litura for48 h were detected by high performance liquid chromatography?HPLC?.The results showed that free amino acids in the feeding region and new roots were significantly increased compared with that in control.Nitrate was increased while ammonium was decreased 48 h after feeding.Additionally,the activities of nitrate reductase and glutamine synthase were increased in leaves but decreased in roots.These results indicated that herbivore infestation could lead to the alteration of nitrogen metabolism and nitrogen probably transport in the form of free amino acids.6.To investigate possible effects of N allocation by insect herbivory on rice ratoon regeneration,rice plants were infected with S.litura larvae for 7 d,more fresh roots were found in herbivore-treated plants compared with that in control.Additionally,similar results also found in the rice where the above-ground part was removed after infection by the larvae for7 d or mechanical damage for 7 d.These results showed that insect herbivory enhanced the ability of rice regeneration.7.RT-PCR analysis showed that expression of genes involved in JA signal and amino acid transport,such as OsAOS,OsMPK 3,OsMPK 6,OsAAP 6,OoAAT 15 were all upregulated after insect herbivory compared with that in control.This study demonstrated that rice plants transport N from insect-feeding area to roots upon insect herbivory for the morphogenesis of roots and the stored N can be used for growth recovery after insect herbivory,leading to the high efficiency of plant nutrients during insect herbivory.