Early Defense Signal In Arabidopsis Thaliana Against Insect Herbivory

Plants have a limited resource arsenal for growth and defense. When defense reactions become more intense, the growth and development suffer. JA or the interaction of JA and IAA play important roles in regulating the plant defense and maintain the plant growth after insect herbivory or wounding. The changes of transmembrane ion flux and the membrane potential established by ion flux is the key to start the plant defense response. However, the original signal induced by JA and how JA affects the root growth is not completely understood.In this thesis, the non-invasive micro-test technique (NMT), confocal laser technology, HPLC-MS were used to investigate the insect herbivory induced transmembrane ion signal network and the mechanism of insect herbivory caused slow growth rate. The main conclusions are as follows:(1) Jasmonate is crucial plant hormone that involves in mediating the plant development and defense. When Arabidopsis thaliana seedlings were attacked by Plutella xyllostella L., the free JA and IAA content was elevated, however, the content of ABA and SA was not significantly affected by Plutella xyllostella L. herbivory, suggested that JA and IAA involved in regulating the plant defense response and growth after insect herbivory.(2) Plutella xyllostella L. herbivory elevated the plant defense response. Feeding the larvae of Plutella xyllostella L. with Col-0 and coi1-1 plants, the larvae infer the coil-1 plants. The plant stomatal can play an active role as part of the plant innate immune system. Similar to the previous researches, we obsereved that MeJA induced the stomatal closure in WT Arabidopsis thaliana plants. However, the Ga mutant gpa1-1 and gpa1-2 impaired the MeJA induced stomatal closure, suggested that the heterotrimeric G-proteins might involved in the signal pathway of MeJA induced stomatal closure. Further, we found that the MeJA insensitive mutant coi1-1 and the PM H+-ATPase mutant aha1-6, aha1-7 also impaired the MeJA induced stomatal closure, suggested the role of COI1 and PM H+-ATPase in guard cells response to MeJA. MeJA treatment and insect herbivory also reduced the seedlings’ growth rate, suggested that the enhancement of plant defense response might reduce the plant growth rate.(3) Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). To futher investigate the role of G protein in te signal pathway of MeJA induced stomatal closure, the transmembrane ion flux and ROS accumulation was examined. We showed that MeJA induced transmembrane H+ efflux, Ca2+ influx, and K+ efflux across the PM of WS guard cells. Also, MeJA treatment promoted the accumulation of H2O2 in guard cells. However, GDP-β-S pre-treatment and gpal mutants impaired the MeJA-activated H+ efflux, Ca2+ influx and K+ efflux. The accumulation of ROS in gpa1-1 and gpa1-2. guard cells was also lower than that in WS guard cells under MeJA treatment. These results suggested that Ga subunits are involved in regulating the signal events in JA signal pathway and stomatal closure.CORONATINE INSENSITIVE 1 (COI1) perceives jasmonate-isoleucine, its mutant was insensitive to MeJA. The transmembrane ion flux results showed that the MeJA caused H+ efflux, Ca2+ influx and K+ efflux was abolished in coi1-1 guard cells, suggesting that MeJA-induced transmembrane ion flux requires COI1. Furthermore, the H+ efflux and Ca2+ influx in Col-0 guard cells was impaired by vanadate pretreatment or PM H+-ATPase mutation, which suggested that the rapid H+ efflux mediated by PM H+-ATPases could function upstream of the Ca2+ channels. After the rapid H+ efflux, the Col-0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H+-ATPase was reduced. Finally, the elevation of cytosolic Ca2+ concentration and the depolarized PM drive the efflux of K+ from the cell, resulting in loss of turgor and closure of the stomate.(4) Oral secretions of Pieris rapae L. treatment caused large H+ and Ca2+ efflux in mesophyll cells of Col-0. However, when H+-ATPase mutant, the oral secretions of Pieris rapae L. induced H+ efflux was significantly inhibited, suggested that this H+ efflux was mediated by PM H+-ATPase. In the mutant of glutamate receptor-like (GLR) genes, the oral secretions induced H+ flux was similar to that in Col-0 mesophyll cells, suggested that the PM H+-ATPase may function upstream of the GLR. Oral secretions of Pieris rapae L. treatment also induced the glutamate efflux, which suggested that the distribution of glutamate involved in the signal pathway of insect herbivory.(5) The inhibition of root growth is a common phenomenon, however, the physiological and molecular mechanisms of how JA or insect herbivory affects root growth is not completely understood. In this study, the influence of MeJA treatment on the auxin and proton flux rates in the root apex region of Col-0, coi1-1,pin2 and auxl-7 mutant lines was examined using a non-invasive micro-test technique (NMT). The auxin and H+ flux profiles taken from coil-1 mutants suggest that the modulation of JA on the auxin flux and H+ flux along the roots require the function of COI1. The auxin and H+ flux in pin2 and auxl mutants with and without JA treatment suggest that JA can affect polar auxin transport by regulating PIN-mediated auxin efflux and the AUX-mediated influx pathways. Furthermore, the expression levels of PIN1, PIN2, PIN3, PIN7, AUX1, and TIR1 genes were reduced, and the auxin biosynthesis related genes CYP79B2 and CYP79B3 was elevated under MeJA treatment, suggesting that JA signaling may modulate the auxin signaling pathways by regulating the expression of key genes during auxin biosynthesis and transport. Similar to MeJA treatment, insect hervivory also redued the IAA efflux and promoted the H+ efflux in in transition zone, suggested that as a long distance signal molecular, JA synthesis by Plutella xylostella L. herbivory down regulated the plant growth rate vis reduced the polar auxin transport in the root.