The Identification Of Jasmonate Transporters And Their Essential Role Of Signal Relay In Systemic Wound Resistance

Analogous to animal acquired immunity and innate immunity, the plant systemic resistances,triggered by the prior/primary local challenges, provide the whole plant broad-spectrum and long-lasting defenses against secondary challenges. Systemic resistances can by divided into systemic acquired resistance (SAR), induced systemic resistance (ISR) and systemic wound resistance (SWR),which are triggered by pathogens, non-pathogenic rhizosphere microbes and mechanical wounding/herbivore attacks, respectively. The oxylipin phytohormone jasmonates (JAs) play a vital role in defense against the bio-/abiotic stresses in both local and systemic challenged sites, and are essential for plant systemic resistances. In JA-mediated systemic resistances, the nature and the relay of the systemic signal(s) remain elusive.Based on our findings that exogenous jasmonate inhibits yeast cell growth, a heterologous system was established to screen putative jasmonate transporters. Utilizing this heterologous screening system, two jasmonate transporters, AtJAT1 and AtJAT2 was identified. 3H-JA transport assessment in yeast cells shows that AtJATI and AtJAT2 functions as jasmonate exporters. In addition,their transport activities were pH-dependent, with decreased activity at pH 6.5, indicating that the protonated JAH is their preferential transport substrate.The promoters of both AtJAT1 and AtJAT2 are principally active in the vascular tissues of roots,hypocotyls, cotyledons and leaves, and in stomatal guard cells of seedlings as shown by promoter-GUS fusion analysis. At the flowering stage, GUS activity was evident in reproductive organs including veins of sepals, stamen filaments and stigmas. The transcript level of both AtJAT1 and AtJAT2 was decreased in opr3 seedlings, showing that endogenous JA is essential for their expression. The promoter activity of AtJATl and AtJAT2 is identical to that of OPR3, which encode a key enzyme in JA biosynthesis. In addition, AtJAT1- and AtJAT2-GFP were also localized in the plasma membrane of phloem cells of vascular tissue and guard cells. These results are in accord with a role of AtJAT1 and AJAT2 of JA transport.We further characterized AtJATl and AtJAT2 in Arabidopsis suspension cells derived from calli induced from root segments of atjatl-1,atjat2-1 and atjatl-1;2-1 mutants, in which expression of AtJATl or/and AtJAT2 was disrupted by T-DNA insertions, as well as the transgenic plants over expressing AtJAT1 (JAT10X) or AtJAT2 (JAT20X). Compared to wild-type cells, atjatl-1,atjat2-1,atjatl-1;2-1 cells exhibited lower, whereas JATIOX and JAT20X cells exhibited higher 3H-JA retention, indicating that AtJAT1 and AtJAT2 functions as JA importers in Arabidopsis cells. In addition, AtJAT1 and AtJAT2 exhibited higher affinity to JA with a Km about 537.1±155.8 nM,416.8±107.5 nM respectively. Substrate competition shows that AtJATl and AtJAT2 have a broad specificity to JAs (MeJA, JA-Ile and Coronatine) and precursors (Linolenic acid and OPDA) but not general aliphatic acids.To clarify a role of AtJATl and AtJAT2 in systemic wound resistance (SWR), we measured the production of JA and JA-Ile in Arabidposis by HPLC-MS/MS. Compared to wild-type plants, the JA and JA-Ile production were reduced in systemic leaves in atjatl-1;2-1 plants, but increased in JATIOX and JAT20X plants, whereas those in local wounded leaves remained comparable. In consistence, the inducibility of the primary wound-responsive genes in the JA biosynthetic (OPR3)and signaling pathway (JAZ5 and JAZ7) was also reduced in systemic leaves but not local leaves of atjatl-1;2-1 plants. These results collectively indicate that AtJATland AtJAT2 play a vital role in maintaining a critical systemic JA concentration and modulating the systemic gene expression.To unravel the mechanism by which AtJAT1/2 proteins are involved in modulating systemic stress responses and systemic JA upsurge, we next examined a role of AtJAT1/2 in long-distance JA transport.Our results suggest that AtJAT1/2 modulate systemic stress responses and systemic JA upsurge by affecting long-distance JA transport. We proposed that AtJAT1 and AtJAT2 could maintain a critical JA concentration for establishing the cascade of de novo JA biosynthesis along the phloem by retrieving JAs to the phloem cells in SWR.In summary, we first indentified jasmonate tranporters AtJATl and AtJAT2 in Arabidopsis and show that they are critical for systemic singal relay in plant SWR. The heterologous system we established to screen JA trasnporters can provide a new platform for screening the transporters of other phytohormones. Furthermore, the model we proposed on systemic JA signal dispersion can also be extrapolated to other weak acid signals in plant systemic resistance.