Cloning, characterization, and subcellular localization of a novel JAZ repressor from Eleusine coracana

Abstract

Jasmonate ZIM domain (JAZ) proteins are key regulators of the jasmonic acid (JA) signaling pathway. Repressors of JAZ remain bound to the myelocytomatosis 2 (MYC2) or MYC3/MYC4 transcription factors in the absence of JA and negatively regulate transcription of the JA responsive genes. In the presence of JA, JAZ proteins interact with coronative insensitive 1 (COI1), the recognition molecule of E3 ubiquitin ligase SCFCOI1 (COI1 stabilized by Skp, cullin, F-box containing complex), get ubiquitinated, and subsequently degraded by the 26S proteasome. However, there is a dearth of knowledge about this gene family in monocot cereals, specifically its role in finger millet is unknown till date. Here we present the isolation and characterization of a novel JAZ family repressor gene from nonsequenced Eleusine coracana (EcJAZ) utilizing available genome information of Oryza, Sorghum, and Setaria. The EcJAZ sequence showed the presence of a conserved ZIM domain, the Jas motif, and N-terminal motif 7 like other Group1 TIFY sequence containing proteins. We observed coronatine (an analog of JA-Ile) dependent and time dependent degradation of recombinant EcJAZ that thereby fulfilled the basic characteristic of the JAZ proteins. We found a proteasome inhibitor N-(phenylmethoxy) carbonyl-L-leucyl-N-[(1R)-1-formyl-3-methylbutyl]-L-leucinamide) (MG132) mediated degradation inhibition of EcJAZ that supported its 26S proteasome mediated degradation. Our study shows the nuclear localization of GFP-EcJAZ by Agrobacterium mediated transient transformation of onion scale epidermal cells. In Eleusine leaves, transcription of EcJAZ increased 4.2-fold by salt stress and 5.5-fold by coronatine application; thus ascertained its inducibility by the abiotic stress as well as by bioactive JA-Ile. Taken together, all these results contribute to our understanding of the JA signaling pathway in Eleusine coracana.

Keywords

• agrobacterium transformation
• coronatine
• finger millet
• jasmonic acid
• salt stress