Third Messenger And Its Downstream Substrates Of Abscisic Acid Signaling Pathway In Maize

Abscisic acid?ABA?is one of the five major hormones in plants,and plays an important role in the regulation of plant development and response to environmental stress.Until 2009 after 46 years of its first discovery in 1963,pyrabactin-like protein?PLY?was identified as the direct receptor of ABA.After that,serine/threonine protein phosphatase type 2C?PP2C?and sucrose non-fermenting protein kinase 2?SnRK2?were indentified as the second and third messengers of ABA signaling,respectively.Under abiotic stresses,ABA accumulates in plants and regulates the expression of resistance-related genes through signal transduction.Compared with Arabidopsis,rice and other model plants,the maize genome is not only large but also has a large number of repeat and recombination sequences.Structure and function are diversified among the members of the ZmPP2C and ZmSnRK2 gene subfamilies.These memebers catalyze dephosphorylation and phosphorylation of many downstream proteins.Therefore,the downstream pathways of ABA signal transduction is divergent.On the basis of the 238 proteins with up-regulated phosphorylation level in response to ABA induction indentified by phosphorylation proteomics analysis in the previous study,the probable substrate proteins were predicted for SnRK2 by sequence alignment with the known substrate proteins of SnRK2 in Arabidopsis and analysis of conserved phosphorylation motif of SnRK2,and evaluated for their interaction with ZmSnRK2 subfamily members by in vitro phosphorylation assay and yeast two-hybrid,in order to reveal ABA signaling pathway downstream of ZmSnRK2,and provide reference for further elucidation of mechanism of ABA-mediated abiotic tolerance.The main results are as follows:?1?By sequence alignment with the known substrate proteins of SnRK2 in Arabidopsis and analysis of conserved phosphorylation motif of SnRK2,ten proetins?XP₀08666965.1,NP₀01183680.1,XP₀08662247.1,NP₀01167942.1,XP₀08656995.1,NP₀01152904.1,NP₀01145831.1,NP₀01149268.1,NP 001132279.1 and NP₀01142137.1?were predicted as the substrates of ZmSnRK2 in response to ABA induction from the 238 proteins with up-regulated phosphorylation level in response to ABA induction.?2?By reverse transcription PCR,ten and eight sequences of the open reading frames were amplified for the eleven members of the ZmSnRK2 gene subfamily predicted by genome-wide serach,and the encoding genes of the ten predicted substrate proteins of ZmSnRK2.No fragment was amplified for the ZmSnRK2.9 gene and for the encoding genes of proteins XP 008662247.1 and NP₀01152904.1.Combined with the previous reports,ZmSnRK2.9 was recognized as a pseudogene without expression.The maize ZmSnRK2 gene subfamily actually contains a total of ten members of ZmSnRK2.1/2/3/4/5/6/7/8/10/11.?3?In in vitro phosphorylation assay,relative luciferase activity of the protoplasts of triple mutant SnRK2.2/3/6 of Arabidopsis respectively transfected by the ZmSnRK2.1/2/8/10 genes under ABA intuction was significantly higher than the negative control,and ten times higher than the unstranfected mutant.Therefore,it is resulted that ZmSnRK2.1/2/8/10 transduct ABA signal from PP2C and catalyze phosphorylation of downstream proteins.However,no significant difference of relative luciferase activity was detected in the protoplasts of the same mutant transfected by the ZmSnRK2.4/6/7/11 genes between ABA induction,the genitive control and the untransfected mutant.These four members of the ZmSnRK2 subfamily may not be involved in ABA signal transduction.?4?Proteins XP₀08666965.1,NP₀01142137.1 and XP₀08656995.1 were excluded from yeast two-hybrid assay for their self phosphorylation.From the other five predicted substrate proteins,interaction was detected between NP₀01183680.1 and ZmSnRK2.1/2/4/5/7/8/10/11,and between NP₀01167942.1 and ZmSnRK2.1/10 in the yeast two-hybrid.Therefore,these two proteins were identified as the substrate of ZmSnRK2 in the downstream pathway of ABA signal transduction.In summary,members ZmSnRK2.1/2/8/10 of the ZmSnRK2 subfamily conduct ABA signal from PP2C and catalyze phosphorylation of the downstream proteins,whereas members ZmSnRK2.4/6/7/11 are not involved in this pathway.Of the two substrates of ZmSnRK2 identified by the yeast two-hybrid assay,one is homologous to a serine/threonine protein kinase in Arabidopsis,and the other is a SAM domain-containing protein.These two proteins involved in many different physiological and biochemical processes,suggesting that the pathway of ABA signal transduction downstream of SnRK2 is divergent.It will be more and more difficult for further investigation of the pathway downstream of the these substrate proteins.