Analysis Of HY1 Promoter In Arabidopsis And HO-1 Participating In The Alleviation Of Programmed Cell Death In Wheat Aleurone Layers

Heme oxygenase?HO,EC 1.14.99.3?,the rate-limiting enzyme of heme metabolism,has been characterized in a wide variety of organisms including animals,plants,and microbes.HO catalyzes the oxidative cleavage of heme to produce biliverdin IXa?BV?,carbon monoxide?CO?,and free iron?Fe2+?,in the present of reducing agent?NADPH/FNR/Fd?.Up to now,the function of plant HO has been well concerned.A great deal of previous evidence with pharmacological approach using activators or inhibitors,and the genetic approach with the over-expressed or knock out/down the indicated gene,has indicated that HO-1 is not only involved in the signaling transduction of photomorphogenesis,lateral and adventitious roots development and stomatal closure,but also response to hormone and abiotic stimuli.However,the functional cis-acting elements existing in HY1 promoter are not well elucidated yet.Meanwhile,the aleurone layer is nutrition cereal crops,playing an important role in the process of germination of seeds of cereal crops.It has been found that,HO-1,cobalt,gibberellin?GA?and hydrogen peroxide?H₂O₂?are associated with the programmed cell death?PCD?in the cereal aleurone layer.Nevertheless,the relationship between HO-1 and GA-induced PCD in wheat aleurone layers,regulated by cobalt or H₂O₂,has not been fully expounded.Here,to investigate the previously unclear characterization of the Arabidopsis HY1?HO-1?promoter,an approximately 1.8 kb of HY1 promoter?pHY1,-1666 to +132?and its deletion fragments?5D1,-1528 to +132;5D2,-1109 to +132;5D3,-688 to +132;5D4,-169 to +132;3D1,-1666 to +100;3D2,-1666 to-1;and 3D3,-1666 to-170?,were fused to?-glucuronidase?GUS?reporter gene and stably transformed into Arabidopsis.The transgenic plants were subjected to several environmental stimuli?especially to mild salinity,iron deficiency,and mercury exposure?.The results showed that the region from +1 to +100 in the 5'-untranslated region?5'-UTR?was essential for basal activity of HY1 promoter.The sequence between-1666 and-688 was,at least partly,required for the promoter basal activity.Moreover,the possibility of negative regulatory elements located between-688 and-169 could not be easily ruled out.The induced GUS activities under mild salinity and hydrogen peroxide?H₂O₂?treatments,were slowed down by the progressive 5' deletion?from-1666 to-688?.which were correlated with the reduced numbers of MBS elements?-1542,-1333,-1078,and-177?.The MBS-free promoter construct 5D4?-169 to +132?,however,fully lost the inducibility of promoter activity.Therefore,we proposed that the MBS elements existing in HY1 promoter might be crucial for salinity-induced HY1 up-regulation in an H₂O₂-dependent fashion.Moreover,the regions from-169 to-1 and-688 to-169 were deduced as the regulatory region of HY1 promoter in response to iron deficiency and mercury exposure,respectively.In conclusion,our results provided useful knowledge for the understanding transcriptional regulation mechanism of Arabidopsis HY1 gene.In the further study,we found that 1)pretreatment with 50 ?M CoCl2 or CoPP alleviated the GA-induced PCD,influencing endogenous H₂O₂ content,and increased the transcripts of superoxide dismutase?SOD?gene;2)pretreatment with 100 ?M H₂O₂ improved the livability of aleurone layer and increased the transcript level of HO-1;3)co-pretreatent with the inhibitor of H₂O₂ synthesis,diphenyleneiodonium?DPI?with CoCl2 or CoPP,inhibited the alleviating effect of CoCl2 or CoPP in wheat aleurone layer PCD,and reduced the expression of HO-1 induced by CoCl2 or CoPP.Together,the above results showed that,HO-1 might be involved in the cobalt-alleviated GA-induced PCD in wheat aleurone layer,H₂O₂ being the downstream signal molecule.