Cloning And Functional Analysis Of GUN4in Rice (Oryza Sativa L.)

GENOMES UNCOUPLED4(GUN4) is originally found in Arabidopsis thaliana, which is involved in the regulation of chlorophyll biosynthesis by activating Mg-chelatase and plastid-to-nucleus retrograde signaling pathway, but the specific mechanisms are still unknown. Here, by using map-based cloning and a yellow-leaf-color mutant Huangyu B(HYB), we identified a knockdown epi-allele of OsGUN4, epi-GUN4, in rice (Oryza sativa L.). We found that HYB had no nucleotide sequence changes but a hypomethylated segment containing an antioxidant response element (ARE) in the promoter region of OsGUN4. Demethylating treatment partially restored the function of HYB and reverted seedlings into normal green leaf color. We further determined OsGUN4was a chloroplast-localized protein and analyzed the response pathways of OsGUN4to low-to-high light-and H2O2-tolerance. The main results are elaborated as following.HYB mutant is sensitive to light intensity. Under paddy field conditions, the mutant HYB exhibited a stable yellow-leaf-color phenotype throughout the development stages and contained only15.0%of the chlorophyll contents as found in its wild type (WT) parental variety LTB. We also found HYB showed leaf color differences under various environmental conditions, especially for light intensity. Under low light (LL) conditions, HYB plants produced yellow-green leaves and accumulated95.0%chlorophyll that of LTB. However, after transferred to the high light (HL) conditions, HYB developed yellow leaves and contained only12.0%chlorophyll content of LTB.OsGUN4is the cadidated gene of the xantha trait in HYB. To identify the gene underpinning the xantha (XNT) phenotype in HYB, we performed map-based cloning using3,173F2recessive xantha plants from crosses of HYB and three wild-type plant lines. On the basis of previous mapping, we further mapped the XNT locus to a57-kb region, between molecular markers IDS7and SNP18, containing eight open reading frames (ORFs) on the short arm of rice chromosome11. ORF2(Osllg1267000) encodes OsGUN4(Genomes Uncoupled4), a GUN4-like domain containing protein. Surprisingly, sequencing of the whole region revealed no nucleotide alternations between LTB and HYB. To determine the cause of the xantha phenotype of HYB, we further performed qRT-PCR expression analysis of the eight ORFs. Although ORF2was highly expressed in the leaves of LTB, nearly no ORF2transcript was detected in HYB plants. The other seven ORFs had no differences in expression level between HYB and LTB.The XNT locus in HYB is allele of OsGUN4. To further prove mutation(s) of OsGUN4might have generated the xantha phenotype in HYB, we identified four xantha mutant alleles from～30,000M3F1individual plant lines derived from crossing gamma rays mutagenized M2population of a temperature-sensitive genic male sterile line GS113with HYB. By DNA sequencing, three recessive deletion mutations of OsGUN4(gun4-1～gun4-3) were identified with a yellow leaf color phenotype similar to that of HYB. These results suggested the XNT trait of HYB was conditioned by an unknown mutation of OsGUN4.The xantha leaf color mutation was caused by epi-genetic mutation of OsGUN4. To clarify whether the xantha phenotype was caused by possible epigenetic mutation(s) that regulated the expression state of the OsGUN4gene, we performed bisulfite sequencing of the entire OsGUN4gene and its downstream promoter sequence and found that HYB had a methylation change at-2386bp loci that contained an antioxidant response element (ARE). Demethylating treatment of5-aza-dC restored28.0-56.0%HYB and its derived xantha lines into normal green leaf color. These results further confirmed that the methylation caused the downregulation of OsGUN4and the yellow-leaf-color mutation was conditioned by an epi-allele of OsGUN4, epi-GUN4.The expression of OsGUN4was induced by reactive oxygen species. The OsGUN4gene, contains only one exon of1071bp length cDNA, encodes a273amino acids protein with a143-aa-length (from87th to229th aa) GUN4-like domain. After treatment of herbicide norflurazon (NF), the expressions of LHCB and PsaA of photosynthesis-associated nuclear genes were significantly down-regulated in LTB plants, whereas the expressions still remained high level in HYB. These indicated that epi-GUN4owned a typtical characteristic of genome uncoupled (GUN) genes, which showed that mutation or down-regulation of OsGUN4could not reinstrain the expression of PhANGs. Besides, after high light and H2O2treatments, the expressions of OsGUN4were significantly increased in LTB plants, whereas there were no significant changes in HYB, which suggested the ARE element was essential for the ROS-induced expression of OsGUN4. The expressions of OsGUN4in both CHX-treatment and CHX and H2O2co-treatment wild-type seedlings showed similar dynamic changes and were lower than that of H2O2-treatment seedlings, so OsGUN4was induced by ROS indirectly. By the subcellular localization, OsGUN4was confirmed as a chloroplast-localized protein. By qutatative RT-PCR analysises, OsGUN4showed a photoperiod expression under high light condition both in japonica variety Nipponbare and indica variety LTB.The OsGUN4mutation affected the expressions of many nuclear genes. HYB owned similar chlorophyll concentrations as in LTB, this indicated that OsGUN4was not essential for chlorophyll biosynthesis but might play very important roles in regulating ROS-induced gene expression of plastid-to-nucleus signaling. Both of high light-and H2O2-induced stress would activate the response from anti-oxidative system of Nrf2-ARE/Keap1, but the OsGUN4mutation could affect the activation. Also, the expression of Nrf2and Keapl were also induced by ROS indirectly. Besides, the mutation of OsGUN4would also reduce the expression of NodL、OCT3、BAP1、 OCT3、FER2and WRKY, but had little effect on TRFL4、APX1and no effect on DREB2A. Finally, we proposed a preliminary hypothesis to elabrate the functions of OsGUN4in the plastid-to-nucleus signaling pathway.In conclusion, antioxidant response element (ARE) in the promoter region of OsGUN4is not only essential for the OsGUN4expression, but also important for OsGUN4to response to oxidantive stresses. The OsGUN4protein in rice has multiple functions; OsGUN4may mainly participate in shielding and attenuating ROS from collisions with O2under highlight conditions, whereas it may focus on promoting chlorophyll biosynthesis in low light environments. Besides, HYB is a novel epigenetic mutant of the OsGUN4locus with distinct characteristics in rice. First of all, unlike others, the DNA hypomethylation site in HYB is located at an antioxidant response element (ARE) in the promoter region of OsGUN4, suggesting that methylation of ARE downregulates expression of OsGUN4. Secondly, compared with the wild type, the adjacent genes up-and downstream of OsGUN4exhibit normal expression level in HYB, so the methylation mark only affects the expression of OsGUN4. Finally, the epigenetic phenotype in HYB is very stable and can be selected multiple generations, so it can be used as a quickly selectable trait for the rice breeding productions. Overall, epi-GUN4is a uniquely rice epigenetic mutation, and provides some opportunities for reveling roles of the methylation modification of regulation elements, including ARE.