Studies On The Regulation Of OsEIL1/2 In ROS-Related Gene Expression And Coleoptile Elongation Of Rice

Rice（Oryza sativa L.）is an important staple food crop in China,its yield is tightly related to food security.As a semi-aquatic plant,rice lives in a water-saturated environment during most of its life cycle.The breeding of rice cultivars with enhanced vigor and seedling emergence rate is important for seedling survival and establishment in natural environment.After the rice seeds germinate under the soil,they will first undergo etiolation growth,which promotes the rapid emergence of seedlings via promoting the rapid elongation of the coleoptile and mesocotyl.The coleoptile is a conical structure which is essential to protect the plumule as it moves through the soil layers.It grows fast to enable rapid elongation of seedlings and stops growing until it reaches the surface of water/soil,and then senesces and withers.Thus rice seedlings with longer coleoptiles exhibit better emergence rate and seedling establishment.Coleoptile growth is affected by various factors,such as soil depth,flooding,hypoxia,and light.To cope with the environment cues,plants have evolved an elaborate etiolation process that enables seedlings to emerge from the soil and acquire autotrophic ability,and ethylene is an important growth regulator in this process.Ethylene promotes coleoptile growth in rice,whereas inhibits it in other monocot crops,such as maize,wheat,and sorghum,which might be related to the semi-aquatic living environment of rice.Until now,the molecular mechanism of ethylene in rice coleoptile growth is largely unclear.Here,we used genetic and biochemical analyses to uncover that ethylene modulates the accumulation of reactive oxygen species（ROS）to promote the growth of coleoptile in rice,thereby improving the emergence capacity of rice seedlings.Through mimicking the subterranean environments of rice seeds germination,we found that ethylene production and OsEIL1/2 protein levels gradually decreased as rice seedlings grow under dark conditions（1,2 and 3 days after seeds germination）;Correspondingly,the expression of GY1,which is inhibited by ethylene,gradually increased with the growth of coleoptile,indicating that ethylene is involved in regulating coleoptile growth in rice seedlings.In addition,soil can further promote ethylene accumulation and regulate coleoptile growth.In order to identify whether ethylene signaling components are involved in regulating rice coleoptile growth,we examined the longitudinal sections of osein2 and oseil1oseil2 mutant coleoptiles,and found that the cell length and coleoptile length of osein2and oseil1oseil2 were shorter than those of wild-type（WT）,indicating that the cell elongation of coleoptile is inhibited in osein2 and oseil1oseil2 mutants.We also examined the radial sections of osein2 and oseil1oseil2 mutant coleoptile tips and found that the apical cell expansion was promoted in osein2 and oseil1oseil2 coleoptiles.Corresponding to the phenotype of coleoptile,the emergence rate of osein2 and oseil1oseil2 mutants was significantly decreased as compared to that of WT.Taken together,the above results indicate that OsEIL1 and OsEIL2 are core regulators in rice coleoptile growth,and ethylene promotes cell elongation and inhibits tip cell expansion in rice coleoptiles,resulting in longer and thinner coleoptile to facilitate seedling emergence from the soil.To dissect the molecular mechanism of OsEIL1/2 regulating the growth of rice coleoptile,we examined changes in gene expression in Nip,osein2 and oseil1oseil2 coleoptiles using transcriptome and found that many OsEIL1/OsEIL2-regulated genes are also regulated by OsEIN2 in coleoptile.These co-regulated genes were involved in redox processes,transcriptional binding,carbohydrate metabolism,lipid metabolism,and oxidative stress response.We analyzed genes associated with ROS generation and scavenging in the transcriptome data and found that multiple ROS generation genes were up-regulated and ROS scavenging genes were down-regulated in osein2 and oseil1oseil2 coleoptiles,this regulation was further confirmed by RT-q PCR.These results indicate that ethylene may affect ROS accumulation in the coleoptile.Among the genes associated with ROS scavenging,OsVTC1-3 and OsPRXs gene were significantly down-regulated.These genes encode GDP-D-mannose pyrophosphorylase（ascorbic acid synthesis）and peroxidase（POD）,respectively,and are capable of scavenging ROS through non-enzymatic and enzymatic pathways.Determination the As A content and the activity of POD enzyme showed that As A content and POD enzyme activity was significantly lower in osein2 and oseil1oseil2 coleoptiles compared to Nip.Correspondingly,the 3,3-diaminobenzidine（DAB）staining and H₂O₂determination showed that the ROS content of osein2 and oseil1oseil2mutants was much higher than that of Nip,suggesting that ROS may affect rice coleoptile growth.Ethylene treatment significantly induces the expression of OsVTC1-3 and OsPRXs.Both OsEIL1 and OsEIL2 were able to bind OsVTC1-3 and OsPRXs gene promoters which were detected by Ch IP-PCR.Dual-luciferase reporter assays also showed that the presence of OsEIL1/2 significantly increased LUC activity driven by the OsVTC1-3 and OsPRXs promoters.In addition,OsEIL1 and OsEIL2 specifically bind to EBS（ATGTA）sites on OsVTC1-3 and OsPRXs promoters identified by electrophoretic mobility shift assay.These results demonstrate that OsEIL1 and OsEIL2 could directly activate the expression of OsVTC1-3 and OsPRXs,increase Ascorbic acid content and POD enzyme activity to decrease ROS accumulation in coleoptile,and disruption of ethylene signaling leads to the accumulation of ROS in the coleoptile,which further inhibits coleoptile growth.Next,we explored how ROS affects the growth of rice coleoptile.Our results showed that the exogenous application of H₂O₂significantly inhibited coleoptile growth and substantially overcame the ethylene-promoted coleoptile elongation,indicating that ROS negatively regulates coleoptile growth and development.In addition,exogenous application of H₂O₂significantly inhibited the expression of ethylene synthesis genes OsACS1 and OsACO7,indicating that ROS may has a certain feedback inhibition effect on ethylene synthesis.The effect of endogenous ROS on rice coleoptile growth was analyzed by OsRboh H-OX and OsVTC1-3-OX rice materials.Our results showed that OsRboh H-OX seedlings had higher ROS level,shorter coleoptile cell length and coleoptile length,and OsVTC1-3-OX seedlings had lower ROS level,longer coleoptile cell length and coleoptile length,than those of WT.In addition,the OsRboh H-OX seedlings exhibited increase in coleoptile apices thickness,and OsVTC1-3-OX seedlings exhibited decrease in coleoptile apices thickness,as compared with WT coleoptiles.Correspondingly,the OsRboh H-OX seedlings emerged slower than WT,whereas the OsVTC1-3-OX seedlings emerged faster than WT.These results indicate that ROS inhibits rice coleoptile elongation and promotes rice coleoptile apical expansion,thereby delaying seedling emergence from soil.In genetic relationship,we also found that OsVTC1-3-OX could significantly restore the coleoptile growth defect of oseil1,indicating that OsVTC1-3 was located downstream of OsEIL1 to regulate the growth of rice coleoptile.In summary,coleoptile growth is tightly associated with seedling emergence in rice,and ROS negatively regulates coleoptile growth by inhibiting coleoptile cell elongation and promoting coleoptile apical expansion,thereby delaying seedling emergence from soil.The ethylene signaling transcription factors OsEIL1 and OsEIL2 directly activate the expression of OsVTC1-3 and OsPRXs to increase Ascorbic acid content and POD enzyme activity to reduce ROS accumulation in coleoptile,thus promoting coleoptile elongation and inhibiting coleoptile apical expansion.This dual role of ethylene on coleoptile growth accelerates to push the shoot tip above the soil surface and reduces mechanical impedance as the seedlings emerge from soil,which facilitates the emergence of rice seedlings from soil and guarantees rice seedling establishment.Thus,our findings enrich the molecular mechanisms by which ethylene regulates coleoptile growth in rice,and provide guidance for breeders to cultivate rice varieties suitable for direct-seeding.