Functional Analysis Of Maize GRAS Transcription Factor ZmSCL14 Gene

One of the essential differences between plants and animals is that plants can be fixed in the soil by the root system,and the root system is also an important organ for the plant to absorb and transport water,inorganic salts and other nutrients.Maize(Zea mays L.)as a main food crop plays a major role in national economic development.Identification and excavation of new genes related to root agronomic traits in maize and studying their regulatory mechanisms have strategic significance in breeding new maize varieties.First,we performed a phenotypic screening from the existing maize transgenic Ds insertion mutant library in the laboratory,and selected a short-root mutant zmscl14-1 whose exon was inserted by the Ds.Through sequence analysis,it was confirmed that the gene belongs to the LISCL subfamily of the transcription factor GRAS family,and has not been reported in maize.We named this gene ZmSCL14,and focused on its functional analysis subsequently.(1)In order to determine the accuracy of the mutant gene,we purchased an EMS mutant zmscl14-2 whose translation is terminated early.By phenotypic analysis of the two allelic mutants,we found that the phenotype of the zmscl14 mutant was mainly on the shorter primary roots at the seedling stage,and there were more lateral roots distributed on the primary roots.The nodal roots have developmental defects at the stocking stage resulting in a smaller total root system.The zmscl14-2 mutant has a slightly severer phenotype probably due to an earlier termination of translation.Comparing to zmscl14-1,the zmscl14-2 plant was even smaller than WT.No significant changes were observed in other developmental processes of the zmscl14 mutants.(2)Tissues from different stages of the maize plants were collected and analyzed by q RT-PCR,which showed that the ZmSCL14 gene was expressed in all organs,but the expression level was higher in young organs.At the same time,we used PEG6000,Na Cl,and various hormones such as NAA to treat wild-type plants,and sampled tissue in the time course: 0h,1h,3h,6h,12 h,and 24 h for q RT-PCR analysis.The results showed that the gene responded strongly to PEG6000,Na Cl and NAA treatments,suggesting that the ZmSCL14 gene is involved in the regulation of internal hormones and external stress signals in plants.(3)Because the gene responded strongly to PEG6000 stress,we used PEG6000 to simulate drought stress and we found that germination of zmscl14-1 was more severely affected than WT.Under real drought conditions,zmscl14-1 seedlings also showed more sensitivity to drought than WT.In order to further verify whether this gene is involved in the regulation of maize drought resistance,Ubi::ZmSCL14 vector was constructed and transformed into Brachypodium distachyon.Drought analysis showed that the drought tolerance of ZmSCL14 over-expressing lines was significantly improved.At the same time,we used CRISPR / Cas9 technology to obtain BdSCL14 loss-of-function mutants.Drought analysis showed that bdscl14 mutants also showed super sensitivity to drought stress.These results suggest that the SCL14 gene might commonly be involved in drought stress for the grass family.(4)q RT-PCR analysis of stress-related genes in WT and zmscl14-1 showed that the expression levels of stress-related genes such as ZmRD20 and ZmNH3 were significantly lower than those of WT,which was consistent with the phenotypic results of drought analysis.(5)In the same way,we analyzed the expression levels of genes related to antioxidation and stress in the ZmSCL14 over-expression transgenic B.distachyon and WT by q RT-PCR.The results showed that there was no difference in the expression of antioxidation-related genes such as BdFe-SOD,BdAPX1,BdCAT between the transgenic line and WT.But the expression of stress-related genes such as BdCBF1,BdCBF2,BdWRKY36 increased significantly in the transgenic line compared with WT.The results were consistent with the phenotype of drought analysis.(6)Sub-cellular localization of ZmSCL14 was observed through constructing the ZmSCL14-GFP fusion gene under the control of the 35 S promoter and transiently expressing it in the tobacco epidermal cells.Results showed that ZmSCL14 was located in nucleus.(7)While the drought tolerance of ZmSCL14-overexpressing B.distachyon was increased significantly,we also found a strong late-flowering effect in the trangenic plants.Hence the ZmSCL14-overexpressing B.distachyon plants had vigorous vegetative growth,produced more branches and spike branches,but had smaller seeds.The results of q RT-PCR of flowering-related genes showed a significant down-regulation of BdVRN1 which might lead to late flowering.Our research in maize and the gramineae model species B.distachyon confirmed that the SCL14 gene is involved in plant root development and is an important regulatory gene responsive to drought stress.