Identification Of Semi-Dwarf Genes And Molecular Mechanism Of Plant Height Regulation In Maize

Maize is a major staple crop in China,and increasing maize yield plays a vital role in safeguarding China’s food security.Increasing the planting density per unit land is the most effective measure to increase the yield of maize.However,high density planting could trigger shade avoidance response,which negatively impacts many important agronomic traits,including increased plant height and lodging.Thus,optimal plant height and ear height are of great significance for lodging resistance and planting density in maize.Since,the 1960s,production of rice and wheat has benefited from utilization of the semi-dwarf genes,so called‘Green Revolution’genes.However,there is no similar report and application of‘Green Revolution’gene in maize.At present,almost all dwarf mutants or genes are difficult to be used in maize breeding because most dwarf mutants are seriously dwarfed and associated with poor agronomic traits.This greatly limits the breeding and improvement of maize plant height traits.To tackle this,we explored new plant height regulators and artificially created new semi-dwarf alleles.The main results and conclusions are listed as followings:1.ZmSPL12 is an inhibitor of D1,the key gene of gibberellin synthesis:（1）In this study,we performed co expression analysis,correlation analysis,promoter analysis,and yeast one-hybrid assay,and obtained the transcription factor ZmSPL12,which could directly inhibit the expression of D1.（2）ZmSPL12 knockout lines exhibited increased plant height,ear height and internods length compared to the nontransgenic plants.On the contrary,the ZmSPL12 over-expression plants displayed reduced plant height,ear height,and tassel branch number and increased hundred-grain weight and stalk strength.The expression level of D1 was significantly decreased in ZmSPL12-OE lines.（3）ZmSPL12-OE can respond to the treatment of exogenous GA₃.After GA₃ treatment,the plant height of ZmSPL12-OE could restore to the nontransgenic plants level.The endogenous bioactive GAs levels decreased in the ZmSPL12-OE plants.These results indicate that ZmSPL12 can reduce the level of endogenous GAs and plant height by inhibiting D1 expression.（4）We planted ZmSPL12-OE and nontransgenic control plants（CK）under three different planting densities.Under all of three planting densities,the plant height,ear height and tassel branch number of the ZmSPL12-OE line decreased significantly compared with those of the CK line.Under planting densities of 135,000 plants per hectare,the ZmSPL12-OE showed higher grain yield and stronger stalk strength than the CK.The above results show that ZmSPL12-OE can decrease plant height and increase grain yield and stalk strength under high plant densities.And ZmSPL12-OE has high breeding value.（5）We introgressed the ZmSPL12-OE conferring semi-dwraf plant height and ear height,through repeated backcrossing,into the six elite parental inbred lines,PH4CV,PH6WC,Jing724,Jing92,WK798-2,and Chang7-2 of maize hybrids widely planted in China and obtained six BC₅F₁ population.The improved inbred lines had significantly reduced plant height and ear height.These results suggest that the ZmSPL12 can both reduce the plant height and ear height and improve lodging resistance.2.Selective editing of auxin transport gene Br2 to create a series of semi-dwarf alleles:（1）Previous studies reported that the mutation of the fifth exon of Br2 can effectively reduce plant height and have little negative impact on yield traits.We designed four conserved target sites in the fifth exon of Br2,which can edit as many inbred lines as possible.Through genome editing technology,we created at least 9 allele types with different plant heights.These nine allelotypes can be used to improve maize plant height and tolerance to wind lodging for specific needs.（2）The ZC01 transgenic lines carrying the Br2-Cas9 cassette were used to pollinate the haploid inducer line CAU5.Using Haploid-Inducer Mediated Genome Editing（IMGE）technology,we obtained14 improved haploid lines in seven inbred lines backgrounds.Homozygous pure DH lines with the desired plant height improvement could be generated within two generations.In summary,our results elucidated the molecular mechanism of how ZmSPL12 regulates plant height and ear height by inhibiting D1 expression and enriched the molecular regulatory network of gibberellin synthesis in maize.ZmSPL12 could mimic the function of‘Green Revolution’genes to confer reduced plant height and increased lodging resistance,thus facilitating high-density planting and increased yield in maize.In combinatiomn with IMGE,the various allele types of Br2 created constitute an excellent germplasm pool of plant heights,which could be used for plant height improvement in future molecular breeding of maize.