Cloning Of Maize Dwarf Gene D2014 And Its Mechanism Of Regulating Internode Elongation

Plant height is an important agronomic trait that determines crop yield.Crop dwarfing not only improves lodging resistance,but also increases planting density and harvest index,which is very beneficial to crop production.As the main grain and forage crop,studying the genetic mechanism of maize plant height has very important theoretical and applied value.In this study,we found a natural dwarf mutant,dwarf2014(d2014),from the inbred line HL9047.Plant height and ear height were reduced by 1/4 and 1/2 of WT,respectively;while other agronomic traits were almost unaffected.Through gene mapping,DNA sequence analysis and allelic test,d2014 was identified as a new type of br2 allele.The mutation of d2014 affected the normal splicing of BR2 gene,leading to massive expression of spliceosome BR2-T02 in vivo instead of BR2-T01 in the WT state.Previous studies only focused on BR2-T01,and indicated that BR2 encodes an auxin exporter PGP1,which belongs to the ABC transporter family.The prediction analysis of protein domain showed that the protein encoded by BR2-T01 is a complete ABC transporter,containing two TMDs and two NBDs;while BR2-T02 encodes a half of ABC transporter protein,which only contains one TMD and one NBD.Heterologous expression in yeast analysis indicated that PGP1-T01 and PGP1-T02 both play a role in auxin export.Therefore,splicing variant d2014 with alternative splicing variation enriches the br2 mutation types.Moreover,we deduced that functional PGP1-T02 may partially compensate for the deficiency of PGP1-T01,giving rise to a mild dwarfing phenotype without other undesirable agronomic traits and making d2014 having potential in maize breeding.Plant height development is closely related to internode elongation.The d2014,similar to other br2 mutants,displayed shortened lower internodes but nearly normal upper parts,which indicated that br2 regulates plant height development in a unique manner.However,its underlying mechanism has not been well explained.Here,the dynamic observation of stem development showed that the internode elongation of d2014 displays the variation pattern of inhibition-acceleration-transient inhibition(ear-internode)during several key development stages(6-leaf to 12-leaf to 14-leaf stage).Through cytological analysis,the number of vascular bundle in shortened internode of d2014 was significantly reduced in comparison with WT,indicating that the intercalary meristem function may be affected.The analysis of BR2 expression pattern in stem showed that BR2 is mainly expressed in the vascular bundle of node and internode,indicating that PGP1 should be responsible for the auxin export in these tissues.By analyzing the auxin concentration of stem tissue in several key periods of internode elongation variation,we found that the auxin concentration of the d2014 stem apex was significantly higher than that of WT at the 6-leaf stage;while the auxin concentration of the stem apex gradually decreased at the 12-leaf and 14-leaf stages,and there was no difference between d2014 and WT;in addition,the auxin concentration of node region in d2014 ear-internode was significantly higher than that in WT,as well as in itself internode region.In context,we proposed that PGP1 functions in auxin efflux from node to internode through the vascular bundles,and excessive auxin accumulation in the node region suppresses internode elongation.The stem contains the intercalary meristem immediately above the node region,and internode elongation depends on intercalary meristem function.Therefore,low auxin levels mediated by PGP1 in the intercalary meristem region are crucial for internode elongation.