Map-based Cloning Of A High Tillering Dwarf Gene OsIAA16 And Regulation Mechanism Of Shoot Branching In Rice

Plant architecture is the key determinant in crop yield and considered as the main driving force for selection and domestication events in major crop species.Compact and semi-dwarf plant architecture with short and thick internodes dramatically increases harvest index and crop yield in many crop species like rice and wheat.Studying plant architecture is important for developing high-yielding varieties and hybrids to increase crop yield.In this quest,we identified a high-tillering dwarf mutant hd103 through EMS?Ethyl Methane Sulphonate?mutagenesis.We identified the underlying gene through map-based cloning approach which encodes an AUX/IAA family protein OsIAA16 involved in auxin signaling pathway.The over expression as well as RNAi mediated knock-down and CRISPR Cas-9 mediated knock-out further confirmed that OsIAA16 is involved in tiller formation in rice.The C-to-T substitution at the 185^th nucleotide in the coding sequence?C185T?changed the amino acid proline at the 62ⁿdd position to leucine?P62L?.This substitution disrupted the conserved degron motif from GWPPV to GWLPV,leading to reduced affinity between OsIAA16 and auxin receptor TIR1?TRANSPORT INHIBITOR RESPONSE-1?.Consequently,degradation of the OsIAA16 protein was repressed,leading to auxin-related growth defects with reduced sensitivity to external auxin application.Auxin exerts its action through Auxin Response Factors?ARFs?and AUX/IAA proteins works as repressors of auxin signaling pathway.We identified ARF6,ARF11 and ARF17 as the positive interacting partners of the OsIAA16 protein.In addition to the increased tillering phenotype,the hd103 mutant displays multiple growth defects like increased mesocotyl elongation,delayed leaf senescence and insensitivity for external strigolactones?SL?application resembling the SL signaling mutants.The expression of the key SL pathway genes D3,D14,D10 and the downstream target TB1 were down-regulated in the hd103mutant.The D53 protein is known as repressor of SL signaling.The GR24?a synthetic analog of SL?mediated degradation of D53 protein was delayed in the hd103 mutant.Auxin application can also induce D53 degradation and this response was compromised in the hd103 and the SL biosynthesis mutant d10.This suggests that normal auxin signaling is required for SL-mediated D53 degradation and vice versa.Another breakthrough observed while working with the hd103 mutant was its hyper responsiveness to sucrose application.We observed that SL and sucrose works antagonistically in tiller development process and high sucrose level abolishes the suppressive action of GR24 on tiller development as well as in D53 protein degradation response.The expression of SL signaling component D3 is repressed by sucrose application and over-expression of D3 abolishes the sucrose-induced tiller development and D53 stabilization.Sucrose also stabilizes D14 protein levels where D3 is responsible for its proteasomal degradation.This again supports that sucrose antagonizes D3 expression which may lead to less D3protein levels resulting in slowdown of SL signaling.The hyper responsiveness of the d3 mutant to sucrose treatment compared to the WT and d14 mutant suggests that D3 could be the target of sucrose-mediated tiller bud development in rice.These results suggest that a possible cross-talk between auxin,SL and sucrose exists and they modulate each other's levels according to environmental signals to achieve perfect plant architecture.