Modulation Of Tillering By MiR156/miR529-OsSPL14 Mediated Auxin Polar Transport In Rice

Rice is one of the most important food crops for half population of the world.Improvement of rice production is significant for ensuring the sustainable development of agriculture and food security.Tiller angle and tiller number are two major factors in ideal plant architecture foundation and crop genetic improvement.OsSPL14 is one of the key genes determining rice architecture.However,the mechanistic understanding of rice architecture regulation through OsSPL14 needs to be furthure explored.Here,we reported the findings that OsSPL14 is the target of miR156 and miR529.Further investigation showed that OsSPL14 binds to the promoters of OsPIN1b and OsPIN13,respectively.The molecular pathway mediated by OsSPL14 influences auxin transport and distribution,thus playing a vital role in regulating rice architecture.The main results are described as follows:1.OsSPL14 are targeted by miR156 and miR529.MiR156 and miR529 belong to the highly conserved miRNA family in plants.Sequence analysis showed that miR529 shares 14-16 overlapping nucleotides with miR156 to recognize their common target gene OsSPL14.Overexpression of miR156 or miR529 decreased OsSPL14 transcription levels,resulting in an increased tiller number,decreased plant height and smaller panicle.Stem-loop PCR and RLM-RACE results suggested that miR529 and miR156 effectively regulate OsSPL14 transcription in the reproductive and vegetable organs to precisely mediate rice architecture development.2.Effects of OsSPL14 on tillering number and panicle sizeOsSPL14 encodes a plant specific transcription factor with a highly conserved SBP-box.qRT-PCR results showed that OsSPL14 is a gene of constitutive expression,but RNA in situ hybridization indicated that the expression is relatively specific in developing young panicles and axillary buds.OsSPL14 overexpression lines exhibited an obvious decrease in tiller number and increase in panicle size,while OsSPL14 antisense lines showed an increased tiller number but reduced plant height,panicle size and panicle branch number,indicating that OsSPL14 play opposite roles in tillering and panicle branch development.3.OsSPL14 activates the expression of OsPIN1b and OsPIN13OsSPL14 is a novel specific and primary auxin-response gene which directly target the core sequence of GTAC in the promoter regions of OsPIN1b and OsPIN13.qRT-PCR and RNA in situ hybridization analyses revealed that the expression levels of OsPIN1b and OsPIN13 were obviously increased in OsSPL14 overexpression lines,whereas were decreased in ds-OsSPL14 lines.These results strongly indicate the role of OsSPL14 in regulating auxin transport through activating the expression of OsPIN1b and Os PIN1 3 in rice.4.OsPIN1b and OsPIN13 negatively regulate rice tillering.OsPIN1b and OsPIN13 have ubiquitous expression.The CRISPR/Cas9 knockout mutants of OsPIN1b or OsPIN13 showed a significantly increased tiller number in the T1 homozygotes.Gain-of-function of OsPIN1b or OsPIN13 could delayed outgrowth of axillary buds,then reduce tiller number and rescue the bushy phenotype of ds-OsSPL14 lines.5.OsSPL14 modulates rice tiller development by polar auxin transportThe indoleacetic acid content was not obviously changed in the axillary buds or root of miR529 or miR156 overexpression lines,neither in OsSPL14 overexpression or ds-OsSPL14 lines.DR::GUS signal was mainly detected at the apex area in both roots and Abs of miR529 or miR156 overexpression lines,which are similar to ds-OsSPL14 lines.In OsSPL14 overexpression lines,the GUS expression was more intense and covered a much broader area in base of the Abs.In addition,the OsSPL14 overexpression plants showed an obvious increase in the basipetal IAA transport,indicating that OsSPL14 involved in auxin transport through activating OsPIN1b and OsPIN13,plays a vital role in regulating axillary bud outgrowth.Taken together,our study established the molecular pathway,miRNA/OsSPL14/OsPIN/PAT(miSPA),for the regulation of auxin distribution and tiller development in rice.These results would provide a support on cultivation for high yield and optimal design of ideal plant type in rice.