Maternal Control Of Suspensor Programmed Cell Death Via Gibberellin Signaling

Embryo is the beginning of a new life.The embryogenesis and embryo development are subject to extremely strict and delicacy regulation to ensure successful reproduction.In both animals and plants,embryos are wrapped in layers of maternal tissue,which makes a stable and nutritional microenvironment.In animals,years of researches have revealed that early embryogenesis is extremely dependent on maternal tissues,and maternal defects could lead to abnormal embryo,indicating that the maternal-embryonic communication is essential for embryo development.However,in plants,little was known about maternal-embryonic communication.Plant suspensor connects the mother and the embryo,not only supporting and fixing the location of the embryo,but also transporting the necessary nutrients and hormones for it,similar to the mammalian umbilical cord.Therefore,suspensor is a unique model for the study of maternal-embryonic communication.Nevertheless,unlike umbilical cord of animal,plant suspensor begin to degrade at very early stage of embryogenesis,thereby disconnecting the direct communication between the mother and the embryo.In this work,we try to illucidate the molecular mechanism underlying the suspensor timely degenerartion.Using tobacco(Nicotiana tabacum)as a model,early experiments revealed that Nt CYS-Nt CP14 is a pair of molecular switches that regulate suspensor programmed cell death(PCD).Based on the previous work,the signaling pathway of Nt CYS-Nt CP14-mediated susceptor PCD and nature of the signal,as well as the tissue originating of the signal,were extensively studied.We reveal that the mother tissue regulates suspensor programmed cell death via the gibberellin(GA)signaling pathway.The main findings are as follows:1.Nt CRF1 is a regulator of Nt CYS and it directly binds to the Nt CYS promoter.We demonstrated that Nt CRF1 directly binds to the M3 motif of the Nt CYS promoter.Nt CRF1 could interact with itself and may act as a dimer in vivo to bind DNA in the same way as other GRAS proteins.2.Nt CRF1 and Nt CYS have the similar expression pattern and dynamic changes in the basal suspensor cell(SC).We generated transgenic plants carrying the p Nt CRF1::Nt CRF1-GFP construct.Nt CRF-GFP was detected in both basal and apical cell lineages,confirming the expression of both Nt CRF-GFP and Nt CYS-GFP in the basal suspensor cell.We also observed that Nt CRF1-GFP fluorescence intensity was high in the basal cell lineage until stage 2,sharply declined at stage 3,and was scarcely detectable at stage 4.These expressional changes coincided with that of Nt CYS-GFP.3.Nt CRF1 regulates Nt CYS expression and induce suspensor PCD.Both Nt CRF1 knockdown and knockout lines down-regulated Nt CYS m RNA expression levels.Oppositely,the expression of Nt CYS was enhanced in the Nt CRF1 overexpression line.The TUNEL assay of the Nt CRF1 knockdown and knockout lines showed that PCD started at the two-celled proembryo stage,which further proved that Nt CRF1 is a direct regulator of Nt CYS and a key regulator of suspensor PCD.In addition,premature degradation of the suspensor also leads to changes in the developmental patterns of embryos and reduced seed fertility.4.Nt CRF1 acts as a DELLA protein in response to bioactive GA.Firstly,it was found by NCBI sequence alignment that Nt CRF1 has the sequence characteristics of DELLA protein,and has a DELLA domain at the N-terminus and a highly conserved GRAS domain at the C-terminus.Secondly,Nt CRF1 responds to bioactive GA.In the p Nt CRF1::Nt CRF1-GFP lines,the proembryos were treated with bioactive GA and the Nt CRF1-GFP fluorescence was weakened or disappeared.The GA synthesis inhibitor(PAC)treated the seeds to the 32-cell embryo stage in vitro,and the Nt CRF1-GFP signal remained in the suspensor.Thirdly,Nt CRF1 relies on bioactive GA to interact with the GA receptor Nt GID1 s.5.Bioactive GA4 rises in a timely manner before suspensor PCD.The results of RT-PCR showed that GA3 ox,a key enzyme of GA biosynthesis,immediately increased before susceptor PCD,which may cause a transient increase of bioactive GA.At the same time,a GA marker GPS1 also indicated a transient GA increase.By measuring the levels of each GAs in the seeds during early embryo development,we found that GA1 was not detected before the suspensor PCD,and only GA4 could be detected.Thus,consistent with previous results,it is the GA4 that was instantly increasing before suspensor PCD.6.Bioactive GA could induce suspensor PCD in vitro.Based on the embryo culture system in vitro,the wild-type two-cell proembryos were treated with bioactive GA4,and it was found by PI-FDA and TUNEL assay that GA could induce the earlier occurrence of suspensor PCD.At the same time,GA can reduce the expression level of Nt CYS in vitro.These results suggest that GA4 as a signal triggers the suspensor PCD by downregulating the expression level of Nt CYS.7.Maternal GA triggers suspensor PCD.In two-celled proembryos,while genes related to GA signaling expressed,those associated with GA biosynthesis and metabolism not,neither in four-celled proembryos.We generated three p Nt GA3ox::H2B-GFP constructs to determine the location of GA synthesis.Nt GA3ox1,Nt GA3ox2 and Nt GA3ox3 were not expressed in the endosperm and proembryo of the transgenic lines before stage 3.However,these genes were expressed in the seed coat and especially expressed towards the micropyle region at stage 4.This suggests that the GA4 that triggers PCD likely originates from maternal tissue,particularly the micropyle part of the seed coat.Accordingly,we found that bioactive GA exhibited a polar distribution in developing seeds: the mean nls GPS1 emission ratio increased significantly from the chalaza to the micropylar ends.This is in agreement with the polar activation of Nt GA3 ox at the same stage.Before stage 2,we observed a strong Nt CRF1-GFP signal in the basal SC,which rapidly diminished as the GA level in the micropyle endothelium increased,indicating activation of GA signaling in the basal SC.Interestingly,the GFP signal gradually declined from the basal SC to the vertically adjacent cell,as did the occurrence of PCD.Therefore,we confirm that maternal GA entrys to the basal cell of and triggers suspensor PCD.8.To further confirm the induction of suspensor PCD by maternal GA in vivo,we ectopically expressed Nt GA3ox1 in the endothelium,endosperm,proembryo,and seed coat under the control of four tissue-specific promoters,respectively.Ectopic expression of Nt GA3ox1-GFP in the seed coat,endosperm,and proembryo did not influence Nt CYS expression.However,ectopic expression of Nt GA3ox1-GFP in the endothelium,to which the basal SC is connected,downregulated Nt CYS expression.We also evaluated proembryo development in the p TPE8::Nt GA3ox1-GFP(TG)lines.As expected,we detected PCD at the two-celled proembryo stage.Therefore,suspensor PCD is triggered,via Nt CRF1,by bioactive GA4 from maternal endothelial cells.In summary,maternal signal GA4 and proembryo responsor Nt CRF1 mediate maternal-proembryo communication via the GA-GID1-CRF1-CYS-CP14 signaling cascade to precisely control the maternal-embryo connection.The discovery not only fills in the blank of the upstream molecular mechanism of suspensor PCD,but also reveals a relatively complete signaling pathway of the maternal-embryo communication for the first time,which may have universal significance for understanding the mechanism of maternal tissue communicate with embryos.