Research On The Mechanism Of Seeds Abortion In Paeonia Ludlowii

Paeonia ludlowii（Stern et Taylor）D.Y.Hong is unique in China.And it is a GradeⅡnational protected plant with high ornamental,medicinal,breeding and economic value.Seed abortion is one of the key factors limiting the natural reproduction,cultivation,introduction,conservation,and explotation of P.ludlowii.In this paper,we investigated the seed abortion mechanism of P.ludlowii from the aspects of resource competition,physiological mechanism,sexual reproduction and abortion related genes through field investigation,field experiment,physiological and biochemical indicators,endogenous hormone determination and microscopic observations,combined with transcriptome sequencing and gene cloning techniques.It provides a theoretical basis for cultivating and propagating,germplasm innovation,ex-situ protection,development and utilization of P.ludlowii.The main results are as follows.（1）Observation on seed abortion in P.ludlowii.（1）The seed abortion rates of P.ludlowii in original site（Nyingchi）and cultivated site（Luanchuan）were 62.68-66.32%and 69.14-74.01%,respectively.（2）10-30th day after flowering was the period of peak fruit growth and the main period of seed abortion.At 13 d after flowering,the external morphology of normal seeds and aborted seeds was significantly different,and the smaller ovules were aborted.（3）The abortion of P.ludlowii was a random abortion whih no obvious positional effect.（4）P.ludlowii bear fruits in both selfing and outcrossing with（32.33±9.82）%and（30.80±12.02）%seed-setting rates,respectively.（2）Resource competition between secondary branching and seed development in P.ludlowii.（1）Around the flowering period,the branches sprout twice at the axillary buds of flower/fruit branches of P.ludlowii,flowered and fruied on the secondary branches the next year.（2）The differentiation process of the apical bud on the secondary branches was the same in cultivated and original site.The apical bud finally formed a compound bud with a top flower bud,multiple lateral flower buds,axillary bud primordiums and leaf primordiums though six differentiation stages.（3）The competition among the secondary branches,lateral flowers and top flowers could reduce the rate of seed-setting of P.ludlowii,which could be improved by removing parts of secondary branches or lateral flowers.（3）Physiological mechanism of seed abortion in P.ludlowii.（1）The low content of soluble sugar,starch,soluble protein and SOD activity were the physiological causes of ovule abortion.At 12-13 d after flowering,the starch content of aborted seeds was the lowest and then increased constantly,while the starch content of normal seeds was the highest at 14 d after flowering,and then decreased rapidly.At 13-14 d after flowering,the content of soluble sugar in normal seeds increased,while that in aborted seeds decreased.At 13-15 d after flowering,the soluble protein content of aborted seeds was increased and then decreased,and the changing trend of that in normal seeds was opposite.The soluble protein content of aborted seeds was the lowest and that of normal seeds was the highest at 13 d after flowering.At 13-50 d after flowering,SOD activity in normal and aborted seeds both increased,and SOD activity in normal seeds was always higher than in aborted seeds.（2）Seed abortion was also related to the abnormal endogenous hormones in the seed.At 13-20 d after flowering,the contents of IAA,GA₃,ZR and the ratio of（GA₃+IAA+ZR）/ABA,GA₃/ABA and IAA/ABA in normal seeds were significantly higher than in aborted seeds,while the contents of ABA and the ratio of ABA/（GA₃+IAA）were lower than aborted seeds.（4）Meiosis of pollen mother cells,pollen viability,pistil structure,pollination and fertilization in P.ludlowii.（1）Abnormal phenomena such as asynchrony,coexistence of multiple division periods,backward chromosomes,micronucleus,and unequal separation of microspore mother cells of P.ludlowii occurred in the process of meiosis,but they did not affect the formation of pollen overall.After the flowers opened,the pollen was full.The rate of full pollen reached 91.2%.And pollen has high vitality.（2）The carpel was composed of stigma,style and ovary.The stigma was a wet stigma,mostly 360°curved,with a large pollen surface.The style was hollow,and the inner epidermal cells had the characteristics of glandular cells.And the ovary contained two rows of longitudinally arranged stigmas,ovule with double integuments and thick nucculus.Most ovules could develop into normal polygonum embryo sacs,and only a few sterile ovules existed.（3）Within 48 h after flowering,a large number of pollen grains germinated on the stigma and extended into the stigma to enter the ovary.At 48-60 h after flowering,the pollen tube passed through the nuculus and entered the embryo sac.Double fertilization is completed after 60-144 h after flowering.（5）Seed development pattern in P.ludlowii.Normal seed form zygotes after fertilization,and then it took 80-100 days from free nuclear embryo,cellular embryo,globular embryo,rod embryo,heart-shaped embryo,torpedo embryo to cotyledon embryo.The formation of endosperm went through free nuclear stage,cellular stage and growth and maturation stage,and the endosperm in mature seeds was solid.（6）Types of seed abortion in P.ludlowii.（1）The abortion of P.ludlowii could be divided into two types:embryo and endosperm abortion and female infertility（including abnormal pistil,sterile ovules and abnormal embryo sac）.Among them,embryo and endosperm abortion was the main type.（2）Embryo and endosperm abortions could be divided into early abortion and middle abortion stages,which all occured at the stage of free nuclear embryo and endosperm.And the early abortion was the main one.Early embryo and endosperm abortion mainly occured at 9-12 d after flowering.The aborted seeds stopped growing,the volume was smaller than normal seeds,the endosperms developed abnormally,the free nucleus of the endosperm abnormal division or continued to degenerate,and the embryo sac became narrow.Mid-stage embryo and endosperm abortion:it mainly occured around 20 d after flowering.The aborted seeds were slightly larger than other early-stage aborted seeds,and slightly smaller than normal seeds.The embryo and endosperm free nuclei in the embryo sac degenerated.（3）Abnormal pistil:during the flowering stage,some pistils had abnormal phenomena such as ovary rupture,ovule bursting,stigma exposure before flowering,and carpel deformity.（4）Sterile ovules:these ovules were small or deformed.Some ovules had no embryo sac,and there was no obvious gap between the outer integument and the inner integument,Some had embryo sac structure,but the space was small,and there was no obvious organization in the embryo sac.（5）Abnormal embryo sac:the embryo sac develops normally,but fails to reach the physiological state of fertilization in time.（7）There were three main forms of aborted seeds in P.ludlowii in the later stage.（1）The embryo sac,nucellus and integument gradually degenerate and did not take shape.（2）The embryo sac was filled with large cells,with only a narrow gap in the middle.（3）The embryo,endosperm and inner integument in the seed were completely degenerated,and finally formed an empty shell.（8）Transcriptome sequencing of aborted seeds in P.ludlowii.（1）A total of 114 350 Unigenes were obtained and 22 703 DEGs were screened.（2）In GO enrichment analysis,DEGs were abundantly enriched in the pathways related to nutrient metabolism such as metabolic process,single-organism process and biosynthesis process.And in KEGG enrichment analysis,DEGs were abundantly enriched in metabolic pathways such as starch and sucrose metabolism,glycolysis/gluconeogenesis,amino sugar and nucleotide sugar metabolism,and phenylpropanoid biosynthesis.Meanwhile,Meanwhile,a large number of DEGs encoding glycolysis/gluconeogenesis,TCA cycle and PPP pathway were down regulated in aborted seeds.It was confirmed that the abortion of P.ludlowii was related to the decomposition and accumulation of nutrients and energy metabolism.（3）Fifteen ovule/seed development and abortion-related genes were screened.Among them,the expression of eight ovule/seed development-related genes（Pl ANT1,Pl AGL11-like,Pl AGL62（two）,Pl OVA8,Pl ERS1,Pl OVA6 and Pl OVA7）in aborted seeds were down-regulated compared with that in normal seeds,and the expression of seven seed abortion-related genes（Pl Ces A8,Pl EIL1,Pl JMT,Pl SMT2,Pl HB14,Pl HB12L and Pl HB7）were up-regulated.（9）The candidate genes Pl HB7 and Pl SMT2 were cloned.（1）The full length of Pl HB7 gene was997 bp,the longest ORF was 432 bp,and it encoded 143 amino acids.The amino acids sequence homologous to HB7 and HB12 proteins of other species.（2）The full length of Pl SMT2 gene was1 843 bp,the longest ORF was 1 083 bp,and it encoded 360 amino acids.The CDS sequence of Pl SMT2homologous to the SMT2 genes of other species.（3）The overexpression vectors of Pl HB7 and Pl SMT2genes were successfully constructed and transformed into Arabidopsis.（4）Compared with WT,Pl HB7transgenic Arabidopsis had slower growth,delayed flowering,less seed germination,reduced plant height,increased number of leaves,and reduced seed setting rate;Pl SMT2 transgenic Arabidopsis had slower growth,delayed flowering,less seed germination,and larger leaves.The pollen fertility of P.ludlowii was good and the germination rate was high.Abnormal embryo sacs were less,normal embryo sacs were more and entered the physiological state of fertilization earlier.And the pistil stigma was large,and the double fertilization was normal.Seed abortion was mainly caused by poor embryo development after pollination.Resource competition,hormone metabolism and nutrition supply were the factors of seed abortion.Fifteen regulatory genes related to ovule/seed development and seed abortion were successfully discovered by transcriptome sequencing,and the functions of Pl HB7 and Pl SMT2 were preliminarily verified by gene cloning technology.However,the functions of the related genes still needed further observation and studies.