Transcription Factor MEE45 Mediates Seed Size By Auxin Biosynthesis In Ovule Tissues Of Arabidopsis

Seed size is the essential factor which determines seed yield.Revealing the mechanisms controlling seed size is therefore crucial for developing new strategies to increase crop seed production.The seed size is determined by the coordinated development of maternal and zygotic tissues.Although several genes have been identified to regulate seed size through maternal tissues in Arabidopsis,it is still unclear about the genetic and molecular mechanisms that maternally control seed size.Here,we identified MEE45 as a novel B3 transcription factor involved in controlling seed size.The main results are as follows:(1)MEE45 maternally controls the seed size.Seeds of the mee45 mutants were much smaller and lighter than the wild-type ones.In contrast,the small seed phenotypes of mee45 were rescued by the MEE45 overexpressing line.Furthermore,MEE45 overexpressing plants produced seeds that were significantly larger and heavier than those of the wild type controls,indicating that MEE45 plays important roles in controlling seed size.We further found a significant decrease in the cell number of the cotyledons in the mee45 embryo comparing with those in the wild type,implying that mutation of MEE45 limits cell proliferation in the embryo development.By performing reciprocal cross experiments between mee45 and the wild type,we observed the effects of MEE45 mutation on seed size when maternal plants are homozygous mee45 mutants.Seeds produced by maternal homozygous mee45 mutants,regardless of the genotypes of the pollen donor,were consistently smaller than those produced by maternal wild-type plants.The result indicates that the embryo genotypes of mee45 do not affect seed size but MEE45 maternally controls seed size.(2)MEE45 mediates the development of ovules.By analyzing the expression patterns of MEE45,we observed that it is expressed in the ovule,the female gametophyte and later in the embryos.The initiation and development of both the inner and the outer integuments in mee45 were much delayed than those in the wild type,but finally the mutant integuments can enclose the nucellus following the normal pattern as the wild type.As a result,mature mee45 ovules were obviously smaller than the wild-type ones.The length of the outer integuments in mee45 were much shorter than the wild type.The number of outer integument cells in mee45 ovules was significantly decreased compared with that in wild-type ones.In the mature ovules of the wild type,strong pCYCB1;1::GUS signals were detected in the nucellus and integument cells enclosing it.By contrast,GUS signals were hardly detected in the ovule integument cells of mee45 mutants,as well as much weak GUS expression signals detected in the nucellus compared with the wild type.Thus,our results suggest that the mutation of MEE45 inhibits cell proliferation in the ovule.(3)MEE45 directly binds the ANT promotor in the ovule integument.Previously,it was shown that ANT maternally control seed size by promoting cell proliferation in the integuments of ovules.The transcript level of ANT was significantly decreased in mee45 but increased in the MEE45 overexpressing line compared with the wild type.Through analyzing the expression pattern of ANT,we observed that ANT expression was severely decreased in the mature ovule integuments of the mee45 mutants.We found that MEE45 binding to the site of CATGCA motif(-623bp/-628bp)in the ANT promoter.Furthermore,the expression of YFP reporter genes driven by the ANT promoter with the mutated MEE45-binding site was strongly reduced in the ovule integuments compared to that driven by the native ANT promoter,indicating that the MEE45-binding is required for normal ANT promoter activity in the ovule integuments.In order to determine the genetic relationship between MEE45 and ANT in the control of ovule development and seed size,we identified a new T-DNA insertion mutant of ant and designed it as ant-T,which is a weaker mutant.We generated the mee45 ant-T double mutants and found that the seed size and mass of the double mutants are both comparable to those of the ant-T single mutants,and was significantly reduced compared with the mee45 single mutants.Furthermore,introducing ectopically overexpressed ANT completely rescued the small seed size phenotypes of mee45,and mutation of ANT in weak ant-T mutants suppressed the seed size increase of MEE45 overexpressing lines.These results suggest that ANT functions in the same pathway as a direct target gene of MEE45 in the maternal control of seed size.(4)MEE45-ANT pathway acts on YUC-mediated auxin biosynthesis.Through the analysis of transcriptomic data,some auxin biosynthetic genes YUCCAs(YUCs)were identified among the downregulated genes in the mee45 mutants compared with those in the wild type,including YUC1,YUC2,YUC4,YUC5 and YUC10.We further detected the expression patterns of YUC4 in the ovules of the mee45 mutants and MEE45 overexpressing lines.YUC4 expression was severely decreased in the ovule integuments of mee45 mutants,but was significantly enhanced in the ovule integuments of the MEE45 overexpressing lines.Thus,MEE45 activates YUCs-mediated auxin biosynthesis in the ovules.As the expression of both ANT and YUC genes are activated by MEE45,and ANT is a direct target of MEE45,we detected whether the YUC genes act in the downstream of MEE45-ANT pathway.The expression levels of YUC genes were significantly decreased in the ant-T ovules but increased in the ovules of the ANT overexpressing lines.Through performing ChIP-qPCR,we found that ANT directly binds to promotor region of YUC4 in the ovule integuments.These results suggest that MEE45 directly activates the expression of ANT in the same genetic pathway which regulates YUC genes-mediated auxin biosynthesis in the ovule integuments.(5)Auxin biosynthesis in the ovule integuments is required for the MEE45-mediated maternal control of seed size.As mee45 mutants exhibited the significantly decreased auxin biosynthesis in the ovule,we examined the auxin contents in the mature ovule of both the mutants and the wild type.The auxin contents in mee45 ovules were significantly lower than those of the wild type.iaaM was ectopically overexpressed in the mee45 mutants using the 35 S promoter and lead to the seed size and mass of mee45 increased to the wild-type levels.YUC4 was ectopically overexpressed in the integuments of the mee45 mutants using the INO promoter,and caused a strong increase of seed size in mee45 mutants.These results indicating that MEE45 controls seed size through regulating auxin biosynthesis in the ovule integuments.Taken together,our results provided the important data on the molecular mechanisms of MEE45 controlling the development of both ovules and seeds.MEE45 regulates cell proliferation of ovule integument and finally maternally controls seed size.Auxin production in the maternal ovule contributes to the MEE45-ANT pathway that controls seed size in Arabidopsis.