Polycomb Proteins And REF6 Control Stem Cell Fate And Differentiation In Arabidopsis Thaliana

As sessile organisms,plants produce their whole life new organs,in a very structured way,through meristems that sustain undifferentiated stem cells.Stem cell populations are defined by their ability to renew themselves and to divide infrequently.On the other hand,plants demonstrate a remarkable developmental plasticity that can regenerate new cells,tissues,or even complete organs in response to injury or wounding.Nevertheless,de novo organogenesis requires de novo formed meristems.In meristems,plant stem cells are maintained in a specific niche by signal transduction like CLAVATA 3(CLV3)signaling and transcription factors like WUSCHEL(WUS)and SHOOTMERISTEMLESS(STM),which balance self-renewal and differentiation of the stem cells.In mature plant organs,the fully differentiated cells are not dividing.In specific conditions,these differentiated cells can transform to a less differentiated or stem cell-like stage called dedifferentiation.This term also refers to callus formation,a cellular process associated with reentry into the cell cycle,upfront of de novo organogenesis.A complex process of establishing and maintaining new gene expression patterns by epigenetic mechanisms is essential for reprogramming of cell fates during(de)differentiation.In Arabidopsis thaliana,the histone methyltransferase CURLY LEAF(CLF)forms together with other Polycomb group(Pc-G)proteins the Polycomb Repressive Complex 2(PRC2)that catalyzes the repressive Histone-3 Lysine-27 Tri-methylation(H3K27me3)mark at its target genes.The H3K27me3 mark can be actively removed by the JUMONJI-C(JmjC)histone demethylases EARLY FLOWERING 6(ELF6),RELATIVE OF EARLY FLOWERING 6(REF6),and JUMONJI 13(JMJ13).Recent publications demonstrate the important role of these histone transferases and histone demethylases in normal development including lateral root initiation and flower development.However,the role of Pc-G and JmjC proteins in regulating gene expressions that control stem cell fate and(de)differentiation is not thoroughly investigated.De novo organogenesis,in which adventitious shoots and roots are regenerated from isolated or injured tissues or organs,is one of the survival strategies of plants.De novo shoot regeneration is enhanced in tissue culture by incubating the isolated tissue or organ explants on callus inducing medium(CIM)and,subsequently,shoot-inducing medium(SIM).Most plant species do not readily regenerate shoots,which makes an initial callus formation step crucial for shoot regeneration in tissue cultures.In the first part of this study,I analyzed the role of Pc-G and JmjC proteins during callus formation and de novo shoot regeneration using several Pc-G mutants and JmjC mutants.My results show that the clf-81 single mutant enhances callus formation,whereas ref6 single mutants decrease callus formation,but both elf6 and jmj13 single mutants show no difference compared to wild-type.It indicates that the less callus formation is mainly caused by the loss of REF6.The less callus formation is a common phenotype of ref6 mutant by investigating the loss of REF6 in different alleles.Contrary to the common doctrine that callus cells are stem cell-like,loss of the stem cell factor WUS did not impair callus formation per se.This suggests that callus cells only partially acquire meristematic characteristics which include the ability to divide.My expression analyses suggest that CLF represses callus formation likely by controlling gene expression on several regulation levels during callus formation.REF6 promotes callus formation likely by direct activation of genes encoding the auxin transporter LIKE AUXIN RESISTANT 1(LAX1)and PIN-FORMED 7(PIN7).Loss of REF6 results also in decreased expression of the downstream genes AUXIN RESPONSE FACTOR 7(ARF7),ARF19,WUSCHEL-RELATED HOMEOBOX 11(WOX11),LATERAL ORGAN BOUNDARIES DOMAIN 16(LBD16),LBD18,and LBD29.These findings suggest that reduced auxin transport could cause the decreased expression levels of the ARF-WOX11-LBDs auxin response module in ref6 mutants.Furthermore,loss of REF6 results in reduced cell division,indicated by decreasing expression of the cell cycle markers CYCB1;1 and KNOLLE(KN)but also leads to reduced vacuolization of callus cells.Surprisingly,the reduced callus formation had no negative effect on shoot regeneration.ref6 mutants showed even enhanced shoot regeneration,while more callus in clf-81 was accompanied by reduced shoot regeneration.This finding indicates that Pc-G proteins and REF6 play important but largely antagonistic roles in both,callus formation and de novo shoot regeneration.The size of the shoot stem cell domain is maintained by the negative feedback loop between WUS and CLV3 functions.WUS encodes a homeodomain transcription factor that non-cell autonomously promotes stem cell fate and CLV3 expression that,in turn,represses WUS.STM is another key regulator of shoot meristem formation and maintenance,which functions in parallel to the WUS-CLV3 feedback loop.Loss of WUS or STM function leads to the lack of shoot stem cells causing meristem arrests.In the second part of this study,I analyzed the role of Pc-G proteins in shoot stem cell maintenance and de novo stem cell regeneration in seedlings.The expression of STM,WUS and CLV3 is strongly increased in Pc-G mutant seedlings indicating that Pc-G proteins restrict the expression of these meristem regulators.My phenotype analysis of CLV3 overexpression lines(35S::CLV3)with and without Pc-G,clf-60,mutation shows that the shoot meristem arrest phenotype of 35S::CLV3 is partially repressed in clf-60 background.Similar,the loss of Pc-G function in emf2-10 vrn2-l and clf-28 can partially repress the meristem arrest phenotype in Pc-G wus mutants,which is indicated by the production of more leaves and earlier shooting than in wus single mutant.These results suggest that the induction of organs may be due to regeneration of stem cells in the absence of WUS.In contrast to wus single mutants,the stem cell marker CLV3 is temporarily expressed in Pc-G wus mutant indicating that de novo stem cell regeneration can occur in a WUS-independent manner but is normally repressed by Pc-G proteins.The expression of the Pc-G target MIR156A is increased in Pc-G wus mutants,which possibly causes the earlier organ initiation.Furthermore,Pc-G stm mutant seedlings can initiate leaves,whereas stm mutants cannot.Notably,WUS is not expressed in both stm and Pc-G stm mutants suggesting that ectopic organ formation in Pc-G stm mutants is largely WUS-independent.Similar to my results in seedlings,Pc-G wus mutant callus can initiate leaf organs during shoot regeneration but does not develop complete shoots.This result suggests that WUS is not essential for initiation but is needed for the progression of de novo shoot regeneration in the loss of Pc-G activity.In summary of the second part,my findings indicate that Pc-G proteins repress de novo stem cell regeneration also in a WUS-independent manner.In shoot apical meristems,stem cells need to be continuously maintained,but normal flower development requires the termination of the floral stem cell pool to a certain time point.In the third part of this study,I analyzed the role of Pc-G proteins in the termination of stem cell activity during early flower development.My gene expression analysis reveals that STM,WUS and CLV3 are misexpressed in emf2-10 vrn2-1 inflorescences.This and other results indicate that these meristem genes are generally misexpressed in Pc-G mutants.The flowers of emf2-10 vrn2-1 mutants carry extra flower organs and an additional whorl inside the gynoecium.The enlarged floral and inflorescence meristems of emf2-10 vrn2-1 resemble the phenotype of clv3-2 mutants.The meristem enlargement is synergistically enhanced in emf2-10 vrn2-1 clv3-2 triple mutants suggesting that CLV3 signaling and Pc-G proteins restrict meristem size in parallel.The expression of STM and WUS is temporally extended beyond stage 6 flower of emf2-10 vrn2-1 mutants.Loss of STM or WUS in emf2-10 vrn2-1 background is sufficient to rescue the number of flower organs to near wild-type in some but not all floral whorls.These results suggest that the extra flower organ numbers are caused to some extent by increased STM and WUS expression.Furthermore,the floral meristem identity gene AGL24,encoding a repressor of AGAMOUS(AG),is strongly misexpressed in emf2-10 vrn2-1 mutant inflorescences.AG encodes a MADS domain transcription factor that is essential for WUS silencing,and therefore,for floral stem cell termination.The number of flowers with the fifth whorl can be significantly reduced by removing AGL24 functions in emf2-10 vrn2-1 mutants.The expression of STM but not WUS is synergistically increased in emf2-10 vrn2-1 agl24-1 triple mutants suggesting that increased AGL24 expression represses partially the ectopic STM expression in Pc-G mutants.In summary of the third part,Pc-G proteins promote floral meristem termination by repression of genes encoding transcription factors working on different regulation levels of flower development including WUS,STM and AGL24.In conclusion,this study shows that Pc-G proteins and REF6 are essential for the control of stem cells fate and differentiation.Callus and de novo shoot regeneration that involves de novo stem cell specification are antagonistically regulated by Pc-G proteins and REF6.Pc-G proteins repress de novo stem cell regeneration in a partially WUS-independent manner.On the other hand,Pc-G proteins promote stem cell termination in floral meristem in a WUS-dependent manner.This study provides new knowledge in the functions of Pc-G proteins and REF6 in differentiation and dedifferentiation processes during plant development.This new knowledge will contribute to a better understanding of cell fate reprogramming,which will enhance commercial tissue culture and agriculture techniques,but it will also push basic research forward.