Introduction - Draft
Plants are sessile organisms; they do not have the capacity to move around as Animals do to in order to attain resources and to respond to changes in the environment, therefore they must use other strategies in order to survive. They have the capacity to develop continuously by growing new organs, such as leaves, flowers and roots, to facilitate this problem (Schmitt et al, 2005). The formation of these new organs are as the result of activity of meristems where pluripotent stem cells produce undifferentiated cells which eventually expand and differentiate to produce the plants organs. To maintain the stability of the meristem its various actions; cell proliferation and organ initiation, must be regulated (Mayer. K.F.X et al, 1998). Plants rely heavily on transcriptional regulation to control gene expression within these meristems and to control their activities when changes to the environment occur (Krogan. K.T and Long J.A, 2009).
Transcription factors are proteins which regulate the transcription of genes (Li-Jia Qu and Yu-Xian Zhu, 2006). They form complex networks at a transcription level and through Protein-protein interactions in order to regulate and control developmental processes (Reichmann J. L and Ratcliffe. O.J, 2000). They are capable of controlling gene expression through many pathways including; chromatin remodelling and through the activities of the RNA polymerase II transcription-initiation complex (Singh. K. B, 1998).
The Arabidopsis genome contains 1510 to 1581 transcription factors which are split into large families, such as MYB and MADS. However, only a small proportion of these genes have been molecularly or genetically characterised (Li-Jia Qu and Yu-Xian Zhu, 2006). By examining the expression patterns of these gene families it has become clear that a family of transcription factors are involved in specific developmental processes such as flower development and different combinations of these genes are present at different times and areas of development (Schmid. M et al, 2005).
Transcription factors function by binding directly to specific DNA sequences on the promoters of target genes and in doing so either activate or repress the expression of the gene. This gives rise to specific regulation of the gene (Singh. K.B, 1998). The activation or repressive activities of different transcription factors allow them to be characterised into their functions as either repressors or activators. Coactivators and corepressors mediate the activities of repressors and activators through specific protein-protein interactions, allowing them to also be specific to a promoter without themselves binding to DNA. These interactions result in the formation of an Enhanceosome (Singh. K.B, 1998) or a Repressosome (Courey and Jia, 2001) which are a protein complexes formed when multiple proteins interact in order to regulate a specific gene and these can either activate or repress respectively (Singh. K.B, 1998). The composition of these complexes may change as the result of environmental or developmental signals (Singh. K.B, 1998).
In the past the study of transcriptional regulation has focused on the activation of genes, however, repression is just as important. In fact mechanisms of gene regulation through activation are constantly being shown to be impeded by transcriptional repressors (Cowel. I.G, 1994) and so the old view that without activation the gene would be fixed and silent without any other cues is fast becoming redundant.
There are two types of repressors; active and passive. Active repressors inhibit initiation of transcription directly through binding to the promoter of the gene. In contrast to passive repressors, which, downregulate the activity of other transcription factors (Cowel. I.G, 1994). Active repressors contain defined repression domains which are sequence-specific enabling them to bind to corepressors and other regulators (Krogan.K.T and Long J.A, 2009). These could be chromatin remodelling factors which result in chromatin tightening and therefore repression of the gene (Krogan.KT and Long J.A, 2009).
The Activities of the WUSCHEL Transcription Factor
The homeodomain transcriptional regulator WUSCHEL (WUS) is required to maintain the undifferentiated state of stem cells in the shoot apical meristem (Ikeda et al, 2009) and the floral meristem (Laux. T et al, 1996) and is an excellent example of a gene which has both activation and repression capabilities. These functions are exemplified as it activates AGAMOUS (AG) and LEAFY (LFY) therefore terminating stem cell maintenance (Lenhard. M et al, 2001) and also represses the expression of the type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, which in turn, repress cytokinin signalling which is involved in cell proliferation (Leifried et al, 2005).
The size of the pool of stems cells which the WUS gene creates is controlled by a negative feedback loop involving the CLAVATA (CLV) gene (Leifried et al, 2005). WUS activates the expression of the small peptide CLV3 which, in some way, acts as a ligand for the CLV1-CLV2 heterodimer which represses WUS expression, therefore maintaining the stem cell number.
Leifired et al (2005) showed that after WUS induction by ethanol levels of ARR5, ARR6, ARR7 and ARR15 mRNA were decreased. By transiently repressing WUS through inducing CLV3 Leifried et al (2005) indicated that ARR genes are repressed by WUS, as after this transient repression of WUS, ARR levels increased. In connection with this ARR5, ARR6, ARR7 and ARR15 promoters were found to be active in the meristem, consistent with their interaction with WUS in this area of the plant.
The former description of WUSCHEL, as an activator of AG, began as the result of analysis of the Arabidopsis WUSCHEL mutant, wus-1. In the wus-1 mutant stem cells were misspecified and instead of remaining pluripotent experienced differentiation thus resulting in the collapse of the Shoot Apical Meristem (SAM) and the Floral Meristem (Mayer. K. F. X et al, 1998). The root apical meristem, however, is unaffected (Laux. T et al 1996). Wildtype Arabidopsis flowers four sepals, four petals, six stamens and two fused carpels, however, the wus-1 mutant does not have most of the central organs and ends in a carpel (Laux. T et al 1996). Wus floral meristems and SAMs were found to terminate prematurely and organ conformation was generally indistinguishable from the wild-type (Laux. T et al 1996). WUS was characterised as specifying stem cell fate and not as a repressor of organ formation as cells were only misspecified and not incorporated into organs (Mayer. K. F. X et al, 1998). The WUS protein is expressed in a small number of meristem cells forming an organising centre just below the stem cells and not in the stem cells themselves (Mayer. K. F. X et al, 1998).
In contrast to Shoot Apical Mersitems (SAMs) which are indeterminate, floral meristems are determinate which means that once the flower structure is complete stem cell maintenance is stopped (Lenhard. M et al, 2001). As both these meristems contain an organizing centre to maintain the stems cells as the result of WUS expression, they also contain identity genes APETALA1 (AP1) or LEAFY (LFY) which are one aspect which ensure the difference between them. The determinacy of the floral meristem is the result of a negative autoregulatory mechanism which requires the activation of AG, the MADS domain transcription factor, by WUS and the repression of WUS by AG. Therefore, once AG is activated by WUS, AG represses the expression of WUS thus preventing the maintenance of the stem cells and preventing further growth (Lenhard. M et al, 2001). This mechanism is further complicated as in the mutant lfy, AG is not sufficiently activated by WUS and therefore termination of the meristem cannot take place. It appears that WUS requires the addition of LFY to activate AG expression. As LFY is only expressed in the floral meristem and not in SAM, SAM remains indeterminate in the presence of both AG and WUS (Lenhard. M et al, 2001). Similarly, AG does not have the capacity to repress WUS alone and therefore must require other transcription factors in the same way as WUS requires LFY (Lenhard. M et al, 2001).
The activation and repressive activities of WUS are due to its possession of various conserved sequence motifs at the C-terminal end of the protein (Kieffer et al, 2006). Excluding the homeodomain the WUS protein contains; an acidic domain, a WUS box and an EAR-like domain each of which have specific functions in either the repression or activation of other proteins. The acidic domain (LEGHGEEEECGGDA) is a common domain conserved in plant transcription factors and it is the only activation domain present on the WUS protein (Kieffer et al, 2006). The EAR motif (L/FDLNL/FxP or LXLXL) is a repression domain that is conserved in plants (Ohta et al, 2001). It is required for the repressive function of many repressors such as; the class II ethylene-repressive element binding factors (ERFs), TFIIA-type zinc repressors and the AUX/IAA proteins (Kieffer et al, 2006). As the WUS protein possesses an EAR-like motif (ASLELTLN), through the evidence that it is present in other transcription factors with repressive function, we are able to suggest that it has a repressive function within the WUS protein. However, mutation within this EAR-like motif does not interfere with the repressive activity of WUS leading to the idea that the WUS box (TLPLFPMH) is also a repression domain (Ikeda et al, 2009). The WUS Box is present in all the WUSCHEL-RELATED HOMEBOX (WOX) genes, except WOX 13, of which some members have been found to control aspects of plant development (Kieffer et al, 2006). Therefore it is speculated that this domain is important in the function of the entire family. This is further enhanced by the fact that the EAR-like domain is not present in all of the WOX family of proteins.
Experiments into the bifucntionality of the WUS transcription factor by Ikeda. M et al (2009) has shown that the WUS Box is a crucial repression domain. They performed experiments involving loss of function and complementation analysis of the three domain structures. Transient expression analysis showed that wild type WUS is, indeed, a strong repressor. As previously mentioned the mutation of the EAR-like domain in WUS does not interfere with the repressive activities of WUS, however, mutations in both the EAR-like motif and the WUS box was shown in this paper by transient expression assays to have no repressive function. This construct did, however, have weak activation activity and when the acidic domain was also mutated the triple mutant had neither repressive nor activation potential thus leading to the proposal that the acidic domain is the only activation domain in WUS. Ikeda. M et al (2006) also found that mutations in the EAR-like domain nor the acidic domain resulted in any deformation of the transgenic Arabidopsis to produce stem cell identity. However, plants with a defective WUS box did have malfunctioning stem cell identity in the Shoot Apical Meristem which is consistent with the wus-1 phenotype. Further experimentation by Ikeda. M et al (2006) into the activation of AG by WUS indicated that when WUS is ectopically expressed so is AG, even in the absence of the acidic domain. Which is surprising as it is the only activation domain on the protein. Further to this the WUS Box is certainly essential for the expression of AG as only the WUS box is capable of the expression of AG in mutants were the EAR-like motif is mutated. These results were obtained by the expression of the reporter gene Gus (Ikeda. M et al, 2006). Through complementation analysis of the different domain mutants and the wus-1 mutant they showed that the WUS box is required to the maintenance of stem cell identity in SAMs and for floral formation in floral meristems with the aid of the EAR-like motif.
As well as these involvements with the activation of AG, WUS, as previously mentioned, repressed the activity of the type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes; ARR5, ARR6, ARR7 and ARR15. Ikeda. M et al (2006) concluded that these interactions were, too, as the result of the WUS box as levels of these ARR genes were increase with both the WUS box mutant and the EAR-like and WUS box mutant. These results point towards the EAR-like motif having no involvement with this reaction. This is in contrast to the AG interaction where the EAR-like motif does have some contribution. These series of experiments have shown that the WUS box is vital for all the activities of WUS including its activation capabilities despite it repressive function (Ikeda. M et al, 2009).
The Regulation of WUSCHEL
Although the mechanism for the prevention of stem cell differentiation by WUSCHEL is not yet completely understood these conserved domain structures have been found to be used as tools for the interaction between two members of a family of Arabidopsis corepressors, TPL and TPR4 (Kieffer et al, 2006). These protein corepressors were found as the result of a yeast two-hybrid screen and were found with various full-length and truncations of the WUS gene. Interactions between TPL (TOPLESS) and TPR4 (TOPLESS-RELATED) and the WUS protein only occurred when the three conserved domains were present (Kieffer et al, 2006). These two corepressors were found to be expressed throughout the development of the plant by microarray analysis (Schmid et al., 2005). TPL and TPR4 contain N-terminal LisH and CTLH domains, a pro-rich region, and two domains containing WD repeat motifs which are vital for their interaction with WUS. (Kieffer et al, 2006). The LisH domain is a dimerization domain present in all plant TPL/TPRs and LEUNIG (LUG) family proteins (Liu and Karmerkar, 2008). WD repeats provide a structure where protein-protein interactions can take place (Smith et al., 1999) indicating that this structure provides a site for WUS to interact with TPL/TPR corepressors. Many other unrelated transcriptional corepressors, such as LUG and the human TBL1 and TBLR1, contain an N-terminal LisH and these WD repeats suggesting these are common motifs in transcriptional corepressors. Therefore this contributes to the confirmation that these two proteins are also corepressors. Also, it is possible that as the WOX family has a similar domain structure to WUS that these, too, may interact with the two corepressors providing a network of interactions between these two families in order to control plant meristem maintenance and consequently plant development in general (Kieffer et al, 2006). In this vein a model is proposed by Kieffer et al (2006) in which WUS recruits the TPL and TPR4 corepressors in order to provide repression of genes required for the differentiation of stem cells in SAM. In the floral meristem this WUS-TPL complex would, somehow, be disrupted and result in WUS switching from a repressor to an activator of AG. Leading on from this it is possible that this disruption could be caused by LEAFY and as LEAFY is only present in the floral meristem and not SAM this disruption would cause only floral meristem maintenance to be disrupted. The mechanism for repression of stem cell differentiation genes by WUS-TPL/TPR is unknown but it is speculated that it may occur as a result of histone deacetylation which is shown to occur in other transcriptional repressors such as the Drosophila gene GROUCHO (Courey and Jia, 2001).
The GROUCHO gene is a transcriptional corepressor which also contains these WD tandem repeats and a tetramerization domain but it does not have a LisH domain as in the aforementioned corepressors (Courey and Jia, 2001). The similarity between the GROUCHO WD repeats and those in the GROUCHO yeast homolog, TUP1 has led to the grouping of these genes into the GROUCHO/TUP1 superfamily. The GROUCHO/TUP1 superfamily have been revealed to cause chromosomes to become silent through histone deacetylation and therefore cause genes in that area to become repressed. This occurs through the interaction of GROUCHO/TUP1 proteins with Histone Deacetylases (HDACs).
A nulceosome consists of eight histone subunits, these subunits are composed of a globular C-terminal domain and a N-terminal tail which projects outward. The deacetylation by HDACs of this tail causes the chromatin structure to become more compact resulting in either transcriptional machinery becoming unable to transcribe the genes due to their inaccessibility or the action of deacetylation could cause the recruitment of chromatin remodelling factors. The actual mechanism through which this occurs is still unresolved (Courey and Jia, 2001). Both histone deacetylases (HDACs) and histone acetyltransferases (HATs) were found to interact with TPL by genetic approaches as the mutant hdac19 had tpl like phenotype (Long, J.A et al, 2006).
Long. J. A et al (2006) have revealed that TPL interacts with the HISTONE DEACETYLASE 19 (HDA19). Mutations in the HDA19 gene increases the mutant tpl-1 phenotype. Therefore, it is suggested that it works with TPL to regulate the repression of genes although HDA19 was also found to have other functions which do not involve TPL.
The Topless-1 Phenotype
A mutation in the Arabidopsis TOPLESS gene, topless-1 (tpl-1), results in the transformation of the shoot pool into a second root pole (Long, J.A et al, 2006). The structure of the apical root in the extreme phenotype is similar to the wild-type root although it has more cells and is wider. It also demonstrates normal gravitropism and has a root cap (Long. J.A et al, 2002).
This phenotype is dramatic as the only mutants to be isolated which affect specific patterning of the plant thus far have not been able to switch one organ into another. In some of these mutants, eg monopteros, bodenlos and axr6 there are disturbances to the root of the plant but the overall polarity of the entire plan is not disrupted (Long. J.A et al, 2002). This suggests that the TOPLESS gene works at a higher level of transcriptional regulation than these other mutants (Long, J.A et al, 2006). This extreme phenotype only occurs at the restrictive temperature of 29°C at lower temperature other phenotypes of this mutant occur. At lower temperature the plant has no hypocotyls, cotyledons or shoot apical meristem (Long. J.A et al, 2002). Other characteristics may arise where the plant has a cup-shaped cotyledon where as others may have no cotyledon at all and only make a hypocotyls. A common characteristic of the mutant seedlings is the nonexistent SAM, however, in some seedlings a SAM can be formed after germination resulting in a relatively normal fertile plant (Long. J.A et al, 2002).
In the extreme topless-1 phenotype the mutant embryo begins morphologically indistinguishable from the wildtype. However, at the heart shaped stage they become more oblong in shape and do not have cotyledon primordial. In the apical half of the tpl-1 embryo there is reduced expression of genes normally expressed in that area and ultimately genes expressed in the basal half are expanded into the apical half resulting in the change in axis formation (Long, J.A et al, 2006). The suggestion that to begin with the axis formation of the embryo is correct and then is lost during the transition stage is shown by the original expression of WUS in the apical half of the embryo and then its loss (Long, J.A et al, 2006).
Therefore, Long, J.A et al (2006) suggest a model where at the transition stage of embryogenesis TPL and other TPR proteins repress this expansion of basal gene expression in the apical half of the embryo and that this repression involves the histone deacetylase HDA19.
The TOPLESS and the regulation Auxin
It is commonly recognised that Auxin and Cytokinin are associated with plant growth and development. In relation to this a treatment of high auxin to cytokinin levels given to plants results in encouraged root development and in contrast High cytokinin to auxin levels results in encouraged shoot development (Skoog and Miller,1957). The mutant axr6 suggest that these ratios are also relevant within the embryo as they fail to respond normal to high levels of auxin (Long. J.A et al, 2002).
As previously mentioned there are other mutants which cause disruption to the root as well as the topless mutant. One of these the, Monopteros mutant, does not develop roots or hypocotyls during embryogenesis. The MONOPTEROS gene encodes a member of the ARF (Auxin Response Factors) family of transcription factors which transduce auxin signals which regulate root development (Long. J.A et al, 2002). ARF Transcription factors possess a DNA-binding domain (DBD) which is used to bind to TGTCTC auxin response elements within the promoter of target genes (Guilfoyle and Hagen, 2007).
ARFs have either activation or repression domains which confer its capacity to regulate. Of the 22 known ARFs which encode proteins there are five known ARF activators (ARF 5-8 and 19) the others are all repressors (Guilfoyle and Hagen, 2007). ARF transcription factors dimerize with the AUX/IAA (Auxin/Indole acetic acid) corepressors using a dimerization domain in order to regulate the transcription of auxin response genes. Without this dimerisation with AUX/IAA proteins ARF proteins are unable to regulate genes in response to auxin (Guilfoyle and Hagen, 2007). The monopteros-7 mutant is a truncated version of the wildtype protein but it does not contain this domain and is therefore exhibits the monopteros phenotype (Long. J.A et al, 2002).
AUX/IAAs are degraded in the presence of Auxin and therefore, Auxin response genes seem to be regulated as AUX/IAA repressors bind to ARF Activators which exist on the promoters of target genes resulting in the repression of the gene. In the presense of Auxin AUX/IAAs are degraded resulting in activation of the target genes as no repression is present (Guilfoyle and Hagen, 2007). In the presence of an ARF repressor at the promoter of a target gene there would be no need for AUX/IAA repression and so in this case the regulation of the target gene would be Auxin independent (Guilfoyle and Hagen, 2007).
AUX/IAA proteins contain the EAR-motif (LXLXL) which is required for the binding of TPL corepressors. A direct interaction between the CTLH domain of TPL and the EAR domain of IAA12 suggests that TPL has some involvement in the AUX/IAA interaction with ARFs and that they use their corepressive function with AUX/IAA to repress ARF activators thus repressing target genes (Szemenyei, H. et al, 2008).
In a yeast-two hybrid assay performed with TPL a number of ARF proteins (ARF2 and ARF 9) were pulled out and as a result were also found to have an EAR-Like domains (LSLKL and RLFGI respectively). As these are repressive ARFs it is suggested that these have this function as the result of the corepressive activity of the TPL interactor. This would explain that aforementioned auxin independent repressive function of these ARFs as it is the degradation of AUX/IAAs which causes the regulation in the presence of auxin. In skipping this step and having only TPL repressing ARF function then the presence of Auxin would be superfluous.
The TOPLESS Family of genes
As previously mentioned the TOPLESS (TPL) gene has a family of TOPLESS-RELATED (TPR) genes which have considerable amino acid similarity with itself (Long, J.A et al, 2006). The four TOPLESS-RELATED genes have similar corepressive function as TPL and are thought to have the same function as TPL with its involvement with WUS and WOX genes. As tpl-1 acts as a type of dominant negative allele for multiple TPR family members (Long, J.A et al, 2006) it is considered that they must have similar genetic functions. ????
