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The TIR1/AFB family of auxin receptors
One key initial step in auxin signalling is the degradation of AUX/IAA repressors which triggers derepression of ARFs allowing auxin-regulated gene transcription. The degradation is initiated when AUX/IAA build a transient complex with the TIR1/AFB proteins, auxin and the members of the SCF complex (Salehin et al. 2015). There are 6 TIR1/AFBs in Arabidopsis thaliana genome: TIR1 and AFB1-5 (Dharmasiri et al. 2005b). The single mutants of TIR1/AFBs have only mild phenotypes (Dharmasiri et al. 2005b, Ruegger et al. 1998) whereas higher order mutants show auxin resistance phenotypes and strong developmental defects which can lead to complete growth arrest (Dharmasiri et al. 2005b).
TIR1 and AFB1-5 proteins function as auxin receptors (Dharmasiri et al. 2005a, b, Prigge et al. 2016). They share a similar structure with an F-BOX domain close to the N-terminus and 18 leucine-rich repeats (LRR) composing the rest of the protein. The AUX/IAA proteins interact with TIR1 through their F-box domain. Another domain or TIR1, the LRR, contains a binding pocket for auxin (Tan et al. 2007). It appears that auxin acts to stabilize interactions between TIR1 and AUX/IAA proteins (Calderon-Villalobos et al. 2010).
There are 6 TIR1/AFB receptors and 29 AUX/IAA repressors in A. thaliana which results in multiple possible combinations of the coreceptor complex. Indeed, different receptor-repressor combinations have variable affinities to auxin (Calderon-Villalobos et al. 2012, Winkler et al. 2017). In addition, the TIR1/AFB receptors and AUX/IAA proteins show tissue-specific expression patterns (Dharmasiri et al. 2005b, Vernoux et a. 2011) which adds another level of specificity to auxin signalling.
The AUX/IAA repressors of auxin signalling
AUX/IAA proteins consist of three conserved domain: domains I, II and the PB1 domain (previously known as domains III and IV). Among them, domain I is the repressor EAR domain which facilitates interaction between AUX/IAA protein and the plant co-repressor TOPLESS; this interaction is required for the repression of the ARF transcriptional activity (Szemenyei et al. 2008). Domain II is responsible for the interaction with TIR1/AFB auxin receptors (Kepinski et al. 2005). The PB1 domain allows for interactions between AUX/IAA repressors and ARFs, a majority of which share this domain (Guilfoyle 2015). The PB1 domain is a well-described protein interaction module found in fungi, amoebas, animals and plants that mediates protein-protein interactions between PB1 domain-containing proteins (Sumimoto et al. 2007). The conserved residues in the N- and C-terminal ends of the PB1 domain confer electrostatic and hydrogen-bond interactions between two proteins with PB1 domains; these interactions are oriented front-to-back enabling formation of oligomers (Guilfoyle 2015, Korasick et al. 2014, Nanao et al. 2014).
At low auxin levels AUX/IAA build oligomers with ARFs and recruit the co-repressor TOPLESS to attenuate auxin signalling. At high auxin, TIR1/AFB and AUX/IAA proteins build a complex which results in ubiquitination and degradation of AUX/IAAs by 26S proteasome and the subsequent derepression of the auxin signalling pathway (Luo et al. 2018). Most of AUX/IAAs are short-lived proteins with half-life ca. 5-12 minutes (Abel et al. 1994).
Majority of the AUX/IAA proteins are able to interact with each other and with ARF activators (Vernoux et al. 2011). There are multiple possibilities to build variable oligomer complexes between AUX/IAA and ARFs (Korasick et al. 2014, Nanao et al. 2014). In addition, the tissue-specific expression of individual AUX/IAAs contributes to the complexity of ARF repression (Vernoux et al. 2011).
ARF transcription factors
Auxin Response Factors mediate auxin signalling by directly transmitting auxin response through the activation or repression of auxin-induced genes. Most of the ARFs share a similar structure with a B3 DNA-binding domain (B3 DBD) at the N-terminus which is responsible for the interactions with the AuxRe element in the promoters of their target genes (Fig. In-10A) (Tiwari et al. 2003). ARFs also contain a dimerization domain (DD) at the N-terminus which facilitates homodimerization of ARFs and allows cooperative binding of ARF dimers to their target AuxRe element; this domain consists of two parts surrounding the DNA-binding domain (Boer et al. 2014). The flanking domains (FD) are found adjacent to the DD at the N-terminus; the function of these domains remains unclear (Boer et al. 2014, Guilfoyle 2015). A variable middle region (MR) inside the ARF protein determines if the ARF is a transcriptional activator or a repressor (Tiwari et al. 2003). The C-terminal domain PB1 is involved in homo- and heterooligomerization between ARFs and AUX/IAAs (Fig. In-10A) (Korasick et al. 2014, Nanao et al. 2014).
The phylogenetical analysis of the ARF gene family leads to division of these genes into 3 classes (Fig. In-10B) (Okushima et al. 2005). Class II genes include ARF5, 6, 7, 8 and 19; it has been argued that these are transcriptional activators due to the presence of the Q-rich middle region located between the N-terminus and the C-terminus. This middle region was shown to act as an activator domain in carrot protoplast assays (Tiwari et al. 2003, Ulmasov et al. 1999). More recent data suggest that at least ARF5 is able to act as both transcriptional activator (Cole et al. 2009, Fig. In-10. The structure of the auxin response factors (ARFs) with the B3 DNA-binding domain (B3 DBD), the dimerization domain (DD), the flanking domain (FD), the middle region (MR) and the PB1 domain (A) (Guilfoyle 2015). The ARF Gene Family of Arabidopsis thaliana (B) (Okushima et al. 2005).
Konishi et al. 2015, Schlereth et al. 2010, Yamaguchi et al. 2013) and transcriptional repressor (Zhang et al. 2014, Zhao et al. 2010). Several members of Class I (15 members) and Class III (3 members) were shown to act as transcriptional repressors (Tiwari et al. 2003, Ulmasov et al. 1999). The focus of my thesis lies in Class II ARFs in Arabidopsis and for this reason I will describe these ARFs in more details below.
Among the Class II ARF family genes ARF5 (also called MONOPTEROS) is the most well studied. Loss of function mutations in the ARF5 gene result in severe phenotypes that include the inability to initiate root growth during embryonic development, variable arrangement of cotyledons and defects in vascular pattern in the leaves (Berleth and Jurgens 1993, Hardtke and Berleth 1998). The arf5 mutant seedlings show defects in the embryonic development already at the triangular stage where the lower tier of cells show erratic patterns of cell division leading to incorrect development of suspensor cells. Consequently, the hypocotyl and the primary root are missing in the mutant seedling (Fig. In-11A) (Berleth and Jurgens 1993).
Cytokinin signalling pathway
Cytokinin is another plant hormone which together with auxin regulates plant development and acts notably in the regulation of meristem activity as seen before. Cytokinin is involved in growth at the shoot and the root apical meristems, formation of lateral roots, leaf senescence, apical dominance, biotic and abiotic stress, and other developmental processes (Argueso et al. 2009, Hwang et al. 2012).
Naturally occurring cytokinins are adenine derivates with a side chain attached at N6 Cytokinin is thought to be biosynthesized from AMP and dimethylallyl pyrophosphate (DMAPP) (Kieber and Schaller 2014, Frebort et al. 2011). The first biosynthesis step is catalyzed by the enzyme isopentenyltransferase (IPT). The subsequent enzymatic reactions are catalyzed by a cytochrome P450 enzyme (CYP735A in Arabidopsis thaliana) and LONELY GUY (LOG) family of enzymes (Fig. In-13B). Cytokinins can be produced in various parts of the plant and then transported shootwards in xylem in form of tZ-riboside or rootwards in phloem as iP type cytokinins (Hirose et al. 2008, Kudo et al. 2010, Sakakibara 2006). The active cytokinins can be reduced to inactive forms by either irreversible cleavage by the cytokinin oxidase/dehydrogenase enzymes (CKX) or reversible conjugation to glucose by cytokinin glycosyltransferases (Fig. In-13B) (Kieber and Schaller 2014, Frebort et al. 2011).
Cytokinin signalling is similar to the bacterial two-component phosphorelay system (Kieber and Schaller 2014). Signalling is initiated by active cytokinins binding to histidine kinase receptors (AHKs) which then undergo autophosphorylation. In turn, the autophosphorylated AHKs can pass the phosphate group to the phosphotransfer proteins (AHPs) thus activating them. AHPs are mobile proteins that shuttle between the nucleus and the cytoplasm. Inside the nucleus, an activated AHP protein phosphorylates B-type response regulators (B-type ARRs). These B-type transcription factors propagate the cytokinin response by direct regulation of numerous cytokinin-responsive genes. This signalling cascade is negatively regulated by A-type ARRs and AHP6 (Fig. In-13C) (Kieber and Schaller 2014; Hwang et al. 2012, To and Kieber 2008). AHP6 is a non-functional phosphotransfer protein which lacks the phosphorylation site and acts as an inhibitor of cytokinin signalling in the root vasculature and in the SAM (Besnard et al. 2014, Bishopp et al. 2011, Mahonen et al. 2006).
Cytokinin signalling can be visualized in planta using a TCS synthetic reporter which consists of B-type ARR binding motif concatemers constitutively driving a reporter gene such as GFP (Muller and Sheen 2008, Zurcher et al. 2013) (Fig. In-3C, 7B).
Cytokinin Response Factors (CRFs)
The well-described cytokinin signalling pathway is based on a phosphorelay system which involves membrane-bound AHK cytokinin receptors, mobile AHP proteins and the B-type ARR transcription factors. Together these three components mediate cytokinin response in various tissues. Nevertheless it was shown recently that the expression of a subset of cytokinin-induced genes is directly regulated by Cytokinin Response Factors (CRFs).
CRFs are a gene family of transcription factors belonging to a subset of AP2/ERF family of transcription factors and consisting of 12 members in Arabidopsis thaliana (Fig. In-14A). They are characterised by a presence of conserved CRF domain at the N-terminus and by a single AP2 domain in the middle region of the protein (Fig. In-14B). The AP2 domain is a well-studied DNA-binding domain; it is present not only in CRF genes but generally in members of the AP2/ERF superfamily of transcription factors (Licausi et al. 2013). The CRF domain is specific for the CRF gene family; it was shown to be involved in protein-protein interactions (Cutcliffe et al. 2011).
Tissue-specific expression of ARF activators provides insights into specificity and redundancy of their functions in plant development
Plant development relies heavily on hormonal signals. Among them auxin is particularly important because it is involved in a myriad of various developmental processes associated with cellular growth, organ initiation and development, responses to biotic and abiotic stresses. The auxin-induced changes in plant body structure are precise, timely and correspond well to plant’s environment. It is astonishing that a great scope of different developmental changes can be regulated by a single basic molecule such as IAA. This raises the question of how the precision and specificity of auxin response is achieved.
The answer could be explained by considering the abundance and diversity of molecules which mediate auxin response. Alone, the presence of 23 ARFs in Arabidopsis thaliana can indicate that these ARFs could be involved in specific independent or redundant auxin responses. Indeed, from 5 ARF activators described in this study each of them shows involvement in both overlapping and independent auxin responses as indicated by single and double mutant phenotypes (Berleth and Jurgens 1993, Nagpal et al. 2005, Okushima et al. 2005, Przemek et al. 1996). Additionally, the ARF activators were shown to control different sets of downstream target genes (Nagpal et al. 2005, Okushima et al. 2005, Schlereth et al. 2010).
The functional diversity and specificity could be in part explained by tissue-specific expression of these ARFs. Accordingly, this study explored the diversity of ARF activator expression patterns in both the root and the shoot with a goal to draw new hints on the function of each ARF activator. In fact, each ARF activator shows distinct expression patterns. In particular, when looking globally at the ARF activator expression in the RAM we see a clear specification in different domains where different combinations of ARFs are expressed. Here the expression patterns can be separated into blocks which can consist of a single tissue type or, alternatively, of multiple autonomous tissue types (Fig. 1-14). For example, columella initials and the mature columella cells are all enriched in ARF5 and ARF6 but deprived of other ARF activators. Similarly, endodermis and cortex both contain only ARF6 and ARF7 transcripts. This could indicate that the endodermis and cortex are able to react to auxin in the same manner even though they are functionally different tissues, thus forming a block of specific auxin response. On the contrary, we expect that cortex/endodermis on one side and columella on the other side would show a different reaction to auxin because of enrichment of different ARF activators in these two tissues.
Post-transcriptional movement or degradation of ARF proteins provides another level of control in the auxin signaling pathway
Often it is presumed that the tissues where the gene is expressed and the tissues where the gene acts are identical. In many papers the expression of a gene is sufficient to justify the tissue-specific function of this gene. Post-transcriptional modifications are often neglected. Similarly, for the majority of the ARFs post-transcriptional regulation has not been studied in details.
In embryos, ARF5 protein does not move but remains in the same tissues where it is expressed and acting non-cell-autonomously through a secondary signal, the mobile transcription factor TMO7 (Schlereth et al. 2010; Weijers et al. 2006). On the contrary, the presence of ARF5 protein in the central zone of the SAM where its not expressed is published in Zhao et al. 2010; nevertheless the potential protein movement is not considered in this paper. In this thesis the cell movement of ARF5 protein in the SAM is confirmed. Further, in the RAM, ARF5 protein is shown to either move towards the xylem or to be degraded in the surrounding vasculature tissues. Thus the function of ARF5 in the xylem development might be reinforced by the accumulation of its protein specifically in this tissue.
ARF6 and ARF8 were never been shown to be involved in non-cell-autonomous cell signaling which could require a protein movement. Indeed, in my study I could not detect any protein movement or degradation for ARF6 in the RAM or the SAM. It was shown that ARF6 and ARF8 undergo a different type of post-transcriptional regulation: their mRNA can be degraded by microRNA167 affecting expression pattern specifically in flowers (Wu et al. 2006). This type of regulation would also be reflected in the differences between the transcriptional and the translational reporters. Since I see no difference in case of ARF6, I conclude that in the RAM and the SAM microRNA-mediated degradation of ARF6 mRNA cannot be detected under my growth conditions.
ARF7 and ARF19 proteins provide puzzling results: both N-terminal and C-terminal protein fusions result in relocation of their proteins in non-nuclear compartments. It appears that the proteins aggregate. Could this aggregation be due to addition of a fluorescent protein tag and the subsequent misfolding of the proteins? Although this explanation cannot be formally discarded, the fact that aggregation persists despite changing the terminus of the fusion speaks against it. Is it possible that ARF7 and ARF19 proteins are intrinsically unstable and undergo constant degradation unless stabilized by an unknown factor? Interestingly, both ARF7 and ARF19 show broad expression patterns in many tissues such as in the older root upwards from the meristematic zone (Fig. 1-4 and 1-6) and to some extend in the SAM (Fig. 1-7). Could their excessive expression be compensated by constant protein degradation rendering ARF7- and ARF19- mediated auxin response inactive by default? In that case which triggers could lead to stabilization and activate normal function of these proteins? Currently no answers can be provided to these questions.
Yeast one-hybrid assay revealed an elaborate network of transcription factors controlling expression of ARF activators
The transcriptional regulators of ARF activator expression were identified using a yeast one-hybrid assay (Fig. 2-1A). The ARF promoter fragments (bait) were fused to the lacZ or HIS3 reporter genes and screened against a library of transcription factors fused to a Gal4 activation domain (preys) in yeast. The physical interaction between the bait and the prey resulted in expression of these reporter genes which then enabled the selection of successful interactions through an X-gal assay (in case of lacZ) or by growing the yeast on a medium deficient in histidine amino acid (in case of HIS reporter gene) (Fig. 2-1B and C). An interaction was considered as positive when either one of the assays (or both) showed positive result.
An important point was to decide on the size of the DNA fragments from the ARF locus to use as a bait in this approach. Ideally these fragments should contain all binding sites for the regulatory transcription factors. The ARF activators all share a similar structure: within their coding sequence they contain 11-14 introns (Suppl. Fig. 1-1). In particular, for ARF5, 7 and 19 the first intron is 2-3 times larger than the other introns. Because this first intron was suspected to be important for the transcriptional regulation, the sequences screened in the yeast one-hybrid included both upstream promoter region and a small part of the downstream region containing this first intron. Overall, large promoter regions were screened with ca. 5000 bp promoters for ARF5, 8 and 19 and ca. 3000 bp promoters for ARF6 and ARF7. The length and the content of the bait fragments gives confidence that a majority of the regulatory interactions would be identified even though there is a chance that some interactions might still be missed. Due to the limitations of the assay, the larger promoters of ARF5, 8 and 19 were screened in two separate fragments (Fig. 2-3). The promoter size and Fig. 2-4. Yeast one-hybrid promoter- transcription factor interaction network for ARF activators. Green boxes are five ARF activators; pink boxes are transcription factors binding to the ARFs. sequence corresponds precisely to the promoters used in the construction of the transcriptional reporter lines described in chapter I. In this regard, the transcriptional reporter lines reveal the expression patterns generated, in part, by the transcriptional regulators identified in this study. In the chapter the reporter lines were shown to have a generally good consistency between the observed expression pattern in the SAM and the published in situ hybridization results; this shows that the fragments used for the bait constructs contain most of the regulatory sequences required to recapitulate in planta expression patterns.
The yeast one-hybrid screen for promoters of ARF5, 6, 7, 8, and 19 was conducted in collaboration with Siobhan Brady’s lab at the UC Davis. The Siobhan Brady’s lab possesses a library of ca. 800 root-specific stele enriched transcription factors used in the yeast one-hybrid screen. In addition to that, I cloned 87 more transcription factors that I added to this collection in this study (see Suppl. Table 2-3). The additional genes include shoot-specific transcription factors which were shown to play important roles in the shoot development. Additionally, a number of genes from signalling pathways of various hormones were added. The goal was to find regulators of ARFs involved in both root and shoot development and to find any potential cross talk between auxin and other hormones. At the time of conducting this experiment, the final prey library represented approximately half of all predicted A. thaliana transcription factors.
A summary of results of the yeast one-hybrid screen are displayed in Figure 2-4. Overall 42 transcription factors bound to the promoters of ARF activators in my assay. The network includes 47 interactions because a few of the transcription factors regulate multiple ARFs. The ARF activators were not uniform in the amount and type of interaction partners. Numerous interactions were identified for ARF8. On the other hand, ARF5 had 4, ARF6 had 2, ARF7 had 6 and ARF19 only 4 interaction partners.
Most of the transcription factors originated from the root-specific library. Only four transcription factors come from the 87 shoot-specific transcription factors that I added to the collection: WUS and KNU which bind to ARF8, KNAT1 binding to ARF5 and CRF10 binding to ARF7.
Table of contents :
Introduction
Plant embryonic development
Root growth and the root apical meristem
Root apical meristem morphology
Regulation of root apical meristem development
Lateral root development
Shoot apical meristem
The plant hormone auxin
The TIR1/AFB family of auxin receptors
The AUX/IAA repressors of auxin signalling
ARF transcription factors
ARF5
ARF6 and ARF8
ARF7 and ARF19
The plant hormone cytokinin
Cytokinin signalling pathway
Cytokinin Response Factors (CRFs)
Aims of the thesis
Chapter I: The tissue-specific expression of ARF activators during growth and development in the RAM and the SAM
Introduction
Results
ARF activators are differentially expressed in the root and the shoot
ARF7 expression in the RAM is regulated at the first intron
ARF activator distribution can be post-transcriptionally regulated
Discussion
Tissue-specific expression of ARF activators provides insights into specificity and redundancy of their functions in plant development
Expression patterns of ARF activators fits their well-studied functions and suggests new potential functions
Which elements are important to correctly recapitulate expression patterns of genes?
Post-transcriptional movement or degradation of ARF proteins provides another level of control in the auxin signaling pathway
Material and methods
Cloning and generation of ARF reporter lines
Root microscopy
Shoot microscopy
Supplementary information
Chapter II: Auxin Response Factor (ARF) activators are transcriptionally regulated by gene-specific repressor network
Introduction
Results
Yeast one-hybrid assay revealed an elaborate network of transcription factors controlling expression of ARF activators
The majority of the ARF activator transcriptional regulators act as repressors
Publically available databases can be used to further validate and explore the interactions
Mutants of the regulatory transcription factors indicate co-regulation of multiple ARF activators
Expression of the several transcription factors is regulated by auxin
Mutants of the regulatory transcription factors show auxin-related defects in root and shoot development
Discussion
Does the yeast one-hybrid gene regulatory network reflect in planta gene interactions?
ARF activator expression is predominantly regulated by transcriptional repressors
Regulation of ARF8 by multiple transcription factors might indicate its special significance in auxin signaling
Gene regulatory network motifs describe regulations that could be important during development
Expression of ARF activators is co-regulated in planta
Material and methods
Plant material
Y1H assay
Transient expression in Arabidopsis thaliana protoplasts
Expression analysis with qRT-PCR
Expression analysis of crosses between ARF transcriptional reporter lines and T-DNA mutants
Shoot phenotype analysis of the TF mutants
Root phenotype analysis of the TF mutants
Growth on NPA for shoot phenotype analysis
Supplementary information
Chapter III: Cytokinin Response Factor 10 regulates ARF7 expression to control plant development
Introduction
Results
Genetic characterization of the crf10 mutant
CRF10 is expressed in various tissues
crf10 mutant has an ARF7-dependant early senescence phenotype
crf10 mutant shows perturbation in hypocotyl response to blue light
crf10 mutant has a defect in root apical meristem morphology
Discussion
Is crf10-1 a loss-of-function mutant?
Does CRF10 act in cytokinin signaling?
CRF10 and ARF7 antagonistically control leaf senescence
CRF10 and ARF7 are acting together in hypocotyl phototropic response
CRF10 and ARF7 are involved in the maintenance of the root apical meristem
Material and methods
Plant material
Analysis of the conserved regions within the CRF10 protein across A. thaliana ecotypes
Genetic analysis of the crf10-1 mutant
Expression analysis with qRT-PCR
Cloning and generation of transgenic lines
Root microscopy
Shoot microscopy
Shoot phenotype analysis
Early senescence phenotype analysis
Chlorophyll measurement
Hypocotyl phototropism assay
Root phenotype analysis
Supplementary information
General discussion
ARF activators in control of plant development
ARF activators diversify and specialize during evolution
Component of auxin signaling pathway show specificity in expression patterns
ARF activator expression is predominantly regulated by gene repression mechanism
Auxin and cytokinin interactions regulate many aspects of plant development
Appendix
Overview of primers used in this study
List of primers
References .
