The Arabidopsis thaliana CLAVATA3/EMBRYO-SURROUNDING REGION 26 (CLE26) peptide is able to alter root architecture of Solanum lycopersicum and Brassica napus

Optimal development of root architecture is vital to the structure and nutrient absorption capabilities of any plant. We recently demonstrated that AtCLE26 regulates A. thaliana root architecture development, possibly by altering auxin distribution to the root apical meristem via inhibition of protophloem development. In addition, we showed that AtCLE26 application is able to induce a root architectural change in the monocots Brachypodium distachyon and Triticum aestivum. Here, we showed that application of the synthetic AtCLE26 peptide similarly affects other important agricultural species, such as Brassica napus and Solanum lycopersicum

Functional characterisation of small signalling peptides and a receptor kinase involved in root architecture development in Arabidopsis and crop species

Post-embryonic root development is a plastic process by which plants are able to interface with the rhizosphere in order to provide anchorage, and increase surface area available for acquisition of nutrients and water. While root growth is governed primarily by auxin/cytokinin interactions, roots are able to sense the presence of surrounding nutrient deposits and changes to environmental conditions – directing growth accordingly – due to the action of signalling cascades. This thesis presents data pertaining to the characterisation of two signalling elements involved in governing root architecture; the small signalling peptide CLAVATA/EMBRYO SURROUNDING REGION 26 (CLE26), and the receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4). Initially, the origins and evolutionary history of root architecture are explored, and an overview of signalling elements involved in root architectural development is provided, before discussing the potential benefits that manipulation of signalling events may allow in targeted crop improvement. To provide background on peptide signalling, the physiological and biochemical effects of small signalling peptides are discussed in view of the current literature, demonstrating the diverse range of developmental processes which are known to be regulated by these ligands and their known receptors. Following this, functional characterisation analyses indicate CLE26 as a novel, potent inhibitor of primary root growth and protophloem development in Arabidopsis, and is also shown to induce a similar effect upon exogenous application to several crop species. Furthermore, data is presented demonstrating the clear requirement for functional analysis during the development phase of creating antagonistic peptides, as a previously described antagonistic peptide technology was not applicable in all cases. Concluding the exploration of CLE26 signalling, a phosphoproteomics screen was conducted to probe further into CLE26 function, determining 23 putative effectors of CLE26 signalling, which are discussed in view of their potential to mediate CLE26 signalling, according to current literature. Next, the known roles of ACR4 and its orthologues are reviewed, demonstrating the importance of ACR4 signalling in many developmental processes, including regulation of asymmetric cell division during postembryonic root development. Although ACR4 activity is known to regulate asymmetric cell division in both columella stem cells and lateral root primordia, little is known about the downstream mediators of ACR4 signalling. In an attempt to fill this gap in knowledge, yeast 2-hybrid and co-immunoprecipitation approaches were employed. These two parallel proteomics screens together resulted in identification of 19 putative interactors of ACR4 signalling (PAIPs), which are discussed as potential mediators of ACR4 signalling in view of current literature. Of the identified PAIPs, three were further characterised by loss of function analysis, demonstrating that loss of PHOSPHOLIPASE Iγ2 and a PROTEIN OF UNKNOWN FUNCTION (At1g49840/UNK) was able to affect root architecture. Further in-silico characterisation of UNK reveals its similarity to soluble phospholipase receptors, which, alongside PLA-Iγ2, may potentially implicate ACR4 as a key player in a novel mechanism involved in regulation of bioactive lipid production

DEF6, a novel substrate for the tec kinase ITK, contains a glutamine-rich aggregation-prone region and forms cytoplasmic granules that co-localize with P-bodies

Localization of DEF6 (SLAT/IBP), a Rho-family guanine nucleotide exchange factor, to the center of the immune synapse is dependent upon ITK, a Tec-family kinase that regulates the spatiotemporal organization of components of T cell signaling pathways and Cdc42-dependent actin polymerization. Here we demonstrate that ITK both interacts with DEF6 and phosphorylates DEF6 at tyrosine residues Tyr210 and Tyr222. Expression of a GFP-tagged Y210E-Y222E phosphomimic resulted in the formation of DEF6 cytoplasmic granules that co-localized with decapping enzyme 1 (DCP1), a marker of P-bodies; sites of mRNA degradation. Similarly treatment of cells with puromycin or sodium arsenite, reagents that arrest translation, also resulted in the accumulation of DEF6 in cytoplasmic granules. Bioinformatics analysis identified a glutamine-rich, heptad-repeat region; a feature of aggregating proteins, within the C-terminal region of DEF6 with the potential to promote granule formation through a phosphorylation-dependent unmasking of this region. These data suggest that in addition to its role as a GEF, DEF6 may also function in regulating mRNA translation

Modulation of Arabidopsis and monocot root architecture by CLAVATA3/EMBRYO SURROUNDING REGION 26 peptide

Plant roots are important for a wide range of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface with the soil environment. Several small signalling peptides and receptor kinases have been shown to affect primary root growth, but very little is known about their role in lateral root development. In this context, the CLE family, a group of small signalling peptides that has been shown to affect a wide range of developmental processes, were the focus of this study. Here, the expression pattern during lateral root initiation for several CLE family members is explored and to what extent CLE1, CLE4, CLE7, CLE26, and CLE27, which show specific expression patterns in the root, are involved in regulating root architecture in Arabidopsis thaliana is assessed. Using chemically synthesized peptide variants, it was found that CLE26 plays an important role in regulating A. thaliana root architecture and interacts with auxin signalling. In addition, through alanine scanning and in silico structural modelling, key residues in the CLE26 peptide sequence that affect its activity are pinpointed. Finally, some interesting similarities and differences regarding the role of CLE26 in regulating monocot root architecture are presented

A phylogenetic approach to study the origin and evolution of the CRINKLY4 family

Cell cell communication plays a crucial role in plant growth and development and relies to a large extent on peptide ligand receptor kinase signaling mechanisms. The CRINKLY4 (CR4) family of receptor-like kinases is involved in a wide range of developmental processes in plants, including mediating columella stem cell identity and differentiation in the Arabidopsis thaliana root tip. Members of the CR4 family contain a signal peptide, an extracellular part, a single-pass transmembrane helix and an intracellular cytoplasmic protein kinase domain. The main distinguishing features of the family are the presence of seven "crinkly" repeats and a TUMOR NECROSIS FACTOR RECEPTOR (TNER)-like domain in the extracellular part. Here, we investigated the evolutionary origin of the CR4 family and explored to what extent members of this family are conserved throughout the green lineage. We identified members of the CR4 family in various dicots and monocots, and also in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. In addition, we attempted to gain insight in the evolutionary origin of different CR4-specific domains, and we could detect "crinkly" repeat containing proteins already in single celled algae. Finally, we related the presence of likely functional CR4 orthologs to its best described signaling module comprising CLAVATA3/EMBRY0 SURROUNDING REGION-RELATED 40 (CLE40), WUSCHEL RELATED HOMEOBOX 5 (W0X5), CLAVATA 1 (CLV1), and ARABIDOPSIS CR4 (ACR4), and established that this module likely is already present in bryophytes and lycophytes

Antagonistic peptide technology for functional dissection of CLE peptides revisited

Information collected using antagonistic peptide approaches can be very useful, but these approaches do not work in all cases and require insight on ligand-receptor interactions and peptide ligand structur

Phosphite treatment can improve root biomass and nutrition use efficiency in wheat

Phosphite represents a reduced form of phosphate that belongs to a class of crop growth-promoting chemicals termed biostimulants. Previous research has shown that phosphite application can enhance root growth, but its underlying mechanism, especially during environmental stresses, remains elusive. To uncover this, we undertook a series of morphological and physiological analyses under nutrient, water and heat stresses following a foliar application in wheat. Non-invasive 3D imaging of root system architecture directly in soil using X-ray Computed Tomography revealed that phosphite treatment improves root architectural traits and increased root biomass. Biochemical and physiological assays identified that phosphite treatment significantly increases Nitrate Reductase (NR) activity, leaf photosynthesis and stomatal conductance, suggesting improved Nitrogen and Carbon assimilation, respectively. These differences were more pronounced under heat or drought treatment (photosynthesis and photosystem II stability) and nutrient deficiency (root traits and NR). Overall our results suggest that phosphite treatment improves the ability of plants to tolerate abiotic stresses through improved Nitrogen and Carbon assimilation, combined with improved root growth which may improve biomass and yield

The growing story of (ARABIDOPSIS) CRINKLY 4

Receptor kinases play important roles in plant growth and development, but only few of them have been functionally characterized in depth. Over the past decade CRINKLY 4 (CR4)-related research has peaked as a result of a newly discovered role of ARABIDOPSIS CR4 (ACR4) in the root. Here, we comprehensively review the available (A)CR4 literature and describe its role in embryo, seed, shoot, and root development, but we also flag an unexpected role in plant defence. In addition, we discuss ACR4 domains and protein structure, describe known ACR4-interacting proteins and substrates, and elaborate on the transcriptional regulation of ACR4. Finally, we address the missing knowledge in our understanding of ACR4 signalling

Functional characterisation of small signalling peptides and a receptor kinase involved in root architecture development in Arabidopsis and crop species

Post-embryonic root development is a plastic process by which plants are able to interface with the rhizosphere in order to provide anchorage, and increase surface area available for acquisition of nutrients and water. While root growth is governed primarily by auxin/cytokinin interactions, roots are able to sense the presence of surrounding nutrient deposits and changes to environmental conditions – directing growth accordingly – due to the action of signalling cascades. This thesis presents data pertaining to the characterisation of two signalling elements involved in governing root architecture; the small signalling peptide CLAVATA/EMBRYO SURROUNDING REGION 26 (CLE26), and the receptor kinase ARABIDOPSIS CRINKLY 4 (ACR4). Initially, the origins and evolutionary history of root architecture are explored, and an overview of signalling elements involved in root architectural development is provided, before discussing the potential benefits that manipulation of signalling events may allow in targeted crop improvement. To provide background on peptide signalling, the physiological and biochemical effects of small signalling peptides are discussed in view of the current literature, demonstrating the diverse range of developmental processes which are known to be regulated by these ligands and their known receptors. Following this, functional characterisation analyses indicate CLE26 as a novel, potent inhibitor of primary root growth and protophloem development in Arabidopsis, and is also shown to induce a similar effect upon exogenous application to several crop species. Furthermore, data is presented demonstrating the clear requirement for functional analysis during the development phase of creating antagonistic peptides, as a previously described antagonistic peptide technology was not applicable in all cases. Concluding the exploration of CLE26 signalling, a phosphoproteomics screen was conducted to probe further into CLE26 function, determining 23 putative effectors of CLE26 signalling, which are discussed in view of their potential to mediate CLE26 signalling, according to current literature. Next, the known roles of ACR4 and its orthologues are reviewed, demonstrating the importance of ACR4 signalling in many developmental processes, including regulation of asymmetric cell division during postembryonic root development. Although ACR4 activity is known to regulate asymmetric cell division in both columella stem cells and lateral root primordia, little is known about the downstream mediators of ACR4 signalling. In an attempt to fill this gap in knowledge, yeast 2-hybrid and co-immunoprecipitation approaches were employed. These two parallel proteomics screens together resulted in identification of 19 putative interactors of ACR4 signalling (PAIPs), which are discussed as potential mediators of ACR4 signalling in view of current literature. Of the identified PAIPs, three were further characterised by loss of function analysis, demonstrating that loss of PHOSPHOLIPASE Iγ2 and a PROTEIN OF UNKNOWN FUNCTION (At1g49840/UNK) was able to affect root architecture. Further in-silico characterisation of UNK reveals its similarity to soluble phospholipase receptors, which, alongside PLA-Iγ2, may potentially implicate ACR4 as a key player in a novel mechanism involved in regulation of bioactive lipid production