J. Peter Etchells

The PXY-CLE41 receptor ligand pair defines a multifunctional pathway that controls the rate and orientation of vascular cell division

Controlling the orientation of cell division is fundamental to the development of complex body plans. This is particularly apparent in plants, where development is determined by differential growth that results solely from changes in cell expansion and orientation of the cell division plane. Despite the fundamental importance of cell division orientation to plant development, the mechanisms regulating this process remain almost completely unknown. During vascular development, the meristematic cambial cells divide down their long axis in a highly orientated manner to generate clear files of cells. The receptor kinase PXY has previously be shown to be essential for this orientation. Here, we demonstrate that the division plane is determined by the interactions of PXY and its peptide ligand, CLE41. PXY is expressed within dividing meristematic cells of the procambium, whereas CLE41 localises to the adjacent phloem cells. Altering the pattern of CLE41 expression leads to a loss of cell division orientation and a dramatic loss of ordered vascular tissue development. By contrast, increasing phloem-specific expression of CLE41 results in more cell divisions, but the orientation of cell division is retained, leading to both increased and well-ordered vascular development. We demonstrate that PXY signalling is down-regulated by CLE41. This feedback mechanism is crucial in integrating the different roles of PXY signalling in controlling xylem differentiation, regulating the rate of vascular cell division and determining the orientation of cell division. Parallels with animal systems indicate that localised signalling from adjacent cells is a general mechanism for defining the plane of cell division.

Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins

Leaves are determinate organs that arise from the flanks of the shoot apical meristem as polar structures with distinct adaxial (dorsal) and abaxial (ventral) sides. Opposing regulatory interactions between genes specifying adaxial or abaxial fates function to maintain dorsoventral polarity. One component of this regulatory network is the Myb-domain transcription factor gene ASYMMETRIC LEAVES1 (AS1). The contribution of AS1 to leaf polarity varies across different plant species; however, in Arabidopsis, as1 mutants have only mild defects in leaf polarity, suggesting that alternate pathways exist for leaf patterning. Here, we describe three genes, PIGGYBACK1 (PGY1), PGY2 and PGY3, which alter leaf patterning in the absence of AS1. All three pgy mutants develop dramatic ectopic lamina outgrowths on the adaxial side of the leaf in an as1 mutant background. This leaf-patterning defect is enhanced by mutations in the adaxial HD-ZIPIII gene REVOLUTA (REV), and is suppressed by mutations in abaxial KANADI genes. Thus, PGY genes influence leaf development via genetic interactions with the HD-ZIPIII-KANADI pathway. PGY1, PGY2 and PGY3 encode cytoplasmic large subunit ribosomal proteins, L10a, L9 and L5, respectively. Our results suggest a role for translation in leaf dorsoventral patterning and indicate that ribosomes are regulators of key patterning events in plant development.

WOX4 and WOX14 act downstream of the PXY receptor kinase to regulate plant vascular proliferation independently of any role in vascular organisation

In plants, the cambium and procambium are meristems from which vascular tissue is derived. In contrast to most plant cells, stem cells within these tissues are thin and extremely long. They are particularly unusual as they divide down their long axis in a highly ordered manner, parallel to the tangential axis of the stem. CLAVATA3-LIKE/ESR-RELATED 41 (CLE41) and PHLOEM INTERCALATED WITH XYLEM (PXY) are a multifunctional ligand-receptor pair that regulate vascular cell division, vascular organisation and xylem differentiation in vascular tissue. A transcription factor gene, WUSCHEL HOMEOBOX RELATED 4 (WOX4) has been shown to act downstream of PXY. Here we show that WOX4 acts redundantly with WOX14 in the regulation of vascular cell division, but that these genes have no function in regulating vascular organisation. Furthermore, we identify an interaction between PXY and the receptor kinase ERECTA (ER) that affects the organisation of the vascular tissue but not the rate of cell division, suggesting that cell division and vascular organisation are genetically separable. Our observations also support a model whereby tissue organisation and cell division are integrated via PXY and ER signalling, which together coordinate development of different cell types that are essential for normal stem formation.

Plant Vascular Cell Division Is Maintained by an Interaction between PXY and Ethylene Signalling

The procambium and cambium are meristematic tissues from which vascular tissue is derived. Vascular initials differentiate into phloem towards the outside of the stem and xylem towards the inside. A small peptide derived from CLV-3/ESR1-LIKE 41 (CLE41) is thought to promote cell divisions in vascular meristems by signalling through the PHLOEM INTERCALLATED WITH XYLEM (PXY) receptor kinase. pxy mutants, however, display only small reductions in vascular cell number, suggesting a mechanism exists that allows plants to compensate for the absence of PXY. Consistent with this idea, we identify a large number of genes specifically upregulated in pxy mutants, including several AP2/ERF transcription factors. These transcription factors are required for normal cell division in the cambium and procambium. These same transcription factors are also upregulated by ethylene and in ethylene-overproducing eto1 mutants. eto1 mutants also exhibit an increase in vascular cell division that is dependent upon the function of at least 2 of these ERF genes. Furthermore, blocking ethylene signalling using a variety of ethylene insensitive mutants such as ein2 enhances the cell division defect of pxy. Our results suggest that these factors define a novel pathway that acts in parallel to PXY/CLE41 to regulate cell division in developing vascular tissue. We propose a model whereby vascular cell division is regulated both by PXY signalling and ethylene/ERF signalling. Under normal circumstances, however, PXY signalling acts to repress the ethylene/ERF pathway.

Wood Formation in Trees Is Increased by Manipulating PXY-Regulated Cell Division

Summary The woody tissue of trees is composed of xylem cells that arise from divisions of stem cells within the cambial meristem. The rate of xylem cell formation is dependent upon the rate of cell division within the cambium and is controlled by both genetic and environmental factors [1, 2]. In the annual plant Arabidopsis, signaling between a peptide ligand CLE41 and a receptor kinase PXY controls cambial cell divisions [3–5]; however, the pathway regulating secondary growth in trees has not been identified. Here, we show that an aspen receptor kinase PttPXY and its peptide ligand PttCLE41 are functional orthologs and act to control a multifunctional pathway that regulates both the rate of cambial cell division and woody tissue organization. Ectopic overexpression of PttPXY and PttCLE41 genes in hybrid aspen resulted in vascular tissue abnormalities and poor plant growth. In contrast, precise tissue-specific overexpression generated trees that exhibited a 2-fold increase in the rate of wood formation, were taller, and possessed larger leaves compared to the controls. Our results demonstrate that the PXY-CLE pathway has evolved to regulate secondary growth and manipulating this pathway can result in dramatically increased tree growth and productivity.

A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps.

BackgroundThe progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs.ResultsHere, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated.ConclusionThe PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.

Identification of cyst nematode B-type CLE peptides and modulation of the vascular stem cell pathway for feeding cell formation.

Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular procambium/cambium are regulated by CLE-receptor kinase-WOX signaling modules. Previous data showed that cyst nematode CLE-like effector proteins delivered into host cells through a stylet, act as ligand mimics of plant A-type CLE peptides and are pivotal for successful parasitism. Here we report the identification of a new class of CLE peptides from cyst nematodes with functional similarity to the B-type CLE peptide TDIF (tracheary element differentiation inhibitory factor) encoded by the CLE41 and CLE44 genes in Arabidopsis. We further demonstrate that the TDIF-TDR (TDIF receptor)-WOX4 pathway, which promotes procambial meristem cell proliferation, is involved in beet cyst nematode Heterodera schachtii parasitism. We observed activation of the TDIF pathway in developing feeding sites, reduced nematode infection in cle41 and tdr-1 wox4-1 mutants, and compromised syncytium size in cle41, tdr-1, wox4-1 and tdr-1 wox4-1 mutants. By qRT-PCR and promoter:GUS analyses, we showed that the expression of WOX4 is decreased in a clv1-101 clv2-101 rpk2-5 mutant, suggesting that WOX4 is a potential downstream target of nematode CLEs. Exogenous treatment with both nematode A-type and B-type CLE peptides induced massive cell proliferation in wild type roots, suggesting that the two types of CLEs may regulate cell proliferation during feeding site formation. These findings highlight an important role of the procambial cell proliferation pathway in cyst nematode feeding site formation.

A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes

BackgroundHomeodomain transcription factors play critical roles in metazoan development. BELLRINGER (BLR), one such transcription factor, is involved in diverse developmental processes in Arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. BLR also has a redundant role in meristem maintenance. Cell wall remodelling underpins many of these processes, and BLR has recently been shown to regulate expression of PECTIN METHYL-ESTERASE 5 (PME5), a cell wall modifying enzyme in control of phyllotaxy. We have further explored the role of BLR in plant development by analysing phenotypes and gene expression in a series of plants over-expressing BLR, and generating combinatorial mutants with blr, brevipedicellus (bp), a member of the KNOX1 family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta (rev), required for radial patterning of the stem.ResultsPlants over-expressing BLR exhibited a wide range of phenotypes. Some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. Other lines with more extreme phenotypes failed to generate lateral organs, consistent with BLR repressing transcription in the shoot apex. Cell wall dynamics are also affected in blr mutant plants, and BLR has previously been shown to regulate vascular development in conjunction with BP. We found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. In these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening.ConclusionsOur data support a role for BLR in ordering the shoot apex and, in conjunction with BP and REV, playing a part in determining the composition and organisation of the vascular system. Microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing BLR result from the misregulation of a suite of genes, targets of BLR in wild type plants, that determine cell size and structure in the developing vasculature.

Class III HD-ZIPs govern vascular cell fate: an HD view on patterning and differentiation

Plant vasculature is required for the transport of water and solutes throughout the plant body. It is constituted of xylem, specialized for transport of water, and phloem, that transports photosynthates. These two differentiated tissues are specified early in development and arise from divisions in the procambium, which is the vascular meristem during primary growth. During secondary growth, the xylem and phloem are further expanded via differentiation of cells derived from divisions in the cambium. Almost all of the developmental fate decisions in this process, including vascular specification, patterning, and differentiation, are regulated by transcription factors belonging to the class III homeodomain-leucine zipper (HD-ZIP III) family. This review draws together the literature describing the roles that these genes play in vascular development, looking at how HD-ZIP IIIs are regulated, and how they in turn influence other regulators of vascular development. Themes covered vary, from interactions between HD-ZIP IIIs and auxin, cytokinin, and brassinosteroids, to the requirement for exquisite spatial and temporal regulation of HD-ZIP III expression through miRNA-mediated post-transcriptional regulation, and interactions with other transcription factors. The literature described places the HD-ZIP III family at the centre of a complex network required for initiating and maintaining plant vascular tissues.

A brief history of the TDIF‐PXY signalling module: balancing meristem identity and differentiation during vascular development

474 I. 474 II. 475 III. 475 IV. 477 V. 477 VI. 477 VII. 479 VIII. 481 482 References 482 SUMMARY: A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue. Xylem is required for water transport, and is present in the vascular tissue with a second conductive tissue, phloem, required primarily for nutrient transport. Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, including cell division in the vascular meristem, vascular organization and differentiation of vascular cell types, is a signalling module characterized by a peptide ligand called TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) and its cognate receptor, PHLOEM INTERCALATED WITH XYLEM (PXY). In this review, we explore the literature that describes signalling components, phytohormones and transcription factors that interact with these two central factors, to control the varying outputs required in vascular tissues for normal organization and elaboration of plant vascular tissue.