Michael A Djordjevic

• Australian National University

A growing understanding is emerging of the roles of peptide hormones in local- and long-distance signalling that coordinates plant growth and development as well as responses to the environment. C-TERMINALLY ENCODED PEPTIDE (CEP) signalling triggered by its interaction with CEP RECEPTOR 1 (CEPR1) is known to play roles in systemic nitrogen (N)-dema...

Root system architecture (RSA) influences the acquisition of heterogeneously dispersed soil nutrients. Cytokinin and C-TERMINALLY ENCODED PEPTIDE (CEP) hormones affect RSA, in part by controlling the angle of lateral root (LR) growth. Both hormone pathways converge on CEP DOWNSTREAM 1 (CEPD1) and CEPD2 to control primary root growth, however, a rol...

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormones act over short and long distances to control plant responses to environmental cues. CEP and cytokinin pathway mutants share phenotypes, however, it is not known if these pathways intersect. We show that CEP and cytokinin signalling converge on CEP DOWNSTREAM (CEPD) glutaredoxins to inhibit p...

Legumes acquire soil nutrients through nitrogen-fixing root nodules and lateral roots. To balance the costs and benefits of nodulation, legumes negatively control root nodule number by autoregulatory and hormonal pathways. How legumes simultaneously coordinate root nodule and lateral root development to procure nutrients remains poorly understood....

C-TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormones act over short and long distances to control plant responses to environmental cues. CEP and cytokinin pathway mutants share phenotypes, however, it is not known if these pathways intersect. We show that CEP and cytokinin signalling converge on CEP DOWNSTREAM (CEPD) glutaredoxins to inhibit p...

C-TERMINALLY ENCODED PEPTIDEs (CEPs) control diverse responses in plants including root development, root system architecture, nitrogen demand signalling, and nutrient allocation influencing yield and there is evidence that different ligands impart different phenotypic responses. Thus, there is a need for a simple method that identifies bona fide C...

Plant transmembrane proteins (TMPs) are essential for normal cellular homeostasis, nutrient exchange, and responses to environmental cues. Commonly used bottom–up proteomic approaches fail to identify a broad coverage of peptide fragments derived from TMPs. Here, we used mass spectrometry (MS) to compare the effectiveness of two solubilization and...

Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remain...

The interaction of C-Terminally Encoded Peptides (CEPs) with CEP RECEPTOR 1 (CEPR1) controls root growth and development, as well as nitrate uptake, but has no known role in determining yield. We used physiological, microscopic, molecular and grafting approaches to demonstrate a reproductive tissue-specific role for CEPR1 in controlling yield and s...

Root system architecture (RSA) influences the effectiveness of resources acquisition from soils but the genetic networks that control RSA remain largely unclear. We used rhizoboxes, X‐ray computed tomography, grafting, auxin transport measurements and hormone quantification to demonstrate that Arabidopsis and Medicago CEP (C‐TERMINALLY ENCODED PEPT...

Lateral root (LR) proliferation is a major determinant of soil nutrient uptake. How resource allocation controls the extent of LR growth remains unresolved. We used genetic, physiological, transcriptomic, and grafting approaches to define a role for C-TERMINALLY ENCODED PEPTIDE RECEPTOR 1 (CEPR1) in controlling sucrose-dependent LR growth. CEPR1 in...

CEPs (C-TERMINALLY ENCODED PEPTIDEs) inhibit Arabidopsis primary root growth by unknown mechanisms. We investigated how CEP3 levels control primary root growth. CEP3 peptide application decreased cell division, S-phase cell number, root meristematic cell number and meristem zone (MZ) size in a dose- and CEP RECEPTOR1-dependent manner. Grafting show...

Excessive or insufficient angiogenesis is associated with major classes of chronic disease. Although less studied, small molecules which can promote angiogenesis are being sought as potential therapeutics for cardiovascular and peripheral arterial disease and stroke. Here we describe a bioassay-directed discovery approach utilising size exclusion a...

UV absorbance of HPLC fractions containing FK1, FK2 and P6. FK1 (A) and FK2 (B). UV absorbance spectra were essentially identical with λmax = 265 nm and a shoulder at 298 nm. The UV spectra of P6 (C) was characterised by a λmax at 283 and 343 nm. (TIF)

Positive mode ESI-MS of FK1 and FK2. (A) FK1, erythro guaiacylglycerol-8-O-4´-(coniferyl alcohol) ether, and (B) FK2, threo guaiacylglycerol-8-O-4´-(coniferyl alcohol) ether). (TIF)

Effect of synthetic FK1 (eGGCE) on HMEC wound healing assay. Synthetic FK1, at concentrations ranging from 5 × 10−6 M to 5 × 10−12 M, was added to cultures and wound healing was measured as % wound confluence from 0 to 26 hr relative to the initial wound mark. Control cultures contained the same diluent dilution as the test compounds. Data analysis...

Effect of eGGCE on HMEC tube formation on Matrigel as measured by circle formation. Microscopic view of isolated endothelial cells cultured for 6 hr on Matrigel (A) in the absence of compound or (B) following addition of 5 × 10−6 M eGGCE which enhanced circle formation, with examples of completed circles indicated (*). (C) Effect of eGGCE, at conce...

Optimisation of fibronectin and vitronectin concentrations used to coat wells for the Rose Bengal adhesion assay using HMEC. HMEC adhesion to fibronectin (A) and vitronectin (B) was measured as optical density of Rose Bengal staining of adherent cells. Error bars represent SEM (n = 6). The background binding in the absence of fibronectin and vitron...

Comparison of elemental formulas derived from the observed negative ion mode ESI MS/MS spectra with the calculated elemental formulas for FK1 and FK2. (DOCX)

NMR Spectra of FK1 and FK2. Overlay of 13C-HSQC spectra for the (A) aliphatic and (B) aromatic region, highlighting the similarity of spectra between FK1 (red/pink) and FK2 (blue/green). Along with coupling constants measured from 1D 1H NMR spectra, assignments were easily transferred from FK1 to FK2. Negative peaks (green and pink) indicate CH2 re...

Comparison of elemental formulas derived from the observed positive ESI MS/MS spectra with the calculated elemental formulas for FK1 and FK2. (DOCX)

Chemical shifts and coupling constants for FK1 and FK2. (DOCX)

Secreted peptide hormones play pivotal roles in plant growth and development. So far, CEPs (C-TERMINALLY ENCODED PEPTIDEs) have been shown to act through CEP receptors (CEPRs) to control nitrogen (N)-demand signalling, nodulation, and lateral root development. Secreted CEP peptides can enter the xylem stream to act as long-distance signals, but evi...

MtCLE12 and MtCLE13 encode CLAVATA3/EMBRYO‐SURROUNDING REGION RELATED (CLE) peptides which regulate autoregulation of nodulation (AON) in Medicago through the shoot receptor, SUNN (SUPER NUMERIC NODULES). Genetics suggests RDN1 (ROOT‐DETERMINED NODULATION 1) arabinosylates MtCLE12 to enable SUNN perception. The functional structures of MtCLE12 and...

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics appr...

Optimized root system deployment should enable moreefficient nutrient acquisition and increased crop yields. C-TERMINALLY ENCODED PEPTIDE (CEP) hormones and their receptors, which regulate root growth, could be important in research with this aim. Roberts et al. (pages 4889-4899 in this issue) suggest that the full extent of CEP function and signal...

C-TERMINALLY ENCODED PEPTIDEs (CEPs) control root system architecture in a non-cell-autonomous manner. In Medicago truncatula, MtCEP1 affects root development by increasing nodule formation and inhibiting lateral root emergence by unknown pathways. Here, we show that the MtCEP1 peptide-dependent increase in nodulation requires the symbiotic signali...

Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized pro...

Encouraging more students to embrace plant science research is a global priority. We have evolved a second year undergraduate course from a standard lecture/practical format into an innovative research-led learning design that gives students hands-on experience of cutting-edge plant science research and specialist instrumentation. By making tangibl...

Many legumes have the capacity to enter into a symbiotic association with soil bacteria generically called ‘rhizobia’ that results in the formation of new lateral organs on roots called nodules within which the rhizobia fix atmospheric nitrogen (N). Up to 200 million tonnes of N per annum is fixed by this association. Therefore, this symbiosis play...

Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two dom...

Plant adaptive potential is critically dependent upon efficient communication and co-ordination of resource allocation and signalling between above- and below-ground plant parts. Plant roots act as gatekeepers that sense and encode information about soil physical, chemical and biological factors, converting them into a sophisticated network of sign...

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathwa...

Background Small, secreted signaling peptides work in parallel with phytohormones to control important aspects of plant growth and development. Genes from the C-TERMINALLY ENCODED PEPTIDE (CEP) family produce such peptides which negatively regulate plant growth, especially under stress, and affect other important developmental processes. To illumin...

The role of MtCEP1, a member of the CEP (C-terminally encoded peptide) signaling peptide family, was examined in Medicago truncatula root development. MtCEP1 was expressed in root tips, vascular tissue, and young lateral organs, and was up-regulated by low nitrogen levels and, independently, by elevated CO2. Overexpressing MtCEP1 or applying MtCEP1...

The C-terminally Encoded Peptide (CEP) family of regulatory peptides controls root development in vascular plants. Here, we present the first NMR structures of CEP. We show that root-knot nematode (RKN: Meloidogyne spp.) also encodes CEP, presumably to mimic plant CEP as part of their stereotypic, parasitic interaction with vascular plants. Molecul...

The manifestation of repetitive developmental programmes during plant growth can be adjusted in response to various environmental cues. During root development, this means being able to precisely control root growth and lateral root development. Small signalling peptides have been found to play roles in many aspects of root development. One member...

Nitrogen, particularly nitrate is an important yield determinant for crops. However, current agricultural practice with excessive fertilizer usage has detrimental effects on the environment. Therefore, legumes have been suggested as a sustainable alternative for replenishing soil nitrogen. Legumes can uniquely form nitrogen-fixing nodules through s...

Hox proteins are one of the best studied sets of transcription factors in developmental biology. They are major determinants for establishing morphological differences along the anterior-posterior axis of animals and are generally regarded as highly conserved in function. This view is based on experiments comparing a few (anterior) Hox proteins, ho...

Plant root systems arise de novo from a single embryonic root. Complex and highly coordinated developmental networks are required to ensure the formation of lateral organs maximizes plant fitness. The Arabidopsis root is well-suited to dissection of regulatory and developmental networks due to its highly ordered, predictable structure. A myriad of...

Subtilisin-like serine protease (MtSBT), serine carboxypeptidase (MtSCP), MtN5, non-specific lipid transfer protein (MtnsLTP), early nodulin2-like protein (MtENOD2-like), FAD-binding domain containing protein (MtFAD-BP1), and rhicadhesin receptor protein (MtRHRE1) were among 34 proteins found in the supernatant of M. truncatula 2HA and sickle cell...

Flavonoids have broad cross-kingdom biological activity. In Arabidopsis, flavonoid accumulation in specific tissues, notably the root elongation zone and root/shoot junction modulate auxin transport, affect root gravitropism, and influence overall plant architecture. The relative contribution made by aglycones and their glycosides remains undetermi...

Plant root architecture is regulated by the initiation and modulation of cell division in regions containing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organogenesis at the site of lateral root or, in legumes, nodule formation. Root meri...

This study investigated the changes in the fatty acid content and composition in the nitrogen-starved Chlamydomonas reinhardtii starchless mutant, BAF-J5, grown at different temperatures. The optimal temperature for vegetative growth under nitrogen sufficient conditions was found to be 32°C. Shifting temperature from 25 to 32°C, in conjunction with...

Hox proteins are one of the best studied sets of transcription factors in developmental biology. They are major determinants for establishing morphological differences along the anterior-posterior axis of animals and are generally regarded as highly conserved in function. This view is based on experiments comparing a few (anterior) Hox proteins, ho...

A subset of CLAVATA3/endosperm-surrounding region-related (CLE) peptides are involved in autoregulation of nodulation (AON) in Medicago truncatula (e.g. MtCLE12 and MtCLE13). However, their linkage to other components of the AON pathways downstream of the shoot-derived inhibitor (SDI) is not understood. We have ectopically expressed the putative pe...

CLE (CLAVATA3/ESR-related) peptides are developmental regulators that are secreted into the apoplast. Little is known about the role of the sequences that flank CLE peptides in terms of their biological activity or how they are targeted by proteases that are known to liberate the final active CLE peptides from their precursor sequences. The biologi...

The root apical meristem (RAM) is responsible for the growth of the plant root system. Because of the importance of root architecture in the performance of crop plants, we established a proteome reference map of the soybean root apex and compared this with the proteome of the differentiated root zone. The root apex samples contained the apical 1 mm...

Transposon mutagenesis was used to generate nodulation (Nod−) and nitrogen fixation (Fix−) mutants of Rhizobium trifolii SU843. Total DNA isolated from mutant clones was digested with EcoR1 and cloned into plasmid pBR322. Recombinants carrying the Tn5 transposon and flanking Rhizobium DNA sequences were used to probe a gene fbank prepared from wild...

Medicago truncatula Gaertn. can generate roots in vitro through the formation of root stem cells from leaf explants cultured with auxin. To identify key genes involved in the early processes of root initiation, we compared gene expression in root-forming cultures (RFC) enriched for root stem cells with non-root-forming cultures (NRFC) and untreated...

CLANS links to save-files. A short text file providing web-links to the CLANS program and the CLANS save-files for each of the similarity maps used in our analysis. (0.00 MB TXT)

Seed sequences. The FASTA-format sequences used to seed the PSI-BLAST searches (also see Table1). (0.02 MB TXT)

Extended homeodomain alignment. The multiple sequence alignment of extended homeodomains from which a Profile-Hidden-Markov-Model (HMM) was derived. This HMM was subsequently used to identify the extended homeodomains of the sequences in our set of interest. (0.00 MB TXT)

CLANS sequences. A Zip archive containing the various groups of sequences used in our CLANS analyses. The archive provides one file with FASTA-format sequences for each of the similarity maps displayed in figures 3–9. In addition, the archive also contains the set of FASTA-format sequences used for our comparison of Hox15, NOB-1 and EGL-5. (2.41 MB...

Homeodomain alignment. The multiple sequence alignment of homeodomains from which a Profile-Hidden-Markov-Model (HMM) was derived. This HMM was subsequently used to identify the homeodomains of the sequences in our set of interest. (0.00 MB TXT)

CLANS network-clustering. Overview of the “network-clustering” approach as implemented in CLANS. Aim of this approach is to automatically identify groups of sequences with greater similarity to each other than to the rest. (0.24 MB PDF)

CLANS parameters. A Zip archive containing text files specifying the parameters used for each of the generated CLANS cluster maps. (0.01 MB ZIP)

The family of Hox-proteins has been a major focus of research for over 30 years. Hox-proteins are crucial to the correct development of bilateral organisms, however, some uncertainty remains as to which Hox-proteins are functionally equivalent across different species. Initial classification of Hox-proteins was based on phylogenetic analysis of the...

Climate change is a growing concern for society and the focus of numerous research initiatives across multiple fields of science. These initiatives often need to capitalize on the cross-specialized knowledge contributed by researchers from very different fields. The diversity of worldviews among key stakeholders requires an effective overall design...

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions including defense, UV protection, auxin transport inhibition, allelopathy, and flower coloring. Interestingly, these compounds also have considerable biological activity in plant, animal and bacterial systems - such broad activity is accomplished by few compounds. Yet, f...

Seeds of genetically modified (GM) peas (Pisum sativum L.) expressing the gene for alpha-amylase inhibitor-1 (alphaAI1) from the common bean (Phaseolus vulgaris L. cv. Tendergreen) exhibit resistance to the pea weevil (Bruchus pisorum). A proteomic analysis was carried out to compare seeds from GM pea lines expressing the bean alphaAI1 protein and...

Flavonoids are low molecular weight secondary plant metabolites with a myriad of functions. As flavonoids affect auxin transport (an important growth-controlling hormone) and are biologically active in eukaryotes, flavonoid mutants were expected to have undescribed architectural phenotypes. The Arabidopsis thaliana transparent testa (tt) mutants ar...

Legumes develop root nodules from pluripotent stem cells in the root pericycle in response to mitogenic activation by a decorated chitin-like nodulation factor synthesized in Rhizobium bacteria. The soybean genes encoding the receptor for such signals were cloned using map-based cloning approaches. Pluripotent cells in the root pericycle and the ou...

Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleave...

Molecular events occurring in the plant apoplast contribute to important developmental and defense responses. To define the secretome of Medicago, we used suspension cultures to isolate and identify secreted proteins as a first step to determining their functions. Proteins in the extracellular medium of the suspension cultures were examined using S...

The NARK (nodule autoregulation receptor kinase) gene, a negative regulator of cell proliferation in nodule primordia in several legumes, encodes a receptor kinase that consists of an extracellular leucine-rich repeat and an intracellular serine/threonine protein kinase domain. The putative catalytic domain of NARK was expressed and purified as a m...

Flavonoid synthesis is modulated by developmental and environmental signals that control the amounts and localization of the diverse flavonoids found in plants. Flavonoids are implicated in regulating a number of physiological processes including UV protection, fertilization, auxin transport, plant architecture, gravitropism and pathogenic and symb...

Pongamia pinnata (L.) Pierre is a fast-growing leguminous tree with the potential for high oil seed production and the added benefit of the ability to grow on marginal land. These properties support the suitability of this plant for large-scale vegetable oil production required by a sustainable biodiesel industry. The future success of P. pinnata a...

Nodulation and concomitant symbiotic nitrogen fixation are critical for the productivity of the legume, yielding food, feed and fuel. The nodule number in legumes is regulated by numerous factors including the number and efficiency of the interacting Rhizobium bacteria and abiotic stresses as well as endogenous processes involving phytohormones, no...

Protein-based interactions occurring in plant extracellular spaces (the apoplast) have attracted increased attention because they are critical for defence and development (Boller, 2005). Important apoplast-located self- and non-self responses include the recog-nition of (i) bioactive peptides from phytobacteria and (ii) endogenous peptides. The bio...

Sinorhizobium meliloti is an agriculturally and ecologically important microbe due to its capacity to establish nitrogen-fixing symbiosis with plant legumes. Two-dimensional gel electrophoresis of total cellular protein was used to establish a proteome reference map for the model microsymbiont Sinorhizobium meliloti strain 1021. The extent of chang...

Flavonoids are synthesized in response to developmental and environmental signals and perform many functions in plants. Arabidopsis (Arabidopsis thaliana) roots grown in complete darkness do not accumulate flavonoids since the expression of genes encoding enzymes of flavonoid biosynthesis is light dependent. Yet, flavonoids accumulate in root tips...

Molecular signaling interactions in the plant apoplast are important for defense and developmental responses. We examined the soybean proteome of the apoplastic conduit of root-to-shoot communication, the xylem stream, using gel electrophoresis combined with two types of tandem mass spectrometry. We examined soybeans for the presence of a Bradyrhiz...

The small protein and native peptide component of plant tissues is a neglected area of proteomic studies. We have used fractionation techniques for denatured and nondenatured protein preparations combined with 2-D LC tandem mass spectrometry to examine the sequences of small proteins and peptides in four tissues of the model legume, Medicago trunca...

The clover-nodulating Rhizobium leguminosarum bv. trifolii ANU794 initiates normal root-nodule development with abnormally low efficiency on the Trifolium subterraneum cv. Woogenellup. The cellular and developmental responses of Woogenellup roots to the site- and dose-defined inoculation of green fluorescent protein (gfp)-labeled cells of ANU843 (n...

A proportion of the Nod factors of some Rhizobium leguminosarum bv. trifolii strains is characterized by the presence of highly unsaturated fatty acyl chains containing trans double bonds in conjugation with the carbonyl group of the glycan oligosaccharide backbone. These fatty acyl chains are C-18:3, C-20:3, C-18:4, or C-20:4 and have UV-absorptio...

A report on the 3rd International Legume Genetics and Genomics Conference, Brisbane, Australia, 9-13 April 2006.

The proteome of the model symbiotic bacterium, Sinorhizobium meliloti was examined to determine the enzymatic reactions and cell processes that occur when S. meliloti occupies the root nodules of Medicago truncatula and Melilotus alba. The proteomes of the nodule bacteria were compared to that of S. meliloti grown under laboratory cultured conditio...