Michael A. Djordjevic

FLAVONOIDS: NEW ROLES FOR OLD MOLECULES

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, for all the research over the last three decades, many of the cellular targets of these secondary metabolites are unknown. The many mutants available in model plant species such as Arabidopsis thaliana and Medicago truncatula are enabling the intricacies of the physiology of these compounds to be deduced. In the present review, we cover recent advances in flavonoid research, discuss deficiencies in our understanding of the physiological processes, and suggest approaches to identify the cellular targets of flavonoids.

Establishment of a root proteome reference map for the model legume medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting

We have established a proteome reference map for Medicago truncatula root proteins using two‐dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation‐related genes and proteins. Over 2500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4–7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105 000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii

Rhizobium trifolii mutants containing Escherichia coli lac gene fusions to specific nodulation (nod) genes were used to characterise phenolic compounds secreted from the roots of white clover (Trifolium repens) plants. These compounds either had stimulatory or inhibitory effects upon the induction of the nod genes. The stimulatory compounds were hydroxylated flavones and the most active compound was 7,4′‐dihydroxyflavone. The inhibitory compounds present in white clover root exudates were umbelliferone (a coumarin) and formononetin (an isoflavone). Transcriptional activation of nod gene promoters in response to short exposures (3 h) of 7,4′‐dihydroxyflavone was growth phase dependent; cells in early log phase were highly responsive to flavone additions in vitro and nod gene induction could be detected within 20 min of exposure at 5 x 10−7 M. Cells in other growth phases were generally unresponsive. A 10‐fold molar excess of umbelliferone to 7,4′‐dihydroxyflavone resulted in complete inhibition of nod gene induction. Some commercially‐obtained flavones were found to have weak stimulatory activity but could also inhibit nod gene induction by more effective stimulatory compounds. Strong stimulatory and inhibitory compounds all possessed a 7‐hydroxy moiety and showed other structural similarities. This suggested that there was one binding site for these compounds. Because the response to these compounds was rapid, we propose that these phenolics act at the bacterial membrane or that an active uptake system is involved.

Pongamia pinnata : An Untapped Resource for the Biofuels Industry of the Future

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 as a sustainable source of feedstock for the biofuels industry is dependent on an extensive knowledge of the genetics, physiology and propagation of this legume. In particular, research should be targeted to maximizing plant growth as it relates to oil biosynthesis. This review assesses and integrates the biological, chemical and genetic attributes of the plant, providing the basis for future research into Pongamia’s role in an emerging industry.

Proteome Analysis of Differentially Displayed Proteins As a Tool for the Investigation of Symbiosis

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.

Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation

The Chlamydomonas reinhardtii starch-less mutant, BAF-J5, was found to store lipids up to 65% of dry cell weight when grown photoheterotrophically and subjected to nitrogen starvation. Fourier transform infrared spectroscopy was used as a high-throughput method for semi-quantitative measurements of protein, carbohydrate and lipid content. The fatty acids of wild-type and starch mutants were identified and quantified by gas chromatography mass spectrometry. C. reinhardtii starch mutants, BAF-J5 and I7, produce significantly elevated levels of 16:0, 18:1(Δ9), 18:2(Δ9,12) and 18:3(Δ9,12,15) fatty acids. Long-chain saturated, mono- and polyunsaturated fatty acids were found under nitrogen starvation. Oleosin-like and caleosin-like genes were identified in the C. reinhardtii genome. However, proteomic analysis of isolated lipid bodies only identified a key lipid droplet associated protein. This study shows it is possible to manipulate algal biosynthetic pathways to produce high levels of lipid that may be suitable for conversion to liquid fuels.

Flavonoids Are Differentially Taken Up and Transported Long Distances in Arabidopsis

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 of plants with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain, and localized aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating uptake and movement systems. This was confirmed by time-course fluorescence experiments and high-performance liquid chromatography. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application to midroot or cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol, and dihydroquercetin were taken up at the root tip, midroot, or cotyledons and traveled long distances via cell-to-cell movement to distal tissues, followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Using ATP-binding cassette (ABC) transporter and H+-ATPase inhibitors suggested that a multidrug resistance-associated protein ABCC transporter facilitated flavonoid movement away from the application site.

Tn5 mutagenesis of Rhizobium trifolii host-specific nodulation genes result in mutants with altered host-range ability

SummaryA 14 kb DNA fragment from the Sym plasmid of the Rhizobium trifolii strain ANU843, known to carry common nodulation nod and host specific nodulation hsn genes, was extensively mutagenised with transposon Tn5. A correlation between the site of Tn5 insertion and the induced nodulation defect led to the identification of three specific regions (designated I, II, III) which affected nodulation ability. Twenty-three Tn5 insertions into region I (ca. 3.5 kb) affected normal root hair curling ability and abolished infection thread formation. The resulting mutants were unable to nodulate all tested plant species. Tn5 insertions in regions II and III resulted in mutants which showed an exaggerated root hair curling (Hac++) response on clover plants. Ten region II mutants which occurred over a 1.1 kb area showed a greatly reduced nodulation ability on clovers and produced aborted, truncated infection threads. Tn5 insertions into region III (ca. 1.5 kb) altered the outcome of crucial early plant recognition and infection steps by R. trifolii. Seven region III mutants displayed host-range properties which differed from the original parent strain. Region III mutants were able to induce marked root hair distortions, infection threads, and nodules on Pisum sativum including the recalcitrant Afghanistan variety. In addition region III mutants showed a poor nodulation ability on Trifolium repens even though the ability to induce infection threads was retained on this host. The altered host-range properties of region III mutants could only be revealed by mutation and the mutant phenotype was shown to be recessive.

A global analysis of protein expression profiles in Sinorhizobium meliloti: Discovery of new genes for nodule occupancy and stress adaptation

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genomes coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosomes coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

Plant factors induce expression of nodulation and host-range genes in Rhizobium trifolii

SummaryIn Rhizobium trifolii ANU843, host specific nodulation capability is encoded within a 14kb HindIII fragment of the symbiosis plasmid. To gain a better understanding of the regulation of the nodulation (Nod) genes, we have isolated lac operon transcriptional fusions to several genes within this fragment, using the mini-Mu-lac bacteriophage transposon MudI1734. Using a broad-host-range vector, fragments containing MudI1734 insertions were introduced into R. trifolii ANU845, a derivative of ANU843 which lacks the symbiosis plasmid. Four distinct regions were identified within the Nod fragment, insertions in which resulted in nodulation phenotypes similar to those found previously for Tn5. Region I mutants were Nod- and defective in root hair curling (Hac-) and corresponded to the nodABC and D genes identified by sequence analysis. Region II mutants showed an exaggerated root hair curling (Hac++) response on clover plants and a greatly reduced nodulation ability. Region III mutants were affected in host-range properties, as they gained the ability to nodulate Pisum sativum (peas), but showed only poor nodulation ability on the normal host plant, Trifolium repens (white clover). Region IV mutants showed a delay in the nodulation of Trifolium repens, but only when plants were grown under high light-regimes. When ANU845 strains carrying the above MudI1734 insertions were grown in standard laboratory media, only insertions in nodD expressed β-galactosidase at high levels. However, when cells were placed in medium in which Trifolium repens was growing, insertions in nodA, nodB, region II, region III, and region IV were all induced from 5–10 times above basal levels. This allowed us to determine the directions of transcription in these regions.