Metal speciation in xylem and phloem exudates

Summary Two computer programs based on simultaneous chemical equilibria were compared for calculation of chemical species in xylem exudates. The first program, CHELATE, was developed to calculate the chemical species in xylem exudates while GEOCHEM was developed to calculate the speciation of natural aquatic systems. The output of the two programs should be similar since they are based on similar calculations. Data input to the programs consisted of concentration data for Ca, Cu, Fe, Mg, Mn, Zn, NH₄, PO₄, pH and 28 organic ligands reported for xylem exudates from soybean (Glycine max (L.) Merr.) and tomato (Lycopersicon esculentum Mill.) plants grown in nutrient solution¹⁷. The organic ligands included amino acids and low molecular weight organic acids (e.g., citric and malic). With the exception of Fe, there were large differences between CHELATE and GEOCHEM in the calculated speciation of nearly all metals in the xylem exudates. In general, there was better agreement between the programs for the speciation of alkaline earth metals than for transition metals. Discrepancies between the two programs were attributed to differences in 1) species considered and 2) stability constants. GEOCHEM considered a greater number of possible complexes. In addition, stability constants for some complexes differed by as much as 10 fold between the two programs. When the data base for GEOCHEM and CHELATE were the same, the output from CHELATE and GEOCHEM was almost identical. Thus, computations performed by the two programs are equally valid, but it is essential that the data base used in chemical models be verified before interpreting the output. Average concentration data for Al, Au, Ca, Cu, Fe, K, La, Mg, Mn, Na, Rb, Zn, Cl, MoO₄, PO₄, SO₄, HVO₄, pH and 18 organic ligands in phloem exudates from Yucca (Yucca flaccida Haw.) were complied from available literature and analyzed by GEOCHEM. Amino acids were the predominant organic ligand analyzed. Calculations revealed that alkali metals existed almost totally as the free ionic species (≥99%) whereas alkaline earth metals were transported as complexes with organic acids (oxalic, malic, and asparagine). Aluminum and Fe were present as hydroxyl species while <1% of micronutrients were transported as the free ion. Major micronutrient species were Cu-glutamine, Mn-asparagine and Zn-alanine. Information on calculated species present in phloem exudates could be useful to guide studies for isolation of metal-ligand complexes in phloem exudates.     

Highly sensitive immunoadsorption procedure for detection of low-abundance proteins

A procedure that virtually eliminates nonspecific adsorption of radiolabeled proteins during immunoprecipitation was devised utilizing staphylococcal cells containing protein A (Staph A). Immunoprecipitates (antigen-antibody complexes) were solubilized from Staph A pellets into detergent micelles by incubation in a small volume of 1% sodium dodecyl sulfate (SDS) at 23 degrees C for 10 min. To allow re-formation of immunocomplexes and rebinding to new Staph A, the SDS-solubilized material was diluted 20-fold in buffer containing 1% Triton X-100 and 0.5% sodium deoxycholate. Specific conductance measurements revealed that this solubilization and subsequent reimmunoadsorption of antibody-antigen complexes occur at SDS concentrations that are first above and then below its critical micelle concentration. This procedure lowered the nonspecific background from approximately 2250 parts per million (ppm) to less than 25 ppm with a final recovery of 30-50% depending on the antigen and antibody. Chaotropic agents such as 2 M urea, 0.2 M KOH, and 3.5 M MgCl2 (as well as combinations of urea and SDS) can substitute for 1% SDS, although the final recovery is somewhat lower. Fluorography of radiolabeled proteins obtained in this manner displays virtually undetectable background even for exposures as long as 2 months. These methods allowed the unambiguous detection of low-abundance antigens at a high level of sensitivity, for example, mouse mammary tumor virus protein products and epidermal growth factor receptor.     

Subunit structure and interactions of the phloem proteins of Cucurbita maxima (pumpkin)

The two major proteins from the phloem exudate of Cucurbita maxima (pumpkin), PP1 and PP2, were stable in the absence of reducing agents after modification of their accessible cysteine residues with iodoacetamide. This permitted their purification without precautions to prevent oxidation. PP2, a lectin specific for oligomers of N-acetyl-D-glucosamine, was shown by sedimentation-equilibrium ultracentrifugation to be a dimer of Mr of 48000. Neither dithiothreitol nor tri-(N-acetyl-D-glucosamine) altered this value. The constituent polypeptides were linked by two buried disulphide bridges. PP2 behaved aberrantly on gel-filtration on both Sephadex and Bio-Gel unless tri-(N-acetyl-D-glucosamine) was added to the elution buffer; the Mr was then measured as 46000. Other proteins which bind oligomers of N-acetyl-D-glucosamine are also retarded on gel-filtration. Soluble phloem filaments were prepared by collection of exudate into deaerated buffer containing iodoacetamide but no reducing agent. Oxidative gellation of the filaments was prevented by rapid modification of their many accessible cysteine residues, and is assumed to have maintained the degree of polymerisation found in vivo. Those disulphide bridges which were present allowed the incorporation of approximately 60% of the PP1 and 80% of the PP2 into polymeric material. It is concluded that PP1 and PP2 are both structural proteins present in the filaments observable in vivo. PP2 had an elongated binding-site for oligomers of N-acetyl-D-glucosamine. It is suggested that this lectin immobilises bacteria and fungi to the cross-linked filaments which seal wounded phloem sieve-tubes, and thus maintains sterility.     

N content of phloem and xylem exudates during the transition to flowering in Sinapis alba and Arabidopsis thaliana

The involvement of nitrogenous substances in the transition to flowering was investigated in Sinapis alba and Arabidopsis thaliana (Columbia). Both species grown in short days (SD) are induced to flower by one long day (LD). In S. alba, the phloem sap (leaf and apical exudates) and the xylem sap (root exudate) were analysed in LD versus SD. In A. thaliana, only the leaf exudate could be analysed but an alternative system for inducing flowering without day‐length extension was used: the displaced SD (DSD). Significant results are: (i) in both species, the leaf exudate was enriched in Gln during the inductive LD, at a time compatible with export of the floral stimulus; (ii) in S. alba, the root export of amino acids decreased in LD, whereas the nitrate remained unchanged – thus the extra‐Gln found in the leaf exudate should originate from the leaves; (iii) extra‐Gln was also found very early in the apical exudate of S. alba in LD, together with more Glu; (iv) in A. thaliana induced by one DSD, the leaf export of Asn increased sharply, instead of Gln in LD. This agrees with Asn prevalence in C‐limited plants. The putative role of amino acids in the transition to flowering is discussed.     

Determination of choline in phloem exudates of trees

Summary Choline, a necessary compound in an artificial diet for phloem-feeding aphids, was determined quantitatively in the phloem exudates of 16 tree species. The method used was a combination of choline oxidase action and oxygen determination with an oxygen electrode. Choline was found in all species, the concentration ranging between 36 and 5340 M.     

Protein profile of Lupinus texensis phloem sap exudates: Searching for Fe‐ and Zn‐containing proteins

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI‐MS and ESI‐MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19–21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe‐containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe‐binding proteins in phloem sap: a metallothionein‐like protein type 2B identified in the Fe‐affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem‐specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn‐binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.     

Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.     

Long-distance phloem transport of glucosinolates in Arabidopsis

Glucosinolates are a large group of plant secondary metabolites found mainly in the order Capparales, which includes a large number of economically important Brassica crops and the model plant Arabidopsis. In the present study, several lines of evidence are provided for phloem transport of glucosinolates in Arabidopsis. When radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) and sucrose were co-applied to the tip of detached leaves, both tracers were collected in the phloem exudates at the petioles. Long-distance transport of [(14)C]p-OHBG was investigated in wild-type and transgenic 35S::CYP79A1 plants, synthesizing high amounts of p-OHBG, which is not a natural constituent of wild-type Arabidopsis. In both wild-type and 35S::CYP79A1 plants, radiolabeled p-OHBG was rapidly transported from the application site into the whole plant and intact p-OHBG was recovered from different tissues. The pattern of distribution of the radioactivity corresponded to that expected for transport of photoassimilates such as sucrose, and was consistent with translocation in phloem following the source-sink relationship. Radiolabeled p-OHBG was shown to accumulate in the seeds of wild-type and 35S::CYP79A1 plants, where p-OHBG had been either exogenously applied or endogenously synthesized from Tyr in the leaves. p-OHBG was found in phloem exudates collected from cut petioles of leaves from both wild-type and 35S::CYP79A1 plants. Phloem exudates were shown to contain intact glucosinolates, and not desulphoglucosinolates, as the transport form. It is concluded that intact glucosinolates are readily loaded into and transported by the phloem.     

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta , were analysed by two-dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20-day-old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa , and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta . Crosses were made between this line and the wild type Landsberg erecta . F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.     

Elimination of affinity reagent interference for the mass spectrometric detection of low-abundance proteins following immunoprecipitation

The presence of affinity reagents such as immunoglobulin in preparations for sensitive mass spectrometry analyses can preclude the identification of low-abundance proteins of interest. We report a method whereby antisera are purified and biotinylated prior to use in immunoprecipitation that allows for its efficient removal from proteomic samples via streptavidin capture. This method can similarly be extended to other affinity reagents such as recombinant fusion proteins for enhanced identification of interacting proteins.     

The compounds in Aloë leaf exudates: a review

The drug Bitter Aloes has been used for several thousands of years because of its purgative properties and later several species of the plant have been cultivated for horticultural interest. In addition, many more local therapeutic properties have been recorded. Research has been largely directed towards the physiologically active components during the 50s and 60s. Extensions to this work in the last decade have been made in Germany, Japan and South Africa. Compounds have been characterized from the dried exudate prepared for drug purposes and from a few species. A number of anthraquinones have been recognized both in the free state and as glycosides. Other compounds with phenolic groups include chromones and a phenyl-pyranone. Chromatographic methods have been developed for recognizing some of these compounds and estimating the levels of those of pharmacological interest. Distribution of some of the compounds among a number of species is described showing that among some groups it has taxonomic significance. The biology of secretion and exudation from Aloë leaves remains largely unknown although fragmentary evidence points to interesting and potentially useful activity in some of the components.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.     

Studies onArtemisia afra Jacq.: The phloem in stem and leaf

Summary The structure of the phloem was studied in stem and leaf ofArtemisia afra Jacq., with particular attention being given to the sieve element walls. Both primary and secondary sieve elements of stem and midvein have nacreous walls, which persist in mature cells. Histochemical tests indicated that the sieve element wall layers contained some pectin. Sieve element wall layers lack lignin. Sieve elements of the minor veins (secondary and tertiary veins) lack nacreous thickening, although their walls may be relatively thick. These walls and those of contiguous transfer cells are rich in pectic substances. Transfer cell wall ingrowths are more highly developed in tertiary than in secondary veins.     

The Arabidopsis thaliana natriuretic peptide AtPNP-A is a systemic regulator of leaf dark respiration and signals via the phloem

Plant natriuretic peptides (PNPs) belong to a novel class of peptidic signaling molecules that share some structural similarity to the N-terminal domain of expansins and affect physiological processes such as water and ion homeostasis at nano-molar concentrations. Here we show that a recombinant Arabidopsis thaliana PNP (AtPNP-A) rapidly increased the rate of dark respiration in treated leaves after 5 min. In addition, we observed increases in lower leaves, and with a lag time of 10 min, the effect spread to the upper leaves and subsequently (after 15 min) to the opposite leaves. This response signature is indicative of phloem mobility of the signal, a hypothesis that was further strengthened by the fact that cold girdling, which affects phloem but not xylem or apoplastic processes, delayed the long distance AtPNP-A effect. We conclude that locally applied AtPNP-A can induce a phloem-mobile signal that rapidly modifies plant homeostasis in distal parts.     

Purification of low-abundance Arabidopsis plasma-membrane protein complexes and identification of candidate components

Purification of low-abundance plasma-membrane (PM) protein complexes is a challenging task. We devised a tandem affinity purification tag termed the HPB tag, which contains the biotin carboxyl carrier protein domain (BCCD) of Arabidopsis 3-methylcrotonal CoA carboxylase. The BCCD is biotinylated in vivo , and the tagged protein can be captured by streptavidin beads. All five C-terminally tagged Arabidopsis proteins tested, including four PM proteins, were functional and biotinylated with high efficiency in Arabidopsis. Transgenic Arabidopsis plants expressing an HPB-tagged protein, RPS2::HPB, were used to develop a method to purify protein complexes containing the HPB-tagged protein. RPS2 is a membrane-associated disease resistance protein of low abundance. The purification method involves microsomal fractionation, chemical cross-linking, solubilization, and one-step affinity purification using magnetic streptavidin beads, followed by protein identification using LC-MS/MS. We identified RIN4, a known RPS2 interactor, as well as other potential components of the RPS2 complex(es). Thus, the HPB tag method is suitable for the purification of low-abundance PM protein complexes.     

Effect of root exudates and extracts on rhizosphere fungi

Summary The effects of root exudates and extracts of Cassia tora L. and Crotalaria medicaginea Lamk., on some dominant rhizosphere fungi isolated from the plants were studied. Root extracts induced a high degree of stimulation in rate of growth of a majority of the fungi tested. Root exudates caused only a marginal effect which was positive on all the fungi except Trichoderma lignorum which was inhibited by the root exudate of C. medicaginea.     

A sensitive and high-throughput assay to detect low-abundance proteins in serum

The ability to detect antigens immunologically is limited by the affinity of the antibodies and the amount of antigens. We have now succeeded in creating a modular, facile amplification system, termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). Such a system can detect protein targets specifically at subfemtomolar levels ( approximately 0.08 fM). We describe here the detection of Her2 (also known as Neu) from rodent and human sera. FACTT is adaptable to high-throughput screening and automation and provides a practical method to enhance current ELISAs in medical practice.