A comparative analysis of phloem exudate proteins from Cucumis melo,Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing

Summary Proteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.Abbreviations DTT dithiothreitol - Bis N,N-methylenebisacrylamide - SDS Sodium dodecyl sulphate - TCA trichloroacetic acid     

The heterogeneity of phloem exudate proteins from different plants: A comparative survey of ten plants using polyacrylamide gel electrophoresis

Summary Proteins in sieve tube exudate from Ricinus communis L., Acer pseudoplatanus L., Aesculus hippocastanum L., Cucumis melo L., and two cultivars each of Cucumis sativus L., Cucurbita pepo L. and Cucurbita maxima Duchesne were fractionated and compared using polyacrylamide gel electrophoresis. Striking differences in major exudate proteins were displayed among the genera and species examined. Even cultivars within a single species, although showing general similarities, differed in some prominent proteins. Estimated molecular weights of the major exudate proteins from each plant are presented. The effects of reducing and chaotropic agents on the aggregation and subunit composition of exudate proteins from Cucumis sativus have been investigated. The problems involved in relating structure, function and biochemistry of P-protein are discussed.     

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

Mass determination of low-molecular-weight proteins in phloem sap using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The low-molecular-weight (LMW), low-abundance protein composition of lupin and pea phloem exudates was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)> Phloem sap was collected from lupin inflorescence stalks and pods (using shallow incisions) or pea seedlings (by placing cut stems in an EDTA solution). Western blot analysis of phloem exudate proteins with either a polyclonal antibody raised against Ricinus communis sieve-tube exudate proteins or pea Rubisco antibody revealed that the collected exudates contained phloem sap, and that contamination with other plant fluids was negligible. Three matrix combinations were tested to assess their ability to facilitate protein ionization. Sinapinic acid in combination with trifluoroacetic acid yielded the cleanest mass spectra, and revealed an array of LMW proteins ranging from 2 to 10 kDa. For pea phloem exudate, the addition of protease inhibitors to the exudate collection solution prevented proteolysis of endogenous proteins; the inhibitors did not interfere with the detection of proteins. The sensitivity of this technique was sufficient to detect changes in LMW phloem peptides throughout plant development in lupin, or to detect differences in the phloem peptide composition of two genotypes of pea. Because only limited sample preparation is required, MALDI-TOF-MS is a useful technique for characterizing complex fluids such as phloem sap.     

Determination of phytohormones in phloem exudate from tree species by radioimmunoassay

A sensitive and specific radioimmunoassay was used to determine quantitatively four of the most important phytohormones in the phloem exudate from 14 different tree species of 8 genera. For cytokinins and indole-3-acetic acid (IAA) we found higher concentrations than those reported previously for other species. The gibberellin values were of the same order of magnitude as in earlier analyses (with different methods) of tree phloem exudates, but lower than the ones reported for Ricinus. Free abscisic acid (ABA) was found in tree phloem exudates in similar concentrations as before in Yucca or palm phloem exudate, but at considerably lower ones than reported for Ricinus and in Lupinus phloem exudate.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA gibberellic acid     

Specific proteins in the sieve-tube exudate of Ricinus commuais L. seedlings: separation,characterization and in-vivo labelling

Ricinus communis L. seedlings exuded pure phloem sap from the cut hypocotyl for several hours. Throughout the entire exudation period proteins were present in the phloem exudate at a constant concentration ranging from 0.11 to 0.41 mg·ml^–1 depending on the culture conditions and the age of the seedlings. Manipulation of the nutrient supply at the cotyledons after removal of the endosperm did not change the protein concentration in the exudate. Comparison of sieve-tube exudate proteins (STEPs) with soluble proteins extracted from the hypocotyl and the cotyledons showed a unique abundance of small proteins in the exudate, with molecular weights ranging from 10 to 25 kDa. Bands at 18, 19 and 20 kDa were especially dominant. The proteins found transiently in the xylem exudate, which might represent proteins secreted at the wound surface, were different in pattern. Two-dimensional separation of STEPs revealed that more than 100 distinct polypeptides occurred in the sieve-tube exudate, most of them slightly acidic with isoelectric points ranging from 4 to 6 and a few basic ones around 8. [³⁵S]Methionine fed to the cotyledons led to labelling of STEPs, demonstrating their rapid synthesis. It is concluded that there is a continuous synthesis and translocation of specific sieve-tube proteins, whose function is unknown.Abbreviations IEF isoelectric focussing - pI isoelectric point - STEP sieve-tube exudate protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid We wish to thank Pia Großmann and Libuse Badewitz for technical help.     

Characterization of Ricinus communis phloem profilin,RcPRO1

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.     

Comparison of gibberellin-like substances from cotyledons and phloem exudate collected from cotyledons of Pharbitis nil in relation to photoperiodic flowering

Gibberellin (GA)-like substances were analyzed in extracts from cotyledons and phloem exudate collected from cotyledons in photoinduced and vegetative seedlings of the short-day plant Pharbitis nil Chois. var. Violet, using high performance liquid chromatography (HPLC) and the dwarf rice bioassay, to see whether any specific GA-like substances were transported from the photoinduced cotyledons via phloem. Cotyledon extracts exhibited five peaks of free GA-like activity in HPLC, whereas only one or two active peaks were detected in phloem exudate extracts. The level of free GA-like activity was considerably lower in phloem exudate than in the cotyledons. In five out of six analyses of cotyledons and phloem exudate, there were substantially higher levels of free GA-like substances in photoinduced plants. Conjugated GA-like substances were present in much higher levels than free GA-like substances in the cotyledon extracts but the levels were not influenced by daylength. In phloem exudate extracts there was no conjugated GA-like substances. The free GA-like substances that are transported via phloem cochromatographed with GA_5/20 and GA₁₉ on HPLC. These were significantly higher in photoinduced plants and thus could have some influence on the photoperiodically-induced flowering in P. nil.     

Towards the proteome of Brassica napus phloem sap

The soluble proteins in sieve tube exudate from Brassica napus plants were systematically analyzed by 1-DE and high-resolution 2-DE, partial amino acid sequence determination by MS/MS, followed by database searches. 140 proteins could be identified by their high similarity to database sequences (135 from 2-DE, 5 additional from 1-DE). Most analyzed spots led to successful protein identifications, demonstrating that Brassica napus, a close relative of Arabidopsis thaliana, is a highly suitable model plant for phloem research. None of the identified proteins was formerly known to be present in Brassica napus phloem, but several proteins have been described in phloem sap of other species. The data, which is discussed with respect to possible physiological importance of the proteins in the phloem, further confirms and substantially extends earlier findings and uncovers the presence of new protein functions in the vascular system. For example, we found several formerly unknown phloem proteins that are potentially involved in signal generation and transport, e.g., proteins mediating calcium and G-protein signaling, a set of RNA-binding proteins, and FLOWERING LOCUS T (FT) and its twin sister that might be key components for the regulation of flowering time.     

Calcium-binding protein in sieve tube exudate

A calcium-binding macromolecule, with an estimated molecular weight greater than 100,000, was detected in phloem exudate from Cucurbita maxima and related species. The macromolecule was a component of sieve tube sap, rather than a contaminant leached from cell walls or cut parenchyma cells during exudate collection. The protein nature of this macromolecule was deduced from its size, lability, susceptibility to proteolytic digestion, and by the dependence of calcium-binding activity on thiol-protecting agents. This protein is distinct from the major proteins of exudate and does not appear to be related to calmodulin.Abbreviations SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - CBP calcium-binding protein     

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.     

Phloem‐specific resistance in Brassica oleracea against the whitefly Aleyrodes proletella

The cabbage whitefly [Aleyrodes proletella L. (Hemiptera: Aleyrodidae)] is becoming a serious pest in Brassica oleracea L. (Brassicaceae) crops. However, almost nothing is known about the interaction of this insect with its host plants. Previous studies have shown differences in the natural occurrence of adults, eggs, and nymphs on the closely related B. oleracea cultivars Christmas Drumhead and Riviera grown in the field. In this study, we aimed to identify the nature of these differences and to gain insight into the resistance mechanisms against A. proletella. We used no‐choice experiments on field‐ and greenhouse‐grown plants to show that the differences between the two cultivars are mainly based on antibiosis (traits that reduce herbivore performance) and not on antixenosis (traits that deter herbivory). This was further supported by laboratory choice experiments that indicated little or no discrimination between the two cultivars based on plant volatiles. We showed that resistance is dependent on plant age, that is, resistance increased during plant development, and is mainly independent of environmental factors. Analysis of probing behaviour revealed that the resistance trait affects A. proletella at the phloem level and that morphological differences between the two cultivars are most likely not involved. We suggest that compounds present in the phloem reduce sap ingestion by the whitefly and that this explains the observed resistance.     

Movement of protein and macromolecules between host plants and the parasitic weed Phelipanche aegyptiaca Pers.

Little is known about the translocation of proteins and other macromolecules from a host plant to the parasitic weed Phelipanche spp. Long-distance movement of proteins between host and parasite was explored using transgenic tomato plants expressing green fluorescent protein (GFP) in their companion cells. We further used fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite. Accumulation of GFP was observed in the central vascular bundle of leaves and in the root phloem of transgenic tomato plants expressing GFP under the regulation of AtSUC2 promoter. When transgenic tomato plants expressing GFP were parasitized with P. aegyptiaca, extensive GFP was translocated from the host phloem to the parasite phloem and accumulated in both Phelipanche tubercles and shoots. No movement of GFP to the parasite was observed when tobacco plants expressing GFP targeted to the ER were parasitized with P. aegyptiaca. Experiments using fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite demonstrated that Phelipanche absorbs dextrans up to 70 kDa in size from the host and that this movement can be bi-directional. In the present study, we prove for the first time delivery of proteins from host to the parasitic weed P. aegyptiaca via phloem connections, providing information for developing parasite resistance strategies.     

How nutritionally imbalanced is phloem sap for aphids?

Aphids harbour intracellular symbionts (Buchnera) that provide their host with amino acids present in low amounts in their diet, phloem sap. To find out the extent to which aphids depend on their symbionts for synthesis of individual essential amino acids, we have evaluated how well phloem sap amino acid composition matches the aphids' needs. Amino acid compositions of the ingested phloem sap were compared to amino acids in aphid body proteins and also to available information about optimal diet composition for other plant feeding insects. Phloem sap data from severed stylets of two aphid species, Rhopalosiphum padi (L.) (Homoptera: Aphididae) feeding on wheat, and Uroleucon sonchi (L.) (Homoptera: Aphididae) feeding on Sonchus oleraceus (L.), together with published information on phloem sap compositions from other plant species were used.Phloem sap has in general only around 20% essential amino acids, with a range from 15–48%. Aphid body proteins and optimal diets for two other plant feeding insects have around 50%. The phloem sap of early flowering S. oleraceus ingested by U. sonchi contained 48%, which seems to be exceptional. Aphids feeding on different plants appear to be very differently dependent on their symbionts for their overall essential amino acid synthesis, due to the large variation in proportion of essential amino acids in phloem sap from different plants.The profile of the essential amino acids in phloem sap from different plant species corresponds rather well to profiles of both aphid body proteins and optimal diets determined for plant feeding insects. However, methionine and leucine in phloem sap are in general low in these comparisons, suggesting a higher dependence on the symbiont for synthesis of these amino acids. Concentrations of several essential amino acids in phloem from different plant species seem to vary together, suggesting that levels of symbiont provisioning of different amino acids are adjusted in parallel.     

Actin in Pumpkin Phloem Exudate

When analyzing cytoskeletal proteins in Cucurbita pepo phloem exudate by immunoblotting, we detected actin in an amount comparable to that in some plant tissues and a small amount of -tubulin. Electron-microscopic examination of the exudate permitted us to observe filaments that were capable of interacting with the myosin subfragment S1 from rabbit skeletal muscle and with phalloidin conjugated with colloidal gold. The addition of 0.5 mM phalloidin to the exudate in the medium containing 20 mM dithiothreitol (DTT) resulted in an increased number of filaments. Since high DTT concentrations induce a breakdown of filaments of the phloem protein PP1, it seems likely that the produced filaments were composed of actin. The addition of 50 mM MgCl₂ to the exudate resulted in the formation of dense bundles and paracrystals, which resembled those produced by muscle actin under similar conditions. Our results demonstrated that actin in phloem sap was capable of polymerization with filament formation.     

Endophytic bacterial and fungal microbiota in different cultivars of cassava (Manihot esculenta Crantz)

Endophytes colonize tissues of healthy host plants and play a crucial role in plant growth and development. However, little attention has been paid to the endophytes of tuber crops such as cassava, which is used as a staple food by approximately 800 million people worldwide. This study aimed to elucidate the diversity and composition of endophytic bacterial and fungal communities in different cassava cultivars using high-throughput sequencing. Although no significant differences in richness or diversity were observed among the different cassava cultivars, the community compositions were diverse. Two cultivars (SC124 and SC205) tolerant to root rot exhibited similar community compositions, while two other cultivars (SC10 and SC5), which are moderately and highly susceptible to root rot, respectively, harboured similar community compositions. Proteobacteria, Firmicutes, and Ascomycota dominated the endophyte assemblages, with Weissella, Serratia, Lasiodiplodia, Fusarium, and Diaporthe being the predominant genera. The differentially abundant taxonomic clades between the tolerant and susceptible cultivars were mainly rare taxa, such as Lachnoclostridium_5, Rhizobium, Lampropedia, and Stenotrophomonas. These seemed to be key genera that affected the susceptibility of cassava to root rot. Moreover, the comparison of KEGG functional profiles revealed that ‘Environmental adaptation’ category was significantly enriched in the tolerant cultivars, while ‘Infectious diseases: Parasitic’ category was significantly enriched in the susceptible cultivars. The present findings open opportunities for further studies on the roles of endophytes in the susceptibility of plants to diseases.

     

Comparative proteomics of cucurbit phloem indicates both unique and shared sets of proteins

Cucurbits are well‐studied models for phloem biology but unusually possess both fascicular phloem (FP) within vascular bundles and additional extrafascicular phloem (EFP). Although the functional differences between the two systems are not yet clear, sugar analysis and limited protein profiling have established that FP and EFP have divergent compositions. Here we report a detailed comparative proteomics study of FP and EFP in two cucurbits, pumpkin and cucumber. We re‐examined the sites of exudation by video microscopy, and confirmed that in both species, the spontaneous exudate following tissue cutting derives almost exclusively from EFP. Comparative gel electrophoresis and mass spectrometry‐based proteomics of exudates, sieve element contents and microdissected stem tissues established that EFP and FP profiles are highly dissimilar, and that there are also species differences. Searches against cucurbit databases enabled identification of more than 300 FP proteins from each species. Few of the detected proteins (about 10%) were shared between the sieve element contents of FP and EFP, and enriched Gene Ontology categories also differed. To explore quantitative differences in the proteomes, we developed multiple reaction monitoring methods for cucumber proteins that are representative markers for FP or EFP and assessed exudate composition at different times after tissue cutting. Based on failure to detect FP markers in exudate samples, we conclude that FP is blocked very rapidly and therefore makes a minimal contribution to the exudates. Overall, the highly divergent contents of FP and EFP indicate that they are substantially independent vascular compartments.     

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.     

A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L

The transport of metal micronutrients to developing organs in a plant is mediated primarily by the sieve elements. Ligands are thought to form complexes with the free ions in order to prevent cellular damage, but no binding partners have been unequivocally identified from plants so far. This study has used the phloem-mediated transport of micronutrients during the germination of the castor bean seedling to identify an iron transport protein (ITP). It is demonstrated that essentially all (55)Fe fed to seedlings is associated with the protein fraction of phloem exudate. It is shown that ITP carries iron in vivo and binds additional iron in vitro. ITP was purified to homogeneity from minute amounts of phloem exudate using immobilized metal ion affinity chromatography. It preferentially binds to Fe(3+) but not to Fe(2+) and also complexes Cu(2+), Zn(2+), and Mn(2+) in vitro. The corresponding cDNA of ITP was cloned using internal peptide fragments. The deduced protein of 96 amino acids shows high similarity to the stress-related family of late embryogenesis abundant proteins. Its predicted characteristics and its RNA expression pattern are consistent with a function in metal ion binding. The ITP from Ricinus provides the first identified micronutrient binding partner for phloem-mediated long distance transport in plants and is the first member of the late embryogenesis abundant protein family shown to have such a function.