Phloem translocation of gibberellins in three species of higher plants

Phloem sap was collected from white lupin (Lupinus albus L.), cowpea (Vigna unguiculata L.) and castor bean (Ricinus communis L.) and analysed for gibberellins (GAs) using gas chromatography-mass spectrometry (GC-MS). A large number of GAs were found in the phloem exudate of all three species, particularly where the sap was collected from pods (white lupin and cowpea) and in these legumes GAs representing both the early C-13-hydroxylation and non-hydroxylation pathways of biosynthesis were identified. In the sap collected from the vegetative tissues of castor bean the number of GAs identified was fewer than that in the other species, representing mainly the non-hydroxylation pathway. Data from sap collected from the pedicel and stylar ends of pods and by making feeds of radiolabelled GAs to seeds in situ in white lupin indicate that the GAs present in the phloem are derived mainly from the vegetative tissues of the plant. No evidence for metabolism of GAs in the phloem could be found.     

Free amino Acid composition of leaf exudates and Phloem sap : a comparative study in oats and barley

Comparisons were made between the free amino acid composition in leaf exudates and that in pure phloem sap, using twin samples taken from a single leaf of two oat (Avena sativa L.) and three barley (Hordeum vulgare L.) varieties. Leaf exudate was collected in a 5 mm EDTA-solution (pH 7.0) from cut leaf blades and phloem sap was obtained through excised aphid (Rhopalosiphum padi L.) stylets. Fluorescent derivatives of amino acids were obtained using 9-fluorenylmethyl chloroformate and were separated by means of high performance liquid chromatography. The total concentration of free amino acids varied considerably in the exudate samples. There was no correlation between the total amino acid content in the exudate samples and that of the corresponding phloem sap samples, but the amino acid composition of the corresponding samples was highly correlated (median R²-value 0.848). There was only limited between-plant variation in phloem sap amino acid composition. Nevertheless, in comparisons involving all samples, many of the amino acids showed significant correlations between their relative amounts in exudate and phloem sap. The results presented here indicate that the exudate technique holds great promise as an interesting alternative to the laborious and time-consuming stylet-cutting technique of obtaining samples for comparative studies of phloem sap.     

A combinatory approach for analysis of protein sets in barley sieve-tube samples using EDTA-facilitated exudation and aphid stylectomy

This study investigated advantages and drawbacks of two sieve-tube sap sampling methods for comparison of phloem proteins in powdery mildew-infested vs. non-infested Hordeum vulgare plants. In one approach, sieve tube sap was collected by stylectomy. Aphid stylets were cut and immediately covered with silicon oil to prevent any contamination or modification of exudates. In this way, a maximum of 1muL pure phloem sap could be obtained per hour. Interestingly, after pathogen infection exudation from microcauterized stylets was reduced to less than 40% of control plants, suggesting that powdery mildew induced sieve tube-occlusion mechanisms. In contrast to the laborious stylectomy, facilitated exudation using EDTA to prevent calcium-mediated callose formation is quick and easy with a large volume yield. After two-dimensional (2D) electrophoresis, a digital overlay of the protein sets extracted from EDTA solutions and stylet exudates showed that some major spots were the same with both sampling techniques. However, EDTA exudates also contained large amounts of contaminative proteins of unknown origin. A combinatory approach may be most favourable for studies in which the protein composition of phloem sap is compared between control and pathogen-infected plants. Facilitated exudation may be applied for subtractive identification of differentially expressed proteins by 2D/mass spectrometry, which requires large amounts of protein. A reference gel loaded with pure phloem sap from stylectomy may be useful for confirmation of phloem origin of candidate spots by digital overlay. The method provides a novel opportunity to study differential expression of phloem proteins in monocotyledonous plant species.     

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.     

Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit,leaves and phloem exudate

Abscisic acid (ABA) was identified by combined gas liquid chromatography-mass spectrometry in sieve-tube exudate collected from the cut stylar ends of white lupin fruit. Water stress caused an increase in ABA levels in leaf, seed and pod tissues and phloem exudate. When compared with levels in extracts of these tissues, the concentration of ABA in sieve-tube sap was very high. It is suggested that ABA is actively transported out of mature leaves in the phloem and this finding is discussed in terms of the ABA balance of the plant.Abbreviations ABA abscisic acid - GLC gas liquid chromatography     

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.)

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.     

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     

Xylem sap proteins

Xylem sap from apple (Malus domestica Borkh), peach (Prunus persica Batsch), and pear (Pyrus communis L.) twigs was collected by means of pressure extrusion. This sap contained a number of acidic peroxidases and other proteins. Two other sources of xylem sap used in this study were stem exudates and guttation fluid. Similar peroxidases were also found in stem exudates and guttation fluids of strawberry (Fragaria x ananassa Duch.), tomato (Lycopersicum esculentum L.), and cucumber (Cucumis sativus L.). Isoelectric focusing activity gels showed that two peroxidases (isoelectric point [pl] 9 and pl 4.6) were present in initial stem exudates collected in the first 30 minutes after excision. Subsequent samples of stem exudate collected contained only the pl 4.6 isozyme. The pl 4.6 peroxidase isozyme was also found in root tissue and guttation fluid. These observations suggest that roots produce and secrete the pl 4.6 peroxidase into xylem sap. Cucumber seedlings were treated with 100 microliters per liter ethylene for 16 hours and the exudate from decapitated hypocotyl stumps was collected over a 3 hour period. Ethylene increased the peroxidase activity of stem exudates and inhibited the amount of exudate released. These observations suggest that xylem sap peroxidase may play a role in plugging damaged vascular tissue.     

Synthesis, transport and accumulation of quinolizidine alkaloids in Lupinus albus L. and L. angustifolius L

Each of the principal quinolizidine alkaloids (QA) found in both xylem and phloem exudates together with extracts from all component organs collected from bitter (cv. Lupini) and sweet (cv. Ultra) cultivars of Lupinus albus L. were quantified by gas chromatographic analyses throughout reproductive development. In addition to establishing the major translocated QA species estimates for fluxes of QA to developing fruits based on their sap composition and water economy showed that around half of the QA that accumulated in fruit tissues was due to synthesis in situ and half to translocation principally by phloem. Detailed analyses of QA in transport fluids and component organs were extended to reciprocal homo- and hetero-grafts using bitter (cv. Fest) and sweet (cv. Danja) cultivars of L. angustifolius L. These data confirmed that the majority of QA were synthesized in shoot tissues. In both lupin species feeding and analysis of deuterated QA (lupanine and 13-hydroxylupanine) were used as tracers to demonstrate direct redistribution of alkaloids by translocation from mature leaves in phloem.     

Presence and identification of polyamines in xylem and Phloem exudates of plants

Polyamines were identified by high performance liquid chromatography (benzoylation) and by thin layer chromatography (dansylation) in xylem exudates from stems of sunflowers (Helianthus annuus [L.]), mung bean (Vigna radiata [L.] Wilczek), grapevine (Vitis vinifera [L.] cv Grenache), and orange (Citrus sinensis [L.] Osbeck, cv Valencia), as well as in phloem sap (using elution into EDTA) of sunflower and mung bean plants. Putrescine was the major polyamine detected, ranging in concentrations of 150 to 9200 picomoles per milliliter exudate, whereas only trace amounts of spermine were detected. High amounts of putrescine and spermidine were found in EDTA eluates (possibly phloem sap) as compared with elution into water. Concentrations of putrescine and spermidine in xylem exudates were related to the physiological conditions of the plants prior to exudate collection. More putrescine was found in exudates of older than in younger sunflower plants, and salt stress applied to sunflower plants resulted in a higher concentration of putrescine and spermidine in the exudate. The presence and abundance of putrescine and spermidine in xylem and phloem exudates indicate that polyamines may be translocated in plants. This long-distance translocation further supports the hypothesis that polyamines have a regulatory role in plant growth and response to stress.     

Spontaneous phloem exudation accompanying abscission in Lupinus mutabilis (Sweet)

Like some of the Mediterranean members of the genus Lupinus, the New World lupin, Lupinus mutabilis (Sweet; cv. Inti), exhibits prolonged (20-40 min) exudation of phloem sap from incisions made in stems, in the raceme and at the tips and sutures of developing fruits. Just prior to or immediately following abscission of flowers of L. mutabilis there was also spontaneous exudation from the proximal face of the abscission zone at the base of the pedicel. This is not a recorded feature of other lupins. Analysis of solutes in this exudate was consistent with its having been derived directly from phloem. The major solutes were sucrose (0.940.04 M), amino acids (18811 mM, 45% as asparagine and 15% as glutamine), K ion (52 mM), and total phosphorus (17 mM). Microscopic examination of the proximal face of the pedicel abscission zone at or following abscission showed little or no breakage of the cells at the zone. The major solutes of spontaneous exudate were similar to those in exudates collected from incisions made in the supporting raceme, upper stem and branches, at the tips and sutures of developing fruits and in the mid- and basal stem regions. However, there were significant compositional differences among minor constituents. The spontaneous exudate had a higher level of Ca ion and, consequently, a narrower Mg/Ca ratio (2.8) than exudate from incisions in the adjacent raceme (9.3) or fruits (15.7). There were also higher concentrations of trace elements (Mn, Zn and Cu) but lower concentrations (3 ng m^-1) of cytokinins compared to exudates collected from incisions (20 ng ml^-1). The relative contents of K and Na ions in exudates from incisions at different sites on the plant showed evidence of selective phloem loading and downward translocation of Na ion and selective loading and upward translocation of K ion.     

Comparison of peptides in the phloem sap of flowering and non-flowering Perilla and lupine plants using microbore HPLC followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Physiological evidence indicates that flower formation is hormonally controlled. The floral stimulus, or florigen, is formed in the leaves as a response to an inductive photoperiod and translocated through the phloem to the apical meristem. However, because of difficulties in obtaining and analyzing phloem sap and the lack of a bioassay, the chemical nature of this stimulus is one of the major unsolved problems in plant biology. A combination of microbore high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to compare the contents of the phloem sap from flowering and non-flowering plants. Instead of using one- or two-dimensional gel electrophoresis, microbore HPLC separations allowed us to detect proteins/peptides that were very small and present at very low levels. We detected more than 100 components in the phloem sap of Perilla ocymoides L. and Lupinus albusL. Sequences for 16 peptides in a mass range from 1 to 9 kDa were obtained. Two of these could be identified, 11 showed similarity to known or deduced protein sequences, and three showed no similarity to any known protein or translated gene sequence. Four of these peptides were specific to, modified, or increased in plants that were flowering, indicating their possible role in flower induction. The sequences of these peptides showed similarities to two purine permeases, a protein with similarity to protein kinases, and a protein with no similarities to any known protein.     

Amino Acid Release from the Seed Coat of Developing Seeds of Vicia faba and Pisum sativum

After removal of the embryo from developing ovules of Viciafaba L. and Pisum sativum L., seed-coat exudates were collectedand the amino acid fraction of the exudate was analyzed. InV. faba, alanine was the most important compound of the aminoacid fraction. In P. sativum, alanine and glutamine were thetwo most important components, whereas only small amounts ofasparagine were present. Comparison with published data suggeststhat seed-coat exudates may differ from phloem sap in the relativeimportance of these amino acids. Pisum sativum, pea, Vicia faba, broad bean, amino acid transport, amino acid unloading, seed-coat exudate, seed development     

Phloem sap intricacy and interplay with aphid feeding

Aphididae feed upon the plant sieve elements (SE), where they ingest sugars, nitrogen compounds and other nutrients. For ingestion, aphid stylets penetrate SE, and because of the high hydrostatic pressure in SE, phloem sap exudes out into the stylets. Severing stylets to sample phloem exudates (i.e. stylectomy) has been used extensively for the study of phloem contents. Alternative sampling techniques are spontaneous exudation upon wounding that only works in a few plant species, and the popular EDTA-facilitated exudation technique. These approaches have allowed fundamental advances on the understanding of phloem sap composition and sieve tube physiology, which are surveyed in this review. A more complete picture of metabolites, ions, proteins and RNAs present in phloem sap is now available, which has provided large evidence for the phloem role as a signalling network in addition to its primary role in partitioning of photo-assimilates. Thus, phloem sap sampling methods can have remarkable applications to analyse plant nutrition, physiology and defence responses. Since aphid behaviour is suspected to be affected by phloem sap quality, attempts to manipulate phloem sap content were recently undertaken based on deregulation in mutant plants of genes controlling amino acid or sugar content of phloem sap. This opens up new strategies to control aphid settlement on a plant host.     

Identification of Zn-nicotianamine and Fe-2'-Deoxymugineic acid in the phloem sap from rice plants (Oryza sativa L.)

In higher plants, the supply of metals such as Zn and Fe via phloem is important for the growth and physiology of young organs. However, little information is available on the speciation (chemical forms) of these metals in the phloem fluids. Because the pH of phloem fluids is slightly alkaline and the concentration of phosphate, which may bind to metals, is high, Zn and Fe in phloem fluids could be precipitated if these metals do not form complexes with some ligand compounds. In the present experiment, we examined the chemical forms of Zn and Fe in phloem sap collected from rice (Oryza sativa L.) by separating the phloem sap using size-exclusion and anion-exchange chromatography, and identifying the contents using electrospray ionization time-of-flight mass spectrometry. The low molecular weight chemical forms of Zn and Fe were identified as Zn-nicotianamine and Fe(III)-2'-deoxymugineic acid complexes, respectively. This report is the first to identify metal-chelate complexes in rice phloem sap.     

Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.)

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C₁₈ reversed-phase high performance liquid chromatography → NH₂ normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.