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.     

Biochemical Changes in Root Exudate and Xylem Sap of Tomato Plants Infected with Meloidogyne incognita

Under two monoxenic culture techniques of growing plants (filter paper and silica sand cultures), sugar in root exudate from Meloidogyne incognita-infected tomato increased 133 to 836% over controls. In contrast, amino acids were moderately reduced 52 to 56%. Chromatographic analysis showed that galled root exudate contained three sugars, twelve amino acids, and three organic acids, whereas healthy root exudate contained four sugars, fifteen amino acids, and four organic acids. Polysaccharide was responsible for the large increase of sugars in galled root exudates. The concn and the absolute amount of total sugars in the infected plant xylem sap were greater than in healthy plant xylem sap up to 6 wk after inoculation, whereas amino acids were moderately lower than in controls throughout the test period. Chromatographic analysis showed that xylem sap from both healthy and infected plants at 4 wk after inoculation contained four sugars and five organic acids. We identified 18 and 17 amino acids in the healthy and infected plant xylem sap, respectively. The concn of sugar increased as the nematode inoculum increased at 2, 4 and 6 wk after inoculation. The amino acids in all samples from the infected plant moderately decreased with an increase of nematode inoculum. We suggest that changes in total sugars and amino acids, of infected plant xylem sap and root exudate are a probable mechanism by which tomato plants are predisposed to Fusarium wilt.     

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.     

Composition of Amino Compounds Transported in Xylem of Casuarina sp.

Seedlings (180-d-old) of Casuarina cunninghamianaM L., C. equisetifoliaMiq. and C. glauca Sieber inoculated with each of two differentsources of Frankia, were analysed for translocated nitrogenouscompounds in xylem sap. Analyses were also made on sap fromnodulated and non-nodulated plants of C. glauca grown with orwithout a range of levels of combined nitrogen. Xylem exudateswere collected from stems, roots, and individual nodules ofnodulated plants and from stems and roots of non-nodulated plants.While the proportional composition of solutes varied, the samerange of amino compounds was found in xylem sap from the threedifferent symbioses. In C. glauca asparagine was the major aminoacid in the root sap followed by proline, while in symbioticC. cunninghamiana arginine accounted for more than 25% of theamino compounds. Citrulline was the major translocated productfound in the stem exudate of symbiotic C. equisetifolia. Increasingconcentrations of ammonium nitrate in the nutrient solutionresulted in increasing levels of free ammonia and glutaminein xylem sap from stems of nodulated and non-nodulated C. glauca,but there was relatively little change in the prominent solutes,e.g. citrulline, proline, and arginine. The composition of nitrogenoussolutes in stem or root exudates of C. glauca was similar tothat of exudate collected from individual nodules and on thisbasis it was not possible to distinguish specific products ofcurrent N₂ fixation in xylem. The main differences in N solutecomposition between the symbioses were apparently due to hostplant effects rather than nodulation or the levels of combinedN. Also, the data indicate that the use of the proportion ofN in sap as citrulline (or indeed any other organic N solute)could not be used as an index of nitrogen fixation.     

The Composition of Phloem Exudate and Xylem Sap from Tree Tobacco (Nicotiana glauca Grah.)

The composition of xylem sap and exudate from stem incisionsof Nicotiana glauca Grah. was compared in detail. Exudationfrom stem incisions occurred over a 5 min period in certainplants, enabling collection of 5–30 µl of sap. Therate of exudation showed an exponential decline. Exudate hada high dry matter content (170–196 mg ml^–1) andhigh sugar (sucrose) levels. Xylem sap had a low pH (5.8) andexudate a pH of 7.9. Glutamine dominated the amino compoundsin xylem sap and exudate, and K⁺ was the major cation. Totalamino compounds in stem exudate reached 10.8 mg ml^–1 whereasxylem sap contained much lower levels (0.28 mg ml^–1).All mineral elements and amino compounds with the exceptionof calcium were more concentrated in stem exudate than in xylemsap. Sucrose was labelled heavily in stem exudate following pulsingof an adjacent leaf with ¹⁴CO₂. A concentration gradient ofsugar (2.1 bar m^–1) was recorded for stems. Levels ofsucrose, amino compounds and K⁺ ions in stem exudate showeda diurnal periodicity. Each commodity reached maximum concentrationat or near noon and minimum concentration about dawn. The evidencesuggests that exudate from stem incisions of N. glauca is arepresentative sample of solutes translocated in the phloem. Nicotiana glauca Grah., phloem sap, xylem sap, sucrose, amino compounds, mineral ions     

Changes in the Nitrogen Compounds of Xylem Sap of Mulberry (Morus alba L.) during Regrowth after Pruning

Changes in fresh and dry weights and total nitrogen in stemsections of mulberry (Morus alba L.) and seasonal fluctuationin the amounts of exudate, pH and nitrogen compounds in xylemsap from cut stems of the plant after pruning have been studied.The amount and concentrations of nitrogen compounds in the sapchanged during the experimental period, but nitrate-nitrogenand ammonia-nitrogen were constant constituents, and asparaginewas quantitatively the major organic nitrogen compound. Smalleramounts of glutamine, aspartic acid and glutamic acid were alsofound, but no ureides or alkaloids were detected. Relationshipsbetween growth and nitrogen in xylem sap of mulberry and therole and importance of asparagine in nitrogen metabolism arediscussed. Morus alba L., mulberry, asparagine, exudates, nitrogen, amino-acids, xylem sap, stem growth, aspartic acid, glutamic acid, ureides     

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.     

Metal Complexation in Xylem Fluid : I. CHEMICAL COMPOSITION OF TOMATO AND SOYBEAN STEM EXUDATE

Xylem fluid was analyzed for numerous solutes to characterize chemically the sap as a medium for forming and transporting metal complexes. The stem exudate was collected hourly for 8 hours from topped 31-day-old soybean (Glycine max L. Merr.) and 46-day-old tomato (Lycopersicon esculentum Mill.) plants grown in normal (0.5 micromolar) and Za-phytotoxic nutrient solutions. Soybean plants were grown in the normal and high-Zn solutions for 24 days; tomato plants were grown for 32 days. The exudate was analyzed for seven organic acids, 22 amino acids, eight inorganic solutes, apparent ionic strength, and pH. Significant changes in many solutes occurred over the 8-hour sampling period. These fluctuations depended on plant species, individual solute, and Zn treatment, and demonstrated that extrapolation of xylem-fluid analyses to whole-plant xylem sap is valid only for sap samples collected shortly after topping a plant. Exudate pH decreased over the 8-hour period for both species; exudate ionic strength increased for tomato and decreased for soybean. At the normal-Zn treatment (0 to 1 hour), the highest acid micromolar concentrations in soybean exudate were: asparagine, 2,583; citric, 1,706; malic, 890; and malonic, 264. Under the same conditions, the highest acid micromolar concentrations in tomato exudate were: maleic, 1,206; malic, 628; glutamine, 522; citric, 301; and asparagine, 242. Cysteine and methionine were above detection limits only in soybean exudate. Zinc phytotoxicity caused significant changes in many solutes. The analyses reported here provide a comprehensive data base for further studies on metal-complex equilibria in xylem fluid.     

Rice cationic peroxidase accumulates in xylem vessels during incompatible interactions with Xanthomonas oryzae pv oryzae.

A cationic peroxidase, PO-C1 (molecular mass 42 kD, isoelectric point 8.6), which is induced in incompatible interactions between the vascular pathogen Xanthomonas oryzae pv oryzae and rice (Oryza sativa L.), was purified. Amino acid sequences from chemically cleaved fragments of PO-C1 exhibited a high percentage of identity with deduced sequences of peroxidases from rice, barley, and wheat. Polyclonal antibodies were raised to an 11-amino acid oligopeptide (POC1a) that was derived from a domain where the sequence of the cationic peroxidase diverged from other known peroxidases. The anti-POC1a antibodies reacted only with a protein of the same mobility as PO-C1 in extracellular and guttation fluids from plants undergoing incompatible responses collected at 24 h after infection. In the compatible responses, the antibodies did not detect PO-C1 until 48 h after infection. Immunoelectron microscopy was used to demonstrate that PO-C1 accumulated within the apoplast of mesophyll cells and within the cell walls and vessel lumen of xylem elements of plants undergoing incompatible interactions.     

Translocation of nickel in xylem exudate of plants

Topped plants of tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), corn (Zea mays), carrot (Daucus carota), and peanut (Arachis hypogaea) were treated with 0.5 to 50 micromolar Ni (containing ⁶³Ni) in nutrient solutions. Xylem exudate was collected for 10 hours or, in the case of corn, for 20 hours at 5-hour intervals. Electrophoresis of nutrient solution distributed all Ni cathodically as inorganic Ni²⁺. Low concentrations of Ni in tomato exudate migrated anodically, presumably bound to organic anion (carrier). However, this carrier became saturated at about 2 micromolar Ni in exudate, and excess Ni ran cathodically. Most of the Ni in cucumber, corn, carrot, and peanut exudate ran anodically, and its migration rate was identical for all exudates. Peanut root sap contained 14 to 735 micromolar Ni. The anodic Ni carriers in root sap and exudate appear identical. The carrier in root sap became saturated near 100 micromolar Ni, as shown by cathodic streaking of Ni exceeding that concentration. It appears that all five species translocate low concentrations of Ni in the same anionic form.     

Chemical forms of aluminum in xylem sap of tea plants (Camellia sinensis L.)

To identify the chemical forms of aluminum (Al) transported from roots to shoots of tea plants (C. sinensis L.), ²⁷Al-nuclear magnetic resonance and ¹⁹F NMR spectroscopy were used to analyze xylem sap.The concentration of Al in collected xylem sap was 0.29 mM, twice as high as that of F. Catechins were not detected in xylem sap. The concentration of malic acid in xylem sap was higher than that of citric acid, whereas the concentration of oxalic acid was negligible.There were two signals in the ²⁷Al NMR spectra of xylem sap, a larger signal at 11 ppm and a smaller one at −1.5 ppm. The former signal was consistent with the peak for an Al-citrate model solution, suggesting that an Al-citrate complex was present in xylem sap. Although the latter signal at −1.5 ppm was thought to indicate the presence of an Al-F complex (at 1.7 ppm) in xylem sap, there was only one signal at −122 ppm in the ¹⁹F NMR spectrum of xylem sap, indicating that the main F complex in xylem sap was F⁻.These results indicate that Al might be translocated as a complex with citrate, while Al-malate, Al-oxalate and Al-F complexes are not major Al complexes in xylem sap of tea plants.     

Changes to the proteome and targeted metabolites of xylem sap in Brassica oleracea in response to salt stress

Root-to-shoot signalling via xylem sap is an important mechanism by which plants respond to stress. This signalling could be mediated by alteration in the concentrations of inorganic and/or organic molecules. The effect of salt stress on the contents of xylem sap in Brassica olarecea has been analysed by mass spectrometry in order to quantify these changes. Subcellular location of arabinogalactan proteins (AGPs) by immunogold labelling and peroxidase isozymes was also analysed by isoelectrofocusing. The xylem sap metabolome analysis demonstrated the presence of many organic compounds such as sugars, organic acids and amino acids. Of these, amino acid concentrations, particularly that of glutamine, the major amino acid in the sap, were substantially reduced by salt stress. The xylem sap proteome analysis demonstrated the accumulation of enzymes involved in xylem differentiation and lignification, such as cystein proteinases, acid peroxidases, and a putative hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase under salt stress. The peroxidase isozyme pattern showed that salt stress induced a high accumulation of an acid isoform. These results suggest that xylem differentiation and lignification is induced by salt stress. The combination of different methods to analyse the xylem sap composition provides new insights into mechanisms in plant development and signalling under salt stress.     

Organic acids and iron translocation in maize genotypes

Translocation of Fe was studied in WF9 (Fe-efficient) and ys₁/ys₁ (Fe-inefficient) maize (Zea mays L.) genotypes. Iron-deficient WF9 translocated more Fe to the tops than Fe-deficient ys₁/ys₁. Malate and citrate contents of root saps increased nearly 2-fold and aconitate increased over 4-fold in both genotypes as Fe of nutrient solutions increased from 0.1 to 3 milligrams per liter. Relative acid contents in root saps were as follows: malate > aconitate > citrate. Citric acid concentrations in stem exudates were nearly the same as in root sap. Malic acid concentrations were considerably lower in exudates than in root saps, and only a trace of aconitic acid was detected in the exudates. The concentration of Fe was 7-fold higher in exudate of WF9 than in exudate of ys₁/ys₁ and the concentration of exudate P was about the same for both genotypes.     

Evaluation of water stress control with polyethylene glycols by analysis of guttation

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of −4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than −4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.     

Modeling C and N transport to developing soybean fruits

Xylem sap and phloem exudates from detached leaves and fruit tips were collected and analyzed during early pod-fill in nodulated soybeans (Glycine max (L.) Merr. cv Wilkin) grown without (−N) and with (+N) NH₄NO₃. Ureides were the predominant from (91%) of N transported in the xylem of −N plants, while amides (45%) and nitrate (23%) accounted for most of the N in the xylem of +N plants. Amino acids (44%) and ureides (36%) were the major N forms exported in phloem from leaves in −N plants, but amides (63%) were most important in +N plants. Based on the composition of fruit tip phloem, ureides (55% and 33%) and amides (26% and 47%) accounted for the majority of N imported by fruits of −N and +N plants, respectively.

C:N weight ratios were lowest in xylem exudate (1.37 and 1.32), highest in petiole phloem (24.5 and 26.0), and intermediate in fruit tip exudate (12.6 and 12.1) for the −N and +N treatments, respectively. The ratios were combined with data on fruit growth and respiration to construct a model of C and N transport to developing fruits. The model indicates xylem to phloem transfer provides 35% to 52% of fruit N. Results suggest the phloem entering fruits oversupplies their N requirement so that 13% of the N imported is exported from fruit in the xylem.

     

Spatial and temporal variations in phloem sap composition of plantation-grown Eucalyptus globulus

Spontaneous bleeding of sugar-rich sap from cambial-deep incisions in the bark of trunks was demonstrated for Eucalyptus globulus and other eucalypts across a range of localities and seasonal conditions in south-west Australia. High levels of sucrose and raffinose (up to 31% w/v total sugars) were present in the exudates, and upward and downward gradients in exudate sugar concentrations were recorded between samples obtained at different heights up trunks of E. globulus. The data indicated a phloem origin for the exudates, with source:sink pressure gradients driving translocation. Concentration ratios of sugars to amino acids were consistently lower in exudate from upper (distal) than basal regions of trunks, suggesting preferential partitioning of nitrogen upwards towards the trunk apex. A comparison of phloem and xylem sap composition from one plantation over a season showed nitrate in xylem but not phloem and substantial amounts of sodium, and high concentrations of chloride and sulphate relative to phosphate in xylem and phloem. Phloem sap sampled across a range of 29 contrasting plantations of E. globulus at peak stress (autumn) showed great inter-site variability in concentrations of amino acids, sulphur, sodium and certain trace elements and in C:N and Na:K ratios of sap. Carbon isotope ratios (δ¹³C) were strongly correlated with sugar concentrations of the sap samples from these and other plantations. Use of sap compositional attributes of phloem and δ¹³C values of translocated carbon is suggested for assessing the current nutritional condition and water status of E. globulus plantings. Received: 9 April 1998 / Accepted: 20 August 1998     

Virus particles in guttate and xylem of infected cucumber (Cucumis sativus L.)

Particles of plant viruses representing ten genera were visualised by electron microscopy in samples of guttate from systemically infected cucumber (Cucumis sativus L.) plants. For the majority of viruses tested, guttation samples tested positively by ELISA and infectivity was also demonstrated. Virus particles of zucchini yellow mosaic potyvirus were also observed in xylem in sections of infected root tissue. Since guttate originates from xylem exudate, this study demonstrates the potential for xylem tissue to act as a conduit for a diverse group of plant viruses in planta     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N₂ fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

Influence of sampling time and sap extraction methodology on xylem pH values in two grapevine varieties grown under drought conditions

The effect of differences in applied pressure and time of sampling on pH values of xylem sap collected using the leaf pressurization technique was examined in two grapevine varieties originating from contrasting habitats (Vitis vinifera L., cvs. Sabatiano and Mavrodafni) after subjecting them to drought. Three fractions of xylem sap exudates were collected from each leaf according to differences in applied pressure; fractions (I), (II) and (III) corresponding to 1 MPa, 2 MPa and 2.5 MPa pressure, respectively. The pH values in fraction (I) were significantly lower than those in fractions (II) and (III). The sap pH values in fraction (III) seemed to better correspond to changes in leaf apoplastic pH. The time of sampling was found to strongly influence xylem pH values. In particular, a positive relationship between predawn xylem pH values and soil drying was observed. Conversely, xylem pH values measured later during the day (i.e. at 8:00, 9:00 and 10:00 am) were not significantly affected by the reduction in soil water availability in both varieties. It is suggested that the most suitable period for sap sampling in order to better discriminate drought effects on xylem sap pH is at predawn. Furthermore, there were significant differences in pH values as well as in the sensitivity of stomatal conductance to pH between the two varieties. These differences might be related to strategy differences between grapevine varieties for adaptation to drought.     

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.