Abstract: Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 mul of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.

Abstract: Cell-to-cell communication is essential for plant development and adaptation to environmental changes. As a strategy for efficient intercellular communication, plants have evolved a plant-specific symplasmic network connected via plasmodesmata that allows a locally restricted information exchange from cell to cell. A rapid information transfer over long distances is enabled via the phloem transport tubes that pervade the complete plant and thus connect even the most distant organs. While communication by small molecules like metabolites and phytohormones is comparably well studied, the intercellular trafficking of proteins and RNAs has only recently emerged as a novel mechanism of cell-to-cell and long-distance signalling in plants. In particular the non-cell-autonomous and systemic transport pathway for specific RNAs seems to play a key role in the co-ordination of important physiological processes, including virus defence, gene silencing, regulation of development, and nutrient allocation. This review is a summary of the current knowledge on RNAs contained in the phloem long-distance transport system, their transport mechanism, and their potential functions.

Abstract: The presence of microRNA species in plant phloem sap suggests potential signaling roles by long-distance regulation of gene expression. Proof for such a role for a phloem-mobile microRNA is lacking. Here we show that phosphate (Pi) starvation-induced microRNA399 (miR399) is present in the phloem sap of two diverse plant species, rapeseed and pumpkin, and levels are strongly and specifically increased in phloem sap during Pi deprivation. By performing micro-grafting experiments using Arabidopsis, we further show that chimeric plants constitutively over-expressing miR399 in the shoot accumulate mature miR399 species to very high levels in their wild-type roots, while corresponding primary transcripts are virtually absent in roots, demonstrating shoot-to-root transport. The chimeric plants exhibit (i) down-regulation of the miR399 target transcript (PHO2), which encodes a critical component for maintenance of Pi homeostasis, in the wild-type root, and (ii) Pi accumulation in the shoot, which is the phenotype of pho2 mutants, miR399 over-expressers or chimeric plants with a genetic knock-out of PHO2 in the root. Hence the transported miR399 molecules retain biological activity. This is a demonstration of systemic control of a biological process, i.e. maintenance of plant Pi homeostasis, by a phloem-mobile microRNA.

Abstract: Systemic signalling is indispensable for the coordination of diverse physiological processes during development, defence and nutrient allocation. Indirect evidence suggests that plant small RNAs (smRNAs) could be involved in long-distance information transfer via the vasculature of the plant. Analyses of the smRNA complements of vascular exudates from oilseed rape (Brassica napus) showed that xylem sap is devoid of RNA, whereas phloem sap contained a large number of smRNAs. In addition to 32 annotated microRNAs (miRNAs) from 18 different families that could be identified and approved, a set of unknown smRNAs, predominantly of 21 and 24 nucleotides in length, was obtained, and selected candidates were found to be highly abundant in phloem sap. Moreover, we could demonstrate that the levels of three miRNAs known to respond to nutrient deprivation in non-vascular tissue, miR395 (sulphate), miR398 (copper) and miR399 (phosphate), were increased in phloem sap during the growth of plants under the respective starvation conditions. Interestingly, only mature miRNA molecules were found to be stress responsive, demonstrating that single-stranded sense miRNAs are most likely to represent the physiologically relevant molecules. The strong responses in the phloem suggest a role of miRNAs in systemic information transfer via this long-distance transport system.

Abstract: Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC-Cd and glutathione-Cd complexes.

Abstract: It is well known that phloem and xylem vessels transport small nutrient molecules over long distances in higher plants. The finding that proteins also occur in both transport fluids was unexpected, and the function of most of these proteins is not yet well understood. This chapter outlines how proteins can be obtained and purified from xylem and phloem saps to perform subsequent proteomic analyses.

Abstract: The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI). With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Deltaycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Deltaycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.

Abstract: 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.

Abstract: The phloem is a well-known target of sucking and piercing insects that utilize the transported fluid as their major nutrient source. In addition to small molecules like sugars and amino acids, phloem sap of higher land plants contains proteins that can accumulate up to high concentrations. Although the knowledge about the identities of these phloem sap proteins is increasing, the functions of most of them are still poorly understood. Since many phloem sap proteins have predicted roles in wound and defence responses, they constitute a class of compounds that can potentially influence plant-insect interactions. However, there are as yet no studies published that have examined direct effects of phloem sap proteins on insect feeding or vice versa. This review summarizes the current knowledge about the identities of phloem sap proteins, focused on polypeptides with probable functions in wound and defence reactions, and their potential impact on plant-insect interactions is discussed.

Abstract: Laser microdissection is a powerful tool to obtain cell-specific isolates from complex tissue samples. This chapter outlines how to prepare plant material for microdissection and methods to extract and measure high-quality RNA suitable for a variety of different downstream applications.

Abstract: Lupinus albus plants can withstand severe drought stress and show signs of recovery 24 h after rewatering (RW). Two-dimensional gel electrophoresis was used to evaluate the effect of water deficit (WD) on the protein composition of the two components of the lupin stem (stele and cortex). This was performed at three distinct stress levels: an early stage, a severe WD, and early recovery. Protein characterisation was performed through mass spectrometric partial sequencing. Modifications in the protein expression were first noticed at 3 days of withholding water, when the plant water status was still unaffected but some decrease in the relative soil water content had already occurred. An increase in serine proteases, possibly associated with WD sensing, was an early alteration induced by WD. When the stress severity increased, a larger number of stem proteins were affected. Immunophilin, serine protease and cysteine protease (well-known components of animal sensing pathways) were some of these proteins. The simultaneous expression of proteases and protease inhibitors that reacted differently to the stress level and to RW was found. Although the level of protease inhibitors was significantly raised, RW did not cause de novo expression of proteins. Many amino acid sequences did not match known sequences of either protein or expressed sequence tag databases. This emphasises the largely unknown nature of stem proteins. Nevertheless, some important clues regarding the way the lupin plant copes with WD were revealed.

Abstract: BACKGROUND: Substance transport in higher land plants is mediated by vascular bundles, consisting of phloem and xylem strands that interconnect all plant organs. While the phloem mainly allocates photoassimilates, the role of the xylem is the transport of water and inorganic nutrients from roots to all aerial plant parts. Only recently it was noticed that in addition to mineral salts, xylem sap contains organic nutrients and even proteins. Although these proteins might have important impact on the performance of above-ground organs, only a few of them have been identified so far and their physiological functions are still unclear. RESULTS: We used root-pressure xylem exudate, collected from cut Brassica napus stems, to extract total proteins. These protein preparations were then separated by high-resolution two-dimensional gel electrophoresis (2-DE). After individual tryptic digests of the most abundant coomassie-stained protein spots, partial peptide sequence information was deduced from tandem mass spectrometric (MS/MS) fragmentation spectra and subsequently used for protein identifications by database searches. This approach resulted in the identification of 69 proteins. These identifications include different proteins potentially involved in defence-related reactions and cell wall metabolism. CONCLUSION: This study provides a comprehensive overview of the most abundant proteins present in xylem sap of Brassica napus. A number of 69 proteins could be identified from which many previously were not known to be localized to this compartment in any other plant species. Since Brassica napus, a close relative of the fully sequenced model plant Arabidopsis thaliana, was used as the experimental system, our results provide a large number of candidate proteins for directed molecular and biochemical analyses of the physiological functions of the xylem under different environmental and developmental conditions. This approach will allow exploiting many of the already established functional genomic resources, like i.e. the large mutant collections, that are available for Arabidopsis.

Abstract: Laser microdissection (LM) allows the collection of homogeneous tissue- and cell-specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryo-sectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.

Abstract: BACKGROUND: Laser microdissection is a useful tool for collecting tissue-specific samples or even single cells from animal and plant tissue sections. This technique has been successfully employed to study cell type-specific expression at the RNA, and more recently also at the protein level. However, metabolites were not amenable to analysis after laser microdissection, due to the procedures routinely applied for sample preparation. Using standard tissue fixation and embedding protocols to prepare histological sections, metabolites are either efficiently extracted by dehydrating solvents, or washed out by embedding agents. RESULTS: In this study, we used cryosectioning as an alternative method that preserves sufficient cellular structure while minimizing metabolite loss by excluding any solute exchange steps. Using this pre-treatment procedure, Arabidopsis thaliana stem sections were prepared for laser microdissection of vascular bundles. Collected samples were subsequently analyzed by gas chromatography-time of flight mass spectrometry (GC-TOF MS) to obtain metabolite profiles. From 100 collected vascular bundles (~5,000 cells), 68 metabolites could be identified. More than half of the identified metabolites could be shown to be enriched or depleted in vascular bundles as compared to the surrounding tissues. CONCLUSION: This study uses the example of vascular bundles to demonstrate for the first time that it is possible to analyze a comprehensive set of metabolites from laser microdissected samples at a tissue-specific level, given that a suitable sample preparation procedure is used.

Abstract: Xylem sap from broccoli (Brassica oleracea L. cv. Calabrais), rape (Brassica napus L. cv. Drakkar), pumpkin (Cucurbita maxima Duch. cv. gelber Zentner) and cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) was collected by root pressure exudation from the surface of cut stems of healthy, adult plants. Total protein concentrations were in the range of 100 microg ml(-1). One-dimensional gel electrophoresis (SDS-PAGE) resulted in 10-20 visible protein bands in a molecular mass range from 10 to 100 kDa. The main bands were cut out, digested with trypsin, and analysed using tandem mass spectrometry. Fifty bands resulted in amino acid sequence information that was used to perform database similarity searches. Sequences from 30 bands showed high homology to proteins present in databases. Among them, we found mostly peroxidases, but could also identify the lectin-like xylem protein XSP30, a glycine-rich protein, serine proteases, an aspartyl protease family protein, chitinases, and a lipid transfer protein-like polypeptide. Sequence analysis predicted apoplastic secretion signals for all database entries similar to the partial xylem protein sequences. This and the lack of cross-reactivity with phloem protein-specific antibodies suggest that the proteins really originate from the xylem and do not result from phloem contamination. Most of the highly similar proteins probably function in repair and defence reactions. Some of the most abundant proteins (peroxidases, chitinases, serine proteases) were present in xylem exudate of all species analysed, often in more than one band. This indicates an important basic role of these proteins in maintaining xylem function.

Abstract: Many different proteins can be separated from the sap of mature sieve tubes of different plant species. To date, only a limited number of those have been identified and functionally characterised. Due to sieve tubes inability of transcription and translation, the proteins are most probably synthesised in the intimately connected companion cells and transported into the sieve elements through plasmodesmata. The specific protein composition of phloem sap suggests an important role of these proteins not only for sieve tube maintenance, but also for whole plant physiology and development. Here we describe a comprehensive analysis of the phloem protein composition employing one- and high-resolution two-dimensional gel electrophoresis and partial sequencing by mass spectrometry. In this study more than 300 partial sequences generated by hybrid mass spectrometry were used to identify a total of 45 different proteins from the phloem exudates of cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) and pumpkin (Cucurbita maxima Duch. cv. Gelber Zentner) plants. In addition to previously described phloem proteins, it was possible to localise proteins with high similarity to an acyl-CoA binding protein, a glyoxalase, a malate dehydrogenase, a rhodanese-like protein, a drought-induced protein, and a beta-glucosidase. The results indicate that the majority of the so far identified proteins are involved in stress and defence reactions.

Abstract: Complexity is a fundamental feature of life. Like animals, higher plants consist of a multitude of different distinct tissues and cell types, each contributing to the overall performance of the whole organism. Our understanding and knowledge of physiology will greatly increase as our ability to spatially resolve molecular and biochemical processes improves. Differential analysis of individual tissues and single cells will eliminate the averaging effect and allow the discovery of detailed differences between various cell types. Recent breakthroughs have been made in tissue-specific DNA, RNA and protein analysis of plants by applying laser-based microdissection techniques.

Abstract: The aim of this work was to examine the consequences of the heterologous expression of a spinach ( Spinacia oleracea L.) sucrose transporter ( SoSUT1) in potato ( Solanum tuberosum L.). Many studies have indicated that reduction of the expression of this class of sucrose transporter has deleterious effects on plant growth and development; however, until now the possibility of improving plant performance by enhancing the expression of this sucrose transporter has not been reported. With this intention we constructed a chimeric construct in which SoSUT1 was cloned in-frame with the myc epitope. We confirmed that this construct, SoSUT1m, was able to mediate sucrose transport by expression in the yeast strain SUSY7. SoSUT1m was expressed in wild-type potato in the sense orientation under the control of the cauliflower mosaic virus 35S promoter to evaluate the effect of an increased constitutive expression of a class-I sucrose transporter. We confirmed that these plants displayed expression of SoSUT1 at both the transcript and protein level and that microsomal fragments isolated from selected lines had an increased sucrose uptake capacity. Analysis of metabolism of these lines indicated that the leaves were characterised by a reduced sucrose level yet exhibited little change in photosynthetic rate. Furthermore, despite the observed increase in sugar (and reduction in amino acid) levels within the tubers, there was little change in either starch content or tuber yield in the transformants. In summary, the genetic manipulation described in this paper resulted in a shift in carbon partitioning in both leaves and tubers and an increased sucrose uptake rate in plasma-membrane vesicles isolated from these lines, but had little impact on tuber metabolism or morphology.

Abstract:

Abstract: The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.

Abstract: Advances in high-throughput genome sequencing demand the development of more efficient ways of examining gene expression at a cellular level. During recent years, polymerase chain reaction (PCR)-based methods have been developed that allow the amplification of mRNA from small amounts of material, even from single animal cells. In parallel, several analytical tools permit a global monitoring of gene expression. To date, high throughput analysis methods have not been accessible for single plant cell samples. In the protocol described here, cDNA array hybridization (expression profiling) and an amplification strategy using reverse transcriptase PCR are merged with high spatial resolution sampling from undamaged plant tissue. This protocol gives us a new tool to examine tissue-specific gene expression patterns on a comprehensive scale. To demonstrate the usefulness of this tool, gene expression patterns in samples from Arabidopsis thaliana L. cv. C24 leaf epidermal and mesophyll cells were measured; several differentially expressed genes were identified when single cell samples were compared. The protocol described has the potential of increasing the efficiency of tissue-specific expression analysis by combining high-throughput profiling with straightforward sampling and amplification procedures.

Abstract: Phloem sap of transgenic Bacillus thuringiensis (Bt) corn expressing a truncated form of the B. thuringiensis delta-endotoxin Cry1Ab, sap sucking aphids feeding on Bt corn and their honeydew were analysed for presence of Cry1Ab using ELISA. Phloem sap of Bt and non-Bt corn was collected using a newly developed technique with a microcapillary being directly inserted into the phloem tubes. Using this technique, no Cry1Ab was detected in the phloem sap. In contrast, measurable concentrations of Cry1Ab in the range of 1 ppb were detected when phloem sap of pooled leaf samples was extracted using EDTA buffer. This was probably because of Cry1Ab toxin released from damaged cells. When analysing apterous adults of Rhopalosiphum padi L. and their honeydew, no Cry1Ab could be detected. In contrast, Cry1Ab was clearly detected in both larvae of the leaf chewing herbivore Spodoptera littoralis (Boisduval) and their faeces, showing that Cry1Ab is detectable after ingestion and excretion by herbivores. These results suggest that R. padi ingests or contains no or only very low concentrations of Cry1Ab in the range of the detection limit. In consequence it is hypothesized that R. padi as an important prey for beneficial insects in corn is unlikely to cause any harm to its antagonists due to mediating Bt toxin.

Abstract: Analysis of organisms using techniques that provide high spatial and temporal resolution is of increasing interest in many disciplines of biomedical research. Although the examination of animal tissues has been the main focus to date, the recent development and improvement of methods for the sampling and handling of single cells and for their biochemical analysis now provide tools for investigating plant as well as animal cells.

Abstract: We have cloned a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) cDNA (AtP5K1) from Arabidopsis thaliana. By the application of cell permeabilization and short-term nonequilibrium labelling we show that expression of AtP5K1 in Baculovirus-infected insect (Spodoptera frugiperda) cells directs synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. The same phosphoinositides were produced by isolated whole-cell membrane fractions of AtP5K1-expressing insect cells. Their synthesis was not affected by adding defined precursor lipids, that is PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, or PtdIns(4,5)P2, in excess, indicating that substrates for the plant enzyme were not limiting in vivo. Enzymatic dissection of lipid headgroups revealed that AtP5K1-directed synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 proceeds via 5-phosphorylation of precursors. Analysis of promoter-reporter gene (beta-glucuronidase) fusions in transgenic plants revealed that expression of the AtP5K1 gene is strongest in vascular tissues of leaves, flowers, and roots, namely in cells of the lateral meristem, that is the procambium. Single-cell sampling of sap from flower stem meristem tissue and neighbouring phloem cells, when coupled to reverse transcriptase--polymerase chain reaction, confirmed preferential expression of AtP5K1 in procambial tissue. We hypothesize that AtP5K1, like animal and yeast PIP5K, may be involved in the control of cell proliferation.

Abstract: A combination of gel electrophoresis and mass spectrometry was used to analyze the soluble proteins from phloem sap of Cucurbita maxima Duch. Phloem proteins were separated using two-dimensional gel electrophoresis. Coomassie-stained spots were cut out and subjected to tryptic digestion. To identify proteins, peptide mass fingerprints were determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. In addition, MALDI-TOF post source decay measurements were used to obtain partial sequence information for the proteins. Results from both approaches were used for database searches. In this study, 17 proteins in the mass range 5-50 kDa were analyzed. Of these proteins six could be clearly identified, seven showed significant homologies to known plant proteins, and four were not significantly homologous to database entries. The present study suggests that the applied method is feasible for a large-scale analysis and identification of phloem proteins derived from different organs or from plants kept under various physiological conditions.

Abstract: In this study 21 amino acid standards, samples of pure phloem sap and samples of pooled mesophyll cells were derivatized with fluorescein isothiocyanate, separated by capillary electrophoresis and detected with laser-induced fluorescence at 488 nm. Two different background electrolytes, a sodium borate buffer containing sodium dodecyl sulfate and a sodium borate buffer containing alpha-cyclodextrin, were used for the separation. Using the sodium dodecyl sulfate buffer, 14 amino acid standards could be separated, spiking identified 12 amino acids in pure phloem sap and 13 amino acids in pooled mesophyll cells. With the alpha-cyclodextrin containing background electrolyte, a resolution of 20 amino acid standards could be attained, 17 amino acids in pure phloem sap and 10 amino acids in mesophyll cells could be assigned. Leucine and isoleucine comigrated in both buffer systems. All separations were performed with a voltage of +20 kV and completed within 30 min. The detection limits obtained were in the fmol range for the sodium dodecyl sulfate and in the pmol range for the alpha-cyclodextrin background electrolyte. Compared to the one published capillary electrophoresis-based method for the determination of amino acids from few plant cells, the procedure described here allows very high sensitivity due to the use of laser-induced fluorescence detection and opens the possibility to dilute and measure pl samples with an fully automated, commercially available CE system.

Abstract: Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS: the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539-1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ.

Abstract: A protocol for the detection of gene transcripts from single plant cells in living, undamaged plant tissue is described. Samples of leaf epidermal, mesophyll and companion cells were extracted by using glass microcapillaries and directly subjected to RT-PCR without any purification steps nor time consuming construction of cDNA libraries. The procedure is not restricted to surface cells or outer cell layers. Even cells from the central region of leaves could be harvested. For identification, companion cells were labelled by expression of the green fluorescent protein under control of a companion cell specific promoter. The described method is applicable to a wide range of plants and genes with different expression levels.

Abstract: Photosynthesis, partitioning of carbohydrates and growth have to be highly orchestrated to enable an efficient performance of plants. To study the diurnal relationships between carbon distribution and growth, we analysed transgenic potato plants with altered carbon allocation patterns. To modify carbohydrate supply of growing sinks, we used plants that accumulated starch as a consequence of inhibition in triose-phosphate export from chloroplasts and plants that were genetically inhibited in starch production. Carbon assimilation was analysed by gas exchange and single cell analysis of source leaves. Export was determined by microanalysis of phloem exudates and internodal growth rates were measured by displacement transducers. Gas exchange measurements showed similar assimilation rates in the wild-type and transgenic plants during the light period. Sugar analysis of phloem exudates and epidermal cells revealed a severe shift of sucrose concentrations in the individual plant lines. Moreover, epidermal cells turned out to be a potential storage site for carbohydrates in potato. Finally, we could demonstrate that changing the diurnal rhythm of carbon allocation results in a change in the diurnal growth pattern.