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.     

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.     

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.     

Melon phloem-sap proteome: developmental control and response to viral infection

In addition to small molecules such as sugars and amino acids, phloem sap contains macromolecules, including mRNA and proteins. It is generally assumed that all molecules in the phloem sap are on the move from source to sink, but recent evidence suggests that the macromolecules' direction of movement can be controlled by endogenous plant mechanisms. To test the hypothesis that the phloem-sap protein profile is affected by local metabolic activities, we analyzed the phloem-sap proteome in young and mature tissues of melon plants. We also examined the effect of cucumber mosaic virus (CMV) infection and expression of CMV movement protein in transgenic melon plants on the phloem protein profile. Sap collected from cut sections of young stems or petioles contained specific proteins that were absent from sap collected from mature stems or petioles. Most of these proteins were involved in defense response and protection from oxidative stress, suggesting that they play a role in maintaining safe activity of the sieve tubes in young tissues. Phloem sap collected from CMV-infected plants and transgenic plants expressing the CMV movement protein contained only a few additional proteins with molecular masses of 18 to 75 kDa. Here again, most of the additional proteins were associated with stress responses. Our study indicated that the proteome of phloem sap is dynamic and under developmental control. Entry and exit of proteins from the sieve tube can be regulated at the tissue level. Moreover, the plant can maintain regulation of protein trafficking from companion cells to sieve elements under viral infection or other perturbations in plasmodesmal function.     

The phloem, a miracle of ingenuity

This review deals with aspects of the cellular and molecular biology of the sieve element/companion cell complex, the functional unit of sieve tubes in angiosperms. It includes the following issues: (a) evolution of the sieve elements; (b) the specific structural outfit of sieve elements and its functional significance; (c) modes of cellular and molecular interaction between sieve element and companion cell; (d) plasmodesmal trafficking between sieve element and companion cell as the basis for macromolecular long‐distance signalling in the phloem; (e) diversity of sieve element/companion cell complexes in the respective phloem zones (collection phloem, transport phloem, release phloem); (f) deployment of carriers, pumps and channels on the plasma membrane of sieve element/companion cell complexes in various phloem zones; and (g) implications of the molecular‐cellular equipment of sieve element/companion cells complexes for mass flow of water and solutes in a whole‐plant frame.     

Faba bean forisomes can function in defence against generalist aphids

Phloem sieve elements have shut‐off mechanisms that prevent loss of nutrient‐rich phloem sap when the phloem is damaged. Some phloem proteins such as the proteins that form forisomes in legume sieve elements are one such mechanism and in response to damage, they instantly form occlusions that stop the flow of sap. It has long been hypothesized that one function of phloem proteins is defence against phloem sap‐feeding insects such as aphids. This study provides the first experimental evidence that aphid feeding can induce phloem protein occlusion and that the aphid‐induced occlusions inhibit phloem sap ingestion. The great majority of phloem penetrations in Vicia faba by the generalist aphids Myzus persicae and Macrosiphum euphorbiae triggered forisome occlusion and the aphids eventually withdrew their stylets without ingesting phloem sap. This contrasts starkly with a previous study on the legume‐specialist aphid, Acyrthosiphon pisum, where penetration of faba bean sieve elements did not trigger forisome occlusion and the aphids readily ingested phloem sap. Next, forisome occlusion was demonstrated to be the cause of failed phloem ingestion attempts by M. persicae: when occlusion was inhibited by the calcium channel blocker lanthanum, M. persicae readily ingested faba bean phloem sap.     

Effects of Fe deficiency on the protein profile of Brassica napus phloem sap

The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2DE (IEF‐SDS‐PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Phloem sap purity was assessed by measuring sugar concentrations. Two hundred sixty‐three spots were consistently detected and 15.6% (41) of them showed significant changes in relative abundance (22 decreasing and 19 increasing) as a result of Fe deficiency. Among them, 85% (35 spots), were unambiguously identified. Functional categories containing the largest number of protein species showing changes as a consequence of Fe deficiency were signaling and regulation (32%), and stress and redox homeostasis (17%). The Phloem sap showed a higher oxidative stress and significant changes in the hormonal profile as a result of Fe deficiency. Results indicate that Fe deficiency elicits major changes in signaling pathways involving Ca and hormones, which are generally associated with flowering and developmental processes, causes an alteration in ROS homeostasis processes, and induces decreases in the abundances of proteins involved in sieve element repair, suggesting that Fe‐deficient plants may have an impaired capacity to heal sieve elements upon injury.     

Scaling phloem transport: elasticity and pressure-concentration waves

Earlier theoretical analyses of the rate of propagation of pressure-concentration waves in the phloem were performed without adequate attention to the elastic expansion of sieve tube walls. Here, it is shown that the rate of propagation of pressure-concentration waves in phloem sieve tubes is not significantly impeded by wall elasticity, but rather, as previously implicated, by the ratio of sap osmotic pressure to the axial drop in sap hydrostatic pressure. It is also shown that pressure-concentration waves move equally well in both the upstream and downstream directions. These results permit future models to ignore elastic effects, and lend additional theoretical support to the "osmoregulatory flow" hypothesis, which argues that efficient molecular control of the phloem is permitted by maintaining sieve sap hydrostatic pressure at a value that is spatially nearly constant, which in turn permits changes in sieve tube state to be rapidly transmitted throughout the sieve tube via pressure-concentration waves.     

Differential distribution of proteins expressed in companion cells in the sieve element-companion cell complex of rice plants

Sieve tubes are comprised of sieve elements, enucleated cells that are incapable of RNA and protein synthesis. The proteins in sieve elements are supplied from the neighboring companion cells through plasmodesmata. In rice plants, it was unclear whether or not all proteins produced in companion cells had the same distribution pattern in the sieve element-companion cell complex. In this study, the distribution pattern of four proteins, beta-glucuronidase (GUS), green fluorescent protein (GFP), thioredoxin h (TRXh) and glutathione S-transferase (GST) were analyzed. The foreign proteins GUS and GFP were expressed in transgenic rice plants under the control of the TRXh gene promoter (PTRXh), a companion cell-specific promoter. Analysis of leaf cross-sections of PTRXh-GUS and PTRXh-GFP plants indicated high accumulation of GUS and GFP, respectively, in companion cells rather than in sieve elements. GUS and GFP were also detected in phloem sap collected from leaf sheaths of the transgenic rice plants, suggesting these proteins could enter sieve elements. Relative amounts of GFP and endogenous phloem proteins, TRXh and GST, in phloem sap and total leaf extracts were compared. Compared to TRXh and GST, GFP content was higher in total leaf extracts, but lower in phloem sap, suggesting that GFP accumulated mainly in companion cells rather than in sieve elements. On the other hand, TRXh and GST appeared to accumulate in sieve elements rather than in companion cells. These results indicate the evidence for differential distribution of proteins between sieve elements and companion cells in rice plants.     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.     

Aphid activities during sieve element punctures

Aphid salivation in sieve elements and phloem sap ingestion were linked to waveforms in the Electrical Penetration Graph (EPG). Non-viruliferousRhopalosiphum padi (L.) (Hemiptera, Aphididae) on barley yellow dwarf virus (BYDV) infected wheat could acquire the virus, which was used as an indication for phloem sap ingestion, whereas virus inoculation by viruliferous aphids on healthy plants was associated with salivation in sieve elements or other phloem cells. Probing was monitored and the waveforms recorded were related to ELISA results of test plants. The EPG patterns A, B, and C are indicative of the stylet pathway phase, whereas patterns E1 and E2 reflect the phloem (sieve element) phase with an unknown activity (E1) or with ingestion and concurrent salivation (E2). Aphids showing pathway and E1 rarely acquired virus, suggesting that little or no phloem sap ingestion can occur during these patterns, whereas those showing additionally pattern E2 did so substantially, indicating phloem sap ingestion. The main pattern related to virus inoculation was E1, although some aphids were able to inoculate plants during pathway. Pattern E1 clearly reflects the most important salivation into sieve elements. Pattern E2 had no clear contribution to virus inoculation, supporting the present hypothesis that during this pattern the saliva is mixed with the phloem sap in the single canal at the stylet tips and ingested immediately, without reaching the plant tissue. Sustained sap ingestion did not affect virus inoculation. So, BYDV inoculation mainly occurs during the first period of a sieve element puncture which is always formed by E1. Implications on persistent virus transmission are discussed.     

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.     

杨柳科(Salicaceae)7种植物次生韧皮部的比较研究

本文研究和比较了杨柳科2属7种植物次生韧皮部解剖结构。结果表明:(1)杨属和柳属植物在次生初皮部解剖上有某些共同特征:次生韧皮部具有明显分层现象;韧皮纤维和含晶细胞与筛管分子、伴胞和韧皮薄壁组织细胞是切向带相间排列;筛管分子均为复筛板,端壁倾斜平均含有7-8个筛域。(2)两属植物在射线和晶体类型上有明显区别:柳属植物次生韧皮部无石细胞;杨属植物不具功能韧皮部中含有石细胞。(3)两属植物均有一些较为原始的韧皮部解剖特征。     

Conductance of needle and twig axis phloem of damaged and intact Norway spruce (Picea abies (L.) Karst.) as investigated by application of14C in situ

Summary Phloem conductance of¹⁴C-labelled assimilates was investigated in natural stands of Norway spruce showing substantial damage from needle yellowing and needle loss disease. Terminal current-year shoots of a branch were allowed to fix¹⁴CO₂ (300–600 ppm in air) and carbon dioxide net uptake was monitored with a gas analyser. The difference between¹⁴C-uptake and the amount of radiocarbon determined in the photosynthesizing needles was interpreted to reflect assimilate export from the needles to the axis of the tree. Compared with an undamaged control tree,¹⁴C-export from the assimilating needles was not impaired in the yellowing tree and only slightly reduced in the tree showing needle loss. Incorporation of¹⁴C into starch increased significantly during autumn particularly in the tree showing needle loss. Import of radiocarbon from the¹⁴C-labelled phloem sap in twig axes and needles older than 1 year was used as a measure of phloem conductivity of older sections of a branch which showed considerable damage. Carbon uptake by these older plant parts was more pronounced than in undamaged twigs. In the case of older needles enhancement of¹⁴C-incorporation suggested an increased sink strength, while the same phenomenon in the twig axes was interpreted as a consequence of partially impaired conductivity of individual sieve elements resulting in an inhomogeneous velocity of phloem transport. The hypothesis is put forward that curtailed viability of the sieve cells is responsible for a delay of transport, which is compensated for by an augmented production of phloem elements from the cambium.     

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     

Immunolocalization of solanaceous SUT1 proteins in companion cells and xylem parenchyma: new perspectives for phloem loading and transport

Leaf sucrose (Suc) transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Suc loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 Suc transporters from tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) were immunolocalized in plasma membranes of enucleate sieve elements. Here, we present data that identify solanaceous SUT1 proteins with high specificity in phloem companion cells. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.