Little is known about molecular responses in plants to phloem feeding by insects. The induction of genes associated with wound and pathogen response pathways was investigated following green peach aphid (Myzus persicae) feeding on Arabidopsis. Aphid feeding on rosette leaves induced transcription of two genes associated with salicylic acid (SA)dependent responses to pathogens (PR-1 and BGL2) 10-and 23-fold, respectively. Induction of PR-1 and BGL2 mRNA was reduced in npr1 mutant plants, which are deficient in SA signaling. Application of the SA analog benzothiadiazole led to decreases in aphid reproduction on leaves of both wild-type plants and mutant plants deficient in responsiveness to SA, suggesting that wild-type SA-dependent responses do not influence resistance to aphids. Twofold increases occurred in mRNA levels of PDF1.2, which encodes defensin, a peptide involved in the jasmonate (JA)-/ethylene-dependent response pathway. Transcripts encoding JA-inducible lipoxygenase (LOX2) and SA/JA-inducible Phe-ammonia lyase increased 1.5to 2-fold. PDF1.2 and LOX2 induction by aphids did not occur in infested leaves of the JA-resistant coi1-1 mutant. Aphid feeding induced 10-fold increases in mRNA levels of a stress-related monosaccharide symporter gene, STP4. Phloem feeding on Arabidopsis leads to stimulation of response pathways associated with both pathogen infection and wounding.

The relationship between phloem-feeding insects (PFIs) and plants offers an intriguing example of a highly specialized biotic interaction. These insects have evolved to survive on a nutritionally imbalanced diet of phloem sap, and to minimize wound responses in their host plants. As a consequence, plant perception of and responses to PFIs differ from plant interactions with other insect-feeding guilds. Transcriptome-wide analyses of gene expression are currently being applied to characterize plant responses to PFIs in crop plants with race-specific innate resistance, as well as in compatible interactions with susceptible hosts. Recent studies indicate that PFIs induce transcriptional reprogramming in their host plants, and that plant responses to PFIs appear to be quantitatively and qualitatively different from responses to other insects or pathogens. Transcript profiling studies also suggest that PFIs induce cell wall modifications, reduce photosynthetic activity, manipulate source-sink relations, and modify secondary metabolism in their hosts, and many of these responses appear to occur within the phloem tissue. Plant responses to these insects appear to be regulated in part by the salicylate, jasmonate, and ethylene signalling pathways. As additional transcript profiling data become available, forward and reverse genetic approaches will be necessary to determine which changes in gene expression influence resistance or susceptibility to PFIs.

Phloem feeding involves unique biological interactions between the herbivore and its host plant. The economic importance of aphids, whiteflies, and other phloem-feeding insects as pests has prompted research to isolate sources of resistance to piercing-sucking insects in crops. However, little information exists about the molecular nature of plant sensitivity to phloem feeding. Recent discoveries involving elicitation by plant pathogens and chewing insects and limited studies on phloem feeders suggest that aphids are capable of inducing responses in plants broadly similar to those associated with pathogen infection and wounding. Our past work showed that compatible aphid feeding on leaves of Arabidopsis thaliana induces localized changes in levels of transcripts of genes that are also associated with infection, mechanical damage, chewing herbivory, or resource allocation shifts. We used microarray and macroarray gene expression analyses of infested plants to better define the response profile of A. thaliana to M. persicae feeding. The results suggest that genes involved in oxidative stress, calcium-dependent signaling, pathogenesis-related responses, and signaling are key components of this profile in plants infested for 72 or 96 h. The use of plant resistance to aphids in crops will benefit from a better understanding of induced responses. The establishment of links between insect elicitation, plant signaling associated with phloem feeding, and proximal resistance mechanisms is critical to further research progress in this area.

Since the first ultrastructural investigations of sieve tubes in the early 1960s, their structure has been a matter of debate. Because sieve tube structure defines frictional interactions in the tube system, the presence of P protein obstructions shown in many transmission electron micrographs led to a discussion about the mode of phloem transport. At present, it is generally agreed that P protein agglomerations are preparation artifacts due to injury, the lumen of sieve tubes is free of obstructions, and phloem flow is driven by an osmotically generated pressure differential according to Mü nch's classical hypothesis. Here, we show that the phloem contains a distinctive network of protein filaments. Stable transgenic lines expressing Arabidopsis thaliana Sieve-Element-Occlusion-Related1 (SEOR1)-yellow fluorescent protein fusions show that At SEOR1 meshworks at the margins and clots in the lumen are a general feature of living sieve tubes. Live imaging of phloem flow and flow velocity measurements in individual tubes indicate that At SEOR1 agglomerations do not markedly affect or alter flow. A transmission electron microscopy preparation protocol has been generated showing sieve tube ultrastructure of unprecedented quality. A reconstruction of sieve tube ultrastructure served as basis for tube resistance calculations. The impact of agglomerations on phloem flow is discussed.

Phloem protein 2 (PP2) is one of the most abundant and enigmatic proteins in the phloem sap. Although thought to be associated with structural P-protein, PP2 is translocated in the assimilate stream where its lectin activity or RNA-binding properties can exert effects over long distances. Analyzing the diversity of these proteins in vascular plants led to the identification ofPP2-like genes in species from 17 angiosperm and gymnosperm genera. This wide distribution of PP2 genes in the plant kingdom indicates that they are ancient and common in vascular plants. Their presence in cereals and gymnosperms, both of which lack structural P-protein, also supports a wider role for these proteins. Within this superfamily, PP2 proteins have considerable size polymorphism. This is attributable to variability in the length of the amino terminus that extends from a highly conserved domain. The conserved PP2 domain was identified in the proteins encoded by six genes from several cucurbits, celery (Apium graveolens), and Arabidopsis that are specifically expressed in the sieve element-companion cell complex. The acquisition of additional modular domains in the amino-terminal extensions of other PP2-like proteins could reflect divergence from its phloem function.

No abstract

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of f luorescently labeled dextrans along with sizefractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual f luorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20-100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

Phloem-specific proteins (P proteins) are particularly useful markers to investigate long-distance trafficking of macromolecules in plants. In this study, genus-specific molecular probes were used in combination with intergeneric grafts to reveal the presence of a pool of translocatable P protein subunits. Immunoblot analyses demonstrated that Cucurbita spp P proteins PP1 and PP2 are translocated from Cucurbita maxima stocks and accumulate in Cucumis sativus scions. Cucurbita maxima or Cucurbita ficifolia PP1 and PP2 mRNAs were not detected in Cucumis sativus scions by either RNA gel blot analysis or reverse transcription-polymerase chain reaction, indicating that the proteins, rather than transcripts, are translocated. Tissue prints of the Cucumis sativus scion, using antibodies raised against Cucurbita maxima PP1 or PP2, detected both proteins in the fascicular phloem of the stem at points distal to the graft union and in the petiole of a developing leaf, suggesting that the proteins move within the assimilate stream toward sink tissues. Cucurbita maxima PP1 was immunolocalized by light microscopy in sieve elements of the extrafascicular phloem of Cucumis sativus scions, whereas Cucurbita maxima PP2 was detected in both sieve elements and companion cells. INTRODUCTIONThe long-distance movement of macromolecules in vascular tissues can impact profoundly normal plant growth and development. The importance of long-distance signaling in response to wounding as well as systemic infections by plant pathogens, such as viruses, has been well documented (Narváez-Vásquez et al., 1995;Schaller and Ryan, 1995;Nelson and Van Bel, 1998). However, little is known about the mechanisms or effects of translocating the numerous proteins that are known to be expressed specifically within the phloem tissue. The phloem of most angiosperms contains proteinaceous structures, collectively called P proteins (phloem proteins), that accumulate in differentiating sieve elements and persist in translocating sieve elements. The P protein is deposited initially into ultrastructurally distinct polymorphous or crystalline bodies during sieve element differentiation (reviewed in Cronshaw, 1975;Cronshaw and Sabnis, 1990;Sabnis and Sabnis, 1995). P protein bodies either persist or more often disperse, forming a filamentous network in the parietal cytoplasm that is thought to be immobilized through interactions with the appressed endomembrane system (Smith et al., 1987). Disruption of sieve elements that occurs during wounding results in the accumulation of P protein filaments at the sieve plate, ostensibly blocking translocation by forming P protein plugs.P protein filaments in Cucurbita maxima (pumpkin) are composed of two very abundant proteins: phloem protein 1 (PP1), a 96-kD phloem filament protein, and phloem protein 2 (PP2), a 48-kD dimeric lectin that specifically binds poly( ␤ -1,4-N -acetylglucosamine) (Beyenbach et al., 1974;Sabnis and Hart, 1978;Allen, 1979;Read and Northcote, 1983b). Analysis of soluble phloem filaments present in phl...