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Gene expression profiling: keys for investigating phloem functions
Review
Gene expression profiling: keys for investigating phloem functions Rozenn Le Hir2, Julie Beneteau1, Catherine Bellini1,2, Franc¸oise Vilaine1 and Sylvie Dinant1 1
INRA UR501 Laboratoire de Biologie Cellulaire, Institut Jean Pierre Bourgin, F78000 Versailles, France Umea˚ Plant Science Center — Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Sciences, 901 83 Umea˚, Sweden 2
Phloem is the major route for transport of carbohydrates, amino acids, and other nutrients from source to sink tissues. Hormones, mRNAs, small RNAs and proteins also are transported by the phloem, and potentially play pivotal roles in communication between organs to coordinate plant development and physiology. A comprehensive understanding of the mechanisms involved in phloem transport and signalling is still lacking. Recent transcript profiling in several plant species has provided new insights to phloem-specialized functions. Here, we review conclusions regarding the unique functions of the phloem and discuss putative roles for mRNAs and small RNA species in long-distance signalling. A superhighway based on highly specialized cells In land plants, the fixation of carbon and its conversion to carbohydrates is restricted to photosynthetic organs. The phloem transports these sugars, as well as other nutrients such as amino acids and electrolytes, to heterotrophic tissues [1]. Hormones, proteins and RNAs also are found in the phloem sap, and potentially are involved in longdistance signalling through the phloem [2,3]. The phloem performs a number of distinct tasks when collecting metabolites and signals from source organs, transporting these molecules and then releasing them to sink organs (Box 1). Therefore, the phloem has great potential to facilitate inter-organ coordination and hence to promote plant growth and development [4]. Long-distance translocation, driven by mass flow, takes place through the phloem sieve elements (SEs), which are enucleated and highly specialized living cells. Other phloem cell types such as companion cells (CCs), phloem parenchyma cells (PPCs) and, in few species, fibres, play important roles in the production, storage and delivery of metabolites and signals. Symplasmic and apoplasmic connections between CCs and SEs are crucial for loading into, retrieval from and release from SEs. Therefore CC–SE complexes are considered to be the functional units of phloem conduits. The structure of CC– SE complexes also reflects their various functions in source or sink organs (Box 1). Our knowledge of phloem functions is currently being extended by novel large-scale approaches to identify genes that are expressed in the phloem. Here, we review data from transcript profiling studies and aim to provide an inventory of the representative gene families that could be associated with specific Corresponding author: Dinant, S. ([email protected]).
functions of CCs, SEs or PPCs, and which could control key features of phloem cells. Our survey is based on currently available gene annotations, with their inherent limitations. We use the word ‘phloem’ to refer to the whole tissue, including PPCs, CCs and SEs, unless otherwise specified. Analyzing phloem transcriptomes Recently improved phloem collection methods (Box 2), together with technical advances in expression profiling, have allowed transcriptional studies of phloem tissue and of individual phloem cell-types such as CCs [5–20]*. These transcriptome studies (Table 1) provide insights into the physiology and formation of the phloem and its involvement in signalling processes, and have the potential to uncover the mechanisms of macromolecule trafficking in the phloem [2]. The ability to collect phloem sap (Box 2) has also allowed the large-scale identification of RNA transcripts and small RNAs that reside in the phloem sap (Table 1; [21–24]), suggesting that phloem could transport RNAs over long distances. Transcript profiling of the phloem: a signature of phloem cells? Transcriptome analyses allow gene discovery through the identification of expressed sequence tags (ESTs) in species whose genome is not sequenced, quantification of transcripts present in the phloem, and comparison with the transcript profiles of other tissues. In addition to work on Arabidopsis [7,13], gene profiling studies on diverse plant species have identified subsets of genes, ranging from one hundred to several thousand RNAs, that are expressed in the phloem (see Table S1 in the online supplementary material). The phloem transcriptome data available from dicots, monocots and a gymnosperm (Table 1) represent a valuable genomic resource for studying the biological diversity of gene families that are expressed in the phloem. They also highlight common features of phloem functions in diverse plant species, providing new clues to unexplored mechanisms. Although one study profiles ESTs from CCs [8], most studies encompass either phloem whole tissue, including CCs, SEs and PPCs [5,6,10,11,15], or *
Note added in proof An alternative to specific sorting of phloem cells relies on transgenic expression of nuclear-targeted GFP and analysis of transcripts in GFP-positive nuclei [77]. This approach was successfully applied to identify in Arabidopsis thaliana additional sets of genes expressed in companion cells of roots.
1360-1385/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2008.03.006 Available online 14 May 2008
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Review
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Box 1. Transport phloem: an intricacy of distinct cell types
Box 2. Tricks and concerns for phloem sampling
Phloem: three main functional compartments
A straightforward approach to analyzing the functions of a particular tissue involves isolating this tissue and analyzing the pools of mRNAs or proteins present, deducing subsets of specific genes that are involved in its functioning. Obstacles to the study of phloem include the inaccessibility of phloem cells in the plant body, the small fraction of phloem within organ tissues, and the small size of phloem cells, especially SEs [1]. Recently, methods for collecting small tissue samples were adapted to species for which the phloem could be isolated, and these methods were used for transcript profiling studies. The availability of phloem tissues varies depending on species and organs. In common plantain or celery, vascular bundles can be peeled from either leaves or petioles [10,15,16]. Dissection has been used to isolate the phloem of species with cellulose fibres, such as flax [14,20] or woody stems [5,11,12], and for Arabidopsis hypocotyls [13]. Laser microdissection has been applied for gene analysis of vascular tissues in both monocots and dicots [6,9]. Flow cytometry and cell sorting of protoplasts from fluorescent reporter lines were successfully applied to lines expressing the green fluorescent protein (GFP) under the control of the companion-cell-specific SUC2 promoter [7,8]*. Alternatively, phloem sap, which also contains RNAs, can be analyzed [2,64]. Phloem sap can be sampled by exudation in few species, such as cucurbits, castor bean and rape. In other major model plant species, including Arabidopsis, the small volume of phloem sap exudates collected precludes analysis. Stylectomy, another sap-sampling method, which yields nanolitre amounts of sap, has not yet been used for large-scale studies of RNAs. A major concern relating to these techniques is that they are invasive and potentially induce local responses to wounding of the phloem tissues, although methods such as exudation or peeling are thought to cause minimal injury. SEs react vigorously to any injury by rapid sealing of damaged tubes [4], and transcripts encoding defence- or stress-related proteins have frequently been found in phloem transcriptome studies. This raises the possibility that transcript profiles of the phloem could include transcripts from nearby or phloem-damaged cells. The effects of sampling method on the induction of stress-related genes have been evaluated in two studies [7,10], which showed that several stress-related genes, including some phloem-specific genes, are expressed in the phloem at a steady-state level.
Collection phloem The site of photoassimilate loading is in the minor veins of mature photosynthetic leaves. Phloem loading proceeds through either apoplasmic or symplasmic pathways. In apoplasmic-loader species, such as Arabidopsis (Figure I), sucrose is loaded actively into the CC–SE by transmembrane carriers. In symplasmic loaders, such as Cucurbitaceae, the loading occurs against a sucrose gradient through the numerous plasmodesmata that connect PPCs and CCs. In both modes of loading, CCs and SEs are connected through pore-plasmodesmata units (PPUs), with highly branched plasmodesmata at the CC side. Transport phloem In main veins, petioles, stems and primary roots transport phloem is responsible for long distance photoassimilate transport from source to sink organs. Active retrieval and release of solutes (lateral transport) also occur through carriers of the SE plasma membrane along the transport phloem. Release phloem Release phloem is responsible for releasing photoassimilates in expanding or accumulating organs, mainly by symplasmic routes. Transport phloem: the best represented in transcriptome profiling studies The most detailed studies are in dicot species and describe mainly the transport phloem [5,7,10,11,13]. The collection phloem has been analysed by EST analysis covering CCs in Arabidopsis [8] and covering leaf vasculature in common plantain [16]. Release phloem has been analyzed in a single study covering the stele of the root of Arabidopsis [7]. A variety of cell types The SEs are enucleated and highly differentiated living cells that characterize the phloem. They are associated with CCs and PPCs. From source to sink, the proportion of CCs to SEs decreases from collection phloem to transport phloem to release phloem [4]. Arabidopsis transport phloem complex involves two other specialized cell types: the S-cells and the M-cells. The first is known to store glucosinolates in the vacuole, whereas the second reportedly possesses myrosinase activity, which hydrolyses glucosinolates [70]. These two cell types constitute the myrosinase–glucosinolate system, which is known to affect the activities of several herbivorous insects.
Figure I. Schematic representation of the main cell types found in Arabidopsis thaliana stem vasculature.
phloem-enriched tissues [7,9,12–14,16–18,20]. With the exception of two studies that involve collection phloem (Box 1; [8,16]) and one looking at the release phloem [7], all data relate to transport phloem (Table 1). The distinct transcript profiles of collection and release phloem, and 274
those of specialized phloem cell types, still require detailed large-scale investigations. Functional pattern of the transport phloem Transcript profiles show that most transcripts found in phloem are also present in other tissues (see Table S1 in the online supplementary material), but a few hundred genes are expressed preferentially in the phloem [5,7,10,13]. These phloem-preferential genes (see Table S2 in the online supplementary material) are involved in basic functions, such as cell wall remodelling, cytoskeleton organization, the ubiquitin pathway, primary metabolism and antioxidant activities. Some of these genes, encoding either markers of CC–SE metabolism or specific structural elements of CCs and SEs, can be considered as key features of CC–SE complexes (see Table S3 in the online supplementary material). For instance, transcripts encoding sucrose synthase (SuSy) have been widely detected [13,20,21] in CCs, consistent with the hypothesis that SuSy plays a central role in the sucrose metabolism in these cells [25]. Similar observations can be drawn for transcripts encoding other classes of phloem-specific enzymes that are involved in carbon primary metabolism and nitrogen remobilisation, or for transcripts that encode CC- or SEspecific structural components, such as sucrose and amino
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Table 1. Sampling methods for transcriptomic analysis of phloem tissue Sample Phloem sap
Phloem tissue
Vascularenriched tissue
Species Castor bean (Ricinus communis) Muskmelon (Cucumis melo) Pumpkin (Cucurbita maxima) Rapeseed (Brassica napus) Arabidopis thaliana
Organ Hypocotyl
Sampling method Exudation
Target tissues or cell type Phloem sap
Phloem type Transport phloem
Refs [21]
Main stem
Exudation
Phloem sap
Transport phloem
[22]
Main stem and petiole Inflorescence stem Leaf blade
Exudation
Phloem sap
Transport phloem
[23]
Exudation
Phloem sap
Transport phloem
[24]
Companion cells
Petiole
Transport and collection phloem Release phloem
[8]
Celery (Apium graveolens) Celery (A. graveolens)
Cell sorting after leaf protoplasting Peeling
Petiole
Peeling
Transport phloem
[15]
Poplar (Populus spp.) Poplar (Populus spp.) Rice (Oryza sativa)
Stem Stem Leaf
Transport phloem Transport phloem Transport phloem
[5] [11] [6]
Arabidopis thaliana
Root
Transport phloem
[7]
A. thaliana
Hypocotyl
Cryo-sectioning Cryo- sectioning Laser-capture microdissection Cell sorting after root protoplasting Dissection
Transport phloem
[19]
A. thaliana
Root and hypocotyl Leaf blade
Dissection
Transport phloem
[20] [16]
Stem
Peeling and dissection
Collection and transport phloem Transport phloem
[17]
Stem Stem
Scraping Peeling
Transport phloem Phloem fibres
[18] [14]
Stem
Peeling
Phloem fibres
[20]
Maize (Zea mays)
Stem
Transport phloem
[9]
White spruce (Picea glauca)
Stem
Laser-capture microdissection Scraping
Transport phloem
[12]
Common plantain (Plantago major) Eucalyptus (Eucalyptus grandis) Eucalyptus (E. gunnii) Flax (Linum usitatissimum) Flax (L. usitatissimum)
Peeling
acid transporters, potassium channels, PP2 phloem proteins or jacalin-related lectins (see Table S3 in the online supplementary material). The transcripts found in transport phloem to some extent act as markers for the CC–SE complex. Phloem specification: the track of transcription factors Vascular development involves the specification of meristematic cells as xylem and phloem before the specification of vascular cell identity. To date, only one gene, ALTERED PHLOEM DEVELOPMENT (APL), encoding a G2-like MYB (Myeloblastosis) transcription factor (TF), has been identified as necessary for phloem specification [26]. KANADI genes are known to alter vascular patterning [27,28]. The transport phloem of Arabidopsis, celery (Apium graveolens) and poplar (Populus spp.) contains transcripts for TFs, some of which are specific to plants [29]. These TFs include the G2-like sub-family of MYB proteins, which includes APL [7,11,13], Dof (DNA-binding with one finger) and NAM (NO APICAL MERISTEM) [5,10,11,13,20]. Some of these TFs might play important roles during phloem development, but their identification is difficult because differentiating SEs are expected to be under-represented in the tissues used for transcript studies and because key genes for phloem specification are likely to be poorly represented in the transcriptome.
Phloem, xylem and non-vascular tissues Phloem of aphid-infested and non-infested plants Phloem, cambium and xylem Phloem, cambium and xylem Phloem Stele (including phloem) and other tissue layers Xylem and bark (including phloem) Phloem and cambium layer, xylem and non-vascular tissue Vascular bundles (including minor veins) Xylem, phloem and cork Xylem and phloem Bark (including phloem fibres) and inner tissues Bark (including phloem fibres and epidermis) Vascular bundles (including phloem) and epidermis Cambium, phloem scrapings and other tissues
[10]
Other approaches, such as the use of cell sorting to target differentiating SEs, should help to elucidate phloem formation. Phloem maturation: programmed semi-cell death in the SE During phloem development, SEs undergo selective autolysis. The nucleus disintegrates, the vacuolar membranes disappear and Golgi bodies and mitochondria decline in number, while the plasma membrane and a thin layer of parietal cytoplasm composed of stacked endoplasmic reticulum (ER) and a few dilated mitochondria remain. This process has been designated ‘programmed semi-cell-death’ [4]. Autophagy plays a major role in regulating developmental programmed cell death (PCD) in plants [30], and interestingly, transcripts encoding ATG (AUTOPHAGY) proteins are represented in the transport phloem of poplar and common plantain (Plantago major) [11,16]. Transcripts encoding vacuolar-processing enzymes (VPEs) [31], candidates for apoptosis-related caspase-like activities [32], were also present in poplar phloem [11]. Moreover, transcripts encoding proteases, several of which are thought to be involved in PCD [33], were detected in the phloem of poplar, common plantain and flax (Linum usitatissimum) [11,16,20], and transcripts encoding protease inhibitors also were found in these tissues [7–10,13,15,21]. It has been suggested that such protease inhibitors 275
Review participate in temporal control of selective autophagy during SE differentiation [34]. Techniques that are commonly used to explore autolytic processes during xylem formation, such as TUNEL (transferase dUTP nick end labelling) staining, could be valuable for identifying processes that occur during the formation of SE and might help to confirm hypotheses that are based on transcript data. The wall of phloem cells The cell wall of SEs is composed of several layers, including an inner layer that exhibits a ‘pearly’ appearance, known as the nacreous layer, which has transversely oriented cellulose-rich microfibrils [35]. The walls of CCs are generally characteristic of primary walls; they are poorly lignified and rich in cellulose and pectin. Phloem transfer cells develop typical wall ingrowths, especially in response to stress [36,37]. Numerous transcripts that are involved in cell wall synthesis and remodelling have been found in the transport phloem of Arabidopsis, celery, common plantain and poplar [7,10,11,13,14,16]. Transcripts for the synthesis of callose, a typical marker for sieve plates, have also been found in the phloem of Arabidopsis [7,13]. Interestingly, transcripts for cell-wall-remodelling enzymes are abundant in flax fibres [14,20], and transcriptome studies of poplar tension wood revealed enrichment in transcripts encoding fasciclin-like arabinogalactan proteins (AGP) [38,39]. Both tension wood and flax bast are composed of cellulose-enriched fibres and the similarities observed in these transcript profiles suggest similar formation processes. Phloem cytoskeleton Cell wall formation is intimately associated with the cytoskeleton [40], which has unusual features in the phloem. Components of the cytoskeleton, including microfilaments and microtubules, are observed in PPCs, CCs and differentiating SEs but disappear in mature SEs, except in the vicinity of sieve plates [35,41,42]. Transcripts encoding atubulins, b-tubulins and microtubule-associated proteins (MAPs), as well as transcripts encoding actin-associated microfilament proteins such as myosin, actin-binding proteins, actin depolymerizing factor (ADF) and profilin, are represented in the published transcriptomes of CCs and transport phloem [8,10,13,21]. This is consistent with the observation that actin and profilin are found in phloem sap [43,44], although the high level of profilin found in the phloem sap was predicted to prevent the formation of microfilaments [45]. Schobert et al. [45] also suggested that microfilaments might be involved in the anchorage of plastids in the parietal layer of SEs. Further studies should explore the organization of the cytoskeleton in plasmodesmata that connect CCs and SEs, near sieve plates and in the peripheral regions of SEs. This might be achieved by taking advantage of recently developed highly sensitive imaging methods, such as confocal laser scanning microscopy, which allow visualization of the cytoskeleton. Subcellular trafficking and cell polarity Although enucleated, SEs contain a variety of proteins including structural proteins, such as membrane proteins or P-proteins, and proteins that are translocated 276
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long-distance by mass flow [4]. Some proteins, such as serpins, are synthesized in differentiating immature SEs [46], but most are thought to be synthesized in the CCs and exported to SEs through plasmodesmata. Macromolecule trafficking classically relies on both the endomembrane system and the cytoskeleton. The endoplasmic reticulum (ER) is in continuity between CCs and SEs [47] and might contribute to delivering proteins into mature SEs, either from the smooth ER or from the Golgi vesicles found in mature SEs. Transcripts encoding regulatory elements of the endomembrane trafficking pathway, such as SNAREs (soluble N-ethylmaleimide sensitive factor attachment receptor proteins), were found in Arabidopsis CCs [8], in the phloem of common plantain [16] and celery [10], and in the phloem sap of castor bean (Ricinus communis) [21]. Interestingly, the discovery that Scarface, a regulator of vascular patterning, encodes an ARF-GAP (ADP-ribosylation factor GTPase-activating protein) [48] supports the hypothesis that endomembrane trafficking plays a role in phloem and vascular organization. Polar localisation of transporters and receptors is expected in the various interfaces between PPCs, CCs and SEs. Accordingly, transcripts that encode auxin facilitators, such as the auxin efflux carrier PIN6 (PINFORMED 6) and the auxin-binding protein ABP1, which are putatively polarly localised, are CC-specific [8]. ABP1 was also reported in the phloem of poplar [5]. Moreover, PIN1 [5], PIN7 [7,16] and the auxin-influx facilitator AUX1 [16] also are represented in other phloem transcript databases. These observations point to phloem as a bi-directional, long-distance pathway for auxin transport. Further, they are in agreement with the existence of a steep radial concentration gradient of the endogenous auxin, indole-3-acetic acid (IAA), across the vascular cambium of Pinus sylvestris [49], which requires a regulatory system based on positional signalling. Transcription factors in phloem: signalling regulatory networks Members of several TF families, such as TGA-bZIP, ERF (ethylene response factor), MYB, Whirly and WRKY, have been implicated in the regulation of defence genes [50]. Because the phloem is known to be involved in long-distance transport of systemic signals in response to stresses, it is not surprising to find that several of these TF genes are highly expressed in the phloem of different species, including poplar, Arabidopsis, castor bean, flax and celery [5,10,11,13,20,21]. Several genes encoding Dof proteins, which are TFs that are associated with plant-specific phenomena including light, phytohormone and defence responses, are also represented in poplar and Arabidopsis transport phloem [7,11,13]. Related TFs are involved in responses to biotic and abiotic stresses, in light signalling regulation or in the control of seed germination through regulation of gibberellic acid (GA) homeostasis [51–53], and so it is tempting to speculate that such TFs are involved in phloem signalling pathways. Evidence for an active auxin-signalling pathway in the phloem Transcripts for the auxin-responsive Aux/IAA transcriptional regulators, which negatively regulate auxin-inducible
Review genes by interacting with a specific class of TFs known as auxin response factors (ARFs) [54], are found in the transport phloem of several species [5,10,13,16,20,21]. This finding suggests that the putative targets of these transcriptional regulators also are likely to be present in the phloem transcriptome. Accordingly, transcripts of putative orthologs of ARF1, ARF2, ARF3 and ARF8 have been detected in the transport phloem of plantain, poplar and flax [11,16,20]. The presence of both auxin-inducible transcripts that correspond to SAUR and GH3-like genes [5,20] and non-canonical auxin-inducible transcripts in all studies strongly suggests that several auxin-related regulatory pathways are active in the phloem. Regulation of ARF activity depends on the ubiquitin-dependent proteolysis of Aux/IAAs, after binding to the ubiquitin-ligase complex, SCFTIR1, in an auxin-dependent manner [55,56]. Ubiquitin-mediated proteolysis appears to be at the centre of hormone signalling cascades, including auxin responses and cross-talk with stress responses [57]. It is interesting to note, therefore, that many transcripts encoding ubiquitination enzymes, including E1 ubiquitin-activating enzyme, E2 ubiquitinconjugating enzyme, RING-H2 finger E3 ubiquitin-ligase and polyubiquitin, are represented in the transport phloem of celery, poplar and Arabidopsis CCs [8,10,11]. The role of the ubiquitin signalling pathway in phloem has not yet been dissected but it is likely to be a key regulator of phloembased signalling. Stress response in the phloem SEs drive the translocation of a range of signals, including hormones, sucrose, calcium and electrical waves. Thus the phloem is involved in the production or amplification of signals that are released into the phloem sap stream in response to stress [58], and in the production and storage of repellent or antimicrobial compounds acting in defence mechanisms. Transcripts annotated as encoding defenceor stress-related proteins have been found in the phloem or in the phloem sap [22,23,59,60]; they encode defensins, gamma-thionins, PR-proteins, universal-stress proteins, proteases and proteins annotated as stress-responsive. Nevertheless, caution is required when interpreting these data. The annotation of stress-related genes is often highly preliminary and can be misleading with regard to the actual function of such genes in the phloem. Moreover, part of the stress response might result from the sampling method (Box 2), although stress-related genes are expressed in the phloem at a steady-state level in the absence of stress [7,10]. Transcripts encoding proteins that are involved in antioxidant responses, such as copper homeostasis factor, metallothioneins, blue copper-binding proteins and enzymes involved in the metabolism of reactive oxygen species, were also found in the phloem of Arabidopsis, celery, and poplar [7,8,10,11,13]. One study investigated the systemic response to an aphid infestation in the phloem of celery [15]. A few stress-related genes were systemically up-regulated in the transport phloem following aphid infestation [15]. This was associated with a complex response, which included both metabolic reprogramming and cell wall modifications, reflecting complex crosstalk between the metabolic status of the plant and stress responses through the phloem. The mechanism by
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which the phloem coordinates long-distance plant responses in plant–pathogen and plant–insect interactions is yet to be understood. Nevertheless, evidence of the expression of stress-related genes in the phloem suggests that unknown post-transcriptional or post-translational regulatory mechanisms are likely to operate in the phloem during stress responses. RNA trafficking in the phloem sap Grafting and transient expression experiments revealed that posttranscriptional gene silencing (PTGS), an RNAbased plant immune system that sequence-specifically down-regulates complementary RNAs, is not cell autonomous [61,62]. This evidence might suggest that mobile RNAs could mediate the systemic PTGS. The discovery that small RNAs are associated with PTGS [63] sheds new light on the potential nature of the mobile RNA molecules involved in systemic PGTS. The presence of RNA transcripts and small RNAs in phloem sap has been investigated in detail in species for which phloem exudation yields a sufficient volume of sap (Table 1, Box 2). Large-scale identification of the mRNAs present in the phloem sap of castor bean, pumpkin (Cucurbita maxima) and melon (Cucumis melo) plants has also been reported ([21–23], Box 3). Transcripts that are related to stress responses are abundant [21,22], but sequences related to general metabolism, transcription factors, ubiquitin-conjugating activities and signal transduction, including abscisic acid (ABA) and auxin signalling components, are also well represented [22,23]. In melon [22], several transcripts were shown to be phloem mobile (Box 3), supporting the hypothesis that RNA participates in long-distance non-cellautonomous signalling processes [2]. Both microRNAs (miRNAs) and small interfering RNAs (siRNAs) have been cloned from the phloem sap of cucurbits, castor bean, Yucca and lupin (Lupinus albus) [64]. Recently, a large-scale description of the small RNAs present in the phloem sap of rapeseed (Brassica napus) [24] provided additional evidence regarding their potential role in long-distance trafficking (Box 3). This study also showed that the amounts of some, although not all, miRNAs increase in the phloem sap in response to stress ([24]; Box 3). The hypothesis that some miRNAs can be transported by the phloem was tested recently. Grafting experiments showed that Arabidopsis miR399 is transported by the phloem and actively downregulates its target mRNAs in recipient tissues, suggesting that phloem mobility could impact some miRNA–target interactions [65]. Caution is warranted, however, when interpreting the presence of small RNAs in the phloem sap because, to some extent, these observations contradict the conclusions from earlier reports suggesting that miRNAs act cell autonomously [66–68]. Therefore, the role of any phloem-mobile miRNA in the systemic control of a biological process might have to be analysed on a case-by-case basis, as is the case for mRNAs. Mechanisms regulating RNA trafficking Cell-to-cell and long-distance trafficking of macromolecules probably also rely on endogenous mechanisms for selecting and docking of RNAs on the transport pathways 277
Review Box 3. Translocation of RNA in phloem sap: still a controversial debate RNA transcripts and small RNAs are present in the phloem sap [21– 24,64]; some are translocated in the phloem sap by mass flow, supporting the notion that phloem could support the long-distance trafficking of a range of RNAs. The discovery that some RNA transcripts and at least one miRNA are graft transmissible [64], together with a recent report suggesting that a significant fraction of mRNAs present in the phloem sap are mobile, indicates that at least some RNAs can be transported long distances through the phloem [2,3,22]. Although the biological relevance of RNA mobility is not known, one exciting possibility is that these RNAs could function as long-distance signals that control plant developmental and physiological processes [2,3]. No long-distance activity for such RNAs has yet been proven, and this constitutes a challenge for researchers. The difficulty resides in the observation that the long-distance trafficking of an mRNA might parallel the long-distance trafficking of its own encoded protein, leading to the possibility that both mRNAs and proteins are active signalling molecules. This is illustrated by the recent controversy regarding the role of FT (FLOWERING LOCUS T) mRNA and protein as the florigen [75]. RNAs present in the phloem sap might also simply reflect the outflow of abundant RNA species from CCs. Indirect evidence suggests there might be selective and controlled translocation of mRNAs from CCs to SEs, and that some, but not all, mRNAs present in the SEs traffic long distances [22,64,76]. The differential accumulation of miRNAs in the phloem sap in response to a stress also suggests selective loading of small RNAs into SEs [24,65]. Future challenges include a more detailed description of mobile RNAs, the identification of mechanisms that are involved in the regulation and selectivity of loading and transport into SEs, and description of their physiological significance.
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One limitation of current approaches is that they look at only abundant and transcriptionally active phloem cells, especially CCs and PPCs, although some structural components of SEs are encoded by transcripts expressed in immature SEs [72]. Immature differentiating SEs, which represent limited fractions of phloem tissue [1], are probably poorly represented. The ability to isolate functional SE protoplasts [73] appears to be a major breakthrough in investigating classes of genes that are expressed in differentiating SEs. Another challenge for the future is the comprehensive description of the diversity of RNAs that are trafficked long-distance in phloem sap. This requires new approaches that can discriminate the role of mRNAs and their encoded proteins, as well as efficient experimental procedures that can demonstrate long distance transport, such as grafting. Studying the subsets of RNAs and proteins present in the phloem sap might uncover not only the mechanisms of transport from CCs to SEs but also the biological relevance of mobile RNAs in long-distance communication pathways. Several studies recently described phloem proteomes [21,44,59,74], allowing the identification of several hundred proteins. A clear identification of phloem-specific proteins, including those trafficked in the phloem sap and those controlling the trafficking of other macromolecules, presents an exciting opportunity for elucidating long-distance signalling processes involving phloem. Acknowledgements
[2]. Transcripts encoding RNA-binding proteins and chaperones are found in Arabidopsis CCs [8], in the phloem of poplar and celery [8,10,11,21,22] and in melon phloem sap [22]. If the corresponding RNA-binding proteins were also found in the phloem, then this would provide additional support for an RNA-based long-distance communication network. The next step for researchers is therefore to inventory which RNA-binding proteins are present in the phloem sap and to identify their RNA targets, they might then better understand how these proteins act as control points for macromolecule trafficking.
We thank the anonymous reviewers for their valuable comments, which improved the manuscript. We also thank Allison Mallory for proofreading the manuscript. Work in our laboratories is supported by INRA (C.B., J.B., S.D. and F.V.), by the Swedish Research Council FORMAS and by the Swedish Foundation for Strategic Research (R.L. and C.B.). This project is also supported by specific grants from the INRA–FORMAS cooperation program (C.B. and S.D.). R.L. is the recipient of a specific fellowship from the Carl Trygger Foundation.
Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.tplants.2008.03.006. References
Future issues The studies described in this review have provided a first description of genes that are preferentially expressed in transport phloem. However, the phloem is composed of a variety of cell types that are involved in the production, storage, retrieval and release of metabolites, ions and macromolecules. Such processes are intricately linked to developmental stage and to source–sink transitions, and there might well be progressive switching between loading, retention, retrieval and release activities. Detailed information on most phloem compartments, including collection and release phloem, is still lacking. An area for focus in the future will be to determine the dynamic spatial and temporal expression patterns of key phloem genes. Such studies will require the optimisation of new tools for phloem-cell sorting, which include flow cytometry, cell sorting based on promoters that are active in only a subset of phloem cells (e.g. minor vein CCs, differentiating SEs, Scells or M-cells [69,70]) and microdissection of phloem tissues [71]. 278
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Gene expression profiling: keys for investigating phloem functions Rozenn Le Hir2, Julie Beneteau1, Catherine Bellini1,2, Franc¸oise Vilaine1 and Sylvie Dinant1 1
INRA UR501 Laboratoire de Biologie Cellulaire, Institut Jean Pierre Bourgin, F78000 Versailles, France Umea˚ Plant Science Center — Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Sciences, 901 83 Umea˚, Sweden 2
Phloem is the major route for transport of carbohydrates, amino acids, and other nutrients from source to sink tissues. Hormones, mRNAs, small RNAs and proteins also are transported by the phloem, and potentially play pivotal roles in communication between organs to coordinate plant development and physiology. A comprehensive understanding of the mechanisms involved in phloem transport and signalling is still lacking. Recent transcript profiling in several plant species has provided new insights to phloem-specialized functions. Here, we review conclusions regarding the unique functions of the phloem and discuss putative roles for mRNAs and small RNA species in long-distance signalling. A superhighway based on highly specialized cells In land plants, the fixation of carbon and its conversion to carbohydrates is restricted to photosynthetic organs. The phloem transports these sugars, as well as other nutrients such as amino acids and electrolytes, to heterotrophic tissues [1]. Hormones, proteins and RNAs also are found in the phloem sap, and potentially are involved in longdistance signalling through the phloem [2,3]. The phloem performs a number of distinct tasks when collecting metabolites and signals from source organs, transporting these molecules and then releasing them to sink organs (Box 1). Therefore, the phloem has great potential to facilitate inter-organ coordination and hence to promote plant growth and development [4]. Long-distance translocation, driven by mass flow, takes place through the phloem sieve elements (SEs), which are enucleated and highly specialized living cells. Other phloem cell types such as companion cells (CCs), phloem parenchyma cells (PPCs) and, in few species, fibres, play important roles in the production, storage and delivery of metabolites and signals. Symplasmic and apoplasmic connections between CCs and SEs are crucial for loading into, retrieval from and release from SEs. Therefore CC–SE complexes are considered to be the functional units of phloem conduits. The structure of CC– SE complexes also reflects their various functions in source or sink organs (Box 1). Our knowledge of phloem functions is currently being extended by novel large-scale approaches to identify genes that are expressed in the phloem. Here, we review data from transcript profiling studies and aim to provide an inventory of the representative gene families that could be associated with specific Corresponding author: Dinant, S. ([email protected]).
functions of CCs, SEs or PPCs, and which could control key features of phloem cells. Our survey is based on currently available gene annotations, with their inherent limitations. We use the word ‘phloem’ to refer to the whole tissue, including PPCs, CCs and SEs, unless otherwise specified. Analyzing phloem transcriptomes Recently improved phloem collection methods (Box 2), together with technical advances in expression profiling, have allowed transcriptional studies of phloem tissue and of individual phloem cell-types such as CCs [5–20]*. These transcriptome studies (Table 1) provide insights into the physiology and formation of the phloem and its involvement in signalling processes, and have the potential to uncover the mechanisms of macromolecule trafficking in the phloem [2]. The ability to collect phloem sap (Box 2) has also allowed the large-scale identification of RNA transcripts and small RNAs that reside in the phloem sap (Table 1; [21–24]), suggesting that phloem could transport RNAs over long distances. Transcript profiling of the phloem: a signature of phloem cells? Transcriptome analyses allow gene discovery through the identification of expressed sequence tags (ESTs) in species whose genome is not sequenced, quantification of transcripts present in the phloem, and comparison with the transcript profiles of other tissues. In addition to work on Arabidopsis [7,13], gene profiling studies on diverse plant species have identified subsets of genes, ranging from one hundred to several thousand RNAs, that are expressed in the phloem (see Table S1 in the online supplementary material). The phloem transcriptome data available from dicots, monocots and a gymnosperm (Table 1) represent a valuable genomic resource for studying the biological diversity of gene families that are expressed in the phloem. They also highlight common features of phloem functions in diverse plant species, providing new clues to unexplored mechanisms. Although one study profiles ESTs from CCs [8], most studies encompass either phloem whole tissue, including CCs, SEs and PPCs [5,6,10,11,15], or *
Note added in proof An alternative to specific sorting of phloem cells relies on transgenic expression of nuclear-targeted GFP and analysis of transcripts in GFP-positive nuclei [77]. This approach was successfully applied to identify in Arabidopsis thaliana additional sets of genes expressed in companion cells of roots.
1360-1385/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2008.03.006 Available online 14 May 2008
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Box 1. Transport phloem: an intricacy of distinct cell types
Box 2. Tricks and concerns for phloem sampling
Phloem: three main functional compartments
A straightforward approach to analyzing the functions of a particular tissue involves isolating this tissue and analyzing the pools of mRNAs or proteins present, deducing subsets of specific genes that are involved in its functioning. Obstacles to the study of phloem include the inaccessibility of phloem cells in the plant body, the small fraction of phloem within organ tissues, and the small size of phloem cells, especially SEs [1]. Recently, methods for collecting small tissue samples were adapted to species for which the phloem could be isolated, and these methods were used for transcript profiling studies. The availability of phloem tissues varies depending on species and organs. In common plantain or celery, vascular bundles can be peeled from either leaves or petioles [10,15,16]. Dissection has been used to isolate the phloem of species with cellulose fibres, such as flax [14,20] or woody stems [5,11,12], and for Arabidopsis hypocotyls [13]. Laser microdissection has been applied for gene analysis of vascular tissues in both monocots and dicots [6,9]. Flow cytometry and cell sorting of protoplasts from fluorescent reporter lines were successfully applied to lines expressing the green fluorescent protein (GFP) under the control of the companion-cell-specific SUC2 promoter [7,8]*. Alternatively, phloem sap, which also contains RNAs, can be analyzed [2,64]. Phloem sap can be sampled by exudation in few species, such as cucurbits, castor bean and rape. In other major model plant species, including Arabidopsis, the small volume of phloem sap exudates collected precludes analysis. Stylectomy, another sap-sampling method, which yields nanolitre amounts of sap, has not yet been used for large-scale studies of RNAs. A major concern relating to these techniques is that they are invasive and potentially induce local responses to wounding of the phloem tissues, although methods such as exudation or peeling are thought to cause minimal injury. SEs react vigorously to any injury by rapid sealing of damaged tubes [4], and transcripts encoding defence- or stress-related proteins have frequently been found in phloem transcriptome studies. This raises the possibility that transcript profiles of the phloem could include transcripts from nearby or phloem-damaged cells. The effects of sampling method on the induction of stress-related genes have been evaluated in two studies [7,10], which showed that several stress-related genes, including some phloem-specific genes, are expressed in the phloem at a steady-state level.
Collection phloem The site of photoassimilate loading is in the minor veins of mature photosynthetic leaves. Phloem loading proceeds through either apoplasmic or symplasmic pathways. In apoplasmic-loader species, such as Arabidopsis (Figure I), sucrose is loaded actively into the CC–SE by transmembrane carriers. In symplasmic loaders, such as Cucurbitaceae, the loading occurs against a sucrose gradient through the numerous plasmodesmata that connect PPCs and CCs. In both modes of loading, CCs and SEs are connected through pore-plasmodesmata units (PPUs), with highly branched plasmodesmata at the CC side. Transport phloem In main veins, petioles, stems and primary roots transport phloem is responsible for long distance photoassimilate transport from source to sink organs. Active retrieval and release of solutes (lateral transport) also occur through carriers of the SE plasma membrane along the transport phloem. Release phloem Release phloem is responsible for releasing photoassimilates in expanding or accumulating organs, mainly by symplasmic routes. Transport phloem: the best represented in transcriptome profiling studies The most detailed studies are in dicot species and describe mainly the transport phloem [5,7,10,11,13]. The collection phloem has been analysed by EST analysis covering CCs in Arabidopsis [8] and covering leaf vasculature in common plantain [16]. Release phloem has been analyzed in a single study covering the stele of the root of Arabidopsis [7]. A variety of cell types The SEs are enucleated and highly differentiated living cells that characterize the phloem. They are associated with CCs and PPCs. From source to sink, the proportion of CCs to SEs decreases from collection phloem to transport phloem to release phloem [4]. Arabidopsis transport phloem complex involves two other specialized cell types: the S-cells and the M-cells. The first is known to store glucosinolates in the vacuole, whereas the second reportedly possesses myrosinase activity, which hydrolyses glucosinolates [70]. These two cell types constitute the myrosinase–glucosinolate system, which is known to affect the activities of several herbivorous insects.
Figure I. Schematic representation of the main cell types found in Arabidopsis thaliana stem vasculature.
phloem-enriched tissues [7,9,12–14,16–18,20]. With the exception of two studies that involve collection phloem (Box 1; [8,16]) and one looking at the release phloem [7], all data relate to transport phloem (Table 1). The distinct transcript profiles of collection and release phloem, and 274
those of specialized phloem cell types, still require detailed large-scale investigations. Functional pattern of the transport phloem Transcript profiles show that most transcripts found in phloem are also present in other tissues (see Table S1 in the online supplementary material), but a few hundred genes are expressed preferentially in the phloem [5,7,10,13]. These phloem-preferential genes (see Table S2 in the online supplementary material) are involved in basic functions, such as cell wall remodelling, cytoskeleton organization, the ubiquitin pathway, primary metabolism and antioxidant activities. Some of these genes, encoding either markers of CC–SE metabolism or specific structural elements of CCs and SEs, can be considered as key features of CC–SE complexes (see Table S3 in the online supplementary material). For instance, transcripts encoding sucrose synthase (SuSy) have been widely detected [13,20,21] in CCs, consistent with the hypothesis that SuSy plays a central role in the sucrose metabolism in these cells [25]. Similar observations can be drawn for transcripts encoding other classes of phloem-specific enzymes that are involved in carbon primary metabolism and nitrogen remobilisation, or for transcripts that encode CC- or SEspecific structural components, such as sucrose and amino
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Table 1. Sampling methods for transcriptomic analysis of phloem tissue Sample Phloem sap
Phloem tissue
Vascularenriched tissue
Species Castor bean (Ricinus communis) Muskmelon (Cucumis melo) Pumpkin (Cucurbita maxima) Rapeseed (Brassica napus) Arabidopis thaliana
Organ Hypocotyl
Sampling method Exudation
Target tissues or cell type Phloem sap
Phloem type Transport phloem
Refs [21]
Main stem
Exudation
Phloem sap
Transport phloem
[22]
Main stem and petiole Inflorescence stem Leaf blade
Exudation
Phloem sap
Transport phloem
[23]
Exudation
Phloem sap
Transport phloem
[24]
Companion cells
Petiole
Transport and collection phloem Release phloem
[8]
Celery (Apium graveolens) Celery (A. graveolens)
Cell sorting after leaf protoplasting Peeling
Petiole
Peeling
Transport phloem
[15]
Poplar (Populus spp.) Poplar (Populus spp.) Rice (Oryza sativa)
Stem Stem Leaf
Transport phloem Transport phloem Transport phloem
[5] [11] [6]
Arabidopis thaliana
Root
Transport phloem
[7]
A. thaliana
Hypocotyl
Cryo-sectioning Cryo- sectioning Laser-capture microdissection Cell sorting after root protoplasting Dissection
Transport phloem
[19]
A. thaliana
Root and hypocotyl Leaf blade
Dissection
Transport phloem
[20] [16]
Stem
Peeling and dissection
Collection and transport phloem Transport phloem
[17]
Stem Stem
Scraping Peeling
Transport phloem Phloem fibres
[18] [14]
Stem
Peeling
Phloem fibres
[20]
Maize (Zea mays)
Stem
Transport phloem
[9]
White spruce (Picea glauca)
Stem
Laser-capture microdissection Scraping
Transport phloem
[12]
Common plantain (Plantago major) Eucalyptus (Eucalyptus grandis) Eucalyptus (E. gunnii) Flax (Linum usitatissimum) Flax (L. usitatissimum)
Peeling
acid transporters, potassium channels, PP2 phloem proteins or jacalin-related lectins (see Table S3 in the online supplementary material). The transcripts found in transport phloem to some extent act as markers for the CC–SE complex. Phloem specification: the track of transcription factors Vascular development involves the specification of meristematic cells as xylem and phloem before the specification of vascular cell identity. To date, only one gene, ALTERED PHLOEM DEVELOPMENT (APL), encoding a G2-like MYB (Myeloblastosis) transcription factor (TF), has been identified as necessary for phloem specification [26]. KANADI genes are known to alter vascular patterning [27,28]. The transport phloem of Arabidopsis, celery (Apium graveolens) and poplar (Populus spp.) contains transcripts for TFs, some of which are specific to plants [29]. These TFs include the G2-like sub-family of MYB proteins, which includes APL [7,11,13], Dof (DNA-binding with one finger) and NAM (NO APICAL MERISTEM) [5,10,11,13,20]. Some of these TFs might play important roles during phloem development, but their identification is difficult because differentiating SEs are expected to be under-represented in the tissues used for transcript studies and because key genes for phloem specification are likely to be poorly represented in the transcriptome.
Phloem, xylem and non-vascular tissues Phloem of aphid-infested and non-infested plants Phloem, cambium and xylem Phloem, cambium and xylem Phloem Stele (including phloem) and other tissue layers Xylem and bark (including phloem) Phloem and cambium layer, xylem and non-vascular tissue Vascular bundles (including minor veins) Xylem, phloem and cork Xylem and phloem Bark (including phloem fibres) and inner tissues Bark (including phloem fibres and epidermis) Vascular bundles (including phloem) and epidermis Cambium, phloem scrapings and other tissues
[10]
Other approaches, such as the use of cell sorting to target differentiating SEs, should help to elucidate phloem formation. Phloem maturation: programmed semi-cell death in the SE During phloem development, SEs undergo selective autolysis. The nucleus disintegrates, the vacuolar membranes disappear and Golgi bodies and mitochondria decline in number, while the plasma membrane and a thin layer of parietal cytoplasm composed of stacked endoplasmic reticulum (ER) and a few dilated mitochondria remain. This process has been designated ‘programmed semi-cell-death’ [4]. Autophagy plays a major role in regulating developmental programmed cell death (PCD) in plants [30], and interestingly, transcripts encoding ATG (AUTOPHAGY) proteins are represented in the transport phloem of poplar and common plantain (Plantago major) [11,16]. Transcripts encoding vacuolar-processing enzymes (VPEs) [31], candidates for apoptosis-related caspase-like activities [32], were also present in poplar phloem [11]. Moreover, transcripts encoding proteases, several of which are thought to be involved in PCD [33], were detected in the phloem of poplar, common plantain and flax (Linum usitatissimum) [11,16,20], and transcripts encoding protease inhibitors also were found in these tissues [7–10,13,15,21]. It has been suggested that such protease inhibitors 275
Review participate in temporal control of selective autophagy during SE differentiation [34]. Techniques that are commonly used to explore autolytic processes during xylem formation, such as TUNEL (transferase dUTP nick end labelling) staining, could be valuable for identifying processes that occur during the formation of SE and might help to confirm hypotheses that are based on transcript data. The wall of phloem cells The cell wall of SEs is composed of several layers, including an inner layer that exhibits a ‘pearly’ appearance, known as the nacreous layer, which has transversely oriented cellulose-rich microfibrils [35]. The walls of CCs are generally characteristic of primary walls; they are poorly lignified and rich in cellulose and pectin. Phloem transfer cells develop typical wall ingrowths, especially in response to stress [36,37]. Numerous transcripts that are involved in cell wall synthesis and remodelling have been found in the transport phloem of Arabidopsis, celery, common plantain and poplar [7,10,11,13,14,16]. Transcripts for the synthesis of callose, a typical marker for sieve plates, have also been found in the phloem of Arabidopsis [7,13]. Interestingly, transcripts for cell-wall-remodelling enzymes are abundant in flax fibres [14,20], and transcriptome studies of poplar tension wood revealed enrichment in transcripts encoding fasciclin-like arabinogalactan proteins (AGP) [38,39]. Both tension wood and flax bast are composed of cellulose-enriched fibres and the similarities observed in these transcript profiles suggest similar formation processes. Phloem cytoskeleton Cell wall formation is intimately associated with the cytoskeleton [40], which has unusual features in the phloem. Components of the cytoskeleton, including microfilaments and microtubules, are observed in PPCs, CCs and differentiating SEs but disappear in mature SEs, except in the vicinity of sieve plates [35,41,42]. Transcripts encoding atubulins, b-tubulins and microtubule-associated proteins (MAPs), as well as transcripts encoding actin-associated microfilament proteins such as myosin, actin-binding proteins, actin depolymerizing factor (ADF) and profilin, are represented in the published transcriptomes of CCs and transport phloem [8,10,13,21]. This is consistent with the observation that actin and profilin are found in phloem sap [43,44], although the high level of profilin found in the phloem sap was predicted to prevent the formation of microfilaments [45]. Schobert et al. [45] also suggested that microfilaments might be involved in the anchorage of plastids in the parietal layer of SEs. Further studies should explore the organization of the cytoskeleton in plasmodesmata that connect CCs and SEs, near sieve plates and in the peripheral regions of SEs. This might be achieved by taking advantage of recently developed highly sensitive imaging methods, such as confocal laser scanning microscopy, which allow visualization of the cytoskeleton. Subcellular trafficking and cell polarity Although enucleated, SEs contain a variety of proteins including structural proteins, such as membrane proteins or P-proteins, and proteins that are translocated 276
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long-distance by mass flow [4]. Some proteins, such as serpins, are synthesized in differentiating immature SEs [46], but most are thought to be synthesized in the CCs and exported to SEs through plasmodesmata. Macromolecule trafficking classically relies on both the endomembrane system and the cytoskeleton. The endoplasmic reticulum (ER) is in continuity between CCs and SEs [47] and might contribute to delivering proteins into mature SEs, either from the smooth ER or from the Golgi vesicles found in mature SEs. Transcripts encoding regulatory elements of the endomembrane trafficking pathway, such as SNAREs (soluble N-ethylmaleimide sensitive factor attachment receptor proteins), were found in Arabidopsis CCs [8], in the phloem of common plantain [16] and celery [10], and in the phloem sap of castor bean (Ricinus communis) [21]. Interestingly, the discovery that Scarface, a regulator of vascular patterning, encodes an ARF-GAP (ADP-ribosylation factor GTPase-activating protein) [48] supports the hypothesis that endomembrane trafficking plays a role in phloem and vascular organization. Polar localisation of transporters and receptors is expected in the various interfaces between PPCs, CCs and SEs. Accordingly, transcripts that encode auxin facilitators, such as the auxin efflux carrier PIN6 (PINFORMED 6) and the auxin-binding protein ABP1, which are putatively polarly localised, are CC-specific [8]. ABP1 was also reported in the phloem of poplar [5]. Moreover, PIN1 [5], PIN7 [7,16] and the auxin-influx facilitator AUX1 [16] also are represented in other phloem transcript databases. These observations point to phloem as a bi-directional, long-distance pathway for auxin transport. Further, they are in agreement with the existence of a steep radial concentration gradient of the endogenous auxin, indole-3-acetic acid (IAA), across the vascular cambium of Pinus sylvestris [49], which requires a regulatory system based on positional signalling. Transcription factors in phloem: signalling regulatory networks Members of several TF families, such as TGA-bZIP, ERF (ethylene response factor), MYB, Whirly and WRKY, have been implicated in the regulation of defence genes [50]. Because the phloem is known to be involved in long-distance transport of systemic signals in response to stresses, it is not surprising to find that several of these TF genes are highly expressed in the phloem of different species, including poplar, Arabidopsis, castor bean, flax and celery [5,10,11,13,20,21]. Several genes encoding Dof proteins, which are TFs that are associated with plant-specific phenomena including light, phytohormone and defence responses, are also represented in poplar and Arabidopsis transport phloem [7,11,13]. Related TFs are involved in responses to biotic and abiotic stresses, in light signalling regulation or in the control of seed germination through regulation of gibberellic acid (GA) homeostasis [51–53], and so it is tempting to speculate that such TFs are involved in phloem signalling pathways. Evidence for an active auxin-signalling pathway in the phloem Transcripts for the auxin-responsive Aux/IAA transcriptional regulators, which negatively regulate auxin-inducible
Review genes by interacting with a specific class of TFs known as auxin response factors (ARFs) [54], are found in the transport phloem of several species [5,10,13,16,20,21]. This finding suggests that the putative targets of these transcriptional regulators also are likely to be present in the phloem transcriptome. Accordingly, transcripts of putative orthologs of ARF1, ARF2, ARF3 and ARF8 have been detected in the transport phloem of plantain, poplar and flax [11,16,20]. The presence of both auxin-inducible transcripts that correspond to SAUR and GH3-like genes [5,20] and non-canonical auxin-inducible transcripts in all studies strongly suggests that several auxin-related regulatory pathways are active in the phloem. Regulation of ARF activity depends on the ubiquitin-dependent proteolysis of Aux/IAAs, after binding to the ubiquitin-ligase complex, SCFTIR1, in an auxin-dependent manner [55,56]. Ubiquitin-mediated proteolysis appears to be at the centre of hormone signalling cascades, including auxin responses and cross-talk with stress responses [57]. It is interesting to note, therefore, that many transcripts encoding ubiquitination enzymes, including E1 ubiquitin-activating enzyme, E2 ubiquitinconjugating enzyme, RING-H2 finger E3 ubiquitin-ligase and polyubiquitin, are represented in the transport phloem of celery, poplar and Arabidopsis CCs [8,10,11]. The role of the ubiquitin signalling pathway in phloem has not yet been dissected but it is likely to be a key regulator of phloembased signalling. Stress response in the phloem SEs drive the translocation of a range of signals, including hormones, sucrose, calcium and electrical waves. Thus the phloem is involved in the production or amplification of signals that are released into the phloem sap stream in response to stress [58], and in the production and storage of repellent or antimicrobial compounds acting in defence mechanisms. Transcripts annotated as encoding defenceor stress-related proteins have been found in the phloem or in the phloem sap [22,23,59,60]; they encode defensins, gamma-thionins, PR-proteins, universal-stress proteins, proteases and proteins annotated as stress-responsive. Nevertheless, caution is required when interpreting these data. The annotation of stress-related genes is often highly preliminary and can be misleading with regard to the actual function of such genes in the phloem. Moreover, part of the stress response might result from the sampling method (Box 2), although stress-related genes are expressed in the phloem at a steady-state level in the absence of stress [7,10]. Transcripts encoding proteins that are involved in antioxidant responses, such as copper homeostasis factor, metallothioneins, blue copper-binding proteins and enzymes involved in the metabolism of reactive oxygen species, were also found in the phloem of Arabidopsis, celery, and poplar [7,8,10,11,13]. One study investigated the systemic response to an aphid infestation in the phloem of celery [15]. A few stress-related genes were systemically up-regulated in the transport phloem following aphid infestation [15]. This was associated with a complex response, which included both metabolic reprogramming and cell wall modifications, reflecting complex crosstalk between the metabolic status of the plant and stress responses through the phloem. The mechanism by
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which the phloem coordinates long-distance plant responses in plant–pathogen and plant–insect interactions is yet to be understood. Nevertheless, evidence of the expression of stress-related genes in the phloem suggests that unknown post-transcriptional or post-translational regulatory mechanisms are likely to operate in the phloem during stress responses. RNA trafficking in the phloem sap Grafting and transient expression experiments revealed that posttranscriptional gene silencing (PTGS), an RNAbased plant immune system that sequence-specifically down-regulates complementary RNAs, is not cell autonomous [61,62]. This evidence might suggest that mobile RNAs could mediate the systemic PTGS. The discovery that small RNAs are associated with PTGS [63] sheds new light on the potential nature of the mobile RNA molecules involved in systemic PGTS. The presence of RNA transcripts and small RNAs in phloem sap has been investigated in detail in species for which phloem exudation yields a sufficient volume of sap (Table 1, Box 2). Large-scale identification of the mRNAs present in the phloem sap of castor bean, pumpkin (Cucurbita maxima) and melon (Cucumis melo) plants has also been reported ([21–23], Box 3). Transcripts that are related to stress responses are abundant [21,22], but sequences related to general metabolism, transcription factors, ubiquitin-conjugating activities and signal transduction, including abscisic acid (ABA) and auxin signalling components, are also well represented [22,23]. In melon [22], several transcripts were shown to be phloem mobile (Box 3), supporting the hypothesis that RNA participates in long-distance non-cellautonomous signalling processes [2]. Both microRNAs (miRNAs) and small interfering RNAs (siRNAs) have been cloned from the phloem sap of cucurbits, castor bean, Yucca and lupin (Lupinus albus) [64]. Recently, a large-scale description of the small RNAs present in the phloem sap of rapeseed (Brassica napus) [24] provided additional evidence regarding their potential role in long-distance trafficking (Box 3). This study also showed that the amounts of some, although not all, miRNAs increase in the phloem sap in response to stress ([24]; Box 3). The hypothesis that some miRNAs can be transported by the phloem was tested recently. Grafting experiments showed that Arabidopsis miR399 is transported by the phloem and actively downregulates its target mRNAs in recipient tissues, suggesting that phloem mobility could impact some miRNA–target interactions [65]. Caution is warranted, however, when interpreting the presence of small RNAs in the phloem sap because, to some extent, these observations contradict the conclusions from earlier reports suggesting that miRNAs act cell autonomously [66–68]. Therefore, the role of any phloem-mobile miRNA in the systemic control of a biological process might have to be analysed on a case-by-case basis, as is the case for mRNAs. Mechanisms regulating RNA trafficking Cell-to-cell and long-distance trafficking of macromolecules probably also rely on endogenous mechanisms for selecting and docking of RNAs on the transport pathways 277
Review Box 3. Translocation of RNA in phloem sap: still a controversial debate RNA transcripts and small RNAs are present in the phloem sap [21– 24,64]; some are translocated in the phloem sap by mass flow, supporting the notion that phloem could support the long-distance trafficking of a range of RNAs. The discovery that some RNA transcripts and at least one miRNA are graft transmissible [64], together with a recent report suggesting that a significant fraction of mRNAs present in the phloem sap are mobile, indicates that at least some RNAs can be transported long distances through the phloem [2,3,22]. Although the biological relevance of RNA mobility is not known, one exciting possibility is that these RNAs could function as long-distance signals that control plant developmental and physiological processes [2,3]. No long-distance activity for such RNAs has yet been proven, and this constitutes a challenge for researchers. The difficulty resides in the observation that the long-distance trafficking of an mRNA might parallel the long-distance trafficking of its own encoded protein, leading to the possibility that both mRNAs and proteins are active signalling molecules. This is illustrated by the recent controversy regarding the role of FT (FLOWERING LOCUS T) mRNA and protein as the florigen [75]. RNAs present in the phloem sap might also simply reflect the outflow of abundant RNA species from CCs. Indirect evidence suggests there might be selective and controlled translocation of mRNAs from CCs to SEs, and that some, but not all, mRNAs present in the SEs traffic long distances [22,64,76]. The differential accumulation of miRNAs in the phloem sap in response to a stress also suggests selective loading of small RNAs into SEs [24,65]. Future challenges include a more detailed description of mobile RNAs, the identification of mechanisms that are involved in the regulation and selectivity of loading and transport into SEs, and description of their physiological significance.
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One limitation of current approaches is that they look at only abundant and transcriptionally active phloem cells, especially CCs and PPCs, although some structural components of SEs are encoded by transcripts expressed in immature SEs [72]. Immature differentiating SEs, which represent limited fractions of phloem tissue [1], are probably poorly represented. The ability to isolate functional SE protoplasts [73] appears to be a major breakthrough in investigating classes of genes that are expressed in differentiating SEs. Another challenge for the future is the comprehensive description of the diversity of RNAs that are trafficked long-distance in phloem sap. This requires new approaches that can discriminate the role of mRNAs and their encoded proteins, as well as efficient experimental procedures that can demonstrate long distance transport, such as grafting. Studying the subsets of RNAs and proteins present in the phloem sap might uncover not only the mechanisms of transport from CCs to SEs but also the biological relevance of mobile RNAs in long-distance communication pathways. Several studies recently described phloem proteomes [21,44,59,74], allowing the identification of several hundred proteins. A clear identification of phloem-specific proteins, including those trafficked in the phloem sap and those controlling the trafficking of other macromolecules, presents an exciting opportunity for elucidating long-distance signalling processes involving phloem. Acknowledgements
[2]. Transcripts encoding RNA-binding proteins and chaperones are found in Arabidopsis CCs [8], in the phloem of poplar and celery [8,10,11,21,22] and in melon phloem sap [22]. If the corresponding RNA-binding proteins were also found in the phloem, then this would provide additional support for an RNA-based long-distance communication network. The next step for researchers is therefore to inventory which RNA-binding proteins are present in the phloem sap and to identify their RNA targets, they might then better understand how these proteins act as control points for macromolecule trafficking.
We thank the anonymous reviewers for their valuable comments, which improved the manuscript. We also thank Allison Mallory for proofreading the manuscript. Work in our laboratories is supported by INRA (C.B., J.B., S.D. and F.V.), by the Swedish Research Council FORMAS and by the Swedish Foundation for Strategic Research (R.L. and C.B.). This project is also supported by specific grants from the INRA–FORMAS cooperation program (C.B. and S.D.). R.L. is the recipient of a specific fellowship from the Carl Trygger Foundation.
Supplementary data Supplementary data associated with this article can be found at doi:10.1016/j.tplants.2008.03.006. References
Future issues The studies described in this review have provided a first description of genes that are preferentially expressed in transport phloem. However, the phloem is composed of a variety of cell types that are involved in the production, storage, retrieval and release of metabolites, ions and macromolecules. Such processes are intricately linked to developmental stage and to source–sink transitions, and there might well be progressive switching between loading, retention, retrieval and release activities. Detailed information on most phloem compartments, including collection and release phloem, is still lacking. An area for focus in the future will be to determine the dynamic spatial and temporal expression patterns of key phloem genes. Such studies will require the optimisation of new tools for phloem-cell sorting, which include flow cytometry, cell sorting based on promoters that are active in only a subset of phloem cells (e.g. minor vein CCs, differentiating SEs, Scells or M-cells [69,70]) and microdissection of phloem tissues [71]. 278
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