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Although the phloem is a highly specialized tissue, certain pathogens, including phytoplasmas, spiroplasmas, and viruses, have evolved to access and live in this sequestered and protected environment, causing substantial economic harm. In particular, Candidatus Liberibacter spp. are devastating citrus in many parts of the world. Given that most phloem pathogens are vectored, they are not exposed to applied chemicals and are therefore difficult to control. Furthermore, pathogens use the phloem network to escape mounted defenses. Our review summarizes the current knowledge of phloem anatomy, physiology, and biochemistry relevant to phloem/pathogen interactions. We focus on aspects of anatomy specific to pathogen movement, including sieve plate structure and phloem-specific proteins. Phloem sampling techniques are discussed. Finally, pathogens that cause particular harm to the phloem of crop species are considered in detail.

Cucurbit phloem is complex, with large sieve tubes on both sides of the xylem (bicollateral phloem), and extrafascicular elements that form an intricate web linking the rest of the vasculature. Little is known of the physical interconnections between these networks or their functional specialization, largely because the extrafascicular phloem strands branch and turn at irregular angles. Here, export in the phloem from specific regions of the lamina of cucumber (Cucumis sativus L.) was mapped using carboxyfluorescein and 14 C as mobile tracers. We also mapped vascular architecture by conventional microscopy and X-ray computed tomography using optimized whole-tissue staining procedures. Differential gene expression in the internal (IP) and external phloem (EP) was analyzed by laser-capture microdissection followed by RNA-seq. The vascular bundles of the lamina form a nexus at the petiole junction, emerging in a predictable pattern, each bundle conducting photoassimilate from a specific region of the blade. The vascular bundles of the stem interconnect at the node, facilitating lateral transport around the stem. Elements of the extrafascicular phloem traverse the stem and petiole obliquely, joining the IP and EP of adjacent bundles. Using pairwise comparisons and weighted gene coexpression network analysis (WGCNA), we found differences in gene expression patterns between the petiole and stem and between IP and EP, and we identified hub genes of tissue-specific modules. Genes related to transport were expressed primarily in the EP while those involved in cell differentiation and development as well as amino acid transport and metabolism were expressed mainly in the IP.

CLas inhibits callose deposition in the sieve pores and the accumulation of reactive oxygen species to favor its cell-to-cell movement.

Huanglongbing (HLB) causes significant economic loss in citrus production worldwide. HLB is caused by Candidatus Liberibacter asiaticus (CLas), a gram-negative bacterium which inhabits the phloem exclusively. CLas infection results in accumulation of callose and reactive oxygen species in the phloem of infected plants, but little is known about the specific processes that take place during infection because of the sparse distribution of bacteria and the inaccessibility of the phloem inside the tree. In this study, we used the seed vasculatures, which accumulate a high number of CLas, as a model tissue to study CLas-host cellular interactions. In vasculature where CLas is abundant, sieve pore callose and H2O2 concentration were reduced compared to healthy seed vasculature. The expression of callose synthases (CalS) and respiratory burst oxidase homolog (RBOH) genes were downregulated in infected seeds compared to healthy ones. In leaves of HLB-infected plants, H2O2 concentration and CalS expression increased compared to uninfected leaves, but cells with CLas had lower levels of sieve plate callose compared to cells without CLas. Our results provide evidence that the bacteria manipulate cell metabolism to disable plant defenses and suggests that HLB disease is the result of a constant arms-race between the pathogen and a defense response, which is ultimately harmful to the host plant.

Made available in DSpace on 2022-04-28T17:20:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 The phloem-limited 'Candidatus Liberibacter asiaticus' (Las) causes huanglongbing, a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in the greenhouse, and the impacts of temperature on plant colonization in growth-chamber experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by quantitative PCR (qPCR) or symptoms at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, maintained at from 8 to 20 degrees C, from 18 to 30 degrees C, or from 24 to 38 degrees C daily range, and assessed by qPCR of samples taken from noninoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to analysis of variance and binomial generalized linear model and logistic regression analyses. Probabilities of detecting Las in greenhouse plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day(-1) average speed of movement. In growth chambers, the temperature impacted plant colonization by Las, new shoot emission, and symptom expression. After a 7-month exposure time, Las was absent in all new shoots in the cooler environment (average three per plant), and present in 70% at the milder environment (six shoots, severe symptoms) and 25% in the warmer environment (eight shoots, no visible symptoms). Temperature of 25.7 degrees C was the optimum condition for plant colonization. This explains the higher impact and incidence of huanglongbing disease during the winter months or regions of milder climates in Brazil. Univ Estadual Paulista, BR-14884900 Jaboticabal, SP, Brazil Fundo Def Citricultura, BR-14807040 Araraquara, SP, Brazil Univ Cambridge, Dept Plant Sci, Cambridge CB2 3EA, England Cornell Univ, Dept Plant Biol, Ithaca, NY 14853 USA Univ Estadual Paulista, BR-14884900 Jaboticabal, SP, Brazil

The phloem-limited '

The phloem of the Cucurbitaceae has long been a subject of interest due to its complex nature and the economic importance of the family. As in a limited number of other families, cucurbit phloem is bicollateral, i.e. with sieve tubes on both sides of the xylem. To date little is known about the specialized functions of the internal phloem (IP) and external phloem (EP). Here, a combination of microscopy, fluorescent dye transport analysis, micro-computed tomography, laser capture microdissection and RNA-sequencing (RNA-Seq) were used to study the functions of IP and EP in the vascular bundles (VBs) of cucumber fruit. There is one type of VB in the peduncle, but four in the fruit: peripheral (PeVB), main (MVB), carpel (CVB) and placental (PlVB). The VBs are bicollateral, except for the CVB and PlVB. Phloem mobile tracers and 14 C applied to leaves are transported primarily in the EP, and to a lesser extent in the IP. RNA-Seq data indicate preferential gene transcription in the IP related to differentiation/development, hormone transport, RNA or protein modification/processing/transport, and nitrogen compound metabolism and transport. The EP preferentially expresses genes for stimulus/stress, defense, ion transport and secondary metabolite biosynthesis. The MVB phloem is preferentially involved in photoassimilate transport, unloading and long-distance signaling, while the PeVB plays a more substantial role in morphogenesis and/or development and defense response. CVB and PlVB transcripts are biased toward development of reproductive organs. These findings provide an integrated view of the differentiated structure and function of the vascular tissue in cucumber fruit.

Most conifer species have needle-shaped leaves that are only a few centimeters long. In general, variation in leaf size has been associated with environmental factors, such as cold or drought stress. However, it has recently been proposed that sugar export efficiency is the limiting factor for conifer needle length, based on the results obtained using a mathematical model of phloem transport. Here, phloem transport rates in long conifer needles were experimentally determined to test if the mathematical model accurately represents phloem transport. The validity of the model’s assumptions was tested by anatomical analyses and sugar quantification. Furthermore, various environmental and physiological factors were tested for their correlation with needle length. The results indicate that needle length is not limited by sugar transport efficiency, but, instead, by winter temperatures and light availability. The identification of factors that influence needle size is instrumental for using this trait as a variable in breeding programs.

Recent heterograft analyses showed that large-scale messenger RNA (mRNA) movement takes place in the phloem, but the number of mobile transcripts reported varies widely. However, our knowledge of the mechanisms underlying large-scale mRNA movement remains limited. In this study, using a Nicotiana benthamiana/tomato (Solanum lycopersicum) heterograft system and a transgenic approach involving potato (Solanum tuberosum), we found that: (1) the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root; (2) increasing the expression levels of nonmobile mRNAs in the companion cells does not promote their mobility; (3) mobile mRNAs undergo degradation during their movement; and (4) some mRNAs arriving in roots move back to shoots. These results indicate that mRNA movement has both regulated and unregulated components. The cellular origins of mobile mRNAs may differ between herbaceous and woody species. Taken together, these findings suggest that the long-distance movement of mRNAs is a complex process and that elucidating the physiological roles associated with this movement is challenging but remains an important task for future research.

Significance Flowering in many plants begins with the perception of daylength in leaves. For example, some plants flower in short days, others in long days. In Arabidopsis thaliana, the leaves then transmit a small protein, FLOWERING TIME T (FT), to the shoot apex in the food-conducting cells, the phloem. Arrival of FT causes the apex to transition from leaf to flower formation. In this paper, we show that only two files of phloem cells in A. thaliana veins synthesize FT. This is also true for the Maryland Mammoth cultivar of tobacco (Nicotiana tabacum). Killing the cells specifically affects downstream, but not upstream, genes, indicating that an extensive intercellular signaling system regulates FT synthesis in the phloem., Flowering is triggered by the transmission of a mobile protein, FLOWERING LOCUS T (FT), from leaves to the shoot apex. FT originates in the phloem of leaf veins. However, the identity of the FT-synthesizing cells in the phloem is not known. As a result, it has not been possible to determine whether the complex regulatory networks that control FT synthesis involve intercellular communication, as is the case in many aspects of plant development. We demonstrate here that FT in Arabidopsis thaliana and FT orthologs in Maryland Mammoth tobacco (Nicotiana tabacum) are produced in two unique files of phloem companion cells. These FT-activating cells, visualized by fluorescent proteins, also activate the GALACTINOL SYNTHASE (CmGAS1) promoter from melon (Cucumis melo). Ablating the cells by expression of the diphtheria toxin gene driven by the CmGAS1 promoter delays flowering in both Arabidopsis and Maryland Mammoth tobacco. In Arabidopsis, toxin expression reduces expression of FT and flowering-associated genes downstream, but not upstream, of FT. Our results indicate that specific companion cells mediate the essential flowering function. Since the identified cells are present in the minor veins of two unrelated dicotyledonous species, this may be a widespread phenomenon.

In many species, sucrose en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e., symplastically. In some of these plants the process is entirely passive, but in others phloem sucrose is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic loading processes. Here we present an integrated model - including local and global transport and the kinetics of oligomerization - for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that: 1) relative to passive loading, oligomerization of sucrose in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of sucrose in the mesophyll; and 2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export.

Intensive investigations have been conducted on the effect of sole drought or salinity stress on the growth of plants. However, there is relatively little knowledge on how plants, particularly woody species, respond to a combination of these two stresses although these stresses can simultaneously occur in the field. In this study, mulberry, an economically important resource for traditional medicine, and the sole food of domesticated silkworms was subjected to a combination of salt and drought stress and analyzed by physiological methods and TMT-based proteomics. Stressed mulberry exhibited significant alteration in physiological parameters, including root/shoot ratio, chlorophyll fluorescence, total carbon, and ion reallocation. A total of 577 and 270 differentially expressed proteins (DEPs) were identified from the stressed leaves and roots, respectively. Through KEGG analysis, these DEPs were assigned to multiple pathways, including carbon metabolism, photosynthesis, redox, secondary metabolism, and hormone metabolism. Among these pathways, the sucrose related metabolic pathway was distinctly enriched in both stressed leaves and roots, indicating an important contribution in mulberry under stress condition. The results provide a comprehensive understanding of the adaptive mechanism of mulberry in response to salt and drought stress, which will facilitate further studies on innovations in terms of crop performance.

Highlight Photosynthesis by cucumber fruits, through direct fixation of atmospheric CO2 and recapture of respired CO2, makes an important contribution to fruit growth., The surface area of a mature green cucumber (Cucumis sativa L.) fruit is comparable with that of a functional leaf, but the characteristics of fruit photosynthesis and its contribution to growth are poorly understood. Here, the photosynthetic properties of two genotypes of cucumber (dark green and light green fruits) were studied using a combination of electron microscopy, immunogold enzyme localization, chlorophyll fluorescence imaging, isotope tracer, and fruit darkening techniques. Chlorophyll content of the exocarp is similar to that of leaves, but there are no distinctive palisade and spongy tissues. The efficiency of PSII is similar to that in leaves, but with lower non-photochemical quenching (NPQ). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is found mainly in the exocarp, while phosphoenolpyruvate carboxylase (PEPC) is primarily localized to vascular bundles and placenta tissue. Rubisco and PEPC expression at both transcriptional and translational levels increases concurrently during fruit growth. The contribution of fruit photosynthesis in exocarp to its own C accumulation is 9.4%, while ~88% of respiratory CO2 in fruit was captured and re-fixed. Photosynthesis by cucumber fruits, through direct fixation of atmospheric CO2 and recapture of respired CO2, as verified by 14CO2 uptake and gas exchange, makes an important contribution to fruit growth.

Despite the crucial role of carbon transport in whole plant physiology and its impact on plant-environment interactions and ecosystem function, relatively little research has tried to examine how phloem physiology impacts plant ecology. In this review, we highlight several areas of active research where inquiry into phloem physiology has increased our understanding of whole plant function and ecological processes. We consider how xylem-phloem interactions impact plant drought tolerance and reproduction, how phloem transport influences carbon allocation in trees and carbon cycling in ecosystems and how phloem function mediates plant relations with insects, pests, microbes and symbiotes. We argue that in spite of challenges that exist in studying phloem physiology, it is critical that we consider the role of this dynamic vascular system when examining the relationship between plants and their biotic and abiotic environment.

Mature leaves export approximately 80% of the carbon they fix by photosynthesis. Although most attention has been devoted to studying export of recently synthesized sugar, this is only one source of nutrients that enter the export stream. The mesophyll also supplies amino acids and other chemical species; ions and compounds are rerouted from the xylem, and companion cells have the capacity to reconfigure metabolites as they pass along the sieve tubes. The extent to which each of these channels contributes to the complexity of sieve tube content is not easy to estimate, an analytical problem made especially acute by the difficulty in obtaining authentic phloem sap.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves (this protocol), (2) the relative rates of carbon loading and transport through the phloem ( Yadav et al., 2017a ), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport ( Yadav et al., 2017b ). We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). In this protocol, Arabidopsis leaf disks isolated from mature rosette leaves are infiltrated with a buffered solution containing [(14)C]Suc. Suc transporters (SUCs or SUTs) load Suc into the phloem and excess, unloaded Suc in the leaf disk is then washed away. Loading of labeled Suc into the veins is visualized by autoradiography of lyophilized leaf disks and quantified by scintillation counting. Results are expressed as disintegration per minute per unit of leaf disk fresh weight or area.

The complex form of higher plants requires continuous, balanced transport of nutrients in the phloem. The initial step of transferring sugars, amino acids, and other materials from photosynthetic cells to the conducting sieve tubes is known as phloem loading. Three phloem loading mechanisms have been described. The first involves release of sucrose into the apoplast and subsequent retrieval by the phloem. The initial release step in this process is now known to be mediated by a new class of transporters, the SWEET proteins. In the other two loading mechanisms, polymer trapping and diffusion, sucrose passes into the phloem through cytoplasmic channels, the plasmodesmata. Recent models have shed additional light on these mechanisms and their ability to sustain the growth of even the tallest trees.

C₄ and C₃ photosynthesis differ in the efficiency with which they consume water and nitrogen. Engineering traits of the more efficient C₄ photosynthesis into C₃ crops could substantially increase crop yields in hot, arid conditions. To identify differences between C₄ and C₃ photosynthetic mechanisms, we profiled metabolites and gene expression in the developing leaves of Zea mays (maize), a C₄ plant, and Oryza sativa (rice), a C₃ plant, using a statistical method named the unified developmental model (UDM). Candidate cis-regulatory elements and transcription factors that might regulate photosynthesis were identified, together with differences between C₄ and C₃ nitrogen and carbon metabolism. The UDM algorithms could be applied to analyze and compare development in other species. These data sets together with community viewers to access and mine them provide a resource for photosynthetic research that will inform efforts to engineer improvements in carbon fixation in economically valuable grass crops.

Sap is driven through phloem sieve tubes by an osmotically generated pressure gradient between source and sink tissues. In many plants, source pressure results from thermodynamically active loading in which energy is used to transfer sucrose (Suc) from mesophyll cells to the phloem of leaf minor veins against a concentration gradient. However, in some species, almost all trees, correlative evidence suggests that sugar migrates passively through plasmodesmata from mesophyll cells into the sieve elements. The possibility of alternate loading mechanisms has important ramifications for the regulation of phloem transport and source-sink interactions. Here, we provide experimental evidence that, in gray poplar (Populus tremula × Populus alba), Suc enters the phloem through plasmodesmata. Transgenic plants were generated with yeast invertase in the cell walls to prevent Suc loading by this route. The constructs were driven either by the constitutive 35S promoter or the minor vein-specific galactinol synthase promoter. Transgenic plants grew at the same rate as the wild type without symptoms of loading inhibition, such as accumulation of carbohydrates or leaf chlorosis. Rates of photosynthesis were normal. In contrast, alfalfa (Medicago sativa) plants, which have limited numbers of plasmodesmata between mesophyll and phloem, displayed typical symptoms of loading inhibition when transformed with the same DNA constructs. The results are consistent with passive loading of Suc through plasmodesmata in poplar. We also noted defense-related symptoms in leaves of transgenic poplar when the plants were abruptly exposed to excessively high temperatures, adding to evidence that hexose is involved in triggering the hypersensitive response.

Vascular plants rely on differences in osmotic pressure to export sugars from regions of synthesis (mature leaves) to sugar sinks (roots, fruits). In this process, known as Munch pressure flow, the loading of sugars from photosynthetic cells to the export conduit (the phloem) is crucial, as it sets the pressure head necessary to power long-distance transport. Whereas most herbaceous plants use active mechanisms to increase phloem sugar concentration above that of the photosynthetic cells, in most tree species, for which transport distances are largest, loading seems, counterintuitively, to occur by means of passive symplastic diffusion from the mesophyll to the phloem. Here, we use a synthetic microfluidic model of a passive loader to explore the non-linear dynamics that arise during export and determine the ability of passive loading to drive long-distance transport. We first demonstrate that in our device, the phloem concentration is set by the balance between the resistances to diffusive loading from the source and convective export through the phloem. Convection-limited export corresponds to classical models of Munch transport, where the phloem concentration is close to that of the source; in contrast, diffusion-limited export leads to small phloem concentrations and weak scaling of flow rates with hydraulic resistance. We then show that the effective regime of convection-limited export is predominant in plants with large transport resistances and low xylem pressures. Moreover, hydrostatic pressures developed in our synthetic passive loader can reach botanically relevant values as high as 10 bars. We conclude that passive loading is sufficient to drive long-distance transport in large plants, and that trees are well suited to take full advantage of passive phloem loading strategies.

Summary Although much is known about the hydraulics of xylem, the hydraulic interconnectivity and dimensional scaling of phloem with respect to xylem in leaves has not been adequately studied to test alternative hydraulic architectural rules such as da Vinci's rule or Murray's rule, or physiological models such as Munch's Pressure Flow hypothesis. Using confocal and electron microscopy as well as mathematical analyses, we examined the hydraulic architecture of the mature leaves of the model species Populus tremula × alba across all seven hierarchical orders of the vascular branching. We show that: phloem and xylem conductive areas increase from minor to major veins; the sum of the conductive areas for each vein order increases exponentially from major to minor veins; the volume of individual sieve tube and vessel members increases from minor to major veins; and phloem conductive area scales isometrically with respect to xylem area across all vein orders. The application of first principles to our data shows that conductive areas scale according to da Vinci's rule and not according to Murray's rule, and that the phloem network in poplar leaves can generate the pressure gradient envisioned in Munch's hypothesis.

More than a quarter of the primary productivity on land, and a large fraction of the food that humans consume, is contributed by plants that fix atmospheric CO(2) by C(4) photosynthesis. It has been estimated that transferring the C(4) pathway to C(3) crops could boost yield by 50% and also increase water use efficiency and reduce the need for fertilizer, particularly in dry, hot environments. The high productivity of maize (Zea mays), sugarcane (Saccharum spp.) and several emerging bioenergy grasses is due largely to C(4) photosynthesis, which is enabled by the orderly arrangement, in concentric rings, of specialized bundle sheath and mesophyll cells in leaves in a pattern known as Kranz anatomy. Here we show that PIN, the auxin efflux protein, is present in the end walls of maize bundle sheath cells, as it is in the endodermis of the root. Since this marker suggests the expression of endodermal genetic programs in bundle sheath cells, we determined whether the transcription factor SCARECROW, which regulates structural differentiation of the root endodermis, also plays a role in the development of Kranz anatomy in maize. Mutations in the Scarecrow gene result in proliferation of bundle sheath cells, abnormal differentiation of bundle sheath chloroplasts, vein disorientation, loss of minor veins and reduction of vein density. Further characterization of this signal transduction pathway should facilitate the transfer of the C(4) trait into C(3) crop species, including rice.

Ultrastructural and molecular studies have provided experimental evidence for the classification of cucurbits as symplastic loaders, mainly translocating the raffinose family oligosaccharides (RFOs) raffinose and stachyose. Earlier studies established that cucumber mosaic virus (CMV) infection causes a significant increase in the sucrose-to-RFO ratio in the phloem sap of melon plants. The alteration in phloem sap sugar composition was associated with upregulation of CmSUT1 transcript within the vascular bundles. The current research aimed to explore the effect of CMV infection on the enzymes involved in symplastic phloem loading and RFO biosynthesis. Viral infection did not affect the activity of either raffinose or stachyose synthases in source leaves, but caused upregulation of the respective transcripts. Interestingly, activity of galactinol synthase was higher in CMV-infected leaves, associated with upregulation of CmGAS2. A significant increase in CmGAS2 expression in source leaves of melon plants exposed to high temperatures indicated that this response is common for both biotic and abiotic stresses. However, the effect of CMV or heat stress on phloem sap sugar composition is not due to alteration in RFO biosynthesis.

Cucurbits exude profusely when stems or petioles are cut. We conducted studies on pumpkin (Cucurbita maxima) and cucumber (Cucumis sativus) to determine the origin and composition of the exudate. Morphometric analysis indicated that the exudate is too voluminous to derive exclusively from the phloem. Cold, which inhibits phloem transport, did not interfere with exudation. However, ice water applied to the roots, which reduces root pressure, rapidly diminished exudation rate. Sap was seen by microscopic examination to flow primarily from the fascicular phloem in cucumber, and several other cucurbit species, but primarily from the extrafascicular phloem in pumpkin. Following exposure of leaves to 14CO2, radiolabeled stachyose and other sugars were detected in the exudate in proportions expected of authentic phloem sap. Most of this radiolabel was released during the first 20 s. Sugars in exudate were dilute. The sugar composition of exudate from extrafascicular phloem near the edge of the stem differed from that of other sources in that it was high in hexose and low in stachyose. We conclude that sap is released from cucurbit phloem upon wounding but contributes negligibly to total exudate volume. The sap is diluted by water from cut cells, the apoplast, and the xylem. Small amounts of dilute, mobile sap from sieve elements can be obtained, although there is evidence that it is contaminated by the contents of other cell types. The function of P-proteins may be to prevent water loss from the xylem as well as nutrient loss from the phloem.

Most herbaceous plants employ thermodynamically active mechanisms of phloem loading, whereas in many trees, the mechanism is passive, by diffusion. Considering the different water transport characteristics of herbs and trees, we hypothesized that water relations play a role in the adoption of phloem loading strategies. We measured whole-plant hydraulic conductance (K p), osmolality, concentrations of polar metabolites, and key inorganic ions in recently mature leaves of 45 dicotyledonous species at midafternoon. Trees, and the few herbs that load passively, have low K p, high osmolality, and high concentrations of transport sugars and total polar metabolites. In contrast, herbs that actively load sucrose alone have high K p, low osmolality, and low concentrations of sugars and total polar metabolites. Solute levels are higher in sugar alcohol-transporting species, both herbs and trees, allowing them to operate at lower leaf water potentials. Polar metabolites are largely responsible for leaf osmolality above a baseline level (approximately 300 mm) contributed by ions. The results suggest that trees must offset low K p with high concentrations of foliar transport sugars, providing the motivating force for sugar diffusion and rendering active phloem loading unnecessary. In contrast, the high K p of most herbaceous plants allows them to lower sugar concentrations in leaves. This reduces inventory costs and significantly increases growth potential but necessitates active phloem loading. Viewed from this perspective, the elevation of hydraulic conductance marks a major milestone in the evolution of the herbaceous habit, not only by facilitating water transport but also by maximizing carbon use efficiency and growth.

Based on the high density of plasmodesmata interconnecting the intermediary cells and their neighboring phloem parenchyma or bundle-sheath cells, and based on the insensitivity to the sucrose transport inhibitor p-chloromercuribenzenesulfonic acid (PCMBS), cucurbits have been concluded to be symplastic loaders. In the present study, we identified and characterized the full-length sequence of sucrose transporter gene (CmSUT1) from melon (Cucumis melo L. cv. Hale's best jumbo). In vitro experiments confirmed that the identified gene product has sucrose transporter activity in baker's yeast. Healthy and cucumber mosaic virus (CMV)-infected melon plants were employed to examine sucrose transporter activity in planta. Pretreatment with PCMBS inhibited loading of newly fixed ¹⁴CO₂ into minor veins of CMV-infected plants. Moreover, CMV infection caused significant increase in CmSUT1 transcripts expression, mainly in vascular bundles of minor veins, which was associated with elevated sucrose content in phloem sap collected from source-leaf petioles. We propose that cucurbit plants contain the machinery for apoplastic phloem loading and that CMV infection causes a quantitative shift in the mode by which photoassimilates are loaded into the sieve tube.

Although transport of organic nutrients and regulatory molecules in the phloem underlies all aspects of growth and development, we still cannot say with confidence that we adequately understand how it is motivated. A major problem is that it is difficult to reconcile the theory of pressure flow

Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 ( SUT1 ), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum , a sucrose transporter, and Verbascum phoeniceum , a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H + symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum , leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species.

The phloem transports nutrients, defensive compounds, and informational signals throughout vascular plants. Sampling the complex components of mobile phloem sap is difficult because of the damage incurred when the pressurized sieve tubes are breached. In this review we discuss sampling methods, the artifacts that can be introduced by different sampling procedures, the intricate pathways by which materials enter and exit the phloem, and the major types of compounds transported. Loading and unloading patterns are largely determined by the conductivity and number of plasmodesmata and the position-dependent function of solute-specific, plasma membrane transport proteins. Recent evidence indicates that mobile proteins and RNA are part of the plant's long-distance communication signaling system. Evidence also exists for the directed transport and sorting of macromolecules as they pass through plasmodesmata. A future challenge is to dissect the molecular and cellular aspects of long-distance macromolecular trafficking in the signal transduction pathways of the whole plant.

Many plants translocate sugar alcohols in the phloem. However, the mechanism(s) of sugar alcohol loading in the minor veins of leaves are debated. We characterized the loading strategies of two species that transport sorbitol (Plantago major and apple [Malus domestica]), and one that transports mannitol (Asarina scandens). Plasmodesmata are abundant at all interfaces in the minor vein phloem of apple, and in one of two types of phloem in the minor veins of A. scandens. Few plasmodesmata are present in the minor veins of P. major. Apple differs from the other two species in that sugar alcohol and sucrose (Suc) are present in much higher concentrations in leaves. Apple leaf tissue exposed to exogenous [(14)C]sorbitol, [(14)C]Suc, or (14)CO(2) did not accumulate radiolabel in the minor veins, as determined by macroautoradiography. P. major minor veins accumulated radiolabel from [(14)C]Suc, [(14)C]sorbitol, and (14)CO(2). A. scandens minor veins accumulated (14)C from [(14)C]Suc and (14)CO(2), but not from [(14)C]mannitol. We conclude that the movement of sugar alcohol from the mesophyll into the phloem in apple and A. scandens is symplastic and passive, but in P. major it involves an apoplastic step and is energized. We also suggest that apple leaves transport sorbitol in high concentrations to avoid the feedback limitation of photosynthesis that would result from driving passive movement of solute into the phloem with high levels of Suc alone. The loading pathways and the mechanisms by which hydrostatic pressure is maintained in the minor vein phloem of these species are discussed.

Prior to the cytosolic synthesis of transport sugars during transitory starch utilization, intermediate products of starch breakdown, such as maltose, must be exported from chloroplasts. Recent work in Arabidopsis indicates that a novel transporter mediates maltose transfer across the chloroplast inner envelope membrane. We cloned a gene from an apple cDNA library that is highly homologous with the Arabidopsis maltose transporter, MEX1. Expression levels of MdMEX determined by real-time PCR were low in the tips of growing shoots, higher in expanding leaves and maximal in mature leaves. Expression was also detected in fruits and roots, indicating a role for MdMEX in starch mobilization in sink tissues. The cDNA from apple was subcloned into an expression cassette between the cauliflower mosaic virus 35S promoter and the sGFP (green fluorescent protein) coding sequence. Plants of the Arabidopsis maltose excess1-1 mutant, which is homozygous for a defective MEX1 allele, were transformed with the 35S:MdMEX:GFP construct. Fluorescence of GFP was localized to chloroplasts, indicating that Arabidopsis recognized the predicted 55 amino acid chloroplast transit peptide in the apple protein. The phenotypes of several independently transformed lines were analyzed. The complemented plants were relieved of the severe stunting and chlorosis characteristic of mex1-1 plants. Furthermore, starch levels and concentrations of soluble sugars, leaf chlorophyll content and maximum quantum efficiency of PSII were restored to wild-type levels. MdMEX (Malus domestica maltose transporter) is the second member of the unique maltose transporter gene family.

Cucurbit phloem is especially interesting and unusual in several respects, which has made these plants the subject of intensive study by microscopists, physiologists and molecular biologists. Early light microscopists were attracted to cucurbits due to the large dimensions of the sieve tubes and sieve pores, which made them relatively easy to see (Esau, The phloem. Berlin/Stuttgart: Gebruder Borntraeger, 1969). Hartig, the forest botanist who discovered the sieve element, turned to Cucurbita in his studies, describing many aspects of phloem structure including sieve plates, callose and “slime plugs.” (See Esau, The phloem. Berlin/Stuttgart: Gebruder Borntraeger, 1969 for the early history of phloem research). Cucurbita has also featured strongly in the analysis of phloem development. For example, Esau et al. (Bot Gaz 123:233–243, 1962) described the development of the sieve pores and the involvement of callose deposition in this process in Cucurbita maxima. A striking feature of cucurbit phloem is the presence of a unique array of extrafascicular (outside the vascular bundles) sieve tubes, which have been studied by several groups in recent years (Zhang et al. Proc Nat Acad Sci U S A 107:13532–13537, 2010; Gaupels et al. Plant Physiol 160:2285–2299, 2012; Zhang et al. Plant Physiol 158:1873–1882, 2012; Gaupels and Ghirardo, Front Plant Sci 4:187, 2013; Gaupels et al. Front Plant Sci 7:154, 2016).

Phloem loading is the initial step in photoassimilate export and the one that creates the driving force for mass flow. It has been proposed that loading occurs symplastically in species that translocate carbohydrate primarily as raffinose family oligosaccharides (RFOs). In these plants, dense fields of plasmodesmata connect bundle sheath cells to specialized companion cells (intermediary cells) in the minor veins. According to the polymer trap model, advanced as a mechanism of symplastic loading, sucrose from the mesophyll diffuses into intermediary cells and is converted there to RFOs. This process keeps the sucrose concentration low and, because of the larger size of the RFOs, prevents back diffusion. To test this model, the RFO pathway was down-regulated in Verbascum phoeniceum L. by suppressing the synthesis of galactinol synthase (GAS), which catalyzes the first committed step in RFO production. Two GAS genes ( VpGAS1 and VpGAS2 ) were cloned and shown to be expressed in intermediary cells. Simultaneous RNAi suppression of both genes resulted in pronounced inhibition of RFO synthesis. Phloem transport was negatively affected, as evidenced by the accumulation of carbohydrate in the lamina and the reduced capacity of leaves to export sugars during a prolonged dark period. In plants with severe down-regulation, additional symptoms of reduced export were obvious, including impaired growth, leaf chlorosis, and necrosis and curling of leaf margins.

The potential role of foliar carbon export features in the acclimation of photosynthetic capacity to differences and changes in light environment was evaluated. These features included apoplastic vs. symplastic phloem loading, density of loading veins, plasmodesmatal frequency in intermediary cells, and the ratio of loading cells to sieve elements. In initial studies, three apoplastic loaders (spinach, pea, Arabidopsis thaliana) exhibited a completely flexible photosynthetic response to changing light conditions, while two symplastic loaders (pumpkin, Verbascum phoeniceum), although able to adjust to different long-term growth conditions, were more limited in their response when transferred from low (LL) to high (HL) light. This suggested that constraints imposed by the completely physical pathway of sugar export might act as a bottleneck in the export of carbon from LL-acclimated leaves of symplastic loaders. While both symplastic loaders exhibited variable loading vein densities (low in LL and high in HL), none of the three apoplastic loaders initially characterized exhibited such differences. However, an additional apoplastic species (tomato) exhibited similar differences in vein density during continuous growth in different light environments. Furthermore, in contrast to the other apoplastic loaders, photosynthetic acclimation in tomato was not complete following a transfer from LL to HL. This suggests that loading vein density and loading cells per sieve element, and thus apparent loading surface capacity, play a major role in the potential for photosynthetic acclimation to changes in light environment. Photosynthetic acclimation and vein density acclimation were also characterized in the slow-growing, sclerophytic evergreen Monstera deliciosa. This evergreen possessed a lower vein density during growth in LL compared to HL and exhibited a more severely limited potential for photosynthetic acclimation to increases in light environment than the rapidly-growing, mesophytic annuals.

Phloem cells adjacent to sieve elements can possess wall invaginations. The role of light and jasmonic acid signaling in wall ingrowth development was examined in pea companion cells (CCs), Arabidopsis thaliana phloem parenchyma cells (PCs), and in Senecio vulgaris (with ingrowths in both cell types). Features characterized included wall ingrowths (from electron microscopic images), foliar vein density and photosynthetic capacity. In Arabidopsis, wall ingrowths were bulky compared with finger-like invaginations in pea and S. vulgaris. Relative to low light (LL), wall invagination in both CCs and PCs was greater in high light (HL). Treatment with methyl jasmonate in LL had no effect on CCs, but increased PC wall ingrowths. LL-to-HL transfer resulted in significantly less wall ingrowth in the fad7-1 fad8-1 (jasmonate-deficient) Arabidopsis mutant relative to the wild type. These results suggest that chloroplast oxidative status, via chloroplast-derived jasmonates, may modulate phloem structure and function. While CC wall ingrowths facilitate phloem loading by expanding the membrane area available for active uptake, one can speculate that phloem PC ingrowths may have two potential roles: to increase the efflux of sugars and/or protons into the apoplast to augment phloem loading; and/or to protect the phloem against pathogens and/or insects.

Plants load sugars from photosynthesizing leaves into the phloem of exporting veins either “apoplastically” (by using H + /sucrose symporters) or “symplastically” (through plasmodesmata). The ability to regulate photosynthesis in response to the light environment was compared among apoplastic loaders (pea and spinach) and symplastic loaders (pumpkin and Verbascum phoeniceum ). Plants were grown under low light (LL) or high light (HL) or transferred from LL to HL. Upon transfer, pea and spinach up-regulated photosynthesis to the level found in HL-acclimated plants, whereas up-regulation in pumpkin and V. phoeniceum was limited. The vein density of pea and spinach was the same in HL and LL. Although spinach did not exhibit anatomical or ultrastructural acclimation to the light environment, in pea, wall invaginations in minor vein companion (transfer) cells were more extensive in HL. Furthermore, upon transfer from LL to HL, these invaginations increased in mature pea leaves. Foliar starch levels in mature leaves of plants transferred from LL to HL were not greater than in HL-acclimated leaves of either apoplastically loading species. In the symplastic loaders, plasmodesmatal frequency per loading cell did not vary with treatment, but vein density and thus total plasmodesmatal frequency were higher in HL. Upon transfer of symplastic loaders, however, vein density remained low, and starch levels were higher than in HL; the incomplete acclimation of photosynthesis upon transfer is thus consistent with a carbon export capacity physically limited by an inability to increase vein and plasmodesmatal density in a mature leaf.

Photoperiod in plants is perceived by leaves and in many species influences the transition to reproductive growth through long-distance signaling. CONSTANS (CO) is implicated as a mediator between photoperiod perception and the transition to flowering in Arabidopsis. To test the role of CO in long-distance signaling, CO was expressed from a promoter specific to the companion cells of the smallest veins of mature leaves. This expression in tissues at the inception of the phloem translocation stream was sufficient to accelerate flowering at the apical meristem under noninductive (short-day) conditions. Grafts that conjoined the vegetative stems of plants with different flower-timing phenotypes demonstrated that minor-vein expression of CO is able to substitute for photoperiod in generating a mobile flowering signal. Our results suggest that a CO-derived signal(s), or possibly CO itself, fits the definition of the hypothetical flowering stimulant, florigen.

The minor-vein phloem of mature leaves is developmentally and physiologically distinct from the phloem in the rest of the vascular system. Phloem loading of transport sugars occurs in the minor veins, and consistent with this, galactinol synthase is expressed in the minor veins of melon (Cucumis melo) as part of the symplastic-loading mechanism that operates in this species. A galactinol synthase promoter from melon drives gene expression in the minor-vein companion cells of both transgenic tobacco (Nicotiana tabacum) and Arabidopsis. Neither of these plants use galactinol in the phloem-loading process, implying that the promoter responds to a minor-vein-specific regulatory cascade that is highly conserved across a broad range of eudicotyledons. Detailed analysis of this promoter by truncation and mutagenesis identified three closely coupled sequences that unambiguously modulate tissue specificity. These sequences cooperate in a combinatorial fashion: two promote expression throughout the vascular system of the plant, whereas the third functions to repress expression in the larger bundles. In a complementary approach, phylogenetic footprinting was used to obtain single-nucleotide resolution of conserved sites in orthologous promoters from diverse members of the Cucurbitaceae. This comparative analysis confirmed the importance of the closely coupled sites but also revealed other highly conserved sequences that may modulate promoter strength or contribute to expression patterns outside of the phloem. The conservation of this regulatory design among species that phloem load by different mechanisms supports a model for organismal development in which tissues and cell types are controlled by relatively ancient and conserved paradigms but expression of genes influencing final form and function are relatively plastic.

Raffinose family oligosaccharides (RFOs) perform several physiological functions in plants. In addition to accumulating during seed formation, raffinose and stachyose are translocated in the phloem and may accumulate in response to low temperatures, drought, or salt stress. Although the synthesis of galactinol, as mediated by galactinol synthase (GAS), is the first committed step in RFO formation, its expression patterns are poorly understood in most species. We have cloned and characterized the expression of two galactinol synthase gene family members in melon (Cucumis melo L. Cantalupensis Group). Both CmGAS1 and CmGAS2 are highly expressed in mature leaves. Galactinol synthase transcription in leaves was not upregulated by either water or low temperature stresses. Transcripts of CmGAS1 were present in developing melon seeds at a time coincident with the formation of raffinose and stachyose. Based on the GAS expression and RFO accumulation patterns, we propose that RFOs in melon function in carbon translocation and seed desiccation tolerance. and ajugose (a hexasaccharide RFO) accumulates in the mesophyll cells of Ajuga reptans after exposure to low temperatures (Bachmann and Keller, 1995; Sprenger and Keller, 2000). These acclimation responses may be critical for the survival of low temperature tolerant species. Although the presence of RFOs has been documented in a variety of systems, there are few studies demonstrating the expression of the GAS genes. Sprenger and Keller (2000) published a study that described the expression patterns of two galactinol synthase genes, GolS-1 and GolS-2, in Ajuga reptans. Ajuga is a frost-hardy evergreen labiate that both accumulates and translocates RFOs. The gene, GolS-1 was primarily expressed in the mesophyll in response to low temperatures while GolS-2 was expressed in intermediary cells and most likely catalyzes the formation of RFOs destined for translocation. The Arabidopsis GAS genes AtGolS1 and AtGolS2 are expressed in response to drought and salt stress while AtGolS3 accumulates in response to low temperature stress (Taji et al., 2002). The expression patterns of other Arabidopsis GAS genes have not been characterized. We have cloned two GAS genes in melon (Cucumis melo L. Cantalupensis Group). One gene, CmGAS1, is highly homologous to the zucchini CpGAS1 gene identified by Kerr et al. (1993). A CmGAS1 promoter-gusA fusion was made and used to transform Arabidopsis and tobacco plants. In these transgenic plants, GUS expression localized to the smallest veins of mature Arabidopsis and tobacco leaves (Haritatos et al., 2000). Although galactinol is not normally synthesized in wild-type tobacco leaves, stain localized to the companion cells and sieve elements in the minor veins (Haritatos et al., 2000), indicating that the CmGAS1 promoter drives vein- specific gene expression. There are no previous reports document- ing the expression pattern of the second melon GAS gene, CmGAS2. In melon, raffinose and stachyose serve as phloem translocation sugars and also accumulate in maturing seeds. Other functions of RFOs in melon have not been documented. We have used RNA blots and RT-PCR techniques to characterize the mRNA expression patterns of the CmGAS1 and CmGAS2 genes in melon.

Despite the crucial role of carbon transport in whole plant physiology and its impact on plant-environment interactions and ecosystem function, relatively little research has tried to examine how phloem physiology impacts plant ecology. In this review, we highlight several areas of active research where inquiry into phloem physiology has increased our understanding of whole plant function and ecological processes. We consider how xylem-phloem interactions impact plant drought tolerance and reproduction, how phloem transport influences carbon allocation in trees and carbon cycling in ecosystems and how phloem function mediates plant relations with insects, pests, microbes and symbiotes. We argue that in spite of challenges that exist in studying phloem physiology, it is critical that we consider the role of this dynamic vascular system when examining the relationship between plants and their biotic and abiotic environment.

C₄ and C₃ photosynthesis differ in the efficiency with which they consume water and nitrogen. Engineering traits of the more efficient C₄ photosynthesis into C₃ crops could substantially increase crop yields in hot, arid conditions. To identify differences between C₄ and C₃ photosynthetic mechanisms, we profiled metabolites and gene expression in the developing leaves of Zea mays (maize), a C₄ plant, and Oryza sativa (rice), a C₃ plant, using a statistical method named the unified developmental model (UDM). Candidate cis-regulatory elements and transcription factors that might regulate photosynthesis were identified, together with differences between C₄ and C₃ nitrogen and carbon metabolism. The UDM algorithms could be applied to analyze and compare development in other species. These data sets together with community viewers to access and mine them provide a resource for photosynthetic research that will inform efforts to engineer improvements in carbon fixation in economically valuable grass crops.

Phylogenetic analysis provides a rational basis for comparative studies of phloem structure and phloem loading. Although several types of minor vein companion cell have been identified, and progress has been made in correlating structural features of these cells with loading mechanisms, little is known about the phylogenetic relationships of the different types. To add to the available data on companion cells, we analyzed the ultrastructure of minor veins in Euonymus fortunei and Celastrus orbiculatis (Celastraceae) leaves and determined that in these species they are specialized as intermediary cells. This cell type has been implicated in symplastic phloem loading. The data were added to published data sets on minor vein phloem characteristics, which were then mapped to a well-supported molecular tree. The analysis indicates that extensive plasmodesmatal continuity between minor vein phloem and surrounding cells is ancestral in the angiosperms. Reduction in plasmodesmatal frequency at this interface is a general evolutionary trend, punctuated by instances of the reverse. This is especially true in the case of intermediary cells that have many plasmodesmata, but other distinguishing characteristics as well, and have arisen independently at least four, and probably six, times in derived lineages. The character of highly reduced plasmodesmatal frequency in minor vein phloem, common in crop plants, has several points of origin in the tree. Thus, caution should be exercised in generalizing results on apoplastic phloem loading obtained from model species. Transfer cells have many independent points of origin, not always from lineages with reduced plasmodesmatal frequency.

Minor vein ultrastructure and phloem loading were studied in leaves of the tulip tree (Liriodendron tulipifera; Magnoliaceae). Plasmodesmatal frequencies leading into minor vein companion cells are higher than in species known to load via the apoplast. However, these companion cells are not specialized as "intermediary cells" as they are in species in which the best evidence for symplastic phloem loading has been documented. Mesophyll cells plasmolyzed in 600 mM sorbitol, whereas sieve elements and companion cells did not plasmolyze even in 1.2 M sorbitol, indicating that solute accumulates in the phloem against a steep concentration gradient. Both [(14)C]sucrose and (14)C-labeled photo-assimilate accumulated in the minor vein network, as demonstrated by autoradiography. [(14)C]sucrose accumulation was prevented by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton cotransport from the apoplast. p-Chloromercuribenzenesulfonic acid largely, but not entirely, inhibited exudation of radiolabeled photoassimilate. The evidence is most consistent with the presence of an apoplastic component to phloem loading in this species, contrary to speculation that the more basal members of the angiosperms load by an entirely symplastic mechanism.

The definition of “minor” veins in leaves is arbitrary and of uncertain biological significance. Generally, the term refers to the smallest vein classes in the leaf, believed to function in phloem loading. We found that a galactinol synthase promoter, cloned from melon (Cucumis melo), directs expression of thegusA gene to the smallest veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves. This expression pattern is consistent with the role of galactinol synthase in sugar synthesis and phloem loading in cucurbits. The expression pattern in tobacco is especially noteworthy since galactinol is not synthesized in the leaves of this plant. Also, we unexpectedly found that expression in tobacco is limited to two of three companion cells in class-V veins, which are the most extensive in the leaf. Thus, the “minor” vein system is defined and regulated at the genetic level, and there is heterogeneity of response to this system by different companion cells of the same vein.

Plant viruses encode movement proteins that are essential for systemic infection of their host but dispensable for replication and encapsidation. BL1, one of the two movement proteins encoded by the bipartite geminivirus squash leaf curl virus, was immunolocalized to unique approximately 40-nm tubules that extended up to and across the walls of procambial cells in systemically infected pumpkin leaves. These tubules were not found in procambial cells from pumpkin seedlings inoculated with BL1 mutants that are defective in movement. The tubules also specifically stained with antisera to binding protein (BiP), indicating that they were derived from the endoplasmic reticulum. Independent confirmation of this endoplasmic reticulum association was obtained by subcellular fractionation studies in which BL1 was localized to fractions that contained both endoplasmic reticulum membranes and BiP. Thus, squash leaf curl virus appears to recruit the endoplasmic reticulum as a conduit for cell-to-cell movement of the viral genome.