Intercellular protein movement: deciphering the language of development.

Development in multicellular organisms requires the coordinated production of a large number of specialized cell types through sophisticated signaling mechanisms. Non-cell-autonomous signals are one of the key mechanisms by which organisms coordinate development. In plants, intercellular movement of transcription factors and other mobile signals, such as hormones and peptides, is essential for normal development. Through a combination of different approaches, a large number of non-cell-autonomous signals that control plant development have been identified. We review some of the transcriptional regulators that traffic between cells, as well as how changes in symplasmic continuity affect and are affected by development. We also review current models for how mobile signals move via plasmodesmata and how movement is inhibited. Finally, we consider challenges in and new tools for studying protein movement.

Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants.

The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response.

Heat Stress Reduces Root Meristem Size via Induction of Plasmodesmal Callose Accumulation Inhibiting Phloem Unloading in Arabidopsis.

The intercellular transport of sugars, nutrients, and small molecules is essential for plant growth, development, and adaptation to environmental changes. Various stresses are known to affect the cell-to-cell molecular trafficking modulated by plasmodesmal permeability. However, the mechanisms of plasmodesmata modification and molecules involved in the phloem unloading process under stress are still not well understood. Here, we show that heat stress reduces the root meristem size and inhibits phloem unloading by inducing callose accumulation at plasmodesmata that connect the sieve element and phloem pole pericycle. Furthermore, we identify the loss-of-function of CALLOSE SYNTHASE 8 (CalS8), which is expressed specifically in the phloem pole pericycle, decreasing the plasmodesmal callose deposition at the interface between the sieve element and phloem pole pericycle and alleviating the suppression at root meristem size by heat stress. Our studies indicate the involvement of callose in the interaction between root meristem growth and heat stress and show that CalS8 negatively regulates the thermotolerance of Arabidopsis roots.

Cell-to-Cell Connection in Plant Grafting-Molecular Insights into Symplasmic Reconstruction.

Grafting is a means to connect tissues from two individual plants and grow a single chimeric plant through the establishment of both apoplasmic and symplasmic connections. Recent molecular studies using RNA-sequencing data have provided genetic information on the processes involved in tissue reunion, including wound response, cell division, cell-cell adhesion, cell differentiation and vascular formation. Thus, studies on grafting increase our understanding of various aspects of plant biology. Grafting has also been used to study systemic signaling and transport of micromolecules and macromolecules in the plant body. Given that graft viability and molecular transport across graft junctions largely depend on vascular formation, a major focus in grafting biology has been the mechanism of vascular development. In addition, it has been thought that symplasmic connections via plasmodesmata are fundamentally important to share cellular information among newly proliferated cells at the graft interface and to accomplish tissue differentiation correctly. Therefore, this review focuses on plasmodesmata formation during grafting. We take advantage of interfamily grafts for unambiguous identification of the graft interface and summarize morphological aspects of de novo formation of plasmodesmata. Important molecular events are addressed by re-examining the time-course transcriptome of interfamily grafts, from which we recently identified the cell-cell adhesion mechanism. Plasmodesmata-associated genes upregulated during graft healing that may provide a link to symplasm establishment are described. We also discuss future research directions.

The Evolutionary Origin of C4 Photosynthesis in the Grass Subtribe Neurachninae.

The Australian grass subtribe Neurachninae contains closely related species that use C3, C4, and C2 photosynthesis. To gain insight into the evolution of C4 photosynthesis in grasses, we examined leaf gas exchange, anatomy and ultrastructure, and tissue localization of Gly decarboxylase subunit P (GLDP) in nine Neurachninae species. We identified previously unrecognized variation in leaf structure and physiology within Neurachne that represents varying degrees of C3-C4 intermediacy in the Neurachninae. These include inverse correlations between the apparent photosynthetic carbon dioxide (CO2) compensation point in the absence of day respiration (C * ) and chloroplast and mitochondrial investment in the mestome sheath (MS), where CO2 is concentrated in C2 and C4 Neurachne species; width of the MS cells; frequency of plasmodesmata in the MS cell walls adjoining the parenchymatous bundle sheath; and the proportion of leaf GLDP invested in the MS tissue. Less than 12% of the leaf GLDP was allocated to the MS of completely C3 Neurachninae species with C * values of 56-61 µmol mol-1, whereas two-thirds of leaf GLDP was in the MS of Neurachne lanigera, which exhibits a newly-identified, partial C2 phenotype with C * of 44 µmol mol-1 Increased investment of GLDP in MS tissue of the C2 species was attributed to more MS mitochondria and less GLDP in mesophyll mitochondria. These results are consistent with a model where C4 evolution in Neurachninae initially occurred via an increase in organelle and GLDP content in MS cells, which generated a sink for photorespired CO2 in MS tissues.

Plasmodesmata at a glance.

Plasmodesmata are cytoplasmic communication channels that are vital for the physiology and development of all plants. They facilitate the intercellular movement of various cargos - ranging from small molecules, such as sugars, ions and other essential nutrients and chemicals, to large complex molecules, such as proteins and different types of RNA species - by bridging neighboring cells across their cell walls. Structurally, an individual channel consists of the cytoplasmic sleeve that is formed between the endoplasmic reticulum and the plasma membrane leaflets. Plasmodesmata are highly versatile channels; they vary in number and structure, and undergo constant adjustments to their permeability in response to many internal and external cues. In this Cell Science at a Glance article and accompanying poster, we provide an overview of plasmodesmata form and function, with highlights on their development and variation, associated components and mobile factors. In addition, we present methodologies that are currently used to study plasmodesmata-mediated intercellular communication.

Direct Comparison of Leaf Plasmodesma Structure and Function in Relation to Phloem-Loading Type.

The export of photosynthetically produced sugars from leaves depends on plasmodesmatal transport of sugar molecules from mesophyll to phloem. Traditionally, the density of plasmodesmata (PD) along this phloem-loading pathway has been used as a defining feature of different phloem-loading types, with species proposed to have either many or few PD between the phloem and surrounding cells of the leaf. However, quantitative determination of PD density has rarely been performed. Moreover, the structure of PD has not been considered, even though it could impact permeability, and functional data are only available for very few species. Here, a comparison of PD density, structure, and function using data from transmission electron microscopy and live-cell microscopy was conducted for all relevant cell-cell interfaces in leaves of nine species. These species represent the three principal phloem-loading types currently discussed in literature. Results show that relative PD density among the different cell-cell interfaces in one species, but not absolute PD density, is indicative of phloem-loading type. PD density data of single interfaces, even combined with PD diameter and length data, did not correlate with the intercellular diffusion capacity measured by the fluorescence loss in photobleaching method. This means that PD substructure not visible on standard transmission electron micrographs may have a strong influence on permeability. Furthermore, the results support a proposed passive symplasmic loading mechanism in the tree species horse chestnut (Aesculus hippocastanum), white birch (Betula pubescens), orchard apple (Malus domestica), and gray poplar (Populus x canescens) as functional cell coupling and PD structure differed from active symplasmic and apoplasmic phloem-loading species.

Interplay between turgor pressure and plasmodesmata during plant development.

Plasmodesmata traverse cell walls, generating connections between neighboring cells. They allow intercellular movement of molecules such as transcription factors, hormones, and sugars, and thus create a symplasmic continuity within a tissue. One important factor that determines plasmodesmal permeability is their aperture, which is regulated during developmental and physiological processes. Regulation of aperture has been shown to affect developmental events such as vascular differentiation in the root, initiation of lateral roots, or transition to flowering. Extensive research has unraveled molecular factors involved in the regulation of plasmodesmal permeability. Nevertheless, many plant developmental processes appear to involve feedbacks mediated by mechanical forces, raising the question of whether mechanical forces and plasmodesmal permeability affect each other. Here, we review experimental data on how one of these forces, turgor pressure, and plasmodesmal permeability may mutually influence each other during plant development, and we discuss the questions raised by these data. Addressing such questions will improve our knowledge of how cellular patterns emerge during development, shedding light on the evolution of complex multicellular plants.

Photoinducible DRONPA-s: a new tool for investigating cell-cell connectivity.

The development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the plant cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables tight control of symplasmic cell-to-cell transport. Here we report on an elegant non-invasive method to quantify the passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA-s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA-s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA-s fluorescence in selected cells of the root meristem. This enabled us to compare movement of DRONPA-s from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the lowest efflux of DRONPA-s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA-s system generates reproducible data and is a valuable tool for studying symplasmic connectivity.

Arabidopsis Class I Formin FH1 Relocates between Membrane Compartments during Root Cell Ontogeny and Associates with Plasmodesmata.

Formins are evolutionarily conserved eukaryotic proteins engaged in actin nucleation and other aspects of cytoskeletal organization. Angiosperms have two formin clades with multiple paralogs; typical plant Class I formins are integral membrane proteins that can anchor cytoskeletal structures to membranes. For the main Arabidopsis housekeeping Class I formin, FH1 (At3g25500), plasmalemma localization was documented in heterologous expression and overexpression studies. We previously showed that loss of FH1 function increases cotyledon epidermal pavement cell shape complexity via modification of actin and microtubule organization and dynamics. Here, we employ transgenic Arabidopsis expressing green fluorescent protein-tagged FH1 (FH1-GFP) from its native promoter to investigate in vivo behavior of this formin using advanced microscopy techniques. The fusion protein is functional, since its expression complements the fh1 loss-of-function mutant phenotype. Accidental overexpression of FH1-GFP results in a decrease in trichome branch number, while fh1 mutation has the opposite effect, indicating a general role of this formin in controlling cell shape complexity. Consistent with previous reports, FH1-GFP associates with membranes. However, the protein exhibits surprising actin- and secretory pathway-dependent dynamic localization and relocates between cellular endomembranes and the plasmalemma during cell division and differentiation in root tissues, with transient tonoplast localization at the transition/elongation zones border. FH1-GFP also accumulates in actin-rich regions of cortical cytoplasm and associates with plasmodesmata in both the cotyledon epidermis and root tissues. Together with previous reports from metazoan systems, this suggests that formins might have a shared (ancestral or convergent) role at cell-cell junctions.

Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement. Consistent with this hypothesis, we show that the MP of CPsV (MPCPsV) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MPCPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MPCPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34KCPsV), but not the 20-kDa fragment (20KCPsV), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MPCPsV (and also the 34KCPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20KCPsV retains the protease activity and is able to cleave a cleavage-deficient MPCPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MPCPsV by an aspartic protease activity, which removes the 20KCPsV protease and thereby releases the 34KCPsV protein for PDLP1-dependent tubule formation at PD.IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34KCPsV and 20KCPsV, that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control.

Going with the Flow: Multiscale Insights into the Composite Nature of Water Transport in Roots.

As water often limits crop production, a more complete understanding of plant water capture and transport is necessary. Here, we developed MECHA, a mathematical model that computes the flow of water across the root at the scale of walls, membranes, and plasmodesmata of individual cells, and used it to test hypotheses related to root water transport in maize (Zea mays). The model uses detailed root anatomical descriptions and a minimal set of experimental cell properties, including the conductivity of plasma membranes, cell walls, and plasmodesmata, which yield quantitative and scale-consistent estimations of water pathways and root radial hydraulic conductivity (k r). MECHA revealed that the mainstream hydraulic theories derived independently at the cell and root segment scales are compatible only if osmotic potentials within the apoplastic domains are uniform. The results suggested that the convection-diffusion of apoplastic solutes explained most of the offset between estimated k r in pressure clamp and osmotic experiments, while the contribution of water-filled intercellular spaces was limited. Furthermore, sensitivity analyses quantified the relative impact of cortex and endodermis cell conductivity of plasma membranes on root k r and suggested that only the latter contributed substantially to k r due to the composite nature of water flow across roots. The explicit root hydraulic anatomy framework brings insights into contradictory interpretations of experiments from the literature and suggests experiments to efficiently address questions pertaining to root water relations. Its scale consistency opens avenues for cross-scale communication in the world of root hydraulics.

Response of plasmodesmata formation in leaves of C4 grasses to growth irradiance.

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C4 leaves is a key requirement for C4 photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C4 species using our PD quantification method, which combines three-dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP-ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 µmol m-2  s-1 ), and high light (1,000 µmol m-2  s-1 ), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C4 leaf PD density depended on plant age and species. The high light treatment resulted in two to four-fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M-BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C4 leaf PD formation to growth irradiance.

Choline transporter-like1 (CHER1) is crucial for plasmodesmata maturation in Arabidopsis thaliana.

Plasmodesmata (PD) are microscopic pores connecting plant cells and enable cell-to-cell transport. Currently, little information is known about the molecular mechanisms regulating PD formation and development. To uncover components of PD development we made use of the 17 kDa movement protein (MP17) encoded by the Potato leafroll virus (PLRV). The protein is required for cell-to-cell movement of the virus and localises to complex PD. Forward genetic screening for Arabidopsis mutants with altered PD binding of MP17 revealed several mutant lines, while molecular genetics, biochemical and microscopic studies allowed further characterisation. Map-based cloning of one mutant revealed a point mutation in the choline transporter-like 1 (CHER1) protein, changing glycine247 into glutamate. Mutation in CHER1 resulted in a starch excess phenotype and stunted growth. Ultrastructure analysis of shoot apical meristems, developing and fully developed leaves showed reduced PD numbers and the absence of complex PD in fully developed leaves. This indicates that cher1 mutants are impaired in PD formation and development. Global lipid profiling revealed only slight modifications in the overall lipid composition, however, altered composition of PD-associated lipids cannot be ruled out. Thus, cher1 is devoid of complex PD in developed leaves and provides insights into the formation of complex PD at the molecular level.

Peeking at a plant through the holes in the wall - exploring the roles of plasmodesmata.

Plasmodesmata (PD) are membrane-lined pores that connect neighbouring plant cells and allow molecular exchange via the symplast. Past studies have revealed the basic structure of PD, some of the transport mechanisms for molecules through PD, and a variety of physiological processes in which they function. Recently, with the help of newly developed technologies, several exciting new features of PD have been revealed. New PD structures were observed during early formation of PD and between phloem sieve elements and phloem pole pericycle cells in roots. Both observations challenge our current understanding of PD structure and function. Research into novel physiological responses, which are regulated by PD, indicates that we have not yet fully explored the potential contribution of PD to overall plant function. In this Viewpoint article, we summarize some of the recent advances in understanding the structure and function of PD and propose the challenges ahead for the community.

Phloem Loading through Plasmodesmata: A Biophysical Analysis.

In many species, Suc 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 Suc 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 kinetics of polymerization-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, polymerization of Suc 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 Suc 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.

Multiple mechanisms for enhanced plasmodesmata density in disparate subtypes of C4 grasses.

Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of two-cell C4 photosynthesis; However, a lack of quantitative data has hampered further exploration and validation of this hypothesis. In this study, we quantified leaf anatomical traits associated with metabolite transport in 18 species of BEP and PACMAD grasses encompassing four origins of C4 photosynthesis and all three C4 subtypes (NADP-ME, NAD-ME, and PCK). We demonstrate that C4 leaves have greater PD density between M and BS cells than C3 leaves. We show that this greater PD density is achieved by increasing either the pit field (cluster of PD) area or the number of PD per pit field area. NAD-ME species had greater pit field area per M-BS interface than NADP-ME or PCK species. In contrast, NADP-ME and PCK species had lower pit field area with increased number of PD per pit field area than NAD-ME species. Overall, PD density per M-BS cell interface was greatest in NAD-ME species while PD density in PCK species exhibited the largest variability. Finally, the only other anatomical characteristic that clearly distinguished C4 from C3 species was their greater Sb value, the BS surface area to subtending leaf area ratio. In contrast, BS cell volume was comparable between the C3 and C4 grass species examined.

A calmodulin-like protein regulates plasmodesmal closure during bacterial immune responses.

Plants sense microbial signatures via activation of pattern recognition receptors (PPRs), which trigger a range of cellular defences. One response is the closure of plasmodesmata, which reduces symplastic connectivity and the capacity for direct molecular exchange between host cells. Plasmodesmal flux is regulated by a variety of environmental cues but the downstream signalling pathways are poorly defined, especially the way in which calcium regulates plasmodesmal closure. Here, we identify that closure of plasmodesmata in response to bacterial flagellin, but not fungal chitin, is mediated by a plasmodesmal-localized Ca2+ -binding protein Calmodulin-like 41 (CML41). CML41 is transcriptionally upregulated by flg22 and facilitates rapid callose deposition at plasmodesmata following flg22 treatment. CML41 acts independently of other defence responses triggered by flg22 perception and reduces bacterial infection. We propose that CML41 enables Ca2+ -signalling specificity during bacterial pathogen attack and is required for a complete defence response against Pseudomonas syringae.

The role of plasmodesma-located proteins in tubule-guided virus transport is limited to the plasmodesmata.

Intercellular spread of plant viruses involves passage of the viral genome or virion through a plasmodesma (PD). Some viruses severely modify the PD structure, as they assemble a virion carrying tubule composed of the viral movement protein (MP) inside the PD channel. Successful modulation of the host plant to allow infection requires an intimate interaction between viral proteins and both structural and regulatory host proteins. To date, however, very few host proteins are known to promote virus spread. Plasmodesmata-located proteins (PDLPs) localised in the PD have been shown to contribute to tubule formation in cauliflower mosaic virus and grapevine fanleaf virus infections. In this study, we have investigated the role of PDLPs in intercellular transport of another tubule-forming virus, cowpea mosaic virus. The MP of this virus was found to interact with PDLPs in the PD, as was shown for other tubule-forming viruses. Expression of PDLPs and MPs in protoplasts in the absence of a PD revealed that these proteins do not co-localise at the site of tubule initiation. Furthermore, we show that tubule assembly in protoplasts does not require an interaction with PDLPs at the base of the tubule, as has been observed in planta. These results suggest that a physical interaction between MPs and PDLPs is not required for assembly of the movement tubule and that the beneficial role of PDLPs in virus movement is confined to the structural context of the PD.

Symplastic phloem loading in poplar.

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.