Gary A. Thompson

Phloem Ultrastructure and Pressure Flow: Sieve-Element-Occlusion-Related Agglomerations Do Not Affect Translocation

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

Arabidopsis P-Protein Filament Formation Requires Both AtSEOR1 and AtSEOR2

The structure-function relationship of proteinaceous filaments in sieve elements has long been a source of investigation in order to understand their role in the biology of the phloem. Two phloem filament proteins AtSEOR1 (At3g01680.1) and AtSEOR2 (At3g01670.1) in Arabidopsis have been identified that are required for filament formation. Immunolocalization experiments using a phloem filament-specific monoclonal antibody in the respective T-DNA insertion mutants provided an initial indication that both proteins are necessary to form phloem filaments. To investigate the relationship between these two proteins further, green fluorescent protein (GFP)-AtSEO fusion proteins were expressed in Columbia wild-type and T-DNA insertion mutants. Analysis of these mutants by confocal microscopy confirmed that phloem filaments could only be detected in the presence of both proteins, indicating that despite significant sequence homology the proteins are not functionally redundant. Individual phloem filament protein subunits of AtSEOR1 and AtSEOR2 were capable of forming homodimers, but not heterodimers in a yeast two-hybrid system. The absence of phloem filaments in phloem sieve elements did not result in gross alterations of plant phenotype or affect basal resistance to green peach aphid (Myzus persicae).

Expression of Small RNA in Aphis gossypii and Its Potential Role in the Resistance Interaction with Melon

Background The regulatory role of small RNAs (sRNAs) in various biological processes is an active area of investigation; however, there has been limited information available on the role of sRNAs in plant-insect interactions. This study was designed to identify sRNAs in cotton-melon aphid (Aphis gossypii) during the Vat-mediated resistance interaction with melon (Cucumis melo). Methodology/Principal Findings The role of miRNAs was investigated in response to aphid herbivory, during both resistant and susceptible interactions. sRNA libraries made from A. gossypii tissues feeding on Vat+ and Vat− plants revealed an unexpected abundance of 27 nt long sRNA sequences in the aphids feeding on Vat+ plants. Eighty-one conserved microRNAs (miRNAs), twelve aphid-specific miRNAs, and nine novel candidate miRNAs were also identified. Plant miRNAs found in the aphid libraries were most likely ingested during phloem feeding. The presence of novel miRNAs was verified by qPCR experiments in both resistant Vat+ and susceptible Vat− interactions. The comparative analyses revealed that novel miRNAs were differentially regulated during the resistant and susceptible interactions. Gene targets predicted for the miRNAs identified in this study by in silico analyses revealed their involvement in morphogenesis and anatomical structure determination, signal transduction pathways, cell differentiation and catabolic processes. Conclusion/Significance In this study, conserved and novel miRNAs were reported in A. gossypii. Deep sequencing data showed differences in the abundance of miRNAs and piRNA-like sequences in A. gossypii. Quantitative RT-PCR revealed that A. gossypii miRNAs were differentially regulated during resistant and susceptible interactions. Aphids can also ingest plant miRNAs during phloem feeding that are stable in the insect.

Live Imaging of Companion Cells and Sieve Elements in Arabidopsis Leaves

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Phloem: the integrative avenue for resource distribution, signaling, and defense

Research over the past 20 years has revealed new functions of the phloem beyond resource allocation to a system that combines distribution and messaging (Thompson and van Bel, 2013) analogous to the circulatory and nervous systems in animals. Apart from allocating resources for maintenance and growth, the phloem distributes hormonal signals and a broad spectrum of protein- and RNA-based messages throughout the plant to regulate a myriad of physiological and developmental processes. Resources and signals, collectively, coordinate development, and growth as well as integrate responses to both biotic and abiotic environmental challenges. The transport of organic compounds such as sugars, amino acids, and lipidic substances through the phloem are exploited by a vast and diverse range of pathogens such as viruses, fungi, nematodes, aphids, and other phloem-feeding insects. Given its ubiquitous occurrence in land plants, the phloem seems to be the integrative tissue par excellence. The contributions included in this research topic encompass the entire bandwidth of known phloem functions with emphasis on the diversity in structures and functions. Differentiation and development of vascular cells is complex and not well understood. Vascular development partly depends on environmental cues that have also impacted the evolution of vascular systems and phloem transport mechanisms. A novel phylogenetic approach to identify genes involved in vascular development (Martinez-Navarro et al., 2013) shows that several vascular genes are expressed in green algae (Chlorophyta), the ancestors of land plants. Analysis of vascular genes in non-vascular and ancient vascular plants indicates that coordinated expression of gene sets led to the emergence of the present vascular system (Martinez-Navarro et al., 2013). Representatives of the Dof gene family are among the transcription factors involved in vascular differentiation (Le Hir and Bellini, 2013). Nematode saliva has the remarkable ability to induce re-differentiation of phloem cells and their neighbors with the objective to “tap” the sieve-tube sap (Absmanner et al., 2013). Ample attention has been paid to the structural diversity, particularly in the phloem-loading zone, where environmental changes have unbuffered, and profound effects. The impact of diverse environmental conditions on leaf structure and carbohydrate processing is demonstrated with Arabidopsis ecotypes (Adams et al., 2013; Cohu et al., 2013a,b). Within the Asteridae, there is an immense diversity in minor-vein structures and companion cells (Batashev et al., 2013), which promises a higher variety of phloem-loading modes in dicots than previously suspected (Slewinski et al., 2013). It appears that a strict subdivision between apoplasmic and symplasmic phloem-loading species must be abandoned, since many species dispose over the devices to operate both modes in parallel. A structural feature of apoplasmic phloem loading in dicotyledons—the involvement of transfer cells—is highlighted in two contributions: one on the evolutionary trends, function and induction (Andriunas et al., 2013) and the other on transcriptional regulators of cell wall invagination (Chinnappa et al., 2013). A physiological feature of “active” symplasmic phloem loading is the size-selective transfer of sugars through plasmodesmata, which is challenged here using mathematical parameters (Liesche and Schulz, 2013). In grasses, the arrangement and ultrastructure of collection phloem suggest an apoplasmic mode of phloem loading (Botha, 2013; Slewinski et al., 2013). However, the functions of two principal structures in monocotyledonous leaves i.e., thick-walled sieve tubes and transverse veins remain puzzling (Botha, 2013). Thick-walled sieve tubes may be viewed as transformed phloem parenchyma cells (Slewinski et al., 2013) engaged in temporary storage (Botha, 2013). Carbohydrate processing may be more homogeneous in transport phloem (Slewinski et al., 2013) than in the phloem-loading zone. Yet, permanently changing conditions require flexible and diverse solutions for release/retrieval along the pathway (De Schepper et al., 2013). Central to the release/retrieval concept is the intercellular competition for sugars that is revealed with electrical methods for in situ measurement of sucrose uptake parameters (Hafke et al., 2013). The strong influence of environmental impacts on phloem functioning at each level is addressed in one comprehensive review by Lemoine et al. (2013). Long-distance signaling via the phloem can be accomplished by a variety of physiological mechanisms. Electrical signaling along sieve tubes affects both the physiology of distant leaves as well as photosynthate release/retrieval (Fromm et al., 2013). Long-distance effects of hormonal signaling on plant development have been demonstrated by expressing melon Aux/IAA genes into tomato plants (Golan et al., 2013). The translocation of microRNAs through the phloem regulates and coordinates distant processes such as mineral homeostasis (Kehr, 2013). Evidence is emerging for remote control of developmental processes in roots and tubers by phloem-mobile mRNAs (Hannapel et al., 2013). However, providing unambiguous evidence for some of the key processes implicated remains challenging and is open for debate (Hannapel, 2013; Suarez-Lopez, 2013). RNA-species are most likely translocated as complexes with RNA-binding proteins (Pallas and Gomez, 2013). As RNAs, several viruses are translocated as ribonucleoprotein complexes (Hipper et al., 2013) which might protect the viral core against the adverse sieve-tube environment and/or confer tagging for invasion of specific target cells. While the viral complexes move through sieve tubes with the mass flow, phytoplasmas with sizes exceeding the diameters of the sieve pores—that are occluded anyway in response to infection—may be disseminated by alternative mechanisms. Remarkably, some plants species are able to overcome phytoplasma infection through the so-called “recovery reactions” that are mediated through callose degradation in sieve tubes (Santi et al., 2013). The phloem contains attractants and repellents for animal pathogens. Present work indicates that sterols serve as attractants (Behmer et al., 2013) to phloem-feeding insects that have a deficient sterol synthesis, whereas benzylisoquinoline alkaloids serve as lethal repellents (Lee et al., 2013). It appears that phloem cells produce and transport an arsenal of defense compounds. The location of these anti-insect chemicals is significant e.g., for genetic manipulation of plants. Defense compounds against aphids may reside either or both in the pre-phloem pathway or inside the phloem cells themselves (Will et al., 2013). It is postulated that the major function of the extrafascicular phloem in cucurbits is to combat insects (Gaupels and Ghirardo, 2013). In legumes, giant proteins bodies (forisomes) may be involved in plant defense by rapid sieve-pore occlusion in response to an aphid attack (Jekat et al., 2013). The interactions between plants and aphids appear extremely complex (Louis and Shah, 2013) which hinders the development of molecular strategies for insect control. Nonetheless, strategies are being developed to increase the plant resistance against aphids (Will et al., 2013), and in one case by engineering of RFO phloem loading (Cao et al., 2013). The strong increment of RFOs in the sieve-tubes rendered the plants less attractive to aphids. As with other research fields, phloem research reveals an ever receding horizon with undreamed possibilities. This topic shows the amazingly diverse ability of plants to cope with an infinite number of environmental challenges by virtue of the vascular tissues.

miRNA‐mediated auxin signalling repression during Vat‐mediated aphid resistance in Cucumis melo

Resistance to Aphis gossypii in melon is attributed to the presence of the single dominant R gene virus aphid transmission (Vat), which is biologically expressed as antibiosis, antixenosis and tolerance. However, the mechanism of resistance is poorly understood at the molecular level. Aphid-induced transcriptional changes, including differentially expressed miRNA profiles that correspond to resistance interaction have been reported in melon. The potential regulatory roles of miRNAs in Vat-mediated aphid resistance were further revealed by identifying the specific miRNA degradation targets. A total of 70 miRNA:target pairs, including 28 novel miRNA:target pairs, for the differentially expressed miRNAs were identified: 11 were associated with phytohormone regulation, including six miRNAs that potentially regulate auxin interactions. A model for a redundant regulatory system of miRNA-mediated auxin insensitivity is proposed that incorporates auxin perception, auxin modification and auxin-regulated transcription. Chemically inhibiting the transport inhibitor response-1 (TIR-1) auxin receptor in susceptible melon tissues provides in vivo support for the model of auxin-mediated impacts on A. gossypii resistance.

Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles.

Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the piRNA pathway is induced in response to viral infection. Evidence for robust insect RNAi machinery in several hemipteran species is of immense interest and is being actively pursued as a possible tool for insect control. RNAi-induced gene silencing following uptake of exogenous dsRNA was successfully demonstrated in several hemipterans and the presence of sid-1 like genes support the concept of a systemic response in some species.