Symplasmic phloem loading and subcellular transport in storage roots are key factors for carbon allocation in cassava.

Cassava (Manihot esculenta) is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilate unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here, we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and at different timepoints to better understand source-sink carbon allocation. We found that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we showed that tissue-specific changes in anatomy and non-structural carbohydrate (NSC) contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically and is likely mostly regulated by local tissue demand and subcellular compartmentalization.

Locally optimal geometry for surface-enhanced diffusion.

Molecular diffusion in bulk liquids proceeds according to Fick's law, which stipulates that the particle current is proportional to the conductive area. This constrains the efficiency of filtration systems in which both selectivity and permeability are valued. Previous studies have demonstrated that interactions between the diffusing species and solid boundaries can enhance or reduce particle transport relative to bulk conditions. However, only cases that preserve the monotonic relationship between particle current and conductive area are known. In this paper, we expose a system in which the diffusive current increases when the conductive area diminishes. These examples are based on the century-old theory of a charged particle interacting with an electrical double layer. This surprising discovery could improve the efficiency of filtration and may advance our understanding of biological pore structures.

Pavement cells distinguish touch from letting go.

A micro-cantilever technique applied to individual leaf epidermis cells of intact Arabidopsis thaliana and Nicotiana tabacum synthesizing genetically encoded calcium indicators (R-GECO1 and GCaMP3) revealed that compressive forces induced local calcium peaks that preceded delayed, slowly moving calcium waves. Releasing the force evoked significantly faster calcium waves. Slow waves were also triggered by increased turgor and fast waves by turgor drops in pressure probe tests. The distinct characteristics of the wave types suggest different underlying mechanisms and an ability of plants to distinguish touch from letting go.

How Münch's adaptation of Pfeffer's circulating water flow became the pressure-flow theory, and the resulting problems - A historical perspective.

Long-distance transport of photoassimilates in the phloem of vascular plants occurs as bulk flow in sieve tubes. These tubes are arrays of cells that lose nuclei, cytoskeleton, and some organelles when they differentiate into mature sieve elements. Symplasmic continuity is achieved by perforations that turn the cell walls between adjoining sieve elements into sieve plates. These structural features are interpreted as adaptations that reduce the resistance sieve tubes offer to cytoplasmic bulk flow. According to the common reading of Ernst Münch's pressure-flow theory, the driving forces for these flows are osmotically generated gradients of hydrostatic pressure along the sieve tubes. However, the significance of pressure gradients in the flow direction has also been questioned. Münch himself stated that no detectable pressure gradients existed between the linked osmotic cells that he used to demonstrate the validity of his ideas, and the earliest explanation of osmotically driven flows by Wilhelm Pfeffer, on which Münch based his theory, explicitly claimed the absence of pressure gradients. To resolve the apparent contradiction, we here reconstruct the history of the idea that osmotically driven transport processes in organisms necessarily require steps or gradients of hydrostatic pressure along the transport route. Our analysis leads us to conclude that some defects of overly simplifying interpretations of Münch's ideas (such as the sieve plate fallacy) could be avoided if our descriptions of his theory in textbooks and the scientific literature would follow the logics of the theory's earliest formulations more closely.

The Phloem as an Arena for Plant Pathogens.

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.

Diversity of funnel plasmodesmata in angiosperms: the impact of geometry on plasmodesmal resistance.

In most plant tissues, threads of cytoplasm, or plasmodesmata, connect the protoplasts via pores in the cell walls. This enables symplasmic transport, for instance in phloem loading, transport and unloading. Importantly, the geometry of the wall pore limits the size of the particles that may be transported, and also (co-)defines plasmodesmal resistance to diffusion and convective flow. However, quantitative information on transport through plasmodesmata in non-cylindrical cell wall pores is scarce. We have found conical, funnel-shaped cell wall pores in the phloem-unloading zone in growing root tips of five eudicot and two monocot species, specifically between protophloem sieve elements and phloem pole pericycle cells. 3D reconstructions by electron tomography suggested that funnel plasmodesmata possess a desmotubule but lack tethers to fix it in a central position. Model calculations showed that both diffusive and hydraulic resistance decrease drastically in conical and trumpet-shaped cell wall pores compared with cylindrical channels, even at very small opening angles. Notably, the effect on hydraulic resistance was relatively larger. We conclude that funnel plasmodesmata generally are present in specific cell-cell interfaces in angiosperm roots, where they appear to facilitate symplasmic phloem unloading. Interestingly, cytosolic sleeves of most plasmodesmata reported in the literature do not resemble annuli of constant diameter but possess variously shaped widenings. Our evaluations suggest that widenings too small for unambiguous identification on electron micrographs may drastically reduce the hydraulic and diffusional resistance of these pores. Consequently, theoretical models assuming cylindrical symmetries will underestimate plasmodesmal conductivities.

Sugar loading is not required for phloem sap flow in maize plants.

Phloem transport of photoassimilates from leaves to non-photosynthetic organs, such as the root and shoot apices and reproductive organs, is crucial to plant growth and yield. For nearly 90 years, evidence has been generally consistent with the theory of a pressure-flow mechanism of phloem transport. Central to this hypothesis is the loading of osmolytes, principally sugars, into the phloem to generate the osmotic pressure that propels bulk flow. Here we used genetic and light manipulations to test whether sugar import into the phloem is required as the driving force for phloem sap flow. Using carbon-11 radiotracer, we show that a maize sucrose transporter1 (sut1) loss-of-function mutant has severely reduced export of carbon from photosynthetic leaves (only ~4% of the wild type level). Yet, the mutant remarkably maintains phloem pressure at ~100% and sap flow speeds at ~50-75% of those of wild type. Potassium (K+) abundance in the phloem was elevated in sut1 mutant leaves. Fluid dynamic modelling supports the conclusion that increased K+ loading compensated for decreased sucrose loading to maintain phloem pressure, and thereby maintained phloem transport via the pressure-flow mechanism. Furthermore, these results suggest that sap flow and transport of other phloem-mobile nutrients and signalling molecules could be regulated independently of sugar loading into the phloem, potentially influencing carbon-nutrient homoeostasis and the distribution of signalling molecules in plants encountering different environmental conditions.

Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element-specific proteins reveal differentiation of the endomembrane system.

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning.

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Under salt stress guard cells rewire ion transport and abscisic acid signaling.

Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.

Plasmodesmata and the problems with size: Interpreting the confusion.

Plant tissues exhibit a symplasmic organization; the individual protoplasts are connected to their neighbors via cytoplasmic bridges that extend through pores in the cell walls. These bridges may have diameters of a micrometer or more, as in the sieve pores of the phloem, but in most cell types they are smaller. Historically, botanists referred to cytoplasmic bridges of all sizes as plasmodesmata. The meaning of the term began to shift when the transmission electron microscope (TEM) became the preferred tool for studying these structures. Today, a plasmodesma is widely understood to be a 'nano-scale' pore. Unfortunately, our understanding of these nanoscopic channels suffers from methodological limitations. This is exemplified by the fact that state-of-the-art EM techniques appear to reveal plasmodesmal pore structures that are much smaller than the tracer molecules known to diffuse through these pores. In general, transport processes in pores that have dimensions in the size range of the transported molecules are governed by different physical parameters than transport process in the macroscopic realm. This can lead to unexpected effects, as experience in nanofluidic technologies demonstrates. Our discussion of problems of size in plasmodesma research leads us to conclude that the field will benefit from technomimetic reasoning - the utilization of concepts developed in applied nanofluidics for the interpretation of biological systems.

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness.

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein-protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.

Sieve elements rapidly develop 'nacreous walls' following injury - a common wounding response?

Thick glistening cell walls occur in sieve tubes of all major land plant taxa. Historically, these 'nacreous walls' have been considered a diagnostic feature of sieve elements; they represent a conundrum, though, in the context of the widely accepted pressure-flow theory as they severely constrict sieve tubes. We employed the cucurbit Gerrardanthus macrorhizus as a model to study nacreous walls in sieve elements by standard and in situ confocal microscopy and electron microscopy, focusing on changes in functional sieve tubes that occur when prepared for microscopic observation. Over 90% of sieve elements in tissue sections processed for microscopy by standard methods exhibit nacreous walls. Sieve elements in whole, live plants that were actively transporting as shown by phloem-mobile tracers, lacked nacreous walls and exhibited open lumina of circular cross-sections instead, an appropriate structure for Münch-type mass flow of the cell contents. Puncturing of transporting sieve elements with micropipettes triggered the rapid (<1 min) development of nacreous walls that occluded the cell lumen almost completely. We conclude that nacreous walls are preparation artefacts rather than structural features of transporting sieve elements. Nacreous walls in land plants resemble the reversibly swellable walls found in various algae, suggesting that they may function in turgor buffering, the amelioration of osmotic stress, wounding-induced sieve tube occlusion, and possibly local defence responses of the phloem.

The diffusive injection micropipette (DIMP).

The microinjection of fluorescent probes into live cells is an essential component in the toolbox of modern cell biology. Microinjection techniques include the penetration of the plasma membrane and, if present, the cell wall with micropipettes, and the application of pressure or electrical currents to drive the micropipette contents into the cell. These procedures interfere with cellular functions and therefore may induce artifacts. We designed the diffusive injection micropipette (DIMP) that avoids most of the possible artifacts due to the drastically reduced volume of its fluid contents and the utilization of diffusion for cargo delivery into the target cell. DIMPs were successfully tested in plant, fungal, and animal cells. Using the continuity of cytoplasmic dynamics over ten minutes after impalement of Nicotiana trichome cells as a criterion for non-invasiveness, we found DIMPs significantly less disruptive than conventional pressure microinjection. The design of DIMPs abolishes major sources of artifacts that cannot be avoided by other microinjection techniques. Moreover, DIMPs are inexpensive, easy to produce, and can be applied without specific equipment other than a micromanipulator. With these features, DIMPs may become the tool of choice for studies that require the least invasive delivery possible of materials into live cells.

Publisher Correction: Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sphingolipid biosynthesis modulates plasmodesmal ultrastructure and phloem unloading.

During phloem unloading, multiple cell-to-cell transport events move organic substances to the root meristem. Although the primary unloading event from the sieve elements to the phloem pole pericycle has been characterized to some extent, little is known about post-sieve element unloading. Here, we report a novel gene, PHLOEM UNLOADING MODULATOR (PLM), in the absence of which plasmodesmata-mediated symplastic transport through the phloem pole pericycle-endodermis interface is specifically enhanced. Increased unloading is attributable to a defect in the formation of the endoplasmic reticulum-plasma membrane tethers during plasmodesmal morphogenesis, resulting in the majority of pores lacking a visible cytoplasmic sleeve. PLM encodes a putative enzyme required for the biosynthesis of sphingolipids with very-long-chain fatty acid. Taken together, our results indicate that post-sieve element unloading involves sphingolipid metabolism, which affects plasmodesmal ultrastructure. They also raise the question of how and why plasmodesmata with no cytoplasmic sleeve facilitate molecular trafficking.

Symplasmic phloem unloading and radial post-phloem transport via vascular rays in tuberous roots of Manihot esculenta.

Cassava (Manihot esculenta) is one of the most important staple food crops worldwide. Its starchy tuberous roots supply over 800 million people with carbohydrates. Yet, surprisingly little is known about the processes involved in filling of those vital storage organs. A better understanding of cassava carbohydrate allocation and starch storage is key to improving storage root yield. Here, we studied cassava morphology and phloem sap flow from source to sink using transgenic pAtSUC2::GFP plants, the phloem tracers esculin and 5(6)-carboxyfluorescein diacetate, as well as several staining techniques. We show that cassava performs apoplasmic phloem loading in source leaves and symplasmic unloading into phloem parenchyma cells of tuberous roots. We demonstrate that vascular rays play an important role in radial transport from the phloem to xylem parenchyma cells in tuberous roots. Furthermore, enzymatic and proteomic measurements of storage root tissues confirmed high abundance and activity of enzymes involved in the sucrose synthase-mediated pathway and indicated that starch is stored most efficiently in the outer xylem layers of tuberous roots. Our findings form the basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.

Protein structural biology using cell-free platform from wheat germ.

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.