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Long distance transport in plants occurs in sieve tubes of the phloem. The pressure flow hypothesis introduced by Ernst Münch in 1930 describes a mechanism of osmotically generated pressure differentials that are supposed to drive the movement of sugars and other solutes in the phloem, but this hypothesis has long faced major challenges. The key issue is whether the conductance of sieve tubes, including sieve plate pores, is sufficient to allow pressure flow. We show that with increasing distance between source and sink, sieve tube conductivity and turgor increases dramatically in Ipomoea nil. Our results provide strong support for the Münch hypothesis, while providing new tools for the investigation of one of the least understood plant tissues. Plants use energy from sunlight to make sugars in a process called photosynthesis. Most photosynthesis takes place in the leaves and so much of the sugar needs to be transported to other parts of the plant, such as fruits or roots. The sugars are transported by phloem tubes, which form a system that spans the entire plant. In 1930, a German scientist called Ernst Münch proposed a hypothesis for how phloem tubes move sugars and other molecules around the plant. He proposed that the loading of these molecules into phloem tubes in the leaves or other “source\" tissues makes the fluid inside the vessels more concentrated so that water is drawn into the phloem from neighboring “xylem” vessels. This creates pressure that pushes the fluid along the phloem tube towards the fruit, roots and other “sink” tissues. In the sink tissues the sugars are consumed, which reduces their concentration in the phloem and the pressure. Overall, this results in the flow of sugars and other molecules from where they are produced to where they are most needed. However, this hypothesis is still largely untested because it has proved difficult to carry out experiments on phloem. Detaching the source tissues from the sink tissues stops the flow of fluid so only experiments in whole plants can provide meaningful data. Knoblauch et al. have now developed new methods to study phloem in an ornamental plant called morning glory. The experiments show that plants can alter the shape of phloem vessels and the pressure within the vessels to allow them to transport sugars and other molecules over different distances. These findings strongly support the Münch hypothesis and make other alternative hypotheses seem unlikely. Furthermore, the methods developed by Knoblauch et al. will allow others to further investigate phloem transport. New findings in this area may allow plant biologists to direct the flow of sugars and other molecules towards particular plant tissues to improve the nutritional quality of food crops in the future.

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.

Trees present a critical challenge to long-distance transport because as a tree grows in height and the transport pathway increases in length, the hydraulic resistance of the vascular tissue should increase. This has led many to question whether trees can rely on a passive transport mechanism to move carbohydrates from their leaves to their roots. Although species that actively load sugars into their phloem, such as vines and herbs, can increase the driving force for transport as they elongate, it is possible that many trees cannot generate high turgor pressures because they do not use transporters to load sugar into the phloem. Here, we examine how trees can maintain efficient carbohydrate transport as they grow taller by analysing sieve tube anatomy, including sieve plate geometry, using recently developed preparation and imaging techniques, and by measuring the turgor pressures in the leaves of a tall tree in situ. Across nine deciduous species, we find that hydraulic resistance in the phloem scales inversely with plant height because of a shift in sieve element structure along the length of individual trees. This scaling relationship seems robust across multiple species despite large differences in plate anatomy. The importance of this scaling becomes clear when phloem transport is modelled using turgor pressures measured in the leaves of a mature red oak tree. These pressures are of sufficient magnitude to drive phloem transport only in concert with structural changes in the phloem that reduce transport resistance. As a result, the key to the long-standing mystery of how trees maintain phloem transport as they increase in size lies in the structure of the phloem and its ability to change hydraulic properties with plant height. The phloem is the system of ‘blood vessels’ that translocates carbohydrates from the leaves to different plant organs. Here, using new structural imaging and pressure measuring tools, the researchers show interesting phloem structural changes that ensure a passive transport mechanism in tall trees.

Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro-electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage-dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltagedependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage-dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.

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. Plasmodesmata channel substance transportation between neighbouring cells, including the sieve tube elements and the phloem companion cells. Now, the plasmodesmal ultrastructure is shown to be regulated by a newly identified phloem unloading modulator that participates in sphingolipid biosynthesis in Arabidopsis .

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.

Sieve tubes of legumes (Fabaceae) contain characteristic P-protein crystalloids with controversial function. We studied their behavior by conventional light, electron, and confocal laser scanning microscopy. In situ, crystalloids are able to undergo rapid (<1 sec) and reversible conversions from the condensed resting state into a dispersed state, in which they occlude the sieve tubes. Crystalloid dispersal is triggered by plasma membrane leakage induced by mechanical injury or permeabilizing substances. Similarly, abrupt turgor changes imposed by osmotic shock cause crystalloid dispersal. Because chelators generally prevent the response, divalent cations appear to be the decisive factor in crystalloid expansion. Cycling between dispersal and condensation can be induced in opened cells by repetitive exchange of bathing media containing either Ca2+ or chelators. Sr2+ and Ba2+, but not Mg2+, are equally active. In conclusion, the fabacean P-protein crystalloids represent a novel class of mechanically active proteinaceous structures, which provide an efficient mechanism with which to control sieve tube conductivity.

Differentiating sieve elements in the phloem of angiosperms produce abundant phloem-specific proteins before their protein synthesis machinery is degraded. These P-proteins initially form dense bodies, which disperse into individual filaments when the sieve element matures. In some cases, however, the dense protein agglomerations remain intact and are visible in functional sieve tubes as non-dispersive P-protein bodies, or NPBs. Species exhibiting NPBs are distributed across the entire angiosperm clade. We found that NPBs in the model tree, Populus trichocarpa, resemble the protein bodies described from other species of the order Malpighiales as they all consist of coaligned tubular fibrils bundled in hexagonal symmetry. NPBs of all Malpighiales tested proved unresponsive to sieve tube wounding and Ca2+. The P. trichocarpa NPBs consisted of a protein encoded by a gene that in the genome database of this species had been annotated as a homolog of SEOR1 (sieve element occlusion-related 1) in Arabidopsis. Sequencing of the gene in our plants corroborated this interpretation, and we named the gene PtSEOR1. Previously characterized SEOR proteins form irregular masses of P-protein slime in functional sieve tubes. We conclude that a subgroup of these proteins is involved in the formation of NPBs at least in the Malpighiales, and that these protein bodies have no role in rapid wound responses of the sieve tube network.

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of ‘funnel plasmodesmata’. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as ‘batch unloading’. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots. A mechanism called photosynthesis allows plants to use energy from sunlight to make sugars from carbon dioxide gas and water. These sugars can then be used as fuel, or as building blocks for wood and other plant structures. Every part of the plant requires sugars, but most photosynthesis happens in the leaves and stems, so the sugars need to be able to move around the plant to wherever they are needed. Phloem tubes form a network that transports sugar, proteins and other molecules around the plant within a fluid known as sap. Because this network is so extensive, it is very difficult to study, which has left researchers with major questions about how it works. For example, it is not clear how the sugar and other molecules leave the phloem when they reach their destination. Ross-Elliot et al. used a combination of microscopy and mathematical modeling to investigate how sugars and other molecules leave the phloem in the roots of a plant called Arabidopsis thaliana. The experiments show that these molecules move directly into cells within a neighboring tissue called the phloem-pole pericycle via pores known as funnel plasmodesmata. Ross-Elliot et al. incorporated the experimental data into a mathematical model of phloem unloading. This model suggests that sugars and other small molecules move freely through the funnel plasmodesmata, but large proteins pass through these pores in pulses. Future challenges include finding out exactly how plants control phloem unloading and to investigate whether it is possible to modify the delivery of specific molecules to particular parts of the plant.

Epidermal trichomes function as mechanosensors, but how trichome-less plants perceive mechanical forces remains unclear. Touching epidermal pavement cells with micro-cantilevers, we discovered distinct cytosolic calcium waves upon application and release of small forces. Thus, not only do plants perceive forces independently of trichomes, they may also distinguish touch from letting go.