Enucleate Sieve Elements Research Articles

Activity of Arabidopsis Rubisco small subunit promoter in various tissues of chickpea.

The activity of a green tissue-specific promoter of the Rubisco small subunit gene fromArabidopsis(AraSSU) was studied using transgenic chickpea lines. We generated transgenic chickpea lines expressing anAraSSUpromoter-drivencry2Aagene throughthe Agrobacterium-mediatedtransformation method. Lines withAraSSUexpressed the gene in all green tissues at high levels (> 90ng/mg of fresh weight tissue) compared to lines generated usingCaMV35S(< 10ng/mg FW). We used vertical cross sections of various tissues of homozygous progeny using microtome for immunolocalization. The immunolocalization showed the expression of thecry2Aagene in the green mesophyll cells of the leaves of bothAraSSUand CaMV35 chickpea lines. Moreover, the accumulation ofAraSSU-regulatedCry2Aa protein was also observed in vascular tissues, including enucleate sieve elements and their companion cells. However, no expression was observed in the roots ofAraSSUlines. In the case ofCaMV35lines, the transgene expression was observed in all the tissues. Since our data indicated that theAraSSUpromoter is active in non-green tissues such as vascular bundles. Therefore, we validated this by RT-PCR. We found Cry2Aa RNA transcripts in leaves, stems without epidermis (for vascular tissues), and roots with and without epidermis. Thus,the AraSSUpromoter is active in all above-ground tissues of the chickpea plant.

Sieve Elements: The Favourite Habitat of Phytoplasmas.

The sieve elements are the only plant compartments, where phytoplasmas can survive and propagate. Therefore, this chapter is focussed on the specific molecular and cell-biological properties of the sieve element. Sieve element-companion cell complexes arise from (pro)cambial mother cells induced by key genes known to be decisive for sieve-element differentiation. The special anatomy, cell biology, and plasma-membrane outfit of sieve elements allows them to act collectively as a tube system that is able to drive a mass flow against the flow induced by transpiration. Plasmodesmal corridors are vital for the translocation of photoassimilates and systemic signals and for survival of the enucleate sieve elements. Of paramount importance is the Ca2+-dependent gating of plasmodesmata by callose and proteins. Hence, some of the complex, regulatory mechanisms to maintain Ca2+ homoeostasis in sieve elements are presented. Finally, the peculiarities of the chemical and physical sieve-element environment offered to phytoplasmas are discussed.

Functional analysis of Brassica napus phloem protein and ribonucleoprotein complexes.

Summary Phloem sap contains a large number of macromolecules, including proteins and RNAs from different classes. Proteome analyses of phloem samples from different plant species under denaturing conditions identified hundreds of proteins potentially involved in diverse processes. Surprisingly, these studies also found a significant number of ribosomal and proteasomal proteins. This led to the suggestion that active ribosome and proteasome complexes might be present in the phloem, challenging the paradigm that protein synthesis and turnover are absent from the enucleate sieve elements of angiosperms. However, the existence of such complexes has as yet not been demonstrated.In this study we used three‐dimensional gel electrophoresis to separate several protein complexes from native phloem sap from Brassica napus. Matrix‐assisted laser desorption ionization‐time of flight MS analyses identified more than 100 proteins in the three major protein‐containing complexes.All three complexes contained proteins belonging to different ribosomal fragments and blue native northern blot confirmed the existence of ribonucleoprotein complexes. In addition, one complex contained proteasome components and further functional analyses confirmed activity of a proteasomal degradation pathway and showed a large number of ubiquitinated phloem proteins.Our results suggest specialized roles for ubiquitin modification and proteasome‐mediated degradation in the phloem.

The secret phloem of pumpkins

The phloem of higher plants transports the products of photosynthesis and other nutrients over long distances from leaves to sink tissues, such as fruits, tubers, and roots, where they are used in growth or storage. The primary conducting cells of the phloem are the enucleate sieve elements, which are intimately connected to their neighboring companion cells. The sieve elements are joined at their ends by perforated cell walls called sieve plates to form sieve tubes, through which solutes flow as a river of nutrient solution. Many recent studies have focused on the fascinating discovery that the phloem functions not only to transport low-molecular–weight solutes but also macromolecules, including proteins and RNAs. The conducting phloem has been likened to the plant’s “superhighway” (1), delivering a broad range of defense and developmental signals to distant parts of the plant, including the flowering signal “florigen” and the signal for tuberization in potatoes (2, 3). These concepts form the foundation of a unique and exciting field in developmental biology.

Analysis of the Pumpkin Phloem Proteome Provides Insights into Angiosperm Sieve Tube Function

Increasing evidence suggests that proteins present in the angiosperm sieve tube system play an important role in the long distance signaling system of plants. To identify the nature of these putatively non-cell-autonomous proteins, we adopted a large scale proteomics approach to analyze pumpkin phloem exudates. Phloem proteins were fractionated by fast protein liquid chromatography using both anion and cation exchange columns and then either in-solution or in-gel digested following further separation by SDS-PAGE. A total of 345 LC-MS/MS data sets were analyzed using a combination of Mascot and X!Tandem against the NCBI non-redundant green plant database and an extensive Cucurbit maxima expressed sequence tag database. In this analysis, 1,209 different consensi were obtained of which 1,121 could be annotated from GenBank and BLAST search analyses against three plant species, Arabidopsis thaliana, rice (Oryza sativa), and poplar (Populus trichocarpa). Gene ontology (GO) enrichment analyses identified sets of phloem proteins that function in RNA binding, mRNA translation, ubiquitin-mediated proteolysis, and macromolecular and vesicle trafficking. Our findings indicate that protein synthesis and turnover, processes that were thought to be absent in enucleate sieve elements, likely occur within the angiosperm phloem translocation stream. In addition, our GO analysis identified a set of phloem proteins that are associated with the GO term "embryonic development ending in seed dormancy"; this finding raises the intriguing question as to whether the phloem may exert some level of control over seed development. The universal significance of the phloem proteome was highlighted by conservation of the phloem proteome in species as diverse as monocots (rice), eudicots (Arabidopsis and pumpkin), and trees (poplar). These results are discussed from the perspective of the role played by the phloem proteome as an integral component of the whole plant communication system.

Immunolocalization of Solanaceous SUT1 Proteins in Companion Cells and Xylem Parenchyma: New Perspectives for Phloem Loading and Transport

Leaf sucrose (Suc) transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Suc loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 Suc transporters from tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) were immunolocalized in plasma membranes of enucleate sieve elements. Here, we present data that identify solanaceous SUT1 proteins with high specificity in phloem companion cells. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.

Phloem Mobility and Stability of Sucrose Transporter Transcripts

Sucrose transporters are essential membrane proteins for the distribution of photoassimilates in higher plants. In Solanaceous species the proteins of all known sucrose transporters are co-localized in enucleate sieve elements and un- dergo permanent turnover. The mRNA of the sucrose transporter StSUT1 is localized in both, sieve elements and compan- ion cells. Sucrose transporter mRNAs have been detected in the phloem sap of several species. Here, we analyzed the mo- bility of sucrose transporter transcripts in grafted plants and between host and parasitic plants. Phloem-mobility was found when a c-myc tagged SUT1-fusion construct without untranslated regions (UTRs) was expressed under the CaMV 35S promoter. We conclude that neither 3'- nor 5' -UTRs are essential for mRNA transport through plasmodesmata. Cycloheximide, which inhibits translation, has also effects on SUT transcript stability. Whereas SUT1 transcripts are de- stabilized when translation is inhibited, SUT2 and SUT4 transcripts accumulate up to 4fold under these conditions. Inhibi- tor studies revealed post-transcriptional regulation of SUT2 and SUT4 transcript accumulation. A model is proposed ex- plaining the coordination of SUT expression in Solanaceae.

Chloroplast-like organelles were found in enucleate sieve elements of transgenic plants overexpressing a proteinase inhibitor.

SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.

Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor

SaPIN2a encodes a proteinase inhibitor in nightshade ( Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

Expression and localisation analysis of the wheat sucrose transporter TaSUT1 in vegetative tissues.

Previously we reported the isolation of three sucrose transporter genes, TaSUT1A, 1B and 1D, all expressed at high levels in the developing grains of hexaploid wheat ( Triticum aestivum L.), but also in a variety of other tissues. In order to further characterise the expression of the TaSUT1 genes in wheat plants, we have analysed TaSUT1 expression in their vegetative tissues using semi-quantitative reverse transcription-polymerase chain reaction, in situ hybridisation and immunolocalisation. The three TaSUT1 genes, which encode 98% identical SUT proteins, all appeared to be expressed at the same level in leaf blades, leaf sheaths and internodes, as well as developing grains, of hexaploid wheat. In mature leaf blades, TaSUT1 protein localised to the plasma membrane of phloem sieve elements in all classes of veins. In contrast, TaSUT1 mRNA was found to be localised to phloem companion cells. A similar localisation pattern for TaSUT1 protein was observed in veins of leaf sheaths and internodes. These results suggest that the wheat SUT1 has a transport function in enucleate sieve elements, in both veins responsible for loading photoassimilates, and in veins for axial transport. Furthermore, transport of the fluorescent dye carboxyfluorescein was used to investigate symplasmic connectivity between sieve element-companion cell complexes and non-phloem cells. Observations in source leaves indicated that sieve element-companion cell complexes of minor veins were symplasmically restricted, suggesting a role of TaSUT1 in apoplasmic phloem loading. In contrast, the dye was able to move symplasmically out of the phloem in internodes. In these circumstances TaSUT1 may also have a role in retrieving sucrose leaked to the phloem apoplasm.

APL regulates vascular tissue identity in Arabidopsis.

Vascular plants have a long-distance transport system consisting of two tissue types with elongated cell files, phloem and xylem. Phloem has two basic cell types, enucleate sieve elements and companion cells. Xylem has various lignified cell types, such as tracheary elements, the differentiation of which involves deposition of elaborate cell wall thickenings and programmed cell death. Until now, little has been known about the genetic control of phloem-xylem patterning. Here we identify the ALTERED PHLOEM DEVELOPMENT (APL) gene, which encodes a MYB coiled-coil-type transcription factor that is required for phloem identity in Arabidopsis. Phloem is established through asymmetric cell divisions and subsequent differentiation. We show that both processes are impaired by a recessive apl mutation. This is associated with the formation of cells that have xylem characteristics in the position of phloem. The APL expression profile is consistent with a key role in phloem development. Ectopic APL expression in the vascular bundle inhibits xylem development. Our studies suggest that APL has a dual role both in promoting phloem differentiation and in repressing xylem differentiation during vascular development.

Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells

BackgroundPlant phloem consists of an interdependent cell pair, the sieve element / companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.ResultsIn contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.ConclusionA fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

Protein-protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element.

Suc represents the major transport form for carbohydrates in plants. Suc is loaded actively against a concentration gradient into sieve elements, which constitute the conduit for assimilate export out of leaves. Three members of the Suc transporter family with different properties were identified: SUT1, a high-affinity Suc proton cotransporter; SUT4, a low-affinity transporter; and SUT2, which in yeast is only weakly active and shows features similar to those of the yeast sugar sensors RGT2 and SNF3. Immunolocalization demonstrated that all three SUT proteins are localized in the same enucleate sieve element. Thus, the potential of Suc transporters to form homooligomers was tested by the yeast-based split-ubiquitin system. The results show that both SUT1 and SUT2 have the potential to form homooligomers. Moreover, all three Suc transporters have the potential to interact with each other. As controls, a potassium channel and a monosaccharide transporter, expressed in the plasma membrane, did not interact with the SUTs. The in vivo interaction between the functionally different Suc transporters indicates that the membrane proteins are capable of forming oligomeric structures that, like mammalian Glc transporter complexes, might be of functional significance for the regulation of transport.

Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.

A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants.

A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The K(m) for sucrose uptake by AtSUT4 of 11.6 +/- 0.6 mM was approximately 10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.

A New Subfamily of Sucrose Transporters, SUT4, with Low Affinity/High Capacity Localized in Enucleate Sieve Elements of Plants

A New Subfamily of Sucrose Transporters, SUT4, with Low Affinity/High Capacity Localized in Enucleate Sieve Elements of Plants

SUT2, a putative sucrose sensor in sieve elements.

In leaves, sucrose uptake kinetics involve high- and low-affinity components. A family of low- and high-affinity sucrose transporters (SUT) was identified. SUT1 serves as a high-affinity transporter essential for phloem loading and long-distance transport in solanaceous species. SUT4 is a low-affinity transporter with an expression pattern overlapping that of SUT1. Both SUT1 and SUT4 localize to enucleate sieve elements of tomato. New sucrose transporter-like proteins, named SUT2, from tomato and Arabidopsis contain extended cytoplasmic domains, thus structurally resembling the yeast sugar sensors SNF3 and RGT2. Features common to these sensors are low codon bias, environment of the start codon, low expression, and lack of detectable transport activity. In contrast to LeSUT1, which is induced during the sink-to-source transition of leaves, SUT2 is more highly expressed in sink than in source leaves and is inducible by sucrose. LeSUT2 protein colocalizes with the low- and high-affinity sucrose transporters in sieve elements of tomato petioles, indicating that multiple SUT mRNAs or proteins travel from companion cells to enucleate sieve elements. The SUT2 gene maps on chromosome V of potato and is linked to a major quantitative trait locus for tuber starch content and yield. Thus, the putative sugar sensor identified colocalizes with two other sucrose transporters, differs from them in kinetic properties, and potentially regulates the relative activity of low- and high-affinity sucrose transport into sieve elements.

Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleate sieve elements.

The leaf sucrose transporter SUT1 is essential for phloem loading and long-distance transport of assimilates. Both SUT1 messenger RNA (mRNA) and protein were shown to be diurnally regulated and to have high turnover rates. SUT1 protein was detected by immunolocalization in plasma membranes of enucleate sieve elements (SEs) in tobacco, potato, and tomato. Analysis by in situ hybridization showed that SUT1 mRNA localizes mainly to the SE and is preferentially associated with plasmodesmata. Antisense inhibition of SUT1 expression under control of a companion cell (CC)-specific promoter indicated synthesis of SUT1 mRNA in the CC. These results provide evidence for targeting of plant endogenous mRNA and potentially SUT1 protein through phloem plasmodesmata and for sucrose loading at the plasma membrane of SE.

Solute concentrations of the phloem and parenchyma cells present in squash callus

Abstract. Glutaraldehyde fixation was used to determine the solute concentrations in the various cell types present in tissue cultures of squash (Cucurbita pepo). Small pieces of callus were plasmolyzed in a graded series of mannitol solutions and fixed in 20 kg m−3 glutaraldehyde adjusted to be isosmotic with the particular plasmolysing solution. The callus samples were further processed using standard electron microscopy techniques. Using this procedure, mature sieve elements that form in squash callus have an osmotic potentional of ‐2.4MPa. The osmotic potential of the callus sieve elements was comparable to values reported for the sieve tube members of the phloem in intact plants. This ability of callus sieve elements to develop high internal hydrostatic pressures demonstrates that they are capable of phloem loading. However, the osmotic potentials of the surrounding parenchymatous cells and companion cells were only –1.15 and –1.5 MPa, respectively. In contrast to the companion cells of the phloem in intact plant tissues, the osmotic potential of the callus companion cells indicated that they were not directly involved in phloem loading. Several immature sieve elements containing distinct nuclei and vacuoles were observed in the callus granules. These immature sieve elements were plasmolyzed in weaker mannitol solutions (below 0.6kmol m−3) than the enucleate sieve elements (1.01 kmol m−3 mannitol). The low solute concentrations in immature sieve elements indicated that the ability to load sugars occurs concomitantly with the maturation of the sieve element protoplast.

Long‐term functioning of enucleate sieve elements: possible mechanisms of damage avoidance and damage repair

Abstract. The sieve‐tube elements of long‐lived arborescent monocotyledons which lack secondary thickening remain functional for many decades despite lacking a nucleus. A minimal requirement for transport by mass flow powered by the Munch mechanism is maintenance of semi‐permeability of the plasmalemma of the sieve tube elements; loading and re‐loading could be deputed to nucleate cells symplastically linked to the sieve elements. An additional requirement in the long‐term relates to replacement of components damaged by mechanical, chemical or radiation intrusions. Minimizing the damage from radiation and chemical agents can be related to a number of commonly observed features of sieve tubes. Damage from O2 and radicals derived there from is minimized in these essentially aerobic cells by (1) the absence of intercellular gas spaces in the phloem combined with the lower O2 solubility and diffusivity in concentrated disaccharide (or sugar alcohol) solutions, (2) the absence of photosynthetic machinery which could generate singlet oxygen, and (3) the presence of at least some components of scavenging mechanisms (glutathione, peroxidase, abscorbate). Non‐enzymic glycosylation (exacerbated by O2) of proteins is minimized by the low concentrations of reducing sugars in sieve‐tubes. Ultraviolet damage is minimized by UV‐absorbing materials between the plant surface and the sieve tubes, including the selerenchymatous cap of fibres on the vascular bundles. The extent to which repair involves symplastic import of polypeptides from nucleate companion cells, thus breaching the −800‐Da limit on symplastic transport, is unclear, but it could occur in fully differentiated companion cell‐sieve element associations without necessarily upsetting development.