Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition.

Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven ß-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.

Apoplasmic loading in the rice phloem supported by the presence of sucrose synthase and plasma membrane-localized proton pyrophosphatase.

BACKGROUND AND AIMS: Although Oryza sativa (rice) is one of the most important cereal crops, the mechanism by which sucrose, the major photosynthate, is loaded into its phloem is still a matter of debate. Current opinion holds that the phloem loading pathway in rice could involve either a symplasmic or an apoplasmic route. It was hypothesized, on the basis of a complementary body of evidence from arabidopsis, which is an apoplasmic loader, that the membrane specificity of proton pyrophosphatases (H(+)-PPases; OVPs) in the sieve element-companion cell (SE-CC) complexes of rice source leaves would support the existence of either of the aforementioned phloem loading mechanisms. Additionally, it was contended that the presence of sucrose synthase in the SE-CC complexes would be consistent with an apoplasmic sucrose loading route in rice. METHODS: Conventional chemical fixation methods were used for immunohistochemical localization of H(+)-PPases and sucrose synthase in rice and arabidopsis at the light microscopy level, while ultrastructural immunogold labelling of H(+)-PPases and sucrose synthase was performed on high-pressure frozen source leaves of rice. KEY RESULTS: Using immunogold labelling, it was found that OVPs predominantly localize at the plasma membrane (PM) of the SE-CC complexes in rice source leaf minor veins, while in the root meristematic cells, OVPs preferentially localize at the vacuoles. The PM specificity of OPVs in the SE-CC complexes was deemed to support apoplasmic loading in the rice phloem. Further backing for this interpretation came from the sucrose synthase-specific immunogold labelling at the SE-CC complexes of rice source leaves. CONCLUSION: These findings are consistent with the idea that, in the same way as in arabidopsis and a majority of grasses, sucrose is actively loaded into the SE-CC complexes of rice leaves using an apoplasmic step.

Dual localization of the carboxy-terminal tail of GLR3.3 in sieve element-companion cell complex.

Glutamate receptor-like (GLR) 3.3 and 3.6 proteins are required for mediating wound-induced leaf-to-leaf electrical signaling. In the previous study, we found that the carboxy-terminal tail of GLR3.3 contains key residues that are indispensable for its action in electrical signaling. In the present work, we generated plants that expressed the truncated C-tail fraction of GLR3.3. To our expectation, the truncated C-tail itself was not functional in propagating leaf-to-leaf signals. However, we identified that the C-tail-mVENUS fusion proteins had dual localization patterns in sieve elements and companion cells. In companion cells, the fusion proteins overlapped largely with the nucleus. We speculated that a possible nuclear localization signal is present in the C-tail of GLR3.3, paralleling the C-tails of the ionotropic glutamate receptors in animal cells. Our further findings on the C-tail of GLR3.3 open up new possibilities for the regulatory roles of the C-tails to GLR proteins.

The Interplay between Enucleated Sieve Elements and Companion Cells.

In order to adapt to sessile life and terrestrial environments, vascular plants have developed highly sophisticated cells to transport photosynthetic products and developmental signals. Of these, two distinct cell types (i.e., the sieve element (SE) and companion cell) are arranged in precise positions, thus ensuring effective transport. During SE differentiation, most of the cellular components are heavily modified or even eliminated. This peculiar differentiation implies the selective disintegration of the nucleus (i.e., enucleation) and the loss of cellular translational capacity. However, some cellular components necessary for transport (e.g., plasmalemma) are retained and specific phloem proteins (P-proteins) appear. Likewise, MYB (i.e., APL) and NAC (i.e., NAC45 and NAC86) transcription factors (TFs) and OCTOPUS proteins play a notable role in SE differentiation. The maturing SEs become heavily dependent on neighboring non-conducting companion cells, to which they are connected by plasmodesmata through which only 20-70 kDa compounds seem to be able to pass. The study of sieve tube proteins still has many gaps. However, the development of a protocol to isolate proteins that are free from any contaminating proteins has constituted an important advance. This review considers the very detailed current state of knowledge of both bound and soluble sap proteins, as well as the role played by the companion cells in their presence. Phloem proteins travel long distances by combining two modes: non-selective transport via bulk flow and selective regulated movement. One of the goals of this study is to discover how the protein content of the sieve tube is controlled. The majority of questions and approaches about the heterogeneity of phloem sap will be clarified once the morphology and physiology of the plasmodesmata have been investigated in depth. Finally, the retention of specific proteins inside an SE is an aspect that should not be forgotten.

A Reservoir of Pluripotent Phloem Cells Safeguards the Linear Developmental Trajectory of Protophloem Sieve Elements.

Plant cells can change their identity based on positional information, a mechanism that confers developmental plasticity to plants. This ability, common to distinct multicellular organisms, is particularly relevant for plant phloem cells. Protophloem sieve elements (PSEs), one type of phloem conductive cells, act as the main organizers of the phloem pole, which comprises four distinct cell files organized in a conserved pattern. Here, we report how Arabidopsis roots generate a reservoir of meristematic phloem cells competent to swap their cell identities. Although PSE misspecification induces cell identity hybridism, the activity of RECEPTOR LIKE PROTEIN KINASE 2 (RPK2) by perceiving CLE45 peptide contributes to restrict PSE identity to the PSE position. By maintaining a spatiotemporal window when PSE and PSE-adjacent cells' identities are interchangeable, CLE45 signaling endows phloem cells with the competence to re-pattern a functional phloem pole when protophloem fails to form.

Transmission Electron Microscopy of the Phloem with Minimal Artifacts.

It is a universal feature of seed plants that their phloem consists of a continuous sieve-tube system throughout the plant that is highly pressurized by its sugar contents. Cellular continuity and the pressure flow, osmotically generated in the source leaves, allow the assimilates to reach all sinks organs. However, both phloem features, the cellular continuity and the high pressure, are challenges when fixing the phloem for transmission electron microscopy. With very few exceptions, the tissue preparation necessary for the fixation evokes rapid wound responses that eventually result in artifacts.This chapter describes the steps necessary to minimize development of artifacts in the phloem and includes preparation of fixatives, a dissection procedure that optimizes penetration of the fixatives and application to axial and lateral plant organs. Moreover, as alternative to the established fixation of fresh hand sections, we suggest a xylem-assisted perfusion fixation method for herbaceous plants. After the initial fixation, the subsequent dehydration, embedding, and ultrathin sectioning of the material follow routine procedures, which are briefly discussed, as is the orientation of samples for obtaining transverse and longitudinal phloem sections.

Methods of Phloem Visualization: A Clear Future in Sight?

There have been exciting new results in phloem research in recent years, at least in part made possible by the rapid advancement of microscopic techniques. Several methods for visualizing phloem cells are available. The suitability of each method depends on the organ and species being studied, and on the scientific question being addressed. This review will briefly explain the specific challenges associated with phloem cell visualization. It will then focus on common methods currently being used for studying phloem anatomy, development, and function. Emphasis will be placed on the most recent improvements in imaging techniques which had, or most certainly will have, an impact on phloem research.

The Major Floral Promoter NtFT5 in Tobacco (Nicotiana tabacum) Is a Promising Target for Crop Improvement.

The FLOWERING LOCUS T (FT)-like gene family encodes key regulators of flower induction that affect the timing of reproduction in many angiosperm species. Agricultural research has therefore focused on such genes to improve the success of breeding programs and enhance agronomic traits. We recently identified a novel FT-like gene (NtFT5) that encodes a day-neutral floral activator in the model tobacco crop Nicotiana tabacum. However, further characterization is necessary to determine its value as a target for breeding programs. We therefore investigated the function of NtFT5 by expression analysis and mutagenesis. Expression analysis revealed that NtFT5 is transcribed in phloem companion cells, as is typical for FT-like genes. However, high levels of NtFT5 mRNA accumulated not only in the leaves but also in the stem. Loss-of-function mutants (generated using CRISPR/Cas9) were unable to switch to reproductive growth under long-day conditions, indicating that NtFT5 is an indispensable major floral activator during long-days. Backcrossing was achieved by grafting the mutant scions onto wild-type rootstock, allowing the restoration of flowering and pollination by a wild-type donor. The resulting heterozygous Ntft5- /NtFT5+ plants flowered with a mean delay of only ~2 days, demonstrating that one functional allele is sufficient for near-normal reproductive timing. However, this minor extension of the vegetative growth phase also conferred beneficial agronomic traits, including a >10% increase in vegetative leaf biomass on the main shoot and the production of more seeds. The agronomic benefits of the heterozygous plants persisted under various abiotic stress conditions, confirming that NtFT5 is a promising target for crop improvement to address the effects of climate change.

Vascular gene expression: a hypothesis.

The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a "primitive" vascular tissue (a lycophyte), as well as from others that lack a true vascular tissue (a bryophyte), and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non-vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT, and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

Phloem development in nematode-induced feeding sites: the implications of auxin and cytokinin.

Sedentary plant parasitic nematodes such as root-knot nematodes and cyst nematodes induce giant cells or syncytia, respectively, in their host plant's roots. These highly specialized structures serve as feeding sites from which exclusively the nematodes withdraw nutrients. While giant cells are symplastically isolated and obtain assimilates by transporter-mediated processes syncytia are massively connected to the phloem by plasmodesmata. To support the feeding sites and the nematode during their development, phloem is induced around syncytia and giant cells. In the case of syncytia the unloading phloem consists of sieve elements and companion cells and in the case of root knots it consists exclusively of sieve elements. We applied immunohistochemistry to identify the cells within the developing phloem that responded to auxin and cytokinin. Both feeding sites themselves did not respond to either hormone. We were able to show that in root knots an auxin response precedes the differentiation of these auxin responsive cells into phloem elements. This process appears to be independent of B-type Arabidopsis response regulators. Using additional markers for tissue identity we provide evidence that around giant cells protophloem is formed and proliferates dramatically. In contrast, the phloem around syncytia responded to both hormones. The presence of companion cells as well as hormone-responsive sieve elements suggests that metaphloem development occurs. The implication of auxin and cytokinin in the further development of the metaphloem is discussed.

The tie-dyed pathway promotes symplastic trafficking in the phloem.

The tie-dyed1 (tdy1) and tdy2 mutants of maize exhibit leaf regions with starch hyperaccumulation and display unusual genetic interactions, suggesting they function in the same physiological process. Tdy2 encodes a putative callose synthase and is expressed in developing vascular tissues of immature leaves. Radiolabelling experiments and transmission electron microscopy (TEM) revealed symplastic trafficking within the phloem was perturbed at the companion cell/sieve element interface. Here, we show that as reported for tdy2 mutants, tdy1 yellow leaf regions display an excessive oil-droplet phenotype in the companion cells. Based on the proposed function of Tdy2 as a callose synthase, our previous work characterizing Tdy1 as a novel, transmembrane-localized protein, and the present findings, we speculate how TDY1 and TDY2 might interact to promote symplastic transport of both solutes and developmentally instructive macromolecules during vascular development at the companion cell/sieve element interface.

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants.

Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (-130 mV to -110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. -100 mV). In roots, the membrane potential of sieve elements dropped abruptly to -55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H(+)-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie-Hofstee (EH) -transformations pointed at biphasic Michaelis-Menten kinetics (2 MM, EH: K m1 1.2-1.8 mM, K m2 6.6-9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, K m values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher K m values (EH: K m1 10 mM, K m2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (-0.1 to -0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) K m values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of K m values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed.

Multiple cis-regulatory elements are involved in the complex regulation of the sieve element-specific MtSEO-F1 promoter from Medicago truncatula.

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1.