Molecular and phylogenetic characterization of the sieve element occlusion gene family in Fabaceae and non-Fabaceae plants

<p>Abstract</p> <p>Background</p> <p>The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the <it>Fabaceae</it>. Recently, the molecular characterization of three genes encoding forisome components in the model legume <it>Medicago truncatula </it>(<it>MtSEO1</it>, <it>MtSEO2 </it>and <it>MtSEO3</it>; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-<it>Fabaceae</it>.</p> <p>Results</p> <p>We performed a comprehensive genome-wide comparative analysis by screening the <it>M. truncatula</it>, <it>Glycine max</it>, <it>Arabidopsis thaliana</it>, <it>Vitis vinifera </it>and <it>Solanum phureja </it>genomes, and a <it>Malus domestica </it>EST library for homologs of <it>MtSEO1</it>, <it>MtSEO2 </it>and <it>MtSEO3 </it>and identified numerous novel <it>SEO </it>genes in <it>Fabaceae </it>and even non-<it>Fabaceae </it>plants, which do not possess forisomes. Even in <it>Fabaceae </it>some <it>SEO </it>genes appear to not encode forisome components. All <it>SEO </it>genes have a similar exon-intron structure and are expressed predominantly in the phloem. Phylogenetic analysis revealed the presence of several subgroups with <it>Fabaceae</it>-specific subgroups containing all of the known as well as newly identified forisome component proteins. We constructed Hidden Markov Models that identified three conserved protein domains, which characterize SEO proteins when present in combination. In addition, one common and three subgroup specific protein motifs were found in the amino acid sequences of SEO proteins. <it>SEO </it>genes are organized in genomic clusters and the conserved synteny allowed us to identify several <it>M. truncatula </it>vs <it>G. max </it>orthologs as well as paralogs within the <it>G. max </it>genome.</p> <p>Conclusions</p> <p>The unexpected occurrence of forisome-like genes in non-<it>Fabaceae </it>plants may indicate that these proteins encode species-specific P-proteins, which is backed up by the phloem-specific expression profiles. The conservation of gene structure, the presence of specific motifs and domains and the genomic synteny argue for a common phylogenetic origin of forisomes and other P-proteins.</p

Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection.

Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants.We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation.We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants

Native and artificial forisomes : functions and applications

Forisomes are remarkable protein bodies found exclusively in the phloem of the Fabaceae. When the phloem is wounded, forisomes are converted from a condensed to a dispersed state in an ATP-independent reaction triggered by Ca(2+), thereby plugging the sieve tubes and preventing the loss of photoassimilates. Potentially, forisomes are ideal biomaterials for technical devices because the conformational changes can be replicated in vitro and are fully reversible over a large number of cycles. However, the development of technical devices based on forisomes has been hampered by the laborious and time-consuming process of purifying native forisomes from plants. More recently, the problem has been overcome by the production of recombinant artificial forisomes. This is a milestone in the development of forisome-based devices, not only because large quantities of homogeneous forisomes can be produced on demand, but also because their properties can be tailored for particular applications. In this review, we discuss the physical and molecular properties of native and artificial forisomes, focusing on their current applications in technical devices and potential developments in the future

P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing

Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently evidenced to be encoded by the widespread SEO gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing

ATP-independent contractile proteins from plants

Emerging technologies are creating increasing interest in smart materials that may serve as actuators in micro- and nanodevices. Mechanically active polymers currently studied include a variety of materials. ATP-driven motor proteins, the actuators of living cells, possess promising characteristics, but their dependence on strictly defined chemical environments can be disadvantagous. Natural proteins that deform reversibly by entropic mechanisms might serve as models for artificial contractile polypeptides with useful functionality, but they are rare. Protein bodies from sieve elements of higher plants provide a novel example. sieve elements form microfluidics systems for pressure-driven transport of photo-assimilates throughout the plant. Unique protein bodies in the sieve elements of legumes act as cellular stopcocks, by undergoing a Ca2+-dependent conformational switch in which they plug the sieve element. In living cells, this reaction is probably controlled by Ca2+-transporters in the cell membrane. Here we report the rapid, reversible, anisotropic and ATP-independent contractility in these protein bodies in vitro. Considering the unique biological function of the legume 'crystalloid' protein bodies and their contractile properties, we suggest to give them the distinctive name forisome ('gate-body'; from the Latin foris, the wing of a gate)

Effects of DNA and PEG concentrations on transfection efficiency.

<p>Oil palm protoplasts were transfected with 25 µg (A) or 50 µg (B) of CFDV-hrGFP plasmid DNA, and with 50 µg CFDV-hrGFP plasmid DNA in the presence of 25% (C), 40% (D) or 50% PEG (E). Black arrows indicate damaged protoplasts caused by PEG toxicity. The transfection efficiencies represent the mean of three replicates. Scale bar  = 10 µm in (A) and (B), 25 µm in (C)–(F).</p

Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in &asymp; 45% of the tubers being downgraded in quality and value, while &asymp; 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation

Effect of DNA concentration on transformation efficiency.

<p>Protoplasts were injected with 100/µl (A and B), 500 ng/µl (C and D) and 1000 ng/µl (E and F) DNA solutions and monitored for evidence of GFP fluorescence after culture for one month. The transformation efficiencies represent the mean of three replicates. Arrows indicate the surviving injected protoplasts and small dots indicate dead cells. Scale bar  = 100 µm. All cells were injected in the visial field shown in this figure, but uninjected cells also developed into (non-fluorescent) microcalli.</p

Microinjection of DNA into oil palm protoplasts.

<p>Oil palm protoplasts were isolated from a 3-month-old cell suspension culture after subculture for 7 days, mixed with 1% alginate solution in Y3A medium and distributed as a thin layer onto supporting medium (A). The embedded protoplasts were arranged in a single planar layer as confirmed by using the 10× objective (B). The protoplasts were incubated at 28°C in the dark for 3 days (C), and then placed on the microscope stage for DNA microinjection (D). The DNA solution was injected into the protoplast (E) and confirmed by Lucifer yellow fluorescence (F). GFP fluorescence was detected in the cytoplasm after 3 days (G and H). The injected protoplast is indicated by an arrow. Scale bar  = 1 cm in (A), (C) and (D), 100 µm in (B), 25 µm in (E)–(H).</p